GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.     

Generation and characterization of peptide-specific antibodies that inhibit von Willebrand factor binding to glycoprotein IIb-IIIa without interacting with other adhesive molecules. Selectivity is conferred by Pro1743 and other amino acid residues adjacent to the sequence Arg1744-Gly1745-Asp1746

We have generated antibodies against a synthetic peptide corresponding to the sequence of human von Willebrand factor (vWF) between residues Glu1737-Ser1750 which includes the Arg-Gly-Asp sequence common to several adhesive molecules. Two anti-peptide antibodies, one polyclonal, and one monoclonal reacted with native vWF and inhibited its binding to platelet glycoprotein (GP) IIb-IIIa, but showed negligible cross-reactivity with fibrinogen, fibronectin, and vitronectin, three other molecules that contain the sequence Arg-Gly-Asp and bind to platelets. The structural bases for the specificity of the two antibodies were evaluated by testing the ability of peptides homologous to the parent sequence, but with single amino acid substitutions, to neutralize the binding of the two antibodies to vWF. The substitution of Pro1743, the residue immediately adjacent to the Arg-Gly-Asp sequence on the amino-terminal side, with Phe resulted in a peptide that failed to interact with either antibody. Thus, Pro1743 is important for maintaining a peptide conformation recognized by two antibodies specific for the GP IIb-IIIa-binding domain of vWF. Other residues important for optimal peptide reactivity with the polyclonal antibody were Ser1742, Arg1744, and Gly1745, whereas Gly1741, Gly1745, and Asp1746, but not Arg1744, were important for reactivity with the monoclonal antibody. The epitopes of both antibodies, therefore, included at least 2 of the residues in the sequence Arg-Gly-Asp considered the common cell-binding site of adhesive molecules that interact with GP IIb-IIIa. Nevertheless, both antibodies reacted only with vWF. These studies demonstrate that peptide-specific antibodies, unlike the promiscuous GP IIb-IIIa receptor, can recognize distinctive structural characteristics of the cell-binding domain of adhesive molecules imposed by residues adjacent to the sequence Arg-Gly-Asp.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

Reverse genetics system for human rotaviruses

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.     

Anti‐adhesive property of P‐selectin glycoprotein ligand‐1 (PSGL‐1) due to steric hindrance effect

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is an adhesive molecule that is known to be a ligand for P‐selectin. An anti‐adhesive property of PSGL‐1 has not been previously reported. In this study, we show that PSGL‐1 expression is anti‐adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL‐1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL‐1. PSGL‐1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL‐1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL‐1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL‐1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O‐glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL‐1 has Janus‐faced functions, being both adhesive and anti‐adhesive. J. Cell. Biochem. 114: 1271–1285, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of naftopidil,an alpha1D/A-adrenoceptor antagonist,on the urinary bladder in rats with spinal cord injury

AimsAlpha_1D-adrenoceptors (α_1D-ARs) located in the spinal cord are involved in the control of lower urinary tract function. In order to clarify the effect of α_1D-ARs on storage function in the spinal cord, we examined the effect of oral administration and intrathecal injection of the α_1D/A-AR antagonist, naftopidil, on bladder activity, as well as the effect of naftopidil on bladder wall histology, in female rats with spinal cord injury (SCI).Main methodsAdult female Sprague–Dawley rats with Th9–10 spinal cord transection were used. In SCI rats with or without 5 mg/day of naftopidil for 4 weeks, bladder activity was examined via continuous cystometry. In other SCI rats, bladder activity was examined before and after intrathecal injection of naftopidil. In addition, bladder wall histology was compared between SCI rats with or without oral administration of naftopidil for 4 weeks.Key findingsOral administration of naftopidil decreased the number of non-voiding contractions (NVCs). Intrathecal injection of naftopidil prolonged the interval between voiding contractions, decreased the maximum voiding contraction pressure and the number of NVCs, and increased bladder capacity without affecting the residual urine volume. Oral administration of naftopidil also decreased bladder wall fibrosis.SignificanceThe α_1D/A-AR antagonist naftopidil might act on the bladder and spinal cord to improve detrusor hyperreflexia in the storage state in SCI female rats. Naftopidil also suppressed bladder wall fibrosis, suggesting that it may be effective for the treatment of neurogenic lower urinary tract dysfunction after SCI.