A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.     

The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

The greenhouse effect: the impacts of carbon dioxide (CO2), ultraviolet-B (UV-B) radiation and ozone (O3) on vegetation (crops)

Man's influence on the greenhouse effect, the heating of the atmosphere due to increasing concentrations of tropospheric trace gases, is of much international concern. Among the climatic variables, elevated levels of carbon dioxide (CO₂), ultraviolet-B (UV-B) radiation and ozone (O₃) are known to have a direct effect on vegetation. Our current knowledge of these effects is mainly based on studies involving single stress mode. Thus, the joint effects of CO₂, UV-B and O₃ on vegetation are poorly understood. Nevertheless, based on the literature analysis of plant response to individual stress factors, it can be concluded that sorghum, pea, bean, potato, oat, lettuce, cucumber, rice and tomato are among the crop species potentially sensitive to the joint effects of the aforementioned three variables. Similar information for tree species is essentially lacking.At least with some climatic variables such as O₃, present modeling efforts of cause-effect relationships have proven to be controversial. While at a regional geographic scale ambient CO₂ concentrations appear to be relatively homogeneous, ambient concentrations of O₃ exhibit significant temporal and spatial variability. Because of the protective action of O₃ against UV-B, similar but inverse temporal and spatial variability is expected in the surface levels of UV-B. Thus, future experimental designs should consider these exposure dynamics and modeling cuase-effect relationships should be directed to stochastic processes.     

Lifelong exercise and stroke.

OBJECTIVES--To examine the potential of lifelong patterns of increased physical activity to prevent stroke. DESIGN--Case-control study. SETTING--11 general practices in west Birmingham. SUBJECTS--125 men and women who had just had their first stroke and were aged 35-74 and 198 controls frequency matched for age and sex recruited over 24 months during 1988-90. Exclusion criteria were a previous history of stroke, mitral valvular heart disease combined with atrial fibrillation, primary or metastatic cerebral neoplasm, or coagulation disorder or myeloproliferative disease. MAIN OUTCOME MEASURES--Odds ratios for stroke related to lifetime history of exercise after 15 years of age. RESULTS--A history of vigorous exercise during the ages 15-25 appeared to protect from stroke: odds ratio adjusted for age and sex 0.33 (95% confidence interval 0.2 to 0.6). This effect was independent of other potential risk factors. Increasing years of participation in vigorous exercise between the ages of 15 and 55 produced an increasing protection from stroke (p < 0.001). In the 65 cases and 169 controls who were free of cardiac ischaemia, peripheral vascular disease, and poor health recent vigorous exercise and walking were protective against stroke: odds ratios of 0.41 (0.2 to 1.0) for recent vigorous exercise and 0.30 (0.1 to 0.7) for recent walking. CONCLUSIONS--Appreciable protection from stroke in later life is conferred by vigorous exercise in early adulthood. This increased level of physical activity should, if possible, be continued lifelong.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.