Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR-31 and MonR-32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR-31 and MonR-32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR-31 or MonR-32 cells required more insulin than CHO cells. Specific binding of [125I]insulin was observed in these cell lines. The two MonR clones bound about one-half or less the [125I]insulin bound by CHO cells. Scatchard analysis for [125I]insulin binding at 4 degrees C and 37 degrees C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down-regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 microgram/ml insulin. A 50-60% decrease in levels of insulin surface binding capacities was observed in CHO after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2-[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.     

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.     

Characterization and partial purification of keratinocyte growth factor from the hypothalamus

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.     

The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase

A 110-kDa protein present in chicken intestinal brush-border microvilli is believed to laterally link the actin filament bundle that forms the structural core of the microvilli with the microvillar plasma membrane. We have purified a 110-kDa protein to greater than 95% homogeneity by extraction of brush borders with solution containing 0.6 M KCl and 5 mM ATP, followed by gel filtration chromatography, sedimentation as a complex with exogenous actin, and hydroxylapatite chromatography. The 110-kDa protein-calmodulin complex bound F-actin in the absence but not the presence of ATP and had K+,EDTA-ATPase (0.2 mumol/min/mg) and Ca2+-ATPase (0.2 mumol/min/mg) activities and Mg2+-ATPase activity (0.03 mumol/min/mg) that was not activated by F-actin. The actin-binding and ATPase activities of the complex were similar to those of purified brush-border myosin. However, immunoblot analysis showed no reactivity between the 110-kDa protein and polyclonal antibody against purified chicken brush-border myosin. Also, peptide maps of 110-kDa protein and myosin obtained by limited proteolysis with chymotrypsin and Staphylococcus aureus V8 protease had few, if any, peptides in common. Immunoblot analysis also showed that myosin heavy chain was stable under the conditions of the preparation.     

Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface

After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.     

Pleiotropic effects of a butyrolactone-type autoregulator on mutants of Streptomyces griseus blocked in cytodifferentiation

Mutants of Streptomyces griseus blocked in cytodifferentiation regained their capacity to form differentiated mycelia and/or anthracycline pigments in the presence of butyrolactone-type autoregulatory effectors such as trans-2-(6'-methylheptanol-1'-yl)-3-hydroxymethyl-4-butanolide+ ++. In the pertinent indicator strains, the effect has been correlated with the increase of lipid synthesis, with changes in the composition of lipid fraction and with the restoration of the production of neutral proteinases. The results suggest that autoregulatory butyrolactones from streptomycetes stimulate cytodifferentiation of their producers at an early stage of development.     

The effects of mating,exogenous juvenile hormone and a juvenile hormone analogue on pheromone titre,calling and oviposition in the omnivorous leafroller moth (Platynota stultana)

Mating in Platynota stultana resulted in the termination of calling, the gradual reduction of pheromone in the pheromone glands to non-detectable levels (<0.1 ng/♀) within 14 h, and oviposition of the first batch of eggs 20–24 h after copulation. Decapitation of virgin females resulted in a similar decline in pheromone titre, and also eliminated oviposition and calling. Pheromone production appears to be controlled via the head. Mating probably terminates neural or hormonal input required for pheromone production and/or removes neural or hormonal inhibition of pheromone degradation. A juvenile hormone analogue (ZR-512) and juvenile hormones I, II and III applied exogenously to virgin females elicited oviposition comparable to mated females and terminated calling within 48 h. The juvenile hormone analogue also appeared to block pheromone production in virgin females. These results suggest that juvenile hormone may be involved in the switch from virgin to mated behaviour in this species.     

Uptake and nature of the intracellular binding of cyclosporin A in a murine thymoma cell line, BW5147

In a survey of malignant cell lines including a variety of leukemias and lymphomas, BW5147, a T lymphoma from the spontaneous virus-associated thymoma in AKR mice, was found to be the most sensitive to growth inhibition by cyclosporin A (Cs A). Inhibition of growth was cell cycle phase-independent and inhibition of macromolecular precursor uptake was relatively nonspecific. Uptake of radiolabeled Cs A by these cells was characterized by two components: one that appeared saturable at low drug concentrations (0.03 to 1.0 microgram/ml), and another that was nonsaturable at higher drug concentrations (1.0 microgram/ml or higher). Most of the drug concentrated by cells (70 to 80%) was located in the cytosol (100,000 X G supernatant of lysed cells). The apparent m.w. of the drug-macromolecule complex was 15,000 to 20,000 as determined by m.w. exclusion columns. This complex could also be formed by adding drug to cytosol prepared from unexposed cells. The low m.w. complex migrated on a preparative isoelectric focusing column to form two peaks with isoelectric points of 6.8 and 8.5. A method was developed to assay for the binding component, and a sequence of m.w. exclusion columns and isoelectric focusing was used to effect partial purification of the Cs A binding component.     

Yeast regulatory gene PPR1. II. Chromosomal localization, meiotic map, suppressibility, dominance/recessivity and dosage effect

The Saccharomyces cerevisiae gene PPR1 encodes a positive regulator of the expression of the two unlinked structural genes URA1 and URA3. The gene has been mapped to a position 6.5 cM from the centromere of chromosome XII. Uninducible alleles have been selected and used to establish a meiotic map. Suppressible alleles have been identified. The sequencing of a suppressible allele confirms the nonsense nature of the mutation as well as the reading frame deduced from the nucleotide sequence. No evidence of intracistronic complementation was found, and enzymatic analysis of leaky mutants did not reveal any mutations dissociating regulation of URA1 from that of URA3. Three in vitro-constructed deletions of PPR1 have been integrated at the chromosomal locus, giving strains with a completely negative phenotype. These deletion mutants display the wild-type basal level of URA1 and URA3 expression and show a semi-dominant phenotype in heteroallelic ppr1+/ppr1-delta diploids. Amplifying PPR1 by introduction into yeast on a multicopy vector increases the induction factor of URA1 and URA3 expression. These results show that the extent of regulation of the two structural genes is dependent on the concentration of the active PPR1 protein.     

Recognition of super-secondary structure in proteins

A procedure to recognize super-secondary structure in protein sequences is described. An idealized template, derived from known super-secondary structures, is used to locate probable sites by matching with secondary structure probability profiles. We applied the method to the identification of βαβ units in β/α type proteins with 75% accuracy. The location of super-secondary structure was then used to refine the original (Garnier et al., 1978) secondary structure prediction resulting in an 8.8% improvement, which correctly assigned 83% of secondary structure elements in 14 proteins. Slight modifications to the Garnier et al. method arc suggested, producing a more accurate identification of protein class and a better prediction for β/α. type proteins. A method for the incorporation of hydrophobic information into the prediction is also described.