Distribution of legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates

A hospital warm water system was monitored for the presence and distribution of legionellae. Subtyping of ten selected Legionella pneumophila isolates, originating from four different sites in the system by using serogroup specific antisera in an indirect immunofluorescence test, revealed that nine of the ten isolates belong to serogroup 6, while the remaining one was serogroup 10. Two monoclonal antibodies (mAbs) specific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reacted with these mAbs. Genome analysis by elaborating NotI profiles using the pulsed field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates derived from different sites, including a new building connected by a ring pipe, were identical according to restriction fragment patterns. The patterns were distinguishable from those of the two L. pneumophila serogroup 6 reference strains, and from that of the L. pneumophila serogroup 10 isolate. These data argue for a relatively homogeneous L. pneumophila serogroup 6 population in the entire water system.     

Generality in multispecies responses to ocean acidification revealed through multiple hypothesis testing

Decades of research have demonstrated that many calcifying species are negatively affected by ocean acidification, a major anthropogenic threat in marine ecosystems. However, even closely related species may exhibit different responses to ocean acidification and less is known about the drivers that shape such variation in different species. Here, we examine the drivers of physiological performance under ocean acidification in a group of five species of turf‐forming coralline algae. Specifically, quantitating the relative weight of evidence for each of ten hypotheses, we show that variation in coralline calcification and photosynthesis was best explained by allometric traits. Across ocean acidification conditions, larger individuals (measured as noncalcified mass) had higher net calcification and photosynthesis rates. Importantly, our approach was able to not only identify the aspect of size that drove the performance of coralline algae, but also determined that responses to ocean acidification were not dependent on species identity, evolutionary relatedness, habitat, shape, or structural composition. In fact, we found that failure to test multiple, alternative hypotheses would underestimate the generality of physiological performances, leading to the conclusion that each species had different baseline performance under ocean acidification. Testing among alternative hypotheses is an essential step toward determining the generalizability of experiments across taxa and identifying common drivers of species responses to global change.     

Improved process conditions for increasing expression of MHC class II protein from a stable <Emphasis Type="Italic">Drosophila</Emphasis> S2 cell line

Objectives

To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line.

Results

When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR1_2xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested.

Conclusions

Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

     

Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans , and its use as a reporter of gene regulation

Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicansACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the non-canonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans. Received: 27 June 1997 / Accepted: 26 September 1997     

Transfer ribonucleic acid methylases of bone. Studies on vitamin A and D deficiency

Methods were devised for the assay of tRNA methylases of rat bone. The activities of bone tRNA methylases are similar to those from other mammalian tissues. However, unlike reports on liver methylases, no inhibitors were found in the supernatant fraction from pH5 precipitate of bone extracts. The effects of vitamins A and D on the methylation of tRNA by cell-free extracts of rat bone were studied. Deficiency of either vitamin resulted in a decrease in the rate and extent of tRNA methylation, whereas the administration of vitamin A to hypovitaminotic-A rats and vitamin D to hypovitaminotic-D rats increased the rate and extent of tRNA methylation. These effects appear to be apart from changes in ribonuclease activity or in concentrations of calcium or magnesium. No evidence of inhibitors of tRNA methylases was found in bone extracts from vitamin-deficient rats nor of activators in bone extracts from deficient rats given vitamin A or D. The pattern of tRNA methylation under conditions of vitamin A or D deficiency was not changed, suggesting a generalized cellular deficiency. It was of significance to find that the specificity for methylation of specific bases in tRNA was different after the administration of vitamin A as contrasted with the effects of vitamin D. The possible significance of tRNA methylation to the biochemical action of the vitamins on bone is discussed.     

DAip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility.

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes.We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions.Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.     

Response of the elm leaf beetle to host plants induced by oviposition and feeding: the infestation rate matters

We investigated by olfactometry and feeding‐ and oviposition‐choice‐tests how the highly specialised elm leaf beetle, Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae), responds to conspecifically induced defences in the field elm Ulmus minor Miller (Ulmaceae). While egg deposition of the beetle induced elms to release volatiles attractive to the egg parasitoid Oomyzus gallerucae Fonscolombe (Hymenoptera: Eulophidae), feeding alone did not. In the present study, females of the elm leaf beetle showed preferences for the odours of twigs induced by low egg deposition and feeding over odours from uninfested twigs. In contrast, heavy infestation rendered elm odours less attractive to the beetles. Feeding and oviposition bioassays revealed an oviposition preference for leaves from uninfested twigs when compared to locally infested leaves. However, beetles preferred to feed upon systemically induced leaves compared to uninfested ones. The different preferences of the elm leaf beetle during host plant approach might be explained by a strategy that accounts for both gaining access to high quality nutrition and avoiding competition or parasitism.