Preparation and structural characterization of large heparin-derived oligosaccharides

Porcine mucosal heparin was partially depolymerized with heparinlyase I and then fractionated into low-molecularweight (<5000)and high-molecular-weight (>5000) oligosaccharides by pressurefiltration. The high-molecular-weight oligosaccharide mixture({small tilde}50 wt% of the starting heparin) also containedintact heparin. This intact polymer complicates oligosacsharidepurification. Thus, the low-molecular-weight fraction was usedto prepare homogeneous oligosaccharides for structural characterization.The low-molecular-weight oligosaccharide mixture was first fractionatedby low pressure gel permeation chromatography into size-uniformmixtures of disaccharides, tetrasaccharides, hexasaccharides,octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharidesand higher oligosaccharides. Each size-fractionated mixturewas then purified on the basis of charge by repetitive semi-preparativestrong-anion-exchange high-performance liquid chromatography.This approach has led to the isolation of 14 homogeneous oligosaccharidesfrom disaccharide to tetradecasaccharide. The purity of theseheparin-derived oligosaccharides was determined by gradientpolyacrylamide gel electrophoresis, analytical strong-anion-exchangehigh-performance liquid chromatography, capillary electrophoresisand one-dimensional nuclear resonance spectroscopy. The structureof these oligosaccharides was established using 600 MHz two-dimensionalnuclear resonance spectroscopy . The spectral methods used includedhomonuclear correlation spectroscopy, nuclear Overhauser effectspectroscopy and heteronuclear multiple quantum coherence spech-clscopy.The ¹H/¹H connectivities of the protons of each sugar residuein an oligosaccharide were established by two-dimensional homonuclearcorrelation spectroscopy, while ¹H/¹³C assignments were madeusing ¹H inverse detection. One- and two-dimensional nuclearresonance spectroscopic analysis of these heparin oligosaccharidesshowed two closely related groups of heparin-oligosaccharidesare afforded by enzymatic depolymerization of heparin. One groupis fully sulphated, having the structures     

Characteristics of synthesized RNA in the isolated and perfused rat liver

The incorporation of 3H-orotic acid into nuclear and microsomal RNA from isolated perfused rat liver has been studied. The specific radioactivity of nuclear RNA indicates that the efficiency for RNA synthesis in the perfused liver is similar to that of the liver 'in vivo'. In contrast, the microsomal RNA specific radioactivity is well below that observed 'in vivo'. This may indicate a slower transport of the labelled RNA from the nucleus to the cytoplasm. Labelling pattern of total nuclear RNA, nuclear poly(A) containing RNA and microsomal RNA appear to be in line with these assumptions.     

Water relation parameters of the CAM plant Kalanchoë daigremontiana in relation to diurnal malate oscillations

Summary In the CAM plant Kalanchoë daigremontiana, kept in an environmental rhythm of 12 h L: 12 h D in a growth chamber at 60% relative humidity and well watered in the root medium, decreasing water potentials and osmotic potentials of the leaves are correlated with malate accumulation in the dark. In the light increasing water and osmotic potentials ( _W and _S ) are associated with decreasing malate levels. Transpiratory H₂O loss is high in dark and low in light.In continuous light, the CAM rhythm rapidly disappears in the form of a highly damped endogenous oscillation. Malate levels, and water and osmotic potentials of the leaves remain correlated as described above. However, transpiration is very high as malate levels decrease and water and osmotic potentials increase.It can concluded, that water relation parameters like total water potential ( _W ) and osmotic potential ( _S ) change in close correlation with changes of malic acid levels. As an important osmotically active solute in CAM plants, malic acid appears to affect water relations independently of and in addition to transpiration. The question remains open, whether turgor ( _P ) is involved in CAM regulation in intact plants in a similar way as it determines malate fluxes in leaf slices.Abbreviations CAM Crassulacean Acid Metabolism - L Light - D Dark     

Infectious primate type C virus proviral DNA in productively infected cells.

Cell lines competent to infection by DNA from cultures chronically infected by type C viruses of the simian sarcoma virus and baboon endogenous virus groups were identified. Significant differences were observed in the relative susceptibility of some cell lines to infection by a given proviral DNA. Practical applications of transfection techniques for the separation of viruses from dually infected cultures and to free virus stocks from mycoplasmal contamination are described.     

Characterization of Rauscher murine leukemia virus envelope glycoprotein receptor in membranes from murine fibroblasts.

Plasma membrane preparations from KA31 (mouse) cells contained receptors for the binding of Rauscher murine leukemia virus (R-MuLV) envelope glycoprotein, gp70. This binding was demonstrated by gel filtration of a mixture of the microsomal fraction of the cells and 125I-labeled gp70. A rapid and convenient assay was developed to measure the complex formation between the membrane receptors and gp70 involving specific precipitation of the complex by 3 to 4% polyethylene glycol. The complex formation was responsive to the concentrations of both the receptor and gp70 and also to changes in temperature and pH. The gp70 binding was a noncooperative, saturable process, and an association constant of 3.5 X 10(8) M-1 was estimated from the binding data. The complex formation was reversible and a near-total exchange of 125I-labeled gp70 in the complex was achieved by incubation with excess of unlabeled gp70. The complex formation was inhibited by protein denaturing agents, guanidine-hydrochloride and urea. Pretreatment of the membrane fractions with either chymotrypsin or phospholipase C led to a loss of the membrane-associated receptor activity, indicating that a lipoprotein structure was important for the receptor function, consistent with the observation that nonionic detergents strongly inhibited the complex formation.     

Hasel-Pollengehalt der Luft in Mitteleuropa

Jahreszeitliche und tageszeitliche Veränderungen des Haselpollengehalts der Luft nach Untersuchungen aus den Jahren 1967 bis 1972 an elf mitteleuropäischen Meßstellen. Vergleich mit einigen anderen europäischen Daten über den Haselpollengehalt der Luft. In Mitteleuropa kamen die meisten Haselpollen zu Beginn oder gegen Ende des Monats März vor. Stündliche Höchstwerte wurden in den Tag- und Abendstunden gemessen. Morgens kamen keine hohen Stundenwerte vor. Temperaturmessungen lassen vermuten, daß für hohe Haselpollengehalte der Luft in Norddeutschland höhere Temperaturen nötig sind als in südwestdeutschen Gebieten.     

Cold hardiness of the green gemmules of Spongilla lacustris L. (Porifera: Spongillidae)

Most green gemmules of Spongilla lacustris survived enclosure in ice at –20 °C for up to 30 days; however, their rate of germination at 20 °C was less rapid than that of control gemmules. The length of time spent at low temperature had little effect on gemmule survival. In contrast, repeated cooling to –20 °C and warming to 4 °C led to a progressive decline in gemmule viability. These results indicate that cold injury occurs primarily during transitions between high and low temperatures.     

Human immunodeficiency virus type 1 envelope gene structure and diversity in vivo and after cocultivation in vitro.

Nested-primer polymerase chain reaction (PCR) has been applied to the molecular cloning of 4.6-kb half-genome fragments of human immunodeficiency virus type 1 (HIV-1) taken directly from the peripheral blood mononuclear cells (PBMC) of an individual with neurological symptoms of HIV-1 infection. In a similar manner, gp120-coding portions of the envelope gene were cloned after PBMC from the same blood sample were cocultivated with uninfected PBMC for 28 days. The complete 1.6-kb nucleotide sequence of the gp120 gene was determined from each of 35 clones examined. Two of 13 (15%) PBMC-derived gp120 genes and 3 of 22 (14%) coculture-derived gp120 genes were defective as a result of frameshifts and an in-frame stop codon(s). Mean diversity between individual gp120-coding sequences in PBMC was fivefold greater (3.24%) than after coculture (0.65%). A predominant sequence of "strain" was found after coculture that was distinct from the diverse viral genotypes detected in vivo and therefore was selectively amplified during in vitro propagation. Multiple distinct third variable (V3) regions encoding the principal neutralizing domain of the envelope protein were detected in PBMC-derived genes, suggesting the presence of immunologic diversity of HIV env genes in vivo not reflected in the cocultured virus sample. The large size of the HIV fragments generated in this study will permit analysis of the diversity of immunologic reactivity, gene function, and pathogenicity of HIV genomes present within infected individuals, including the functional significance of the loss of diversity that occurs upon coculture.     

Effects of cytokines from activated immune cells on vascular cell growth and HIV-1 gene expression. Implications for AIDS-Kaposi's sarcoma pathogenesis.

Kaposi's sarcoma (KS) arises more frequently in homosexual and bisexual men than in other groups of HIV-1 infected individuals. Clinico-epidemiologic data indicate that homosexuals often are infected with multiple microbial agents and/or subjected to other antigenic stimuli, preceding or accompanying HIV-1 infection. Signs of immune activation, in fact, frequently have been detected in these individuals, and the onset of KS can precede any sign of immunodeficiency. These data have suggested that products from activated immune cells may affect the development of AIDS-KS. Here we report that conditioned media from activated or dysregulated T cells contain a variety of cytokines that promote the growth of spindle cells derived from KS lesions of AIDS patients (AIDS-KS cells) and induce normal vascular cells, potential cell progenitors of the AIDS-KS cells, to acquire features of the KS cell phenotype ("spindle" cell morphology and growth responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein). The same conditioned media or cytokines promote HIV-1 gene expression and rescue defective HIV-1 proviruses, interrupting HIV-1 latency and increasing Tat production. The cellular and viral effects of cytokines are increased in an additive or synergistic manner by picomolar concentrations of extracellular Tat. These data suggest that cytokines produced by activated immune cells cooperate with HIV-1 infection in AIDS-KS pathogenesis.