Inhibition of a Membrane-Bound Enkephalin-Degrading Aminopeptidase by Bestatin Analogs

Abstract: A variety of bestatin analogs were examined as potent inhibitors of a membrane-bound enkephalin-degrading aminopeptidase that was purified from monkey brain. Bestatinyl amino acid derivatives showed strong inhibition of this enzyme. The most effective was bestatin- l -Arg AcOH, with a K _i value of 0.21 × 10⁻⁸ M with Leu-enkephalin as substrate. It exhibited competitive kinetics and was about 100-fold more potent than bestatin. This compound seems to be useful for pharmacological and other studies.     

Loss of lysophospholipase 3 increases atherosclerosis in apolipoprotein E-deficient mice

Human LCAT-like lysophospholipase (LLPL), or lysophospholipase 3, was first identified in vitro, in foam cells derived from THP-1 cells. We demonstrated that LLPL was present in foam cells in the severe atherosclerotic lesions that develop in apolipoprotein E-null (apoE(-/-)) mice. This indicated that LLPL might affect lipid metabolisms in foam cells and, therefore, atherogenesis. Accordingly, we created LLPL-knockout mice by gene targeting and crossed them with apoE(-/-) mice. We showed that the absence of LLPL increased lesion formation markedly in apoE(-/-) mice but had little effect on the plasma-lipid profile. In addition, LLPL-deficient peritoneal macrophages were more sensitive to apoptosis induced by exposure to oxidized low-density lipoprotein. LLPL might provide a link between apoptosis in macrophages and atherogenesis. Our data demonstrate that LLPL activity is anti-atherogenic and indicate that the regulation of this enzyme might be a novel drug target for the treatment of atherosclerosis.     

Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development

Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine involved in both acquired and innate immunity. MIF also has many functions outside the immune system, such as isomerase and oxidoreductase activities and control of cell proliferation. Considering the involvement of MIF in various intra- and extracellular events, we expected that MIF might also be important in vertebrate development. To elucidate the possible role of MIF in developmental processes, we knocked down MIF in embryos of the African clawed frog Xenopus laevis, using MIF-specific morpholino oligomers (MOs). For the synthesis of the MOs, we cloned a cDNA for a Xenopus homolog of MIF. Sequence analysis, determination of the isomerase activity, and x-ray crystallographic analysis revealed that the protein encoded by the cDNA was the ortholog of mammalian MIF. We carried out whole mount in situ hybridization of MIF mRNA and found that MIF was expressed at high levels in the neural tissues of normal embryos. Although early embryogenesis of MO-injected embryos proceeded normally until the gastrula stage, their neurulation was completely inhibited. At the tailbud stage, the MO-injected embryos lacked neural and mesodermal tissues, and also showed severe defects in their head and tail structures. Thus, MIF was found to be essential for axis formation and neural development of Xenopus embryos.     

X-ray structures of NADPH-dependent carbonyl reductase from Sporobolomyces salmonicolor provide insights into stereoselective reductions of carbonyl compounds

The X-ray structures of red yeast Sporobolomyces salmonicolor carbonyl reductase (SSCR) and its complex with a coenzyme, NADPH, have been determined at a resolution of 1.8A and 1.6A, respectively. SSCR was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=54.86 A, b=83.49 A, and c=148.72 A. On its cocrystallization with NADPH, isomorphous crystals of the SSCR/NADPH complex were obtained. The structure of SSCR was solved by a single wavelength anomalous diffraction measurement using a selenomethionine-substituted enzyme, and that of the SSCR/NADPH complex was solved by a molecular replacement method using the solved structure of SSCR. The structures of SSCR and the SSCR/NADPH complex were refined to an R-factor of 0.193 (R(free)=0.233) and 0.211 (R(free)=0.238), respectively. SSCR has two domains, an NADPH-binding domain and a substrate-binding domain, and belongs to the short-chain dehydrogenases/reductases family. The structure of the NADPH-binding domain and the interaction between the enzyme and NADPH are very similar to those found in other structure-solved enzymes belonging to the short-chain dehydrogenases/reductases family, while the structure of the substrate-binding domain is unique. SSCR has stereoselectivity in its catalytic reaction, giving rise to excessive production of (S)-alcohols from ethyl 4-chloro-3-oxobutanoate. The X-ray structure of the SSCR/NADPH complex and preliminary modeling show that the formation of the hydrophobic channel induced by the binding of NADPH is closely related to the stereoselective reduction by SSCR.     

Appropriate preoperative membranous urethral length predicts recovery of urinary continence after robot-assisted laparoscopic prostatectomy

Purpose

We investigated that preoperative membranous urethral length (MUL) would be associated with the recovery of urinary continence after robot-assisted laparoscopic prostatectomy (RALP).

Patients and methods

We studied 204 patients who underwent RALP between May 2013 and March 2016. All patients underwent pelvic magnetic resonance imaging (MRI) preoperatively to measure MUL. Urinary continence was defined as the use of one pad or less (safety pad). The 204 patients were divided into two groups: continence group, those who achieved recovery of continence at 3, 6, and 12?months after RALP, and incontinence group, those who did not. We retrospectively analyzed the patients in terms of preoperative clinical factors including age, body mass index (BMI), estimated prostate volume, neurovascular bundle salvage, history of preoperative hormonal therapy, and MUL.

Results

The safety pad use rate was 69.6%, 86.9%, and 91.1% at 3, 6, and 12?months, respectively. On univariate and multivariate analyses, MUL were significant factors in every term of recovery of urinary continence in both groups. According to the receiver operating characteristic (ROC) curve analysis, the preoperative MUL that could best predict early recovery of urinary continence at 3 months after RALP was 12?mm.

Conclusions

We suggest that preoperative MUL >?12?mm would be a predictor of early recovery of urinary continence after RALP.

     

Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.     

Recent progress in studies on the health benefits of pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ), an aromatic tricyclic o-quinone, was identified initially as a redox cofactor for bacterial dehydrogenases. Although PQQ is not biosynthesized in mammals, trace amounts of PQQ have been found in human and rat tissues because of its wide distribution in dietary sources. Importantly, nutritional studies in rodents have revealed that PQQ deficiency exhibits diverse systemic responses, including growth impairment, immune dysfunction, and abnormal reproductive performance. Although PQQ is not currently classified as a vitamin, PQQ has been implicated as an important nutrient in mammals. In recent years, PQQ has been receiving much attention owing to its physiological importance and pharmacological effects. In this article, we review the potential health benefits of PQQ with a focus on its growth-promoting activity, anti-diabetic effect, anti-oxidative action, and neuroprotective function. Additionally, we provide an update of its basic pharmacokinetics and safety information in oral ingestion.     

Oxidative Deamination of Serum Albumins by (-)-Epigallocatechin-3-O-Gallate: A Potential Mechanism for the Formation of Innate Antigens by Antioxidants

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies.