A monoclonal Fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse human immunodeficiency virus type 1 strains

A monoclonal Fab (Fab 3674) selected from a human nonimmune phage library by panning against the chimeric construct N_CCG-gp41 (which comprises an exposed coiled-coil trimer of gp41 N helices fused in the helical phase onto the minimal thermostable ectodomain of gp41) is described. Fab 3674 is shown to neutralize diverse laboratory-adapted B strains of human immunodeficiency virus type 1 (HIV-1) and primary isolates of subtypes A, B, and C in an Env-pseudotyped-virus neutralization assay, albeit with reduced potency (approximately 25-fold) compared to that of 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N helices of gp41 that is exposed between two C helices in the fusogenic six-helix bundle conformation of gp41. Bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C helix of gp41) are shown to act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.     

Seroprevalence and isolation of Toxoplasma gondii from free-range chickens in Ghana, Indonesia, Italy, Poland, and Vietnam

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of the parasite's oocysts in soil because chicken feed from the ground. The prevalence of T. gondii in free-range chickens from Ghana, Indonesia, Italy, Poland, and Vietnam was determined using the modified agglutination test (MAT). Antibodies to T. gondii were found in 41 (64%) of 64 chickens from Ghana, 24 (24.4%) of 98 chickens from Indonesia, 10 (12.5%) of 80 chickens from Italy, 6 (30%) of 20 chickens from Poland, and 81 (24.2%) of 330 chickens from Vietnam. Hearts and brains of chickens were bioassayed for T. gondii. Viable T. gondii was isolated from 2 chickens from Ghana, 1 chicken from Indonesia, 3 chickens from Italy, 2 chickens from Poland, and 1 chicken from Vietnam. Toxoplasma gondii isolates from 9 chickens were genotyped using 10 PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. A total of 7 genotypes was identified; the 3 isolates from chickens from Italy were clonal type II, and the others were nonclonal. This is the first report of genetic characterization of T. gondii isolates from animals from these countries.     

alpha-Glucosidase inhibitors from the seeds of Syagrus romanzoffiana

Bioassay-guided fractionation against alpha-glucosidase resulted in isolation and characterization of eight active compounds from the EtOH extract of the seeds of Syagrus romanzoffiana. Of these, seven are stilbenoids, and two of them, 13-hydroxykompasinol A (1) and scirpusin C (4), possess potent inhibitory activity against alpha-glucosidase type IV from Bacillus stearothermophilus with the IC50 value of 6.5 and 4.9 microM, respectively. The in vivo assay on normal Wistar rats using oral sucrose challenge also demonstrated that kompasinol A (2) and 3,3',4,5,5'-pentahydroxy-trans-stilbene (5) possess significant effect in reducing the postprandial blood glucose level (10.2% and 12.1% at 10mg/kg, respectively). These results suggest that stilbenoids might be explored for their therapeutic potential as hypoglycemic agents.     

Planktonic bacteria and fungi are selectively eliminated by exposure to marine macroalgae in close proximity

To test whether macroalgae affect microbial colonizers in close proximity in a phylum-specific fashion, the community richness of planktonic bacteria and fungi was analyzed with selective oligonucleotide probes targeting the Cytophaga/Flavobacterium/Bacteroides (CFB), Alphaproteobacteria and Roseobacter group and the ITS1 region of marine fungi. Naturally occuring planktonic microorganisms were incubated in the presence of macroalgae or in seawater previously conditioned with macroalgal metabolites. The red algae Ceramium rubrum and Mastocarpus stellatus as well as seawater conditioned with these algae reduced the community composition of bacteria to a greater extent than the brown alga Laminaria digitata, indicating that metabolites differed among macroalgae or that the susceptibility of planktonic bacteria towards alga-derived antimicrobials correlated with their phylogenetic affiliation. The most affected phylotypes belonged to the CFB and the Roseobacter clade. The planktonic fungal community was only affected in the presence of macroalgae and not in algal-conditioned water, but with a specificity different from that observed for bacteria. The macroalgae L. digitata and M. stellatus exhibited more pronounced antifungal effects than C. rubrum. This study demonstrates macroalgal defenses against epiphytic microorganisms based on natural delivery mechanisms of allelochemicals utilizing a culture-independent approach, thus minimizing the ecological bias inherent to culture-dependent studies based on few microbial isolates.     

A low-sodium-salt formulation for the fermentation of salinosporamides by <Emphasis Type="Italic">Salinispora tropica</Emphasis> strain NPS21184

In this paper, we described the development of a potassium-chloride-based-salt formulation containing low sodium concentrations (5.0 to 11 mM) to support the growth of Salinispora tropica strain NPS21184 and its production of salinosporamide A (NPI-0052). The sodium present in the media was essentially derived from the complex nitrogen sources Hy Soy, yeast extract, and peptone used in the media. We demonstrated that good growth rate and yield of S. tropica strain NPS21184 were detected in both agar and liquid media containing the potassium-chloride-based-salt formulation with sodium concentration as low as 5.0 mM, significantly less than the critical seawater-growth requirement concentration of 50 mM sodium for a marine microorganism. We also observed good production of NPI-0052 (176 to 243 mg/l) by S. tropica strain NPS21184 grown in production media containing the potassium chloride-based-salt formulation. The production of deschloro analog, salinosporamide B (NPI-0047), was significantly lower in the low-sodium-salt-formulation medium than in the high-sodium-salt-formulation media. We demonstrated that while S. tropica strain NPS21184 is a novel marine actinomycete that requires high salt content for growth, it does not require sodium-chloride-based seawater-type media for growth and production of NPI-0052.     

Central lactate metabolism regulates food intake

The central nervous system regulates food intake (FI) and body weight (BW), but the associated mechanisms remain to be elucidated. Here we report that central injections of lactate reduced FI and BW in rodents. Inhibition of central lactate metabolism to pyruvate with the lactate dehydrogenase inhibitor oxamate abolished the central effects of lactate on FI and BW. Conversely, central injections of pyruvate recapitulated the effects of lactate. Finally, inhibition of central lactate metabolism prevented the ability of circulating lactate to lower FI and BW. Together, the data indicate that activation of central lactate metabolism lowers FI and BW.     

Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL

Memory B (B(MEM)) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting B(MEM) cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived B(MEM) cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, B(MEM) cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity.     

Airway house dust extract exposures modify allergen-induced airway hypersensitivity responses by TLR4-dependent and independent pathways

TLR ligands and other allergen-nonspecific immunostimulatory molecules are ubiquitous in ambient air and have profound modulatory activities in animal models of allergic asthma. However, several of these molecules have been shown to promote exaggerated Th2-biased airway hypersensitivity responses (AHRs), whereas others attenuate the asthmatic phenotype. Therefore, it has proven difficult to extrapolate experimental results with purified molecules toward a more general understanding of the allergen-nonspecific immunomodulatory influence of living environments on the natural history of allergic asthma. These investigations determined how regular and intermittent airway exposures to an unpurified, but sterile house dust extract standard (HDEst) affected the OVA-specific AHR and immune status of previously Th2-sensitized mice. Low-dose daily and high-dose intermittent HDEst exposures modulated ongoing AHRs considerably, reducing eosinophil recruitment and methacholine responsiveness, while increasing neutrophilic inflammation. However, only daily airway delivery of low-dose HDEst attenuated OVA-specific Th2 cytokine production and Th2-biased AHRs to allergen challenge 1 mo later. Finally, whereas LPS mimicked many of the immunomodulatory characteristics of HDEst in this murine asthma model, daily airway HDEst delivery was highly effective in attenuating the AHR of OVA/alum-sensitized TLR4-deficient mice. Taken together, these investigations provide direct evidence that living environments contain allergen-nonspecific immunostimulatory molecules that influence the airway hypersensitivity status of allergen-sensitized mice by TLR4-dependent and independent mechanisms.     

Vimentin supports mitochondrial morphology and organization

Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.     

Degradation of the tumor suppressor PML by Pin1 contributes to the cancer phenotype of breast cancer MDA-MB-231 cells

Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.