An oral Salmonella vaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.     

Formation of a lamellar compound by reaction of acrylic acid crystallosolvated in highly crystalline beta-chitin

A lamellar compound resulted from reaction of acrylic acid inside crystalline beta-chitin and the structure was investigated. Beta-chitin acts like a layered crystal, having stacked molecular sheets composed of parallel chains bound in one direction by intermolecular amide hydrogen bonding. Small guest molecules can be inserted between the molecular sheets, and a crystallosolvate can be formed. By immersion of beta-chitin in acrylic acid, a crystallosolvate was formed, which was then changed into the more stable lamellar compound by heat treatment at 105 degrees C. NMR measurement and IR spectroscopy showed that during the heat treatment there was a reaction between acrylic acid and the beta-chitin molecular sheet, but the sheet structure was maintained. By IR with deuteration, it was shown that the accessibility of solvents to this lamellar compound was greater than that for the initial beta-chitin. The lamellar compound is considered a kind of "pillared" structure related to the lamellar crystal.     

Decreased caveolin‐1 levels contribute to fibrosis and deposition of extracellular IGFBP‐5

Our previous studies have demonstrated increased expression of insulin‐like growth factor binding protein‐5 (IGFBP‐5) in fibrotic tissues and IGFBP‐5 induction of extracellular matrix (ECM) components. The mechanism resulting in increased IGFBP‐5 in the extracellular milieu of fibrotic fibroblasts is unknown. Since Caveolin‐1 (Cav‐1) has been implicated to play a role in membrane trafficking and signal transduction in tissue fibrosis, we examined the effect of Cav‐1 on IGFBP‐5 internalization, trafficking and secretion. We demonstrated that IGFBP‐5 localized to lipid rafts in human lung fibroblasts and bound Cav‐1. Cav‐1 was detected in the nucleus in IGFBP‐5‐expressing fibroblasts, within aggregates enriched with IGFBP‐5, suggesting a coordinate trafficking of IGFBP‐5 and Cav‐1 from the plasma membrane to the nucleus. This trafficking was dependent on Cav‐1 as fibroblasts from Cav‐1 null mice had increased extracellular IGFBP‐5, and as fibroblasts in which Cav‐1 was silenced or lipid raft structure was disrupted through cholesterol depletion also had defective IGFBP‐5 internalization. Restoration of Cav‐1 function through administration of Cav‐1 scaffolding peptide dramatically increased IGFBP‐5 uptake. Finally, we demonstrated that IGFBP‐5 in the ECM protects fibronectin from proteolytic degradation. Taken together, our findings identify a novel role for Cav‐1 in the internalization and nuclear trafficking of IGFBP‐5. Decreased Cav‐1 expression in fibrotic diseases likely leads to increased deposition of IGFBP‐5 in the ECM with subsequent reduction in ECM degradation, thus identifying a mechanism by which reduced Cav‐1 and increased IGFBP‐5 concomitantly contribute to the perpetuation of fibrosis.     

Neuroendocrine roles of the brain in the regulation of 20-hydroxyecdysone responsiveness in two types of diapause pupae of the cabbage armyworm, Mamestra brassicae

The cabbage armyworm, Mamestra brassicae, has winter-and aestival- diapause pupae (WD- and AD-pupae) showing differences in the strength of diapause. We tried to quantify diapause-strength by measuring the doses of 20-hydroxyecdysone (20-E) required to induce adult development in WD-, AD- and decerebrated non-diapause pupae (ND-pupae). The role of the brain in the regulation of diapause-strength was studied through the decerebration and brain-reimplantation of WD-and AD-pupae. The 20-E doses required for adult development were small within the first 2 days of pupation, and increased thereafter to reach a constant level about 10 days after pupation in AD- and decerebrated ND-pupae. The required 20-E doses in WD-pupae increased for more than 40 days after pupation. When 0-day-old WD- and 0-day-old AD-pupae were decerebrated, required 20-E doses increased after pupation and reached a constant about 10 days later. The required 20-E dose reached a constant level in decerebrated WD-pupae that was smaller than that observed for decerebrated ND- and WD-pupae. Furthermore, the required doses increased when 0-day-old WD-pupal brains were reimplanted into decerebrated WD- and decerebrated ND-pupae. In WD-, AD- and decerebrated ND-pupae, diapause-strength can be represented as the 20-E dose required for adult development. Diapause-strength is weak after pupation, increases thereafter, and reaches a constant about 10 days later in AD- and decerebrated ND-pupae. In WD-pupae, diapause-strength increases for more than 40 days after pupation and reaches a level that is twice that estimated for AD-pupae. Brains of diapausing WD-pupae may secrete a factor that suppresses the 20-E responsiveness of pupal organs, for the purpose of maintaining winter-diapause.     

Change in enantioselectivity in bufuralol 1''-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6

The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1'-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1'-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF > S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1'R-OH-BF < 1'-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1'-hydroxylation by CYP2D6.     

Stable-isotope dilution gas chromatography-mass spectrometric measurement of 3-hydroxyglutaric acid, glutaric acid and related metabolites in body fluids of patients with glutaric aciduria type 1 found in newborn screening

We developed a simple and sensitive stable-isotope dilution method for the quantification of 3-hydroxyglutaric acid (3HGA) and glutaric acid (GA) in body fluids. In our method, tert-butyldimethylsilyl (tBDMS) derivatives of 3HGA and GA were measured with a conventional electron-impact ionization (EI) mode in gas chromatography-mass spectrometry (GC-MS). The control values for 3HGA in nmol/ml were 0.15+/-0.08 (serum; n=10) and 0.07+/-0.03 (CSF; n=10). In addition, glutarylcarnitine and free carnitine were quantified by electrospray tandem mass spectrometry. Using these methods, we monitored 3HGA, GA, and glutarylcarnitine in the body fluids of three patients with glutaric aciduria type 1 found during newborn screening. None of the patients had experienced neurological strokes, which are possibly caused by the accumulation of 3HGA, at 15-24 months of age under a disease-specific treatment, including carnitine supplementation. Our data showed that 3HGA levels were relatively high in some serum samples with lower glutarylcarnitine and carnitine levels, suggesting that carnitine supplementation may play a role in preventing the accumulation of 3HGA in patients with this disease.     

Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant

A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.     

Cerebroside sulfotransferase deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.     

Differential Anti-APOBEC3G Activity of HIV-1 Vif Proteins Derived from Different Subtypes

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.