ALBERT

Notes: We have shown previously that adenosine 3′5′ cyclic monophosphate (cAMP) increased the transport of thymidine into monkey cells (CV-1) without affecting DNA synthesis (Roller et al., '74). Using techniques of cell synchronization, cell separation and DNA synthesis inhibitors in CV-1 and other cell lines, we now report that thymidine transport at low thymidine concentrations increases during the S phase of the cell cycle and that initiation of DNA synthesis is necessary for thymidine transport, although continuation of DNA synthesis is not necessary. Cyclic AMP alters nucleoside transport by increasing the Vmax for the transport reaction, but has no effect on the Km. Cyclic AMP is found not to affect the time in the cell cycle when thymidine transport occurs or the quality of the transport reaction, and only affects the amount of thymidine transported.

Notes: The binding of tritium-labelled cytochalasin B (3H-CB) to a variety of mammalian cells was investigated. Binding studies revealed near-equilibrium binding of 3H-CB within 5 to 10 minutes, but the equilibrium level was influenced by 3H-CB concentration. Binding kinetics revealed strong temperature dependence. Rapid release of up to 70% of cell-bound 3H-CB molecules occurred when cells were washed and returned to fresh medium without CB. The remaining 30% of cell-bound 3H-CB molecules dissociated more slowly. Equilibrium binding studies on a variety of diploid, heteroploid and transformed cells treated with 1 μg/ml 3H-CB revealed between 1.7 X 107 to 5.3 X 107 3H-CB binding sites per cell. Cellular binding of 3H-CB was not affected by inhibition of cellular energy metabolism, RNA or protein synthesis. Modification of the cell surface by proteases, neuraminidase, hyaluronidase, ribonuclease, or occupation of cell surface saccharide residues by a variety of plant lectins did not significantly alter the pattern of 3H-CB binding. Surface pressure measurements on CB-treated lipid monolayers indicated that CB can interact with lipid molecules. The partition of CB in hydrophobic lipid regions of cell membrane systems as a possible mechanism of cellular binding of CB is discussed. Fractionation of 3H-CB-treated cells revealed binding of 3H-CB to both the plasma membrane and by intracellular membranes.

Notes: Nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN) have been proposed as intermediates in the biosynthesis of nicotinamide adenine dinucleotide (NAD) from nicotinic acid and nicotinamide respectively. Although NaMN and NMN were not detected in acid extracts from whole D98/AH2 cells following pulses with 3H-nicotinic acid and 3H-nicotinamide, they were observed following pulses of enucleated cells. Several experiments indicate that enucleated cells are incapable of synthesizing NAD and that biosynthesis stops with mononucleotide formation. These results provide support for the idea, based upon fractionation studies, that NAD pyrophosphorylase (which catalyzes the reaction NMN or NaMN + ATP → NAD or desamido-NAD) is localized exclusively in the nucleus.Acid extraction and chromatography were also used to compare the stability of 3H-NAD in enucleated and whole cells. In the presence of the glutamine analog, azaserine, the intracellular concentration of 3H-NAD decreased exponentially with a half-life of 4 hours in whole cells. In enucleated cells the intracellular concentration of 3H-NAD decreased with a half-life greater than 14 hours. The increased stability of NAD in enucleated cells was confirmed by autoradiography. These results demonstrate the key role of the nucleus in both the biosynthesis and turnover of NAD.

Notes: The discharge of Aplysia pacemaker neurons varies with temperature over the range 10 to 22°C. Three types of frequency-temperature plots are found, with maximal discharge at lowest, intermediate or highest temperatures. In the presence of ouabain, however, all cells show maximal discharge at the highest temperature, suggesting that the steady state activity of an electrogenic sodium pump is an important determinant of membrane excitability. The average magnitude of pump current, as indicated by the applied current necessary to restore discharge to control values after ouabain application, was about 4 namps at 20°C but near zero at 10°C. These neurons may be excellent models of mammalian thermoreceotprs.

Notes: Intranuclear Na, K and Mg concentrations were determined in cells of salivary glands incubated for 1h in selected NaCl/KCl/MgCl2 media. By variation of the external milieu beyond “physiological” limits the intranuclear electrolytes can be shifted between ca 100 and 280 mM [K]i, between ca 8 and 100 mM [Na]i and between ca 5 and 75 mM [Mg]i. No significant competition or interactions of the 3 ionic species are apparent. The relationships [K]e : [K]i and [Na]e : [Na]i can best be described by a positive and linear, that between [Mg]e : [Mg]i by a negative and exponential function. Regression parameters are given which permit a computation of intranuclear [Na], [K] and [Mg] as induced by NaCl/KCl/MgCl2 in any binary or triple combination that is tolerated by the explanted gland without visible damage.

Notes: Mouse bone marrow forms colonies of granulocytes and monocytic phagocytes when cultured in the presence of human plasma, urine or “feeder layers” prepared from human leukocytes. By contrast, human marrow produces colonies in the presence of leukocyte feeder layers but not in the presence of plasma or urine. It has been tacitly assumed that the response of mouse marrow to human blood leukocyte feeder layers is a measure of physiological substances released by those leukocytes which might control human granulopoiesis. This assumption however, has never been put to the test by comparing the response of mouse and human marrow to stimulation by leukocytes from the same individual. This has been done in the present study by using leukocytes from normal and leukemic subjects. Different human marrows responded similarly to stimulation by the same normal feeder layers, but there was no quantitative or qualitative correlation between the response of human and mouse marrows. Feeder layers from patients with acute granulocytic leukemia did not stimulate colony growth in normal human marrow but were as potent in stimulating mouse marrow colony growth as were feeder layers of normal leukocytes.We conclude that different factors may stimulate human and mouse marrows and that assays of granulopoietic factors of human origin should in future be carried out in human rather than mouse marrows.

Notes: Strong evidence is given that a nucleoside diphosphate kinase is present to some extent on the surface of intact neoplastic cells in culture. Experiments could be performed with cells cultured in a few plates to which an incubation medium was added. The cells were firmly attached to the supporting medium and remained viable during the incubation procedure.Determinations of lactate dehydrogenase were carried out to rule out any possible contamination from the culturing medium as well as from the cell interior. From these analyses, a procedure was developed which easily removed the last traces of the culturing medium and which showed that there was no leakage of intracellular lactate dehydrogenase during the incubation procedure. There was a rather insignificant diffusion of nucleoside diphosphate kinase into the incubation medium. In common with other nucleoside diphosphate kinases, the glioma cell surface enzyme seemed to be nonspecific with regard to nucleotide substrates.

Notes: A perfusion technique is described for the study of melanosome response in ventral tailfin melanophores of Xenopus laevis tadpoles. The melanosomes remain aggregated (punctate melanophores) in Ringer's. Theophylline (15 mM) and caffeine (30 mM) cause a reversible dispersion (stellate melanophores) of melanosomes which is partly blocked by cytochalasin B (10 μg/ml). When added with theophylline or caffeine to stellate cells, cytochalasin B causes a disrupted distribution of pigment granules, characterized by a melanosome free central region. C-AMP (20 mM) and dibutyryl c-AMP (1 mM) cause a reversible dispersion of melanosomes which is partly inhibited by cytochalasin. When cytochalasin plus a nucleotide are added to stellate cells, some show the disrupted distribution of melanosomes. Colchicine (5 mM) causes irreversible, while griseofulvin (0.2 mM) causes a slight, but reversible dispersion of melanosomes, and cytochalasin has little effect on these reactions. Perfused tailfin melanophores remain capable of responding to reversible reagents for at least 12 hours and are unresponsive to changes in illumination.

Notes: The effect of cAMP on a monkey kidney cell line (CV-1) noninfected and infected by SV40 was studied. No effect was found on either growth rate or cell morphology when concentrations up to 1 mM of 3′5′ cAMP were used. However, cAMP was found to increase the incorporation of 3H-thymidine into both cellular and viral DNA without a net increase in DNA synthesis. This increased incorporation was found to be related to an enhanced uptake of thymidine into the nucleotide pool which is reflected in an increase of phosphorylated nucleotides. This, coupled with a lack of effect of cAMP on endogenous deoxyribonucleotide production, produces an increased specific activity of the deoxyribonucleotide triphosphates, with a resultant increase in specific activity of DNA.

Notes: Exposure of early sea urchin embryos to 5-bromodeoxyuridine (at concentrations up to 100 μg per ml) severely decreases the uptake of exogenous 3H-uridine into RNA. However, the actual gross rate of DNA or RNA synthesis in these embryos appears not to be affected by the presence of 5-bromodeoxyuridine.

Notes: The macromolecular reguirements for the initiation and maintenance of macronuclear DNA replication were studied in heat synchronized Tetrahymena pyriformis GL-C. Previous work had established that macronuclear S periods could occur in a consecutive fashion without intervening cell divisions during a multiple heat shock treatment, as well as immediately following the synchronized cell divisions. Cycloheximide treatment prior to or during the S period which follows the first synchronized cell division resulted in abolition of the initiation of DNA synthesis or an almost immediate cessation of DNA synthesis in progress. Temporary inhibition of DNA synthesis occurred when cycloheximide was added late in the S period. Treatment with actinomycin D was found to block the initiation of DNA synthesis but did not appreciably affect the continuation of the S period. It was concluded that RNA synthesis was required for the initiation but not the maintenance of DNA replication, whereas protein synthesis was necessary for both processes.The dependency of the initiation of an S period on prior RNA and protein synthesis was also shown to exist when a second consecutive S period was initiated without a preceding cell division. Treatment with actinomycin or cycloheximide prior to a supernumerary S period during a multiple heat shock treatment completely abolished the initiation of DNA synthesis. In T. pyriformis the synthesis of RNA and protein related to the initiation of the S period is tightly coupled to each cycle of DNA replication.

Notes: Enzymatic activities involved in the transformations of uridine nucleotides by intact erythrocytes and their subfractions have been studied and the following enzymatic activities have been identified: UTPase, UDPase, UMPase and uridylate kinase.UTPase activity was present exclusively in the stromal fraction while UMPase and uridylate kinase activities were specific in the soluble fraction. UDPase was present in both the stromal and soluble fractions.This compartmentation in erythrocytes differs from that reported for the enzymes involved in adenine nucleotides transformations. Due to its sensitivity to Zn2+, uridylate kinase could be differentiated from adenylate kinase.

Notes: Monolayer cultures of a mouse teratocarcinoma were established in vitro. These cultures contained embryonal carcinoma, the malignant stem cell, and its differentiated progeny: parietal yolk sac, neuroepithelial, and mesenchymal cells. Tissues such as squamous epithelium, cartilage, striated muscle, neuroepithelium, and glands were produced from embryonal carcinoma that was maintained under conditions of long term culture. Frequent subcultivation with pancreatin allowed the establishment of cell lines of embryonal carcinoma which have been maintained for more than 18 months in vitro and continue to produce differentiated cells under specific culture conditions. Chromosomally these lines of embryonal carcinoma have a stem line of 39 chromosomes. Two lines of parietal yolk sac cells have been established which produce basement membrane, are not tumorigenic, and chromosomally are hypotetraploid. This system may yield information concerning neoplastic differentiation and its possible use in therapy for cancer.

Notes: Variants or “mutants” temperature-sensitive (ts) for growth have been isolated by selection from a near-diploid mouse cell line. Thus far. 10 ts mutants which grow normally at 33° C, but not at 39° C, have been isolated. These ts mutants were then studied to determine if any manifested their defect at a unique point or stage in the cell cycle. This type of ts mutant is termed a “cell cycle” mutant.The first screen involves observing individual cells of an asynchronous culture for residual division after a shift from 33° C (permissive temperature) to 39° (nonpermissive temperature). A cell cycle mutant should show some fraction of the cells dividing only once at a normal rate after the shift. The ts variant B54 met this first criterion for a cell cycle mutant (i.e., 50% residual division) and was further analyzed.The second screening technique monitors (1) the rate of entry into S, (2) the length of G2, and (3) the rate and duration of cells entering mitosis after a shift of an asynchronous culture to 39°. This experiment with B54 revealed that cells in G1 at the time of the shift to 39° failed to enter S while cells already into S completed the cycle at 39°. These results suggest that B54 is defective in a G1 function which is required for entry into S, but which is no longer needed once cells have entered S. Other results are presented which also support this hypothesis.In addition the ts function of B54 is apparently required for recovery from a “high density” G1 arrest.

Notes: Explant cultures were prepared from the slow anterior latissimus dorsi muscle and the fast posterior latissimus dorsi muscle of 15 day chick embryos. The morphology and growth pattern of myotubes from the two types of muscle were very similar. Intracellular microelectrode studies did not reveal consistent differences between the myotube types in regard to resting potential, input resistance, input time constant, or ability to produce active electrogenic responses. It is suggested that specific differentiation of the two muscles is determined by their innervation.

Notes: Changes in the number and erythropoietin sensitivity of erythropoietic progenitor cells (CFU-E or E) capable of erythropoietin-dependent colony formation in culture have been measured in mice subjected to a variety of manipulations known to affect erythropoiesis. Injection of erythropoietin into mice whose E levels had been reduced by transfusion-induced plethora stimulated a striking increase in this population within 24 hours in both spleen and marrow. Following irradiation and marrow transplantation E were found to regenerate in the spleen with a population doubling time of 6 hours. Evidence of erythropoietin-dependent stimulation of E in vivo under conditions of hemopoietic regeneration was also obtained, although substantial numbers of E were detected in both regenerating spleen and marrow of plethoric mice even without erythropoietin administration, In the same experiments comparable effects of erythropoietin on pluripotent stem cells or the progenitors of granulopoietic colonies in culture were not observed. Detailed studies of the dependence of colony formation on erythropoietin concentration in culture showed variations in erythropoietin sensitivity of E from normal, regenerating and plethoric, erythropoietin-stim-ulated spleen and marrow. This finding provides the basis for an extremely fine mechanism regulating the flow of erythropoietic differentiation at the level of the production of E.The number of E present in the marrow of untreated W/WV mice was normal, but their sensitivity to erythropoietin in culture was decreased to a similar extent as E from normal mice exposed to a heightened erythropoietic demand. Plethora had a more marked effect in reducing E numbers in W/WV mice than in +/ + controls and stimulation of E following subsequent erythropoietin administration was highly defective. The results of these studies provide further evidence that cells identified as E represent a stage of differentiation along the erythropoietic pathway that is several steps removed from pluripotent stem cells and support the view that erythropoiesis, granulopoiesis and stem cell self-renewal are regulated to a major degree by independent mechanisms.

Notes: The Chinese hamster cell line K12 is temperature sensitive for the initiation of DNA synthesis and for the programming of rounding up to enter mitosis. Viability is lost expontentially after twelve hours at the non-permissive temperature. This temperature sensitivity is reversed following exposure of K12 cells to SV40 virus.

Notes: A transient increase in the number of peritoneal colony-forming cells in agar (PCFC) was noted in the peritoneal cavity of mice given one intraperitoneal injection of thioglycollate medium. This increase of PCFC was not accompanied by a similar degree of increase in the number of either pluripotent hemopoietic stem cells or committed hemopoietic stem cells for granulocytes and/or macrophages that are normally present in the peritoneal cavity in small numbers. Other substances, such as glycogen, starch, Freund's adjuvant, sheep red blood cells and lectins, were also capable of increasing PCFC, but to different degrees. The ability of thioglycollate medium as well as other stimulatory agents to increase PCFC appeared to be dose-dependent.

Notes: Multiplication-stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum-free medium conditioned by the growth of a line of rat liver cells (CRL), The biological activities of purified CRL MSA for chicken embryo fibroblasts were compared with those of calf serum to determine which activities are important for the stimulation of DNA synthesis and mitosis. In a balanced salt solution, only glucose and amino acids were needed in addition to purified CRL MSA to stimulate DNA synthesis maximally. Purified CRL MSA stimulated the rates of uptake of glucose and α-aminoisobutyric acid. Only the stimulation of the rate of glucose uptake appeared to be a primary response to purified CRL MSA since the stimulation was not inhibited by actinomycin D or cycloheximide. The stimulation of the rate of uptake of α-aminoisobutyric acid was inhibited by actinomycin D.CRL MSA differed from calf serum in its inability to commit cells irreversibly to synthesize DNA after the removal of CRL MSA and in its lack of the ability to stimulate the migration or prolong the survival of chicken embryo fibroblasts.Comparative studies indicated that purified CRL MSA had functional similarities to insulin and somatomedin. CRL MSA may be representative of a family of small polypeptide hormones having insulin-like activity which are involved in the control of cell multiplication.

Notes: A microphotometric method is introduced that allows measurement of the contraction-relaxation kinetics of Spirostomum in response to electrical stimulation. The time course of contraction includes a rapidly contracting phase of some 4-5 mS during which cells shorten at a rate in excess of 100 cell lengths sec-1. While a stimulus strength-duration curve determines the threshold of the response, the response to above threshold stimuli of different strengths and to trains of stimuli suggest that contraction of Spirostomum may not be an all-or-none event. The kinetics of relaxation following high stimulating voltages and repetitive after contractions also induced by high voltages are explained by excitation-contraction coupling through a stimulus-dependent intermediate effector, possibly the release of calcium ions. Changes in resting membrane potential detected by intracellular recording do not influence the initiation of contraction, while microinjection of calcium buffers above 10-5 M Ca2+ invariably induces contraction.

Notes: More than 80% of the intracellular pyridine metabolite pool of human culture cells is trapped by OsO4 fixation. The fixed pyridine metabolites fully exchange with nicotinamide and nicotinic acid but not with nicotinamide adenine dinucleotide. Yet, chromatography of the exchanged compounds reveals that NAD and NADP constitute more than 95%. of the fixed material. Although the mechanism of OsO4 fixation is not fully understood, such fixation has permitted the autoradiographic detection of intracellular pyridine metabolites. Cells of the human cell line, D98/AH2, synthesize pyridine nucleotides during all phases of the cell cycle at rates which do not vary by more than six-fold. There is no difference in the apparent concentration of pyridine metabolites between nucleus and cytoplasm after ten minute or three day pulses with 3H-nicotinic acid. The 3H-labeled pyridine ring is lost from D98/AH2 cells upon transfer to unlabeled medium. In general, the rate of loss is uniform among cells in the population. However, in a small proportion of cells there is little or no loss. Non-dividing cells lose the pyridine ring at approximately the same rate as dividing cells, yet the intracellular concentration of pyridine metabolites is 50% greater in non-dividing cells.

Notes: L1210 murine leukemia cells after treatment with Cl. perfringens neuraminidase at pH 7.0 incorporated six times more N-acetylneuraminic acid-[C14] than control cells when incubated for 30 minutes with cytidine 5′-monophosphate N-acetylneuraminic-[C14] acid and three times more galactose-[C14] when incubated with uridine diphosphate galactose-[C14]. These sugars were incorporated in a 10% trichloracetic acid insoluble fraction and more than 75% of the incorporated N-acetylneuraminic acid- [C14] could be removed by further treatment of these cells with neuraminidase. The incorporation of N-acetylneuraminic acid- [C14] as a function of time was divided into two rates: a rapid one, active during the first 30 minutes followed by a slower one, similar to the rate observed with untreated cells. The addition of Ba++ and Ca++ ions at 8.3 mM increased the incorporation of N-acetylneuraminic acid- [C14] by 25% while 8.3 mM EDTA decreased activity by 58% . The addition of Zn++ or Hg++ at similar concentrations abolished the incorporation almost completely. The optimal pH for the incorporation of N-acetylneuraminic acid- [C14] by these neuraminidase treated cells was 6.5. These data suggest that ectoglycosyltransferases are present on the outer surface of the plasma membrane of L1210 cells and are able to catalyze the addition of radiolabeled nucleotide sugars onto macromolecular acceptors (cell surface glycoproteins and glycolipids) prepared by prior incubation of the cells with neuraminidase. Use of these procedures for labeling outer cell surfaces may also prove to be valuable for the study of plasma membrane glycoprotein and glycolipid structure, synthesis, and turnover.

Notes: Hemopoietic colonies were counted macroscopically and microscopically in spleens of hybrid mice seven or eight days after they had been irradiated and given parental bone marrow in donor-host combinations exhibiting poor growth. Colonies counted microscopically were classified as to differentiation pathway. Lymphocytes from the thymus or lymph nodes were injected into some recipients at several different dosages and lymphocyte: bone marrow (L:B) ratios. In confirmation of earlier work it was found that thymocytes increased the number and size of colonies in recipients of marrow. A shift of differentiation toward granulopoiesis was also seen when thymocytes were given, although erythropoietic colonies were still the most frequently seen type except at very high L:B ratios. Lymph node lymphocytes shifted the pattern more markedly toward granulopoiesis, even at low L:B ratios. When lymphocytes from either source were given without marrow, only a few colonies could be found in recipients, and if differentiated they were almost exclusively granulopoietic. Irradiation (900 R) of lymphocyte donors reversed the shift so that a normal pattern of differentiation, like that resulting from marrow alone, was seen; irradiated lymphocytes were nonetheless capable of augmenting the size and total number of hemopoietic colonies.

Notes: Bone cells were isolated by collagenase-hyaluronidase digestion of 19-20 day-old fetal rat calvaria. It is shown that contamination from calcified matrix can give erroneously high values for total cell calcium and can mask intracellular calcium exchange.Filtration of the cell suspension through 35 μ mesh nylon net and a five minute treatment with a cold pH 6.0 isotonic salt solution removed the contamination. Total calcium for cells prepared in this manner was 16.8 mmoles/kg wet weight. 45Ca uptake studies revealed an active component of calcium exchange.

Notes: The effects of elevated temperatures upon protein biosynthesis were determined in L5178Y murine leukemic lymphoblasts. The rate of protein synthesis was inhibited proportionately to the increase in temperature. Efforts were made to determine the mechanism of heat inactivation of protein synthesis by studying the requirements for recovery of activity after the cells were returned to 37°C. The ability of actinomycin to block the recovery process suggests that elevated temperatures destroy or inactivate a species of RNA required for protein synthesis. Loss of RNA during heating of the cells is apparently at least partially dependent on protein synthesis, since the presence of cycloheximide during heat shock, is capable of ameliorating the effects of short duration heat treatment.

Notes: A temperature-sensitive growth mutant derived from the BHK 21 cell Line, ts AF8, was found to have greatly reduced DNA synthesis at the nonpermissive temperature. This reduction is mainly due to a decrease in the frequency of cells synthesizing DNA. Upon shift up, ts AF8 becomes blocked in the G1 phase of the cell cycle. The cells acquire elevated cAMP levels and a unimodal distribution of DNA content, equivalent to that of G1 cells at the permissive temperature, Ts AF8 cells blocked at the G1/S boundary with hydroxyurea will enter S when shifted to the nonpermissive temperature. On the other hand, ts AF8 cells arrested m G1 by serum deprivation and shifted to the nonpermissive temperature at the moment of serum addition do not enter S, while those synchronized by isoleucine deprivation and shifted at the time of isoleucine addition will enter S. These data suggest that the cycle arrest point of the ts AF8 mutation is located in G1 between the blocks induced by serum starvation and isoleucine deprivation.The reduction in DNA synthesis caused by the ts AF8 mutation is not reversed by infection or transformation with Polyoma virus. Mitochondrial DNA continues to be synthesized at wild-type levels at the nonpermissive temperature.

Notes: Exposure of a thymic lymphocyte population (suspended in serum-free synthetic medium) to the phytomitogen concanavalin A (Con A) causes brief (within the first 8 to 12 minutes) rises in the cellular contents of cyclic AMP and cyclic GMP. However, the rise in the cyclic GMP level is calcium (extracellular)-dependent, but the cyclic AMP rise is not. These changes are followed during the next hour by the initiation of DNA synthesis by a large fraction of the lymphoblast subpopulation which, like the preceding cyclic GMP rise, is calcium-dependent. The stimulated lymphoblasts eventually progress into mitosis. Additional observations indicate that Con A operates by sensitizing lymphoblasts to calcium ions which, in turn, cause the initiation of DNA synthesis by a process mediated by cyclic GMP, but not cyclic AMP.

Notes: Differential centrifugation of homogenates of Harding-Passey melanoma demonstrated that aryl sulfatase A and β-glucuronidase sediment with particles (i.e., lysosomes) distinct from those particles bearing tyrosinase (i.e., melanosomes). The sedimentation curves for the lysosomal enzymes and tyrosinase, however, demonstrated that an adequate separation of these particle types could not be obtained by differential centrifugation. Isopycnic density gradient centrifugation was used to obtain the necessary resolution. The results of the density gradient studies demonstrated that lysosomes and melanosomes could be separated by this technique, as judged by enzyme distribution among the fractions recovered from the gradients and from electron microscopic examination of the melanosome fractions. It was further evident that the purified and washed melanosomes contained significant amounts of both acid hydrolase activities. Indeed 24% to 27% of the total acid hydrolase activities recovered from the density gradients were associated with the melanosome fractions. The acid hydrolases associated with the melanosomes could not be solubilized by treatment with 0.1% (v/v) Triton X-100, nor by exposure to hypo-osmotic shock. The melanoma lysosomes, however, did release most of both their hydrolase activities into soluble form after treatment with the same percentage of detergent. The lysosomes were, however, very resistant to rupture by exposure to hypo-osmotic conditions.

Notes: The attraction of fern spermatozoids by secretions from the female reproductive structures, and by salts of malic acid, has long been known as a classic example of precise chemotactic orientation by motile cells. Spermatozoids of the bracken fern are attracted by the partially ionized form of malic acid, bimalate ion, and also by calcium ions. Both calcium and bimalate ions must be present for chemotactic response and for response to voltage gradients, Spermatozoids swimming up a bimalate concentration gradient swim in helical paths of abnormally small radius; if they accidentally swim down the concentration gradient the radii of their path helices become abnormally large.These observations suggest that changes in the direction of flagellar beating in response to the rate of change of bimalate ion concentration with time may be the basis for chemotactic orientation. A “coupled diffusion” hypothesis for chemo-reception is presented, which postulates a membrane carrier which can only circulate freely in the membrane if it binds both bimalate and calcium ions. This hypothesis could explain time-differentiation of the stimulus, the coupling of a specific stimulus  -  bimalate ions  -  to a general mediator of intracellular response  -  calcium ions  -  and the quantitative relationship between response of the spermatozoids and the chemical potential of “calcium bimalate.”

Notes: Leukemic myeloblasts induced by avian myeloblastosis virus in the chicken formed small compact (type II) colonies in semi-solid agar medium. Normal yolk sac cells from 12-day old embryos formed large diffuse (type I) colonies under the same conditions. Type I colony formation (but not type II) was strictly dependent upon the presence in the medium of a colony stimulating factor (CSF) present in fresh chicken serum or conditioned medium. Serum CSF levels were determined for normal, leukemic, and birds which had spontaneously regressed from myeloblastic leukemia. When type I colony formation was used as the assay, serum CSF levels of leukemic birds were found to be significantly lower than levels in either normal or regressed birds. When the same sera were tested for their ability to induce type II colonies, leukemic birds demonstrated a significantly higher CSF level than either normal or regressed sera. Regressed chickens had serum CSF levels similar to normal birds.

Notes: Balb/3T3 cells transformed in culture by chemical carcinogens were shown to multiply in a medium supplemented with 2% calf serum or with 10% agamma new-born calf serum. The cell lines that multiply well in medium supplemented with 10% agamma serum produced a higher incidence of tumors in X-irradiated weanling mice than the lines that multiply poorly. The difference in 2-deoxy-D-glucose uptake into exponentially growing transformed and un-transformed cells was 50-100%. In crowded cultures untransformed Balb/3T3 cells ceased taking up the sugar, while chemically transformed cells continued at the same rate even at high cell densities; thus, the difference became greater in crowded cultures. When the serum concentration in the media was reduced from 10% to 2%, untransformed Balb/3T3 cells took up the sugar at a reduced rate, while chemically transformed cells were only slightly affected; agamma new born calf serum supplemented medium had no effect on sugar uptake in any of the cells. When the serum concentration was changed from 2% to 10%, untransformed cells increased sugar uptake followed by cell division. The immediacy (within 15 min) of the response in the sugar uptake to 10% serum concentration suggested that the increased uptake rate and the consequent higher concentration of the sugar (D-glucose in normal situation) within Balb/3T3 cells triggered the cell cycle. Chemical carcinogens appear to alter permanently the uptake mechanism for a key nutrient.

Notes: The sensory innervation of the mandibular stylets of the aphid Brevicoryne brassicae (L.) has been examined by electron microscopy. Two groups of sensory neurones are present in the mandible. Each has two neurones, one with a short dendrite extending into the base of the mandible and ending in the base and another with a long microtubular process which extends 500 m̈ down to the distal tip of the mandible. The two neurones are enclosed by an ensheathing cell comparable to the trichogen cell enveloping the group of neurones innervating pegs and hairs. This ensheathing cell is supported by extensive electron-dense filaments to form a scolopale and is embedded in the mass of stylet-forming cells at the base of the mandible. The inner segments of the dendrites are anchored to the ensheathing cell by desmosome junctions. Desmosome junctions also bind the microtubular outer segments of the short and long dendrite to each other. There is no evidence of a dendritic sheath enclosing the distal portion of the short dendrite which ends while still in the extracellular space within the ensheathing cell. The microtubular process of the long dendrite extends down the lumen of the mandible enclosed by a close-fitting extracellular sheath which penetrates and is attached to the cuticular wall of the mandible tip. Distally this sheath is thickened on one side. Deflection of the mandible would therefore deform the dendritic membrane asymmetrically because the thin walls of the sheath would bend more than the thick walls. This would exert an unequal mechanical strain on the dendritic membrane which could result in depolarization in response to deflection in a particular direction. The arrangement of the dendrites and their sheaths within the mandible is such that deflection to the right would distort one dendrite in the same way as deflection to the left would distort the other.

Notes: Ampullary receptor organs of African mormyrids consist of a cavity beneath the epidermis. The wall of the cavity contains embedded receptor cells and two types of supporting cells. A canal extends from the cavity to an opening at the surface. The lumen of the canal and the ampulla are filled with a jelly-like material and dense cylinders apparently secreted by two types of supporting cells. Flattened cells of the canal wall are joined by occluding junctions. Synapses between receptor cells and the afferent nerve fiber are characterized by a presynaptic dense body, but presynaptic vesicles were not observed. Degenerating receptor cells are occasionally seen among normal receptor cells in the base of the organ.

Notes: This paper examines the effect of early thymectomy on the subsequent development of lymphoid tissues in the toad, Xenopus laevis. At the time of thymic removal (8 days post-fertilization) all the lymphoid organ anlagen are at a rudimentary state of differentiation and contain few, if any, small lymphocytes. Despite the absence of any thymic tissue all thymectomized animals grew normally.Thymectomized larvae developed relatively normal lymphoid organs. However, lymphoid depletion was apparent in the splenic red pulp and in the pharyngeal ventral cavity bodies. Examination of the lymphoid organs of post-metamorphic Xenopus revealed reduction in spleen size following thymectomy. Lymphoid depletion was evident in the splenic red pulp of many thymectomized toadlets and reduction in proportion of white to red pulp was also noted in a few of these animals. Absence of the thymus had no apparent effect on the histology of the other lymphoid organs examined.

Notes: Extraocular muscles from representative species of vertebrate groups ranging from amphibians to the higher mammals were examined in serial histological sections for the presence of muscle spindles. These observations and data from the literature indicate that extraocular muscles of the pig, calf, sheep and other even-toed ungulates are richly supplied with well-defined spindles having a generous complement of intrafusal fibers distinguishable as nuclear bag and chain fibers. Spindles in human eye muscles are also numerous. In macaque and chimpanzee muscles a few poorly developed spindles were present in some, but not all, muscles. No encapsulated receptors were found in 20 other mammalian and submammalian species examined in this study. When present, spindles tended to be located in the zone of small muscle fibers found along the orbital surface of the muscle. Rectus and oblique muscles in all species had such a zone, so that its existence did not determine whether spindles would occur.

Notes: Gonadotrophic cells in the pars distalis of Anolis carolinensis often contain juxtanuclear concentrations of filaments with diameters intermediate in size (approximately 100 Å) between microtubules and microfilaments. Their size and their substructure, which gives them a tubular appearance when they are displayed in cross-section, appear to place these filaments in the “intermediate filament” category (Ishikawa et al., '68). In their juxtanuclear position in the intact animal, the intermediate filaments are collected in randomly-oriented tangles. In castrated specimens of Anolis, gonadotrophs degranulate and elongate. During this elongation, increased numbers of microtubules appear in orientation parallel to the long axis of the cell, and the 100 Å filaments reassemble in rod-like masses oriented parallel to the microtubules. This apparent distributional interaction may facilitate the elongation of the cell. Intimate physical associations between the intermediate filaments and secretory granules suggest that the filaments may act in the movement of the granules during the processes of degranulation and secretion.

Notes: The gastric mucosa of both the echidna and platypus is aglandular and the lining epithelium is stratified squamous. The latter exhibits three principle layers: stratum germinativum, stratum spinosum, and stratum corneum. The cytoplasm of cells composing the first two strata of both species shows bundles of tonofibrils and numerous free ribosomes. Cells of the stratum spinosum in the platypus also show numerous dense granules limited to the peripheral cytoplasm. The stratum spinosum of both species is comprised of fusiform-shaped cells whose adjacent cell membranes show extensive interlocking. The stratum spinosum of the echidna in addition shows numerous intercellular bridges. Cells of the stratum corneum become flattened and elongate and in the echidna nuclei near the surface appear to degenerate. Cells comprising the stratum corneum of the platypus exhibit well preserved nuclei and contain scattered large granules of varying electron density. Prior to sloughing, cells near the surface of both species show a separation of adjacent cell membranes. True keratinization is not found in the gastric lining epithelium of either species and the epithelium lining of the stomach of the echidna more closely represents a form of parakeratosis. Delicate papillae containing capillaries extend considerable distances into the overlying epithelium of both species and are thought to contribute to its nutrition.

Notes: Cells in secretory glands of globiferous pedicellariae from Strongylocentrotus purpuratus (Stimpson) were studied with the electron microscope and subjected to preliminary light microscopic, histochemical analysis. Specimens for electron microscopic observation were fixed with chilled 2% glutaraldehyde in sea water postfixed in cold 1.33% osmic acid, and embedded in Araldite 502 epoxy resin Samples for histochemical analysis were fixed in the same manner, and then embedded in n-butylmethacrylate.Secretory cells line and fill partially bifurcated, muscular gland sacs located peripherally on each of three jaw elements comprising the pedicellarial head. Cells from venom glands are typically mucoid in appearance, possessing small volumes of basally displaced, vesiculated cytoplasm and an extensive system of vacuoles dominating the apical nine-tenths of each cell. These vacuoles enclose ground substances of various densities and staining affinities. Despite their extensive vacuolation, gland cells contain numerous cytomembrane complexes indicating metabolic activity just prior to fixation. Deciduous endoplasmic reticulum, Golgi complexes, large vacuoles, and various species of vesicles associated with these membrane systems are found in spatial proximity which indicates an apparent biosynthetic association.Preliminary histochemical tests on sections embedded in acrylic plastic indicate vacuolar products may consist of protein and nonsulfated acid mucosubstances.Gland cells are probably holocrine in function, releasing their vacuolar complement upon constriction of the muscular gland sac. There is no evidence indicating delivery of non-membrane bounded, granular secretion to an acellular lumen within the gland sac.

Notes: Transmission and scanning electron microscopic studies demonstrate the stimulatory effect of synthetic salmon calcitonin on the fine structure of fibroblasts and on collagen formation in cutaneous wounds experimentally induced in rabbits. Long-term administration of calcitonin enhances fibroblast growth and collagen synthesis. The fibroblasts hypertrophy and exhibit a highly developed rough endoplasmic reticulum (RER), several polyribosomes, large nuclei, hypertrophic Golgi complex, and many dense granules and lysosomes. Mitochondria are elongate and ramify; intracellular as well as extracellular synthesis of collagen increases. Fibrils appear tightly packed, in large heaps or spicula, with a characteristic periodicity and striation.Scanning electron micrographs of topography and relationships with collagen fibers and fibrils and cells surface changes demonstrate an extensive network of fine fibrils between collagen fibers, marked ruffling of cell membranes as well as numerous blebs on the cell surface. The latter are significant in collagen formation and egestion.

Notes: Sperm enter the anterior vas deferens individually in the spider crab male. There they become surrounded by secretion products from the cells of the vas deferens, and are compartmentalized into spermatophores of varying size. The anterior vas deferens can be divided into three regions. The epithelium of the anterior vas deferens varies regionally from low to high columnar. The cytoplasm contains vast arrays of rough endoplasmic reticulum and Golgi complexes but few mitochondria. Intercellular spaces contain septate junctions, gap junctions and vesicles.Once the spermatophores have been formed in the anterior vas deferens, they are moved posteriorly to the middle vas deferens where they are stored and surrounded by seminal fluids. The epithelial cells of the middle vas deferens contain large amounts of rough endoplasmic reticulum and Golgi complexes. Numerous micropinocytotic vesicles appear, forming at the cell surface and within the apical cytoplasm. Their suggested function is the resorption of secretion products of the anterior vas deferens which initiated compartmentalization of the spermatozoa into spermatophores.The posterior vas deferens functions primarily as a storage center for spermatophores until they are released at the time of copulation. Seminal fluid surrounding the spermatophores is produced in this region as well as in the middle vas deferens. The cells of this region contain vast arrays of vesicular rough endoplasmic reticulum and Golgi complexes. The cells are multinucleate. Microtubules are numerous throughout the length of the cells and appear to insert on the plasma membrane.

Notes: Basicranial anatomy of mammalian carnivores is significant in the determination of carnivore evolution. One of the least understood yet most often studied features of the basicranium is the auditory bulla, a bony capsule enclosing the middle ear. Although previously believed to be formed by a tympanic bone alone, or by tympanic and entotympanic, it is shown here that the carnivore bulla is formed by three and in some cases four ontogenetic elements: tympanic, rostral entotympanic, caudal entotympanic(s). These elements in Carnivora appear to correspond to bulla elements discovered over 50 years ago by Van der Klaauw in representative species of several other orders of mammals.Increased auditory sensitivity appears to have been attained in various carnivore lineages by increase in the volume of the middle ear cavity, principally by hypertrophy of the caudal entotympanic, but also by different yet less common anatomical strategies such as invasion of the mastoid bone by the middle ear space. Five basic types of bulla among living Carnivora can be recognized, based largely on the relationship of the caudal entotympanic to the other bulla elements.Closely associated with the bulla in the auditory region are the median and promontory branches of the internal carotid artery. These branches exhibit a precise relationship to the bulla elements, particularly the rostral entotympanic. The promontory branch is much reduced or completely lacking in all living carnivores. Reduction or loss of the median branch is explained as the result of the development of arterial retia on the anastomotic artery in the orbital region. Reduction of the median branch is particularly characteristic of the aeluroid Carnivora, in which a greater proportion of the cerebral blood supply passes to the brain in the external carotid artery via the anastomotic branch, and is cooled in the orbital region by a countercurrent heat exchange mechanism formed by arterial retia closely associated with venous blood in surrounding sinuses. The arctoid and cynoid Carnivora are characterized by only rudimentary development of retia along the anastomotic artery, and the median branch is much better developed than in the aeluroids. Among arctoids, ursids parallel the aeluroids in the development of a countercurrent mechanism but employ the median branch of the internal carotid rather than the anastomotic branch of the external carotid artery.Bulla structure and nature of the carotid circulation in the auditory region can be determined from fossil Carnivora as well as for living forms. This study attempts to define basicranial anatomical patterns characteristic of living Carnivora which then can be used to trace the evolution of lineages in the fossil record.

Notes: The termite gut flagellates are of interest because of their unusual motile organelles, their ability to digest cellulose, and their symbiotic relationship with prokaryotes inhabiting the insect gut. This report provides a detailed ultrastructural description of Pyrsonympha from the hind-gut of Reticulitermes flavipes.The motile axostyle is composed of 2,000-4,000 microtubules connected by cross-bridges. At its anterior end, the axostyle is associated with a “primary row” of microtubules which is associated with a fibrous network. The “primary row” is embedded in a large mass of amorphous, electron-dense material occupying the furthest anterior end of the cell. The basal bodies of the eight flagella are also embedded in this presumptive microtubule-organizing center. The flagella are associated with the cell surface throughout their length. Isolation and reactivation of the axostyle has demonstrated that although ATP dependent motility is inherent in the structure of the axostyle, its proper control may be mediated by the attachment of the axostyle to structures at the anterior end of the cell.Pyrsonympha lacks morphologically distinguishable mitochondria and Golgi complexes. The cell surface is covered by unique, previously underscribed, tubular specializations. Symbiotic microorganisms are observed associated with the cell surface and within the cytoplasm.Wood particles are taken up from the gut fluid by large phagocytic vacuoles formed at the posterior end of the cell. Even during the process of breakdown, the wood is always enclosed within the membrane of the phagocytic vacuole.The Pyrsonympha from Reticulitermes flavipes are not attached to the lining of the hind-gut and do not contain an attachment organelle, unlike the Pyrsonympha from other species of Reticulitermes.

Notes: The gecko ear was studied in 36 species belonging to 24 genera. This receptor has attained an advanced level of structure and performance in this group of lizards, but there are many variations among species. To a large extent these variations follow subfamily lines as represented in Kluge's system of classification.Brief consideration is given to features of the outer and middle ear, but chief concern is with inner ear structures and their relations to auditory sensitivity as represented by the cochlear potentials.The auditory papilla is segmented, with a dorsal portion whose hair cells have their ciliary tufts attached to a tectorial membrane, and a ventral portion in which these cells form tow assemblages, one with tectorial connections and the other with connections to a line of sallets.The dorsal segment varies greatly in length and in the form of ciliary orientation. In Eublepharinae and most Gekkoninae the ciliary orientation is unidirectional, and the degree of sensitivity relates to the length of this segment. In Diplodactylinae and Sphaerodactylinae the orientation is bidirectional, and this segment functionally hardly differs from the ventral segment.Auditory sensitivity as measured in terms of the cochlear potentials shows close relations with subfamily groupings, except for the Gekkoninae in which considerable diversity is found.The evidence from structural differentiation, along with that derived from the forms of the cochlear potential functions, leads to the suggestion that these ears possess a high degree of pitch discrimination and capability for the analysis of complex sounds.

Notes: Mature mouse and cat peripheral nerve fibers have been examined in vitro by time-lapse photography. Some Schmidt-Lanterman clefts which were open at the start closed later; other were seen to open and then to close, some of them more than once. The implications of these movements are considered, especially in regard to the question of the passage of materials from the endoneurial connective tissue spaces to the axon.Myelin movements other than those occurring at the Schmidt-Lanterman clefts consisted primarily of the development and frequent regression of indentations of the myelin sheath. A single evagination was seen to develop and then to recede. These myelin movements suggest that previously described invaginations and evaginations of the myelin sheath, including flaps of “redundant myelin”, are not static but rather that they are in a state of movement, forming and regressing at intervals.The possible functional significance of the development and regression of myelin sheath indentations in relationship to axoplasmic flow is discussed.

Notes: The authors describe the spermiogenesis of Polyxenus lagurus, a diplopod, in the male genital ducts and the transformations the spermatozoon successively undergoes in the spermatheca.The spermatozoon in the male genital ducts looks like a little barrel devoid of centriole and of any kind of rudimentary flagellum whatever. The organelles are markedly modified; cross sections present an elongated, flattened nucleus, an X-shaped body running parallel to it on the opposite side and two longitudinal mitochondrial strips interposed between them. The rest of this barrel-shaped spermatozoon is filled with peculiar Golgi formations, the spongy chambers, which open outwards through little vents.In the spermatheca the spermatozoon is quite different: it is shaped like a long ribbon. The basic structure of the spermatozoon is formed by the double folding of part of the cortical layer of the barrel-shaped spermatozoon. On the central part of this endo-skeleton are longitudinally ranged the nucleus and the acrosome flanked on both sides by a thread of mitochondria. Even in this phase the sperm has no flagellum.

Notes: Several secretory and nonsecretory enzymes were localized histochemically in the main venom gland of 13 viperid snakes. All secretory cells show the intracellular oxidative enzymes succinate dehydrogenase and monoamine oxidase. The granular reactions obtained for both enzymes resemble mitochondria in distribution. Distinctive cells with a very high succinate dehydrogenase activity are dispersed among the secretory cells of all species except Atractaspis.Nonspecific acid phosphatase activity is found in the supranuclear region of the secretory cells in species that do not secrete this enzyme and throughout the cytoplasm in snakes that secrete the enzyme. Nonspecific alkaline phosphatase activity occurs in the secretory cells of those snakes whose venom shows this activity. Leucine amino peptidase (aryl amidase) activity is found in the venom and in the secretory cells of all the species.In Vipera palaestinae both the venom and the secretory cells of the main venom gland contain nonspecific esterase, L-amino acid oxidase and phosphodiesterase activities. The localization of phosphodiesterase and L-amino acid oxidase do not show major differences between glands at different intervals from an initial milking.Adenosine-monophosphate phosphatase activity is localized in the supranuclear region of the secretory cells in the glands of Vipera palaestinae and Aspis cerastes. Its activity is found in the venom of Aspis only.

Notes: The male genital systems of two mysids, Archaeomysis grebnitzkii (subfamily Gastrosaccinae) and Neomysis awatschensis (subfamily Mysinae) are described. The testes of both species include a pair of central cords (spermatogonial cells), two sets of lateral sacs, and a set of dorsal-lateral spermatidic pouches in which spermiogenesis takes place. Mature sperm exit dorsally from the pouches into a common U-shaped seminal vesicle, the arms of which extend posteriorly as the right and left vasa deferentia.The emphasis of this study was on the follicle cell-spermatid relationship. Spermatids retain their own cell membranes as do the follicle cells. A bundle of sperm tails extends toward the follicle cell nucleus making several revolutions about the nucleus. Masses of rodlets occupy this tract with the spermatid tails. The tail consists of an outer cylinder with banding in a periodicity similar to collagen protein, and an inner cavity filled with a structureless, dark-staining material. Heads of the spermatozoa differ considerably between the two species. The elongate, reflexed head of Neomysis with its central filament joins the tail at an acute angle. The short cylindrical head of Archaeomysis with its anterior hood-like extension joins the tail directly. The distinctiveness of the sperm types might prove useful for taxonomic purposes. As noted by previous investigators, the number of spermatids per follicle cell shows specificity: Neomysis awatschensis has around 16 spermatids per follicle cell, Archaeomysis grebnitzkii about 100.

Notes: Relationships between the cribriform plate of the ethmoid, the olfactory bulb, and olfactory acuity were explored using material from 13 of the 17 bat families.All megachiropteran cribriform plates were entirely perforated. In contrast, microchiropteran plates showed distinct perforated portions dorsally and nonperforated portions ventrally. The plates of frugivorous species had more foramina than those of insectivorous ones. Bats with mixed dietary habits were intermediate. Our data suggest that the Chilonycterinae were originally frugivorous, and have only secondarily reverted to an insectivorous diet.Trend analyses show that wherever dietary preference appears to favor a more acute sense of smell, bulb diameter tends to be larger. In general, frugivorous bats tend to have bulbs exceeding 2 mm in diameter; insectivorous bats tend to have bulb diameters of 2 mm or less. The number of foramina in the plates and total cribriform plate area tends to increase as a function of bulb area, but the plate area the foramina occupied increases as a function of bulb volume. The ratio of the size of the bulb to the size of the cerebral hemisphere does not predict olfactory acuity in bats.

Notes: Mast cells were demonstrated in eight species of snakes, using special fixation techniques to prevent solubilization of cytoplasmic granules. Toluidine blue O and azure A were the major stains and observations were made under light microscope including cytophotometric analysis.The mast cells of snakes were shown to be relatively small (7-11 μ in diameter) when compared to mast cells of a lizard (8-15 μ), dog and rat (9-15 μ).Among the various organs examined, mast cells were particularly numerous in the mesentery, tongue, underneath the serosa of the digestive tract and in the heart, between muscle fibers and in the epicardium.Although under the light microscope some snake mast cells seemed to be orthochromatically stained, when analysed by cytophotometry they were demonstrated to be actually metachromatic.Snake mast cells granules were demonstrated to have an amphoteric behavior, since they were stained with both basic (toluidine blue O and azure A) and acid dyes (eosin and ponceau-acid-fuchsin).

Notes: The number, location, size, shape and microstructure of the parathyroid glands of Agama agama agama, Hemidactylus brooki angulatus and Pytodactylus hasselquisti hasselquisti was investigated using approximately 250 specimens of each species from the Zaria area of Nigeria.Only parathyroid III was found. Additional patches of tissue in Hemidactylus, though possibly parathyroid IV, are considered to be derived from parathyroid III. It was found that the amount of parathyroid tissue per gram of body weight was similar in the three species used, and that females had more parathyroid tissue than males. The same situation seems possible in other species.The structure of the parathyroid glands could not be related to taxonomic grouping within the Sauria, but the general picture was found to be more similar to that of birds and mammals than to that of amphibians.

Notes: Retinal projections were studied experimentally in the Northern water snake using modifications of the Nauta silver impregnation technique. Contralaterally, the retina projects to nucleus geniculatus lateralis pars dorsalis and pars ventralis, nucleus lentiformis mesencephali and nucleus geniculatus pretectalis. A sparse projection was also observed to nucleus ovalis. An additional afferent thalamic projection to nucleus ventrolateralis was found in two cases. The retina projects ipsilaterally to the dorsolateral portion of nucleus geniculatus lateralis pars dorsalis, and sparsely to nucleus lentiformis mesencephali and nucleus geniculatus pretectalis. Nucleus posterodorsalis receives dense bilateral retinal projections. Contralaterally, the retina also projects to the superficial layers of the tectum (layers 8-13 of Ramón) and to nucleus opticus tegmenti. Armstrong's findings that the retinal projections in Natrix are qualittatively similar to those in lizards were confirmed. However there are marked quantitative differences among the various pathways and their corresponding nuclei. These differences are particularly striking in comparing the visual projections to the dorsal thalamus, the retino-tecto-rotundal and the retino-geniculate systems. The first is reduced in volume and the second is markedly increased in volume in comparison with lizards. These data lend support to the theories of Walls that snakes evolved from fossorial lizards and of Underwood that the eyes of these lizards underwent reduction but not complete degeneration. Qualitatively the retinal projections are conservative among lizards and snakes, but a history of reduction of these pathways in ancestral snakes with a selective increase in the retino-geniculate system as a surface niche was reattained is reflected in the anatomy of this ophidian visual system.

Notes: Fine structure of the torus semicircularis of the loach, carp, common eel and rainbow trout was studied by light and elecron microscopy. The torus semicircularis of each species is divided into four layers. The subependymal first layer comprises numerous unmyelinated fibers and their terminals which contain cored vesicles. The second and the third layers are composed of small cell bodies and their dendrites respectively. These layers develop equally in the four species and contain the usual axodendritic synapses. On the other hand, the fourth layer varies in different species. The mediumsized cells in this layer, which are inferred to be of the same origin as the small cells from their configuration and size, show differences in lamination in each species. Compared with the usual axodendritic synapse of the small cells, the medium-sized cells have quite different synaptic patterns, which include inhibitory and electrical as well as the usual excitatory chemical synapses. From these findings, the medium-sized cells are surmized to receive sound of different degrees of intensity from that received by the small cells, which may have an effect on feeding behaviors of the species. In the deepest portion of the torus semicircularis of all species, there are large multipolar cells on which numerous axon terminals synapse in much the same way as they do on the medium-sized cells. These findings suggest that the synaptic patterns in the torus semicircularis may depend not on the receptive cells in each layer but on the various characteristics of the afferent fibers.

Notes: Abdominal extra-adrenal chromaffin tissue, or paraganglia, was examined at the ultrastructural level to elucidate the innervation of this adrenal medullary homologue. Paraganglia display unmyelinated nerve fibers surrounded by Schwann cell cytoplasm. These nerves are separated from the paraganglion Type I (granule-containing) cells by cytoplasmic projections of paraganglion Type II (satellite) cells. However, serial sections show that the nerves eventually make synaptic contact with the Type I cell. At the axon-chromaffin cell junction, only the outer aspect of the nerve is covered by the satellite cell. The presynaptic endings contain numerous synaptic vesicles, mitochondria and glycogen particles. The vesicles are predominantly of the clear-cored variety, but a few possess centers which are elecron opaque. The pre- and postsynaptic membranes are separated bya subsynaptic space and occasionally exhibit the membranal densities usually associated with synaptic areas. These ultrastructural studies establish definite evidence that abdominal paraganglion cells are innervated.

Notes: The gland cells of Lyonet's gland, which is accessory to the silk gland in the silkworm larva, is characterized by the presence of complicated canaliculi bearing microvilli on their inner surface, large numbers of mitochondria and remarkably convoluted basal plasma membrane. On the other hand, the cell lacks the well-developed cytoplasmic membrane system such as rough- and smooth-surfaced endoplasmic reticula and Golgi bodies, though free ribosomes are numerous. Secretory vesicles are absent, and the canaliculi contain no dense material. From such ultrastructural observations, it was suggested that a possible role of the gland may be the exchange of the small molecules such as water and ions, rather than the hitherto supposed secretory role of a cementing sunstance of silk proteins. The lumen of the proximal part of the glandular duct contains a kind of proteinaceous substance which can be demonstrated histochemically and is regarded as similar to one of the silk proteins in the silk gland, not to the real product of the Lyonet's gland.

Notes: Specimens, representative of each of the major taxa of mosquitoes, were fixed in copula and the external genitalia examined by scanning electron microscopy. The periphery of the basin-like everted aedeagus of Aedus aegypti precisely matches that of the everted atrial membrane of the female. These structures are appressed during coitus and sealed by pressure of the paraprocts, aedeagal pouch and proctiger. When everted, the aedeagus of male Culex pipiens reveals a ridged dome that surrounds the genital opening. This dome seals itself laterally into a gutter formed by pad-like extensions of the female's genital lips and is sealed dorsally by pressure of the aedeagal apodeme. The aedeagus of another culicine species, Wyeomyia smithii, bears the gonopore at the apex of a spined tube. This tube is inserted between the female's genital lips and is sealed within the genital atrium. The aedeagus of the toxorhynchitine species Toxorhynchitis brevipalpus is immobile and is inserted deep within the genital atrium of the female where it is sealed by pressure of the atrial walls. Males of each of these mosquitoes deliver a mixture of semen and sperm to the copulatory bursa of the female. After withdrawal of the aedeagus, sperm is transferred to the spermathecae.In contrast, sperm of Anopheles quadrimaculatus are delivered directly to the spermathecal duct. The tube-like aedeagus is positioned by its leaflets during sperm transfer and is driven deep into the atrium, where a mixture of semen and sperm is ejaculated.The significance of mechanical barriers to mating between species is discussed.

Notes: The gross and microscopic anatomy of the venom producing parotoid glands of Bufo marinus has been studied by light and electron microscopy and reactions for the presence of glycoprotein and mucopolysaccharides, the catechollamines, 5-hydroxytryptamine or dopamine, glucose-6-phosphatase, adenosine triphosphatase and the steroid nucleus and cholesterol and its esters have been performed. The gland is composed of numerous individual lobules, each lobule surrounded by a double cell layer. The interior surface of the outer layer is thrown into small cytoplasmic projections which traverse an intercellular space and interdigitate with microvilli formed by the outer plasmalemma of the inner layer. The outer layer resembles smooth muscle-like cells, is rich in adenosine triphosphatase, contains many pinocytotic vesicles and various organelles and may function in some aspect of venom synthesis, active cellular transport and contraction in the discharge of the secretory product. The inner layer shows a positive chromaffin reaction, contains various organelles, appears devoid of a plasmalemma on its inner surface and is involved in venom formation and release via an apocrine type of secretion. The intercellular space is rich in PAS positive materials, while the secretory product, itself, demonstrates a positive chromaffin reaction. The significance of these findings is discussed.

Notes: The synganglion of Dermacentor variabilis Say is a single nerve mass, condensed around the esophagus and within the periganglionic sinus of the ciculatory system. Protocerebral, cheliceral (including stomodeal bridge), and pedipalpal ganglia lie in the pre-esophageal portion of the nerve mass and bear optic, cheliceral, and pedipalpal nerves respectively. The unpaired stomodeal and the recurrent nerve which forms the hyper-esophageal ganglion arise from the stomodeal bridge. Paired primary and accessory nerves to the retrocerebral organ complex have mixed protocerebral-cheliceral origins. Pedal ganglia (including ventral olfactory lobes of pedal ganglia I) and composite opisthosomal ganglion lie in the post-esophageal nerve mass and bear pedal nerve trunks and two pairs of opisthosomal nerves respectively.Internally, the synganglion consists of cellular rind and fibrous core. A welldefined neurilemma with a laminar matrix covers nerve mass and peripheral nerves. The rind contains the somata of ganglionic neurons and ensheathing glial cells and is restricted to the synganglion mass. It is limited by two specialized glial layers, the external perineurium and internal subperineurium. Discrete glomerular formations are present within the protocerebrum and olfactory lobes. Olfactory glomeruli located in pedal ganglia I are associated with a pair of globuli cell groups.Possible physiological relationships between anatomical specializations of the synganglion, extraneural sinuses and circulating hemocytes are considered. The evolutionary significances of condensation in the stomatogastric neuropile regions and throughout the synganglion, together with the simplification and loss of glomerular formations, are discussed.

Notes: The intraspecific mean length of medullary cones in avian kidneys is analogous to medullary thickness in the mammalian kidney. Hence, relative medullary thickness (based upon kidney volume) can be calculated for birds as was done in mammals years ago. Comparative figures are given for 26 species from nine avian orders. The organizational pattern of cortex and medulla in the bird kidney is reviewed, and a simplified diagram of this relationship is presented. With some exceptions, urine concentrating ability and relative medullary thickness are directly proportional in mammals. Contrarily, no similar trend was evident in birds when current information on water economy was compared to relative medullary thickness in various species. There are a number of factors (such as the respective functional roles of reptilian and mammalian-type nephrons, interspecific variations in ion transport, etc.) which require study before the significance of relative thickness in the avian medulla can be evaluated more thoroughly.

Notes: The stylocyte (Gr. stylos; pillar) of Corvomeyenia carolinensis Harrison (Spongillidae), a previously undescribed proiferan cell type, was examined using phase contrast microscopy, histochemistry and electron microscopy. The stylocyte, an anucleolate amoebocyte, is characterized by a rhomboidal intranuclear crystal. The crystal, lacking an investing membrane, is embedded directly into the nucleoplasm. It is homogenous with no demonstrable crystalline subunits. Histochemical studies suggest that the crystal is proteinaceous, containing no DNA or RNA. Cytoplasmically, the stylocyte contains promienent homogenous smooth membrane-bound inclusions which contain high levels of neutral (PAS-positive) and polycarboxylated mucopolysaccharides but low levels of glycogen and no significant phosphatase activity. The granular endoplasmic reticulum is poorly developed. Correspondingly, with various histochemical methods, little or no cytoplasmic RNA is demonstrated. Because electron microscopic studies of C. carolinensis indicate the probable absence of viral inclusions in the sponge and because the crystal contains no histochemically demonstrable nucleic acid, the evidence appears to suggest that the crystal neither represents an assemblage of mature virus units nor a virus-induced structure. The stylocyte cell type may play a role in nutrient cycling in C. carolinensis with the crystal acting either as a site of protein storage or as an excretory product.

Notes: Regions of the crayfish kidney were examined by electron microscopy. Coelsmosac cells are loosely bound together by desmosome-like spot junctions, and connected to the basal lamina via characteristic pedicels. The cytoplasm contains numerous vesicles and vacuoles of various sizes and is often crowded with large, lysosome-like granules or dense bodies. The morphology suggests a filtration mechanism with reabsorption of materials such as protein from the filtrate and secretion of other substances into the lumen.The labyrinth is composed of cuboidal to columnar cells which possess a brush border, long and narrow intercellular spaces, basal plasmalemmal invaginations and typical cytoplasmic components. Two sub-regions are distinguishable. The morphology of labyrinth I suggests that these cells move fluid isotonically across the epithelium. Labyrinth II, in addition to isotonic transport, appears to be more active in the endocytic uptake and intracellular digestion of large molecules such as protein.The nephridial canal consists of cells which lack a brush border, but display extensive basal invaginations associated with elongated mitochondria. A proximal and distal region are cytologically distinguishable. Proximally, the cells are small and filled with mitochondria throughout. Scattered within the cytoplasm are vesicles, vacuoles, diffuse glycogen, free ribosomes, dense bodies and some rough endoplasmic reticulum. Distally, the cells are less compact, larger, and cuboidal to columnar in shape. The cytoplasm is similar to that of the proximal cells, but the basal invaginations are even larger and more extensive. The morphology of cells in both regions of the nephridial canal is highly suggestive of active solute reabsorption, probably occurring against an osmotic gradient.

Notes: The distal depression of the ventral pedal groove of Mytilus californianus was investigated by scanning and transmission electron microscopy. This part of the byssus forming system is responsible for the formation of the attachment plaque of the byssus thread.The longitudinal pedal ducts open into this area and the floor of the distal depression is covered by specialized cilia which terminate as biconcave flattened discs or “paddles.” The disc is formed by a 360° curvature of the axoneme tip within the ciliary membrane. The diameter of the disc is about 1.33 μ while that of the shaft portion is 0.24 μ. There are about 11 cilia per square micron of surface area and the necks of the cilia are separated from each other by a web-like extension of apical cytoplasm extending from the epithelial cells.It is proposed that these specialized cilia function as microscopic spatulas for the application of the adhesive plaque material to substrate surfaces. The pattern of surface convection currents seen in vivo tends to support this hypothesis.

Notes: Light and electron microscope studies of development of the ultraviolet-reflecting scales of male Colias eurytheme butterflies show that basic developmental processes are similar to those of other scales. The ridges form between bundles of microfilaments and as they form they buckle to produce the lamellae seen in the adult scales. There is evidence that the buckling may be purely in response to mechanical stress and that some of the bundles of microfilaments may produce such stresses.

Notes: A large extrachromosomal mass of Feulgen positive material, the DNA body, has been visualized in early prophase oocytes of crickets (Orthoptera: Gryllidae) representative of the closely related subfamilies Gryllinae and Nemobiinae. A similar structure is present in oocytes of representatives of two subfamilies of crickets (subfamilies Oecanthinae and Gryllotalpinae) which taxonomically and phylogenetically are quite separate from those mentioned previously. In situ hybridization demonstrates that the body contains amplified copies of genes coding for ribosomal RNA. Unlike the DNA body in early diplotene oocytes of representatives of the subfamily Gryllinae, which is closely associated with the developing nucleolar apparatus, the DNA body in oocytes of the Oecanthinae and Gryllotalpinae cannot be demonstrated during diplotene. In the Oecanthinae, the nucleolar apparatus of early diplotene stage oocytes is composed of four to seven separate structures, the ribonucleoprotein of which has a characteristically lamellated appearance. During late diplotene, these nucleoli give rise to many smaller structures which are distributed throughout the germinal vesicle. In early diplotene stage oocytes of Scapteriscus acletus (Subfamily: Gryllotalpinae), the nucleolar apparatus consists of a single compact mass of ribonucleoprotein. In contrast to the oocytes of all other crickets that have been studied, the nucleolus of S. acletus remains single throughout diplotene. In situ hybridization analysis indicates that the amplified genes coding for rRNA which are localized in the DNA body of early prophase oocytes become incorporated into this compact nucleolar mass. Differences in nucleolar structure appear to reflect differences in the organization of amplified genes coding for rRNA.

Notes: An arbitrary classification scheme is presented for the thirteen distinct types of secretory cells distinguished within the central nervous system of Dermacentor variabilis by several specific and general neurosecretory staining techniques. Comparisons to classic arthropod neurosecretory cell types are made and the histochemical implications of the chromophilic response of various secretory products are discussed. Dermacentor cells of Types I, VII, IX and X may be considered neurosecretory on the basis of intracellular elaboration and discharge of secretory product. Type II, III, IV, V, VI, XI and XII cells are considered as putative neurosecretory cells although secretory products were detected only within the perikarya. The large Type XII cells are also similar to motor neurones reported from other arachnids. Cells of Types VIII and XIII appear to be glial elements. The secretory products of Type XIIIA are distributed within trabecular processes in the subperineurium. These products may play a trophic role or they may have some endocrine function as a form of “gliosecretion”.

Notes: Rostral pores are epidermal invaginations which occur on the internarial region of most chelonians. Representatives of all Recent chelonian families and 67 of the 74 extant genera were examined grossly and/or histologically. Pores are absent only in the families Carettochelyidae, Cheloniidae, and Dermochelyidae. The number and microscopic structure of pores vary markedly within and between taxa. Morphological data suggest that rostral pores could function in mechanoreception. The possible origin and evolution of rostral pores are discussed in the context of other chelonian integumentary speializations.

Notes: This study of the testicular capsule of rat, dog, cat and human has confirmed the presence of three layers, viz., the tunica vaginalis, the tunica albuginea proper and an innermost tunica vasculosa. Smooth muscle cells are present in the tunica albuginea of all four species and are more prominent at the posterior pole of the testis where the capsule merges with the mediastinum testis. In the rat and the dog, a few striated muscle fibers also are present.While the tunica albuginea is to be considered as a dense connective tissue, the arrangement of the collagen bundles and the presence of a relatively high content of elastic fibers probably permits changes in size of the testis following spontaneous contractions of the muscle elements, which are known to occur. The role of the testicular capsule in sperm transport is discussed in relation to other factors, the spontaneous contractions of the capsule presumably having a “pumping” action and aiding the movement of non-motile spermatozoa from the testis to the epididymis.The presence of striated muscle fibers in two species is of interest and, while these may function in a similar manner to the smooth muscle, they may represent simply an unusual differentiation of embryonic myoblasts.

Notes: The surface structures of the antennular flagella of Pagurus alaskensis are described in detail. Attention is directed towards the surface morphology of two types of possible sensilla: (1) exoskeletal pores (1.0-3.0 μm in diameter); (2) setae of various kinds. In addition, small (0.1-0.2 μm) pits occur in the exoskeleton which are not considered to be sensory in function. The exoskeletal pores are found at fairly specific locations on both the inner and outer flagella, particularly on the short segments of the outer flagella. Neither the inner nor the outer flagella are bilaterally symmetrical with respect to their setal armature. On the outer flagellum six groups of setae may be distinguished: lateralmesial; dorsal; ventral; accessory; aesthetasc; setae of the distal segment. On the inner flagellum setae of the mesial and lateral rows form distinctive groups. The morphology, orientation and locations of all the flagellar setae are defined and where possible the numbers of the various morphological types within the specific setal groups are given. It is noteworthy that many setal types have obvious apical pores and yet no pores could be found in the chemoreceptive aesthetasc setae. The functions of the various setae are discussed in relation to their topographical position and to existing electrophysiological and behavioral data. Some suggestions are made about future experiments to demonstrate the central connections of specific sensilla or groups of sensilla and to show their significance in the whole animal.

Notes: Blood vessels in Nereis japonica were studied by electron microscopy. It was found that blood vessels regardless of location were similar in the basic organization of the basal lamina and the usual presence of collagen fibrils on the vessel wall. Differences arise, depending on whether the outside of the basal lamina is covered by peritoneal cells, by gut epithelium, or by epidermis. These relate to the location of the vessels in mesenteries, gut or epidermis, but do not reflect basic structural differences in the vessels themselves. Furthermore, it was concluded that true endothelial cells do not exist in the circulatory system of Nereis japonica and that, in this respect, the system is essentially different from that of vertebrates, in which endothelial cells line the vessels of a closed circulatory system. These considerations lead to the further conclusion that the vascular lumen in Nereis is essentially interstitial space and that the system, which has been known as a typical “closed” circulatory system in annelids, is actually an open circulatory system. Peritoneal cells covering the walls of internal vessels show various degrees of muscular differentiation and those possessing myofilaments may be called “myomesothelial cells.”

Notes: The nymphal and adult cervicothoracic skeleton of Euborellia annulipes (Lucas) is described and discussed.

Notes: The mean number (± S. D.) of nucleoli per hepatic cell was determined for inbred mice of the BALB/c and C57BL/6J strains, using azure B bromide. Because thyroid hormones have been reported to increase nucleolar number in cultured human cells, certain of the males received parenteral triiodothyronine (T3). Hormone effect was demonstrated by a two-fold increase in hepatic mitochondrial α-glycerophosphate dehydrogenase activity. Results included the following: (1) In the BALB/c strain, the nucleolar number of the T3 treated males (5.0 ± 2.4) was slightly but significantly higher (p 〈 0.001) than that of the controls (4.8 ± 2.3). For the C57BL/6J strain, however, the nucleolar number of the treated males (4.0 ± 1.7) was significantly lower than the controls (4.2 ± 1.8; p 〈 0.001). (2) The difference in nucleolar numbers of untreated males of both inbred strains was highly significant (p 〈 0.001). (3) The nucleolar number in BALB/c female cells (4.1 ± 1.8) was significantly lower than that of male BALB/c cells (p ≪ 0.001).

Notes: Each silk gland of Calpodes ethlius consists of five distinct regions: the duct, the green, anterior, middle and posterior regions. Although the gland increases approximately tenfold in length during the larval life, the number of cells remains constant with a concomitant increase in ploidy which is not constant either throughout larval life or in the different regions of the gland. Histochemistry on the glands of the mid-fifth instar larva shows that progressively more mucosubstances are deposited in the lumen, so that while in the distal regions there is only one weakly acidic deposit, this is increased to three more acidic bands in the proximal regions. These bands can be correlated with materials of different electron density. All five regions have characteristic secretory ultrastructure, with prominent secretory vesicles or granules and microvilli. However, the posterior and middle regions have electron-translucent vesicles and relatively short microvilli, while the other three regions have electron dense granules and a more complex, microvillate apical surface. This complexity is greatest in the duct which suggests that it may function in water reabsorption.

Notes: A band of flexible cuticle encircles the deutonymph, separating the dorsal and ventral plates. The coxae are large, flat and fused with one another to form most of the ventor. Individual coxal margins are redefined as sternites, epimerites or simply apodemes according to which margins fuse with which others. A given area of cuticle may have patches of dark or light cuticle not corresponding to particular structures or cuticular contours; this is a source of confusion to taxonomists.Each leg has a dicondylic coxal-trochantal (adduction-abduction) and trochantal-femoral (promotion-remotion) joint with opposing muscles. The three more distal monocondylic joints (flexion-extension) have only flexor muscles; extension is by increased haemolymph pressure.The five apodemes of the sucker plate provide rigidity; the four suckers attach by a flexible cuticular ring to a solid flange or socket in the sucker plate. The sucker muscles attach to the center of each sucker. The flat, external face of the sucker plate apodemes may complement sucker action by adhesion. Coxal discs and sucker plate discs are identical, contain birefringent cuticular elements, and are considered modified setae.Functional mouthparts and a pharynx are lacking, but a cheliceral anlage is present. The esophagus, midgut and caecae, and malpighian tubules are lumenless and the cells small. The hindgut has a lumen, larger cells and opens externally via the anus. Whereas the digestive tract is regressed, the reproductive system is yet incompletely developed. In older deutonymphs anlagen of ducts, accessory glands and gonads are discernible. The nature of the haemocoel and peritoneum remains nuclear.The central nerve mass is conspicuously large for the size of the deutonymph. The supraesophageal ganglion gives rise to the cheliceral nerves; all other nerves arise from the subesophageal ganglion. Most major nerves were traced to the effector organs.The muscles are divided into leg, dorso-ventral (derived from coxal muscles), dorsal, sucker, and anogenital muscles. The trochantal adductor muscles originate on an endosternite, which is supported by muscles running to the dorsal hysterosoma. The dorso-ventral and propodosomal retractor muscles affect haemolymph pressure. The massive sucker retractor muscles are unique to this instar. Anogenital muscles are not well developed.

Notes: The pattern of transport and distribution of rabbit embryos in the oviduct and uterus was studied 15 to 168 hours post coitum (p. c.). The reproductive tract was frozen in liquid nitrogen, thawed, and cleared in benzyl-benzoate solution using Orsini's technique. The location of the eggs and the ampullary-isthmic junction were identified using transmitted light from a dissecting microscope. Accumulation of the eggs in the oviduct occured in two phases. In the first phase the eggs were retained above the ampullaryisthmic junction, 3-12 hours after ovulation. In the second phase, the eggs were retained 36-60 hours after ovulation, above the uterotubal junction (at a distance approximately 12 % of the oviductal length). The rate of transport of individual eggs in the oviduct, and the time of the entry of eggs into the uterus were variable. Au 78 hours p. c. most blastocysts occupied the proximal half of the uterine horn, although some appeared very close to the internal os of the cervix. Spacing of blastocysts in the uterus, 114 to 120 hours p. c., involved movement of blastocysts away from the cervix. Unfertilized eggs remained in the uterus, along with developing blastocysts 168 hours p. c. Few eggs were retained in the oviduct at 108 and 115 hours p. c.

Notes: An ultrastructural study of the hindgut, associated hepatopancreatic lobes and hepatopancreatic ducts of the terrestrial isopod, Armadillidium vulgre was undertaken. The simple epithelium which lines the heptopancreas consists of two cell types, S and B. Both cell types have numerous microvilli. A simple epithelium consisting of one cell type lines the hepatopancreatic ducts and the hindgut. Microvilli are not evident in the duct. The cells of the duct and the hindgut are covered with a cuticle. Dense granular bodies are characteristic of the cytoplasm of the duct cells. The hindgut epithelial cells have pronounced apical and basal-lateral cytoplasmic infoldings. Apical infoldings from large subcuticular spaces, especially in the posterior hindgut. Microtubules and large numbers of mitochondria are associated with the cytoplasmic infoldings. Large microtubular bundles are seen in the peripheral cytoplasm, and residual bodies are present in the central cytoplasm. Lateral plasma membranes form septate desmosomes in the apical region of cells, while zonulae adherentes and intercellular spaces are found basal to the septate desmosomes. The cellular organization of the hindgut is suggestive of cells active in water and ion transport.

Notes: The ultrastructure of the phyllobranchiate type gill of the shrimp, Caridina japonica, was studied. The most characteristic feature of the open circulatory system of Cardina is the vascular lumen of the gill capillaries which is considered to be the interstitial space. The following observations substantiate this view: (1) a thin fibrous layer forms the innermost structure of the walls of gill capillaries and is in direct contact with the blood stream; (2) filaments in the fibrous layer are assumed to correspond to the reticular fibers in the interstitial space of the alveolar wall of mammals; (3) the absence of the endothelium as well as the endothelial basal lamina which are the essential structural components of the closed circulatory system in vertebrates. The gill epithelium contains intermediate, septate and tight junctions. The first two form a junctional complex near the apical cell border and may function as a permeability barrier by occluding the intercellular space as well as functioning in electrical coupling and cellular adhesion. The tight junction is spot-like and may serve no role in the function of the permeability barrier.

Notes: The hermit crab, Pagurus pollicarus, has the same organization in its fourth abdominal ganglion as its macruran relatives in spite of the reduction in abdominal muscles, sensory receptors, and appendages. Connective axons are grouped into discrete bundles between which five groups of commissural fibers run to connect left and right sides. The neurites of ventral cell bodies run dorsally in characteristic groups between the connective bundles. The hermit crab fourth ganglion has two thirds as many cells as the crayfish and is laterally compressed. This reduction appears related to the reduction in the sizes of the ganglionic roots. The ventral fine fibered neuropil is larger on the left than the right side reflecting the loss of pleopods on the right side. The basic organization of decapod abdominal ganglia appears to permit considerable integrative flexibility within a relatively conservative morphological framework.

Notes: Comparative histological observations of the eyes of Typhlotriton spelaeus and several epigean, plethodontid species indicate that the principal postembryonic degenerative changes in the eyes of T. spelaeus involve the eyelids and cornea, visual cells, outer plexiform layer, and the pigment epithelium. Ordinarily these changes were initiated after metamorphosis, before attainment of sexual maturity, but a few larvae had degenerating retinae.The corneal epithelium becomes irregular and thin as eyelids develop during and after metamorphosis, but retains its larval structure in animals in which eyelid overlap is incomplete. Disruption and vacuolation of the lens sometimes occurs in postmetamorphic animals with degenerating visual cells. Retinal degeneration involves reduction of the inner and outer segments of visual cells, loss of the outer plexiform layer, and retraction of apical processes of the pigment epithelium. In its earliest stage, retinal reduction is first apparent at the retinal margin where visual cells are normally less well-differentiated, but in its terminal stage reduction has gone to completion over the entire retina. Extent of retinal degeneration in adults is directly related to postmetamorphic age but there is variability in each age group. Females generally have smaller eyes, and more extensive degeneration of visual cells than males. The loss of visual function in adults is correlated with extensive visual cell degeneration.

Notes: This report adds to the available information on comparative histology of mammalian bone and introduces a semi-quantitative approach to its study. Bone samples consisted of large fragments of rib tibia and/or femur from humans, laboratory rodents and from animals that died at the Bronx Zoological Gardens. A total of 44 bone specimens representing six mammalian orders were available. Decalcified paraffin-embedded 10 μm histological cross-sections were examined at × 320. Qualitative observations were supplemented with measurements of the frequency and size distributions of Haversian and non-Haversian canals. The relative distribution of lacunae was also ascertained. The standard textbook description of mammalian bone as consisting mainly of secondary osteons was not generally seen except in Primate (especially human) bone. Rats showed a few scattered osteon-like structures, but bones of Marsupialia, Insectivora, Artiodactyla and Carnivora were entirely devoid of them. Generally, vascular bone with longitudinal canals was seen except in Lorisidae which showed a reticular type of bone. The distribution of primary longitudinal canals and the number of “filled” or “apparently empty” lacunae/unit area of bone varied both inter-species and among different areas in the same bone. Large areas of acellular and non-vascular bone were encountered in all specimens. This preliminary study revealed that species differences i bone microstructure involve the relative distribution of the same basic components which lend themselves to quantitative treatment. Comparative investigations of bone histology at many ontogenetic and phylogenetic levels should yield significant quantitative information in bone biology.

Notes: Spleens from normal, healthy cats, dogs and rabbits were perfused with Ringer solution until only a few red cells remained. After fixation of the intact organ, small pieces of tissue were dried by a camphene method and examined under the scanning electron microscope. In all three species the red cells remaining in the spleen were either reticulocytes, spiculated cells, or cells of tear-drop shape and they were found adhering to macrophages and reticulum cells throughout the red pulp. Elongated masses were found on the sinusal surface of fenestrated endothelium (only in dog and rabbit); some of these appeared to be cells of tear-drop shape emerging from the cords into the sinus. This may perhaps denote a pitting process, as suggested by others, but it cannot be a unique function of fenestrated endothelium for red cells of similar shape were found elsewhere in the pulp. In all three species the network of reticulum fibres presents a very large contact surface area for blood cells and it seems likely that increased cell stickiness, rather than decreased deformability, leads to the trapping of immature red cells in the spleen.

Notes: Allografts of embryonic limb buds were grafted orthotopically on embryos of Chelydra serpentina. Donors were from a different geographic area, the same geographic area, or siblings. The initial indication of rejection was excessive sloughing of epidermis. This was followed by loss of muscle, claws and bone.Early histological changes involved an infiltration of mononuclear or rejection cells primarily associated with small blood vessels of the connective tissue. Subsequently, muscle and bone were lost and they were replaced by connective tissue. Epidermis and nerves persisted. The skeletal cartilages were isolated from immunological activity.Although the incidence of rejection was essentially the same in sibling and non-sibling combinations, the initial external signs of rejection occurred earliest when donor and host were from different geographic areas but not later than two years after hatching. The first signs of rejection in sibling allografts occurred not later than three years after hatching. Animals that survived these periods without rejection did not show subsequent rejection.

Notes: Allografts of skin were observed in Chelydra serpentina. The response to these grafts was modified by a previous transplantation of a limb bud at an early embryonic stage. When the same donor was used for all transplants, the first skin graft was accepted by the host. A second skin graft, however, was rejected at about the rate of a simple first set allograft of skin. The animals were conditioned by the embryonic limb graft; this embryonic graft can be undergoing rejection at the same time a first set skin graft from the same donor was being accepted. The tolerance induced by the embryonic graft was sepcific for its donor.

Notes: As part of program of research into insect cellular immunity, an integrated light and electron microscopic study of the hemocytes of seven members of the Order Dictyoptera has been made. In fresh hemolymph, five cell types, the prohemocytes, plasmatocytes, granular cells, spherule cells and cystocytes, are easilv distinguished. However, in thick Araldite sections and in thin sections in the electron microscope it is sometimes difficult to identify the various cell types. The reasons for this difficulty are discussed.Granules with a microtubular substrcture are found in the plasmatocytes, spherule cells and cystocytes. In the plasmatocytes these granules have a different ultrastructure than those in the spherule cells and cystocytes. The in vitro fragility of these granules in both the spherule cells and cystocytes during coagulation partially explains the previous confusion in distinguishing these two cell types.Evidence is presented which indicates that the plasmatocytes, granular cells and spherule cells represent a developmental series originating from the prohemocytes. Where exactly the cystocytes are derived from is unknown.

Notes: A cardiaca-allatal commissural plexus (CACP) lies between and partly overlapping the postcommissural lobes of the corpora cardiaca (CC), the nervi corpori allati I (NCA I) and the corpora allata (CA). CACP, which is often continuous posteriorly with a complicated postallatal plexus (PAP), comprises a variable number of connectives with neurosecretory processes linking the cardiaca-commissural organ or dorsal cardiac commissure (containig tritocerebral fibres) to the NCA I. the allatal commissure and the CA. Neurosecretory processes are exchanged between the two halves of the cephalic neuroendocrine complex (CNC) both intracerebrally at different locales, possibly to ensure functional synchrony of CNC components.NCA I and CACP are drawn out with their stroma to varying extents over the CA. Histophysiological evidence suggests that part of the stainable secretion stored in, and or in axonal transit through CA may be released through CA surface; NCA I, the nervi cardiostomatogastrici, CACP, perhaps also NCA II may function as neurohaemal areas. A “directed” neurosecretory pathway could be distinguished from PAP to the foregut and the fat body. The degree of spatial intimacy detected between neurosecretory and stomatogastric components of CNC suggests that the two systems may function in an integrated fashion. The recurrent-oesophageal nerve complex serves not only for a direct transport of neurosecretion, but also as one of the sites of its release.

Notes: Four glands of the house sparrow, chicken and turkey were examined histologically and for their content of amylase. These were the external and intermediate mandibular glands, the maxillary gland and glandula anguli oris of the sparrow and the anterior and posterior mandibular, maxillary and anguli oris glands of the chicken and turkey. Amylase was determined by a starch substrate slide method and by biochemical assay. General morphology and mucopolysaccharide staining are described.All four glands of the sparrow demonstrated significant amylolytic activity by the assay. In the external mandibular and anguli oris glands this activity could be traced to mucous and seromucous cells of origin by means of the starch substrate slide procedure. None of the glands of the chicken or turkey displayed significant amylolytic activity.

Notes: A cinematographic analysis of the unrestrained walking, trotting, galloping, jumping and landing movements of 11 adult cats was undertaken to provide previously unavailable information concerning the demands imposed on the nervous system for the control of low and high speed movements and the demands imposed by such natural movements on muscle performance and proprioceptive response.With due regard for the swing (F and E1) and stance (E2 and E3) phases of the step cycle of an individual limb, single frame analysis of the film permitted measurement of instantaneous angles of the lower spine, hip, knee, ankle and metatarsophalangeal joints. Appropriate lever arm measurements were also made on 50 freshly dispatched cats and 25 cadavers such that the Law of Cosines could be used to calculate instantaneous lengths of select hind limb muscles that would apply to the natural movements of adult cats of small (1.5-2.5 Kg), intermediate (2.6-3.5 Kg) and large (3.6-4.5 Kg) size. Muscle displacements were analyzed relative to maximum and minimus in situ lengths and the lengths associated with quiet standing. Use was also made of a previous electromyographic analysis of hind limb muscles during unrestrained locomotion (Engberg and Lundberg, '69).The sequential relations between the four phases of the step cycle are maintained as forward speed increases from walking ( 〈 2 mph) to high speed galloping ( 〉 16 mph). There are significant differences in the time consumed by each phase, however, with a greater reduction in the E3 phase, little reduction in the E2 and E1 phases and virtually no reduction in the F phase. When each phase is expressed as a relative percentage of the duration of the total step cycle, the greatest reduction is again in E3 with little change in the E2 phase. In contrast F and E1 phases increase in the percent of time they occur in each cycle, with the greatest increase in the F phase. For all speeds, analysis of the phase relations between movements of various sections of the hind limb revealed a remarkable unity of knee and ankle joint movement. The hip joint is largely out of phase with the knee and ankle during E1 and E2, all three joints being in phase in F and E3. The digits are essentially out of phase with the other joints except in the stance phase of the gallop.Rates and extents of muscle displacement during natural movements are greater than might be anticipated when expressed in absolute mm's and mm/sec but not when considered in relation to maximum and minimum in situ length and the length associated with quiet standing (Ls). During stepping a progressive increase in forward speed results in: (a) a greater usage of muscles at lengths between Ls and maximum in situ length; (b) for knee and ankle extensors, pronounced increase in the lengthening contraction associated with the E2 (yield) phase of step; and, (c) for both flexor and extensor muscles, an increased active phase of lengthening or near isometric contraction immediately prior to periods of active shortening. In contrast to these changes in active muscle status, the change from walking to galloping has little effect on the extent and rate of passive muscle displacements, particularly the F phase stretch of extensors.For the soleus muscle, calculations were made of the relation between changes in overall muscle length during natural movements and the length of the average muscle fiber and the tendon of insertion. These measurements revealed that the increases in fiber length when passive and decreases in length during active shortening are less than would be anticipated from the extensive liteature on extirpated fibers. In contrast, the increase in fiber length when active is greater than would be expected from the admittedly sparse literature on this subject.The results of this study are discussed largely in relation to two points of neurophysiological interest: the physiological range of muscle stretch as it pertains to the responsiveness of muscle spindles and tendon organs; and those mechanical aspects of lengthening contractions that give insight into the neural control of stepping. For exciting both spindles and tendon organs passive muscle stretch and shortening contractions are shown to be relatively ineffective and lengthening and isometric contractions particularly effective movements. It is suggested that, just as recent literature has emphasized the co-activation of efferent alpha and gamma motoneurons as a muscle becomes active, so too is there a synchronous activation of afferents, particularly the Ia and group II endings of muscle spindles and Ib endings of tendon organs. Finally the thesis is advanced that, while it has been convenient to separate E2 from E3 in the description of the stance phase of the step cycle, extensor muscles are actually undergoing a single mechanical event: an active stretch-shorten cycle for knee and ankle extensors and an active isometric-shorten cycle for hip extensors. This hypothesis has significant implications for the neural control program that regulates the stepping sequence in that it emphasizes the extent to which appropriate changes must be preprogrammed in the mechanical properties of muscles for the smooth execution of stepping.

Notes: Cytological and cytochemical methods were used to identify and characterzie six distinct regions of the crayfish kidney: coelomosac, labyrinth I and II, and nephridial canal I, II, and III. Cells of the coelomosac possess cytoplasm which is strongly PAS-positive, but poor in RNA and protein. Their nuclei possess unusual projections which extend to the basal plasmalemma. Labyrinth I contains columnar cells rich in glycogen. Labyrinth II is characterized by a distended lumen and by shorter cells with high cytoplasmic RNA, many possessing a large intracellular vacuole. A PAS-positive brush border is unique to the two portions of the labyrinth. Cells in the nephridial canal show strong reactions for RNA and Mg++-dependent ATPase. In nephridial canal I and II, cells are flattened to cuboidal with the lumen being more distended in nephridial canal I than anywhere else in the tubule. In nephridial canal III, the cells are large and columnar, and the cytoplasmic RNA concentration is greatest apically. Nuclei in all regions of the tubule epithelium, except coelomosac, are large and react strongly for protein. Coelomosac nuclei and those in blood cells are condensed and contain little protein. The cytoplasm of blood cells displays a significant amount of RNA, and traces of polysaccharide material.These observations demonstrate the presence of highly specialized morphological and histochemical alterations along the length of the kidney tubule and indicate sequential modification of urine in the lumen. Evident morphological and cytochemical likenesses between analogous regions of the mammlian nephron and the crayfish kidney tubule suggest that basic physiological similarities may also exist.

Notes: In the tiger salamnder the distribution of optic fibers, as revealed by the Fink-Heimer method, is compared with the localization of acetylcholinesterase, as revealed by histochemical methods. AChE activity coincides with optic nerve axons in the optic fiber layer of the retina, in the optic nerve, in the optic tracts and in every optic projection. Reginons of optic fiber terminals show heavier activity than optic fibers of passage. Comparison with other vertebrates is also made.

Notes: Light and electron microscope studies indicate that the old ependyma just proximal to the plane of amputation in early lizard tail regenerates shows a sequence of morphological changes which suggests that it as well as the new ependyma growing into the regenerate may play an active role in the initiation and maintenance of early tail regeneration.The old ependyma close to the plane of amputation undergoes hypertrophy and/or hyperplasia causing a partial closure of the central canal and pseudostratification. Its nuclei shift from an original apical position to a basal one. The ependymal processes become more prominent and extend to the pia, a condition not found more rostrally. There is also a significant increase in the amount of Golgi substance and a moderate increase in the rough endoplasmic reticulum. These observations lead to the thought that these cellular changes may be an expression of enhanced secretion and other activities in the old and new ependyma just proximal or distal to the plane of amputation.

Notes: The dorsal proctodeal wall of the Coturnix contains a gland which is especially well-developed in the sexually active male and which secretes a foamy exudate. This gland has not been recorded in any other genus of birds and appears to be unique to members of the genus Coturnix. On the basis of its location in the adult we have named this gland the proctodeal gland (glandula proctodealis). Structure of the gland does not conform to any curently defined category of glands because it is an aggregate of individual glandular units each of which opens independently into the proctodeal cavity but with the entire structure enclosed by a connective tissue capsule. We have thus characterized it as an aggregate gland. Observations on the arrangement and composition of the fibrous capsule and its intimate relationship to the perimysium of M. sphincter cloacae indicate that release of the secretion probably results from contraction of the cloacal musculature. Preliminary data suggest that development of glandular tissue in the female may be a function of age rather than of androgen sensitivity which characterizes the male gland. It is concluded that the chemical nature of the glandular secretion and its function in the sexually active bird, as based on presently available information, remains unknown.