Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.     

Different isoforms of fasciclin II are expressed by a subset of developing olfactory receptor neurons and by olfactory-nerve glial cells during formation of glomeruli in the moth Manduca sexta

During development of the primary olfactory projection, olfactory receptor axons must sort by odor specificity and seek particular sites in the brain in which to create odor-specific glomeruli. In the moth Manduca sexta, we showed previously that fasciclin II, a cell adhesion molecule in the immunoglobulin superfamily, is expressed by the axons of a subset of olfactory receptor neurons during development and that, in a specialized glia-rich "sorting zone," these axons segregate from nonfasciclin II-expressing axons before entering the neuropil of the glomerular layer. The segregation into fasciclin II-positive fascicles is dependent on the presence of the glial cells in the sorting zone. Here, we explore the expression patterns for different isoforms of Manduca fasciclin II in the developing olfactory system. We find that olfactory receptor axons express transmembrane fasciclin II during the period of axonal ingrowth and glomerulus development. Fascicles of TM-fasciclin II+ axons target certain glomeruli and avoid others, such as the sexually dimorphic glomeruli. These results suggest that TM-fasciclin II may play a role in the sorting and guidance of the axons. GPI-linked forms of fasciclin II are expressed weakly by glial cells associated with the receptor axons before they reach the sorting zone, but not by sorting-zone glia. GPI-fasciclin II may, therefore, be involved in axon-glia interactions related to stabilization of axons in the nerve, but probably not related to sorting.     

Functional Conservation of Calreticulin in Euglena gracilis

Calreticulin is the major high capacity, low affinity Ca²⁺ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca²⁺ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca²⁺ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca²⁺ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phytogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca²⁺ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore iii that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca²⁺ binding function of calreticulin from Euglena traces the functional history of Ca²⁺ second messenger signal transduction pathways deep into eukaryotic evolution.     

Chromosome location of the ribosomal genes in Triturus vulgaris meridionalis (Amphibia Urodela)

In Triturus vulgaris meridionalis, the 18S + 28S rDNA sequences have been shown to be located in a number of additional chromosomal sites besides the nucleolus organizing region. The additional ribosomal sites have been found to vary as to their number and chromosomal location in different individuals of the species.—The data presented in this study concern the chromosomal distribution of the ribosomal sequences as analyzed by in situ hybridization technique in two individuals as well as in their offspring. The evidence obtained by this analysis indicates quite clearly that all 18S + 28S rRNA sites present in each individual genome are inherited according to simple mendelian principles.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA DNA coding for 18S+28S rRNA plus the intervening spacers - SSC 0.15M Sodium chloride, 0.015 M Sodium citrate, pH 7 - RNase ribonuclease     

Chromosomal localization of 18S + 28S and 5S ribosomal RNA genes in evolutionarily diverse anuran amphibians

The chromosomal locations of the 18S + 28S and 5S ribosomal RNA genes have been analyzed by in situ hybridization in ten anuran species of different taxonomic positions. The chosen species belong to both primitive and evolved families of the present day Anura. Each examined species has 18S + 28S rRNA genes clustered in one locus per haploid chromosome set: this locus is placed either in an intercalary position or proximal to the centromere, or close to the telomere. The 5S rRNA genes are arranged in clusters which vary in number from one to six per haploid set. The 5S rDNA sites are found in intercalary positions, at the telomeres, and at, or close to, the centromeres. Microchromosomes and small chromosomes in primitive karyotypes have been found to carry 5S rDNA sequences. The results are discussed in relation to ideas on the karyological evolution of Amphibia.     

Identifying specific matrix metalloproteinase-2-inhibiting peptides through phage display-based subtractive screening

Gelatinases A and B, which are members of the matrix metalloproteinase (MMP) family, play essential roles in cancer development and metastasis, as they can break down basal membranes. Therefore, the determination and inhibition of gelatinases is essential for cancer treatment. Peptides that can specifically block each gelatinase may, therefore, be useful for cancer treatment. In this study, subtractive panning was carried out using a 12-mer peptide library to identify peptides that block gelatinase A activity (MMP-2), which is a key pharmacological target. Using this method, 17 unique peptide sequences were determined. MMP-2 inhibition by these peptides was evaluated through zymogram analyses, which revealed that four peptides inhibited MMP-2 activity by at least 65%. These four peptides were synthesized and used for in vitro wound healing using human umbilical vein endothelial cells, and two peptides, AOMP12 and AOMP29, were found to inhibit wound healing by 40%. These peptides are, thus, potential candidates for MMP-2 inhibition for cancer treatment. Furthermore, our findings suggest that our substractive biopanning screening method is a suitable strategy for identifying peptides that selectively inhibit MMP-2.     

Circulating miR-184 is a potential predictive biomarker of cardiac damage in Anderson–Fabry disease

Enzyme replacement therapy (ERT) is a mainstay of treatment for Anderson–Fabry disease (AFD), a pathology with negative effects on the heart and kidneys. However, no reliable biomarkers are available to monitor its efficacy. Therefore, we tested a panel of four microRNAs linked with cardiac and renal damage in order to identify a novel biomarker associated with AFD and modulated by ERT. To this end, 60 patients with a definite diagnosis of AFD and on chronic ERT, and 29 age- and sex-matched healthy individuals, were enrolled by two Italian university hospitals. Only miR-184 met both conditions: its level discriminated untreated AFD patients from healthy individuals (c-statistic = 0.7522), and it was upregulated upon ERT (P < 0.001). On multivariable analysis, miR-184 was independently and inversely associated with a higher risk of cardiac damage (odds ratio = 0.86; 95% confidence interval [CI] = 0.76–0.98; P = 0.026). Adding miR-184 to a comprehensive clinical model improved the prediction of cardiac damage in terms of global model fit, calibration, discrimination, and classification accuracy (continuous net reclassification improvement = 0.917, P < 0.001; integrated discrimination improvement [IDI] = 0.105, P = 0.017; relative IDI = 0.221, 95% CI = 0.002–0.356). Thus, miR-184 is a circulating biomarker of AFD that changes after ERT. Assessment of its level in plasma could be clinically valuable in improving the prediction of cardiac damage in AFD patients.Subject terms: Prognostic markers, Cardiovascular diseases     

Characterization of ISRgn1, a Novel Insertion Sequence of the IS3 Family Isolated from a Bacteriocin-Negative Mutant of Ruminococcus gnavus E1

ISRgn1, an insertion sequence of the IS3 family, has been identified in the genome of a bacteriocin-negative mutant of Ruminococcus gnavus E1. The copy number of ISRgn1 in R. gnavus E1, as well as its distribution among phylogenetically E1-related strains, has been determined. Results obtained suggest that ISRgn1 is not indigenous to the R. gnavus phylogenetic group but that it can transpose in this bacterium.     

Sperm structure and spermiogenesis in Coletinia sp. (Nicoletiidae, Zygentoma, Insecta) with a comparative analysis of sperm structure in Zygentoma

The spermatozoon of Coletinia sp. has a bilayered acrosome, a short nucleus (4 microm) and a relatively short sperm tail with two mitochondrial derivatives. The chromatin is uniformly dense except for several electron-lucid channels or strands which permeate the nucleus and which originate in the spermatid as invaginations of the nuclear envelope. The invaginations occur mostly or exclusively along two meridians of the spermatid that are also characterized by the presence of a longitudinal rod of medium electron density. The two rods (designated as 'mid-spermatid rods') evidently are instrumental in the formation of the electron-lucid channels. The significance of this elaborate system of intranuclear channels is not understood. The sperm tail has a 9 + 9 + 2 axoneme with each of the nine microtubular doublets accompanied by an accessory microtubule; scant intertubular material can also be distinguished. Hence, the tail axoneme resembles that of many pterygote insects. Each of the two mitochondrial derivatives contains a crystalline inclusion that has periodically spaced layers going in different directions on either side of the midline. Two synapomorphic traits appear to be shared by Ateluridae and Nicoletiidae, namely the invaginations of the nuclear membrane along two meridians of the nucleus and the shape of the crystalline inclusions of the mitochondrial derivatives. Four species from the family Lepismatidae were also examined as to their sperm ultrastructure. Three of them, Allacrotelsa kraepelini, Ctenolepisma longicaudata and Ctenolepisma sp., were found to be very similar to the two previously examined lepismatids, Thermobia domestica and Lepisma saccharina. On the other hand, spermatozoa of Tricholepisma aurea were aggregated in small groups rather than pairwise joined as seen in the other lepismatids. Sperm characters are also used to reconstruct a phylogenetic hypothesis which suggests a close relationship between Ateluridae and Nicoletiidae.     

Three new observational scales for use in Dutch nursing homes: scales from the Resident Assessment Instrument for Activities of Daily Living, cognition and depression

The reliability and validity of three MDS scales for ADL, cognition and depression are described. The scales consist of items of the Minimum Data Set of the Resident Assessment Instrument and are available just after an MDS assessment. Data collection took place in nine Dutch nursing homes (N = 227) and consisted of three MDS assessments within one month to determine reliability. Several criterion measures were assessed in order to determine convergent validity. Intra- and inter-rater reliability and internal consistency were determined as well as correlation coefficients of the criterion measures and the MDS scales. All three MDS scales appear reliable, especially the ADL-Hierarchy has very good psychometric properties (intra- and inter-rater Intra Class Correlation were 0.81 and 0.83, respectively). Convergent validity of the ADL-Hierarchy and the Cognitive Performance Scale is good, the Depression Rating Scale appears valid in residents with moderate cognitive disorders at the most, but the results are more difficult to interpret in residents with severe cognitive disorders. The MDS scales appear useful in clinical practice and for research purposes in the Dutch nursing homes.