Inhibition of polyamine synthesis reduces the growth rate and delays the expression of differentiated phenotypes in primary cultures of embryonic mesoderm from chick.

Inhibition of polyamine synthesis in early chick embryos blocks their development at gastrulation. Analyses of arrested embryos show that mesodermal outgrowth and differentiation are drastically impaired. To study these effects in greater detail, we have used primary cultures of embryonic mesoderm from chick. The cultures were treated with alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, the first and rate-limiting enzyme in polyamine synthesis. In control culture medium, mesodermal cells retained their in ovo outgrowth behavior and differentiation pattern. Addition of 10 mM DFMO to the culture medium, however, retarded attachment and outgrowth, and reduced the rate of proliferation of the mesodermal cells. Furthermore, the expression of differentiated phenotypes, such as beating heart tissue, erythroid cells, and adipocyte-like cells, was delayed. Simultaneous addition of 100 microM putrescine prevented or reduced the effects of DFMO, showing that these were indeed caused by polyamine deficiency. In the DFMO-treated mesoderm, DNA synthesis was markedly suppressed by the first day. Similar effects on RNA and protein synthesis developed at a later time. Our data suggest that a reduction in the concentrations of the polyamines decreases the rate of mesodermal cell proliferation, and as a consequence delays the expression of differentiated phenotypes.

Tissue distribution of sulfolipids in the rat. Restricted location of sulfatoxygalactosylacylalkylglycerol.

Eleven rat tissues (excluding brain) have been assayed for their ability to synthesize sulfatoxygalactosylacylalkylglycerol (SGG) from Na235SO4 in vivo. These tissues were also assayed for the presence of SGG by an indirect immunofluorescence procedure using rabbit anti-SGG and frozen tissue sections. By both procedures SGG was found to be restricted to the testis; several novel sulfolipids were detected during this study.

Investigations on myelination in vitro. Regulation of sulfolipid synthesis by thyroid hormone in cultures of dissociated brain cells from embryonic mice.

L-3,5,3'-Triiodothyronine (T3) has been shown to influence the synthesis of myelin-associated lipids in cultures of cells dissociated from brains of embryonic mice (Bhat, N. R., Sarlieve, L., Subba Rao, G., and Pieringer, R. A. (1979) J. Biol. Chem. 254, 9342-9344). This culture system was used in the present study to gain additional information on the regulation of the synthesis of myelin lipids by thyroid hormone. The rate of synthesis of the myelin associated sulfolipids remained drastically diminished throughout a 70-day developmental period when cells were grown in the presence of hypothyroid calf serum (T3 < 25 ng/100 ml; thyroxine (T4), 1.2 microgram/ml). However, the activity could be restored to normal levels after 72 h of exposure to deficient medium supplemented with exogenous T3. Half-maximal effects were obtained with 2 X 10(-9) M T3 and 6.25 X 10(-7) M T4. T3 does not alter the synthesis of sulfated mucopolysaccharides, which share adenosine 3'-phosphate, 5'-phosphosulfate (PAPS), as a common precursor, with sulfolipids. This observation argues against the hormone altering the entry of sulfate or the synthesis of PAPS. Rather, T3 acts by changing the activity of the glycolipid:PAPS sulfotransferase(s) in direct proportion to the concentration of T3 in the growth medium. The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase, another myelin marker was also found to be T3 dependent. The response of sulfolipid synthesis to varying amounts of T3 was also observed in a serum-free medium, which suggests that T3 can function independently of other hormones and serum factors in exerting a relatively specific effect on the regulation of myelination.

Differential response of articular chondrocyte populations to thromboxane B2 and analogs of prostaglandin cyclic endoperoxides.

The effects of two unsaturated fatty acids, protaglandin E2, thromboxane B2 (TxB2) and 2 analogs of PG endoperoxide on monolayer cultures of rabbit articular chondrocytes have been studied. Arachidonic and linoleic acids had no effect on either DNA or sulfated-glycosaminoglycan biosynthesis, while 13,14 dihydro-PGE2 and PGE2 markedly inhibited the former. Two epoxymethano analogs of endoperoxide PGH2 (Em-;pgh2) at concentrations of 2.5 and 24 microgram/ml stimulated cell proliferation while reducing 35SO4 incorporation. By contrast, Em-PGH2 at lower concentrations (0.25 - 250 ng/ml) inhibited DNA synthesis in a dose-dependent manner. TxB2 at 2.5 microgram/ml did not alter cellular proliferation. At lower concentrations, 2.5 and 25 ng/ml, TxB2 significantly stimulated sulfated-glycosaminoglycan biosynthesis in at least one of the chondrocyte populations tested. The results also demonstrated marked differences in the effects of TxB2 and the Em-PGH2 analogs on the partitioning of newly synthesized sulfated-proteoglycan between the cells and medium of these cell cultures.

Assay of bacterial copper leaching from covellin at alkaline initial pH.

Copper sulphide CuS was leached by bacteria. Beginning with an initial pH of 9.1, the bacteria acidified the environment and at the same time leached up to 5 g copper per liter. It is characteristic that the pH may sometimes stabilize at 4.0--5.0 and not fall below this value.

Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells.

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.

Degradation of oil shale by sulfur-oxidizing bacteria.

Approximately 40% of oil shale can be solubilized by the action of sulfur-oxidizing bacteria. Thiobacillus thiooxidans and Thiobacillus concretivorous are equally effective in solubilization. Continuous leaching experiments show that this process can be completed within 14 days. The growth of Thiobacillus and the production of acid were measured under several conditions. Almost all of the CaMg(CO(3))(2) was removed by this process, leaving a complex of silica and kerogen that could be burned as low-energy fuel. The silica-kerogen complex had not yet been biologically degraded.

Histochemical changes in gastric mucosubstances in patients with acute and chronic ulcer disease.

The several types of epithelial cells in human gastric mucosa produce different mucosubstances. The surface epithelium largely forms a neutral mucosubstance except that in about two-thirds of the specimens the deep foveolar cells produce a slight to moderate amount of a mucosubstance apparently containing sulfate esters and carboxyl groups. Mucous neck cells often exhibit a neutral mucosubstance but in about onehalf of the stomachs reveal a slight to moderate reactivity indicative of sulfated mucosubstance. Chief cells contain a sulfated mucosubstance with unique histochemical properties. Mast cells vary widely in prevalence but those in the gastric mucosa appear depleted of stored mucosubstances when compared with those in the gastric submucosa or the esophagus. The sulfated mucosubstance normally abundant in human as in canine chief cells appears consistently depleted in patients with stress ulcer or hemorrhagic gastritis. In addition, mucus often appears depleted in the surface epithelium and interstitial edema is present in the superficial mucosa of these patients. These findings appear consistent with the view that biosynthetic activity in chief cells and surface epithelial cells is impaired perhaps secondary to shock-induced circulatory changes in gastric mucosa of patients with stress ulcer or hemorrhagic gastritis.

The formation of choline O-sulphate by Pseudomonas C12B and other Pseudomonas species.

Pseudomonas C(12)B and other Pseudomonas species released larger amounts of a (35)S-labelled metabolite into the medium when cultured on growth-limiting concentrations of Na(2)SO(4) as opposed to growth in SO(4) (2-)-sufficient media. The metabolite was found at all stages of the culture cycle of Pseudomonas C(12)B and maximum quantities occurred in stationary-phase culture supernatants. The metabolite was not detected when the bacterium was cultured on growth-limiting concentrations of potassium phosphate. The amount of the metabolite present in the medium greatly exceeded that which could be extracted from intact cells and, except for choline chloride, it was independent of the carbon source used for growth. If choline chloride was present in high concentration, then larger amounts of the metabolite were found in the culture medium. The metabolite was not detected extracellularly or intracellularly when the bacterium was grown in SO(4) (2-)-deficient media containing 5mm-l-cysteine. The same metabolite was also synthesized in vitro only when Pseudomonas C(12)B extracts were incubated with choline chloride, ATP, MgCl(2) and Na(2) (35)SO(4). The metabolite-forming system was not subject to repression by Na(2)SO(4) and was completely inhibited by 0.5mm-l-cysteine and activated by Na(2)SO(4) (up to 1.0mm). The metabolite was identified as choline O-sulphate by electrophoresis, chromatography and isotope-dilution analysis. Another (35)S-labelled metabolite was also detected in culture supernatants, but was not identified.