Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

A novel constitutively active mutation in the second cytoplasmic loop of metabotropic glutamate receptor

G protein-coupled receptors have a common structural motif of seven transmembrane alpha-helices and are classified into different families showing no sequence similarity. Extensive studies have been conducted on the structure-function relationship in family 1 receptors, but those in other families have not been well studied. In this study, to investigate the molecular basis leading to the G protein activation by metabotropic glutamate receptor (mGluR), the member of family 3, we searched for the amino acid residues responsible for the G protein activation in the second cytoplasmic loop, which was thought to be the main G protein binding region. Analyses of the systematical mutations of Gi/Go-coupled mGluR8 revealed the presence of a constitutively active mutation in the C-terminal region of the second loop. The corresponding mutation in the second loop of Gq-coupled mGluR1 also exhibited high agonist-independent activity. These results indicate that there is a common constitutive active mutation site regardless of mGluR subtypes, suggesting that the structural change of the junction between the second cytoplasmic loop and helix IV is strongly linked to the formation of the active state.     

The effect of high glucose on NO and O2- through endothelial GTPCH1 and NADPH oxidase

Although endothelial dysfunction deteriorates diabetic angiopathy, the mechanisms are obscure. We revealed that high glucose augmented eNOS through stimulation of eNOS mRNA in cultured BAECs. NO was decreased and O2- was increased simultaneously. NOS inhibitor, inhibited O2- release, so did NADPH oxidase inhibitor. The effects were synergistic. Both intracellular BH4 level and GTPCH1 activity were decreased by high glucose, in line with decrease of GTPCH1 mRNA. HMG-CoA reductase inhibitor, atorvastatin increased GTPCH1 mRNA and activity, and BH4 level. Conclusively, high glucose leads to eNOS dysfunction by inhibiting BH4 synthesis and atorvastatin stimulate BH4 synthesis directly, and it may work as atherogenic process.     

Identification and characterization of a protostome homologue of peropsin from a jumping spider

Peropsin, a member of the opsin family, has characteristics of two functionally distinct opsin-groups, that is, amino acid residues conserved among opsins for light-sensing and a retinal-photoisomerase-like molecular property. Although such a bilateral feature of peropsin seems to be important for understanding the diversity of the opsin family, previous studies have been limited to higher deuterostome, vertebrate and amphioxus peropsins. Here, we report a protostome peropsin homologue from a jumping spider. We found a spider opsin that shares amino acid homology and conserved amino acid residues with known peropsins. The spider opsin-based pigment heterologously expressed in cultured cells exhibited photoisomerase-like isomerization characteristics and a bistable nature. Based on the characteristics of both the amino acid homology and its photochemical properties, we concluded that the spider opsin is the first protostome peropsin homologue. These results show that peropsin existed before the deuterostome–protostome split like other members of the opsin family. In addition, the spider peropsin was localized to non-visual cells in the retina, and fluorescence from reduced retinal chromophore was also observed in the region where peropsin was localized. These findings provide the first demonstration that the peropsin can form a photosensitive pigment in vivo and underlie non-visual function.     

Photochemical nature of parietopsin

Parietopsin is a nonvisual green light-sensitive opsin closely related to vertebrate visual opsins and was originally identified in lizard parietal eye photoreceptor cells. To obtain insight into the functional diversity of opsins, we investigated by UV-visible absorption spectroscopy the molecular properties of parietopsin and its mutants exogenously expressed in cultured cells and compared the properties to those of vertebrate and invertebrate visual opsins. Our mutational analysis revealed that the counterion in parietopsin is the glutamic acid (Glu) in the second extracellular loop, corresponding to Glu181 in bovine rhodopsin. This arrangement is characteristic of invertebrate rather than vertebrate visual opsins. The photosensitivity and the molar extinction coefficient of parietopsin were also lower than those of vertebrate visual opsins, features likewise characteristic of invertebrate visual opsins. On the other hand, irradiation of parietopsin yielded meta-I, meta-II, and meta-III intermediates after batho and lumi intermediates, similar to vertebrate visual opsins. The pH-dependent equilibrium profile between meta-I and meta-II intermediates was, however, similar to that between acid and alkaline metarhodopsins in invertebrate visual opsins. Thus, parietopsin behaves as an "evolutionary intermediate" between invertebrate and vertebrate visual opsins.     

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.     

Efficacy of liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, on body weight, eating behavior, and glycemic control, in Japanese obese type 2 diabetes

ABSTRACT: BACKGROUND: We recently reported that short-term treatment with liraglutide (20.0 +/- 6.4 days) reduced body weight and improved some scales of eating behavior in Japanese type 2 diabetes inpatients. However, it remained uncertain whether such liraglutide-induced improvement is maintained after discharge from the hospital. The aim of the present study was to determine the long-term effects of liraglutide on body weight, glycemic control, and eating behavior in Japanese obese type 2 diabetics. METHODS: Patients with obesity (body mass index (BMI) >25 kg/m2) and type 2 diabetes were hospitalized at Osaka University Hospital between November 2010 and December 2011. BMI and glycated hemoglobin (HbA1c) were examined on admission, at discharge and at 1, 3, and 6 months after discharge. For the liraglutide group (BMI; 31.3 +/- 5.3 kg/m2, n = 29), patients were introduced to liraglutide after correction of hyperglycemic by insulin or oral glucose-lowering drugs and maintained on liraglutide after discharge. Eating behavior was assessed in patients treated with liraglutide using The Guideline For Obesity questionnaire issued by the Japan Society for the Study of Obesity, at admission, discharge, 3 and 6 months after discharge. For the insulin group (BMI; 29.1 +/- 3.0 kg/m2, n = 28), each patient was treated with insulin during hospitalization and glycemic control maintained by insulin after discharge. RESULTS: Liraglutide induced significant and persistent weight loss from admission up to 6 months after discharge, while no change in body weight after discharge was noted in the insulin group. Liraglutide produced significant improvements in all major scores of eating behavior questionnaire items and such effect was maintained at 6 months after discharge. Weight loss correlated significantly with the decrease in scores for recognition of weight and constitution, sense of hunger, and eating style. CONCLUSION: Liraglutide produced meaningful long-term weight loss and significantly improved eating behavior in obese Japanese patients with type 2 diabetes.