Variant subclones of the rat hepatoma cell line FU5-5 have been isolated that are altered in their production of rat serum albumin. Three of these variants, isolated in a random screening, have been categorized as high, intermediate, and low producers. They secrete albumin into the culture medium at different rates: 16, 1.7, and 0.3 microgram/mg cell protein/48 h. A fourth variant, isolated on the basis of altered morphology, secretes no detectable albumin. Unlike the albumin-producing variants, this null variant is also deficient in the level and inducibility of tyrosine aminotransferase activity. Albumin biosynthesis as determined in pulse-labeling experiments is affected similarly in the four variants, yielding albumin synthetic rates of 0.24, 0.035, 0.006, and less than 0.002% of total protein synthesis. The translatable albumin messenger RNA content in these variants was measured using a rabbit reticulocyte lysate system. The null variant contains no detectable mRNA, and the three quantitative variants contain levels of translatable albumin messenger RNA corresponding to 0.07, 0.03, and 0.005% of total stimulated polypeptide synthesis. The highest producing variant contains less translatable albumin mRNA than expected on the basis of cellular biosynthetic measurements, suggesting a translation efficiency difference in this clone. Cell hybrids constructed by fusing the high-producing clone and the null variant produce little or no albumin. This extinction indicates that the null variant contains a diffusible regulatory factor capable of decreasing albumin gene expression. The relatively stable and discrete heritable phenotypic changes exhibited by these clones may serve as a model for similar changes that occur during hepatic differentiation.
The cytochalasins stimulate steroid secretion of Y-1 adrenal tumor cells two-to threefold. The order of potencies is cytochalasin E > D > B, but the maximum response is the same and always less than with ACTH. Like that with ACTH, the stimulation has a rapid onset, is easily reversible, is inhibited by cycloheximide and aminoglutethimide, and occurs at a stage before pregnenolone. Although the cytochalasins, like ACTH, produce cell rounding, it is shown that this morphological change is not necessarily coupled to steroidogenesis. Unlike ACTH, cytochalasin B does not measurably increase cellular levels of cAMP at concentrations that lead to maximal steroidogenesis.The cytochalasin B-induced stimulation of steroidogenesis, unlike the shortterm ACTH effect, fails to occur in the absence of serum. This lack of response can be corrected by even low concentrations of human high density lipoproteins (HDL) but not by low density lipoproteins (LDL). We, therefore, propose that cytochalasin B enhances the availability of cholesterol bound to HDL for steroidogenesis by Y-1 adrenal cells.
Synoviocyte adhesion, migration, and proliferation are essential in the growth of pannus and the subsequent invasion of cartilage in inflammatory arthritis. These cellular functions are regulated by the extracellular matrix.' In vitro studies of synoviocytes have previously demonstrated that they display abnormal phenotypic properties, including a transformation-like morphology manifested by cell overlapping and focus formation.2 3 Abnormal synoviocyte phenotype has been shown to be strongly influenced by extracellular matrix; synoviocytes exhibit extensive focus formation when plated on exogenous fibronectin rich matrices or when primed to switch endogenous matrix production from a collagen enriched to a fibronectin enriched type.3 To determine if the influence of matrix on synoviocyte morphology is associated with effects on synoviocyte adhesion and growth, we studied the effect of purified matrix components and a cell free matrix preparation isolated from human synovium on the attachment, spreading, and proliferation of human synoviocytes in vitro. Materials and methods CELL CULTURE IN SERUM FREE MEDIUMSynovial samples obtained from patients undergoing total knee replacement surgery for rheumatoid arthritis were placed in explant culture in serum free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 supplemented with insulin 5 ,ug/ml, hydrocortisone 5 ,ug/ml, epidermal growth factor 5 ng/ml, transferrin 20 1Lg/ml, bovine serum albumin (BSA) 2-5 mg/ml, and penicillin/streptomycin. Cells labelled with carbon-14 amino acids (1 ,uCi/ml; New England Nuclear NEC-44SE) were harvested with trypsin as above, washed in DMEM containing 0-25% BSA and plated at 2 x 104 cells/well in 24 well dishes for 60 minutes at 37°C. Non-adherent cells were removed by washing with phosphate buffered saline (PBS) and adherent cells were lysed with 0 1 ml of 1% sodium dodecyl sulphate. One hundred microlitres of lysate was added to 1 ml of Ecoscint and counted using a Beckman LS-9000 scintillation counter. EXTRACELLULAR MATRIX PREPARATIONSFibronectin was isolated from outdated, citrated human plasma as previously described.5 Type I collagen was purchased from Collaborative Research. Microtitre plate wells were coated with fibronectin (100 ,ug/ml) or type I collagen (2.5 mg/ml) in PBS for 16 hours at 4°C. Fibronectincollagen complexes were formed by adding fibronectin solution to type I collagen coated wells for 60 minutes at 23°C. Extracellular matrix fibres were isolated from rheumatoid synovial tissue by a modification6 of the method of Rojkind et al.7 The peptides Arg-Gly-Asp-Ser (RGDS) and ArgGly-Glu-Ser (RGES) were obtained from Sigma.
Multiple low-dose injections of streptozotocin (STZ) induce a delayed but progressively increasing state of hyperglycemia in mice. Different inbred strains of mice show different susceptibility to this treatment. We examined whether genetic factors associated with the H-2 complex influence the susceptibility or resistance, using a selected group of 12 inbred and 5 congenic resistant strains of mice. We found that different congenic strains differed significantly in their susceptibility to STZ-induced diabetes, suggesting that H-2-associated genes do influence the susceptibility. However, at least some inbred strains sharing the same H-2 haplotype also differed in their susceptibility, indicating that genes outside the H-2 complex may also affect the susceptibility. Therefore, there appear to be at least two genes, one within and one or more outside the H-2 complex, that determine the susceptibility to multiple low doses of STZ.
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