Bovine alpha-2-HS-glycoprotein functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and SIVmac239 envelope glycoprotein

The presence of anti-CCR5 and anti-HIV-1 envelope glycoprotein (ENV) gp41 antibodies (Abs) at sites of HIV-1 exposure was effective in preventing its transmission to HIV-1-exposed seronegative (ESN) subjects. Here, we design an immunogen that can induce Abs against CCR5 and SIVmac239 ENV simultaneously and show that bovine alpha-2-HS-glycoprotein (bAHSG) functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and ENV. Initially, we generated a rhesus CCR5-derived cyclopeptide (cDDR5) conjugated with a recombinant trimeric SIVmac239 Env. When inguinally administered to rhesus macaques, the immunogen simultaneously induced both anti-CCR5 and anti-ENV Abs in sera, and the purified serum IgG fraction exerted an inhibitory effect on SIVmac239 infection in vitro. When further boosted with bAHSG, the responses of both Abs were significantly enhanced. To examine the cross-reactivity of bAHSG, it was administered to naïve cynomolgus macaques. The results showed a statistically significant increase in IgG response against cynomolgus CCR5 and SIVmac239 ENV, and the induction of neutralizing activity against SIVmac239. These findings suggest that bAHSG is useful for immune strategies aimed at generating Abs against CCR5 and ENV simultaneously to confer HIV-protective immunity.     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-kappaB but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.     

Development of cell-expressed and virion-incorporated CCR5-targeted vaccine

Our previous study demonstrated that the immunization with a cycloimmunogen derived from extracellular loop-2 (ECL-2) of CCR5 (cDDR5) attenuated acute phase of CCR5-tropic simian-human immunodeficiency virus (SHIV)_SF162P3 replication in vivo. Although the study showed that the antisera raised against cDDR5 reacted with cell-expressed CCR5, we have not yet demonstrated whether the antisera can react with virion-incorporated CCR5. Here, we show that rhesus cDDR5 (rcDDR5)-specific antibodies react with not only cell-expressed but also virion-incorporated simian CCR5s (siCCR5s), but may predominantly exert their inhibitory effects on simian immunodeficiency virus (SIV) infection by the binding of cell-expressed rather than virion-incorporated CCR5s. These results suggest that the virion-incorporated CCR5 may contribute to the reactivation of the anti-rcDDR5 antibody-producing B-cells by SIV particles after rcDDR5 immunization, although the binding of anti-rcDDR5 antibody to virion-incorporated CCR5 results in a partial inhibitory effect on SIV infection.     

p-NITROPHENYL PHOSPHATASES OF A MEMBRANE FRACTION FROM BOVINE CEREBRAL CORTEX

Abstract— Three different types of p -nitrophenyl phosphatases (NPPases) were solubilized by deoxycholate treatment from a membrane fraction of bovine cerebral cortex, and their characteristics were determined. Of these three NPPases (acid, Mg²⁺-activated, and K⁺, Mg²⁺-activated), only K-Mg NPPase was stimulated about two-fold by phospholipid and was inhibited by unsaturated neutral lipids and fatty acids. Unlike Na⁺-K⁺-Mg^a+-activated ATPase, the enzyme did not absolutely require phospholipid for its activity, but was similarly thermolabile and was protected by phospholipid from thermal inactivation. Acid NPPase was separable from the other two NPPases by ammonium sulphate fractionation, and partly solubilized by dialysis against ATP-mercaptoethanol solution. Hg²⁺ inhibited equally all three NPPases, but Ca²⁺ inhibited only Mg and K-Mg NPPases. Ouabain was effective on K-Mg NPPase alone.     

Bidding Customs and Habitat Improvement for Matsutake (<Emphasis Type="Italic">Tricholoma matsutake</Emphasis>) in Japan

Bidding Customs and Habitat Improvement for Matsutake (Tricholoma matsutake) in Japan. A brief history of Japan’s matsutake production, use, and management is presented, with emphasis on habitat improvement efforts and the land use tradition of iriai. Bidding systems for allotment of matsutake gathering rights are discussed. Three villages with different bidding systems are compared to see what effects the bidding systems have on village finances, matsutake production, and matsutake habitat enhancement. Contrary to expectations, habitat for matsutake was not improved when land owners were guaranteed the gathering and selling rights to matsutake growing on their own lands. Instead, habitat improvement was most successful and matsutake production was highest on community-owned lands in Oka Village, where the iriai tradition is strongest.     

Anionic complexes of MWCNT with supergiant cyanobacterial polyanions

Multi‐walled carbon nanotubes (MWCNTs) were well dispersed in an aqueous solution of the cyanobacterial polysaccharide, sacran, with an ultra‐high molecular weight >10 million g/mol. MWCNTs powder was put into aqueous solutions of various polysaccharides including sacran and was dispersed under sonication. As a result of the turbidity measurement of the supernatant, it was found that sacran showed the highest MWCNT‐dispersion efficiency of all the polysaccharides used here. Cryogenic transmission electron microscopic (Cryo‐TEM) studies directly demonstrated the existence of MWCNTs in the supernatant, and high‐resolution TEM observation revealed that MWCNTs covered by sacran chains made their efficient dispersion in water. Raman spectroscopy demonstrated the existence of MWCNT in dried sample from supernatant and the interaction between MWCNT and sacran. The ζ‐potential measurement of the dispersion indicated the negative surface charges of the sacran/MWCNT complexes. Then the MWCNT complexes were able to fabricate by ionic interaction; electrophoresis of the anionic complex formed the sacran/MWCNT gels on the anode while the droplet of sacran/MWCNT dispersion formed gel beads in the presence of the lanthanoid cations. © 2012 Wiley Periodicals, Inc.     

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1–3), domains I and II (D1–2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1–3, D1–2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1–3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.