Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

X射线对根尖细胞分裂和分化的作用

用30—70GyX射线照射小麦幼苗(浸种后5天)后发现根毛区到根尖的距离缩短,根毛变密,根毛长度为对照的2—3倍。照射后2—3天就可看到该现象。根毛着生处到根尖的距离随剂量增加而减少,甚至根尖全为根毛所复盖,另外还看到有分叉的根毛。根尖纵切片表明根尖分生区随剂量增加而缩小,分生区后接着就出现输导组织。玉米和黄瓜也有类似现象。这现象说明根细胞的分裂过程对射线很敏感,而分化过程则相当耐辐射,细胞分裂停止后立即转向细胞分化过程。     

Differentiation of Extracutaneous Melanocytes in Embryos of the Turtle,Trionyx sinensis japonicus

The present study investigates the mode of differentiation of neural crest-derived melanocytes in the embryos of the soft-shell turtle, Trionyx sinensis japonicus. DOPA reaction-positive melanoblasts were first detected in 10-day-old embryos. Melanocyte differentiation in terms of pigmentation takes place from the day 16 of development. Melanin pigments were found in the dorsal integument as well as in various extracutaneous tissues such as skeletal muscle, dorsal aorta, peritoneum, blood vessels, choroid, lung, bone marrow, fat tissues and in the connective tissue of the nose. These results suggest the presence of a specific environmental regulation of the melanoblast differentiation in the soft-shell turtle.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

Heparin-binding growth factors and their receptors

Heparin-binding growth factors modulate diverse biological activities including cellular proliferation, cellular differentiation, morphogenesis, and angiogenesis. Biochemical characterization for two members of the heparin-binding growth factor family, acidic and basic fibroblast growth factors, is extensive, while characterization of the remaining five members is forthcoming. Cell surface receptors have been identified for acidic and basic fibroblast growth factors, but little is known concerning their sites of action in vivo or the mechanisms involved in transducing the energy of growth factor binding to a biological response. An understanding of the biological basis for the diversity of the heparin binding growth factor family and the in vivo actions of these factors will prove a major challenge to future research efforts.     

Isozyme differentiation among sibling species and among populations of the Echinogammarus berilloni-group (Crustacea, Amphipoda)

The sibling species of the Echinogammarus berilloni-group are endemic for the Iberian Peninsula and southern France. These species show wide morphological variability with some overlap in their dianostic characters making their distinction difficult. Reroductive isolation and enzmatic jivergence amon allopatric and sympatric populations of four species sharing the same chromosome number has been studied. The results show a clear genetic differentiation of E. longiserosus and E. calvus versus the other two species. However, E. margalefi and E. echinosetosus show no clear genetic differentiation between them, confirming their crose relationship. All four species often coexist in the same drainage system. Isozme analysis was employed to check the hypothesis Of Margalef that sympathy would occur age, long-term phenomena of speciation inside of a given basin with subsequent contact and overlap between the differentiated forms. Electrophoretic data were also used to determine whether one flow among gammarids populations exists. A model proosed by other authors according to which the heterozyosity decreases towards the headwaters foes not fit to the data we have obtained from E. calvus. Thus, populations of this species from sources and springs of the Duero basin show the hiFhest values of mean heterozygosity. The differentiation in this basin can be explained by drift. Migration between populations of different rivers is prevented by natural barriers. The lowest river stretches are without amhipods interrupting the gene flow amon populations. A correction between genetic and geographic fistances among subbasins and basins was found applying a double logarithmic model. A model of migration of E. calvus in the Duero basin is proposed on the basis of allelic frequencies and on the distribution of mean hetero-zygosities.     

Catabolic Regulation of the Expression of the Major Myelin Glycoprotein by Schwann Cells in Culture

Previous studies have suggested that neonatal Schwann cell cultures deprived of axonal contact do not express components of the myelin membrane, including the major myelin glycoprotein, P0. In contrast, Schwann cells from permanently transected, adult nerve exhibit continued biosynthesis of P0 after culture, suggesting that the ability to express the myelin glycoprotein may depend on the degree of cellular differentiation. To examine further the ability of Schwann cell cultures to express P0 as a function of age, we have performed precursor incorporation studies on endoneurial explants from 4- to 12-day-old rat sciatic nerves after 5 days in culture. The data reveal that explants from 12-day-old animals synthesize detectable levels of this integral myelin protein when assayed by [3H]mannose incorporation, even though there is no apparent myelin assembly in the cultures. Pulse-chase analysis of cultures from 12-day-old rats demonstrates that [3H]mannose-labeled P0 is substantially degraded within 3 h. This catabolism largely can be prevented by the addition of swainsonine, ammonium chloride, or L-methionine methyl ester to the pulse-chase media. The former agent alters oligosaccharide processing whereas the latter two compounds inhibit lysosomal function. The P0 synthesized by the 12-day explant cultures following the addition of swainsonine is readily fucosylated, implying that the protein has progressed at least as far as the medial Golgi before its exit and subsequent catabolism. If cultures from 4-, 6-, and 8-day-old animals are analyzed for P0 biosynthesis by [3H]mannose incorporation in the presence of swainsonine, detectable levels of the glycoprotein are seen.(ABSTRACT TRUNCATED AT 250 WORDS)