ALBERT

Notes: Scanning electron microscopy revealed spores of Nosema apis and Thelohania fibrata to be egg-shaped, but only the mature spore of T. fibrata was shown to possess a horseshoe-like concavity at the posterior pole. Freezeetched preparations indicated that this concavity was due to a thin area of the spore coat. Freeze-etching studies also show spores of N. apis do possess an umbrella-shaped polaroplast, and a polar filament which is arranged in a double layer with over 30 coils. The spore of T. fibrata contains a pear-shaped arrangement of the polaroplast membrane, and a polar filament arranged in a single layer of 22 coils.

Notes: The fiber constituents and connections of the calyces  -  the input-receiving regions  -  of the corpora pedunculata (“mushroom bodies”) were studied in reduced silver preparations from the American cockroach, Periplaneta americana (L.). In the outer synaptic layer of the calyces five fiber classes were distinguished, the first three of which arise outside the mushroom body. (1) Four highly similar neurons with somata near the optic lobe branch into different parts of the ipsiateral protocerebrum, including both calyces. Their fibers are highly constant in arrangement and position and contain small nucleus-like bodies. (2) The tractus olfactorio-globularis (sensu lato) emits fiber groups which course along the calycal walls as “calycal tracts” before ultimately dissipating into the synaptic layer. Variability within these tracts is described. (3) Fibers of undertermined origin outside the mushroom body radiate from the calycal center outwards through the synaptic layer. (4) From the inner calycal layer of neurites belonging to intrinsic mushroom-body neurons, perpendicular collaterals enter the synaptic layer. (5) Intrinsic-neuron somata near the calycal rim emit fibers which course tangentially within the synaptic layer from calycal rim to center. These fibers form a special peripheral zone in the pedunculus.The predominant presumably afferent calycal fiber class is that derived from the tractus olfactorio-globularis. No evidence was found for tracts from optic lobe to calyces. On this basis, and in light of the experimental and comparative anatomical literature, it is suggested that the corpora pedunculata of P. americana and other pterygotes are fundamentally second-order antennal sensory processing centers.Conflicting observations in earlier reports are critically discussed.

Notes: The epidermis of Eisenia is covered by a cuticle and rests on a basement lamella. The cuticle, which is resistant to a variety of enzymes, is composed of non-striated, bundles of probable collagen fibers that are orthogonally oriented and are embedded in a proteoglycan matrix. The basement lamella consists of striated collagen fibers with a 560 Å major periodicity. Proximity and morphology suggest that the epidermis may contribute to both the cuticle and the basement lamella  -  that is, the single tissue may synthesize at least two types of collagen. The epidermis is a pseudostratified epithelium containing three major cell types (columnar, basal and gland) and a rare fourth type with apical cilia. The esophagus is lined by a simple cuticulated epithelium composed predominantly of a single cell type, which resembles the epidermal columnar cell. Rare gland cells occur in the esophageal epithelium, but basal cells are lacking.

Notes: Gill development begins on the sixth day of incubation at 10°C and is complete by 31 days (hatching). Gill arches are formed by fusion and perforation of ectoderm and endoderm across the pharyngeal wall. A primary branchial artery forms within each arch and a second branchial artery forms as a branch from its ventral end. A series of filament loop vessels forms connecting the two arteries and when several are patent a unidirectional blood flow is established via afferent (second) branchial artery, filament loop vessels to efferent (primary) branchial artery. Part of the efferent branchial artery just above its junction with the afferent branchial artery constricts and occludes. It is suggested that this change in the pattern of blood flow is dependent on differences in resistance of the two branchial arteries. A later extension of the gill ventrally is thought not to be homologous with similar regions in elasmobranchs and Acipenser.

Notes: Scanning and transmission electron microscopy studies were conducted on the antennal sensory sensilla of the hymenopteran parasitoid, Cardiochiles nigriceps Viereck, of the family Braconidae. Distinct morphological differences were found between the chemoreceptors of the male and female. Curved, non-fluted, thin-walled sensilla were found to be very abundant on the male and restricted in location and number on the female. Trichoid, placoid and fluted basiconic sensilla were numerous on the antennal flagella of both sexes. Smooth basiconic sensilla were restricted in number to one per flagellar segment in both sexes. Behavioral data suggest that bent-tipped, thick-walled sensilla unique to the female are involved in detecting a chemical(s) emitted from the host, Heliothis virescens (Fab.).

Notes: The biomechanical role of the mammalian clavicle and the functional significance of the aclaviculate condition were investigated. Shoulder movements in rats (Rattus norvegicus) with excised clavicles were compared to those of normal rats by biplanar plate radiography. Shoulder movements during walking of the claviculate American opossum (Didelphis marsupialis), and aclaviculate raccoons (Procyon lotor) and cats (Felis domestica) were compared by biplanar cineradiography.The mammalian clavicle, where present in its complete form, exerts both a “spoke” and a “strut” effect on shoulder movement. By maintaining a fixed distance between the acromion and manubrium, the clavicle ensures that relative movement between these structures is arcuate. Aclaviculate mammals, in contrast, have linear shoulder excursions that are nearly parallel or slightly oblique to the median plane, depending on the conformation of the thorax. Medial collapse of the shoulder in aclaviculate rats demonstrates that the clavicle is under compression, and thus acts as a strut.Reduction or loss of the clavicle, which has occurred independently in numerous mammalian phylogenies, has been regarded as an adaptation for greater shoulder movement and hence increased stride. However, on present evidence clavicular reduction in cursorial mammals appears to be more directly related to a linear excursion of the shoulder joint and a restriction of limb movements to a sagittal plane.

Notes: The tail of Teratoscincus scincus has dorsal scales that have tubercles on their dorsal and ventral surfaces. Sounds are produced when these rub past each other as the excited animal moves its tail. The relative movement of scales is intensified by caudal torsion. The frequencies of the sounds cover a range from 9 to 25 kops and thus, differ from those produced during vocalizations.

Notes: The locomotor function of the caudal muscle cells of ascidian larvae is identical with that of lower vertebrate somatic striated (skeletal) muscle fibers, but other features, including the presence of transverse myomuscular junctions, an active Golgi apparatus, a single nucleus, and partial innervation, are characteristic of vertebrate myocardial cells.Seven stages in the development of the compound ascidian Distaplia occidentalis were selected for an ultrastructural study of caudal myogenesis. A timetable of development and differentiation was obtained from cultures of isolated embryos in vitro.The myoblasts of the neurulating embryo are yolky, undifferentiated cells. They are arranged in two bands between the epidermis and the notochord in the caudal rudiment and are actively engaged in mitosis.Myoblasts of the caudate embryo continue to divide and rearrange themselves into longitudinal rows so that each cell simultaneously adjoins the epidermis and the notochord. The formation of secretory granules by the Golgi apparatus coincides with the onset of proteid-yolk degradation and the accumulation of glycogen in the ground cytoplasm.Randomly oriented networks of thick and thin myofilaments appear in the peripheral sarcoplasm of the muscle cells of the comma embryo. Bridges interconnect the thick and thin myofilaments (actomyosin bridges) and the thick myofilaments (H-bridges), but no banding patterns are evident. The sarcoplasmic reticulum (SR), derived from evaginations of the nuclear envelope, forms intimate associations (peripheral couplings) with the sarcolemma.Precursory Z-lines are interposed between the networks of myofilaments in the vesiculate embryo, and the nascent myofibrils become predominantly oriented parallel to the long axis of the muscle cell.Muscle cells of the papillate embryo contain a single row of cortical myofibrils. Myofibrils, already spanning the length of the cell, grow only in diameter by the apposition of myofilaments. The formation of transverse myomuscular junctions begins at this stage, but the differentiating junctions are frequently oriented obliquely rather than orthogonally to the primary axes of the myofibrils.With the appearance of H-bands and M-lines, a single perforated sheet of sarcoplasmic reticulum is found centered on the Z-line and embracing the I-band. The sheet of SR establishes peripheral couplings with the sarcolemma.In the prehatching tadpole, a second collar of SR, centered on the M-line and extending laterally to the boundaries with the A-bands, is formed. A single perforated sheet surrounds the myofibril but is discontinuous at the side of the myofibril most distant from the sarcolemma. To produce the intricate architecture of the fully differentiated collar in the swimming tadpole (J. Morph., 138: 349, 1972). the free ends of the sheet must elevate from the surface of the myofibril, recurve, and extend peripherally toward the sarcolemma to establish peripheral couplings.Morphological changes in the nucleus, nucleolus, mitochondria, and Golgi bodies are described, as well as changes in the ground cytoplasmic content of yolk, glycogen, and ribosomes.The volume of the differentiating cells, calculated from the mean cellular dimensions, and analyses of cellular shape are presented, along with schematic diagrams of cells in each stage of caudal myogenesis. In an attempt to quantify the differences observed ultrastructurally, calculations of the cytoplasmic volume occupied by the mqjor classes of organelles are included.Comparison is made with published accounts on differentiating vertebrate somatic striated and cardiac muscles.

Notes: Anolis embryos have limb buds at the time eggs are laid and require about 39 days to complete development at 28°C. Rathke's pouch is present at five days, and the subdivisions of the adenohypophysis are differentiated by ten days after oviposition. The cells of the rostral half of the pars distalis (PD) are derived from the anterior face of Rathke's pouch; cells of the caudal half from the posterior face. Lateral lobe cells differentiate on the lateral margins of the developing caudal PD, and knob-like outgrowths of this tissue attach to the walls of the diencephalon to form the pars tuberalis (PT). Subsequently, the cells of the PT lose their connection with the PD and become a pair of flattened oblong plaques. They reach maximal size in midincubation, and are gradually invaded by nervous elements and incorporated into the walls of the hypothalamus. Electron micrographs demonstrate that the embryonic PT is secretory.Ultrastructurally the pars intermedia (PI) and PD are composed of parenchymous secretory cells in a framework of stellate cells. Stellate cells surround the lumen of Rathke's pouch and are connected laterally by complex junctions that exclude the secretory cells from the luminal surface. They extend in sheet-like processes among the secretory cells to the outer margin of the gland where they form a partial sheath within the basal lamina around the secretory tissue. As development proceeds, the lumen becomes subdivided and the resulting reduced lumina are recognizable as the forerunners of the follicles of the adult adenohypophysis.The cells of the PI are differentiated into secretory or stellate cells halfway through incubation. At this time only half of the cells of the PD can be so classified. Four of the five granulated cell types described in the adult are recognizable by mid-incubation; the fifth cell type (prolactin cell) becomes distinguishable within ten days thereafter, and at hatching appears to be actively synthesizing secretory products.

Notes: The hydranth of the gymnoblastic hydroid Syncoryne tenella is invested by a cuticle approximately 530 mμ thick which is continuous with the periderm of the hydrocaulus. The ectodermal cells of the hydranth possess regularly spaced microvilli orientated with their long axis perpendicular to the ectodermal surface. The microvilli project into the cuticle, and probably serve to anchor the cuticle to the ectoderm. In the hydrocaulus the periderm is loosely applied to the ectoderm: in this region microvilli are absent from ectodermal cells. The periderm is a layered structure composed of finely filamentous material. No structural basis is found for the previously reported differential staining of peridermal layers in the hydrocaulus.

Notes: The nuchal organs of polychaetes from four different families (Nereidae, Nephtyidae, Phyllodocidae and Glyceridae) were examined with the light and electron microscopes. In each case, the organ consists of ciliated cells and primary sensory elements. The ciliated cells are similar to the cells of the adjacent epidermis but bear motile cilia. Primary sensory neurons are situated within the organs in Nephtyidae and Phyllodocidae, but are located within the brain in Nereidae and Glyceridae. Each sensory cell gives rise to a distal process which penetrates between the ciliated cells to form an apical sensory bulb bearing modified cilia. Apically these processes are lined with juxtamembranous plaques. The ciliated cells are innervated by efferent axons from the brain, and in Nereis the axons appear to be peptidergic. The elements comprising the nuchal organs closely resemble those of the vertebrate olfactory mucosa.

Notes: The cuticle of five species of Oligochaeta, chosen to represent differences in size and a variety of biotopes, was studied electron microscopically after fixation with the acrolein-TAPO-osmium tetroxide method. Five distinct layers in the cuticle of all studied species were found. Staining with lead and uranyl ions or with silver proteinate visualized basically the same structural components of the cuticle, but the degree of electron opacity and the distribution of the electron-opaque stain in these components differed according to the staining method used. Since the acrolein-TAPO-osmium tetroxide method visualized the cuticular zones preferentially stained by Thiéry's silver proteinate method, it was concluded that the TAPO method may be considered suitable for the visualization of polysaccharides. Staining with phosphotungstic acid provided some information on the composition of the cuticle of Oligochaeta not obtained by staining ultrathin sections with lead and uranyl ions nor with silver proteinate. The conclusion is that phosphotungstic acid binds to polysaccharides which do not contain vicglycol groups nor active sites responsible for the positive reaction with lead and uranyl salts. Structural components in the cuticle of the oligochaetes studied were characteristic for each species. The taxonomic value of such components, however, must be confirmed by examination of a larger number of species of oligochaetes.

Notes: Laboratory-reared outgrowths of the freshwater sponge Corvomeyenia carolinensis Harrison were examined using histological and histochemical techniques, supplemented by phase contrast observations of cellular behavior. The tissue and cellular components of the spongillid outgrowth region were defined in terms of function and morphogenic state. Archeocytes differ considderably, in both histochemical and morphological characteristics, from other cell types of the adult sponge, being histochemically similar to stem cells reported from a variety of developmental series. Archeocytes exhibit cytological characteristics of unspecialized cells capable of high levels of synthetic activity while other cell types of C. carolinensis, for the most part, can be characterized as fully differentiated cells displaying more restricted synthetic capabilities but often accumulating neutral mucoproteins. The presence of aggregates of amebocytes, not identifiable as archeocytes and possibly engaged in gemmule formation, is discussed in terms of current concepts of gemmulation and cellular developmental capabilities in sponges.

Notes: The long antennal flagellum of Neoconocephalus ensiger is covered with many sharp-tipped hairs that appear to be non-innervated; thick-walled chemoreceptors, that may also have a tactile function; thin-walled chemoreceptors of several kinds and coeloconic chemoreceptors. All of the chemoreceptors are innervated by small groups of neurons. The first flagellar subsegment is unusual in that it bears a small protuberance on its latero-ventral surface. This marks the site of the attachment, internally, of a scoloparium containing about eleven scolopales in which the dendrites of some 23 sensory neurons terminate. The most distal subsegment lacks the scoloparium reported earlier for the grasshopper. No conspicuous difference between the antennae of males and of females was found.

Notes: The antennae of two species of thrips, Bagnalliella yuccae (Hinds) and Frankliniella tritici (Fitch), have been examined with the light and electron microscopes. The antennal flagellum of both species is provided with tactile hairs, thick-walled chemoreceptors and thin-walled chemoreceptors. In addition, B. yuccae, but not F. tritici, has a single coeloconic chemoreceptor on the dorsal surface of the pedicel. Observations were made on the fluids in the lumen of the antennae of E. yuccae in the living insect. The movement of the fluids probably has an important physiological significance.

Notes: The cervicothoracic muscles of nymphal and adult Euborellia annulipes (Lucas) are described, the former for the first time. In the adult, eight new muscles are identified, while the nymphs possess a further seven muscles that disappear at maturation or before. Otherwise the same muscles occur in nymphs as in adults, though some nymphal muscles are less clearly separated from one another than their adult homologs. The attachment sites of certain muscles show a number of slight differences between nymphs and adults. The work emphasizes the necessity of taking the immature musculature into account in assessing the muscular pattern represented in an insect order.

Notes: Some of the cytological characteristics of the hemocytes of the scorpion, Palamnaeus swammerdami, were studied. The morphological features of the arachnid hemocytes were observed to be similar to those of hemocytes of insects, millipedes and isopods. Jones' system of hemocyte classification was extended to the arachnid hemocytes. The six classes of hemocytes indentified in the scorpion correspond to prohemocytes, plasmatocytes, granular hemoocytes, cystocytes, spherule cells and adipohemocytes of insects. A cell type comparable to oenocytoids of insects and crustaceans is absent. The prohemocytes can be subdivided into four categories that probably represent the precursors of the major cell types. The cytological characteristics of the major cell classes and the occurrence of the miniatures of some of these major cell types support the concept of Jones (62) that these cell types might have different cell lineages and might not be capable of transforming into one another. Some of the prohemocytes, plasmatocytes and granular hemocytes were amoeboid. The nature of the granules and the vacuoles of plasmatocytes and granular hemocytes were compared with the granules and vacuoles of corresponding hemocytes of other arthropods. Cystocytes did not bring about any visible coagulation similar to their counterparts in millipedes and crustaceans. Plasmatocytes, granular hemocytes and spherule cells were observed to occur in conglomerates. The cell types noted in the present study were compared with the hemocytes of other arachnids.

Notes: The morphological features of the hemocytes of the crustacean Ligia exotica are similar to hemocytes of insects and millipedes. Jones system of hemocyte classification is extended to crustacean hemocytes. As in insects, seven classes of hemocytes, identified as prohemocytes, plasmatocytes, granular hemocytes, cystocytes, oenocytoids, spherule cells and adipohemocytes, occur. The prohemocytes can be subdivided into five categories that probably represent the precursor of major cell types. The structural and chemical features of other major cell classes are distinct and support the concept of Jones ('62) that these types might have different lineages and might not be capable of transforming into one another. Some of the prohemocytes, plasmatocytes and granular hemocytes are amoeboid. Cystocytes do not bring about any visible plasma coagulation similar to their counterpart in millipedes. Oneocytoids and adipohemocytes are rare. Plasmatocytes, cystocytes and oenocytoids occur in conglomerates, the significance of which is discussed. The cell types are compared with those of the hemocytes of other crustaceans. It is suggested that the nomenclature based on morphological characters is more suited for crustacean hemocytes than a nomenclature based on behavioural and physiological characters.

Notes: Well preserved transitional cells were found between differentiated cells and horny cells of the frog epidermis, thus facilitating the study of the sequential events involved in horny cell formation. Autolysosomes appear to play an important role in the formation of horny cells. These structures preferentially digest those cytoplasmic components which are not necessary constituents of the terminal horny cell. The release of the contents of the small mucous granules into the intercellular spaces is one of the initial events in horny cell formation. Filaments and large mucous granules seem to be resistant to the lytic digestion and contribute to the bulk of the horny cell. Loss of fluids through the plasma membrane and consolidation of the remaining constituents, results in a flattened horny cell. The appearance of a thickened membrane around the horny cell signifies the completion of the transformation process.

Notes: The three dorsal ocelli of worker honeybees have been studied by light and electron microscopy. Each ocellus has a single flattened spheroidal lens and about 800 elongated retinular cells. Retinular cells are paired and form a two-part plate-like rhabdom between their distal processes. Each rhabdomere comprises parallel microvilli projecting laterally from the apposed retinular cells. Primary receptor cell axons synapse within the ocellus with ocellar nerve fibers of two different calibers. Each ocellus has eight thick fibers ca 10 m̈m in diameter and several thinner ones less than 3 m̈m in diameter. Fine structural evidence suggests that retinular axons end presynaptically on both types of ocellar nerve fibers. Since all retinular cells apparently synapse repeatedly with the thick fibers this involves a convergence of about 100:1. Thick fibers always terminate postsynaptically within the ocellus while thin fibers terminate presynaptically on other thin fibers, thick fibers or retinular axons. Structural evidence for synaptic polarization indicates that retinular cells and thick fibers are afferent, thin fibers efferent. Thus complex processing of the ocellar visual input can occur before the secondary neurons of the three ocelli converge to form the single short ocellar nerve which runs to the posterior forebrain.

Notes: The functional morphology of the forelimbs of the following African Viverridae was studied, Atilax paludinosus, Bdeogale crassicauda, Civettictis civetta, Genetta genetta, G. tigrina, Helogale parvula, Herpestes ichneumon, H. sanguineus, Ichneumia albicauda, Mungos mungo, Nandinia binotata. Their locomotory behaviour has been previously studied and described and is related to morphological differences. The osteology of all the species and the myology of three species is described. The species have been assigned to primary locomotor categories on the basis of their locomotion. These are 1, climbing, arboreal walking; 2, arboreal and terrestrial walking and jumping; 3, general terrestrial walking and scrambling; and 4, trotting. In the climbing arboreal walking category the most distinctive morphological adaptations are powerful flexors and extensors as well as a flexible plantigrade manus with retractile claws. In the arboreal and terrestrial walking category the shoulder, elbow and carpal joints are flexible and the manus has retractile claws, though the flexor and extensor musculature is insufficiently developed for controlled climbing. The trotting category is characterised by a high humero-radial index and a rigid antibrachium. The foot is digitigrade with the claws short and stout. Species in the general walking and scrambling category show many differences in the morphology of their feet, even though the proximal parts of the forelimb appear similar. Due to the restricted nature of the adaptations, these species have been assigned to secondary locomotor categories. Morphological characters typical of the locomotor categories are summarized in the discussion.

Notes: The relationship between alkaline phosphatase activity and cell growth has been studied in hamster cells transformed by different carcinogens. About 90% of normal hamster embryo cells were constitutively positive for alkaline phosphatase activity (AP+). However, there were no AP+ cells in cell lines transformed after treatment with the chemical carcinogens dimethylnitrosamine or 4-nitro-quinoline-N-oxide and 0.02% and 4% AP+ cells in cell lines transformed by polyoma virus or Simian virus 40. The glucocorticoid hormone, prednisolone, induced alkaline phosphatase activity in 12% and 44% of the enzyme-negative (AP-) cells in cell lines transformed by polyoma or Simian virus 40, but this hormone did not induce alkaline phosphatase activity in AP- cells from cell lines transformed after treatment with the chemical carcinogens. Treatment of polyoma transformed AP- cells with the mutagen N-methyl-N′-nitro-N-nitro-soguanidine produced AP+ cells, whereas no AP+ cells were found after mutagen treatment of AP- cells from the chemically transformed cell lines. Studies on spontaneous segregation in the polyoma transformed cell line has shown that AP+ cells segregated AP- cells both in vitro and in vivo, although no spontaneous segregation was observed from AP- to AP+ cells.AP+ cells, compared to AP- cells, showed a decrease in DNA synthesis, cell multiplication, the ability to form colonies in soft agar and tumorogenicity in animals. AP- cells induced for alkaline phosphatase activity by prednisolone, showed the same growth properties in vitro as uninduced AP- cells. The decreased cell growth found in AP+ cells which were constitutive for alkaline phosphatase activity was therefore not found in the hormone induced AP- cells. The results indicate that constitutive alkaline phosphatase activity appears to be related to the regulation of cell growth and that AP- cells have a selective advantage over AP+ cells.

Notes: The pattern of changing activities of three lysosomal enzymes, N-acetyl-β-D-glucosaminidase, aryl sulfatase, and DNase II, and that of DNA polymerase was followed in homogenates of 3T3 cells during the logarithmic phase of growth and in stationary cultures. The change in activities of the polymerase and the lysosomal enzymes is antiparallel. DNA polymerase exhibits highest activity in growing cultures, and shows a three-fold decline of the specific activity in stationary cultures. The lysosomal enzymes show a very marked increase in their specific activity after density saturation is reached, which can be prevented by the addition of cycloheximide. Colcemid added to logarithmically growing cultures also causes an increase in the specific activities of lysosomal enzymes.

Notes: A series of Chinese hamster cell lines were tested and found to be able to proliferate in the absence of added bicarbonate and carbondioxide if hypoxanthine and uridine were present in the medium. Conversely, cells incapable of salvaging one of these precursors, such as hypoxanthine-guanine phosphoribosyltransferase (HGPRT-) deficient cells did not multiply under these conditions.We describe another variant capable of utilizing hypoxanthine and uridine which has an absolute requirement for exogenous CO2/NaHCO3 for growth. These cells appear to be defective in the complete oxidation of pyruvate to carbondioxide, and indications are that the entry of pyruvate into the Krebs cycle is affected.

Notes: Elevated calcium and magnesium concentrations promoted mitotic activity in rat thymic lymphocyte cultures. Oestradiol inhibited calcium- but not magnesium-induced mitogenesis. One prerequisite for the mitogenic action of calcium is a raised intracellular concentration of cyclic adenosine 3′5′ monophosphate (cyclic AMP) but cyclic AMP-induced mitogenesis was insensitive to oestradiol. This suggests that the steroid blocks the mitogenic process at a stage preceding the endogenous cyclic AMP elevation. Furthermore the mitogenic actions of adrenaline, which stimulates adenylate cyclase (the enzyme responsible for cyclic AMP biosynthesis), and caffeine, which inhibits phosphodiesterase (the enzyme which degrades cyclic AMP) were also insensitive to oestradiol inhibition. This precludes a direct effect of the steroid on these enzymes. However, oestradiol did inhibit the mitogenic action of parathyroid hormone (PTH). Since the mitogenic action of PTH probably involves increased calcium entry to the cell, oestradiol may block this ion influx.The inhibition of calcium- and PTH-induced mitogenesis must be attributable to some structurally specific action of oestradiol. The steroids cholesterol, progesterone and testosterone all failed to reduce calcium-induced mitogenesis, whereas both α and β oestradiol were effective.In addition to its insensitivity to oestradiol inhibition, magnesium-stimulated mitosis was unaffected by both imidazole and calcitonin at concentrations which significantly reduced calcium-stimulated proliferation. These findings are compatible with the thesis that magnesium-induced mitogenesis does not involve the elevation of cyclic AMP concentrations.

Notes: The transport of various deoxyribonucleosides by cultured Novikoff rat hepatoma cells (subline N1S1-67) follows normal Michaelis-Menten kinetics. The transport reactions are competitively inhibited by most heterologous deoxy- and ribonucleosides and by Persantin and Cytochalasin B. Comparisons of the transport kinetics of the various deoxyribonucleosides (Km and Vmax ) and of the Km/Ki ratios for the inhibitions indicate that deoxythymidine, deoxyuridine and 5-fluordeoxyuridine are transported by a single system, whereas deoxycytidine and the purine deoxyribonucleosides are transported by other systems. The data suggest that deoxyadenosine, deoxyguanosine and deoxyinosine, are not transported by a single system, but the number of transport systems involved could not be established unequivocally. Similar comparisons also suggest that the deoxyribonucleosides are transported by different systems than the ribonucleosides. All deoxyribonucleoside transport systems are inhibited to about the same extent by Persantin (Ki = 1-2 μM) and Cytochalasin B (Ki = 4-12 μM). The inhibitions of deoxynucleoside transport resulted in corresponding apparent competitive inhibitions of their incorporation into nucleic acids.

Notes: The incorporation of 3H-labeled deoxyadenosine and deoxyguanosine into nucleic acids by cultured Novikoff rat hepatoma cells is about 80% into RNA and 20% into DNA. The pathways of incorporation have been elucidated in studies with whole cells and cell-free extracts. Deoxyadenosine is very rapidly deaminated to deoxyinosine. Most of the deoxyinosine formed by whole cells is transported out of the cells and accumulates in the medium. A portion of the deoxyinosine, and deoxyguanosine are phosphorolyzed by purine nucleoside phosphorylase to hypoxanthine and guanine, respectively. The latter are subsequently converted by hypoxanthine-guanine phosphoribosyl transferase to IMP and GMP, respectively. Incorporation of the purine deoxyribonucleosides into DNA is mainly via this pathway and the subsequent reduction of ADP and GDP by ribonucleoside reductase, although a small proportion of the deoxyadenosine and deoxyguanosine taken up by the cells seems to be directly phosphorylated to dAMP and dGMP, respectively. Deoxyguanosine is incorporated only into guanine residues of RNA and DNA. Deoxyadenosine is also mainly incorporated into guanine residues of RNA and DNA, although the radioactivity of deoxyadenosine in the acid-soluble pool is almost exclusively associated with ATP. A similar labeling pattern is observed with labeled deoxyinosine, inosine or hypoxanthine. The pyrimidine deoxyribonucleosides, on the other hand, are specific precursors for their respective bases in DNA.Hydroxyurea inhibits the incorporation of all deoxyribonucleosides into DNA. Results from pulse-chase experiments indicate that the inhibition of DNA synthesis is prevented by the presence of high concentrations of deoxyadenosine plus deoxyguanosine in the medium. Either purine deoxyribonucleoside alone or deoxycytidine, hypoxanthine or inosine alone or in combination with deoxyadenosine or deoxyguanosine are ineffective. The results are consistent with the conclusion that the inhibition of DNA synthesis is due to a depletion of the dATP and dGTP pools as a result of the hydroxyurea treatment. On the other hand, hydroxyurea causes an increased incorporation of thymidine and deoxycytidine into the dTTP and dCTP pools, respectively. Evidence is presented to indicate that this effect of hydroxyurea is due to an increased synthesis of dTTP and dCTP rather than to an inhibition of their turnover.

Notes: HTC cells incubated in the absence of serum for more than 14 hours have very low levels of ornithine decarboxylase, the first enzyme on the pathway of polyamine synthesis. Readdition of serum causes an increase in the activity of ODC, reaching a maximum on average 17 times above the basal level after five hours. This increase is due in part to a decrease in the apparent rate of degradation of ODC, and also to a stimulation of its synthesis. Within the first two hours the serum induction of ODC is resistant to Actinomycin D. Insulin at 5 μm/ml alsocauses an increase in ODC activity but only after a delay of two hours, in contrast to its more rapid stimulation of tyrosine transaminase activity.

Notes: Net fluxes of sodium and potassium were studied in Ehrlich mouse ascites tumor cells during contact with the agglutinating protein, concanavalin A. This lectin altered cation transport markedly at concentrations of 20-105 μg/ml (6-47 μg/mg cell protein). Whereas control cells extruded sodium and maintained or accumulated potassium against electrochemical gradients, in the presence of concanavalin A there was rapid net sodium entry and potassium loss. After 10-20 minutes in concanavalin A, sodium extrusion began and potassium loss diminished but these events were prevented by ouabain. The alterations in cation content induced by concanavalin A are unlikely to be the result only of agglutination since soybean agglutinin caused much smaller changes although it agglutinated the cells equally well.

Notes: The effect of cell density on morphological transformation of chick embryo cells by Rous Sarcoma Virus (RSV) was examined in this study, and a cell density optimum for transformation was found. Less than 10% of the transformed foci appearing at the optimum density (2.5 × 104 cells per cm2) developed at high cell densities, and the diameters of the foci (an indication of the number of cells per focus) decreased with increasing cell density. No correlation was found between the decrease in transformation at high cell densities and the effect of cell density on the initial rate of cell proliferation, although dissociation of transformation from incorporation of radioactive precursors into nucleic acids could not be established. Redistribution of cells infected at high density showed that only a small proportion of successfully infected cells developed into foci. The results indicate that transformation of cells containing the RSV genome can be suppressed by physiological factors accompanying high cell density.

Notes: Ehrlich cells subjected to anisoosmolar media show very rapid volume changes. In hypertonic media they shrink. In hypotonic media they swell but the rapid initial swelling is followed by a regulatory shrinkage lasting ca. 30 minutes.Cells suspended in media with identical ionic concentrations but different total osmolarity (adjusted by sucrose) were compared. These studies revealed that swollen cells adjust their volume by decreasing the amount of intracellular K+ and ninhydrin positive substances. Intracellular Na+ and ATP concentrations were unchanged. Accordingly 42K+ flux analysis showed that the (passive) cell membrane permeability for K+ is increased to a minor degree and the Na+ permeability unaffected. The increased K+ permeability could not be correlated to an increase in 45Ca2+ influx.

Notes: The membrane potentials of human embryonic lung fibroblasts have been measured in different cellular environments. Sparse cells on plastic have a mean membrane potential of -8.5 mV. As the cells progress to dense culture, the mean membrane potential rises to -14.7 mV. The mean membrane potential of fibroblasts in human embryonic lung fragments by comparison was found to be -16.5 mV. Sparse cells on collagen, at the same density as the sparse cells on plastic, have mean membrane potentials of -10.8 mV.Sparse cells on plastic migrating from dense cellular areas, following a cut being made in a thick sheet of cells, have mean membrane potentials of -5.9 mV.The significance of these results in relation to cellular environments has been discussed.

Notes: Using a combination of DNA-cytophotometry and tritiated thymidine-autoradiography, we have shown that the majority of nondividing cells in serially propagated human diploid cell populations have the 2C DNA content consistent with their being arrested in the G1 phase of the diploid cell cycle. Unlabeled 4C cells appear increasingly with time in culture. These may be arrested G2 diploids or they may be G1 tetraploids, since there is an associated increase in polyploidy in older cultures as evidenced by the appearance of labeled 8C cells.

Notes: Secondary cultures of human diploid fibroblasts were used to study the effect of pH on cellular proliferation. In nonconfluent cultures, the growth rate at pH 7.1 was similar to that at pH 7.7 regardless of serum concentration. However, the saturation density achieved at pH 7.7 at any serum concentration was always 2-4 times that achieved at pH 7.1, although the greatest differences in saturation density were observed at the higher serum levels. The results suggest that the effect of pH on saturation density is due to two factors. One, cells at pH 7.1 seem to have a greater ability to undergo contact-inhibition than at pH 7.7, independent of any serum functions; and, two, confluent cells in medium at pH 7.1 are somewhat less sensitive to growth stimulation by increasing serum concentration than are confluent cells raised in medium at pH 7.7.

Notes: In phototrophic culture of Euglena gracilis, good synchrony was found only under rather restricted programs of light-dark cycles, and rather narrow ranges of temperature and light intensity, when cultures were flushed with air fortified with adequate amounts of CO2. When flushed with air alone, CO2 was found to be limiting, and while cell divisions were rhythmic, less than a doubling of cell number occurred in division bursts. With air as gas phase, rhythmic division activity was maintained over wide ranges of temperature, light intensity, and the ratio of light:dark in a given program; all these factors affected the amplitude of the division burst, however.

Notes: Embryoid bodies are produced when a transplantable testicular teratoma from strain 129 mice is serially passaged in the peritoneal cavity of these mice. These bodies are roughly spherical containing two morphologically distinct cell types. Scanning and transmission electron microscopy have been employed to show that the endodermal cells of embryoid bodies, like those of the mouse embryo, are on the outer surface and have highly convoluted surfaces containing numerous microvilli-like projections. The inner, embryonal carcinoma cells, are the pluripotent stem cells of this tumor.Intracisternal A-type particles have been observed in electron micrographs and are almost exclusively located in the endodermal cells of the embryoid bodies. The A-type complement-fixing antigen has been identified in extracts prepared from this tumor.When embryoid bodies are placed in culture and allowed to attach to the surface of a petri dish, a large number of new morphologically distinct cell types appear. Attachment to the petri dish surface is required for the generation of these new cell types. Cells of similar morphology in culture, display a distinctly “clonal” distribution on the petri dish surface.

Notes: Following enucleation of a portion of human culture cells containing 3H-pyridine nucleotides, autoradiography revealed no difference in the grain density over enucleated and whole cells. These results provide evidence that the concentration of pydine nucleotides does not differ by more than threefold between nucleus and cytoplasm and probably does not vary at all.

Notes: A single hematocytoblast in the yolk sac of the chick embryo has been shown previously to give rise on the average to a clone of 128 erythrocytes. Furthermore, in any given generation the erythroid cell synthesizes a characteristic amount of hemoglobin (Hb). In these experiments day 4 embryos were treated with FUdR for 12 hours, and then reversed with thymidine. We have monitored both the passage of these erythroblasts through the cell cycle, and the effect of this perturbation on the Hb content of single cells. As a result of this disruption the amount of Hb synthesized in a given generation can be varied, but the final amount of Hb/cell in the mature erythrocyte is the same as in the untreated controls. Apparently the total amount of the Hb/cell does not in itself influence the passage of the cell through the cycle. The coefficients of variation of the Hb values in the mature erythrocytes from both normal an perturbed embryos are similar.

Notes: This paper reports the isolation of a phenotypically stable line of Chinese hamster ovary cells which exhibits a temperature dependent alteration in the transport of some purines. The alteration manifests itself for the uptake of guanine, hypoxanthine, azaguanine and guanosine but not for adenine, adenosine or thymidine. Studies with crude cell extracts suggest that, at the temperature the alteration is being expressed, the HGPRT activity is within the normal range. In cell-cell hybridization studies the alteration behaves as a recessive genetic trait.

Notes: Mammalian cell populations may be enriched for temperature-sensitive (ts) mutants by the tritiated thymidine (3H-TdR) suicide procedure (Thompson et al., '71). Such procedures were carried out on the near-diploid Chinese hamster ovary (CHO) cell line and on a line made “tetraploid” by Colcemid treatment. Clones of ts mutants were obtained from the diploid line, but in spite of repeated attempts, no ts mutants were isolated from the tetroploid line. The phenotypes of the new diploid ts mutants recovered were consistent with the particular selection regime employed.

Notes: In cultured fibroblasts, a mutation resulting in deficiency of a pyrimidine salvage enzyme leads to excretion of related pyrimidines. For example, absence of thymidine kinase led to loss of thymidine and deoxyuridine, and absence of deoxycytidine kinase to loss of deoxyuridine. Both wild type and mutant cells excreted uracil; if established lines are representative in this respect, a fully adequate salvage system for uracil does not seem to be present in the fibroblast.

Notes: Ornithine decarboxylase activity in high density, stationary phase rat hepatoma (HTC) cells in suspension culture has an extremely short half-life of between 5 and 15 minutes, as measured after inhibiting protein synthesis. Following dilution of these cells into fresh medium there is a large increase in ornithine decarboxylase activity, reaching a peak often several hundred times the initial level at about four hours. At least part of this stimulation is due to an increase in the apparent half-life of the enzyme, to between 30 and 90 minutes. Evidence is presented that the supply of amino acids can control the turnover of ODC under some conditions. For example supplementing high density cells with glutamine, asparagine, serine, glycine and proline, either singly or together, increases ODC activity and decreases its apparent turnover. The stimulation by amino acids is enhanced by serum.

Notes: Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%-10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10-14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.

Notes: Calcium is required for ACTH stimulated steroidogenesis in adrenal tumor cells in tissue culture. In the absence of calcium, the dose of ACTH required to induce half maximum steroidogenesis was increased 30 fold. In contrast to intact adrenal glands or isolated adrenal cells, high doses of ACTH (50 mU/ml) maximally stimulated steroidogenesis in the absence of calcium. Growth for up to six days in medium with low calcium did not affect basal or ACTH induced steroidogenesis. The addition of calcium to cells incubated with ACTH produced a maximum steroidogenic response in 15 minutes. In contrast to intact adrenal glands, calcium is not required for adenosine-3′,5′-cyclic monophosphate (cyclic AMP) stimulated steroidogenesis in adrenal tumor cells. These experiments support the concept that calcium is important at the level of ACTH-membrane receptor site interaction or activation of adenyl cyclase in adrenal tumor cells.

Notes: Redistribution of surface immunoglobulins (Ig), H-2b, Thy-1.2 and TL. 1,2,3 alloantigens, and concanavalin A (Con A) receptors on mouse thymus, lymph node and spleen cells into “caps” induced by bivalent antibodies or ligands was compared by immunofluorescence. Surface Ig was capped rapidly following attachment of anti-Ig antibody at 37°. Capping of alloantigens and Con A receptors occurred very slowly following attachment of alloantibody or Con A, but much more rapidly after addition of a secondary bivalent antibody. An inverse relationship between the number of surface component sites per cell and the extent of capping of that component was observed. Capping of alloantigens sparsely represented on the cell surface was not inhibited by high concentrations of alloantibody, in contrast to capping of alloantigens present in greater quantities. These results suggest that factors in addition to molecular cross-linking may be involved in ligand-induced redistribution of cell surface components.

Notes: The potassium content of single human red cells was measured with an electron probe. Cells were placed on beryllium discs and coated with a thin layer of dibutyl pthalate to prevent loss of cellular contents. Samples were stable under the electron beam during analysis for more than 15 minutes and could be stored for long periods of time. Primary standards were prepared by loading red cells with varying known amounts of potassium in order to circumvent the corrections for absorption. The X-ray intensity was found to be directly proportional to the potassium content of the cells.

Notes: The cAMP levels of the 3T3 cell line and its transformed derivatives were determined. The level was found to be lower in transformed cell lines than in parental 3T3 and to decrease at confluence in all cell lines tested. SV40 transformed 3T3 cell lines which are temperature sensitive in growth control were found to have lower cAMP levels than 3T3, but these levels were not temperature dependent. Retransformation of these cell lines by murine sarcoma virus did not markedly affect their cAMP levels.

Notes: Regulation of the proliferation of transplanted colony forming units (CFUs) was investigated in lethally irradiated mice, pretreated by methods known to accelerate hemopoietic recovery after sublethal irradiation. Prospective recipients were exposed to either hypoxia, vinblastine or priming irradiation and at different intervals thereafter lethally irradiated and transplanted with bone marrow. Repopulation of CFUs was determined by counting the number of splenic colonies in primary recipients or by retransplantation.Regeneration of grafted CFUs was greatly accelerated and their self-renewal capacity increased in mice grafted within two days after hypoxia. Also the number of splenic colonies formed by grafted syngeneic CFUs as well as by C57BL parent CFUs growing in BC3F1 hosts was significantly increased. The effect was not dependent on the seeding efficiency of CFUs and apparently resulted from hypoxia induced changes in the hosts physiological environment. Proliferative capacity of grafted CFUs increased remarkably in hosts receiving vinblastine two or four days prior to irradiation. Priming irradiation given six days before main irradiation accelerated, given two days before impaired regeneration of CFUs. The increased rate of regeneration was not related to the cellularity of hemopoietic organs at the time of transplantation. The growth of CFUs in diffusion chambers implanted into posthypoxic mice was only slightly improved which does indicate that the accelerated regeneration of CFUs in posthypoxic mice is mainly due to the changes in the hemopoietic microenvironment. A short conditioning of transplanted CFUs by host factor(s) was sufficient to improve regeneration. The results might suggest that the speed of hemopoietic regeneration depends on the number of CFUs being induced to proliferate shordy after irradiation, rather than on the absolute numbers of CFUs available to the organism.

Notes: During the early developmental stages of the toad, Bufo arenarum, Hensel. up to the stage of gill circulation (150 hr of development at 20-25°C) the total phospholipids content as well as that of phosphoglycerides of choline and of ethanolamine were found unchanged. The subfraction of both phosphoglycerides were separated according to the number of double bonds on silver-ion chromatography and were also found to be unchanged up to the tail bud stage. The distribution of non-polar side chains in the subfractions varied in both phosphoglycerides showing a structural heterogeneity. In the phosphatidylethanolamines predominate the polyenoic containing subfractions.In contrast with the constant concentration of polar lipids, during early embryogenesis a steady increase in 32P incorporation into phospholipids takes place when oocytes labeled during oogenesis are used. These changes were also correlated with the DNA content up to gill circulation stage.It is proposed that most of the nascent membrane polar lipids during early embryogenesis may be derived from a storage site through an active and specific intracellular redistribution process. At the arrival of the polar lipid to the nascent membrane a change in their covalent structure by introduction of a phosphorylbase from a highly labeled pool may explain the raise in specific activity. This change may be necessary to make possible the assembly of the lipid into the membrane structure.

Notes: A recently described pituitary chondrocyte growth factor (CGF) is a contaminant of several related glycoprotein hormones (TSH, LH and HCG). Chondrocytes were cultured from rabbits two to three months old. Bovine TSH (NIH) 69.5 μg/ml, used as the source of CGF, reduced the generation time from 16 to 10 hours through a virtual effacement of the G1 period. Incorporation of 3H-thymidine declined rapidly after 48 hours from maximal values (control 300 cpm/μg DNA; CGF, 679). Total DNA accumulated thereafter until 116 hours when the figures were 36 and 98 μg/flask, respectively. Little growth response occurred in spinner cultures. CGF lowered plating efficiency from 4.5 to 2.3%. The stimulatory effect diminished when CGF was removed from the medium. The treated cells were smaller and contained less protein and RNA than controls. They synthesized smaller quantitites of sulfated mucopolysaccharides (chondroitin sulfates 4,6 and doubly sulfated chondroitin as well as some dermatan sulfate) but hyaluronate production was not diminished.

Notes: A flow-system multiparameter cell analyzer that simultaneously measures and processes fluorescence and cell volume signals from single cells was used to study the binding of fluorescein-conjugated Concanavalin A (Con A-F) to the cell surface. Cells reacted with Con A-F were passed through a flow chamber where sensors measured both cell volume and fluorescence of each individual cell. Sensor signals were electronically processed by first converting the cell volume signals to two-thirds power (proportional to surface area) and then forming the fluorescence-to-surface area ratio. These ratios, which were considered as estimates of the surface density of binding sites, were displayed as frequency distribution histograms using a multichannel pulse-height analyzer for various cell populations differing in cell size. Comparisons between cell lines showed characteristic differences in binding site density. Cell cycle dependent changes were not found for CHO cells synchronized by mitotic selection. An important benefit of this analysis method was the ability to quantitate very weak cell surface fluorescence.

Notes: The factors involved during the pumping of a particle suspended in an electrolyte through a small cylindrical orifice across which exists an electric field are reviewed, and their practical application to cell volume measurement in terms of orifice geometry and particle properties is considered. Procedures are described to determine whether a particular cell type satisfies the operational criteria of a rigid, non-conducting sphere, for which the theoretical expressions can be reduced to an especially simple form under appropriate conditions.Experimental results are presented for the case of chick, mouse and human lymphocytes, all of which are shown to satisfy the above requirements. Volume distributions are provided for chick blood lymphocytes and compared with those from various lymphatic organs; representative data are also reported for different types of mouse lymphocytes. Human blood lymphocytes are found to have normally-distributed diameters (p 〉 98%), a property not shared by their volumes or their surface areas, nor by blood lymphocytes from the chick or from patients with chronic lymphatic leukemia. A possible implication of this finding is mentioned briefly.

Notes: Metabolic cooperation is a form of cell communication in which the mutant phenotype of enzyme deficient cells, as determined by incorporation of labeled substrates, is corrected in culture by contact with normal cells. Previous studies showed that metabolic cooperation between normal and hypoxanthine phosphoribosyl transferase deficient cells (HPRT-) was the result of transfer of product of the enzyme, nucleotide or nucleotide derivative, from normal to mutant cells rather than transfer of enzyme or informational macromolecules leading to the synthesis of the enzyme.In the present study the nature and mechanisms involved in these cell interactions were investigated. Effective communication is observed within one hour of cell contact. Modifications of the extracellular environment including changes in osmolarity, concentration of sodium and divalent ions failed to interfere significantly with transfer. Changes of cell shape induced by cyclic nucleotides, hormones and urea also did not affect communication. Cytochalasin B which dissociates microfilaments and binds to cell membranes reduced metabolic cooperation while colcemide which dissociates microtubules had little effect. Enzymatic oxidation and iodination of cell surface structures abolished metabolic cooperation. The subcellular localization of label in donor cells is important in determining efficiency of transfer. Metabolic cooperation is efficient when radioactive label is primarily located in the nucleus and inefficient if the label is cytoplasmic. Cell lines previously classified as “non-communicating” because they lack gap junctions, ionic coupling and metabolic cooperation were shown in the present study to communicate when incubated with labeled substrates for 20 hours rather than 3. Cell communication is a more generalized phenomenon among cells in contact than previously appreciated.

Notes: When embryoid bodies are grown in suspension culture in vitro, they undergo only a limited amount of morphological development. When these same embryoid bodies are permitted to attach to the surface of a culture dish, a wide variety of new morphological cell types appear.Suspension cultures of embryoid bodies do not contain significant detectable levels of acetylcholine esterase or creatine phosphokinase. These same enzymes however are produced in cell cultures derived from embryoid bodies attached to the culture dish surface. Polyacrylamide gel electrophoresis has been employed to demonstrate that the electrophoretic form of creatine phosphokinase produced by teratoma cells in culture is the brain form of the enzyme.Solid transplantable tumors containing only embryonal carcinoma cells (stem cells) do not contain either of these enzymatic activities. Well differentiated transplantable teratomas contain both enzymes.

Notes: Agglutinability with Concanavalin was studied as function of cell cycle transition in normal and SV40 virus transformed 3T3 cells. In synchronized cultures of normal cells, agglutinbility was high during mitosis and disappeared rapidly. Agglutinability of transformed cells remained high in G1 phase but diminished gradually upon entering S phase and reached minimum in G1 phase. Decreased agglutinability a the end of the cell cycle was also observed in synchronous SV3T3 cultures by a combined technique of haemadsorption and density gradient centrifugation. In normal 3T3 cells, similar variations in agglutin ability during interphase could not be observed.

Notes: An “overlay” method for rapidly and synchronously inducing contact inhibition in normal cultured cells has been developed. Using this method, disaggregation of cytoplasmic polyribosomes has been observed to occur within a matter of hours after overlay, followed by a decrease in cellular ribosomal RNA. Polysome disaggregation was influenced by the extent of cell-cell interaction and was inhibited by pretreatment of overlay cells with cycloheximide. Treatment of underlay cells with cytosine arabinoside also induced polysome disaggregation, but only after an appreciable lag as compared to that observed in overlaid cultures. Disaggregation could be induced by this method in cultured cells derived from normal tissue but not in cells derived from cancerous tissue. Polysome synthesis in growing “normal” cells (as measured by incorporation of tracer uridine into RNA) was markedly decreased when a cell surface membrane preparation was added to cultures.

Notes: Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G1, middle S and G2 phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G1 through S to G2 phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G1, S and G2 phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.

Notes: Hydroxyurea and guanazole were used as selective agents in tissue culture to obtain independent Chinese hamster ovary cell lines resistant to the cytotoxic effects of hydroxyurea or guanazole. In all cases tested a cell line selected for resistance to one of the antitumor agents exhibited resistance to both drugs. This result supports the view that these two drugs act at a common site.

Notes: Thymocytes incubated as cell suspensions in vitro are known to be markedly dependent upon added glucose for maintenance of maximal rates of incorporation of radiolabelled amino acids into protein. This requirement is only partially satisfied by other added substrates, such as pyruvate. Evidence is presented that incorporation of amino acids into protein associated with the nuclear fraction isolated from these cells is more dependent upon added glucose than is labelling of protein found in the rest of the cell.The dependence of the labelling of nuclear protein upon glucose is shown by comparing the ability of glucose and pyruvate to stimulate the incorporation of [14C-L] valine into the protein of nuclear and cytoplasmic fractions of thymus cells. The fractions are isolated on sucrose gradients after incubating suspensions of cells in substrate-free medium for two hours, adding carbohydrates and labelled L-valine for 30 min and then stopping the incubation by breaking the cells with hypotonic shock. When the protein-synthetic stimulatory effects of glucose and pyruvate are compared, glucose is almost equally capable (90%) at stimulating rates of protein synthesis in nuclear compared to cytoplasmic fractions. Pyruvate is much less effective in nuclear than in cytoplasmic fractions (30%).Evidence is also presented from pulse-chase experiments that the glucosedependent labelling of protein associated with the nuclear fraction occurs within that fraction, as opposed to migration to the nuclear fraction after being synthesized elsewhere.It is suggested from these and other data that a unique ability of glucose to provide non-mitochondrial ATP to the nucleus may be central to the dependence of the labelling of nuclear protein on this substrate.

Notes: Respiration as an index of oxidative energy production was investigated in a L-cell suspension culture system previously shown to exhibit density-dependent inhibition of growth. It was found that as cultures progressed from exponential growth to high density nongrowing populations (6-10 × 106 cells/ml) over a 2-week period, the respiratory rate determined from the total amount of oxygen consumed during the daily medium renewal cycle, declined from 5.4 to 1.8 fmoles O2/cell/min. There are two components in this decrement. The first consists of a daily recurrent decline of oxygen uptake resulting from decreased availability of medium oxygen and glutamine and is readily reversed by medium supplementation. The second component which is refractory to medium supplementation and accounts for approximately 50% of the total respiratory decline, is considered to indicate an adaptive change of the respiratory capacity of the cells. This change is reversed during the lag period which precedes resumption of exponential growth upon subculture to low cell densities. The significance of these results is discussed in relation to recent reports indicating a marked depression of respiratory activity in nongrowing dense attached cultures as well.

Notes: Skin fibroblast cultures from six patients with Down's syndrome (Trisomy 21) were compared with four in vitro age-matched normal fibroblast cultures. Growth rates were calculated from increases in cell number and total protein during exponential growth, early in culture lifetime (less than 20 doublings). The Down's syndrome (D.S.) cultures had an average population doubling time of 35.6 ± 1.1 hours and average mass doubling time of 38.6 ± 3.2 hours, significantly lower (p〈0.005) than the corresponding normal culture values of 23.0 ± 0.7 hours, and 23.3 ± 1.9 hours. D. S. Cells also contained 4.46 ± 0.19 ± 10-4 μg protein/cell as compared to 3.06 ± 0.13 × 10-4 μg/cell (p〈0.001) for normal fibroblasts.Similar in vitro observations of increased doubling time and protein content have been reported in normal fibroblasts from older donors, and from individuals with premature aging syndromes, as well as in normal fibroblasts near the end of their in vitro lifetime. The present results, obtained from cultures young in vitro, may therefore suggest that D.S. fibroblast cultures age prematurely. This hypothesis is consistent with clinical manifestations of premature aging in D.S. patients and points to a defect in growth regulation, both in vivo and in vitro, resulting from an extra copy of chromosome 21.

Notes: High density L cell suspension cultures were previously shown to remain viable for indefinite periods of time and to exhibit marked inhibition of DNA synthesis and mitosis while the fraction of total protein synthesis represented by collagen is increased. The present study demonstrates that regulation in this system extends to the activity of acetylcholinesterase found to be approximately 100-fold greater in the high density populations than in low density exponentially growing cultures. Kinetic studies of the increase of the activity, its fluctuation over an extended period of time and its decrease upon resumption of exponential growth after dilution of the cultures were performed. The data obtained indicate that the enzyme does not accumulate in high density populations merely as a result of the absence of net protein synthesis and cell division but that changes of its rates of synthesis and possibly degradation are involved. The expression of regulated acetylcholinesterase activity in a cell line of connective tissue origin is considered in relation to phenotype reprogramming and to cell membrane associated growth control mechanisms.

Notes: Sera from different strains of mice injected with endotoxin induced clones (D+) from a cultured line of myeloid leukemic cells to undergo normal differentiation to mature granulocytes and macrophages. Other clones (D-) derived from the same cell line were not inducible by these sera to undergo normal cell differentiation. Sera from the same strains of mice that had not been injected with endotoxin, increased the cloning efficiency of D+ and D - clones but did not induce differentiation. Endotoxin serum induced differentiation in D+ cells at dilutions up to 1:64, but increased the cloning efficiency of these cells at dilutions up to 1:2048. The end point of the dilution of endotoxin serum that induced differentiation in D+ cells, was also the end point that induced the formation of colonies with differentiation from normal bone marrow cells. The results indicate that serum from endotoxin treated animals can serve as a good in vivo source to induce normal differentiation in D+ myeloid leukemic cells; that the progeny of a single leukemic cell was induced to undergo differentiation to both macrophages and granulocytes; that endotoxin serum contained two activities, one that increased cloning efficiency and the other that induced cell differentiation; and that the same material in endotoxin serum induced cell differentiation in normal and leukemic cells.

Notes: Viral infection by vesicular stomatitis virus (VSV) as well as cellular protection against it were studied in cultured human diploid fibroblast cells (WI38 strain) of varying senescence (passage) level. Full protection against viral infection can be achieved by pretreatment of cells with an interferon inducer (complex of polycytidylate and polyinosinate) or by pretreatment with concentrates of interferon itself. The late passages (over 45) require higher concentration of both agents than the medium passages (25-45); a complete failure of protective mechanisms occurs only close to cellular death. Furthermore, effects of confluency and of duration of induction impulse were studied. WI38 cells are sensitive to VSV infection through their entire life span; however, during the last passage before their death by senescence, VSV replication is significantly decreased. It is concluded that even in relatively senescent cells (a) protection mechanisms which were not used previously can be activated, (b) synthesizing machinery necessary for efficient support of viral replication is sustained, and (c) release of lysosomal enzymes ending the viral replication does not begin sooner than with cells of earlier passage level.