Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors.

Reconstituted influenza virus envelopes were obtained following solubilization of intact virions with Triton X-100. Quantitative determination revealed that the hemolytic and fusogenic activities of the envelopes prepared by the present method were close or identical to those expressed by intact virions. Hemolysis as well as virus-membrane fusion occurred only at low pH values, while both activities were negligible at neutral pH values. Fusion of intact virions as well as reconstituted envelopes with erythrocyte membranes--and also with liposomes--was determined by the use of fluorescently labeled viral envelopes and fluorescence dequenching measurements. Fusion with liposomes did not require the presence of specific virus receptors, namely sialoglycolipids. Under hypotonic conditions, influenza virions or their reconstituted envelopes were able to fuse with erythrocyte membranes from which virus receptors had been removed by treatment with neuraminidase and pronase. Inactivated intact virions or reconstituted envelopes, namely, envelopes treated with hydroxylamine or glutaraldehyde or incubated at low pH or 85 degrees C, neither caused hemolysis nor possessed fusogenic activity. Fluorescence dequenching measurements showed that only fusion with liposomes composed of neutral phospholipids and containing cholesterol reflected the viral fusogenic activity needed for infection.     

Unusual sequence element found at the end of an amplicon.

In a polyomavirus-transformed rat cell line, designated LPT, the polyomavirus DNA is integrated into a single chromosomal site. Treatment of LPT cells with carcinogens induces amplification of the integrated virus DNA and flanking cellular sequences. We show that the amplification is arrested within a specific cell DNA segment that maps 1.3 to 1.85 kilobases beyond one virus-cell DNA junction, defined as the left junction. We also present the sequence of an 897-base-pair fragment spanning the arrest site. This fragment contains an unusual sequence element, which consists of two contiguous components, a potential cruciform with stems of 6 base pairs and a d(G-A)27 X d(T-C)27 tract, and maps 1,497 to 1,564 nucleotides beyond the left junction. The possibility that this unusual sequence plays a role in the arrest of the amplification process is discussed.     

Microbial production of L-[15N]glutamic acid and its gas chromatography-mass spectrometry analysis

L-[15N]Glutamic acid was prepared in high yields via a fermentative process. Brevibacterium lactofermentum, growing on a medium containing 97% enriched 15NH4Cl as a sole isotopic precursor, excreted mostly L-[15N]glutamic acid. The L-[15N]glutamic acid was purified and identified. Gas chromatography-mass spectrometry analysis was performed to demonstrate its usefulness in clinical studies.     

Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

Enzymatic hydrolysis of n-substituted met-tRNA(M) and met-tRNA(F)

By addition of 1-(14)C-sodium acedate to the growth medium of Nocardia asteroides, it can be shown that the lipid content increases during the exponential phase, but does not vary during the stationary phase of the growth. Nocardic acid biosynthesis from the medium molecular weight fatty acids occurs chiefly during te stationary phase. As these compounds are localised in the cell walls, it becomes evident that the lipid envelope of the walls is still increasing when the cell growth and division have stopped.     

Fusion-mediated microinjection of liposome-enclosed DNA into cultured cells with the aid of influenza virus glycoproteins

Influenza viruses were able to mediate fusion of DNA-loaded liposomes with living cultured cells such as monkey COS-7 cells. This was inferred from the appearance of CAT activity in recipient cells incubated with the combination of influenza viruses and liposomes loaded with the plasmid pSV2CAT. Influenza virions were found to be as efficient as intact Sendai virions in mediating microinjection of foreign DNA into living cells. Also, reconstituted envelopes bearing either influenza glycoproteins or the combination of Sendai and influenza glycoproteins were highly efficient in promoting fusion of loaded liposomes with recipient cells. Introduction of DNA into cultured cells required the presence of an active influenza fusion protein; namely, an active HA glycoprotein. Very little or no CAT activity was observed in cells incubated with loaded liposomes and unfusogenic influenza viruses. The virus-induced fusion event probably occurs within intracellular organelles such as endosomes following receptor-mediated endocytosis of virus-liposome complexes. This is due to the fact that the viral fusion glycoprotein is activated only at acidic pH values such as those which characterize the intraendosomal environment. Results of the present work demonstrate for the first time microinjection of foreign DNA via fusion with membranes of intracellular organelles. The potential of the present system to serve as a biological carrier for in vivo use is discussed.     

Purification of peptidyl-tRNA on benzoylated DEAE-cellulose columns.

The o-nitrophenylsulfenyl group, and amino protecting group in the chemical synthesis of peptidyl-tRNA was used to attach newly synthesized peptidyl-tRNA to a benzoylated DEAE-cellulose column. This facilitated the isolation of a highly purified specific tRNA with a well defined peptide chain.     

GeneCards 2002: towards a complete,object-oriented,human gene compendium

MOTIVATION: In the post-genomic era, functional analysis of genes requires a sophisticated interdisciplinary arsenal. Comprehensive resources are challenged to provide consistently improving, state-of-the-art tools. RESULTS: GeneCards (Rebhan et al., 1998) has made innovative strides: (a). regular updates and enhancements incorporating new genes enriched with sequences, genomic locations, cDNA assemblies, orthologies, medical information, 3D protein structures, gene expression, and focused SNP summaries; (b). restructured software using object-oriented Perl, migration to schema-driven XML, and (c). pilot studies, introducing methods to produce cards for novel and predicted genes.     

DNA ploidy as a prognostic factor in rhabdomyosarcoma. Analysis of 35 cases with image cytometry

OBJECTIVE: To correlate DNA ploidy in rhabdomyosarcoma (RMS) with other prognostic factors and patient survival and to search for possible reasons for inconsistent conclusions in similar, published studies. STUDY DESIGN: DNA content was measured in archival specimens obtained from 35 patients (23 children and 12 adults) with RMS. Cell suspensions were prepared by the modified Hedley technique, stained by the modified Feulgen-thionin method and analyzed by automated high-resolution image cytometry. DNA ploidy was assessed on the basis of DNA index values. We used the chi 2 test to correlate DNA ploidy with other prognostic factors, Kaplan-Meier procedure to estimate overall survival in terms of individual prognostic factors, log-rank test to calculate differences in survival between groups and Cox multivariate regression analysis to determine the independence of variables in relation to survival. RESULTS: A statistically significant correlation was found only between DNA ploidy and histologic subtype of RMS, patient sex and patient age. A hyperdiploid DNA pattern predominated among patients with embryonal RMS, and a tetraploid pattern dominated among patients with alveolar RMS. The highest 5-year survival rate was seen among patients with hyperdiploid RMS, followed by those with diploid, tetraploid and hypertetraploid RMS. Although DNA ploidy was a significant prognostic factor in univariate analysis, it did not retain its independent prognostic value in multivariate analysis, in which patient age, tumor size and histologic subtype were the only significant factors. We found 12 articles reporting on the association between DNA ploidy and survival of patients with RMS: 6 found a correlation, and 6 did not. The main reasons for the discrepancies seem to be the inclusion of chemotherapy-treated and nontreated patients, low number of patients and differences in grouping DNA histograms. CONCLUSION: The precise prognostic value of DNA ploidy in RMS remains equivocal. Larger, cooperative studies could give statistically more reliable results.