Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.     

Altered infant feeding patterns in boys with acquired nonsyndromic cryptorchidism

BACKGROUND : Genetic and environmental factors likely influence susceptibility to nonsyndromic cryptorchidism, a common disease presenting at birth or in later childhood. We compared cases and controls to define differential risk factors for congenital versus acquired cryptorchidism. METHODS : We compared questionnaire and clinical data from cases of congenital cryptorchidism (n = 230), acquired cryptorchidism (n = 182) and hernia/hydrocele (n = 104) with a group of healthy male controls (n = 358). Potential predictor variables (p < 0.2 in univariable analysis) were included in stepwise multivariable logistic regression models. RESULTS : Temporary (odds ratio [OR], 0.5; 95% confidence interval [CI], 0.4–0.8) or exclusive (OR, 0.6; 95% CI, 0.4–0.9) breastfeeding was reduced and soy formula feeding increased (OR, 1.8; 95% CI, 1.2–2.9) in acquired but not congenital or hernia/hydrocele groups. The highest risk estimates were observed for primary soy formula feeding with limited or no breastfeeding (OR 2.5; 95% CI, 1.4–4.3; adjusted OR, 2.7; 95% CI, 1.4–5.4) in the acquired group. Primary feeding risk estimates were equivalent or strengthened when multivariable models were limited to age greater than 2 years, full‐term or not small for gestational age, or Caucasian subjects. Pregnancy complications and increased maternal exposure to cosmetic or household chemicals were not consistently associated with either form of cryptorchidism in these models. CONCLUSIONS : Our data support reduced breastfeeding and soy formula feeding as potential risk factors for acquired cryptorchidism. Although additional studies are needed, hormonally active components of breast milk and soy formula could influence the establishment of normal testis position in the first months of life, leading to apparent ascent of testes in childhood. Birth Defects Research (Part A), 2012. © 2012 Wiley Periodicals, Inc.     

Crystal structure of the serine protease domain of prophenoloxidase activating factor-I

A family of serine proteases (SPs) mediates the proteolytic cascades of embryonic development and immune response in invertebrates. These proteases, called easter-type SPs, consist of clip and chymotrypsin-like SP domains. The SP domain of easter-type proteases differs from those of typical SPs in its primary structure. Herein, we report the first crystal structure of the SP domain of easter-type proteases, presented as that of prophenoloxidase activating factor (PPAF)-I in zymogen form. This structure reveals several important structural features including a bound calcium ion, an additional loop with a unique disulfide linkage, a canyon-like deep active site, and an exposed activation loop. We subsequently show the role of the bound calcium and the proteolytic susceptibility of the activation loop, which occurs in a clip domain-independent manner. Based on biochemical study in the presence of heparin, we suggest that PPAF-III, highly homologous to PPAF-I, contains a surface patch that is responsible for enhancing the catalytic activity through interaction with a nonsubstrate region of a target protein. These results provide insights into an activation mechanism of easter-type proteases in proteolytic cascades, in comparison with the well studied blood coagulation enzymes in mammals.     

Occurrence and molecular identification of the zoysiagrass mite Aceria zoysiae (Acari: Eriophyidae) in turfgrasses in Korea

Mites are one of the serious pests of turfgrass. Our survey of turfgrass fields from 2013 to 2015 in Korea showed that the occurrence of leaf chlorotic symptom has gradually extended to at least 60% of the examined golf courses. We identified the zoysiae mite Aceria zoysiae in most damaged zoysiagrasses. Artificial infestation of A. zoysiae into zoysiagrasses in pots resulted in symptoms of chlorosis and marginal rolling of the leaves within 3 weeks. We firstly determined the nucleotide sequence of mitochondrial cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA region of A. zoysiae. The variations in COI and ITS2 between A. zoysiae and other species of the genus were 20.9%–43.0% and 7.5%–67.3%, respectively, suggesting significant genetic divergence within the genus. Our study provides valuable information for the rapid diagnosis and infestation monitoring of A. zoysiae in turfgrass fields.     

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.     

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1^‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l ‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1^‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.     

Modulation of auxin signalling through DIAGETROPICA and ENTIRE differentially affects tomato plant growth via changes in photosynthetic and mitochondrial metabolism

Auxin modulates a range of plant developmental processes including embryogenesis, organogenesis, and shoot and root development. Recent studies have shown that plant hormones also strongly influence metabolic networks, which results in altered growth phenotypes. Modulating auxin signalling pathways may therefore provide an opportunity to alter crop performance. Here, we performed a detailed physiological and metabolic characterization of tomato (Solanum lycopersicum) mutants with either increased (entire) or reduced (diageotropica—dgt) auxin signalling to investigate the consequences of altered auxin signalling on photosynthesis, water use, and primary metabolism. We show that reduced auxin sensitivity in dgt led to anatomical and physiological modifications, including altered stomatal distribution along the leaf blade and reduced stomatal conductance, resulting in clear reductions in both photosynthesis and water loss in detached leaves. By contrast, plants with higher auxin sensitivity (entire) increased the photosynthetic capacity, as deduced by higher V_cmax and J_max coupled with reduced stomatal limitation. Remarkably, our results demonstrate that auxin‐sensitive mutants (dgt) are characterized by impairments in the usage of starch that led to lower growth, most likely associated with decreased respiration. Collectively, our findings suggest that mutations in different components of the auxin signalling pathway specifically modulate photosynthetic and respiratory processes.