Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.     

Die Ausdifferenzierung des Vakuolensystems in Blüten- und Laubblattepidermen vonPrimula kewensis

Ohne Zusammenfassung     

Influence of serum albumin on fecundity and weight of the progeny of the tsetse fly Glossina palpalis palpalis

Females of a membrane-fed colony of G. p. palpalis (food source: fresh defibrinated bovine blood) were fed on derivatives of bovine blood, in which the red cell fraction remained unchanged while the serum fraction was replaced by an artificial solution. This solution consisted of a simple saline, isotonic to serum and enriched by bovine serum albumin with increasing concentrations. Physical parameters such as pH and osmolarity of all media were similar to controls. The absence of serum albumin in the food medium resulted in sterility of the females and caused high mortality. The presence of increased levels of serum albumin in the medium increased the fecundity until it was similar to that of flies fed on the standard diet of fresh defibrinated bovine blood. The offspring size was positively correlated with the concentration of the serum albumin in the mother's diet. However, a high concentration of albumin (>6%) resulted in increased adult mortality. Thus, the presence of optimum amounts of free serum albumin in bovine blood appears necessary for the development and production of viable larvae of the tsetse fly G. p. palpalis.

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Résumé Les femelles d'une population de G. p. palpalis élevées sur membrane (aliment: sang frais défibriné de bovin) ont été nourries de dérivés de sang bovin dont les globules rouges n'avaient pas été modifiés, mais dont le sérum avait été remplacé par une solution artificielle. Cette solution était simple, saline, isotonique au sérum et enrichie d'albumine de sérum bovin en concentrations croissantes. Les témoins ont montré l'identité des paramètres physiques, tels que le pH et l'osmolarité, pour toutes les solutions. L'absence d'albumine de sérum dans l'aliment donné aux femelles a provoqué leur stérilité et une mortalité élevée. L'augmentation des quantités d'albumine de sérum a accru la fécondité jusqu'à ce que celle-ci soit identique à celle des glossines avec un régime normal composé de sang frais défibriné de bovin. Il y avait une corrélation directe entre la taille des descendants et la concentration d'albumine de sérum dans l'alimentation de la mère. Toutefois, une forte concentration d'albumine (plus de 6%) a augmenté la mortalité imaginale. La présence de quantités optimales d'albumine de sérum libre dans le sang bovin semble donc nécessaire au développement et à la production de larves viables de la mouche tsé-tsé, G. p. palpalis.

     

Constraints on flowering phenology in a phryganic (East Mediterranean shrub) community

The phenological pattern of flowering at the community level was studied in a Greek phryganic ecosystem near Athens for 4 consecutive years. Flowering is strongly seasonal: 80% of the insect-pollinated flora, which consists of 133 species, blooms between February and June. There is a variably expressed secondary flowering period in autumn. The pollinating fauna follows a strongly correlated pattern of abundance. Two types of plants were distinguished: pauciflorous species bearing <10 flowers that are large compared to the plant body, and multiflorous species with many small flowers. Pauciflorous species flower in the winter half of the year, while multiflorous species flower mainly in the summer half. The mean flower life spans are 9 and 3 days, respectively. The duration of flowering (DF) for each species is 55 days on average, which is long compared to other communities. The DF shows year-to-year variations, concomitant with the vicissitudes of the climate. The start of flowering of a species is statistically correlated with the temperature in the previous month, not with rainfall; its end date of flowering only partly compensates for the time gained or lost. DF is maximal in winter. The average flower life span of species flowering at any given date varies strongly and independently of the average DF. We tested the hypothesis that flowering phenology is set by phylogenetic and life form constraints. This could not be corroborated for phylogeny, evidently because of the overriding influence of the mediterranean climate, and probably also for biogeographical reasons. In contrast, life forms and multiflorous and pauciflorous species show strong differences. Many (51) of the species are therophytes; we tested the hypothesis that because of their annual habit they would be more dependent on pollination than perennials. Thus we anticipated that therophytic species would be differentiated from perennials in their flowering phenologies. This is not corroborated. We therefore conclude that the seed bank plays a role that is analogous to that of a perennial plant body.     

An IGF-I promoter polymorphism modifies the relationships between birth weight and risk factors for cardiovascular disease and diabetes at age 36

BACKGROUND: Biochemical testing for pheochromocytoma by measurement of fractionated plasma metanephrines is limited by false positive rates of up to 18% in people without known genetic predisposition to the disease. The plasma normetanephrine fraction is responsible for most false positives and plasma normetanephrine increases with age. The objective of this study was to determine if we could improve the specificity of fractionated plasma measurements, by statistically adjusting for age. METHODS: An age-adjusted metanephrine score was derived using logistic regression from 343 subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as part of investigations for suspected pheochromocytoma at Mayo Clinic Rochester (derivation set). The performance of the age-adjusted score was validated in a dataset of 158 subjects (including patients 23 with pheochromocytoma) that underwent measurements of fractionated plasma metanephrines at Mayo Clinic the following year (validation dataset). None of the participants in the validation dataset had known genetic predisposition to pheochromocytoma. RESULTS: The sensitivity of the age-adjusted metanephrine score was the same as that of traditional interpretation of fractionated plasma metanephrine measurements, yielding a sensitivity of 100% (23/23, 95% confidence interval [CI] 85.7%, 100%). However, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3% (22/135, 95% CI, 11.0%, 23.4%) and that of the age-adjusted score was significantly lower at 3.0% (4/135, 95% CI, 1.2%, 7.4%) (p < 0.001 using McNemar's test). CONCLUSION: An adjustment for age in the interpretation of results of fractionated plasma metanephrines may significantly decrease false positives when using this test to exclude sporadic pheochromocytoma. Such improvements in false positive rate may result in savings of expenditures related to confirmatory imaging.     

EFFECTS OF UV-B-INDUCED DNA DAMAGE AND PHOTOINHIBITION ON GROWTH OF TEMPERATE MARINE RED MACROPHYTES: HABITAT-RELATED DIFFERENCES IN UV-B TOLERANCE

The sensitivity to UV-B radiation (UVBR: 280–315 nm) was tested for littoral (Palmaria palmata[L.] O. Kuntze, Chondrus crispus Stackhouse) and sublittoral (Phyllophora pseudoceranoides S. G. Gmelin, Rhodymenia pseudopalmata[Lamouroux] Silva, Phycodrys rubens[L.] Batt, Polyneura hilliae[Greville] Kylin) red macrophytes from Brittany, France. Algal fragments were subjected to daily repeated exposures of artificial UVBR that were realistic for springtime solar UVBR at the water surface in Brittany. Growth, DNA damage, photoinhibition, and UV-absorbing compounds were monitored during 2 weeks of PAR + UV-A radiation (UVAR) + UVBR, whereas PAR + UVAR and PAR treatments were used as controls. The littoral species showed a higher UV tolerance than the sublittoral species. After 2 weeks, growth of P. palmata and C. crispus was not significantly affected by UVBR, and DNA damage, measured as the number of cyclobutane-pyrimidine dimers per 10⁶ nucleotides, was negligible. Photoinhibition, determined as the decline in optimal quantum yield, was low and decreased during the course of the experiment, coinciding with the production of UV-absorbing compounds in these species. In contrast, no UV-absorbing compounds were induced in the sublittoral species. Growth rates of P. pseudoceranoides and R. pseudopalmata were reduced by 40% compared with the PAR treatment. Additionally, constant levels of DNA damage and pronounced photoinhibition were observed after the UVBR treatments. Growth was completely halted for Phycodrys rubens and Polyneura hilliae, whereas DNA damage accumulated in the course of the experiment. Because Phycodrys rubens and Polyneura hilliae showed the same degree of photoinhibition as the other sublittoral species, it appears that the accumulation of DNA damage may have been responsible for the complete inhibition of growth. The results suggest an important role of DNA repair pathways in determining the UV sensitivity in red macrophytes.     

Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability

Thermus thermophilus proline dehydrogenase ( TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N‐terminal helices: αA, αB, and αC. Using maltose‐binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP‐TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion‐pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion‐pairs improve the thermostability of TtProDH and a trade‐off is made between thermostability and catalytic activity.     

Increased exopolysaccharide production in Lactococcus lactis due to increased levels of expression of the NIZO B40 eps gene cluster

Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products. This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels. A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level. It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels. However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.     

Why do proteins use selenocysteine instead of cysteine?

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.     

Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.