Density-dependent inhibitory effect of transforming growth factor-β on human fibroblasts involves the down-regulation of platelet-derived growth factor α-receptors

We have previously found that transforming growth factor-β1 (TGF-β1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-β1 on the PDGF α-receptor, which binds all PDGF isoforms, as well as on the β-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-β1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-β1, there was an complete inhibition of ³H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-β1 was seen in PDGF-BB treated cells, although less pronounced. The binding of ¹²⁵I-labeled PDGF-AA and PDGF-BB to the α-receptor was significantly reduced after treatment with TGF-β1 in dense cultures, whereas the sparse cultures were less affected. A decrease of α-receptor mRNA was also seen. The levels of β-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-β1 is cell density-dependent and involves down-regulation of PDGF α-receptors. © 1993 Wiley-Liss, Inc.     

Inhibition of benzoyl peroxide/Cu2+-dependent lipid peroxidation by manganese

1. Formation of peroxides by benxoyl peroxide (BPO) and CuCl2 was examined in the human red blood cell ghost. 2. Amounts of peroxides formed increased with the amount of the ghost solution added. 3. Of all the cations tested only manganese ion inhibited the formation of peroxides in BPO-CuCl2 reaction system. 4. The formation of peroxides was inhibited approx. 50% with 0.4 microM manganese. 5. The inhibitory manner of manganese was non-competitive against copper.     

Bacterial reduction of hexavalent molybdenum to molybdenum blue

A bacterium that was able to tolerate and reduce as high as 50 mM of sodium molybdate to molybdenum blue has been isolated from a metal recycling ground. The isolate was tentatively identified as Serratia sp. strain Dr.Y8 based on the carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. ANOVA analysis showed that isolate Dr.Y8 produced significantly higher (P < 0.05) amount of Mo-blue with 3, 5.1 and 11.3 times more molybdenum blue than previously isolated molybdenum reducers such as Serratia marcescens strain Dr.Y6, E. coli K12 and E. cloacae strain 48, respectively. Its molybdate reduction characteristics were studied in this work. Electron donor sources such as sucrose, mannitol, fructose, glucose and starch supported molybdate reduction. The optimum phosphate, pH and temperature that supported molybdate reduction were 5 mM, pH 6.0 and 37°C, respectively. The molybdenum blue produced from cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Metal ions such as chromium, silver, copper and mercury resulted in approximately 61, 57, 80, and 69% inhibition of the molybdenum-reducing activity at 1 mM, respectively. The reduction characteristics of strain Dr.Y8 suggest that it would be useful in future molybdenum bioremediation.     

Seasonal changes in the cecal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus)

The dominant cecal bacteria in the high-arctic Svalbard reindeer were characterized, their population densities were estimated, and cecal pH was determined in summer, when food quality and availability is good, and in winter, when it is very poor. In summer the total culturable viable bacterial population was (8.9 +/- 5.3) X 10(8) cells ml-1, whereas in winter it was (1.5 +/- 0.7) X 10(8) cells ml-1, representing a decrease to 17% of the summer population density. Of the dominant species of cultured bacteria, Butyrivibrio fibrisolvens represented 23% in summer and 18% in winter. Streptococcus bovis represented 17% in summer and 5% in winter. Bacteroides ruminicola represented 10% in summer and 26% in winter. In summer and winter, respectively, the proportion of the viable population showing the following activities was as follows: fiber digestion, 36 and 48%; cellulolysis, 10 and 6%; xylanolysis, 33 and 48%; and starch utilization, 77 and 71%. The most abundant cellulolytic species in summer was Butyrivibrio fibrisolvens, representing 62% of the total cellulolytic population, and in winter it was Ruminococcus albus, representing 80% of the total cellulolytic population. The most abundant xylanolytic species in summer was Butyrivibrio fibrisolvens, and in winter it was Bacteroides ruminicola, representing 59 and 54% of the xylanolytic isolates in summer and winter, respectively. The cecal bacterial of the Svalbard reindeer have the ability to digest starch and the major structural carbohydrates of the diet that are not digested in the rumen. The cecum in these animals has the potential to contribute very substantially to the digestion of the available plant material in both summer and winter.     

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).     

Change in the ultraviolet spectrum of solubilized Ca2+-dependent ATPase from sarcoplasmic reticulum due to binding with Ca2+ ions.

Solubilized sarcoplasmic reticulum (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The UV absorption of SSR decreased reversibly at about 286 and 292 nm on removal of free Ca2+ ions, while no change in the fluorescence spectrum was detectable. On the other hand, the fluorescence intensity of FSR decreased 3-4% on removal of free Ca2+ ions, as previously reported by Dupont [(1976) Biochem. Biophys. Res. Commun. 71, 544-550]. The UV absorption of FSR increased reversibly at about 270-280 nm on removal of free Ca2+ ions, but the rate of the change was very slow (k = about 0.1 min-1).     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.     

On the toroidal condensed state of closed circular DNA

The influence of double helix torsional elasticity on the compaction and structure of circular DNA compact form is studied theoretically in the case when the compact (globular) form has torus shape. For closed circular DNA the topological invariant, the linking number, yields a strict connection between conformation of the double helix considered as unifilar homopolymer and elastic energy of torsional twisting. The contribution of torsional elasticity to the free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superhelical density. Allowance of the torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Import and processing of the precursor of cytochrome P-450(SCC) by bovine adrenal cortex mitochondria

Isolated bovine adrenal cortex mitochondria imported in vitro synthesized pre-P-450(SCC) and processed it to the mature form. Partial radio-sequencing of the processed P-450(SCC) gave a result identical with that for authentic P-450(SCC). Rat liver mitochondria also imported pre-P-450(SCC) and processed it to the mature form, whereas bovine heart mitochondria were unable to import and process pre-P-450(SCC) although both mitochondrial preparations imported and processed pre-adrenodoxin. The pre-P-450(SCC) processing activity of bovine adrenal cortex mitochondria was associated with the matrix side surface of the inner membrane. The processing protease could be solubilized by sodium cholate and partially purified by ammonium sulfate fractionation. The partially purified processing protease cleaved pre-P-450(SCC) at the correct position. It was also active in processing pre-P-450(11 beta) but inactive toward pre-adrenodoxin. Bovine heart mitochondria lacked the processing activity to pre-P-450(SCC). The localization of pre-P-450(SCC) and mature P-450(SCC) in bovine adrenal cortex mitochondria was examined. Mature P-450(SCC) processed by the mitochondria was found associated with the matrix-side surface of the inner membrane, which is the correct location of P-450(SCC) in the cell. In the presence of o-phenanthroline, pre-P-450(SCC) was imported into the organelles without being processed and remained soluble in the matrix. The incorporation of newly processed mature P-450(SCC) into the inner membrane was also observed when pre-P-450(SCC) was incubated with inner membrane vesicles. Mature P-450(SCC) generated in vitro from pre-P-450(SCC) by the partially purified processing protease was incorporated not only into the inner membrane vesicles but also into bovine adrenal cortex microsomes. These findings suggested that the processing of pre-P-450(SCC) occurred prior to the incorporation of mature-P-450(SCC) into the inner membrane.