Serine Incorporation Research Articles

Influence of cytochalasin d-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes

There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. We have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35SO 4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35S0 4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [ 3H]serine incorporation into core protein was also stimulated. The observed stimulation of proteoglycan synthesis was not due to an overall stimulation of protein synthesis, to inhibition of DNA synthesis, or to accumulation of cells in one phase of the cell cycle. Cytochalasin D-treatment of cells in suspension caused no further stimulation of 35 S0 4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se; nevertheless, we cannot completely rule out other, nonspecific, effects of the drug. Fibroblasts and chondrocytes that had been passaged to stimulate dedifferentiation did not incorporate more 35SO 4 when treated with cytochalasin D, suggesting that increased proteoglycan synthesis in response to rounding may itself be a differentiated property of chondrocytes.

Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by beta-chloroalanine.

The effects of beta-chloroalanine (beta-Cl-alanine) on serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid (5 mM beta-Cl-alanine caused complete inactivation in 10 min), irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with beta-Cl-L-alanine (not beta-Cl-D-alanine) and was blocked by L-serine. These are characteristics of mechanism-based ("suicide") inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with beta-Cl-alanine (complete inhibition occurred in 15 min with 5 mM), and this treatment inhibited [14C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of beta-Cl-L-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [14C]serine uptake was not blocked, total lipid biosynthesis from [14C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [3H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [3H]leucine, which was only decreased by 14%. Although beta-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that beta-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.

Effects of Limited Exposure of Rabbit Chondrocyte Cultures to Parathyroid Hormone and Dibutyryl Adenosine 3′,5′-Monophosphate on Cartilage-Characteristic Proteoglycan Synthesis*

Treatment of rabbit chondrocyte cultures with PTH or (Bu)2cAMP for 30 h increased by 2- to 3-fold the incorporation of [35S]sulfate and 3H radioactivity with glucosamine as the precursor into large chondroitin sulfate proteoglycans characteristically found in cartilage matrix. However, PTH and (Bu)2cAMP did not increase either [35S]sulfate incorporation into small proteoglycans or the incorporation of 3H radioactivity into hyaluronic acid and other glycosaminoglycans. PTH and (Bu)2cAMP also increased the incorporation of [3H] serine into both proteoglycans and total protein. In all cultures described above, the stimulation of [3H]serine incorporation into proteoglycans exceeded that of [3H]serine incorporation into total protein. These data indicate that PTH and (Bu)2cAMP selectively stimulate cartilage proteoglycan synthesis while they increase total protein synthesis. Since cAMP seems to play a mediatory role in the action of PTH, we elected to examine the effects of a limited exposure of chondrocytes to PTH or (Bu)2cAMP on the synthesis of proteoglycans. Treatment with PTH or (Bu)2cAMP for only the initial 2-7 h did not increase the rates of incorporation of [35S]sulfate, the 3H radioactivity with glucosamine, and [3H]serine into proteoglycans, as measured at 30 h, despite the fact that this treatment brought about a rapid and transient rise in the cAMP level. Furthermore, the application of prostaglandin I2 at concentrations that increased cAMP levels in a similar fashion as did PTH did not affect [35S] sulfate incorporation into proteoglycans. These observations suggest that in addition to the transient rise of cAMP, other biochemical changes are required for elaboration of the effect of PTH on proteoglycan synthesis. Although cAMP analogs mimic some of the effects of PTH in chondrocytes, the nucleotides and PTH appear to stimulate proteoglycan synthesis by different mechanisms.

Action of 1,25-dihydroxyvitamin D3 on phospholipid metabolism of human bone cells in culture

It has recently been proposed that the action of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on bone metabolism may be mediated by changes in phospholipid metabolism. The effects of vitamin D metabolites on the incorporation of radiolabelled precursors into corresponding phospholipid classes were investigated using cells arising from cultured explants of normal human bone with osteoblast-like characteristics. Treatment with 1,25-(OH)2D3 increased the incorporation of serine, measured as the ratio of [3H]serine in phosphatidylserine (PS) to [14C]ethanolamine in phosphatidylethanolamine (PE), in a time- and dose-dependent manner. The maximum effect on PS/PE of 141.6 +/- 5.9% over control (P = 0.022) was observed at a dose of 0.1 nmol 1,25-(OH)2D3/l, maintained for 24 h. Incubations with 25-hydroxyvitamin D3 (0.1 mumol/l) and 24,25-dihydroxyvitamin D3 (10 nmol/l) had no effect. Supraphysiological doses (0.1 mumol/l) of 1,24,25- and 1,25,26-trihydroxyvitamin D3 showed similar effects to those of 1,25-(OH)2D3, emphasizing the importance of 1 alpha-hydroxylation. Incorporation of [14C]choline into phosphatidylcholine, calculated as a ratio to PE, was not affected by treatment with vitamin D metabolites. However, [3H]inositol uptake into phosphatidylinositol was almost doubled when compared with control uptake within 2 h of treatment with 1,25-(OH)2D3 (0.1 mumol/l). This may be of relevance, considering the importance of phosphoinositide metabolism in influencing the intracellular calcium concentration. These results support a role for 1,25-(OH)2D3 in the modulation of phospholipid metabolism in human bone cells, which in turn may be involved in the action of 1,25-(OH)2D3 in bone mineralization.

Effects of Nitrous Oxide on Macromolecular Content and DNA Synthesis in Rat Embryos

Exposure of pregnant rats to the anesthetic gas, nitrous oxide (N2O), has been reported to be teratogenic. This gas decreases activity of the enzyme 5-methyltetrahydrofolate homocysteine methyltransferase (methionine synthetase) and alters the level and distribution of folic acid derivatives in embryonic tissue. Because of their role in purine, thymidine and amino acid metabolism, alterations in the levels of various folate forms could affect macromolecular synthesis. The effect of N2O exposure on the number of somites, the content of DNA, RNA and protein as well as the incorporation of thymidine, deoxyuridine or serine into DNA were determined. Pregnant rats were exposed for 24 hr to 50% N2O-50% O2 beginning on day 10 of gestation. Control animals were exposed to 50% N2-50% O2. Embryos were removed and cultured for 4 hr using a rodent whole embryo culture system in medium containing radiolabeled precursors for DNA. Treatment with N2O had no effect on somite number, RNA or protein content. However, there was a significant decrease in DNA content. There was an increase in the incorporation of thymidine into DNA, but the incorporation of deoxyuridine and serine into DNA was decreased. These data demonstrate that treatment of pregnant rats with N2O results in decreased DNA synthesis and content in exposed embryos.

Characterization of primary translation products from ovine and rat salivary gland mRNAs

Ovine and rat salivary gland mRNAs have been prepared and their translation products characterized. A 60 kD translation product from ovine submaxillary and sublingual gland mRNAs is identical in mass to the ovine apomucin. Two additional ovine translation products, 25 and 40 kD, are specific to mucin-producing salivary glands. Four rat mRNA translation products are encoded by mucin-producing salivary glands (38, 44, 67, 69 kD). These polypeptides were not detected in the parotid gland mRNAs, a serous gland. Each of these products has a high level of [3H]serine incorporation, a characteristic of mucins. The nature of these products suggests that they are mucins or mucin-like and that their molecular weights should approximate that of the corresponding apomucins.

Impairment of Photorespiratory Carbon Flow into Rubber by the Inhibition of the Glycolate Pathway in Guayule (Parthenium argentatum Gray)

Cut shoots of guayule (Parthenium argentatum Gray) were treated with four inhibitors of the glycolate pathway (alpha-hydroxypyridinemethanesulfonic acid; isonicotinic acid hydrazide, glycine hydroxamate, and amino-oxyacetate, AOA) in order to evaluate the role of photorespiratory intermediates in providing precursors for the biosynthesis of rubber. Photorespiratory CO(2) evolution in guayule leaves was severely inhibited by AOA. Application of each of the four inhibitors has resulted in a significantly decreased incorporation of (14)C into rubber fractions suggesting that the glycolate pathway is involved in the biosynthesis of rubber in guayule. However, the application of each of the glycolate pathway inhibitors showed no significant effect on photosynthetic CO(2) fixation in the leaves. The inhibitors individually also reduced the incorporation of labeled glycolate, glyoxylate, and glycine into rubber, while the incorporation of serine and pyruvate was not affected. The effective inhibition of incorporation of glycolate pathway intermediates in the presence of AOA was due to an inhibition of glycine decarboxylase and serine hydroxymethyltransferase. It is concluded that serine is a putative photorespiratory intermediate in the biosynthesis of rubber via pyruvate and acetyl coenzyme A.

The modulation of serine metabolism in hepatoma 3924A during different phases of cellular proliferation in culture.

The activities of 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase increased markedly during the transition of hepatoma cells from a resting non-proliferating culture into the proliferating growth phase. Activities declined as cells reached confluency and entered the plateau growth phase. This pattern was paralleled by changes in [14C]serine incorporation into nucleic acids. The experiments support the hypothesis that the biosynthesis of serine is metabolically coupled to its utilization for nucleotide precursor formation in cancer cells.

Effect of 16,16-dimethyl prostaglandin E 2 on mucus glycoprotein biosynthesis in rat stomach and duodenal glands

The present study describes the effect of 16,16-dimethyl prostaglandin E 2 (16,16-dmPGE 2) on mucus glycoprotein biosynthesis in rat stomach and duodenal glands. After in vivo treatment with 16,16-dmPGE 2 (10 μg/kg subcutaneously) for 1 h, the incorporation rate of [ 3H]galactose, [ 3H]glucosamine, and [ 3H]serine in the ex vivo vascularly perfused stomach was determined by light microscopic autoradiography. As was previously found by us for the surface mucous cells in the fundus of 16,16-dmPGE 2-treated rats, the incorporation of [ 3H]galactose and [ 3H]glucosamine (indicative of mucus glycoprotein synthesis) in the isthmus was increased two- to fourfold. Small if any increases were detected in the mucous cells near the base of the glands of the fundus (neck cells), the mucous cells in the antrum and the mucous cells in the duodenal glands. Total protein synthesis as measured by [ 3H]serine incorporation was not increased in any of these cells. We conclude that 16,16-dmPGE 2 has different effects on mucus glycoprotein biosynthesis in various regions of the rat stomach. Increased biosynthesis in the fundus points to a role for mucus in the prostaglandin-induced protection of the gastric mucosa against injury.

2-Chloro-4,6-dimethoxy-1,3,5-triazine. A New Coupling Reagent for Peptide Synthesis

In order to facilitate the purification of peptides, the new coupling reagent 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) is proposed. Due to the weakly basic properties of the triazine ring, the side products and excess coupling reagent are easily removed by washing the crude reaction product with diluted acids. CDMT enables the synthesis of di-,tri-, and pentapeptides in 75-98% yield under mild conditions and without concomitant racemization. Moreover, the reagent diminishes the formation of side products during the incorporation of serine with an unprotected hydroxy group or of N-protected ω-nitroarginine into the peptide chain.

Serine incorporation into the selenocysteine moiety of glutathione peroxidase.

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.

Stereochemistry of incorporation of serine into the oxazole ring of virginiamycin M1

Incorportion of serine into the oxazole ring of virginiamycin M1 proceeds via the (S)-isoemr of serine with loss of the 3-(pro-S) hydrogen, as deduced from feeding studies with various stereospecifically labelled precursors.

Stimulatory effect of calmodulin antagonists on phospholipid base-exchange reactions in rabbit platelet membranes

The properties of Ca 2+-dependent incorporation of [ 3H]serine, [ 3H]ethanolamine and [ 3H]choline into the corresponding phospholipids mediated by base-exchange enzymes in rabbit platelet membranes were studied in the presence or absence of the calmodulin antagonists chlorpromazine, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), all of which markedly activate three base-exchange reactions. The base-exchange activities were dependent on Ca 2+ both in the presence and absence of the drugs. Other metal ions tested did not stimulate the base-exchange reactions, even in the presence of the drugs. Apparent K m values for serine, ethanolamine and choline were not affected significantly by the concentration of Ca 2+, with or without the drugs. [ 3H]Serine incorporation into phospholipid was competitively inhibited by ethanolamine and choline, [ 3H]choline incorporation was competitively inhibited by serine and ethanolamine, whereas [ 3H]ethanolamine incorporation was competitively inhibited by serine and noncompetitively by choline. These competitive and noncompetitive relations between each base were also not affected by the drugs. The amount of 45Ca 2+ binding to platelet membranes was decreased by the drugs dose dependently. A weaker calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), only slightly stimulated the base-exchange reactions, but did clearly inhibit 45Ca 2+ binding to the membranes, in the same manner as that of the other calmodulin antagonists used. The concentration of chlorpromazine, trifluoperazine, W-7 and W-5, required to produce half-maximal inhibition of Ca 2+ binding, was approximately 30 μM. These results suggest that the calmodulin antagonists used activate the base-exchange reactions only in the presence of Ca 2+ without changing the affinity of each free base to base-exchange enzymes. The activation of the base-exchange reactions was not due to the increase in free Ca 2+ caused by the drug-induced inhibition of Ca 2+ binding to platelet membranes.

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 μg/ml tunicamycin) disproportionately to that of general protein synthesis measured by [ 3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [ 35S]sulfate and [ 3H]glucosamine were used as labeling precursors, the ratio of 35S 3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP- N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t 1 2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-β-xyloside, however, lasted longer ( t 1 2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: (a) tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely (b) a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.

Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells.

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.

Mechanism for the decreased biosynthesis of cartilage proteoglycan in the scorbutic guinea pig.

Our previous work showed that vitamin C deficiency caused about a 70-80% decrease in the incorporation of [35S]sulfate into proteoglycan of guinea pig costal cartilage, coordinately with a decrease in collagen synthesis (Bird, T. A., Spanheimer, R. G., and Peterkofsky, B. (1986) Arch. Biochem. Biophys. 246, 42-51). We examined the mechanism for decreased proteoglycan synthesis by labeling normal and scorbutic cartilage in vitro with radioactive precursors. Proteoglycan monomers from scorbutic tissue were of a slightly smaller average hydrodynamic size than normal but there was no difference in the size of the glycosaminoglycan chains isolated after papain digestion. The type of glycosaminoglycans synthesized and the degree of sulfation were unaffected as determined by chondroitinase ABC digestion and duel labeling with [35S]sulfate and [3H]glucosamine. Conversion of [3H]glucosamine to [3H]galactosamine also was unimpaired. There was about a 40% decrease in core protein synthesis, measured by [14C]serine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nevertheless, decreased incorporation of [35S]sulfate into scorbutic tissue persisted in the presence of p-nitrophenyl-beta-D-xyloside and cycloheximide, which indicated that the site of the scorbutic defect was beyond core protein synthesis and xylosylation. Galactosyltransferase activity in scorbutic cartilage decreased to about one-third the levels in control samples in parallel with the decreases in proteoglycan and collagen synthesis. Our results suggest that the step catalyzed by this enzyme activity, the addition of galactose to xylose prior to chondroitin sulfate chain elongation, is the major site of the scorbutic defect in proteoglycan synthesis. Decreased enzyme activity may be related to increased cortisol levels in scorbutic serum.

Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride.

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphatidylserine synthesis in adult dirofilaria immitis females

Phosphatidylserine synthesis in adult Dirofilria immitis females. International Journal for Parasitology 16: 9–11. Phosphatidylserine synthesis in extracts of Dirofilaria immitis females was found to occur exclusively by way of a calcium-stimulated enzyme-catalyzed exchange of l-serine for the base components of preformed phospholipids. Under specified conditions the rate of serine incorporation into phospholipid(s) was around 44 pmol min −1 mg −1 protein.

Changes in proteoglycan composition during development of rat skin. The occurrence in fetal skin of a chondroitin sulfate proteoglycan with high turnover rate.

Extraction of the skin of newborn rat yielded two populations of galactosaminoglycan-containing proteoglycan: a Mr = 111,000-200,000 dermatan sulfate proteoglycan (DS-PG) with a Mr congruent to 55,000 core glycoprotein and a Mr congruent to 10(6) chondroitin sulfate proteoglycan (CS-PGs) composed of two subpopulations with different size core-glycoproteins (Mr congruent to 480,000 and 520,000). Tryptic peptide mapping of chondroitinase-treated DS-PG and CS-PGs indicated that the peptide patterns observed with the two core molecules from CS-PGs were identical with each other but distinct from the peptide pattern of the DS-PG core molecule. It is likely therefore that the two forms of CS-PGs are derived from the same gene product by post-translational modification or partial degradation, but DS-PG is derived from a distinct gene product. Comparison of the concentration (hexuronate/DNA) of the proteoglycans in newborn and fetal rat skin showed an age-related change in proteoglycan composition; at 4 days before birth, the uronic acid proportions, DS-PG:CS-PGs, were about 14:1 and during the next 4 days, DS-PG increased 2.2-fold whereas CS-PGs decreased 4-fold. On a per DNA basis, the rate of [3H]serine incorporation into CS-PGs was 2.5 times the rate for DS-PG at 4 days before birth but decreased by 95% during the next 4 days. The rate for DS-PG also decreased but to a much lesser extent, so that by 2 days before birth, it began to exceed the rate for CS-PGs. The striking change in the concentration and labeling rate of CS-PGs can be interpreted either as a decrease of CS-PGs synthesis, or as an increase of CS-PGs breakdown, or both, a process which might be involved in the transition of extracellular matrix from a fetal type to a newborn or adult type.

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in a clonal osteoblast-like rat osteogenic sarcoma cell line.

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis.