Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma-associated herpesvirus and functions as an RGD-dependent entry receptor

Kaposi's sarcoma-associated herpesvirus (KSHV) envelope-associated glycoprotein B (gB) is involved in the initial steps of binding to host cells during KSHV infection. gB contains an RGD motif reported to bind the integrin α₃β₁ during virus entry. Although the ligand specificity of α₃β₁ has been controversial, current literature indicates that α₃β₁ ligand recognition is independent of RGD. We compared α₃β₁ to the RGD-binding integrin, α_Vβ₃, for binding to envelope-associated gB and a gB(RGD) peptide. Adhesion assays demonstrated that β₃-CHO cells overexpressing α_Vβ₃ specifically bound gB(RGD), whereas α₃-CHO cells overexpressing α₃β₁ did not. Function-blocking antibodies to α_Vβ₃ inhibited the adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to α₃β₁ did not. Using affinity-purified integrins and confocal microscopy, α_Vβ₃ bound to gB(RGD) and KSHV virions, demonstrating direct receptor-ligand interactions. Specific α_Vβ₃ antagonists, including cyclic and dicyclic RGD peptides and α_Vβ₃ function-blocking antibodies, inhibited KSHV infection by 70 to 80%. Keratinocytes from α₃-null mice lacking α₃β₁ were fully competent for infection by KSHV, and reconstitution of α₃β₁ function by transfection with α₃ cDNA reduced KSHV infectivity from 74% to 55%. Additional inhibitory effects of α₃β₁ on the cell surface expression of α_Vβ₃ and on α_Vβ₃-mediated adhesion of α₃-CHO cells overexpressing α₃β₁ were detected, consistent with previous reports of transdominant inhibition of α_Vβ₃ function by α₃β₁. These observations may explain previous reports of an inhibition of KSHV infection by soluble α₃β₁. Our studies demonstrate that α_Vβ₃ is a cellular receptor mediating both the cell adhesion and entry of KSHV into target cells through binding the virion-associated gB(RGD).     

Bacterial and Archaeal Community Structure in the Surface Diatom Sediments of Deep Freshwater Lake Baikal (Eastern Siberia)

Diatom sediment records of large lakes can be used to decipher the history of ancient phytoplankton. The upper layer of the sediment is an important area of remineralization of the sedimenting phytoplankton biomass. It hosts a bacterial community different from those of both the water column and deeper sediment layers. In this work, we analyzed the structure and diversity of the communities of Bacteria and Archaea in the surface sediment core containing valves of diatoms, the major producers in Lake Baikal. Pyrosequencing of the bacterial V3–V4 region of the 16 S ribosomal RNA (rRNA) and archaeal V1–V3 16 S rRNA gene regions yielded 29,168 and 36,997 reads, respectively. In total, we have identified 33 bacterial phyla; uncultured Actinobacteria were the most abundant in the upper layers, while lower sediment was dominated by Firmicutes and Alphaproteobacteria. The composition of the archaeal community changed with depth, but was generally dominated by Crenarchaeota from the classes Marine Group I and Miscellaneous Crenarchaeotic Group, as well as Euryarchaeota from the class Thermoplasmata. These dominant bacterial and archaeal taxa are presumed to participate in the destruction of buried organic matter, which eventually leads to degradation of the diatom valves.     

Mechanism of pyocyanin- and 1-hydroxyphenazine-induced lung neutrophilia in sheep airways

Pyocyanin (Pyo)and 1-hydroxyphenazine (1-HP) are extracellular products ofPseudomonas aeruginosa. To testwhether these products were capable of producing an inflammatoryresponse in the airways, combinations of Pyo and 1-HP at concentrationsof 10⁴ and10⁵ M were instilled intosheep airways, and indexes of inflammation were assessed bybronchoalveolar lavage (BAL) 24 h later. Challenge with the phenazinescaused a significant dose-dependent increase in the number of cells andneutrophils recovered by BAL. Control challenges produced no suchchanges. The lung neutrophilia was accompanied by an increasedconcentration of albumin in BAL. The increases in BAL neutrophils andalbumin could be blocked by treating the sheep with the 5-lipoxygenaseinhibitor zileuton. Neither 1-HP nor Pyo was chemotactic to neutrophilswhen tested in vitro, but when alveolar macrophages (AM) were culturedin vitro in the presence of both Pyo and 1-HP (1 µM), thesupernatants caused neutrophil chemotaxis. Analysis of AM culturesupernatants incubated with the combination of pigments showedsignificant increases in leukotrieneB₄ and interleukin-8, and blockingthese mediators separately or together reduced AM supernatant-inducedneutrophil chemotaxis. We conclude that local instillation of Pyo and1-HP can initiate an inflammatory response in the airways of sheep invivo. This effect can be explained, in part, by the release ofchemotactic factors produced by AM.

     

Increased Brain Levels of Platelet-Activating Factor in a Murine Acquired Immune Deficiency Syndrome Are NMDA Receptor-Mediated

Abstract: Mice infected with the LP-BM5 murine leukemia virus (MuLV) develop an immunodeficiency syndrome (murine AIDS) and an encephalopathy characterized by impaired spatial learning and memory. Because platelet-activating factor (PAF) has been implicated in the pathogenesis of HIV-associated dementia complex, brain PAF levels were measured in LP-BM5 MuLV-infected mice. PAF levels in cerebral cortex and hippocampus were significantly increased at 6 and 12 weeks after LP-BM5 MuLV inoculation, whereas significant increases in striatal and cerebellar PAF levels were observed only at 12 weeks after inoculation. Administration of the NMDA antagonist MK-801 significantly reduced the increased PAF levels in the cerebral cortex and hippocampus of LP-BM5 MuLV-infected mice. These results indicate that the LP-BM5 MuLV-induced increases in brain PAF levels are the result of NMDA receptor activation and are consistent with the hypothesis that elevated CNS PAF levels contribute to the behavioral deficits observed in LP-BM5 MuLV-infected mice.     

Performance evaluation of constructed wetlands in a tropical region

Five emergent plant species were compared for their effectiveness in treating contaminants in a wetland system constructed on a military base in El Salvador. The system consisted of the subsurface flow (SSF), open water (OW) and free surface flow (SF) wetlands with a combined flow capacity of up to 151.4 m³ d^?1. Reliability and consistent performance in extreme conditions, such as those occurring during the tropical dry or wet seasons were important evaluation criteria. The discontinuous flow patterns typical of tropical climates necessitated the use of water balance calculations using climatic data such as rainfall and evapotranspiration. System characterization was achieved by computation of daily input and output mass loading rates for each individual constituent. Results suggest that Phragmites and Brachiaria were the most effective plants in SSF wetland. Brachiaria provided the added benefit of serving as a source of fodder and proved proficient, with N and P uptakes of 1.5–3.14% and 0.17–0.25% per dry plants’ biomass, respectively. Typha yielded the highest dry season removal efficiency within the SF (BOD₅: 80.78 ± 9.35%, COD: 65.18 ± 19.6%, TN: 58.59 ± 19.3%, oil and grease: 78.34 ± 10.55%, total dissolved phosphorus: 66.5 ± 20.7%). Phragmites–Typha treatment subset performed better year-round than either Thalia–Thalia or Brachiaria–Cyperus. Evaluated plants were capable of surviving and proliferating in extreme tropical climates.     

Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ERalpha and ERbeta) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17alpha-ethynyl-17beta-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutrophil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model.     

T-cadherin GPI-anchor is insufficient for apical targeting in MDCK cells

T-cadherin is a 95kDa glycoprotein member of the cadherin family of adhesion molecules attached to the extracellular surface of the cell membrane through a glycosyl-phosphatidylinositol (GPI)-anchor. Whether a T-cadherin ectodomain apical targeting signal or the GPI-anchor itself targets this protein to the apical membrane is not known. Chimeras of the reporter EGFP and T-cadherin have demonstrated that a minimal construct consisting of the C-terminal 25 amino acids including the N690 (omega-site) of T-cadherin was sufficient to GPI-anchor the EGFP protein. However, efficient GPI-anchor with minimal secretion of the protein required an additional 5 residues (omega-1 to omega-5). The GPI-anchored chimeras fractionated to the Triton X-100 detergent insoluble fraction and were released to the cell culture supernatant by phosphoinositide-specific phospho-lipase C digestion. When expressed in MDCK cells, all GPI-anchored chimeras targeted to the basolateral membrane, while the T/N-chimera and the wild-type T-cadherin targeted to the apical membrane. Therefore, T-cadherin is an example of another rare GPI-anchored protein where the anchor itself is not sufficient for apical targeting in MDCK cells.     

The Drosophila fragile X mental retardation protein controls actin dynamics by directly regulating profilin in the brain

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.     

A novel mechanism for mitogen-activated protein kinase localization

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.