Thymidine as a synchronizing agent. I. Nucleic acid and protein formation in synchronous HeLa cultures treated with excess thymidine

Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.     

Nuclear Behavior in Artificially Induced Multi-Nuclear Cells of Dictyostelium discoideum

The culture medium of the strain CK-8 of the cellular slime mold Polysphondylium pallidum contains a cell-fusion induction factor. Cells of the two opposite mating type strains NC-4 and HM1 of Dictyostelium discoideum were treated to induce cell fusion with the diluted fraction of CK-8 cultures, F2, which contains the factor and consequently numerous multinuclear cells were produced. NC-4 and HM1 usually fuse in the sexual cycle and form large multinuclear cells, called giant cells, which develop into macrocysts. These cells are very similar in morphology to the multinuclear cells produced following F2 treatment, however, the latter cells did not develop into macrocysts. In the sexually formed multinuclear cells, only two haploid nuclei fused to form a diploid nucleus and all others degenerate as previously reported. However, in the artificially produced multinuclear cells, no nuclear-fusion and degeneration took place. They stayed as heterokaryons and seem to lyse within 20 h incubation.     

THE HETEROCYSTS OF BLUE-GREEN ALGAE (MYXOPHYCEAE)

1. Heterocysts are found in many species of filamentous blue-green algae. They are cells of slightly larger size and with a more thickened wall than the vegetative cells. 2. Structural details of the heterocyst are: the presence of three additional wall layers, the absence of granules, sparse thylakoid network throughout, except at the poles where a dense coiling of membranes occurs. Other characters include the two pores at opposite poles ‘plugged’ with refractive material called the polar granule. 3. Peculiarities in the pigment composition of the heterocyst include an abundance of carotenoids and absence of phycobilins, and a short-wave form of chlorophyll a. 4. Unique glycolipids and an acyl lipid, not found in the vegetative cells of the algae or in other plant cells, are associated with the heterocyst. The glycolipids constitute the laminated layer of the wall and probably regulate diffusion of substances through it, whereas the acyl lipids are supposed to function as carriers and intermediates in the biosynthesis of the wall. 5. The heterocysts develop from vegetative cells, and the visible changes during differentiation include cell enlargement, synthesis of additional wall layers, disappearance of granules and reorientation and synthesis of the thylakoids. 6. Heterocysts are formed sequentially with characteristic cellular spacing during the growth of cultures in medium free from combined nitrogen. 7. Various sources of combined nitrogen inhibit heterocyst formation when supplied in the culture medium. Ammonium salts are among the most powerful inhibitors. Heterocysts are formed simultaneously and within a short period after transference of ammonia-grown non-heterocystous filaments to ammonia-free medium. 8. Incompletely differentiated heterocysts or proheterocysts are found in cultures grown in the presence of combined nitrogen. If two or more proheterocysts are close together generally a single one develops to maturity after a competitive interaction in medium free from combined nitrogen. This indicates that heterocyst formation is completed in two phases: phase I, synthesis and conservation of macromolecules, which takes place during growth in ammonia-containing medium: and phase 11, morphological differentiation of the heterocyst which is unaccompanied by growth in cell number. In the ammonia-free medium phase 11 quickly succeeds phase 1 and the whole process appears as a continuum. 9. Heterocyst formation shows a definite requirement for light. Red light favours heterocyst formation, whereas green and blue light do not. The effects of light seem to be mainly due to photosynthesis, although some effects may be morphogenetic. 10. Studies with metabolic inhibitors have revealed the involvement of photosynthesis, respiration and protein synthesis in heterocyst formation. Photosynthesis provides carbon skeletons, whereas ATP is most probably supplied by oxidative metabolism. 11. Various functions have been assigned to the heterocyst from time to time. Their role in akinete formation is suggested by (i) the formation of akinetes adjacent to the heterocysts and (ii) prevention of sporulation by detachment of the heterocysts from the vegetative cells (potential akinetes). Despite substantial evidence for such a role, it is not applicable to all akinete-forming genera. 12. Heterocysts are now widely believed to be the site of nitrogen fixation in blue-green algae. The main facts in favour of such a role are: (i) fixation of nitrogen by all heterocystous algae, (ii) inhibition of heterocyst formation by combined nitrogen and (iii) direct observations on acetylene reduction by isolated heterocysts. 13. Some non-heterocystous and unicellular algae, and vegetative cells of heterocystous algae fix nitrogen under microaerophilic conditions suggesting that absence of oxygen favours nitrogenase activity. Heterocysts lack the oxygen-evolving photo-system 11, possess oxidative enzymes, and reduce externally supplied tetrazolium salts - all indicating that they are the most suitable sites for harbouring nitrogenase in aerobic conditions. 14. Heterocysts probably originated in the Precambrian in response to the earth's changing environment and seem to be the first example of morphological differentiation in the plant kingdom.     

Butyric acid causes morphological changes in cultured chondrocytes through alterations in the extracellular matrix

Butyric acid induces characteristic changes in the morphology of chick embryo chondrocytes. Chick embryo chondrocytes when cultured in the absence of butyrate exhibit a spherical morphology and synthesize cartilage-specific chondroitin sulfate proteoglycan (CSPG). When these cultures are initiated and maintained in the presence of butyric acid, chondrocytes exhibit a mesenchymal morphology, a 90% reduction in the synthesis of CSPG, and a 75% reduction in DNA synthesis. The reduced synthesis of CSPG and DNA was shown not to be dependent on the morphological change. Chondrocytes require CSPG in order to express a spherical morphology, since including chondroitinase ABC in the culture media caused the cells to spread. In addition, the treatment of chondrocytes with purified CSPG prior to culture in media containing butyric acid resulted in spherical cells. The butyrate-induced spreading was shown to require either serum or fibronectin and could be prevented with antiserum against chick cell-surface fibronectin (cFn). Cell-surface fibronectin, which was present on both spherical and flattened chondrocytes, organized into fibrils beneath cells which spread. Increased fibronectin synthesis was not responsible for the butyrate-induced morphological change. From this evidence, it is concluded that the mechanism by which butyrate alters the morphology of these cells in culture involves inhibiting CSPG synthesis, thus preventing CSPG accumulation in the extracellular matrix (ECM). The absence of CSPG in the ECM allows fibronectin to mediate spreading of chondrocytes in culture.     

The alkylation of hemoglobin S by nitrogen mustard. High resolution proton nuclear magnetic resonance studies.

Sickle cell hemoglobin (Hb S) treated with nitrogen mustard (bis(beta-chloroethyl)methylamine hydrochloride) gives two reaction products, one labile and one stable. After dialysis against buffer solution, the remaining stable product is found to inhibit the polymerization of deoxyhemoglobin S. High resolution proton nuclear magnetic resonance has been used to study the structure and function of this stable product and to investigate the nature of the binding sites of nitrogen mustard to the hemoglobin molecule. The NMR results suggest that the nitrogen mustard treatment of Hb S does not alter the heme environment or the subunit interfaces of the hemoglobin molecule. Moreover, the NMR spectra have also shown that the nitrogen mustard reacts with the beta2 histidines of the hemoglobin molecule and have suggested that several other surface amino acid residues of the hemoglobin molecule are also affected by the nitrogen mustard alkylation. These NMR findings are in good agreement with the data obtained from biochemical studies of nitrogen mustard-treated Hb S. The NMR spectra also indicate that nornitrogen mustard (which is also effective in inhibiting sickling) binds with the hemoglobin molecule in a manner identical with nitrogen mustard. Sulfur mustard, on the other hand, produces no observable changes in the aromatic proton resonances, which is consistent with the fact that it does not inhibit the polymerization of deoxy-Hb S.     

Nitrogen metabolism in soybean tissue culture: I. Assimilation of urea

Cultured soybean (Glycine max, Kanrich variety) cells grow with 25 mm urea as the sole nitrogen source but at a slower rate than with the Murashige and Skoog (MS) (Physiol. Plant. 15: 473-497, 1962) nitrogen source of 18.8 mm KNO(3) and 20.6 mm NH(4)NO(3). Growth with urea is restricted by 18.8 mm NO(3) (-), 50 mm methylammonia, 10 mm citrate or 100 mum hydroxyurea, substances which are much less restrictive or nonrestrictive in the presence of ammonia nitrogen source. The restrictive conditions of urea assimilation were examined as possible bases for selection schemes to recover urease-overproducing mutants. Since urease has higher methionine levels than the soybean seed proteins among which it is found, such selections may be a model for improving seed protein quality by plant cell culture techniques.Callus will not grow with 1 mm urea plus 18.8 mm KNO(3). Urease levels decrease 80% within two divisions after transfer from MS nitrogen source to 1 mm urea plus 18.8 mm KNO(3). Hydroxyurea is a potent inhibitor of soybean urease and this appears to be the basis for its inhibition of urea utilization by callus cells.Stationary phase suspension cultures grown with MS nitrogen source exhibit trace or zero urease levels. Soon after transfer to fresh medium (24 hours after escape from lag), urease levels increase in the presence of both MS or urea nitrogen source. However, the increase is 10 to 20 times greater in the presence of urea. NH(4)Cl (50 mm) lowers urease induction by 50% whereas 50 mm methylammonium chloride results in more drastic reductions in urea-stimulated urease levels. Citrate (10 mm) completely blocks urease synthesis in the presence of urea.Ammonia and methylammonia do not inhibit soybean urease nor do they appreciably inhibit urea uptake by suspension cultures. It appears likely that methylammonia inhibits urea utilization in cultured soybean cells primarily due to its "repressive" effect on urease synthesis.Citrate does not inhibit urease activity in vitro and exhibits only a partial inhibition (0-50% in several experiments) of urea uptake. It appears likely that the citrate elimination of urease production by cultured soybean cells is due to its chelation of trace Ni(2+) in the growth medium. Dixon et al. (J. Am. Chem. Soc. 97: 4131-4133, 1975) have reported that jack bean (Canavalia ensiformis) urease contains nickel at the active site.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

The structure of methylated xanthines in relation to their effects on DNA synthesis and cell lethality in nitrogen mustard-treated cells.

The variation in cellular response to alkylated xanthines possessing different side chains has been used to evaluate more fully the effect of caffeine on both survival and DNA synthesis in cells with DNA damage. A correlation is observed between the ability of these xanthines to reverse the inhibitory effects of nitrogen mustard damage on DNA synthesis and their ability to enhance nitrogen mustard lethality in human HT-29 cells. These findings are consistent with our theory that regulation of damaged replicon initiation protects against potentially lethal damage in the form of unrepaired DNA alkylations. Enhancement of nitrogen mustard lethality is observed to have a maximum limit, which can be reduced by highly toxic xanthine concentrations. The lethal effects of xanthines alone at higher concentrations are unrelated to the effects of caffeine specific to nitrogen mustard treated cells, and appear to be related to an immediate reduction in thymidine incorporation most likely caused by inhibition of other enzyme systems influencing DNA synthesis such as de novo and salvage pathways for purine biosynthesis.     

Pyruvate kinase isoenzyme transitions in cultures of fetal rat hepatocytes

Changes in the expression of two isoenzymic forms of pyruvate kinase in fetal hepatocyte cultures derived from 15- and 19-day gestation rats are studied by immunocytochemical localization of the respective antigens. Initially, in cultures established from 15-day gestation rats only the ‘embryonic’ form of the enzyme (M2-PK) is detected in all cells. Cells which stain positively for the liver specific form of the enzyme (L-PK) are not observed. After 2 days' culture, a significant number of cells have become positive for L-PK. All the positive cells have a morphology which is typical of liver parenchymal cells. However, the majority of parenchymal cells remain negative for L-PK while retaining M2-PK. In contrast, all cells which display a fibroblastic morphology, as well as clear epithelial cells are M2-PK positive, but L-PK negative. In 5-day-old cultures, all hepatocytes have become L-PK positive. Hepatocytes derived from 19-day gestation rat liver stain positively for L-PK on day 1 of culture in agreement with previously published biochemical data. A minor population of negative cells is non-parenchymal in appearance. All parenchymal cells are negative when the culture is stained with M2-PK specific antibody. Five days after the culture is established, many non-parenchymal cells are present. Such cells are L-PK negative and M2-PK positive and their presence in cultures derived from both 15- and 19-day gestation rats explains the persistence of M2-PK. This study reveals that during enzymic differentiation of fetal hepatocytes, all immature hepatocytes are initially capable of expressing M2-PK while they do not produce L-PK. During culture, a sub-population of these cells initiates synthesis of L-PK, indicating that only a fraction of the cells differentiate. At the same time, hepatocytes which do not stain for M2-PK appear, which suggests that cells which initiate L-PK synthesis have ceased to make M2-PK. Eventually all hepatocytes are L-PK positive and M2-PK negative, indicating that a switchover in expression of the pyruvate kinase isoenzymes has occurred.     

褶纹冠蚌精子发生的研究

光镜和透射电镜研究结果表明：褶纹冠蚌精子发生是非同步的,精子发生经历了一系列重要的形态和结构变化,主要包括：核逐步延长、染色质浓缩、线粒体逐渐发达与融合、胞质消除以及鞭毛的形成。精原细胞胞质中含有许多致密的轴纤丝,它们后来形成鞭毛轴丝。精母细胞质中含有线粒体、中心粒、内质网和电子透明的囊泡。精细胞分化为４个时期。成熟精子属原始类型,由头部、中段和尾部三部分组成。多核结构和细胞间桥自始至终存在于精子     

Novel peripheral blood-derived human cell lines with properties of megakaryocytes

For 18 mo, we derived 18 cell lines from 11 donors with various clinical profiles ranging from normal to leukemic. Suspension cultures were initiated with 1 X 10(6) mononuclear blood cells/ml of nutrient medium containing 10% human serum and 10% lectin-stimulated human lymphocyte conditioned medium. The cultures were monitored weekly by morphological analyses of Wright-Giemsa-stained cell preparations. All successful cultures showed a significant decline in viability during the first 3-4 wk with rate "lymphoid" cells observed in mitosis. Within the next 2 wk, the proliferating cells gave rise to a rapidly expanding population of mononuclear cells. As the cultures expanded, cell morphology became heterogeneous with respect to cell size and nuclear ploidy, with an accumulation of giant multinuclear cells that were suggestive of megakarocytes. Even though the cells did not have the classical morphology of mature platelet-forming megakaryocytes, 90% of the cells within a cell line were positive by direct or indirect immunofluorescence for the platelet membrane glycoproteins IIb and IIIa; for surface markers HLA-Dr and B2-microglobulin; for intracellular platelet-derived growth factor and platelet factor IV; and for membrane affinity or binding with serum platelet-derived growth factor and platelet factor IV. These results suggest that a blood precursor cell, most likely a primitive megakaryoblast, was isolated from the peripheral blood and was provided with an optimal culture environment for sustained growth. These cells did not mature to a more differentiated stage, perhaps owing to regulatory factor deficiencies in this in vitro system. The remarkable frequency of obtaining cell lines with megakaryocyte properties from normal peripheral blood and the capacity of some normal donors to repeatedly yield these cell lines make this cell culture system indeed unique by being selective for putative megakaryocyte precursors.     

Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells.

Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum-starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D-ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L-ascorbic acid had minimal potentiating effect with serum and no effect with EGF present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I-labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf-aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I-labelled EGF was similar in "dense" and "sparse" cultures. Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I-labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.     

中心体异常在咖啡因增强顺铂杀伤人肝癌细胞系中的作用

为了探讨咖啡因是否影响细胞周期检验点而增强顺铂杀伤肿瘤细胞及其作用机制 ,选取同步化于S期的肝癌细胞系SMMC 772 1,用顺铂和咖啡因进行不同方式的处理 ,包括顺铂处理、咖啡因处理以及先经顺铂 ,再用咖啡因处理 .利用相关方法对不同处理的细胞进行了分析 ,包括细胞形态 ,细胞生长速率 ,多核细胞的形成与死亡 ,中心体的异常等 .结果显示 ,顺铂与咖啡因联合处理的细胞出现明显的多核化现象 ,多核细胞占总细胞的百分比可以达到 30 %以上 ,高于用顺铂或用咖啡因处理的细胞 .同时观察到多核细胞生存能力较差 ,它们会通过细胞凋亡的形式死亡 .抗中心体人自身免疫血清的免疫荧光结果显示 ,中心体异常与多核细胞的形成直接相关 .在部分多核细胞的核周围有多个不同强度的荧光点 ,在另部分多核细胞中 ,在其中央有一个大的荧光点 ,被多个细胞核围绕 ,荧光较强 .根据结果推测 ,由多个不完整的中心体导致的多极分裂形成多核细胞 ,随后多个中心体聚集到中央形成大的中心体 ,负责间期微管的组装 .结果表明 ,受到顺铂损伤的细胞由于检验点的作用而使细胞周期阻断 ,咖啡因可消除周期的阻断 ,使细胞在中心体未完成正常复制状态下进入有丝分裂 ,产生大量多核细胞 ,这些多核细胞最终发生凋亡 .     

THE EFFECT OF NITROGEN MUSTARD ON THE HAEMOPOIETIC STEM CELLS OF THE BONE MARROW IN THE RAT

The sensitivity of haemopoietic stem cells to the action of nitrogen mustard has been investigated by transfusion of bone marrow from treated donor rats to recipients whose own haemopoiesis had been reduced to low levels by whole body X-irradiation. By measurement of the resultant erythropoiesis in the recipients with radioactive iron, a comparison of the repopulating ability of nitrogen mustard treated bone marrow with that of normal bone marrow could be made. It was found that although a dose of 0.9 mg/kg body weight reduces bone marrow cellularity to less than 10% of normal, repopulating ability is not decreased to much less than half the normal level. This is in contrast to the effects of X-radiation, which has a more marked effect on the stem cell population than on the differentiated marrow cells. Possible reasons for this difference are discussed. It could be that the proliferative or metabolic state of the cell plays a role, or that some repair mechanism is operative in the stem cells which does not exist in the differentiated cells.     

THE DIFFERENTIAL SENSITIVITY OF CULTURED CHICK MESODERMAL CELLS TO ACTINOMYCIN D

Cells were isolated from the somite mesoderm and from the unsegmented (presomite) mesoderm of early chick embryos and exposed to actinomycin D in single cell culture. Actinomycin D inhibited proliferation in cell cultures derived from the unsegmented mesoderm, although the same concentrations of this antibiotic did not inhibit cultures derived from the somite mesoderm. This differential sensitivity parallels the regionally specific necrosis and degeneration observed in the unsegmented mesoderm of intact chick embryos exposed to actinomycin D. In culture, both cell types exhibited approximately the same permeability to labeled actinomycin D and showed comparable inhibition of RNA, DNA, and protein syntheses in the presence of the antibiotic. However, freshly isolated mesodermal cells from the somite region had a higher content of RNA than did cells from the unsegmented region, and the somite cells maintained a higher rate of macromolecular synthesis in untreated cultures.     

Identification of a Second Locus in DROSOPHILA MELANOGASTER Required for Excision Repair

The mus(2)201 locus in Drosophila is defined by two mutant alleles that render homozygous larvae hypersensitive to mutagens. Both alleles confer strong in vivo somatic sensitivity to treatment by methyl methanesulfonate, nitrogen mustard and ultraviolet radiation but only weak hypersensitivity to X-irradiation. Unlike the excision-defective mei-9 mutants identified in previous studies, the mus(2)201 mutants do not affect female fertility and do not appear to influence recombination proficiency or chromosome segregation in female meiocytes.—Three independent biochemical assays reveal that cell cultures derived from embryos homozygous for the mus(2)^D1 allele are devoid of detectable excision repair. 1. Such cells quantitatively retain pyrimidine dimers in their DNA for 24 hr following UV exposure. 2. No measurable unscheduled DNA synthesis is induced in mutant cultures by UV treatment. 3. Single-strand DNA breaks, which are associated with normal excision repair after treatment with either UV or N-acetoxy-N-acetyl-2-aminofluorene,* are much reduced in these cultures. Mutant cells possess a normal capacity for postreplication repair and the repair of single-strand breaks induced by X-rays.     

Ultrastructural effects of nerve growth factor on PC 12 pheochromocytoma cells in spinner culture

PC12 pheochromocytoma cells treated with nerve growth factor (NGF) for two weeks in spinner cultures quickly begin to form processes after plating on an appropriate substrate, while cells freshly exposed to NGF in monolayer culture initiate neurite outgrowth only after a lag period of several days. The present ultrastructural studies indicate that PC 12 cells treated with NGF in spinner cultures do not form neurites, but do form short extensions comparable to those which have been reported within the first two days of exposure to NGF in monolayer cultures. These extensions contain organelles believed to be required for locomotion and for transport of cytoskeletal and membrane components and neurotransmitters. They also form bulbous distensions in which numerous chromaffin-type granules accumulate. These findings suggest that NGF may affect cells in spinner cultures by promoting development or activation of axonal transport mechanisms, and that the existence of these mechanisms may contribute to the neurite outgrowth which the cells exhibit when plated. NGF-treated PC 12 cells in spinner cultures do not accumulate the agranular synaptic-like vesicles, which are typically found in comparably treated monolayer cultures and which have been hypothesized to be sites of acetylcholine storage. These and other data demonstrate that attachment to a substrate can selectively modulate the responses of PC 12 cells to NGF.     

Acetylcholine Synthesis by Adult Bovine Adrenal Chromaffin Cell Cultures

Adrenal chromaffin cells normally synthesize and release catecholamines. In the present study, [3H]acetylcholine synthesis and another characteristic of cholinergic neurons, [3H]choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells. Chromaffin cell cultures took up [3H]choline from the medium and acetylated the [3H]choline to form [3H]acetylcholine. The rate of [3H]acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture. [3H]Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+. The ability for K+ to stimulate synthesis of [3H]acetylcholine developed only after 28 days in culture. [3H]Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline. [3H]Choline uptake was enhanced by Na+ omission in day-14 cultures, but was at least partially Na+-dependent in day-29 cultures. Hemicholinium-3 (IC50 less than 10 muM) inhibited [3H]choline uptake into chromaffin cell cultures. It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.     

Regulation of the formation of protease inBacillus megaterium

Protease is synthesized by the cultures growing in a glucose-containing mineral medium. However, it is formed even during incubation of the washed cells in a nitrogen free medium. The enzyme synthesis is decreased substantially by the addition of the individual amino acids or their mixture. Threonine, isoleucine, leucine and valine are the most inhibitory. Arginine, cysteine, glycine, lysine and tryptophan in concentrations of 10³ m do not inhibit the production of protease. The growth of the culture is also somewhat inhibited by threonine and isoleucine, the repression of protease being, however, much higher. Concentrations of 10³ m inhibit its synthesis by 80–90%. However, the enzyme activity is not influenced. The inhibition is caused byl,-isomers. Repression of the enzyme synthesis after the addition of threonine into the medium is much greater in a growing culture than in a culture starving in a nitrogen-free medium. However the level of free threonine in the pool is roughly the same in both growing and non-growing cultures. A mixture of 13 amino acids, which themselves are little inhibitory, suppresses the synthesis of protease much more than threonine or isoleucine. The inhibitory effect of the individual amino acids on the enzyme formation is apparently additive.     

Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture

Arterial proteoglycans have been implicated in several important physiological processes ranging from lipid metabolism to regulation of smooth muscle cell growth. Vascular smooth muscle (VSM) cells are the major producers of proteoglycans in the medial layer of blood vessels. To study functional consequences of alterations in VSM proteoglycan metabolism we used 4-methylumbelliferyl-beta-D-xyloside to inhibit proteoglycan synthesis in primary and early passage cultures of rat aortic smooth muscle cells. Biochemical analysis of cultures labeled with 35SO4 showed the drug inhibited synthesis of different classes of proteoglycans by 50 to 62%. Inhibition of proteoglycan synthesis resulted in reduced accumulation of extracellular matrix, as shown by immunofluorescent staining with antibodies to chondroitin sulfate, fibronectin, thrombospondin, and laminin. There was also an inhibition of postconfluent (multilayered) growth of the smooth muscle cells, and a change in the morphology of the cells, with no apparent effect on subconfluent growth. In addition, in drug-treated cells there was a reduction in the number of cytoskeletal filaments that contained alpha-actin, the actin subtype synthesized by differentiated VSM cells. This occurred even though the total content of alpha-actin in the cells was not reduced. The effects of the inhibitor on growth and morphology could be reversed by switching the cultures to normal medium and could be prevented by growing the cells on preformed VSM extracellular matrix. These observations suggest the vascular extracellular matrix may play a role in regulating the growth and differentiation of smooth muscle cells.