Synthesis of HPr(Ser-P)(His-P) by enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius

HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue greatly inhibits the second phosphorylation. However, streptococci contain significant amounts of HPr(Ser-P)(His approximately P) during exponential growth, and recent studies suggest that phosphorylation of HPr(Ser-P) by EI is involved in the recycling of HPr(Ser-P)(His approximately P). We report in this paper a study on the phosphorylation of Streptococcus salivarius HPr, HPr(Ser-P), and HPr(S46D) by EI. Our results indicate that (i) the specificity constant (k(cat)/K(m)) of EI for HPr(Ser-P) at pH 7.9 was approximately 5000-fold smaller than that observed for HPr, (ii) no metabolic intermediates were able to stimulate HPr(Ser-P) phosphorylation, (iii) the rate of HPr phosphorylation decreased at pHs below 6.5, while that of HPr(Ser-P) increased and was almost 10-fold higher at pH 6.1 than at pH 7.9, (iv) HPr(S46D), a mutated HPr alleged to mimic HPr(Ser-P), was also phosphorylated more efficiently under acidic conditions, and, lastly, (v) phosphorylation of Bacillus subtilis HPr(Ser-P) by B. subtilis EI was also stimulated at acidic pH. Our results suggest that the high levels of HPr(Ser-P)(His approximately P) in streptococci result from the combination of two factors, a high physiological concentration of HPr(Ser-P) and stimulation of HPr(Ser-P) phosphorylation by EI at acidic pH, an intracellular condition that occurs in response to the acidification of the external medium during growth of the culture.     

The Src homology 2-containing inositol 5-phosphatase 1 (SHIP1) is involved in CD32a signaling in human neutrophils

Phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P(3)) plays important signaling roles in immune cells, particularly in the control of activating pathways and of survival. It is formed by a family of phosphatidylinositol 3'-kinases (PI3Ks) which phosphorylate PtdIns(4,5)P(2) in vivo. In human neutrophils, the levels of PtdIns(3,4,5)P(3) increase rapidly at the leading edge of locomoting cells and at the base of the phagocytic cup during FcgammaR-mediated particle ingestion. Even though these, and other, data indicate that PtdIns(3,4,5)P(3) is involved in the control of chemotaxis and phagocytosis in human neutrophils, the mechanisms that regulate its levels have yet to be fully elucidated in these cells. We evaluated the potential implication of SHIP1 and PTEN, two lipid phosphatases that utilize PtdIns(3,4,5)P(3) as substrate, in the signaling pathways called upon in response to CD32a cross-linking. We observed that the cross-linking of CD32a resulted in a transient accumulation of PtdIns(3,4,5)P(3). CD32a cross-linking also induced the tyrosine phosphorylation of SHIP1, its translocation to the plasma membrane and its co-immunoprecipitation with CD32a. CD32a cross-linking had no effect on the level of serine/threonine phosphorylation of PTEN and did not stimulate its translocation to the plasma membrane. PP2, a Src kinase inhibitor, inhibited the tyrosine phosphorylation of SHIP1 as well as its translocation to the plasma membrane. Wortmannin, a PI3K inhibitor, had no effect on either of these two indices of activation of SHIP1. Our results indicate that SHIP1 is involved, in a Src kinase-dependent manner, in the early signaling events observed upon the cross-linking of CD32a in human neutrophils.     

A SELDI‐TOF MS procedure for the detection,quantitation, and preliminary characterization of low‐molecular‐weight recombinant proteins expressed in transgenic plants

We describe a SELDI‐TOF MS procedure for the rapid detection and quantitation of low‐molecular‐weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens‐mediated transformation, and then used as test material for the analyses. Real‐time RT‐PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI‐TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.     

TGFβ2-mediated production of hyaluronan is important for the induction of epicardial cell differentiation and invasion

In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFβ2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFβ2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFβ2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFβ2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFβ2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFβ2. Taken together, these findings demonstrate that TGFβ2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFβ2 and HA signals are integrated to regulate changes in epicardial cell behavior.     

Low but structured chloroplast diversity in Atherosperma moschatum (Atherospermataceae) suggests bottlenecks in response to the Pleistocene glacials

Background and Aims

The cool temperate rainforests of Australia were much reduced in range during the cold and dry glacial periods, although genetic evidence indicates that two key rainforest species, Nothofagus cunninghamii and Tasmannia lanceolata, survived within multiple locations and underwent only local range expansions at the end of the Last Glacial. To better understand the glacial response of a co-occurring but wind-dispersed and less cold-tolerant rainforest tree species, Atherosperma moschatum, a chloroplast phylogeographic study was undertaken.

Methods

A total of 3294 bp of chloroplast DNA sequence was obtained for 155 samples collected from across the species'' range.

Key Results

The distribution of six haplotypes observed in A. moschatum was geographically structured with an inferred ancestral haplotype restricted to Tasmania, while three non-overlapping and endemic haplotypes were found on the mainland of south-eastern Australia. Last glacial refugia for A. moschatum are likely to have occurred in at least one location in western Tasmania and in Victoria and within at least two locations in the Great Dividing Range of New South Wales. Nucleotide diversity of A. moschatum was lower (π = 0·00021) than either N. cunninghamii (0·00101) or T. lanceolata (0·00073), and was amongst the lowest recorded for any tree species.

Conclusions

This study provides evidence for past bottlenecks having impacted the chloroplast diversity of A. moschatum as a result of the species narrower climatic niche during glacials. This hypothesis is supported by the star-like haplotype network and similar estimated rates of chloroplast DNA substitution for A. moschatum and the two more cold tolerant and co-occurring species that have higher chloroplast diversity, N. cunninghamii and T. lanceolata.     

Considerations in the development of live biotherapeutic products for clinical use

Food products in the United States (U.S.), including dietary supplements, may contain live microorganisms and can be promoted for general health, nutritional, or structure/function claims. In contrast, such preparations used with the intention of having a preventive or therapeutic effect in humans are regulated by the Food and Drug Administration (FDA) in the U.S. as biological products, specifically as live biotherapeutic products (LBPs). Discussion of considerations in the early development of LBPs may aid in preparation of an Investigational New Drug Application (IND) that is designed to collect clinical data to support marketing approval of a LBP in the U.S. for a specific clinical use. Product information is an important component of an IND to support a proposed clinical study.     

Identification and Characterization of a Two-Peptide Class IIb Bacteriocin in Streptococcus pluranimalium Isolated from the Nasal Cavity of a Healthy Pig

Probiotics and Antimicrobial Proteins - In addition to be an important zoonotic agent, Streptococcus suis serotype 2 causes severe infections in pigs. In this study, we characterized a new...     

Genetic control of flowering time in Eucalyptus globulus ssp. globulus

Understanding the factors affecting variation in phenology within a species is important as flowering time constitutes one of the major barriers to gene flow. We studied the genetic and environmental control of flower initiation and anthesis time in E. globulus ssp. globulus. For 5 years, flower initiation and anthesis were monitored in a seed orchard containing clones of 63 genotypes from four different regions of the species’ natural distribution. Anthesis occurred over a long period each year, spanning as much as 9 months in 2008. This variation was under strong genetic control with little genotype by year interaction (broad-sense heritability, Ĥ ² = 0.78 ± 0.04). There were highly significant differences among regions; anthesis occurred earlier for Furneaux and Tasmania than Strzelecki and Otways each year. Surprisingly though, there was little variation in flower initiation time between regions and genotypes, and this was under weak genetic control (Ĥ ² = 0.06 ± 0.05). The average anthesis time in the orchard varied from year to year, and there was evidence that heat sum was a major driver of this environmental variation. Anthesis time is controlled by both genetic and environmental factors, with the responses to each being predictable to some extent, and unrelated to the timing of flower initiation.