In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.     

Acid denaturation and refolding of green fluorescent protein

Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.     

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.     

Structure of the whole cytosolic region of ATP-dependent protease FtsH

An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.     

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Properties of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae

We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.     

A second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.     

Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor

PGD(2) plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35-55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD(2)-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.     

Molecular analysis of halophilic bacterial community for high-rate denitrification of saline industrial wastewater

A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal-restriction fragment length polymorphism (T-RFLP) analysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the γ-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity conditions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4–10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitrification system for saline wastewater can be designed using acclimated sludge with a halophilic community.     

Identification of novel citrullinated autoantigens of synovium in rheumatoid arthritis using a proteomic approach

Recently, autoantibodies to some citrullinated autoantigens have been reported to be specific for rheumatoid arthritis (RA). However, an entire profile of and autoimmunity of the citrullinated proteins have been poorly understood. To understand the profile, we examined citrullinated autoantigens by a proteomic approach and further investigated the significance of citrullination in antigenicity of one of the autoantigens. Specifically, we detected citrullinated autoantigens in synovial tissue of a patient with RA by two-dimensional electrophoresis and Western blotting by using pooled sera from five patients with RA and anti-citrulline antibodies. After identifying the detected autoantigens by mass spectrometry, we investigated the contribution of citrullination to autoantigenicity by using a recombinant protein with or without citrullination on one of the identified novel citrullinated autoantigens. As a result, we found 51 citrullinated protein spots. Thirty (58.8%) of these spots were autoantigenic. We identified 13 out of the 30 detected citrullinated autoantigenic proteins. They contained three fibrinogen derivatives and several novel citrullinated autoantigens (for example, asporin and F-actin capping protein alpha-1 subunit [CapZalpha-1]). We further analyzed the contribution of citrullination to autoantigenicity in one of the detected citrullinated autoantigens, CapZalpha-1. As a result, frequencies of autoantibodies to non-citrullinated CapZalpha-1 were 36.7% in the RA group tested, 10.7% in the osteoarthritis (OA) group, and 6.5% in healthy donors. On the other hand, those to citrullinated CapZalpha-1 were 53.3% in the RA group, 7.1% in the OA group, and 6.5% in the healthy donors. This shows that autoantigenicity of citrullinated or non-citrullinated CapZalpha-1 is relevant to RA. The antibody titers to the citrullinated CapZalpha-1 were significantly higher than those to the non-citrullinated CapZalpha-1 in 36.7% of patients; however, the other patients showed almost equal antibody titers to both citrullinated and non-citrullinated CapZalpha-1. Therefore, the autoantibodies would target citrulline-related and/or citrulline-unrelated epitope(s) of CapZalpha-1. In conclusion, we report a profile of citrullinated autoantigens for the first time. Even though citrullination is closely related to autoantigenicity, citrullination would not always produce autoantigenicity in RA. Citrullinated and non-citrullinated autoantigens/autoepitopes would have different pathological roles in RA.