In vivo and in vitro fertilization of hamster, rat and mouse eggs after treatment with anti-hamster ovary antiserum.

Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zone pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.     

Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD⁺ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.     

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4–7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.     

HLA-DQB1*03 Confers Susceptibility to Chronic Hepatitis C in Japanese: A Genome-Wide Association Study

Hepatitis C virus (HCV) establishes a chronic infection in 70-80% of infected individuals. Many researchers have examined the effect of human leukocyte antigen (HLA) on viral persistence because of its critical role in the immune response against exposure to HCV, but almost all studies have proven to be inconclusive. To identify genetic risk factors for chronic HCV infection, we analyzed 458,207 single nucleotide polymorphisms (SNPs) in 481 chronic HCV patients and 2,963 controls in a Japanese cohort. Next, we performed a replication study with an independent panel of 4,358 cases and 1,114 controls. We further confirmed the association in 1,379 cases and 25,817 controls. In the GWAS phase, we found 17 SNPs that showed suggestive association (P < 1 × 10^-5). After the first replication study, we found one intronic SNP in the HLA-DQ locus associated with chronic HCV infection, and when we combined the two studies, the association reached the level of genome-wide significance. In the second replication study, we again confirmed the association (P _combined = 3.59 × 10⁻¹⁶, odds ratio [OR] = 0.79). Subsequent analysis revealed another SNP, rs1130380, with a stronger association (OR=0.72). This nucleotide substitution causes an amino acid substitution (R55P) in the HLA-DQB1 protein specific to the DQB1*03 allele, which is common worldwide. In addition, we confirmed an association with the previously reported IFNL3-IFNL4 locus and propose that the effect of DQB1*03 on HCV persistence might be affected by the IFNL4 polymorphism. Our findings suggest that a common amino acid substitution in HLA-DQB1 affects susceptibility to chronic infection with HCV in the Japanese population and may not be independent of the IFNL4 genotype.     

New ellagitannin and galloyl esters of phenolic glycosides from sapwood of Quercus mongolica var. crispula (Japanese oak)

Two novel glycosides, 4,5-dimethoxy-3-hydroxyphenol 1-O-β-(6′-O-galloyl)-glucopyranoside (1) and (+)-2α-O-galloyl lyoniresinol 3α-O-β-d-xylopyranoside (2), as well as a novel ellagitannin named epiquisqualin B (3), were isolated from sapwood of Quercus mongolica var. crispula along with 19 known phenolic compounds. The structures of the novel compounds were elucidated on the basis of chemical and spectroscopic investigation. Compound 2 is the first example of a lignan galloyl ester, and 3 is the oxidation product of vescalagin, which is the major ellagitannin of this plant.