Host--virus interactions in the control of T4 prereplicative transcription. I. tabC (rho) mutants.

In this paper we describe properties of old (Takahashi, 1978) and new tabC^ts and tabC^cs bacterial mutants. We find that under non-permissive conditions they differently inhibit the synthesis of specific T4 prereplicative gene products. Among such products, that we have been able to identify, are P43 and PrIIA. In contrast, P32 and PrIIB are not affected.Inhibition of P43 (T4 DNA polymerase) synthesis is sufficient to account for depressed DNA synthesis in tabC (Takahashi, 1978).In heterodiploids: (1) all tabC mutants are recessive; (2) all tabC mutants do not complement with each other; (3) at least one, tabC^ts-5521, becomes dominant at 42.6 °C if rho mutant ts15 (Tab⁺) (Das et al., 1976) is situated in trans; (4) tabC^ts-5521 also becomes dominant at 42.6 °C if tabC^cs-110 and tabC^cs-18 are situated in trans (42.6 °C is non-permissive for T4 development on tabC^cs-5521 and permissive for T4 development on tabC^cs mutants).We discuss the possibility that in tabC mutants rho protein is altered and insensitive to T4-specific anti-termination functions. We also discuss a model that accounts for the differential effect of tabC mutants on the synthesis of T4 prereplicative proteins.     

The effect of tunicamycin on development of the mammalian embryonic pancreas

The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [³H]mannose, [³H]glucosamine, and [³H]fucose incorporation by 95, 85, and 90%, respectively, while [³H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [³H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [³H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.     

Light and Phosphate Competition Between Cylindrospermopsis raciborskii and Microcystis aeruginosa is Strain Dependent

The hypothesis that outcomes of phosphorus and light competition between Cylindrospermopsis raciborskii and Microcystis aeruginosa are strain dependent was tested experimentally. Critical requirements of phosphorus (P*) and of light (I*) of two strains of each species were determined through monoculture experiments, which indicated a trade-off between species and also between Microcystis strains. Competition experiments between species were performed using the weakest predicted competitors (with the highest values of P* and of I*) and with the strongest predicted competitors (with the lowest values of P* and of I*). Under light limitation, competition between the weakest competitors led C. raciborskii to dominate. Between the strongest competitors, the opposite was observed, M. aeruginosa displaced C. raciborskii, but both strains co-existed in equilibrium. Under phosphate limitation, competition between the weakest competitors led C. raciborskii to exclude M. aeruginosa, and between the strongest competitors, the opposite was observed, M. aeruginosa displaced C. raciborskii, but the system did not reach an equilibrium and both strains were washed out. Hence, outcomes of the competition depended on the pair of competing strains and not only on species or on type of limitation. We concluded that existence of different trade-offs among strains and between species underlie our results showing that C. raciborskii can either dominate or be displaced by M. aeruginosa when exposed to different conditions of light or phosphate limitation.     

Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.     

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species

C₄ photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C₄ and non-C₄ populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C₄ lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C₄ phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C₄ physiology away from its region of origin.     

Exploring the outcome of genetic modifications of glycosylation in cultured cell lines by concurrent isolation of the major classes of vertebrate glycans

In the preceding article (Manzi,A.E., Norgard-Sumnicht,K., Argade,S., Marth,J.D., van Halbeek,H. and Varki.A. [2000] GLYCOBIOLOGY:, 10, 669-688), we reported a comprehensive approach for the extraction, fractionation, and isolation of all of the major classes of sugar chains (glycans) from vertebrate tissues. Here we apply this "Glycan Isolation Protocol" to a variety of cultured mammalian cell lines, including two wild-type Chinese hamster ovary (CHO) cell lines and some of their genetically modified variants that were predicted or known to have defined abnormalities in the biosynthesis of one or more classes of glycans. We also use this approach to characterize clone 489, a new derivative of the GAG-deficient CHO clone pgsA-745, in which sulfation has been restored by transfection of a wild-type CHO cDNA library. By metabolically labeling the cell lines with [6-(3)H]glucosamine we were able to monitor the recovery of all major classes of glycans. The results allow us to reach several conclusions: first, the protocol described in the preceding paper is further validated by finding good recovery of total radioactivity and appropriate distribution of label in the correct glycan classes in the fractions from a variety of cell lines; second, the amount of radioactivity recovered in free glycosylphosphatidylinositol (GPI) lipids is remarkably high when compared to that found in GPI anchors, with the former being the dominant form in some cells; third, cells with known genetic mutations in specific glycosylation pathways are shown to have the expected changes in the distribution of recovered radioactivity in the appropriate fractions; fourth, the N- and O- glycans recovered via the protocol are of adequate quality to demonstrate marked differences in their structural profiles and/or content; fifth, the protocol can pick up unexpected differences of glycan classes not predicted to be affected by the primary defect; finally, the reappearance of sulfation in the novel clone 489 is not due to restoration of GAG sulfation, but rather due to the new expression of sulfation in the fraction enriched in N- and O-linked glycopeptides. These results demonstrate the power of this comprehensive approach for the concurrent exploration and profiling of the different major classes of glycans in cells.     

Biocides formulation of essential oils having antimicrobial activity

Essential oils of fennel, peppermint, caraway, eucalyptus, geranium and lemon were tested for their antimicrobial activities against some plant pathogenic micro-organisms (Fusarium oxysporum, Alternaria alternate, Penicilium italicum Penicilium digitatum and Botyritus cinerea). Essential oils of fennel, peppermint, caraway were selected as an active ingredient for the formulation of biocides due to their efficiency in controlling the tested micro-organisms. Successful emulsifiable concentrates (biocides) were prepared from these oils using different emulsifiers (Emulgator B.L.M. Tween20 and Tween80) and different fixed oils (sesame, olive, cotton and soybean oils). Physico-chemical properties of the formulated biocide (spontaneous emulsification, emulsion stability test, cold stability and heat stability tests as well as viscosity, surface tension and pH) were measured. The prepared biocides were ready to be tested for application in a future work as a safe pesticide against different pathogens.     

Effect of whole body vibration training on lower limb performance in selected high-level ballet students

The aim of this study was to examine the effects of 8 weeks of whole body vibration (WBV) training on vertical jump ability (CMJ) and knee-extensor performance at selected external loads (50, 70, and 100 kg; leg-press exercise) in elite ballerinas. Twenty-two (age, 21.25 +/- 1.5 years) full-time ballerinas were assigned randomly to the experimental (E, n = 11) and control (C, n = 11) groups. The experimental group was submitted to WBV training 3 times per week before ballet practice. During the training period, the E and C groups undertook the same amount of ballet practice. Posttraining CMJ performance significantly increased in E group (6.3 +/- 3.8%, p < 0.001). Furthermore, E group showed significant (p < 0.05-0.001) posttraining average leg-press power and velocity improvements at all the external loads considered. Consequently, the force-velocity and power-velocity relationship shifted to the right after WBV training in the E group. The results of the present study show that WBV training is an effective short-term training methodology for inducing improvements in knee-extensor explosiveness in elite ballerinas.