Haprin‐deficient spermatozoa are incapable of in vitro fertilization

Haprin (TRIM36) is a ubiquitin‐protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin‐deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility‐related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin‐deficient mice having poorer sperm morphology and motility than wild‐type mice. Interestingly, haprin‐deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.     

Methylation dynamics of IG-DMR and Gtl2-DMR during murine embryonic and placental development

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains IG-DMR and Gtl2-DMR, whose methylation patterns are established in the germline and after fertilization, respectively. In this study, we determine that acquisition of DNA methylation at the paternal allele of the Gtl2-DMR is initiated after the blastocyst stage and completed by embryonic day 6.5, and that Gtl2 (approved symbol: Meg3) is monoallelically expressed from the maternal allele as early as the blastocyst. Therefore, DNA methylation at the Gtl2-DMR is not a prerequisite for the imprinted expression of Gtl2, which may be involved in the control of proliferation and differentiation of cells during early gestation. We also reveal that a subregion of the IG-DMR exhibits tissue-specific differences in allelic methylation patterns. These results add to the growing body of knowledge elucidating the mechanism whereby parent-of-origin-dependent DNA methylation at the IG-DMR leads to the imprinted expression of the Dlk1-Dio3 cluster.     

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance

Transforming growth factor-beta 2 (TGF-β2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-β2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-β2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-β2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-β2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm’s canal (MSC) cell monolayers was decreased by TGF-β2 treatment. SAHA inhibited the effects of TGF-β2 on the permeability of these cells. TGF-β2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-β2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-β2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-β signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-β. These results imply that SAHA prevents TGF-β2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-β signaling in TM and MSC cells.     

Discovery and structure–activity relationship study of 1,3,6-trisubstituted 1,4-diazepane-7-ones as novel human kallikrein 7 inhibitors

Compound 1, composed of a 1,3,6-trisubstituted 1,4-diazepane-7-one, was discovered as a novel human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme, SCCE) inhibitor, and its derivatives were synthesized and evaluated. Structure–activity relationship studies of the amidoxime unit and benzoic acid part of this new scaffold led to the identification of 25 and 34, which were more potent than the hit compound, 1. The X-ray co-crystal structure of compound 25 and human KLK7 revealed the characteristic interactions and enabled explanations of the structure–activity relationship.     

Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat)

A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K(m) of 161 microM and a k(cat)/K(m) of 348 s(-1) mM(-1) for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 degrees C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO(4), HgCl(2), AgNO(3), FeCl(3), beta-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k(cat)/K(m)) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.     

Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.     

Genes for an alkaline D-stereospecific endopeptidase and its homolog are located in tandem on Bacillus cereus genome

Alkaline D-peptidase (Adp) from Bacillus cereus DF4-B is a D-stereospecific endopeptidase acting on oligopeptides composed of D-phenylalanine and the primary structure deduced from its gene, adp, shows a similarity with D-stereospecific hydrolases from Ochrobactrum anthropi strains. We have isolated DNA fragments covering the flanking region of adp from DF4-B genome and found an additional gene, adp2, located upstream of adp. The deduced amino acid sequence of Adp2 showed 96% and 85% identity with those of Adp from B. cereus strains AH559 and DF4-B, respectively. The recombinant Adp2 expressed in Escherichia coli was purified to homogeneity and characterized. It had hydrolyzing activity toward (D-Phe)3, (D-Phe)4, and (D-Phe)6 but did not act on (L-Phe)4, D-Phe-NH2, and L-Phe-NH2, some characteristics that are closely related to those of Adp from strain DF4-B. These results indicate that highly homologous genes encoding D-stereospecific endopeptidases are arranged in a tandem manner on the genomic DNA of B. cereus DF4-B.     

Discovery and structure-activity relationship of imidazolinylindole derivatives as kallikrein 7 inhibitors

A series of imidazolinylindole derivatives were discovered as novel kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Structure-activity relationship (SAR) studies led to the identification of potent human KLK7 inhibitors. By further modification of the benzenesulfonyl moiety to overcome species differences in inhibitory activity, potent inhibitors against both human and mouse KLK7 were identified. Furthermore, the complex structure of 25 with mouse KLK7 could explain the SAR and the cause of the species differences in inhibitory activity.     

Drosophila CTLA-2-like protein (D/CTLA-2) inhibits cysteine proteinase 1 (CP1), a cathepsin L-like enzyme

In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine proteinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.     

p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

Glaucoma is an age-related neurodegenerative disease of retinal ganglion cells, and appropriate turnover of the extracellular matrix in the trabecular meshwork is important in its pathology. Here, we report the effects of Rho-associated kinase (ROCK) and p38 MAP kinase on transforming growth factor (TGF)-β2–induced type I collagen production in human trabecular meshwork cells. TGF-β2 increased RhoA activity, actin polymerization, and myosin light chain 2 phosphorylation. These effects were significantly inhibited by Y-27632, but not SB203580. TGF-β2 also increased promoter activity, mRNA synthesis, and protein expression of COL1A2. These effects were significantly inhibited by SB203580, but not Y-27632. Additionally, Y-27632 did not significantly inhibit TGF-β2–induced promoter activation, or phosphorylation or nuclear translocation of Smad2/3, whereas SB203580 partially suppressed these processes. Collectively, TGF-β2–induced production of type 1 collagen is suppressed by p38 inhibition and accompanied by partial inactivation of Smad2/3, in human trabecular meshwork cells.