The polypeptide composition and ultrastructure of nuclear ghosts isolated from mammalian cells

The polypeptide species of non-membranous nuclear ghosts from purified cell nuclei are conserved among a variety of human, hamster and mouse cell types studied, including HeLa, BHK, 3T6 and Hep-2 cell lines. The polypeptide species present in nuclear ghosts from HeLa cells synchronized in various stages of the cell cycle are largely the same with minor variations. The isolated nuclear ghosts are similar, in terms of polypeptide composition, to other residual nuclear structures isolated by independent techniques. The nuclear ghosts appear as flattened sac-like structures when viewed by scanning electron microscopy. Transmission electron microscopy of the nuclear ghosts reveals ring-like structures which may represent the nuclear pores. Also observed are novel rod-shaped structures approximately 260 nm in length and 50 nm in diameter. The latter images either arise by a rearrangement during isolation of the nuclear ghost macromolecules or are a heretofore undescribed structure of intact nuclei.     

Structural and immunological studies on a sialoglycopeptide isolated from human urine.

A sialoglycopeptide is isolated from human pregnancy urine after gel filtration, ion-exchange chromatography, paper chromatography, and high voltage paper electrophoresis. It contains serine, N-acetyl-galactosamine, galactose, sialic acid (1-1-1-2). Structural studies show that the carbohydrate moiety is likely O-glycosidically linked to serine, and related to MN blood group structures. However this glycopeptide does not exhibit any cross antigenic reactivity by the hemagglutination assay using anti-N, anti-M rabbit immune sera or anti-N lectin from Vicia Graminea.     

Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties

Extraction of isolated axonemes from trout (Salmo gairdneri) sperm with 0.6 M NaCl removed 97% of the outer arms, approximately 12% of the protein, and approximately 50% of the MgATPase activity. Fractionation of this high salt extract by sucrose density gradient centrifugation yielded a single peak of ATPase activity with an apparent sedimentation coefficient of 19 S. Electrophoretic analysis showed that this 19 S particle was composed of two heavy chains (termed alpha and beta; Mr 430,000 and 415,000, respectively), five intermediate molecular weight chains (IC1-IC5; Mr 85,000, 73,000, 65,000, 63,000, and 57,000), and six light chains (LC1-LC6; Mr 22,000-6,000). A similar complex was obtained following further purification by DEAE-Sephacel column chromatography. Quantitative densitometry of Coomassie Blue-stained gels indicated that the heavy and intermediate chains were present in equimolar amounts. Electron microscopic examination of the 19 S particles revealed that it consisted of two globular heads joined together by a Y-shaped stem. The 19 S particle had a specific MgATPase activity of 1.1 +/- 0.3 mumol of phosphate released/min/mg and exhibited an apparent Km for MgATP2- of 40 +/- 16 microM. MnATP2- and CaATP2- were hydrolyzed at rates 100 and 80% that of MgATP2-, respectively. The Mg-ATPase activity was inhibited by vanadate, but not by ouabain or oligomycin, and exhibited a high activity between pH 7.0 and 10.0 with a maximum at pH 9.0-9.5. ATP was the preferred nucleotide, although GTP and CTP (but not ITP) did interact with the dynein to a minor extent. Based on its origin, sedimentation coefficient, polypeptide composition, and enzymatic properties, we conclude that this two-headed 19 S particle represents the entire trout sperm axonemal outer arm dynein. This dynein is probably exemplary of the outer arm dyneins of other vertebrates.     

Comparison of transforming growth factor-beta and a macrophage- deactivating polypeptide from tumor cells. Differences in antigenicity and mechanism of action

A factor in medium conditioned by mouse tumor cells was shown previously to suppress the capacity of mouse peritoneal macrophages to undergo a respiratory burst and to kill protozoal pathogens (macrophage deactivation factor, MDF). Recently, pure transforming growth factor-beta (TGF-beta) proved to be a potent macrophage deactivator as well. Two lines of evidence suggest that MDF is not identical with TGF-beta. First, rabbit anti-TGF-beta IgG neutralized the respiratory burst-suppressing activity of TGF-beta without affecting the bioactivity of MDF, even when the latter was treated with acid to activate potentially latent TGF-beta. Second, in contrast to MDF, which decreases the affinity of the NADPH oxidase for NADPH, permeabilized macrophages that had been deactivated with TGF-beta displayed the same Km and Vmax of the oxidase as activated macrophages. As with MDF, TGF-beta had no effect on two other potential control points over the secretion of respiratory burst products, namely, hydrogen peroxide catabolism or glucose uptake. Finally, neither MDF nor TGF-beta affected the extent or affinity of binding of phorbol diesters to macrophages, the activity or cofactor requirements for protein kinase C, or the ability of protein kinase C to translocate quantitatively from cytosol to membrane fractions in response to phorbol diesters. Thus, 1) MDF is not identical with TGF-beta, and 2) in contrast to the activation or deactivation of macrophages by numerous other agents, TGF-beta regulates macrophage respiratory burst capacity at a level other than the apparent affinity of the oxidase for its substrate.     

Isolation, polypeptide composition and properties of aminoacyl-tRNA-synthetase complexes from the rabbit liver

The procedure for isolation of the aminoacyl-tRNA-synthetase complex from rabbit liver based on the affinity chromatography on heparin- and tRNA-Sepharose has been developed. The complex has a Mr of about 1100 kD and is made up of 10 polypeptides, eight of which are aminoacyl-tRNA synthetase. The complex stability was studied under various conditions. The data obtained are discussed in terms of the involvement of hydrophobic domains of aminoacyl-tRNA synthetases in the stabilization of the complex.     

Comparison of the in vitro transforming activities of human papillomavirus types.

The association of certain human papillomavirus (HPV) types with the majority of human cervical carcinomas suggests a role for the virus in the development of this type of cancer. In this paper, we have examined the transforming properties of several HPV types where the early region genes of the virus are under the control of a strong heterologous promoter and show that major differences exist between the HPV types in their ability to transform primary rat kidney epithelial cells in conjunction with an activated ras oncogene. Those HPV types most commonly found in carcinomas--types 16, 18, 31 and 33--are capable of co-operating with ras to transform primary cells, but those types most commonly found in benign lesions--types 6 and 11--are not. We further demonstrate that the E7 gene of HPV16 by itself is sufficient to co-operate with activated ras to produce transformed cells which are tumorigenic in immunocompetent animals.     

Peptidoglycans as promoters of slow-wave sleep. I. Structure of the sleep-promoting factor isolated from human urine

Fast atom bombardment-mass spectrometry (FABMS) has been used to determine the structure of the urinary sleep-promoting factor (FSu), the nature of whose components had been reported earlier. Less than 1 nmol of the underivatized substance sufficed for the FABMS experiments. The major somnogenic constituent of the purified preparation was a peptidoglycan of Mr = 921 with the structure N-acetylglucosaminyl-N -acetylanhydromuramylalanylglutamyldiaminopimelylalanine. The anhydro linkage is between C-1 and C-6 of the muramyl entity. Two additional substances accompanied the above compound. These were the hydrated form (i.e. in which the muramyl entity had a free reducing end, and a free hydroxyl on C-6), and an anhydro analogue lacking the terminal alanine. The Mr values were 939 and 850, respectively. Methyl esters were prepared, and these were also acetylated. The mass spectra of the methyl ester of Mr = 921 displayed an increase in Mr of 42 (i.e. 3 X 14), indicating the presence, originally, of three free carboxyls. Acetylation increased Mr by a further 168 units (i.e. 4 X 42), indicating 4 hydroxyl or amino groups. These data are consistent with the structure cited above for the main entity of FSu. Similar confirmatory results were obtained for the two minor constituents described above. These operations were worked out on natural muramyl peptides of known structure, obtained from other sources, and the data are given for comparison.     

Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods.

Escherichia coli outer membranes were prepared by centrifugation to equilibrium in sucrose gradients and then treated with Sarkosyl in the presence of ethylenediaminetetraacetate. The polypeptide profiles of the two outer membrane preparations were compared by two-dimensional polyacrylamide gel electrophoresis. The patterns obtained were not identical, and Sarkosyl removed several minor proteins from the outer membrane.     

Detection of transforming growth factor alpha in human urine and plasma

A sensitive enzyme-linked immunosorbent assay (ELISA) system for human transforming growth factor alpha (TGF alpha) was developed in combination with polyclonal and monoclonal antibodies. Employing this assay system, we detected TGF alpha like activity in normal human plasma as well as in cancer patients' urine and plasma. These TGF alpha were analyzed by chromatography, immunoreactivity, and EGF-TGF alpha receptor binding assay and found to be identical to authentic human TGF alpha. The presence of TGF alpha circulating in normal adult plasma suggests a new role of TGF alpha in the human body.     

Rotavirus spike structure and polypeptide composition.

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.     

Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm.

Microsomal fractions, glyoxysomes and mitochondria were isolated from homogenates of germinating castor-bean (Ricinus communis) endosperm by sucrose-density-gradient centrifugation. Washed membrane preparations from these cellular fractions were examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. At corresponding developmental stages the endoplasmic-reticulum and glyoxysomal membranes were strikingly similar in polypeptide composition, at least 16 polypeptides being present in membranes isolated from 3-day-old tissue. Supplying [35S]methionine to intact endosperm tissue resulted in the labelling of all membrane polypeptides, the specific radioactivity in the endoplasmic reticulum being greater than for equivalent polypeptides of the glyoxysomal membrane. Washing these membranes with sodium deoxycholate solution extensively solubilized protein components, with the exception of a predominant polypeptide of mol.wt. 55000. Mitochondrial membrane preparations differed from those of the endoplasmic reticulum and glyoxysomes in polypeptide molecular-weight distribution and the [35S]methionine-labelling pattern. The similarity in polypeptide composition between endoplasmic-reticulum and glyoxysomal membranes is discussed in relation to glyoxysome biogenesis.     

Studies on the mode of action of gonadotrophin-inhibiting material (anti-LH) isolated from human urine.

Gonadotrophin-inhibiting material (GIM) was able to inhibit the binding of 125I-hCG to rat Leydig cells, suggesting that its inhibiting properties are due to its ability to complete for the hCG binding sites on Leydig cells. Binding of fluorescein isothiocyanate-tagged GIM to Leydig cells was also seen. The effect of GIM on hCG-induced adenylate cyclase activation and testosterone production was also studied. It was found that there was a dose-dependent inhibition of both these effects.     

Phospholipid, cholesterol, polypeptide and glycoprotein composition of hepatic endosome subfractions.

The composition of hepatic endosome subfractions was compared directly with that of plasma membranes and Golgi-apparatus fractions. The neutral lipid and phospholipid composition of two endosome subfractions separated on Nycodenz gradients from a parent endosome fraction was similar to that of plasma membranes. The phospholipid/cholesterol ratios and the sphingomyelin contents were high, as in plasma membranes. However, the phosphatidylserine content was low. Endosomal subfractions contained a simpler polypeptide profile than did plasma membranes. However, a large number of glycoproteins were common to both fractions. Two endosome-specific glycoproteins, of Mr 59 000 and 38 000, were identified. Sialic acid was present at concentrations higher than in plasma membranes. The results indicate that endosomal membranes have a similar composition to plasma membranes that probably reflects their functional interaction during endocytosis and receptor recycling.     

Comparison of the taxonomy, serology, drug resistance transfer, and virulence of Citrobacter freundii strains from mammals and poikilothermic hosts.

In this study, the phenotypic, antigenic, and virulence characteristics of 32 Citrobacter freundii strains of fish, human, and veterinary origin were comparatively analyzed. In addition, the spread of drug resistance factors by conjugation was investigated. Regardless of the source of isolation, the strains exhibited variable reactions mainly for arginine dihydrolase, ornithine decarboxylase, and fermentation of sucrose, melibiose, amygdalin, and salicin. Total fatty acid methyl ester analysis by gas chromatography proved to be useful for an intratypic differentiation within the C. freundii strains studied. In fact, although all of the isolates exhibited similar fatty acid methyl ester profiles, significant differences in the major fatty acids 16:1 and 16:0 and in the 17:0 delta region were observed between the isolates from salmonids and the remaining strains. Serological studies using agglutination tests, analysis of lipopolysaccharides (LPS), and the corresponding immunoblots with 13 antisera indicated a great antigenic diversity among the strains. Common LPS patterns were shared only by some isolates showing high cross-agglutination titers. In contrast, although all strains exhibited very similar surface protein patterns, only two common outer membrane proteins of 54 and 58 kDa were immunologically related. Infectivity trials performed in mice and rainbow trout indicated that all of the C. freundii strains were not pathogenic for mice (50% lethal dose of > 5 x 10(7)). Although the isolates displayed a low degree of virulence for trout, inoculated strains were always recovered from the survivors in pure culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of polyribonucleotide dependent polypeptide synthesis on isolated ribosomes from two different species of mycobacteria

The characteristics of polyribonucleotide dependent polyphenylalanine synthesis in cell-free systems from slow-growingMycobacterium bovis (BCG) and rapid-growingMycobacterium smegmatis (M 607) are described. Differences between the two strains were found in optimal magnesium ion concentration, the effect of polyU amount and the time of incubation. Observed differences in biological characteristics of the two mycobacterial strains and on the incompleteness of cell-free systems are discussed.     

The polypeptide composition of axoplasm and of neurofilaments from the marine worm Myxicola infundibulum.

1. Axoplasm from Myxicola contains two major polypeptides associated with neurofilaments, together with actin, tubulin and many minor polypeptide components. 2. Some of the minor polypeptides with molecular weights between 140,000 and 50,000 purify with neurofilaments under a variety of conditions and they appear to represent an integral part of the filament structure. 3. Peptide fingerprinting shows that the two major neurofilament polypeptides are almost identical. The fingerprint patterns from these major polypeptides share features with those obtained from the minor components. 4. Peptide fingerprinting has enabled us to propose a scheme for the main sites at which papain cleaves the major neurofilament polypeptides. In addition fingerprinting indicates how the minor components are related to the major polypeptides. 5. It is suggested that many of the minor neurofilament polypeptides could arise by proteolysis in vivo.