Contributors: Motoyasu Satou, Hiroyuki Kaiya, Yoshihiro Nishi, Akio Shinohara, Shin-ichiro Kawada, Mikiya Miyazato, Kenji Kangawa, Hiroyuki Sugimoto
... Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1–27], [1–19], [1–16] and [1–15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5′-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals.
Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk
Contributors: Mari Miyamoto, Hiroshi M. Ueno, Masayuki Watanabe, Yumi Tatsuma, Yasuyuki Seto, Taku Miyamoto, Hadjime Nakajima
... Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120T) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45–50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of β-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from previously reported L. helveticus strains in terms of its optimal temperature and its degradation of β-casein. Therefore, the Mongolian L. helveticus strains differ from other strains of the species in different collections and are specifically suited for the natural lactic acid bacterial population in airag.
Contributors: Na Yu, Guy Smagghe
... Cholecystokinin (CCK) and gastrin are regulatory peptides in vertebrates. Their homologues are widely present in metazoan animals, in form of cionin in tunicates, neuropeptide-like protein 12 in nematodes and sulfakinin (SK) in arthropods. CCK(-like) peptides exert diverse physiological effects through binding their corresponding receptors, which are important members of the hormone-binding G-protein-coupled receptors.
Contributors: F. Jerry Reen, Matthieu Barret, Emilie Fargier, Marcus O’Muinneacháin, Fergal O’Gara
... Signal perception and transduction through tightly coordinated circuits is integral to the survival and persistence of microbes in diverse ecological niches. The capacity to adapt to changes in the environment is central to their ability to thrive under adverse circumstances. Signal dependent transcriptional regulators are a key mechanism through which microbes assimilate environmental cues and mediate the appropriate adaptive response. By far the largest class of transcriptional regulator is the LysR-class, which is universally distributed among bacteria, archaea, and even eukaryotic organisms. The number of LysR-Type Transcriptional Regulators (LTTRs) varies among species with one of the largest repertoires encoded in the genome of the nosocomial pathogen Pseudomonas aeruginosa. To understand the evolutionary basis for this, we undertook to analyse the relationship between the LTTRs, both at the species and genus level. Phylogenetic analysis of the complete Pseudomonas LTTR dataset revealed significant cluster patterns based on full length and domain analysis. Interestingly, evidence of acquisition through horizontal gene transfer was rare, with divergent evolution apparently favoured. Furthermore, genes that appear to have been acquired, as well as those with a non-classical topological arrangement were clustered in distinct groups in the phylogenetic trees, indicating some ancestral association. The conservation within clusters identified in this study will provide a useful platform for future molecular analyses.