the influence of lentinus edodes (shiitake mushroom) preparations on bacteriological and morphological aspects of the small intestine in piglets

Among substances intended to replace growth promoting antibiotics in pig nutrition, non-digestible oligosaccharides or polysaccharides could be potential alternative compounds. Therefore, the influence of β-1,3-1,6 glucans on bacteriological, biochemical and morphological aspects of the small intestine in weaned piglets was investigated. As sources of β-glucans, Lentinan (extract of Lentinus edodes mycelium) or dried L. edodes mycelium were added to the diet. Four homogenous groups of 5 newly weaned piglets (4 weeks of age) received one of four diets: control diet (C), C supplemented with Avilamycin (50?mg/kg, positive control), C supplemented with 0.1% of Lentinan and C supplemented with 5% of dried L. edodes mycelium powder. A first group of 10 piglets was euthanized after 11 days and the remaining 10 on day 12 of the experiment. The gastrointestinal tract was divided in segments and samples taken from digesta (stomach, proximal and distal jejunum, caecum), mucosal scrapings (jejunum) and ring shaped tissue samples (1?cm) of proximal and distal jejunum. Bacterial counts were made with digesta and mucosal samples, and short-chain fatty acids (SCFA), lactic acid and ammonia concentrations were determined. Tissue samples of both jejunal sites were embedded in paraffin wax for morphometrical (villus length, crypt depth) and histological observations (numbers of intraepithelial lymphocytes (IEL), goblet cells, apoptotic enterocytes on villi, mitotic cells in crypts). Only the diet containing 5% of dried L. edodes consistently resulted in lower viable counts (ca. 1?–?2 log₁₀ CFU) of total bacteria, E. coli, streptococci and lactic acid bacteria, and luminal and mucosal effects agreed very well. With this diet, acetate and butyrate concentrations in the distal jejunum were doubled, which is favourable in view of the trophic effect on enterocytes and colonocytes. Villus length (V) was increased with both diets containing β-glucans while crypt depth (C) was not altered, but V/C was higher. IEL counts were decreased by both diets although bacterial numbers, which is only one parameter of bacterial load, were only diminished with the L. edodes feed. The three supplemented feeds lowered the number of apoptotic enterocytes on the villi, but these numbers were very low (control diet?:?44 cells per 100 villi), making clear interpretation difficult. The mitotic index was slightly lower with the L. edodes feed, although not statistically significant. Decreased viable counts observed with the latter diet is a favourable effect as it is accepted that a lower bacterial load causes lower turnover rates of the intestinal epithelial cells, while there is also less competition for specific substrates. A higher V/C ratio, a smaller number of IEL in the epithelium and a lower apoptotic index also indicate slower turnover rate of the mucosa when Lentinan and L. edodes diets were fed. The inconsistent effects observed with Lentinan were probably due to the low amount added to the diet. It should be taken into account that the influence of L. edodes mycelium powder was more likely due to the presence of antibacterial compounds (eg. lenthionine, lentinamycin, terpenoids, polyphenols), rather than to an immunostimulating action of β-glucans with increased release of IgA onto the mucosa surface.     

Characterisation of antimony-resistant Leishmania donovani isolates: biochemical and biophysical studies and interaction with host cells

Recent clinical isolates of Leishmania donovani from the hyperendemic zone of Bihar were characterised in vitro in terms of their sensitivity towards sodium stibogluconate in a macrophage culture system. The resulting half maximal effective concentration (EC(50)) values were compared with those of known sensitive isolates. Fifteen of the isolates showed decreased sensitivity towards SSG with an average EC(50) of 25.7 ± 4.5 μg/ml pentavalent antimony (defined as antimony resistant), whereas nine showed considerable sensitivity with an average EC(50) of 4.6 ± 1.7 μg/ml (defined as antimony sensitive). Out of those nine, seven were recent clinical isolates and the remaining two were known sensitive isolates. Compared with the antimony sensitive, resistant isolates showed enhanced expression of thiol metabolising enzymes in varying degrees coupled with increased intracellular non-protein thiol content, decreased fluorescence anisotropy (inversely proportional with membrane fluidity) and over-expression of the terminal glycoconjugates (N-acetyl-d-galactosaminyl residue). Macrophages infected with resistant but not with sensitive showed up-regulation of the ATP Binding Cassette transporter multidrug resistance protein 1 and permeability glycoprotein, while the supernatant contained abundant IL-10. The above results reinforce the notion that antimony resistant parasites have undergone a number of biochemical and biophysical changes as part of their adaptation to ensure their survival in the host.     

Role of the inositol 1,4,5-trisphosphate receptor/Ca2+-release channel in autophagy

ABSTRACT: Autophagy is an important cell-biological process responsible for the disposal of long-lived proteins, protein aggregates, defective organelles and intracellular pathogens. It is activated in response to cellular stress and plays a role in development, cell differentiation, and ageing. Moreover, it has been shown to be involved in different pathologies, including cancer and neurodegenerative diseases. It is a long standing issue whether and how the Ca2+ ion is involved in its regulation. The role of the inositol 1,4,5-trisphosphate receptor, the main intracellular Ca2+-release channel, in apoptosis is well recognized, but its role in autophagy only recently emerged and is therefore much less well understood. Positive as well as negative effects on autophagy have been reported for both the inositol 1,4,5-trisphosphate receptor and Ca2+. This review will critically present the evidence for a role of the inositol 1,4,5-trisphosphate receptor/Ca2+-release channel in autophagy and will demonstrate that depending on the cellular conditions it can either suppress or promote autophagy. Suppression occurs through Ca2+ signals directed to the mitochondria, fueling ATP production and decreasing AMP-activated kinase activity. In contrast, Ca2+-induced autophagy can be mediated by several pathways including calmodulin-dependent kinase kinase β, calmodulin-dependent kinase I, protein kinase C θ, and/or extracellular signal-regulated kinase.     

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability.     

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles

Introduction

Fibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.

Methods

Citrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.

Results

Five citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit_1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit_1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).

Conclusions

Fibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.     

Automatically extracting functionally equivalent proteins from SwissProt

Background  

There is a frequent need to obtain sets of functionally equivalent homologous proteins (FEPs) from different species. While it is usually the case that orthology implies functional equivalence, this is not always true; therefore datasets of orthologous proteins are not appropriate. The information relevant to extracting FEPs is contained in databanks such as UniProtKB/Swiss-Prot and a manual analysis of these data allow FEPs to be extracted on a one-off basis. However there has been no resource allowing the easy, automatic extraction of groups of FEPs – for example, all instances of protein C.     

Improved genome assembly and evidence-based global gene model set for the chordate Ciona intestinalis: new insight into intron and operon populations

Background

The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly.

Results

We have prepared a less-fragmented assembly on the basis of scaffold-joining guided by paired-end EST and bacterial artificial chromosome (BAC) sequences, and BAC chromosomal in situ hybridization data. The new assembly (115.2 Mb) is similar in length to the initial assembly (116.7 Mb) but contains 1,272 (approximately 50%) fewer scaffolds. The largest scaffold in the new assembly incorporates 95 initial-assembly scaffolds. In conjunction with the new assembly, we have prepared a greatly improved global gene model set strictly correlated with the extensive currently available EST data. The total gene number (15,254) is similar to that of the initial set (15,582), but the new set includes 3,330 models at genomic sites where none were present in the initial set, and 1,779 models that represent fusions of multiple previously incomplete models. In approximately half, 5'-ends were precisely mapped using 5'-full-length ESTs, an important refinement even in otherwise unchanged models.

Conclusion

Using these new resources, we identify a population of non-canonical (non-GT-AG) introns and also find that approximately 20% of Ciona genes reside in operons and that operons contain a high proportion of single-exon genes. Thus, the present dataset provides an opportunity to analyze the Ciona genome much more precisely than ever.     

Monitoring of residues of ochratoxin A in blood and kidney of chicken using HPLC-FLD

Detoxification of ochratoxin A can be achieved by chemical or enzymatic hydrolyzation, the products of such reactions are ochratoxin α and phenylalanine. Ochratoxin α like ochratoxin A, is a fluorescing molecule, therefore sensitive analysis is possible at very low concentration levels. Methods have been established that make it possible to look for residues of ochratoxin A and its main metabolite ochratoxin α in blood and tissues at very low concentration levels. Plasma is extracted by the use of small amounts of chloroform; the extract is cleaned with water and afterwards evaporated to dryness]. The residue is re-dissolved and analysed by HPLC-FLD. Using this method a limit of detection of 0.5μg/l for both ochratoxin A and ochratoxin α can be reached.