Cellular contaminants and structural proteins of Rous sarcoma virus (RSV), studied by polyacrylamide gel electrophoresis]

The number of polypeptides in highly purified preparations of RSV, of two different subgroups, produced in culture, has been compared to the polypeptides present in the supernatant of uninfected cultures and processed in identical manner. The analysis of PAGE-SDS shows that from 13 to 18 polypeptides present in viral preparations may be cellular contaminants. Fewer contaminating polypeptides are found in the myeloblastosis virus purified from plasma of Chicken.     

Molecular study in polyacrylamide gel of polypeptides of several strains of rabies virus

Comparative study of structural polypeptides of five rabies virus strains (serotype 1) and one serotype IV, demonstrates difference in molecular weight of the envelope polypeptides M1 and G of all strains. There is little difference between the structural polypeptides N, M2 and probably L. Some purified strains have been treated by trypsine to localize the position of polypeptides. There was no difference between the four major polypeptides of complete and defective virus. Pasteur strain cultivated on different cells shows very little difference in molecular weight of the four major polypeptides.     

Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells

The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. This effect was prevented by 5-HT2 specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT2 receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.     

Inhibition of granulocyte-macrophage (GM) colony formation by basic polypeptides from mouse Ehrlich ascites tumor cells

Summary The polypeptides isolated from mouse Ehrlich ascites tumor cells were tested for their inhibitory activity against GM colony formation. It was found that basic polypeptides with low molecular weight evidently inhibit colony formation. Our data reveal that the tested polypeptides may show chalone activity.     

Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells

Summary The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [³H]inositol monophosphate, [³H]inositol bisphosphate and [³H]inositol trisphosphate. This effect was prevented by 5-HT₂ specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT₂ receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.Part of the data contained in this paper was presented at the 74th local meeting of the Japanese Society of Pharmacology at Kanagawa.     

Release of plasminogen activator from normal and neoplastic endometrium

Summary In the medium of endometrial carcinoma cultures, anti-urokinase-reacting plasminogen activator was released in contrast to cultures of normal or hyperplastic endometrium.This investigation was supported by grants from Malmö General Hospital, the Council for Tobacco Research, USA and the Swedish Medical Research Council (B79-17X-04523-05B).     

Evidence for the extranuclear localization of thymosins in thymus.

A new radioimmunoassay has been developed for thymosin beta 4 by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1-15 coupled to KLH. The synthetic analogue [Tyr12]-thymosin beta 4 (1-15) was used as tracer. This radioimmunoassay, with a useful range of 10-1000 pmoles, showed cross-reactivity with the second homologous beta-thymosin of man and rat (thymosin beta 10) but not of calf (thymosin beta 9). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin alpha, was employed for the measurement of the levels of thymosin beta 4 and parathymosin alpha in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of alpha- and beta-thymosins.     

Fusion polypeptides in gene cloning: potential problems due to conformational alterations at the junction

Many eukaryotic genes are cloned in bacterial hosts as fusion polypeptides. Prediction of the secondary structures for some common prokaryotic fusion polypeptides shows that many junction sites correspond to important secondary structures. It is suggested that such structures could affect (hinder, etc.) the conformation or drive the folding of the neighboring eukaryotic counterparts. Thus the prokaryotic junction should be better performed in random coil regions, or short fusion prokaryotic polypeptides should be used.     

Hsp70s and lysosomal proteolysis

Confluent cultured cells activate a lysosomal pathway of polypeptide breakdown in response to withdrawal of serum growth factors. The substrates for this proteolytic pathway are a restricted class of cytosolic polypeptides containing peptide sequences biochemically related to lysine-phenylalanine-glutamate-arginine-glutamine, or, in single amino acid abbreviations, KFERQ. The heat shock cognate protein of 73 kD (hsc73) binds to a variety of polypeptides via this molecular determinant and facilitates their lysosomal import and degradation. In addition, a portion of intracellular hsc73 resides within the lysosome and appears to be an essential component of the proteolytic machinery. Several potential mechanisms by which hsc73 mediates selective lysosomal import and degradation of polypeptides are discussed.     

Iclusion proteins and viral proteins of the baculovirus of the dipteran Tipula paludosa (Meigen)]

Examination of the polyhedron protein by polyacrylamide gel electrophoresis shows only one polypeptide with a molecular weight of 25,500 +/- 500 daltons, while that of virion proteins reveals 13 polypeptides. No antigenic community could be demonstrated between the polyhedron protein of the Baculovirus of T. paludosa and the polyhedron protein of several other Baculoviruses.     

The CLE family of plant polypeptide signaling molecules

Polypeptide ligands have long been recognized as primary signaling molecules in diverse physiological processes in animal systems. Recent studies in plants have provided major breakthroughs with the discovery that small polypeptides are also involved in many plant biological processes, indicating that the use of polypeptides as signaling molecules in cell-to-cell communication is evolutionarily conserved. The CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins are currently the best understood family of small polypeptides in plants. The recent isolation of MCLV3 from Arabidopsis and TDIF from a Zinnia cell culture system indicates that biologically active CLE polypeptides are produced by post-translational proteolysis and modification, similar to peptide hormone production in animals and yeast. Here, we review exciting discoveries involving the identification of the CLE proteins and their functions in various aspects of plant development, including restriction of stem cell accumulation by CLV3 and inhibition of xylem differentiation by TDIF.     

Storage of irradiated human blood; a source of error in quantitative chromosome analysis

Human whole blood was irradiated with 2.5 Gy of 220 k Vp X-rays and stored before culture with 9.7 microM BrdU and 19.4 or 38.7 microM BrdU for 0, 24, 48 and 72 h. The frequency of dicentrics and ring chromosomes was determined in cells staining as first division (M1) metaphases with the fluorescence plus Giemsa technique. Storage had no influence on the observed aberration yields in 44 h cultures containing 9.7 microM BrdU. In 66 h cultures at 19.4 microM BrdU the observed yields after 2 and 3 days' storage were significantly lower as compared to cultures from fresh blood. No storage effect was revealed in 66 h cultures containing 38.7 microM BrdU. In cases where cytogenetic radiation dosimetry has to be carried out using blood samples which have been in transit for 2-3 days, the findings are of relevance for a correct determination of the chromosome damage in M1 cells.     

Melatonin antagonizes colchicine-induced mitotic arrest.

Melatonin, in concentrations up to 10(-3) M, showed no effect on mitosis in cultures of HeLa or KB cells. However, when melatonin at 10(-4) M was preincubated with HeLa cells prior to addition of 10(-7) M colchicine, a reduction in the mitotic index, in comparison to colchicine alone, was observed.     

Phosphorylation of cockroach antennal polypeptides: effects of second messengers and pheromonal blend

In insect antennal extracts, Schleicher et al.¹ showed that protein kinase C (PKC) inhibitors abolish the transience of pheromone-induced rapid inositol trisphosphate responses, which suggests that pheromonal signals act on phosphorylation of specific proteins. To confirm this hypothesis, we studied the effects of second messengers and a pheromonal blend on phosphorylation of antennal proteins in the cockroachPeriplaneta americana. Proteins from adult male antennae were phosphorylated in vitro in the presence of [³²P] triphosphate, then separated by SDS-polyacrylamide gel electrophoresis. Numerous phosphopolypeptides were visualized. The presence of Ca⁺⁺/calmodulin in the incubation medium resulted in increased phosphorylation of polypeptides with molecular weights of 38, 48, 51, 54 and 58 kDa. Stimulation of PKC by addition of Ca⁺⁺ phosphatidylserine (PS)/phorbol myristate acetate (PMA) resulted in the appearance of three phosphopolypeptides of 36, 70 and 120 kDa. In the presence of cyclic adenosine monophosphate, two new major polypeptides of 46 and 42 kDa appeared; the latter polypeptide also appeared in the presence of cyclic guanosine monophosphate. Comparison with polypeptide composition of tissue from the cerci, leg, brain and fat body showed that the 36 and 48 kDa polypeptides were specific to antennae, whereas the 120 kDa polypeptide was also present in the adult brain. When antennae are subjected to pheromonal stimulation for 16 seconds prior to homogenization, in vitro phosphorylation of the 120, 70, 64 and 38 kDa polypeptides was inhibited, whereas phosphorylation of the 58, 54, 51 and 48 kDa polypeptides was strongly stimulated. It is noteworthy that a 107 kDa polypeptide was observed only after pheromonal stimulation by Ca⁺⁺/PS/PMA. Our findings suggest that Ca⁺⁺-and PKC-dependent protein phosphorylation systems play an important role in the transduction of pheromonal signals in antennae of male cockroachP. americana. We speculate that specific phosphoproteins may modulate sensitivity and signal amplification during the olfactory transduction process.     

Snake venom action: are enzymes involved in it?

Enzymes were the first clearly recognized components of snake venoms. When several more were discovered, attempts were made to correlate venom action with enzymic functions. The last few years have seen most successful efforts in the identification, isolation and structrual elucidation of highly toxic polypeptides present in snake venoms, in particular of 'neurotoxins' and membrane-active toxins. Following this development the polypeptides were called the true toxic components and the enzymes lost their previous central position in venom pharmacology. The time, therefore, has come re-evaluate the role of enzymes in the complex interaction between snake and prey. While highly active polypeptides indeed dominate the actionof hydrophiid venoms, they appear to play a lesser role in crotalid venom action as compared with enzyme components. Enzymes are involved in many levels of venom action, e.g. by serving as spreading factors, of by producing very active agents, such as bradykinin and lysolecithins in tissues of preys or predators. Some toxins, e.g. the membrane-active polypeptides appear to participate in the interaction between membrane phospholipids and venom phospholipases. The classical neurotoxin, beta-bungarotoxin, has been recognized as a powerful phospholipase. Several instances are known which indicate that some enzymes potentiate the toxic action of others; the analysis of a single enzyme may, therefore, not fully reveal its biofunction. For 3 enzymes,ophidian L-amino acid oxicase, ATPpyrophosphatase, and acetylcholinesterase, some of the problems pertaining to venom toxicity are discussed.     

Electron microscopy of mitochondrial DNA in Podospora anserina and the presence of a multimeric range of circular DNA molecules from senescent cultures]

Mitochondrial DNA from young cultures of race s of Podospora anserina was isolated. Its density in Cesium chloride density equilibrium gradients was 1.694 g/cc. Examination by the electron microscope revealed that ca 1% of this DNA consisted of circles, 31 micrometer in contour length; the remaining DNA was composed of linear molecules ranging in length from 2 to 33 micrometer. In DNA of similar density obtained from senescent cultures of the same race s, about 11% of the molecules consisted of a multimeric set of circles ranging in size from 0.9 to 15 micrometer, with most being in the 1.8 and 2.7 micrometer classes. The similarity of these DNA molecules with the mitochondrial DNA from rho(-) yeast mutants is discussed.     

Fusion polypeptides in gene cloning: Potential problems due to conformational alterations at the junction

Summary Many eukaryotic genes are cloned in bacterial hosts as fusion polypeptides. Prediction of the secondary structures for some common prokaryotic fusion polypeptides shows that many junction sites correspond to important secondary structures. It is suggested that such structures could affect (hinder, etc.) the conformation or drive the folding of the neighboring eukaryotic counterparts. Thus the prokaryotic junction should be better performed, in random coil regions, or short fusion prokaryotic polypeptides should be sued.1 October 1986     

Use of Cowan strain I of Staphylococcus aureus for radio-immunological determination of retrovirus proteins]

The use of Staphylococcus aureus for the radio-immunoassay of C-type virus polypeptides provided very specific results and proved to present several advantages over the classical methods of precipitation of immune complexes.     

Snake venom action: Are enzymes involved in it?

Summary Enzymes were the first clearly recognized components of snake venoms. When several more were discovered, attempts were made to correlate venom action with enzymic functions. The last few years have seen most successful efforts in the identification, isolation and structural elucidation of highly toxic polypeptides present in snake venoms, in particular of neurotoxins and membrane-active toxins. Following this development the polypeptides were called the true toxic components and the enzymes lost their previous central position in venom pharmacology. The time, therefore, has come to re-evaluate the role of enzymes in the complex interaction between snake and prey. While highly active polypeptides indeed dominate the action of hydrophiid venoms, they appear to play a lesser role in crotalid venom action as compared with enzyme components. Enzymes are involved in many levels of venom action, e. g. by serving as spreading factors, of by producing very active agents, such as bradykinin and lysolecithins in tissues of preys or predators. Some toxins, e. g. the membrane-active polypeptides appear to participate in the interaction between membrane phospholipids and venom phospholipases. The classical neurotoxin, -bungarotoxin, has been recognized as a powerful phospholipase. Several instances are known which indicate that some enzymes potentiate the toxic action of others; the analysis of a single enzyme may, therefore, not fully reveal its biofunction. For 3 enzymes, ophidianl-amino acid oxidase, ATPpyrophosphatase, and acetylcholinesterase, some of the problems pertaining to venom toxicity are discussed.