Bovine Milk Proteome in the First 9 Days: Protein Interactions in Maturation of the Immune and Digestive System of the Newborn

In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold). Three relative high abundant enzymes (XDH, LPL, and RNASE1) and cell division and proliferation protein (CREG1) may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.     

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.     

Adsorption of Cationic Peptides to Solid Surfaces of Glass and Plastic

Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant experimental implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use analytical HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical experimental peptide concentrations, 90% or more of the peptides might be lost from solution due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting experiments on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the experimental impact of these adsorption effects.     

SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.     

Diversity of sulfate-reducing bacteria from an extreme hypersaline sediment, Great Salt Lake (Utah)

The diversity of sulfate-reducing bacteria (SRB) inhabiting the extreme hypersaline sediment (270 g L(-1) NaCl) of the northern arm of Great Salt Lake was studied by integrating cultivation and genotypic identification approaches involving PCR-based retrieval of 16S rRNA and dsrAB genes, the latter encoding major subunits of dissimilatory (bi) sulfite reductase. The majority (85%) of dsrAB sequences retrieved directly from the sediment formed a lineage of high (micro) diversity affiliated with the genus Desulfohalobium, while others represented novel lineages within the families Desulfohalobiaceae and Desulfobacteraceae or among Gram-positive SRB. Using the same sediment, SRB enrichment cultures were established in parallel at 100 and at 190 g L(-1) NaCl using different electron donors. After 5-6 transfers, dsrAB and 16S rRNA gene-based profiling of these enrichment cultures recovered a SRB community composition congruent with the cultivation-independent profiling of the sediment. Pure culture representatives of the predominant Desulfohalobium-related lineage and of one of the Desulfobacteraceae-affilated lineages were successfully obtained. The growth performance of these isolates and of the enrichment cultures suggests that the sediment SRB community of the northern arm of Great Salt Lake consists of moderate halophiles, which are salt-stressed at the in situ salinity of 27%.     

Follow-up testing of rodent carcinogens not positive in the standard genotoxicity testing battery: IWGT workgroup report

At the Plymouth Third International Workshop on Genotoxicity Testing in June 2002, a new expert group started a working process to provide guidance on a common strategy for genotoxicity testing beyond the current standard battery. The group identified amongst others "Follow-up testing of tumorigenic agents not positive in the standard genotoxicity test battery" as one subject for further consideration [L. Müller, D. Blakey, K.L. Dearfield, S. Galloway, P. Guzzie, M. Hayashi, P. Kasper, D. Kirkland, J.T. MacGregor, J.M. Parry, L. Schechtman, A. Smith, N. Tanaka, D. Tweats, H. Yamasaki, Strategy for genotoxicity testing and stratification of genotoxicity test results-report on initial activities of the IWGT Expert Group, Mutat. Res. 540 (2003) 177-181]. A workgroup devoted to this topic was formed and met on September 9-10, 2005, in San Francisco. This workgroup was devoted to the discussion of when it would be appropriate to conduct additional genetic toxicology studies, as well as what type of studies, if the initial standard battery of tests was negative, but tumor formation was observed in the rodent carcinogenicity assessment. The important role of the standard genetic toxicology testing to determine the mode of action (MOA) for carcinogenesis (genotoxic versus non-genotoxic) was discussed, but the limitations of the standard testing were also reviewed. The workgroup also acknowledged that the entire toxicological profile (e.g. structure-activity relationships, the nature of the tumor finding and metabolic profiles) of a compound needed to be taken into consideration before the conduct of any additional testing. As part of the meeting, case studies were discussed to understand the practical application of additional testing as well as to form a decision tree. Finally, suitable additional genetic toxicology assays to help determine the carcinogenic MOA or establish a weight of evidence (WOE) argument were discussed and formulated into a decision tree.     

The 1.4-A crystal structure of the S. pombe Pop2p deadenylase subunit unveils the configuration of an active enzyme

Deadenylation is the first and probably also rate-limiting step of controlled mRNA decay in eukaryotes and therefore central for the overall rate of gene expression. In yeast, the process is maintained by the mega-Dalton Ccr4-Not complex, of which both the Ccr4p and Pop2p subunits are 3′–5′ exonucleases potentially responsible for the deadenylation reaction. Here, we present the crystal structure of the Pop2p subunit from Schizosaccharomyces pombe determined to 1.4Å resolution and show that the enzyme is a competent ribonuclease with a tunable specificity towards poly-A. In contrast to S. cerevisiae Pop2p, the S. pombe enzyme contains a fully conserved DEDDh active site, and the high resolution allows for a detailed analysis of its configuration, including divalent metal ion binding. Functional data further indicates that the identity of the ions in the active site can modulate both activity and specificity of the enzyme, and finally structural superposition of single nucleotides and poly-A oligonucleotides provide insight into the catalytic cycle of the protein.     

What is the Value of a Good Map? An Example Using High Spatial Resolution Imagery to Aid Riparian Restoration

Riparian areas contain structurally diverse habitats that are challenging to monitor routinely and accurately over broad areas. As the structural variability within riparian areas is often indiscernible using moderate-scale satellite imagery, new mapping techniques are needed. We used high spatial resolution satellite imagery from the QuickBird satellite to map harvested and intact forests in coastal British Columbia, Canada. We distinguished forest structural classes used in riparian restoration planning, each with different restoration costs. To assess the accuracy of high spatial resolution imagery relative to coarser imagery, we coarsened the pixel resolution of the image, repeated the classifications, and compared results. Accuracy assessments produced individual class accuracies ranging from 70 to 90% for most classes; whilst accuracies obtained using coarser scale imagery were lower. We also examined the implications of map error on riparian restoration budgets derived from our classified maps. To do so, we modified the confusion matrix to create a cost error matrix quantifying costs associated with misclassification. High spatial resolution satellite imagery can be useful for riparian mapping; however, errors in restoration budgets attributable to misclassification error can be significant, even when using highly accurate maps. As the spatial resolution of imagery increases, it will be used more routinely in ecosystem ecology. Thus, our ability to evaluate map accuracy in practical, meaningful ways must develop further. The cost error matrix is one method that can be adapted for conservation and planning decisions in many ecosystems.     

Integrative analysis for finding genes and networks involved in diabetes and other complex diseases

We have developed an integrative analysis method combining genetic interactions, identified using type 1 diabetes genome scan data, and a high-confidence human protein interaction network. Resulting networks were ranked by the significance of the enrichment of proteins from interacting regions. We identified a number of new protein network modules and novel candidate genes/proteins for type 1 diabetes. We propose this type of integrative analysis as a general method for the elucidation of genes and networks involved in diabetes and other complex diseases.     

Assessment of angiogenesis-related parameters in juvenile idiopathic arthritis-associated uveitis

Molecular Biology Reports - Juvenile idiopathic arthritis-associated uveitis (JIAU) may run a chronic and treatment-resistant course, and occasionally, alterations of the iris vasculature may be...