DNA sequence of the coding region of the HLA-B44 gene

An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.     

Effect of cell recycle on continuous butanol-acetone fermentation with Clostridium acetobutylicum under phosphate limitation

Summary An overflow filtration unit for cell recycle with Clostridium acetobutylicum was developed. A cellulose-triacetate ultrafiltration membrane with a cut-off volume of 20 000 MW was found to work best. C. acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH value of 4.4 with cell recycle, the cell dry weight in the culture vessel reached 13.1 g/l at a dilution rate of D=0.10 h^-1 and 37°C. 377 mM of glucose were fermented to 190 mM butanol, 116.2 mM acetone and 25.8 mM ethanol. Total acids were 47.6 mM. The butanol productivity was 1.41 g/l/h. At a dilution rate of 0.40 h^-1 the butanol productivity was increased to 4.1 g/l/h but glucose consumption was decreased to 285 mM and butanol, acetone and ethanol production to 138.2, 97.5, 16.5 mM, respectively.     

Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C

Ten strains representing a novel genus of marine thermophilic archaebacteria growing at between 70 and 103°C with an optimal growth temperature of 100°C and a doubling time of only 37 min were isolated from geothermally heated marine sediments at the beach of Porto di Levante, Vulcano, Italy. The organisms are spherical-shaped, 0.8 to 2.5 m in width and exhibit monopolar polytrichous flagellation. They are strictly anaerobic heterotrophs, growing on starch, maltose, peptone and complex organic substrates. Only CO₂ and H₂ could be detected as metabolic products, the latter being inhibitory to growth at high concentrations. Hydrogen inhibition can be prevented by the addition of S^o, whereupon H₂S is formed in addition, most likely as the result of a detoxification reaction. The GC-content of the DNA of isolate Vc 1 is 38 mol%. The new genus is named Pyrococcus, the fireball. Type species and strain is Pyrococcus furiosus Vc 1 (DSM 3638).     

Formation of plastocyanin and cytochrome c-553 in different species of blue-green algae

Fifteen species from different genera of blue-green algae have been examined for their formation of plastocyanin (PC) and cytochrome c-553 (cyt c-553) in high or low Cu media. In addition to species which contain only cyt c-553 and those which completely exchange their cyt c-553 by PC, a new regulatory type was detected in which this exchange was incomplete. By comparing different species, it could be shown that either this incomplete exchange of cyt c-553 by PC as well as lack of PC in some other blue-green algae is not caused by restricted Cu uptake but is due to different biosynthetic and regulatory properties. Occurrence of PC and cyt c-553 cannot be used as a taxonomic criterium to classify blue-green algae. However, formation of either one or both of these redox components fits well into a line of evolution of the photosynthetic apparatus from the blue-green algae via green algae to higher plants.Abbreviations PC plastocyanin; cyt c-553, cytochrome c-553     

Distribution and abundance of the planktic foraminifer Neogloboquadrina pachyderma in sea ice of the Weddell Sea (Antarctica)

Summary Sea ice cores were obtained from eleven fast ice stations and one floe in the Weddell Sea, Antarctica in January–February 1985. All cores from the north eastern part of the Weddell Sea contained numerous living and dead planktic foraminifers of the species Neogloboquadrina pachyderma (Ehrenberg), while cores drilled in southern parts were barren of foraminifers with one exception. Foraminiferal abundances were variable, with numbers up to 320 individuals per liter melted sea ice. Distribution of foraminifers appears to be patchy, parallel cores taken less than 30 cm apart contained numbers which varied considerably. On the other hand, three cores taken on a transect each more than 3 km apart showed striking similarities. In general, small dead tests were found in the upper parts of the sea ice cores while large living individuals mainly occurred in lower sections. Abundant diatoms probably serve as a food source for the foraminifers. Correlation of foraminiferal abundance with salinity, chlorophyll and nutrient profiles are inconsistent. The possible mechanism of incorporation of N. pachyderma into the ice is discussed.     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

A rapid cell membrane permeability test using flourescent dyes and flow cytometry

A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA dimethyl sulfate - DMSO dimethyl sulfoxide - EB ethidium bromide - F fluorescein - FDA fluorescein diacetate - FS₂₅ concentration of test substance resulting in a F-peak left-shift of 25% from control - PBS phosphate buffered saline - SCT forward light scatter - SDS sodium dodecyl sulfate     

Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza virus A/PR/834-mediated hemolysis.

Exposure of influenza virus to an acidic environment, which is known to be required for viral fusion and hemolysis, has recently been shown to induce a conformational change in the hemagglutinin molecule. In the present study, we examined the effects of acid incubation on the antigenicity, biological activity, and morphology of influenza virus A/PR/8/34 (H1N1). Incubation of PR8 virus at pH 5 in the absence of erythrocytes resulted in a rapid and irreversible loss of viral hemolytic activity and infectivity. Apart from a less distinct appearance of the viral surface projections and slight damage to the envelope structure, acid incubation did not result in gross morphological changes in the viral architecture. The acid-induced change could be detected in the form of greatly increased or decreased binding of many monoclonal antibodies directed to each of the four major antigenic regions of the hemagglutinin. Triggering of viral hemolytic activity and antigenic alterations was similarly pH dependent. In addition, the different pH dependencies of egg-grown and trypsin-treated MDCK-grown viruses coincided with an analogous pH dependence of the antigenic alterations that were observed with these viruses. These observations are compatible with the idea that some of the anti-hemagglutinin antibodies detect conformational changes in the hemagglutinin which are required for the initiation of fusion and hemolysis.     

Processing of yeast mitochondrial RNA: involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion

Revertants have been obtained from six mutants of the box9 cluster, which are supposed to be defective in RNA splicing as a result of alterations in a splice signal sequence. This sequence is in the 5' part of intron 4 of the cob gene, 330 to 340 bp downstream from the 5' splice site. Sequencing reveals that reversion to splicing competence is achieved by restoration of the wild-type box9 sequence; by creation of novel box9 sequences; and by introduction of a second site or suppressor mutation (sup-) compensating for the effect of the primary box9- mutation. The sup- mutation alters a sequence in intron 4,293 bp upstream from the box9- primary mutation. The box9 sequence and this upstream sequence can base pair to form an intramolecular hybrid in intron RNA in which box9- and sup- are compensatory base pair exchanges (G----A and C----U, respectively). Thus intramolecular hybrid structures of intron RNA are essential for RNA splicing.