A sequence-specific protease cleaves a maternal, cortical protein during early embryogenesis in Sciara coprophila (Diptera)

One of the first events of egg activation in Sciara coprophila (Diptera) is the disappearance of an abundant maternal 38-kDa protein (p38) and the simultaneous emergence of an abundant 35-kDa protein (p35). Western blotting experiments using monoclonal antibodies directed against p38 reveal that p38 and p35 are serologically related and indicate that maternal p38 is transformed into p35 during early development. This transition is possibly accompanied by a conformational change in the part of the protein that is common to both protein species. The processing of p38 to p35 can be mimicked by trypsin treatment in vitro, suggesting that a trypsin-like protease is responsible for this conversion in vivo. Immunostaining indicates that the p38 class of antigens is evently distributed in the periplasm of early cleavage embryos. After the arrival of nuclei in the periplasm, the antigens become associated with the infolding cellular membranes. A similar membrane association of actin can be observed with anti-actin antibodies. Nevertheless, p38 and actin are clearly distinct from each other. We presume that p38 is a product of a maternal effect gene necessary for early dipteran development.     

Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy

Two siblings of consanguineous parents had presented with a variety of findings indicative of juvenile metachromatic leukodystrophy (MLD). However, instead of the expected profound deficiency of arylsulfatase A (ARS A), their enzyme levels were about half-normal, and enzyme from fibroblasts had properties identical with the properties of enzyme from normal fibroblasts. Nevertheless, the hydrolysis of cerebroside sulfate by growing fibroblasts was markedly attenuated. Supplementation of the fibroblasts with cerebroside sulfatase activator normalized the response in the loading test. These results imply that the fibroblasts, and by extension the patients, are deficient in activator. Although the defective catabolism of cerebroside sulfate and the clinical manifestations in these patients mimic MLD, the molecular basis is distinct from the classical forms of the disorder.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.     

Effects of retinoic acid on protein synthesis in cultured melanoma cells

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G₁ phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis anlaysis of L-[³⁵S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.     

Role of prostaglandin E2 in the induction of nonspecific T lymphocyte suppressor activity

Activated human monocytes and concanavalin A (Con A)-activated T lymphocytes are known to suppress T and B lymphocyte proliferation and B cell maturation into immunoglobulin-producing cells. We have now shown that monocyte suppressive activity is predominantly mediated through release of prostaglandin E2 (PGE2), which is active only in the presence of a "short-lived," radiosensitive T lymphocyte subset. PGE2, at high concentration, can activate T suppressor lymphocytes (TS), which display the same characteristics as Con A-activated TS lymphocytes. Moreover, Con A activation of TS lymphocytes was obtained only in the presence of PGE2, as specific anti-PGE2 antiserum or indomethacin prevented TS activation; this suggested a double signal as a prerequisite for activation of the nonspecific TS cell subset. We propose that TS lymphocytes modified by Con A become sensitive to small amounts of PGE2 produced by monocytes that must be present during the Con A-stimulated activation phase of suppressive cells.     

Fertilization following mixed insemination with 'cervix-selected' and 'unselected' spermatozoa in the rabbit

The possible selection of spermatozoa for fertilization by the female genital tract was investigated using genetically homogeneous, numerically adjusted 'cervix-selected' and 'unselected' rabbit spermatozoa. Samples of spermatozoa were marked by 15 min exposure to 1.3 mg TEPA/ml and then washed for 15 min. TEPA-treated and control samples were inseminated alternately into the vagina (= 'cervix-selected') or uterine horns (= 'unselected') of prospective donors. After 6 h spermatozoa were recovered from the uterine horns of the donors. Equal numbers of 'selected' and 'unselected' spermatozoa were inseminated either into the uterine horns (24 does) or oviducts (25 does) of recipients. The fertilization rates were 48 and 72%, respectively. Significantly more eggs were fertilized by untreated than by TEPA-treated spermatozoa. Almost identical fertilization rates, however, were observed between 'cervix-selected' and 'unselected' spermatozoa. It is concluded, therefore, that in the rabbit no selection of (preincubated) spermatozoa for fertilization takes place at the cervical level.     

Calmodulin and cyclic nucleotide phosphodiesterase activities in erythrocytes from normal and schizophrenic subjects

Calmodulin and cyclic nucleotide phosphodiesterase activities were measured in hemolysates prepared from 18 normal and 17 schizophrenic subjects. No significant difference between groups was found for either activity. The results suggest that calmodulin is present in normal amounts in patients with schizophrenia. This is compatible with the idea that the interaction of calmodulin with antipsychotic agents is structurally non-specific.     

The origin of the carbon chain in the thiazole moiety of thiamine in Escherichia coli: incorporation of deuterated 1-deoxy-D-threo-2-pentulose

Non growing washed cells of Escherichia coli, derepressed for the biosynthesis of thiamine, have been incubated in the presence of glucose and either 1-deoxy-$\text{D}$-threo-2-pentulose $\text{1}$ or 1-déoxy-$\text{D}$-erythro-2-pentulose $\text{2}$ trideuterated on the methyl group. The incorporation of deuterium into the thiazole moiety of thiamine was measured by mass spectrometry. The label of the threo-compound was found in more than 40% of the thiazole biosynthesized in its presence; the label of the erythro-compound in less than 5%. Hence it is likely that the carbon chain of 1-deoxy-$\text{D}$-threo-2-pentulose is the precursor of the five carbons chain of the thiazole moiety of the thiamine molecule in E. coli.     

Regulation of glutamate dehydrogenase in Bacillus subtilis.

The activity of the nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Bacillus subtilis was influenced by the carbon source, but not the nitrogen source, in the growth medium. The highest specific activity for this enzyme was found when B. subtilis was grown in a minimal or rich medium that contained glutamate as the carbon source. It is proposed that glutamate dehydrogenase serves a catabolic function in the metabolism of glutamate, is induced by glutamate, and is subject to catabolite repression.     

Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV. Trigalactosylglycerophospho-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis Kiel 42172.

Streptococcus lactis Kiel 42172 contains at least six unusually polar glycerophosphoglycolipids. The predominant one was composed of D-galactose, D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 3 : 2 : 2 : 3 : 1. By analysis of the breakdown products of HF hydrolysis and Smith-degradation the structure was established to be [Galp (alpha 1 leads to 6)Galp(alpha 1 leads to 3)-sn-glycero(2 comes from 1 alpha Galp)-1-phospho] leads to 6Glcp(alpha 1 leads to 2), acyl leads to Glcp(alpha 1 leads to 3)-acyl2Gro. By HF hydrolysis the other compounds were shown to be in the main also derivatives of GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro but they released as water-soluble glycosides Gal(alpha 1 leads to 2)Gro, Gal(alpha 1 leads to 3)Gro, Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), Gal(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro and Gal(alpha 1 leads to 6)Gal-(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), respectively. In the lipid extract Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro and GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3) acyl2Gro were also observed. This set of compounds is proposed to constitute a biosynthetic series reflecting the individual steps in the synthesis of the lipoteichoic acid of Streptococcus lactis Kiel 42172 which is made up by the same lipid anchor and a non-classical poly(galabiosyl, galactosyl glycerophosphate)-chain (Koch, H.U. and Fischer, W. (1978) Biochemistry 17, 5275--5281).