ALBERT

Beschreibung: When the dependent (or response) variable response variable in an experiment has direction and magnitude, one approach that has been used for statistical analysis involves splitting magnitude and direction and applying univariate statistical techniques to the components. However, such treatment of quantities with direction and magnitude is not justifiable mathematically and can lead to incorrect conclusions about relationships among variables and, as a result, to flawed interpretations. This note discusses a problem with that practice and recommends mathematically correct procedures to be used with dependent variables that have direction and magnitude for 1) computation of mean values, 2) statistical contrasts of and confidence intervals for means, and 3) correlation methods.

Beschreibung: PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

Beschreibung: We suggest that intrinsic two-dimensional (i2D) features, computationally defined as the outputs of nonlinear operators that model the activity of end-stopped neurons, play a role in preattentive texture discrimination. We first show that for discriminable textures with identical power spectra the predictions of traditional models depend on the type of nonlinearity and fail for energy measures. We then argue that the concept of intrinsic dimensionality, and the existence of end-stopped neurons, can help us to understand the role of the nonlinearities. Furthermore, we show examples in which models without strong i2D selectivity fail to predict the correct ranking order of perceptual segregation. Our arguments regarding the importance of i2D features resemble the arguments of Julesz and co-workers regarding textons such as terminators and crossings. However, we provide a computational framework that identifies textons with the outputs of nonlinear operators that are selective to i2D features.

Beschreibung: Sixteen point probes monitored moisture level in the root medium of the wheat plants grown in greenhouse SVET on the MIR/NASA space science program. The article outlines types of water migration in the absence of gravity. Hydrophysical characteristics of perspective root media have been explored. Results of the water supply monitoring and control in the course of experiment are reported. The authors put forward porous root media to facilitate water migration and aeration.

Beschreibung: Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

Beschreibung: We have proposed previously a computational neural-network model by which the complex patterns of retinal image motion generated during locomotion (optic flow) can be processed by specialized detectors acting as templates for specific instances of self-motion. The detectors in this template model respond to global optic flow by sampling image motion over a large portion of the visual field through networks of local motion sensors with properties similar to those of neurons found in the middle temporal (MT) area of primate extrastriate visual cortex. These detectors, arranged within cortical-like maps, were designed to extract self-translation (heading) and self-rotation, as well as the scene layout (relative distances) ahead of a moving observer. We then postulated that heading from optic flow is directly encoded by individual neurons acting as heading detectors within the medial superior temporal (MST) area. Others have questioned whether individual MST neurons can perform this function because some of their receptive-field properties seem inconsistent with this role. To resolve this issue, we systematically compared MST responses with those of detectors from two different configurations of the model under matched stimulus conditions. We found that the characteristic physiological properties of MST neurons can be explained by the template model. We conclude that MST neurons are well suited to support self-motion estimation via a direct encoding of heading and that the template model provides an explicit set of testable hypotheses that can guide future exploration of MST and adjacent areas within the superior temporal sulcus.

Beschreibung: The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

Beschreibung: Surveys of larval habitats of Anopheles vestitipennis and Anopheles punctimacula were conducted in Belize, Central America. Habitat analysis and classification resulted in delineation of eight habitat types defined by dominant life forms and hydrology. Percent cover of tall dense macrophytes, shrubs, open water, and pH were significantly different between sites with and without An. vestitipennis. For An. punctimacula, percent cover of tall dense macrophytes, trees, detritus, open water, and water depth were significantly different between larvae positive and negative sites. The discriminant function for An. vestitipennis correctly predicted the presence of larvae in 65% of sites and correctly predicted the absence of larvae in 88% of sites. The discriminant function for An. punctimacula correctly predicted 81% of sites for the presence of larvae and 45% for the absence of larvae. Canonical discriminant analysis of the three groups of habitats (An. vestitipennis positive; An. punctimacula positive; all negative) confirmed that while larval habitats of An. punctimacula are clustered in the tree dominated area, larval habitats of An. vestitipennis were found in both tree dominated and tall dense macrophyte dominated environments. The forest larval habitats of An. vestitipennis and An. punctimacula seem to be randomly distributed among different forest types. Both species tend to occur in denser forests with more detritus, shallower water, and slightly higher pH. Classification of dry season (February) SPOT multispectral satellite imagery produced 10 land cover types with the swamp forest and tall dense marsh classes being of particular interest. The accuracy assessment showed that commission errors for the tall, dense marsh and swamp forest appeared to be minor; but omission errors were significant, especially for the swamp forest (perhaps because no swamp forests are flooded in February). This means that where the classification indicates there are An. vestitipennis breeding sites, they probably do exist; but breeding sites in many locations are not identified and could be more abundant than indicated.

Beschreibung: Otolith-induced eye movements of rhesus monkeys were studied before and after the 1989 COSMOS 2044 and the 1992 to 1993 COSMOS 2229 flights. Two animals flew in each mission for approximately 2 weeks. After flight, spatial orientation of the angular vestibulo-ocular reflex was altered. In one animal the time constant of postrotatory nystagmus, which had been shortened by head tilts with regard to gravity before flight, was unaffected by the same head tilts after flight. In another animal, eye velocity, which tended to align with a gravitational axis before flight, moved toward a body axis after flight. This shift of orientation disappeared by 7 days after landing. After flight, the magnitude of compensatory ocular counter-rolling was reduced by about 70% in both dynamic and static tilts. Modulation in vergence in response to naso-occipital linear acceleration during off-vertical axis rotation was reduced by more than 50%. These changes persisted for 11 days after recovery. An up and down asymmetry of vertical nystagmus was diminished for 7 days. Gains of the semicircular canal-induced horizontal and vertical angular vestibulo-ocular reflexes were unaffected in both flights, but the gain of the roll angular vestibulo-ocular reflex was decreased. These data indicate that there are short- and long-term changes in otolith-induced eye movements after adaptation to microgravity. These experiments also demonstrate the unique value of the monkey as a model for studying effects of vestibular adaptation in space. Eye movements can be measured in three dimensions in response to controlled vestibular and visual stimulation, and the results are directly applicable to human beings. Studies in monkeys to determine how otolith afferent input and central processing is altered by adaptation to microgravity should be an essential component of future space-related research.

Beschreibung: The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Beschreibung: Perceived movement of a stationary visual stimulus during head motion was measured before and after adaptation intervals during which participants performed voluntary head oscillations while viewing a moving spot. During these intervals, participants viewed the spot stimulus moving alternately in the same direction as the head was moving during either .25- or 2.0-Hz oscillations, and then in the opposite direction as the head at the other of the two frequencies. Postadaptation measures indicated that the visual stimuli were perceived as stationary only if traveling in the same direction as that viewed during adaptation at the same frequency of head motion. Thus, opposite directions of spot motion were perceived as stationary following adaptation depending on head movement frequency. The results provide an example of the ability to establish dual (or "context-specific") adaptations to altered visual-vestibular feedback.

Beschreibung: The effects of two different duration space-flights on the extent of atrophy, fiber type composition, and myosin heavy chain (MHC) content of rat soleus muscles were compared. Adult male Fisher rats (n=12) were aboard flight STS-57 and exposed to 10 days of microgravity and adult ovariectomized female Spraque-Dawley rats (n=12) were aboard flight STS-62 for 14 days. Soleus muscles were bilaterally removed from the flight and control animals and frozen for subsequent analyses. Muscle wet weights, fiber types (I, IC, IIC, and IIA), cross-sectional area, and MHC content were determined. Although a significant difference was found between the soleus wet weights of the two ground-based control groups, they were similar with regard to MHC content (ca 90% MHCI and ca 10% MHCIIa) and fiber type composition. Unloading of the muscles caused slow-to-fast transformations which included a decrease in the percentage of type I fibers and MHCI, an increase in fibers classified as type IC, and the expression of two fast myosin heavy chains not found in the control rat soleus muscles (MHCIId and MHCIIb). Although the amount of atrophy (ca 26%) and the extent of slow-to-fast transformation (decrease in the percentage of MHCI from 90% to 82.5%) in the soleus muscles were similar between the two spaceflights, the percentages of the fast MHCs differed. After 14 days of spaceflight, the percentage of MHCIIa was significantly lower and the percentages of MHCIId and MHCIIb were significantly higher than the corresponding MHC content of the soleus muscles from the 10-day animals. Indeed, MHCIId became the predominant fast MHC after 14 days in space. These data suggest fast-to-faster transformations continued during the longer spaceflight.

Beschreibung: Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

Beschreibung: The polarity of germinating single-celled spores of the fern Ceratopteris richardii Brogn. is influenced by gravity during a time period prior to the first cellular division designated a "polarity-determination window". After this window closes, control of polarity is seen in the downward (with respect to gravity) migration of the nucleus along the proximal face of the spore and the subsequent downward growth of the primary rhizoid. When spores are germinated on a clinostat the direction of nuclear migration and subsequent primary rhizoid growth is random. However, in each case the direction of nuclear migration predicts the direction of rhizoid elongation. Although it is the most obvious movement, the downward migration is not the first movement of the nucleus. During the polarity-determination window, the nucleus moves randomly within a region centered behind the trilete marking. While the polarity of many fern spores has been reported to be controlled by light, spores of C. richardii are the first documented to have their polarity influenced by gravity. Directional white light also affects the polarity of these spores, but this influence is slight and is secondary to that of gravity.

Beschreibung: This paper presents a method for segmentation and tracking of cardiac structures in ultrasound image sequences. The developed algorithm is based on the active contour framework. This approach requires initial placement of the contour close to the desired position in the image, usually an object outline. Best contour shape and position are then calculated, assuming that at this configuration a global energy function, associated with a contour, attains its minimum. Active contours can be used for tracking by selecting a solution from a previous frame as an initial position in a present frame. Such an approach, however, fails for large displacements of the object of interest. This paper presents a technique that incorporates the information on pixel velocities (optical flow) into the estimate of initial contour to enable tracking of fast-moving objects. The algorithm was tested on several ultrasound image sequences, each covering one complete cardiac cycle. The contour successfully tracked boundaries of mitral valve leaflets, aortic root and endocardial borders of the left ventricle. The algorithm-generated outlines were compared against manual tracings by expert physicians. The automated method resulted in contours that were within the boundaries of intraobserver variability.

Beschreibung: We complied a 5.8S nuclear ribosomal gene sequence database for animals, plants, and fungi using both newly generated and GenBank sequences. We demonstrate the utility of this database as an internal check to determine whether the target organism and not a contaminant has been sequenced, as a diagnostic tool for ecologists and evolutionary biologists to determine the placement of asexual fungi within larger taxonomic groups, and as a tool to help identify fungi that form ectomycorrhizae.

Beschreibung: Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.

Beschreibung: The restoration of body fluid balance following dehydration induced by exercise will occur through regulatory responses which stimulate ingestion of water and sodium ions. A number of different afferent signalling systems are necessary to generate appropriate thirst or sodium appetite. The primary sensory information of naturally occurring thirst is derived from receptors sensing cell volume and the volume of the extracellular fluid compartment. Sensory information from the oropharyngeal region is also an important determinant of thirst. The interaction of these various afferent signalling systems within the central nervous system determines the extent of fluid replacement following dehydration.

Beschreibung: Intramuscular promethazine (PMZ) is used aboard the US Space Shuttle to ameliorate symptoms of space motion sickness. Bioavailability after an oral dose of PMZ during space flight is thought to be impaired because of gastrointestinal disturbances associated with weightlessness and space motion sickness. In an attempt to find an alternative dosage form for use in space, we evaluated two intranasal (i.n.) dosage forms of PMZ in dogs for absorption and bioavailability relative to that of an equivalent intramuscular dose. Promethazine (5 mg kg-1) was administered as two intranasal dosage forms and as an intramuscular (i.m.) dose to three dogs in a randomised cross-over design. Serial blood samples were taken and analysed for PMZ concentrations and the absorption and bioavailability of PMZ were calculated for the three dosage forms. PMZ absorption from the carboxymethyl cellulose microsphere i.n. dosage form was more rapid and complete than from the myverol cubic gel formulation or from an i.m. injection. Bioavailability of the microsphere formulation was also greater than that of the gel formulation (AUC 3009 vs 1727 ng h ml-1). The bioavailability of the two i.n. dosage forms (relative to that of the i.m. injection) were 94% (microsphere) and 54% (gel). The i.n. microsphere formulation of PMZ offers great promise as an effective non-invasive alternative for treating space motion sickness due to its rapid absorption and bioavailability equivalent to the i.m. dose.

Beschreibung: Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

Beschreibung: The mitotic inhibitor, chloral hydrate, induces ciliary loss in the early embryo phase of Lytechinus pictus. It causes a breakdown of cilia at the junction of the cilium and the basal body known as the basal plate. This leaves the plasma membrane temporarily unsealed. The basal apparatus accessory structures, consisting of the basal body, basal foot, basal foot cap, striated side arm, and striated rootlet, are either misaligned or disintegrated by treatment with chloral hydrate. Furthermore, microtubules which are associated with the basal apparatus are disassembled. Mitochondria accumulate at the base of cilia - underneath the plasma membrane - and show alterations in their structural organization. The accumulation of mitochondria is observed in 40% of all electron micrograph sections while 60% show the areas mostly devoid of mitochondria. The microvilli surrounding a cilium and striated rootlet remain intact in the presence of chloral hydrate. These results suggest that deciliation in early sea urchin embryos by chloral hydrate is caused by combined effects on the ciliary membrane and on microtubules in the cilia. Furthermore, it is suggested that chloral hydrate can serve as a tool to explore the cytoskeletal mechanisms that are involved in cilia motility in the developing sea urchin embryo.

Beschreibung: A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

Beschreibung: Many colorectal cancers have high levels of cyclo-oxygenase 2 (COX-2), an enzyme that metabolizes the essential fatty acids into prostaglandins. Since the low-density lipoprotein receptor (LDLr) is involved in the uptake of essential fatty acids, we studied the effect of LDL on growth and gene regulation in colorectal cancer cells. DiFi cells grown in lipoprotein-deficient sera (LPDS) grew more slowly than cells with LDL. LDLr antibody caused significant inhibition of tumor cell growth but did not affect controls. In addition, LDL uptake did not change in the presence of excess LDL, suggesting that ldlr mRNA lacks normal feedback regulation in some colorectal cancers. Analysis of the ldlr mRNA showed that excess LDL in the medium did not cause down-regulation of the message even after 24 hr. The second portion of the study examined the mRNA expression of ldlr and its co-regulation with cox-2 in normal and tumor specimens from patients with colorectal adenocarcinomas. The ratio of tumor:paired normal mucosa of mRNA expression of ldlr and of cox-2 was measured in specimens taken during colonoscopy. ldlr and cox-2 transcripts were apparent in 11 of 11 carcinomas. There was significant coordinate up-regulation both of ldlr and of cox-2 in 6 of 11 (55%) tumors compared with normal colonic mucosa. There was no up-regulation of cox-2 without concomitant up-regulation of ldlr. These data suggest that the LDLr is abnormally regulated in some colorectal tumors and may play a role in the up-regulation of cox-2. Copyright 1999 Wiley-Liss, Inc.

Beschreibung: Following spaceflight, changes in renal function of humans have been suggested. To assess the effects of readaptation on renal function, urine was collected from male rats ( approximately 245 g) over a 2-wk period following a 14-day spaceflight. Rats were assigned to three groups: flight animals (n = 6), flight controls (n = 6) housed in the flight cages on the ground, and vivarium controls (n = 5) housed in standard shoe box cages. Animals were placed into individual metabolic cages for urine collection. Urine output was significantly increased for 3 days following flight. Excretion rates of Na+ and K+ were increased, resulting in an increased osmotic excretion rate. Creatinine excretion rate increased over the first two postflight days. Glomerular filtration rate increased immediately following spaceflight without changes in plasma creatinine, Na+, K+, or osmolality. Increased excretion of solute was thus the result of increased delivery and a decreased percent reabsorption of the filtered load. Osmolal clearance was increased immediately postflight while free water clearance was decreased. In growing rats, the diuresis after short-duration spaceflight is the result of an increase in solute excretion with an accompanying reduction in free water clearance.

Beschreibung: No abstract available

Beschreibung: Extended exposure to weightlessness results in bone loss. However, little information exists as to the precise nature or time course of this bone loss. Bone resorption results in the release of collagen breakdown products, including N-telopeptide and the pyridinium (PYD) cross-links, pyridinoline and deoxypyridinoline. Urinary pyridinoline and deoxypyridinoline are known to increase during bed rest. We assessed excretion of PYD cross-links and N-telopeptide before, during, and after long (28-day, 59-day, and 84-day) Skylab missions, as well as during short (14-day) and long (119-day) bed-rest studies. During space flight, the urinary cross-link excretion level was twice those observed before flight. Urinary excretion levels of the collagen breakdown products were also 40-50% higher, during short and long bed rest, than before. These results clearly show that the changes in bone metabolism associated with space flight involve increased resorption. The rate of response (i.e. within days to weeks) suggests that alterations in bone metabolism are an early effect of weightlessness. These studies are important for a better understanding of bone metabolism in space crews and in those who are bedridden.

Beschreibung: The primary objective of this study was to evaluate the antitumor effects of recombinant vaccinia virus-p53 (rVV-p53) in combination with radiation therapy against the C6 rat glioma, a p53 deficient tumor that is relatively radioresistant. VV-LIVP, the parental virus (Lister strain), was used as a control. Localized treatment of subcutaneous C6 tumors in athymic mice with either rVV-p53 or VV-LIVP together with tumor irradiation resulted in low tumor incidence and significantly slower tumor progression compared to the agents given as single modalities. Assays of blood and spleen indicated that immune system activation may account, at least partly, for the enhance tumor inhibition seen with combined treatment. No overt signs of treatment-related toxicity were noted.

Beschreibung: INTRODUCTION: The current standard for arrhythmic risk stratification is electrophysiologic (EP) testing, which, due to its invasive nature, is limited to patients already known to be at high risk. A number of noninvasive tests, such as determination of left ventricular ejection fraction (LVEF) or heart rate variability, have been evaluated as additional risk stratifiers. Microvolt T wave alternans (TWA) is a promising new risk marker. Prospective evaluation of noninvasive risk markers in low- or moderate-risk populations requires studies involving very large numbers of patients, and in such studies, documentation of the occurrence of ventricular tachyarrhythmias is difficult. In the present study, we identified a high-risk population, recipients of an implantable cardioverter defibrillator (ICD), and prospectively compared microvolt TWA with invasive EP testing and other risk markers with respect to their ability to predict recurrence of ventricular tachyarrhythmias as documented by ICD electrograms. METHODS AND RESULTS: Ninety-five patients with a history of ventricular tachyarrhythmias undergoing implantation of an ICD underwent EP testing, assessment of TWA, as well as determination of LVEF, baroreflex sensitivity, signal-averaged ECG, analysis of 24-hour Holter monitoring, and QT dispersion from the 12-lead surface ECG. The endpoint of the study was first appropriate ICD therapy for electrogram-documented ventricular fibrillation or tachycardia during follow-up. Kaplan-Meier survival analysis revealed that TWA (P 〈 0.006) and LVEF (P 〈 0.04) were the only significant univariate risk stratifiers. EP testing was not statistically significant (P 〈 0.2). Multivariate Cox regression analysis revealed that TWA was the only statistically significant independent risk factor. CONCLUSIONS: Measurement of microvolt TWA compared favorably with both invasive EP testing and other currently used noninvasive risk assessment methods in predicting recurrence of ventricular tachyarrhythmias in ICD recipients. This study suggests that TWA might also be a powerful tool for risk stratification in low- or moderate-risk patients, and needs to be prospectively evaluated in such populations.

Beschreibung: The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

Beschreibung: Nitric oxide is hypothesized to be an inhibitory modulator of central sympathetic nervous outflow, and deficient neuronal nitric oxide production to cause sympathetic overactivity, which then contributes to nitric-oxide-deficient hypertension. The biochemical and neuroanatomical basis for this concept revolves around nitric oxide modulation of glutamatergic neurotransmission within brainstem vasomotor centers. The functional consequence of neuronal nitric oxide in blood pressure regulation is, however, marked by an apparent conflict in the literature. On one hand, conscious animal studies using sympathetic blockade suggest a significant role for neuronal nitric oxide deficiency in the development of nitric-oxide-deficient hypertension, and on the other hand, there is evidence against such a role derived from 'knock-out' mice lacking nitric-oxide synthase 1, the major source of neuronal nitric oxide.

Beschreibung: No abstract available

Beschreibung: The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

Beschreibung: Whole body heat production (HP) and heat loss (HL) were examined to determine their relative contributions to light masking of the circadian rhythm in body temperature (Tb). Squirrel monkey metabolism (n = 6) was monitored by both indirect and direct calorimetry, with telemetered measurement of body temperature and activity. Feeding was also measured. Responses to an entraining light-dark (LD) cycle (LD 12:12) and a masking LD cycle (LD 2:2) were compared. HP and HL contributed to both the daily rhythm and the masking changes in Tb. All variables showed phase-dependent masking responses. Masking transients at L or D transitions were generally greater during subjective day; however, L masking resulted in sustained elevation of Tb, HP, and HL during subjective night. Parallel, apparently compensatory, changes of HL and HP suggest action by both the circadian timing system and light masking on Tb set point. Furthermore, transient HL increases during subjective night suggest that gain change may supplement set point regulation of Tb.

Beschreibung: In the present study, F ratios for both stable chromosome aberrations, i.e. ratios of translocations to pericentric inversions, and unstable aberrations, i.e. dicentrics and centric rings, were measured using fluorescence in situ hybridization. F ratios for stable aberrations measured after exposure to low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 1.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiation were 6.5 +/- 1.5, 4.7 +/- 1.6, 9.3 +/- 2.5 and 10.4 +/- 3.0, respectively. F ratios for unstable aberrations measured after low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiations were 6.5 +/- 1.6, 6.3 +/- 2.3 and 11.1 +/- 3.7, respectively. No significant difference between the F ratios for low- and high-LET radiation was found. Further tests on the models for calculation of the F ratio proposed by Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) showed that the F ratio may not be straightforward as a practical fingerprint for densely ionizing radiation.

Beschreibung: This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of 125I-labeled CaM at both micromolar and nanomolar Ca2+ concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (〈3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free 125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.

Beschreibung: PURPOSE: The objective was to investigate the ability of a glycosteroid (TC002) to increase the oral bioavailability of gentamicin. METHODS: Admixtures of gentamicin and TC002 were administered to the rat ileum by injection and to dogs by ileal or jejunal externalized ports, or PO. Bioavailability of gentamicin was determined by HPLC. 3H-TC002 was injected via externalized cannulas into rat ileum or jejunum, or PO and its distribution and elimination was determined. The metabolism of TC002 in rats was evaluated by solid phase extraction and HPLC analysis of plasma, urine and feces following oral or intestinal administration. RESULTS: The bioavailability of gentamicin was substantially increased in the presence of TC002 in both rats and dogs. The level of absorption was dependent on the concentration of TC002 and site of administration. Greatest absorption occurred following ileal orjejunal administration. TC002 was significantly more efficacious than sodium taurocholate, but similar in cytotoxicity. TC002 remained primarily in the GI tract following oral or intestinal administration and cleared rapidly from the body. It was only partly metabolized in the GI tract, but was rapidly and completely converted to its metabolite in plasma and urine. CONCLUSIONS: TC002 shows promise as a new drug transport agent for promoting intestinal absorption of polar molecules such as gentamicin.

Beschreibung: Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis. The present study evaluated the influence of the metabolic stressor 2-deoxy-D-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF1 mice were injected with 2-DG (500 mg/kg b. wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L. monocytogenes. Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection. In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge. The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments. First, 2-DG did not inhibit listeria replication in trypticase soy broth. Second, replication of L. monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG. Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L. monocytogenes. These results support the contention that the host response to 2-DG-induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens.

Beschreibung: These experiments tested the hypothesis that the ability to change sensorimotor set quickly for automatic responses depends on the time interval between successive surface perturbations. Sensorimotor set refers to the influence of prior experience or context on the state of the sensorimotor system. Sensorimotor set for postural responses was influenced by first giving subjects a block of identical backward translations of the support surface, causing forward sway and automatic gastrocnemius responses. The ability to change set quickly was inferred by measuring the suppression of the stretched antagonist gastrocnemius responses to toes-up rotations causing backward sway, following the translations. Responses were examined under short (10-14 s) and long (19-24 s) inter-trial intervals in young healthy subjects. The results showed that subjects in the long-interval group changed set immediately by suppressing gastrocnemius to 51% of translation responses within the first rotation and continued to suppress them over succeeding rotations. In contrast, subjects in the short-interval group did not change set immediately, but required two or more rotations to suppress gastrocnemius responses. By the last rotation, the short-interval group suppressed gastrocnemius responses to 33%, similar to the long-interval group of 29%. Associated surface plantarflexor torque resulting from these responses showed similar results. When rotation and translation perturbations alternated, however, the short-interval group was not able to suppress gastrocnemius responses to rotations as much as the long-interval group, although they did suppress more than in the first rotation trial after a series of translations. Set for automatic responses appears to linger, from one trial to the next. Specifically, sensorimotor set is more difficult to change when surface perturbations are given in close succession, making it appear as if set has become progressively stronger. A strong set does not mean that responses become larger over consecutive trials. Rather, it is inferred by the extent of difficulty in changing a response when it is appropriate to do so. These results suggest that the ability to change sensorimotor set quickly is sensitive to whether the change is required after a long or a short series of a prior different response, which in turn depends on the time interval between successive trials. Different rate of gastrocnemius suppression to toes-up rotation of the support surface have been reported in previous studies. This may be partially explained by different inter-trial time intervals demonstrated in this study.

Beschreibung: Relatively low levels of expression from naturally occurring promoters have limited the use of muscle as a gene therapy target. Myogenic restricted gene promoters display complex organization usually involving combinations of several myogenic regulatory elements. By random assembly of E-box, MEF-2, TEF-1, and SRE sites into synthetic promoter recombinant libraries, and screening of hundreds of individual clones for transcriptional activity in vitro and in vivo, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters.

Beschreibung: MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Beschreibung: Circadian rhythms of activity (Act) and body temperature (Tb) were recorded from male Syrian hamsters under square-wave (LDSq) and simulated natural (LDSN, with dawn and dusk transitions) light-dark cycles. Light intensity and data sampling were under the synchronized control of a laboratory computer. Changes in reactive and predictive onsets and offsets for the circadian rhythms of Act and Tb were examined in both lighting conditions. The reactive Act onset occurred 1.1 h earlier (P 〈 0.01) in LDSN than in LDSq and had a longer alpha-period (1.7 h; P 〈 0.05). The reactive Tb onset was 0.7 h earlier (P 〈 0.01) in LDSN. In LDSN, the predictive Act onset advanced by 0.3 h (P 〈 0.05), whereas the Tb predictive onset remained the same as in LDSq. The phase angle difference between Act and Tb predictive onsets decreased by 0.9 h (P 〈 0.05) in LDSN, but the offsets of both measures remained unchanged. In this study, animals exhibited different circadian entrainment characteristics under LDSq and LDSN, suggesting that gradual and abrupt transitions between light and dark may provide different temporal cues.

Beschreibung: 1. Soleus biopsies were obtained from four male astronauts 45 days before and within 2 h after a 17 day spaceflight. 2. For all astronauts, single chemically skinned post-flight fibres expressing only type I myosin heavy chain (MHC) developed less average peak Ca2+ activated force (Po) during fixed-end contractions (0.78 +/- 0. 02 vs. 0.99 +/- 0.03 mN) and shortened at a greater mean velocity during unloaded contractions (Vo) (0.83 +/- 0.02 vs. 0.64 +/- 0.02 fibre lengths s-1) than pre-flight type I fibres. 3. The flight-induced decline in absolute Po was attributed to reductions in fibre diameter and/or Po per fibre cross-sectional area. Fibres from the astronaut who experienced the greatest relative loss of peak force also displayed a reduction in Ca2+ sensitivity. 4. The elevated Vo of the post-flight slow type I fibres could not be explained by alterations in myosin heavy or light chain composition. One alternative possibility is that the elevated Vo resulted from an increased myofilament lattice spacing. This hypothesis was supported by electron micrographic analysis demonstrating a reduction in thin filament density post-flight. 5. Post-flight fibres shortened at 30 % higher velocities than pre-flight fibres at external loads associated with peak power output. This increase in shortening velocity either reduced (2 astronauts) or prevented (2 astronauts) a post-flight loss in fibre absolute peak power (microN (fibre length) s-1). 6. The changes in soleus fibre diameter and function following spaceflight were similar to those observed after 17 days of bed rest. Although in-flight exercise countermeasures probably reduced the effects of microgravity, the results support the idea that ground-based bed rest can serve as a model of human spaceflight. 7. In conclusion, 17 days of spaceflight decreased force and increased shortening velocity of single Ca2+-activated muscle cells expressing type I MHC. The increase in shortening velocity greatly reduced the impact that impaired force production had on absolute peak power.

Beschreibung: Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

Beschreibung: Long-term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. In this study a new plasmid-based human tumor necrosis factor-alpha (TNF-alpha) expression vector was synthesized (pGL1-TNF-alpha) and evaluated together with radiation in the aggressive, rapidly growing C6 rat glioma model. pGL1-TNF-alpha was successfully transfected into C6 cells in vitro using a cationic polyamine method. Expression was detected up to 7 days and averaged 0.4 ng of TNF-alpha in the culture medium from 1x10(5) cells. The expressed protein was biologically functional, as evidenced by growth inhibition of L929, a TNF-alpha-susceptible cell line. Using fluorescence-labeled monoclonal antibodies and laser scanning cytometry, we confirmed that both the P55 and P75 receptors for TNF-alpha were present on the C6 cell membrane. However, the receptors were present at low density and P55 was expressed more than the P75 receptor. These findings were in contrast to results obtained with TNF-alpha-susceptible L929 cells. Tests in athymic mice showed that pGL1-TNF-alpha administered intratumorally 16-18 h before radiation (each modality given three times) significantly inhibited C6 tumor progression (P〈0.05). This effect was more than additive, because pGL1-TNF-alpha alone did not slow tumor growth and radiation alone had little effect on tumor growth. These results indicate that pGL1-TNF-alpha has potential to augment the antitumor effects of radiation against a tumor type that is virtually incurable.

Beschreibung: The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.

Beschreibung: Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

Beschreibung: The ectostriatum is a major visual component of the avian telencephalon. The core region of the ectostriatum (Ec) receives visual input from the optic tectum through thalamic nuclei. In the present study, the efferent projections of the ectostriatum were investigated by using the anterograde tracers Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine. Projection patterns resulting from these tracers were confirmed by the retrograde tracer cholera toxin subunit B. When anterograde tracers were injected in Ec, primary projections were seen traveling dorsolaterally to the belt region of the ectostriatum (Ep) and the neostriatal area immediately surrounding Ep (Ep2). Neurons in Ep sent projections primarily to the overlying Ep2. The efferents of Ep2 traveled dorsolaterally to terminate in three telencephalic regions, from anterior to posterior: (1) neostriatum frontale, pars lateralis (NFL), (2) area temporo-parieto-occipitalis (TPO), and (3) neostriatum intermedium, pars lateralis (NIL). A part of the archistriatum intermedium and the lateral part of the neostriatum caudale also received somewhat minor projections. In addition, some neurons in Ec were also the source of direct, but minor, projections to the NFL, TPO, NIL, and archistriatum intermedium. The topographical relationship among the primary (Ec), secondary (Ep and Ep2), and tertiary (NFL, TPO, NIL) areas indicate that the neural populations for visual processing are organized along the rostral-caudal axis. Thus, the anterior Ec sent efferents to the anterior Ep, which in turn sent projections to anterior Ep2. Neurons in the anterior Ep2 sent projections to NFL and the anterior TPO. Similarly, the intermediate and posterior Ec sent projections to corresponding parts of Ep, whose efferents projected to intermediate and posterior Ep2, respectively. The intermediate Ep2 gave rise to major projections to TPO, whereas posterior Ep2 neurons sent efferents primarily to NIL. The organization of this neural circuit is compared with those of other sensory circuits in the avian telencephalon, as well as the laminar arrangement of the mammalian isocortex.

Beschreibung: A number of new methods have been recently developed to quantify complex heart rate (HR) dynamics based on nonlinear and fractal analysis, but their value in risk stratification has not been evaluated. This study was designed to determine whether selected new dynamic analysis methods of HR variability predict mortality in patients with depressed left ventricular (LV) function after acute myocardial infarction (AMI). Traditional time- and frequency-domain HR variability indexes along with short-term fractal-like correlation properties of RR intervals (exponent alpha) and power-law scaling (exponent beta) were studied in 159 patients with depressed LV function (ejection fraction 〈35%) after an AMI. By the end of 4-year follow-up, 72 patients (45%) had died and 87 (55%) were still alive. Short-term scaling exponent alpha (1.07 +/- 0.26 vs 0.90 +/- 0.26, p 〈0.001) and power-law slope beta (-1.35 +/- 0.23 vs -1.44 +/- 0.25, p 〈0.05) differed between survivors and those who died, but none of the traditional HR variability measures differed between these groups. Among all analyzed variables, reduced scaling exponent alpha (〈0.85) was the best univariable predictor of mortality (relative risk 3.17, 95% confidence interval 1.96 to 5.15, p 〈0.0001), with positive and negative predictive accuracies of 65% and 86%, respectively. In the multivariable Cox proportional hazards analysis, mortality was independently predicted by the reduced exponent alpha (p 〈0.001) after adjustment for several clinical variables and LV function. A short-term fractal-like scaling exponent was the most powerful HR variability index in predicting mortality in patients with depressed LV function. Reduction in fractal correlation properties implies more random short-term HR dynamics in patients with increased risk of death after AMI.

Beschreibung: The traditional methods of analyzing heart rate (HR) variability have failed to predict imminent ventricular fibrillation (VF). We sought to determine whether new methods of analyzing RR interval variability based on nonlinear dynamics and fractal analysis may help to detect subtle abnormalities in RR interval behavior before the onset of life-threatening arrhythmias. RR interval dynamics were analyzed from 24-hour Holter recordings of 15 patients who experienced VF during electrocardiographic recording. Thirty patients without spontaneous or inducible arrhythmia events served as a control group in this retrospective case control study. Conventional time- and frequency-domain measurements, the short-term fractal scaling exponent (alpha) obtained by detrended fluctuation analysis, and the slope (beta) of the power-law regression line (log power - log frequency, 10(-4)-10(-2) Hz) of RR interval dynamics were determined. The short-term correlation exponent alpha of RR intervals (0.64 +/- 0.19 vs 1.05 +/- 0.12; p 〈0.001) and the power-law slope beta (-1.63 +/- 0.28 vs -1.31 +/- 0.20, p 〈0.001) were lower in the patients before the onset of VF than in the control patients, but the SD and the low-frequency spectral components of RR intervals did not differ between the groups. The short-term scaling exponent performed better than any other measurement of HR variability in differentiating between the patients with VF and controls. Altered fractal correlation properties of HR behavior precede the spontaneous onset of VF. Dynamic analysis methods of analyzing RR intervals may help to identify abnormalities in HR behavior before VF.

Beschreibung: Color Doppler images of aortic regurgitation (AR) flow acceleration, flow convergence (FC), and the vena contracta (VC) have been reported to be useful for evaluating severity of AR. However, clinical application of these methods has been limited because of the difficulty in clearly imaging the FC and VC. This study aimed to explore new windows for imaging the FC and VC to evaluate AR volumes in patients and to validate this in animals with chronic AR. Forty patients with AR and 17 hemodynamic states in 4 sheep with strictly quantified AR volumes were evaluated. A Toshiba SSH 380A with a 3.75-MHz transducer was used to image the FC and VC. After routine echo Doppler imaging, patients were repositioned in the right lateral decubitus position, and the FC and VC were imaged from high right parasternal windows. In only 15 of the 40 patients was it possible to image clearly and measure accurately the FC and VC from conventional (left decubitus) apical or parasternal views. In contrast, 31 of 40 patients had clearly imaged FC regions and VCs using the new windows. In patients, AR volumes derived from the FC and VC methods combined with continuous velocity agreed well with each other (r = 0.97, mean difference = -7.9 ml +/- 9.9 ml/beat). In chronic animal model studies, AR volumes derived from both the VC and the FC agreed well with the electromagnetically derived AR volumes (r = 0.92, mean difference = -1.3 +/- 4.0 ml/beat). By imaging from high right parasternal windows in the right decubitus position, complementary use of the FC and VC methods can provide clinically valuable information about AR volumes.

Beschreibung: Errors introduced using a tissue equivalent proportional counter to estimate radiation quality of an arbitrary ion field as related to space radiations are examined. This is accomplished by using a generalized analytic model to calculate the effect of energy loss straggling, track structure, and pathlength distribution on the microdosimetric distribution. The error can be as large as a factor of two, but no systematic trend could be found.

Beschreibung: Bone biopsies indicate that during immobilization bone marrow adipose tissue increases while the functional cellular fraction decreases. One objective of our Spacelab flight experiment was to determine, using in vivo volume-localized magnetic resonance spectroscopy (VLMRS), whether bone marrow composition was altered by space flight. Four crew members of a 17 day Spacelab mission participated in the experiment. The apparent cellular fraction and transverse relaxation time (T2) were determined twice before launch and at several times after flight. Immediately after flight, no significant change in the cellular fraction was found. However, the T2 of the cellular, but not the fat component increased following flight, although to a variable extent, in all crew members with a time course for return to baseline lasting several months. The T2 of seven control subjects showed no significant change. Although these observations may have several explanations, it is speculated that the observed T2 changes might reflect increased marrow osteoblastic activity during recovery from space flight.

Beschreibung: Previous observations have shown that neutrophil invasion precedes macrophage invasion during muscle inflammation and that peak muscle injury is observed at the peak of ED1+ macrophage invasion. We tested the hypothesis that neutrophil invasion causes subsequent invasion by ED1+ macrophages and that ED1+ macrophages then contribute significantly to muscle membrane injury during modified muscle use. Rat hindlimbs were unloaded for 10 days followed by reloading by normal ambulation to induce inflammation. Membrane injury was measured by assaying Evans blue-bound serum protein influx through membrane lesions. Muscle neutrophil populations increased significantly during the first 2 h of reloading but ED1+ macrophages did not increase until 24 h. Neutrophil invasion was uncoupled from subsequent macrophage invasion by reloading rat hindlimbs for 2 h to cause neutrophil invasion, followed by resuspension for hours 2-24. This produced similar increases in neutrophil concentration as measured in muscles continuously reloaded for 24 h without causing an increase in macrophages. However, resuspension did not reduce the extent of muscle damage compared with that occurring in muscles that were reloaded continuously for 24 h. Thus, muscle invasion by neutrophils is not sufficient to cause invasion by ED1+ macrophages. In addition, muscle membrane injury that occurs during reloading is independent of invasion by ED1+ macrophages.

Beschreibung: A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

Beschreibung: Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

Beschreibung: This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.

Beschreibung: No abstract available

Beschreibung: Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (〈17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or 〈10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.

Beschreibung: Microgravity provides a unique environment in which to study normal and pathological phenomenon. Very few studies have been done to examine the effects of microgravity on developing skeletal tissue such as growth plate formation and maintenance, elongation of bone primordia, or the mineralization of growth plate cartilage. Embryonic mouse premetatarsal triads were cultured on three space shuttle flights to study cartilage growth, differentiation, and mineralization, in a microgravity environment. The premetatarsal triads that were cultured in microgravity all formed cartilage rods and grew in length. However, the premetatarsal cartilage rods cultured in microgravity grew less in length than the ground control cartilage rods. Terminal chondrocyte differentiation also occurred during culture in microgravity, as well as in the ground controls, and the matrix around the hypertrophied chondrocytes was capable of mineralizing in both groups. The same percentage of premetatarsals mineralized in the microgravity cultures as mineralized in the ground control cultures. In addition, the sizes of the mineralized areas between the two groups were very similar. However, the amount of 45Ca incorporated into the mineralized areas was significantly lower in the microgravity cultures, suggesting that the composition or density of the mineralized regions was compromised in microgravity. There was no significant difference in the amount of 45Ca liberated from prelabeled explants in microgravity or in the ground controls.

Beschreibung: Whole body heat production (HP) and heat loss (HL) were examined to determine if the free-running circadian rhythm in body temperature (Tb) results from coordinated changes in HP and HL rhythms in thermoneutrality (27 degrees C) as well as mild cold (17 degrees C). Squirrel monkey metabolism (n = 6) was monitored by both indirect and direct calorimetry, with telemetered measurement of Tb and activity. Feeding was also measured. Rhythms of HP, HL, and conductance were tightly coupled with the circadian Tb rhythm at both ambient temperatures (TA). At 17 degrees C, increased HP compensated for higher HL at all phases of the Tb rhythm, resulting in only minor changes to Tb. Parallel compensatory changes of HP and HL were seen at all rhythm phases at both TA. Similar time courses of Tb, HP, and HL in their respective rhythms and the relative stability of Tb during both active and rest periods suggest action of the circadian timing system on Tb set point.

Beschreibung: Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.

Beschreibung: The nucleus rotundus is a large thalamic nucleus in birds and plays a critical role in many visual discrimination tasks. In order to test the hypothesis that there are functionally distinct subdivisions in the nucleus rotundus, effects of selective lesions of the nucleus were studied in pigeons. The birds were trained to discriminate between different types of stationary objects and between different directions of moving objects. Multiple regression analyses revealed that lesions in the anterior, but not posterior, division caused deficits in discrimination of small stationary stimuli. Lesions in neither the anterior nor posterior divisions predicted effects in discrimination of moving stimuli. These results are consistent with a prediction led from the hypothesis that the nucleus is composed of functional subdivisions.

Beschreibung: A technique is introduced for the measurement of plantar soft tissue thickness during barefoot walking. Subjects stepped into an adjustable Plexiglas frame which ensured that the required bony landmarks were appropriately positioned relative to a linear ultrasound probe connected to a conventional 7.5 MHz ultrasound scanner. Clear images of the metatarsal condyles or other foot bones were obtained throughout ground contact. Subsequent analysis of the video taped images using a motion analysis system allowed the tissue displacement to be calculated as a function of time. The tissue underneath the second metatarsal head was shown to undergo an average maximum compression of 45.7% during the late stages of ground contact during first step gait in a group of five normal subjects with a mean unloaded tissue thickness of 15.2 mm. The technique has a number of applications, including use in the validation of deformation predicted by finite element models of the soft tissue of the foot, and the study of alterations in the cushioning properties of the heel by devices which constrain the displacement of the heel pad.

Beschreibung: Spatial transformations of the vestibular-optokinetic system must account for changes in head position with respect to gravity in order to produce compensatory oculomotor responses. The purpose of this experiment was to study the influence of gravity on the vestibulo-ocular reflex (VOR) in darkness and on visual-vestibular interaction in the pitch plane in human subjects using two different comparisons: (1) Earth-horizontal axis (EHA) rotation about an upright versus a supine body orientation, and (2) Earth-horizontal versus Earth-vertical (EVA) rotation axes. Visual-vestibular responses (VVR) were evaluated by measuring the slow phase velocity of nystagmus induced during sinusoidal motion of the body in the pitch plane (at 0.2 Hz and 0.8 Hz) combined with a constant-velocity vertical optokinetic stimulation (at +/- 36 degrees/s). The results showed no significant effect on the gain or phase of the VOR in darkness or on the VVR responses at 0.8 Hz between EHA upright and EHA supine body orientations. However, there was a downward shift in the VOR bias in darkness in the supine orientation. There were systematic changes in VOR and VVR between EHA and EVA for 0.2 Hz, including a reduced modulation gain, increased phase lead, and decreased bias during EVA rotation. The same trend was also observed at 0.8 Hz, but at a lesser extent, presumably due to the effects of eccentric rotation in our EVA condition and/or to the different canal input across frequencies. The change in the bias at 0.2 Hz between rotation in darkness and rotation with an optokinetic stimulus was greater than the optokinetic responses without rotation. During EHA, changes in head position relative to gravity preserve graviceptor input to the VVR regardless of body orientation. However, the modifications in VVR gain and phase when the rotation axis is aligned with gravity indicate that this graviceptive information is important for providing compensatory eye movements during visual-vestibular interaction in the pitch plane.

Beschreibung: Small spots, lines and Gabor patterns can be easier to detect when they are superimposed upon similar spots, lines and Gabor patterns. Traditionally, such facilitation has been understood to be a consequence of nonlinear contrast transduction. Facilitation has also been reported to arise from non-overlapping patterns with opposite sign. We point out that this result does not preclude the traditional explanation for superimposed targets. Moreover, we find that facilitation from opposite-sign flanks is weaker than facilitation from same-sign flanks. Simulations with a transducer model produce opposite-sign facilitation.

Beschreibung: Space travel causes rapid and pronounced skeletal muscle wasting in humans that reduces their long-term flight capabilities. To develop effective countermeasures, the basis of this atrophy needs to be better understood. Space travel may cause muscle atrophy indirectly by altering circulating levels of factors such as growth hormone, glucocorticoids, and anabolic steroids and/or by a direct effect on the muscle fibers themselves. To determine whether skeletal muscle cells are directly affected by space travel, tissue-cultured avian skeletal muscle cells were tissue engineered into bioartificial muscles and flown in perfusion bioreactors for 9 to 10 days aboard the Space Transportation System (STS, i.e., Space Shuttle). Significant muscle fiber atrophy occurred due to a decrease in protein synthesis rates without alterations in protein degradation. Return of the muscle cells to Earth stimulated protein synthesis rates of both muscle-specific and extracellular matrix proteins relative to ground controls. These results show for the first time that skeletal muscle fibers are directly responsive to space travel and should be a target for countermeasure development.

Beschreibung: Thirty-three families (71 subjects) were screened for the presence of Candida albicans in mouthwash or stool specimens; 12 families (28 subjects) were culture-positive for this yeast. An enrichment procedure provided a twofold increase in the recovery of C. albicans from mouthwash specimens. Nine of the twelve culture-positive families had two positive members each, two families had three positive members each, and one family had four positive members. Genetic profiles were obtained by three methods: pulsed-field gel electrophoresis; restriction endonuclease analysis, and random amplification of polymorphic DNA analysis. DNA fingerprinting of C. albicans isolated from one body site three consecutive times revealed that each of the 12 families carried a distinct genotype. No two families shared the same strain, and two or more members of a family commonly shared the same strain. Intrafamily genotypic identity (i.e., each member within the family harbored the same strain) was demonstrated in six families. Genotypes of isolates from husband and wife differed from one another in five families. All three methods were satisfactory in determining genotypes; however, we concluded that restriction endonuclease analysis provided adequate resolving power.

Beschreibung: Batch equilibrium adsorption isotherm determination is used to characterize the adsorption of mixed yeast RNA on agarose-immobilized m-aminophenylboronic acid. It is shown that the affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium. This adsorption-promoting influence of barium is suggested to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected. The substitution of barium for the standard magnesium counterion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations. RNA adsorption isotherms exhibit sharp transitions as functions of temperature, and these transitions occur at different temperatures with Mg2+ and with Ba2+. Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process. The stoichiometric displacement parameter, Z, in Ba2+ buffer is three times the value in Mg2+ buffer, and is close to unity.

Beschreibung: Application accuracy is a crucial factor for stereotactic surgical localization systems, in which space digitization camera systems are one of the most critical components. In this study we compared the effect of the OPTOTRAK 3020 space digitization system and the FlashPoint Model 3000 and 5000 3D digitizer systems on the application accuracy for interactive localization of intracranial lesions. A phantom was mounted with several implantable frameless markers which were randomly distributed on its surface. The target point was digitized and the coordinates were recorded and compared with reference points. The differences from the reference points represented the deviation from the "true point." The root mean square (RMS) was calculated to show the differences, and a paired t-test was used to analyze the results. The results with the phantom showed that, for 1-mm sections of CT scans, the RMS was 0.76 +/- 0. 54 mm for the OPTOTRAK system, 1.23 +/- 0.53 mm for the FlashPoint Model 3000 3D digitizer system, and 1.00 +/- 0.42 mm for the FlashPoint Model 5000 system. These preliminary results showed that there is no significant difference between the three tracking systems, and, from the quality point of view, they can all be used for image-guided surgery procedures. Copyright 1999 Wiley-Liss, Inc.

Beschreibung: Intravascular ultrasound (IVUS) provides exact anatomy of arteries, allowing accurate quantitative analysis. Automated segmentation of IVUS images is a prerequisite for routine quantitative analyses. We present a new three-dimensional (3D) segmentation technique, called active surface segmentation, which detects luminal and adventitial borders in IVUS pullback examinations of coronary arteries. The technique was validated against expert tracings by computing correlation coefficients (range 0.83-0.97) and William's index values (range 0.37-0.66). The technique was statistically accurate, robust to image artifacts, and capable of segmenting a large number of images rapidly. Active surface segmentation enabled geometrically accurate 3D reconstruction and visualization of coronary arteries and volumetric measurements.

Beschreibung: Interstellar dust grains intercepted by the dust detectors on the Ulysses and Galileo spacecrafts at heliocentric distances from 2 to 4 astronomical units show a deficit of grains with masses from 1 x 10(-17) to 3 x 10(-16) kilograms relative to grains intercepted outside 4 astronomical units. To divert grains out of the 2- to 4-astronomical unit region, the solar radiation pressure must be 1.4 to 1.8 times the force of solar gravity. These figures are consistent with the optical properties of spherical or elongated grains that consist of astronomical silicates or organic refractory material. Pure graphite grains with diameters of 0.2 to 0.4 micrometer experience a solar radiation pressure force as much as twice the force of solar gravity.

Beschreibung: A substantial body of evidence indicates that aged-related changes in the fluidity and lipid composition of the plasma membrane contribute to cellular dysfunction in humans and other mammalian species. In the CNS, reductions in neuronal plasma membrane order (PMO) (i.e., increased plasma membrane fluidity) have been attributed to age as well as the presence of the beta-amyloid peptide-25-35, known to play an important role in the neuropathology of Alzheimer's disease (AD). These PMO increases may influence neurotransmitter synthesis, receptor binding, and second messenger systems as well as signal transduction pathways. The effects of neuronal PMO on learning and memory processes have not been adequately investigated, however. Based on the hypothesis that an increase in PMO may alter a number of aspects of synaptic transmission, we investigated several neurochemical and behavioral effects of the membrane ordering agent, PF-68. In cell culture, PF-68 (nmoles/mg SDS extractable protein) reduced [3H]norepinephrine (NE) uptake into differentiated PC-12 cells as well as reduced nicotine stimulated [3H]NE release. The compound (800-2400 microg/kg, i.p., resulting in nmoles/mg SDS extractable protein in the brain) decreased step-through latencies and increased the frequencies of crossing into the unsafe side of the chamber in inhibitory avoidance training. In the Morris water maze, PF-68 increased the latencies and swim distances required to locate a hidden platform and reduced the time spent and distance swam in the previous target quadrant during transfer (probe) trials. PF-68 did not impair performance of a well-learned working memory task, the rat delayed stimulus discrimination task (DSDT), however. Studies with 14C-labeled PF-68 indicated that significant (pmoles/mg wet tissue) levels of the compound entered the brain from peripheral (i.p.) injection. No PF-68 related changes were observed in swim speeds or in visual acuity tests in water maze experiments, rotorod performance, or in tests of general locomotor activity. Furthermore, latencies to select a lever in the DSDT were not affected. These results suggest that PF-68 induced deficits in learning and memory without confounding peripheral motor, sensory, or motivational effects at the tested doses. Furthermore, none of the doses induced a conditioned taste aversion to a novel 0.1% saccharin solution indicating a lack of nausea or gastrointestinal malaise induced by the compound. The data indicate that increases in neuronal plasma membrane order may have significant effects on neurotransmitter function as well as learning and memory processes. Furthermore, compounds such as PF-68 may also offer novel tools for studying the role of neuronal PMO in mnemonic processes and changes in PMO resulting from age-related disorders such as AD.

Beschreibung: A new 2.8-3.8 micrometers spectrum of the carbon-rich protoplanetary nebula CRL 618 confirms the previous detection of a circumstellar 3.4 micrometers absorption feature in this object (Lequeux & Jourdain de Muizon). The high resolution and high signal-to-noise ratio of our spectrum allow us to derive the detailed profile of this absorption feature, which is very similar to that observed in the spectrum of the Galactic center and also resembles the strong 3.4 micrometers emission feature in some post-asymptotic giant branch stars. A weak 3.3 micrometers unidentified infrared band, marginally detected in the CRL 618 spectrum of Lequeux & Jourdain de Muizon, is present in our spectrum. The existence of the 3.4 micrometers feature implies the presence of relatively short-chained, aliphatic hydrocarbon materials (-CH2-/-CH3 approximately = 2-2.5) in the circumstellar environment around CRL 618. It also implies that the carriers of the interstellar 3.4 micrometers feature are produced at least in part in circumstellar material, and it calls into question whether any are produced by the processing of interstellar ices in dense interstellar clouds, as has been previously proposed. Other features in the spectrum are recombination lines of hydrogen, rotational and vibration-rotation lines of molecular hydrogen, and a broad absorption probably due to a blend of HCN and C2H2 bands.

Beschreibung: Here we review cases where initiation of morphogenesis, including the differentiation of specialized cells and tissues, has clearly evolved due to cyclical symbiont integration. For reasons of space, our examples are drawn chiefly from the plant, fungal and bacterial kingdoms. Partners live in symbioses and show unique morphological specializations that result when they directly and cyclically interact. We include here brief citations to relevant literature where plant, bacterial or fungal partners alternate independent with entirely integrated living. The independent, or at least physically unassociated stages, are correlated with the appearance of distinctive morphologies that can be traced to the simultaneous presence and strong interaction of the plant with individuals that represent different taxa.

Beschreibung: Inocula from organic-rich black muds immediately underlying intertidal laminated microbial mats dominated by Microcoleus chthonoplastes yielded large, variable diameter spirochetes. These unusual spirochetes, previously reported only from the Alfacs Peninsula at the delta of the Ebro river in northeast Spain, contain striking arrays of cytoplasmic granules packed into their protoplasmic cylinders. On several occasions, both in summer and winter, the huge spirochetes were recognized in samples from mats growing in the Sippewissett salt marsh at Woods Hole Massachusetts. They were also seen in similar samples from microbial mats at North Pond, Laguna Figueroa, Baja California Norte, Mexico. The identity of these spirochetes was confirmed by electron microscopy: number and disposition of flagella, composite structure, measurements of their distinctive cytoplasmic granules. The granules, larger, more conspicuous and present in addition to ribosomes, are hypothesized to contain ATPases. As culture conditions worsen, these spirochetes retract into membrane-bounded round bodies in which they form refractile inclusions. From morphology and behavior we conclude the North American spirochetes from both Atlantic and Pacific intertidal microbial mats are indistinguishable from those at the delta of the Ebro river. We conclude a cosmopolitan distribution for Spirosymplokos deltaeiberi.

Beschreibung: Space provides a unique environment that can affect the interplay between cell cycle controls and environment and can thus modify the processes of cell division, development and growth. It is proposed that the chromosomal and nuclear abnormalities frequently encountered in cells of various plants exposed to space are due to a combination of factors including the biological status of the systems and the way in which they are grown, exposed to, and ultimately, the way in which they experience multiple stresses. The extent to which space-specific changes become manifest is dependent on the extent of pre-existing stresses in the system. This has become evident in a variety of plant species grown in space but has been particularly amenable to study using in vitro systems, especially in developing embryoids. The following observations allow us to harmonize disparate results from a variety of space experiments:- (a) the more completely developed a system, the less likely it is to show cell stress during growth; the less morphologically complex, the greater the vulnerability; (b) the size/"packaging" of the genome (karyotype) are significant experimental variables; plants with larger genomes (e.g. polyploids) seem to be more space-stress tolerant; (c) a single space-associated stress is inadequate to produce a significant adverse response unless the stress is severe, or a biological parameter necessary to 'amplify' it exists. On this view, an appropriate "stress match" with other non-equilibrium determinants, much like a 'tug of war', can result in genomic variations in space. All this emphasizes that fastidiously-controlled growing environments must be devised if one is to resolve the matter of direct versus indirect effects of space. Better understanding of the novel physico-chemical equilibrium phenomena associated with space will allow those interested in space cell and developmental biology to pick and choose procedures best suited to their exploitation for specific objectives.

Beschreibung: When Arabidopsis thaliana seedlings grow embedded in an agar-based medium, their roots grow vertically downward. This reflects their ability to sense the gravity vector and to position their tip parallel to it (gravitropism). We have isolated a number of mutations affecting root gravitropism in Arabidopsis thaliana. One of these mutations, named arg1, affects root and hypocotyl gravitropism without promoting defects in starch content or in the ability of seedlings' organs to respond to plant hormones. The ARG1 gene was cloned and shown to code for a protein with a J domain at its amino terminus and a second sequence motif found in several cytoskeleton binding proteins. Mutations in the AGR1 locus promote a strong defect in root gravitropism. Some alleles also confer an increased root resistance to exogenous ethylene and an increased sensitivity to auxin. AGR1 was cloned and found to encode a putative transmembrane protein which might be involved in polar auxin transport, or in regulating the differential growth response to gravistimulation. When Arabidopsis seedlings grow on the surface of agar-based media tilted backward, their roots wave. That wavy pattern of root growth derives from a combined response to gravity, touch and other surface-derived stimuli. It is accompanied by a reversible rotation of the root tip about its axis. A number of mutations affect the presence or the shape of root waves on tilted agar-based surfaces. One of them, wvc1, promotes the formation of compressed root waves under these conditions. The physiological and molecular analyses of this mutant suggest that a tryptophan-derived molecule other than IAA might be an important regulator of the curvature responsible for root waving.

Beschreibung: The maize Ac-Ds transposable element family has been used as the basis of transposon mutagenesis systems that function in a variety of plants, including Arabidopsis. We have developed modified transposons and methods which simplify the detection, cloning and analysis of insertion mutations. We have identified and are analyzing two plant lines in which genes expressed either in the root cap cells or in the quiescent cells, cortex/endodermal initial cells and columella cells of the root cap have been tagged with a transposon carrying a reporter gene. A gene expressed in root cap cells tagged with an enhancer-trap Ds was isolated and its corresponding EST cDNA was identified. Nucleotide and deduced amino acid sequences of the gene show no significant similarity to other genes in the database. Genetic ablation experiments have been done by fusing a root cap-specific promoter to the diphtheria toxin A-chain gene and introducing the fusion construct into Arabidopsis plants. We find that in addition to eliminating gravitropism, root cap ablation inhibits elongation of roots by lowering root meristematic activities.

Beschreibung: Space flight induces endocrine changes that perturb metabolism. This altered metabolism affects both the astronauts' body composition and the nutritional requirements necessary to maintain their health. During the last 25 years, a combination of studies conducted on Skylab (the first U.S. space laboratory), U.S. Shuttle flights, and Soviet and Russian flights provides a range of data from which general conclusions about energy and protein requirements can be drawn. We have reviewed the endocrine data from those studies and related it to changes in body composition. From these data it appears that protein and energy intake of astronauts are similar to those on Earth. However, a combination of measures, including exercise, appropriate diet, and, potentially, drugs, is required to provide the muscle health needed for long duration space flight.

Beschreibung: The mortality related to cervical cancer can be substantially reduced through early detection and treatment. However, current detection techniques, such as Pap smear and colposcopy, fail to achieve a concurrently high sensitivity and specificity. In vivo fluorescence spectroscopy is a technique which quickly, noninvasively and quantitatively probes the biochemical and morphological changes that occur in precancerous tissue. A multivariate statistical algorithm was used to extract clinically useful information from tissue spectra acquired from 361 cervical sites from 95 patients at 337-, 380-, and 460-nm excitation wavelengths. The multivariate statistical analysis was also employed to reduce the number of fluorescence excitation-emission wavelength pairs required to discriminate healthy tissue samples from precancerous tissue samples. The use of connectionist methods such as multilayered perceptrons, radial basis function (RBF) networks, and ensembles of such networks was investigated. RBF ensemble algorithms based on fluorescence spectra potentially provide automated and near real-time implementation of precancer detection in the hands of nonexperts. The results are more reliable, direct, and accurate than those achieved by either human experts or multivariate statistical algorithms.

Beschreibung: Adaptations in muscle mass stimulated by changes in muscle loading state entail alternations in the synthesis and degradation of myofiber proteins and the modulation of myonuclear number such that the ratio between the number of myonuclei and the size of the myofibers remains relatively constant. As depicted schematically in Figure 2.6, the literature regarding the role of IGF-in mediating muscle adaptation to alterations in loading state suggests the following conclusions: During periods of increased loading, myofibers upregulate the expression and secretion of IGF-I. Acting as an autocrine and/or paracrine growth factor, IGF-I stimulates myofiber anabolic processes. Acting as a paracrine growth factor, IGF-I also stimulates adjacent satellite cells to enter the cell cycle and proliferate. Continued myofiber production of IGF-I stimulates some satellite cells to differentiate and then fuse with myofibers, thus providing additional myonuclei in order to maintain or reestablish the myonucleus to myofiber size ratios of the enlarged myofibers.

Beschreibung: Neuroscientists are often interested in grouping variables to facilitate understanding of a particular phenomenon. Factor analysis is a powerful statistical technique that groups variables into conceptually meaningful clusters, but remains underutilized by neuroscience researchers presumably due to its complicated concepts and procedures. This paper illustrates an application of factor analysis to identify coordinated patterns of whole-body muscle activation during treadmill walking. Ten male subjects walked on a treadmill (6.4 km/h) for 20 s during which surface electromyographic (EMG) activity was obtained from the left side sternocleidomastoid, neck extensors, erector spinae, and right side biceps femoris, rectus femoris, tibialis anterior, and medial gastrocnemius. Factor analysis revealed 65% of the variance of seven muscles sampled aligned with two orthogonal factors, labeled 'transition control' and 'loading'. These two factors describe coordinated patterns of muscular activity across body segments that would not be evident by evaluating individual muscle patterns. The results show that factor analysis can be effectively used to explore relationships among muscle patterns across all body segments to increase understanding of the complex coordination necessary for smooth and efficient locomotion. We encourage neuroscientists to consider using factor analysis to identify coordinated patterns of neuromuscular activation that would be obscured using more traditional EMG analyses.

Beschreibung: This purpose of this study was to examine the spatial coding of eye movements during static roll tilt (up to +/-45 degrees) relative to perceived earth and head orientations. Binocular videographic recordings obtained in darkness from eight subjects allowed us to quantify the mean deviations in gaze trajectories along both horizontal and vertical coordinates relative to the true earth and head orientations. We found that both variability and curvature of gaze trajectories increased with roll tilt. The trajectories of eye movements made along the perceived earth-horizontal (PEH) were more accurate than movements along the perceived head-horizontal (PHH). The trajectories of both PEH and PHH saccades tended to deviate in the same direction as the head tilt. The deviations in gaze trajectories along the perceived earth-vertical (PEV) and perceived head-vertical (PHV) were both similar to the PHH orientation, except that saccades along the PEV deviated in the opposite direction relative to the head tilt. The magnitude of deviations along the PEV, PHH, and PHV corresponded to perceptual overestimations of roll tilt obtained from verbal reports. Both PEV gaze trajectories and perceptual estimates of tilt orientation were different following clockwise rather than counterclockwise tilt rotation; however, the PEH gaze trajectories were less affected by the direction of tilt rotation. Our results suggest that errors in gaze trajectories along PEV and perceived head orientations increase during roll tilt in a similar way to perceptual errors of tilt orientation. Although PEH and PEV gaze trajectories became nonorthogonal during roll tilt, we conclude that the spatial coding of eye movements during roll tilt is overall more accurate for the perceived earth reference frame than for the perceived head reference frame.

Beschreibung: High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude in hy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4 seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4 escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4 mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.

Beschreibung: OBJECTIVES: To develop and validate an automated noninvasive method to quantify mitral regurgitation. BACKGROUND: Automated cardiac output measurement (ACM), which integrates digital color Doppler velocities in space and in time, has been validated for the left ventricular (LV) outflow tract but has not been tested for the LV inflow tract or to assess mitral regurgitation (MR). METHODS: First, to validate ACM against a gold standard (ultrasonic flow meter), 8 dogs were studied at 40 different stages of cardiac output (CO). Second, to compare ACM to the LV outflow (ACMa) and inflow (ACMm) tracts, 50 normal volunteers without MR or aortic regurgitation (44+/-5 years, 31 male) were studied. Third, to compare ACM with the standard pulsed Doppler-two-dimensional echocardiographic (PD-2D) method for quantification of MR, 51 patients (61+/-14 years, 30 male) with MR were studied. RESULTS: In the canine studies, CO by ACM (1.32+/-0.3 liter/min, y) and flow meter (1.35+/-0.3 liter/min, x) showed good correlation (r=0.95, y=0.89x+0.11) and agreement (deltaCO(y-x)=0.03+/-0.08 [mean+/-SD] liter/min). In the normal subjects, CO measured by ACMm agreed with CO by ACMa (r=0.90, p 〈 0.0001, deltaCO=-0.09+/-0.42 liter/min), PD (r=0.87, p 〈 0.0001, deltaCO=0.12+/-0.49 liter/min) and 2D (r=0.84, p 〈 0.0001, deltaCO=-0.16+/-0.48 liter/min). In the patients, mitral regurgitant volume (MRV) by ACMm-ACMa agreed with PD-2D (r= 0.88, y=0.88x+6.6, p 〈 0.0001, deltaMRV=2.68+/-9.7 ml). CONCLUSIONS: We determined that ACM is a feasible new method for quantifying LV outflow and inflow volume to measure MRV and that ACM automatically performs calculations that are equivalent to more time-consuming Doppler and 2D measurements. Additionally, ACM should improve MR quantification in routine clinical practice.

Beschreibung: The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

Beschreibung: The present study was designed to validate our noninvasive ultrasonic technique (pulse phase locked loop: PPLL) for measuring intracranial pressure (ICP) waveforms. The technique is based upon detecting skull movements which are known to occur in conjunction with altered intracranial pressure. In bench model studies, PPLL output was highly correlated with changes in the distance between a transducer and a reflecting target (R2 = 0.977). In cadaver studies, transcranial distance was measured while pulsations of ICP (amplitudes of zero to 10 mmHg) were generated by rhythmic injections of saline. Frequency analyses (fast Fourier transformation) clearly demonstrate the correspondence between the PPLL output and ICP pulse cycles. Although theoretically there is a slight possibility that changes in the PPLL output are caused by changes in the ultrasonic velocity of brain tissue, the decreased amplitudes of the PPLL output as the external compression of the head was increased indicates that the PPLL output represents substantial skull movement associated with altered ICP. In conclusion, the ultrasound device has sufficient sensitivity to detect transcranial pulsations which occur in association with the cardiac cycle. Our technique makes it possible to analyze ICP waveforms noninvasively and will be helpful for understanding intracranial compliance and cerebrovascular circulation.

Beschreibung: Investigative research efforts using a cardiovascular model required the determination of central circulatory haemodynamic and arterial system parameters for the evaluation of cardiovascular performance. These calculations required continuous beat-to-beat measurement of pressure within the four chambers of the heart and great vessels. Sensitivity and offset drift, longevity, and sources of error for eight 3F dual-tipped micromanometers were determined during 21 days of implantation in goats. Subjects were instrumented with pairs of chronically implanted fluid-filled access catheters in the left and right ventricles, through which dual-tipped (test) micromanometers were chronically inserted and single-tip (standard) micromanometers were acutely inserted. Acutely inserted sensors were calibrated daily and measured pressures were compared in vivo to the chronically inserted sensors. Comparison of the pre- and post-gain calibration of the chronically inserted sensors showed a mean sensitivity drift of 1.0 +/- 0.4% (99% confidence, n = 9 sensors) and mean offset drift of 5.0 +/- 1.5 mmHg (99% confidence, n = 9 sensors). Potential sources of error for these drifts were identified, and included measurement system inaccuracies, temperature drift, hydrostatic column gradients, and dynamic pressure changes. Based upon these findings, we determined that these micromanometers may be chronically inserted in high-pressure chambers for up to 17 days with an acceptable error, but should be limited to acute (hours) insertions in low-pressure applications.

Beschreibung: Experiments performed in the period of 1995-1996 cooperatively with US investigators within the MIR/SHUTTLE and MIR/NASA space science projects continued exploration of avian embryogenesis in microgravity. Evaluation of Japanese quail embryos incubated in spaceflight microgravity showed that for the most part they were normally developed and compliant with duration of incubation. One of the major morphometric characteristics of embryo are its mass and size. Comparative analysis of body mass values in the space and laboratory and synchronous control groups pointed to a slight retardation. Body length of space embryos mimicked their mass curve. Data on the dynamics of mass and length of Japanese quail embryos support the well-known theory according to which growth and formation are distinguished by equifinality. No differences were revealed by the investigations of individual parts of embryonic bodies in the space and control groups. However, this finding was true only with regard to the embryos that had no developmental abnormalities. A part of embryos had defective eyes (microphtalmia), limbs (twisted fingers), and beaks.

Beschreibung: A theory of cell wall extension is proposed. It is shown that macroscopic properties of cell walls can be explained through the microscopic properties of interpenetrating networks of cellulose and hemicellulose. The qualitative conclusions of the theory agree with the existing experimental data. The dependence of the cell wall yield threshold on the secretion of the wall components is discussed.

Beschreibung: While there have been few documented permanent neurological changes resulting from space travel, there is a growing literature which suggests that neural plasticity sometimes occurs within peripheral and central vestibular pathways during and following spaceflight. This plasticity probably has adaptive value within the context of the space environment, but it can be maladaptive upon return to the terrestrial environment. Fortunately, the maladaptive responses resulting from neuronal plasticity diminish following return to earth. However, the literature suggests that the longer the space travel, the more difficult the readaptation. With the possibility of extended space voyages and extended stays on board the international space station, it seems worthwhile to review examples of plastic vestibular responses and changes in the underlying neural substrates. Studies and facilities needed for space station investigation of plastic changes in the neural substrates are suggested. Copyright 1998 Elsevier Science B.V.

Beschreibung: Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

Beschreibung: Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence, developmental expression, and quantity do not seem to be affected by a brief exposure to zero gravity in utero.

Beschreibung: The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of alpha-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple alpha-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Beschreibung: The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP3, and IP3 levels on the lower side continued to increase up to approximately 5-fold over basal levels before visible growth. As bending became visible after 8-10 h, IP3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP3 production was accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.

Beschreibung: The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

Beschreibung: Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

Beschreibung: The protein loss in humans during spaceflight is partly due to a normal adaptive response to a decreased work load on the muscles involved in weight bearing. The process is mediated by changes in prostaglandin release, secondary to the decrease in tension on the affected muscles. On missions, where there is a high level of physical demands on the astronauts, there tends to be an energy deficit, which adds to the muscle protein loss and depletes the body fat reserves. While the adaptive response is a normal part of homeostasis, the additional protein loss from an energy deficit can, in the long run, have a negative effect on health and capability of humans to live and work in space and afterward return to Earth.

Beschreibung: PURPOSE: Differences in gene expression underlie the phenotypic differences between irradiated and unirradiated cells. The goal was to identify late-transcribed genes following irradiations differing in quality, and to determine the RBE of 1 GeV/n Fe ions. MATERIALS AND METHODS: Clonogenic assay was used to determine the RBE of Fe ions. Differential hybridization to cDNA target clones was used to detect differences in expression of corresponding genes in mRNA samples isolated from MCF7 cells irradiated with iso-survival doses of Fe ions (0 or 2.5 Gy) or fission neutrons (0 or 1.2 Gy) 7 days earlier. Northern analysis was used to confirm differential expression of cDNA-specific mRNA and to examine expression kinetics up to 2 weeks after irradiation. RESULTS: Fe ion RBE values were between 2.2 and 2.6 in the lines examined. Two of 17 differentially expressed cDNA clones were characterized. hpS2 mRNA was elevated from 1 to 14 days after irradiation, whereas CIP1/WAF1/SDI1 remained elevated from 3 h to 14 days after irradiation. Induction of hpS2 mRNA by irradiation was independent of p53, whereas induction of CIP1/WAF1/SDI1 was observed only in wild-type p53 lines. CONCLUSIONS: A set of coordinately regulated genes, some of which are independent of p53, is associated with change in gene expression during the first 2 weeks post-irradiation.

Beschreibung: The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium. Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect. Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA. The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells. The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins. These results suggest that the lesion of axrl mutants is different from that of axr4.