Length of the linker and the interval between immunizations influences the efficacy of Vibrio cholerae O1, Ogawa hexasaccharide neoglycoconjugates

Ogawa hexasaccharide neoglycoconjugates induce protective antibodies in mice. Similar Ogawa conjugates but with a longer linker that connects the carrier to shorter saccharides are immunogenic, but generally ineffective at inducing vibriocidal or protective antibodies. The efficacy of Ogawa hexasaccharide neoglycoconjugates of different linker lengths were tested. The majority of mice given immunizations separated by a 14-day gap did not produce vibriocidal or protective antibodies. Mice immunized 28 days apart with immunogens containing the shortest or medium length linker, but not the longest, produced vibriocidal and protective antibodies. A nonprotective, priming dose of purified Ogawa LPS followed 5 days later with a booster of the Ogawa neoglycoconjugates (di-, tetra-, or hexasaccharide) resulted in vibriocidal antibodies at day 10.     

ESCRT‐II coordinates the assembly of ESCRT‐III filaments for cargo sorting and multivesicular body vesicle formation

The sequential action of five distinct endosomal‐sorting complex required for transport (ESCRT) complexes is required for the lysosomal downregulation of cell surface receptors through the multivesicular body (MVB) pathway. On endosomes, the assembly of ESCRT‐III is a highly ordered process. We show that the length of ESCRT‐III (Snf7) oligomers controls the size of MVB vesicles and addresses how ESCRT‐II regulates ESCRT‐III assembly. The first step of ESCRT‐III assembly is mediated by Vps20, which nucleates Snf7/Vps32 oligomerization, and serves as the link to ESCRT‐II. The ESCRT‐II subunit Vps25 induces an essential conformational switch that converts inactive monomeric Vps20 into the active nucleator for Snf7 oligomerization. Each ESCRT‐II complex contains two Vps25 molecules (arms) that generate a characteristic Y‐shaped structure. Mutant ‘one‐armed’ ESCRT‐II complexes with a single Vps25 arm are sufficient to nucleate Snf7 oligomerization. However, these oligomers cannot execute ESCRT‐III function. Both Vps25 arms provide essential geometry for the assembly of a functional ESCRT‐III complex. We propose that ESCRT‐II serves as a scaffold that nucleates the assembly of two Snf7 oligomers, which together are required for cargo sequestration and vesicle formation during MVB sorting.     

Stimulation of apical Cl⁻/HCO₃⁻(OH⁻) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl?/HCO??(OH?) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl?/HCO??(OH?) exchange activity (～2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl?/HCO??(OH?) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl?/HCO??(OH?) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (～45%) by NPY compared with control. MβCD significantly decreased Cl?/HCO??(OH?) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive 3?Cl? uptake (～50%). Our results demonstrate that NPY modulates Cl?/HCO??(OH?) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl?/HCO??(OH?) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.     

Ordered assembly of the ESCRT-III complex on endosomes is required to sequester cargo during MVB formation

The sequential action of the Vps27/HRS complex, ESCRT-I, -II, and -III is required to sort ubiquitinated transmembrane proteins to the lumen of lysosomes via the multivesicular body (MVB) pathway. While Vps27/HRS, ESCRT-I, and -II are recruited to endosomes as preformed complexes, the ESCRT-III subunits Vps20, Snf7, Vps24, and Vps2 only assemble into a complex on endosomes. We have addressed the pathway and the regulation for ESCRT-III assembly. Our findings indicate the ordered assembly of a transient 450 kDa ESCRT-III complex on endosomes. Despite biochemical and structural similarity, each subunit contributes a specific function. Vps20 nucleates transient oligomerization of Snf7, which appears to sequester MVB cargo. Vps24 terminates Snf7 oligomerization by recruiting Vps2, which subsequently engages the AAA-ATPase Vps4 to dissociate ESCRT-III. We propose that the ordered assembly and disassembly of ESCRT-III delineates an MVB sorting domain to sequester cargo and complete the last steps of MVB sorting.     

Petrobactin is produced by both pathogenic and non-pathogenic isolates of the Bacillus cereus group of bacteria

Petrobactin is the primary siderophore synthesized by Bacillus anthracis str Sterne and is required for virulence of this organism in a mouse model. The siderophore's biosynthetic machinery was recently defined and gene homologues of this operon exist in several other Bacillus strains known to be mammalian pathogens, but are absent in several known to be harmless such as B. subtilis and B. lichenformis. Thus, a common hypothesis regarding siderophore production in Bacillus species is that petrobactin production is exclusive to pathogenic isolates. In order to test this hypothesis, siderophores produced by 106 strains of an in-house library of the Bacillus cereus sensu lato group were isolated and identified using a MALDI-TOF-MS assay. Strains were selected from a previously defined phylogenetic tree of this group in order to include both known pathogens and innocuous strains. Petrobactin is produced by pathogenic strains and innocuous isolates alike, and thus is not itself indicative of virulence.     

Comparative evaluation of intranasal and subcutaneous route of immunization for development of mucosal vaccine against experimental tuberculosis

Activation of mucosal immunity in the respiratory tract is crucial for protection against respiratory infections. Whether the intranasal route of vaccination imparts better protection against pulmonary tuberculosis than that of subcutaneous vaccination remains a debatable issue. In this study, we have investigated the effect of the routes of immunization on the induction of immunoprotection against experimental tuberculosis employing mycobacterial culture filtrate proteins complexed with dimethyldioctadecylammonium bromide. Vaccination via intranasal and subcutaneous routes triggered immune activation in the spleen and cervical lymph node, while the former route of vaccination lead to higher antigen-specific lymphocyte proliferation, interferon-gamma, interleukin-12 and interleukin-4 responses in cervical lymph node and induction of antigen-specific IgA responses at mucosal level of the respiratory tract. Mice vaccinated via the intranasal route were found to be better protected against experimental tuberculosis particularly in lung compared to subcutaneous-immunized mice. These results emphasize the importance of the intranasal route vaccination in tuberculosis.     

Periprandial changes in growth hormone release in goldfish: role of somatostatin, ghrelin, and gastrin-releasing peptide

In goldfish, growth hormone (GH) transiently rises 30 min after meals, returning to baseline at 1 h postmeal. Somatostatin (SRIF) is the major inhibitor of GH release. Three cDNAs encoding pre-pro-SRIF (PSS) have been previously cloned from goldfish brain: PSS-I, which encodes SRIF-14; PSS-II, which is potentially processed into gSRIF-28 that has [Glu(1),Tyr(7)(,)Gly(10)]SRIF-14 at the COOH terminus; and PSS-III, which encodes [Pro(2)]SRIF-14 at its COOH terminus. In goldfish, bombesin (BBS), mimicking the endogenous gastrin-releasing peptide (GRP), acutely suppresses food intake and also stimulates GH release. Ghrelin was recently characterized in goldfish as a GH secretagogue and an orexigen. In this paper, we studied the changes in SRIF mRNA levels during feeding and analyzed the influences of BBS and ghrelin peptides on forebrain PSS expression. The results showed a 60% reduction in PSS-II mRNA after meals, but no changes in the expression of PSS-I and PSS-III were found. Intraperitoneal injections of 100 ng/g body wt of BBS increased GH secretion and decreased PSS-I and PSS-II gene expression. Intraperitoneal injection of goldfish ghrelin (100 ng/g body wt) transiently increased the serum GH levels and increased PSS-I, while decreasing PSS-II mRNA levels. Ghrelin (50 ng/g body wt) blocked the effects of BBS (100 ng/g body wt) on PSS-I but not on PSS-II expression. Coadministration of BBS and ghrelin decreased only the PSS-II gene expression. We conclude that the interactions between BBS/GRP and ghrelin can account for the postprandial variations in serum GH levels and the forebrain expression of PSS-II. Furthermore, we demonstrate that intraperitoneal administration of BBS reduces the ghrelin expression levels in the gut. Thus the inhibition of production of ghrelin in the gut may contribute to the satiety effects of BBS/GRP peptides.     

Expression and membrane localization of MCT isoforms along the length of the human intestine

Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells. monocarboxylate transporter; short-chain fatty acids; absorption; short-chain fatty acid transport; mammalian colon     

Hepatitis C virus NS3-4A serine protease inhibitors: SAR of P'2 moiety with improved potency

We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.     

Dipeptide sulfonamides as endothelin ETA/ETB receptor antagonists

Endothelin-1 (ET-1) is a potent mitogen and modulator of vascular tone. It is synthesized and released from endothelial cells and acts upon two receptor subtypes designated as ETA and ETB. In this study, a series of potent dipeptide sulfonamide dual-endothelin ETA/ETB receptor antagonists were prepared to investigate their potential benefit in vascular diseases. CGS 31398 inhibited [125I]ET-1 binding to human ETA and ETB receptors expressed in Chinese hamster ovary (CHO) cells (ETA/CHO, ETB/CHO) with respective IC50 values of 0.26 and 0.12 nM. However, in anesthetized rats, this compound markedly potentiated ET-1-induced renal vascular resistance, a response normally observed with selective ETB receptor antagonists. To determine whether species differences account for these results, a direct comparison was made between binding to rat and rabbit aortic membranes versus functional antagonism in isolated rat aortic rings. It was found that CGS 31398 had potent affinity for the ETA receptor in rat and rabbit aorta with IC50 values of 0.87 and 0.79 nM, respectively. Inhibition of ET-1-induced contractions of rat aorta by the compound was considerably weaker than expected (pKB = 6.4), while that of sarafotoxin S6c induced contraction of dog saphenous vein (100% inhibition at 100 nM) was consistent with corresponding binding data. These results suggest that although CGS 31398 is a potent dual inhibitor of ETA/ETB receptor binding, it surprisingly displays potent ETB and weak ETA receptor antagonism in functional assays.