In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis.

We examined the different steps necessary for the enzymatic digestion of proteins in the polyacrylamide matrix after gel electrophoresis. As a result, we developed an improved method for obtaining peptides for internal sequence analysis from 1-2 micrograms of in-gel-digested proteins. The long washing-lyophilization-equilibration steps necessary to eliminate the dye, sodium dodecyl sulfate, and other gel-associated contaminants that perturb protein digestion in Coomassie blue-stained gels have been replaced by washing for 40 min with 50% acetonitrile, drying for 10 min at room temperature, and then rehydrating with a protease solution. The washing and drying steps result in a substantial reduction of the gel slice volume that, when next swollen in the protease solution, readily absorbs the enzyme, facilitating digestion. The Coomassie blue staining procedure has also been modified by reducing acetic acid and methanol concentrations in the staining solution and by eliminating acetic acid in the destaining solution. The peptides resulting from the in-gel digestion are easily recovered by passive elution, in excellent yields for structural characterization. This simple and rapid method has been successfully applied for the internal sequence analysis of membrane proteins from the rat mitochondria resolved in preparative two-dimensional gel electrophoresis.     

Identification of the root of Punica granatum in galenic preparations using TLC

The alkaloids present in the root of "Punica granatum" have been extracted by two different methods: extraction by Soxlet and extraction by steam distillation. Then the extracts have been compared by TLC chromatography using different solvents and specific chromogen reagents. The presence of pseudo-pelletierine has been confirmed in both the extractive solution by reaction with conc. K2Cr2O7. The above results explains the toxic activity of the unsuitable galenic preparations.     

A PTG variant contributes to a milder phenotype in Lafora disease

Lafora disease is an autosomal recessive form of progressive myoclonus epilepsy with no effective therapy. Although the outcome is always unfavorable, onset of symptoms and progression of the disease may vary. We aimed to identify modifier genes that may contribute to the clinical course of Lafora disease patients with EPM2A or EPM2B mutations. We established a list of 43 genes coding for proteins related to laforin/malin function and/or glycogen metabolism and tested common polymorphisms for possible associations with phenotypic differences using a collection of Lafora disease families. Genotype and haplotype analysis showed that PPP1R3C may be associated with a slow progression of the disease. The PPP1R3C gene encodes protein targeting to glycogen (PTG). Glycogen targeting subunits play a major role in recruiting type 1 protein phosphatase (PP1) to glycogen-enriched cell compartments and in increasing the specific activity of PP1 toward specific glycogenic substrates (glycogen synthase and glycogen phosphorylase). Here, we report a new mutation (c.746A>G, N249S) in the PPP1R3C gene that results in a decreased capacity to induce glycogen synthesis and a reduced interaction with glycogen phosphorylase and laforin, supporting a key role of this mutation in the glycogenic activity of PTG. This variant was found in one of two affected siblings of a Lafora disease family characterized by a remarkable mild course. Our findings suggest that variations in PTG may condition the course of Lafora disease and establish PTG as a potential target for pharmacogenetic and therapeutic approaches.     

Holographic imaging of unlabelled sperm cells for semen analysis: a review

Male reproductive health in both humans and animals is an important research field in biological study. In order to characterize the morphology, the motility and the concentration of the sperm cells, which are the most important parameters to feature them, digital holography demonstrated to be an attractive technique. Indeed, it is a label‐free, non‐invasive and high‐resolution method that enables the characterization of live specimen. The review is intended both for summarizing the state‐of‐art on the semen analysis and recent achievement obtained by means of digital holography and for exploring new possible applications of digital holography in this field.

Quantitative phase maps of living swimming spermatozoa.     

<Emphasis Type="Italic">Salinivibrio sharmensis</Emphasis> sp. nov., a novel haloalkaliphilic bacterium from a saline lake in Ras Mohammed Park (Egypt)

A novel haloalkaliphilic, facultative anaerobic and Gram-negative Salinivibrio-like microorganism (designated strain BAG^T) was recovered from a saline lake in Ras Mohammed Park (Egypt). Cells were motile, curved rods, not spore-forming and occurred singly. Strain BAG^T grew optimally at 35°C (temperature growth range 25–40°C) with 10.0% (w/v) NaCl [NaCl growth range 6.0–16.0% (w/v)] and at pH 9.0 (pH growth range 6.0–10.0). Strain BAG^T had phosphatidylethanolamine (PEA) and phosphatidylglycerol (PG) as the main polar lipids, C16:0 (54.0%) and C16:1 (26.0%) as the predominant cellular fatty acids and Q-8 as the major respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BAG^T was a member of Salinivibrio genus, with the highest sequence similarities of 99.1, 98.4 and 98.1% to Salinivibrio siamensis JCM 14472^T, Salinivibrio proteolyticus DSM 19052^T and Salinivibrio costicola subsp. alcaliphilus DSM 16359^T, respectively. DNA–DNA hybridization values of strain BAG^T with members of Salinivibrio genus were lower than 55.0%. DNA G + C content was 51.0 mol%. On the basis of the polyphasic taxonomic results revealed in this study, strain BAG^T should be classified as a novel species of Salinivibrio genus, for which the name Salinivibrio sharmensis sp. nov. is proposed, with the type strain BAG^T (=ATCC BAA-1319^T = DSM 18182^T).     

Heterozygous Germline Mutations in the CBL Tumor-Suppressor Gene Cause a Noonan Syndrome-like Phenotype

RAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies.     

Thirteen type I loci from HSA4q, HSA6p, HSA7q and HSA12q were comparatively FISH-mapped in four river buffalo and sheep chromosomes

Thirteen goat BAC clones containing coding sequences from HSA7, HSA12q, HSA4 and HSA6p were fluorescence in situ mapped to river buffalo (Bubalus bubalis, BBU) and sheep (Ovis aries, OAR) R-banded chromosomes. The following type I loci were mapped: BCP to BBU8q32 and OAR4q32, CLCN1 to BBU8q34 and OAR4q34, IGFBP3 to BBU8q24 and OAR4q27, KRT to BBU4q21 and OAR 3q21, IFNG to BBU4q23 and OAR3q23, IGF1 to BBU4q31 and OAR3q31, GNRHR to BBU7q32 and OAR6q32, MTP to BBU7q21 and OAR6q15, PDE6B to BBU7q36 and OAR6q36, BF to BBU2p22 and OAR20q22, EDN1 to BBU2p24 and OAR20q24, GSTA1 to BBU2p22 and OAR20q22, OLADRB (MHC) to BBU2p22 and OAR20q22. All mapped loci appeared to be located on homologous chromosomes and chromosome bands in both bovids. Comparison between gene orders in bovid (BBU and OAR) and human (HSA) chromosomes revealed complex rearrangements, especially between BBU7/OAR6 and HSA4, as well as between BBU2p/OAR20 and HSA6p.     

Beet necrotic yellow vein virus coat protein-mediated protection in sugarbeet (Beta vulgaris L.) protoplasts

Transformed Beta vulgaris L. suspension cultures were obtained after cocultivation of sugarbeet cells with Agrobacterium tumefaciens harbouring a binary vector containing the coat protein gene of beet necrotic yellow vein virus inserted between the kanamycin resistance gene and a ß-glucuronidase reporter gene. Protoplasts were isolated both from untransformed cells, and from transformed cells expressing the viral coat protein, and both were then infected with beet necrotic yellow vein virus. Comparison of the levels of infectivity shows that the expression of the coat protein gene in sugarbeet protoplasts mediates high levels of protection against infection by beet necrotic yellow vein virus.Abbreviations TMV Tobacco Mosaic Virus - CP Coat Protein - BNYVV Beet Necrotic Yellow Vein Virus - ß-Glu ß-glucuronidase - MS Murashige and Skoog (1962) - PEG Polyethylene glycol - npt neomycin phosphotransferase - nos nopaline synthase - FITC fluoresceine isothiocyanate - IAA indole acetic acid - BAP benzyl amino purine - MES 2-[N-Morpholino]ethane sulfonic acid - IgG Immunoglobulin G - nt nucleotide     

Gut-Microbiota-Metabolite Axis in Early Renal Function Decline

Introduction

Several circulating metabolites derived from bacterial protein fermentation have been found to be inversely associated with renal function but the timing and disease severity is unclear. The aim of this study is to explore the relationship between indoxyl-sulfate, p-cresyl-sulfate, phenylacetylglutamine and gut-microbial profiles in early renal function decline.

Results

Indoxyl-sulfate (Beta(SE) = -2.74(0.24); P = 8.8x10^-29), p-cresyl-sulfate (-1.99(0.24), P = 4.6x10^-16), and phenylacetylglutamine(-2.73 (0.25), P = 1.2x10^-25) were inversely associated with eGFR in a large population base cohort (TwinsUK, n = 4439) with minimal renal function decline. In a sub-sample of 855 individuals, we analysed metabolite associations with 16S gut microbiome profiles (909 profiles, QIIME 1.7.0). Three Operational Taxonomic Units (OTUs) were significantly associated with indoxyl-sulfate and 52 with phenylacetylglutamine after multiple testing; while one OTU was nominally associated with p-cresyl sulfate. All 56 microbial members belong to the order Clostridiales and are represented by anaerobic Gram-positive families Christensenellaceae, Ruminococcaceae and Lachnospiraceae. Within these, three microbes were also associated with eGFR.

Conclusions

Our data suggest that indoxyl-sulfate, p-cresyl-sulfate and phenylacetylglutamine are early markers of renal function decline. Changes in the intestinal flora associated with these metabolites are detectable in early kidney disease. Future efforts should dissect this relationship to improve early diagnostics and therapeutics strategies.