Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.     

Purification and properties of arylmannosidases from mung bean seedlings and soybean cells

Two arylmannosidases (signified as A and B) were purified tohomogeneity from soluble and microsomal fractions of mung beanseedlings. Arylmannosidase A from the microsomes appeared thesame on native gels and on SDS gels as soluble arylmannosidaseA, the same was true for arylmannosidase B. Sedimentation velocitystudies indicated that both enzymes were homogeneous, and thatarylmannosidase A had a molecular mass of 237 kd while B hada molecular mass of 243 kd. Arylmannosidase A showed two majorprotein bands on SDS gels with molecular masses of 60 and 55kd, and minor bands of 79, 39 and 35 kd. All of these bandswere N-linked since they were susceptible to digestion by endo-glucosaminidaseH. In addition, at least the major bands could be detected byWestern blots with antibody raised against the xylose moietyof N-linked plant oligosaccharides, and they could also be labeledin soybean suspension cells with [2–3H]mannose. ArylmannosidaseB showed three major bands with molecular masses of 72, 55 and45 kd, and minor bands of 42 and 39 kd. With the possible exceptionof the 45 and 42 kd bands, all of these bands are glycoproteins.Arylmannosidases A and B showed somewhat different kineticsin terms of mannose release from high-mannose oligosaccharides,but they were equally susceptible to inhibition by swainsonineand mannostatin A. Polyclonal antibody raised against the arylmannosidaseB cross-reacted equally well with arylmannosidase A from mungbean seedlings and with arylmannosidase from soybean cells.However, monoclonal antibody against mung bean arylmannosidaseA was much less effective against arylmannosidase B. Antibodywas used to examine the biosynthesis and structure of the carbohydratechains of arylmannosidase in soybean cells grown in [2–³H]mannose.Treatment of the purified enzyme with Endo H released 50% ofthe radioactivity, and these labeled oligosaccharides were ofthe high-mannose type, i.e. mostly Man9GlcNAc. The precipitatedprotein isolated from the Endo H treatment still contained 50%of the radioactivity, and this was present in modified structuresthat probably contain xylose residues. Mung beans mannosidases glycoproteins -soybean--mannosidases xylose-containing N-linked glycoproteins     

Morpho-functional features in adolescent males under iodine deficiency of the Saratov region

An auxological study of 538 adolescent males (from 12 to 17) from different settlements of the Saratov region with various degrees of industrialization and iodine deficiency was carried out. All subjects have undergone an ultra-sound screening of thyroid volume to reveal the frequency of endemic goiter in each group. The results obtained during investigation showed the existence of deviations in the physical development of boys with goiter in skinfold thickness, body circumferences (chest, shoulder, and forearm), biacromial and biiliocristal diameters, transversal and sagittal chest diameters, body height and weight, BMI, leg length, and corpus length, all of which are greater in healthy adolescents (SD = 1.0, p = 0.000). In 46 subjects with endemic goiter, characteristics of metabolic status were investigated by the method of registration of endogenous intoxication (Malakhova, 1995). In comparison to the control group, a 1.2 times lower LAMM level in erythrocytes (p < 0.05) and 1.1 in urine (p < 0.05), and an increase by 2.3 in the LAMM level in blood plasma (p < 0.01) were detected. The relative percentage of catabolic substances exceeded the control values by 3.2 (p < 0.001). The OP level in erythrocytes is reduced by 2.2 (p < 0.01), in urine (p < 0.01) by 8.4. The OP level is higher in plasma by 3.0 (p < 0.01). The adolescents with endemic goiter have a reallocation of protein matter between erythrocytes, plasma and urine. The spectrogram of erythrocytes shows lowering metabolites on membrane frames, which testifies to the destruction of the structurally functional properties of erythrocytes, and a lowering of absorption properties in glycocalix erythrocytes.     

The use of supramolecular structures as protein ligands

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.     

Direct Regulation of Prokaryotic Kir Channel by Cholesterol

Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by ⁸⁶Rb⁺ uptake. Our results show that ⁸⁶Rb⁺ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses ⁸⁶Rb⁺ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated ⁸⁶Rb⁺ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.Inwardly rectifying potassium channels (Kir) are known to play critical roles in the regulation of multiple cellular functions including membrane excitability, heart rate, and vascular tone (1–3). Kir channels are classified into seven subfamilies (Kir1–7) identified by distinct biophysical properties and sensitivities to different regulators (2). Our earlier studies have shown that Kir2 channels, one of the major subfamilies of Kir that are responsible for maintaining membrane potential in a variety of cell types, are strongly suppressed by the elevation of membrane cholesterol (4, 5). Cholesterol-induced suppression of Kir2 was first observed in aortic endothelial cells (4), in which resting K⁺ conductance is dominated by Kir2.1 and Kir2.2 channels (6), and then when channels were heterologously expressed in Chinese hamster ovary cells (5, 7). Furthermore, the same effect was observed ex vivo in endothelial cells and bone marrow-derived progenitor cells isolated from hypercholesterolemic pigs (8, 9).In terms of the mechanism, the first insights came from comparing the effects of cholesterol and of its chiral analogue, epicholesterol. Although the two sterols are known to have almost identical effects on the biophysical properties of the lipid bilayer (10, 11), their impact on Kir activity is completely different; partial substitution of endogenous cholesterol with epicholesterol resulted in significant increase in Kir current in endothelial cells (4). These observations suggest that specific sterol-protein interactions may be involved in the cholesterol sensitivity of Kir2 channels. However, in the complex environment of the plasma membrane, cholesterol may interact not only with the channels themselves but also with other proteins, which in turn may regulate the activity of the channels. In the cellular environment, therefore, it is impossible to discriminate between direct channel-cholesterol interactions and indirect effects. Moreover, it is impossible to define the actual concentrations of cholesterol in any given membrane compartment. To quantitatively test direct cholesterol-protein interactions, it is necessary to examine sensitivity of pure Kir channels to membrane cholesterol in a membrane of defined lipid composition. To date, only the cytoplasmic domains of several mammalian Kir channels have been purified (Kir2.1, Kir3.1, and Kir3.2) (12–15). We therefore concentrate in this study on the effect of cholesterol on two bacterial K⁺ channels that differ in the level of their homology to mammalian Kir channels, KirBac1.1 and KcsA. KirBac channels have high sequence homology with mammalian Kirs (e.g. 52% homology between KirBac1.1 and Kir2.1; see Fig. 7A) and have now been extensively used as structural models of mammalian Kir channels (3, 16, 17). The sequence similarity between KcsA and mammalian K channels lies mainly in the transmembrane domain (18). The overall sequence homology of KcsA to mammalian Kir channels is relatively low (e.g. 22% homology between KcsA and Kir2.1; see Fig. 7A), with an entirely different cytoplasmic domain structure.Open in a separate windowFIGURE 7.Cholesterol has no effect on KcsA-mediated ⁸⁶Rb⁺ uptake. A, time courses of ⁸⁶Rb⁺ uptake into liposomes reconstituted with 50 μg of cholesterol/mg of PL and as compared with liposomes containing no cholesterol (control). Both batches of liposomes contained 5 μg of KcsA/mg of PL. Blank liposomes contain no protein. The points represent averages of three independent experiments (means ± S.D.). B, normalized time courses of ⁸⁶Rb⁺ uptake in liposomes incorporating 50, 150, and 250 μg of cholesterol/mg of PL. C, maximal uptake of ⁸⁶Rb⁺ after 240 s in liposomes containing 10, 25, 50, 100, 150, 200, and 250 μg of cholesterol/mg of PL normalized to control (means ± S.D. of 3–5 independent experiments; *, p < 0.05). DPM, disintegrations per minute.Here we show that, similarly to Kir2 channels, prokaryotic Kir channels incorporated into liposomes are strongly suppressed by an increase in membrane cholesterol. Furthermore, the sensitivity of prokaryotic Kir to cholesterol is stereo-selective to cholesterol optical analogues. In contrast, KscA channels are insensitive to membrane cholesterol. These observations suggest that cholesterol directly suppresses Kir channels.     

Superiority of Classification Tree versus Cluster,Fuzzy and Discriminant Models in a Heartbeat Classification System

This study presents a 2-stage heartbeat classifier of supraventricular (SVB) and ventricular (VB) beats. Stage 1 makes computationally-efficient classification of SVB-beats, using simple correlation threshold criterion for finding close match with a predominant normal (reference) beat template. The non-matched beats are next subjected to measurement of 20 basic features, tracking the beat and reference template morphology and RR-variability for subsequent refined classification in SVB or VB-class by Stage 2. Four linear classifiers are compared: cluster, fuzzy, linear discriminant analysis (LDA) and classification tree (CT), all subjected to iterative training for selection of the optimal feature space among extended 210-sized set, embodying interactive second-order effects between 20 independent features. The optimization process minimizes at equal weight the false positives in SVB-class and false negatives in VB-class. The training with European ST-T, AHA, MIT-BIH Supraventricular Arrhythmia databases found the best performance settings of all classification models: Cluster (30 features), Fuzzy (72 features), LDA (142 coefficients), CT (221 decision nodes) with top-3 best scored features: normalized current RR-interval, higher/lower frequency content ratio, beat-to-template correlation. Unbiased test-validation with MIT-BIH Arrhythmia database rates the classifiers in descending order of their specificity for SVB-class: CT (99.9%), LDA (99.6%), Cluster (99.5%), Fuzzy (99.4%); sensitivity for ventricular ectopic beats as part from VB-class (commonly reported in published beat-classification studies): CT (96.7%), Fuzzy (94.4%), LDA (94.2%), Cluster (92.4%); positive predictivity: CT (99.2%), Cluster (93.6%), LDA (93.0%), Fuzzy (92.4%). CT has superior accuracy by 0.3–6.8% points, with the advantage for easy model complexity configuration by pruning the tree consisted of easy interpretable ‘if-then’ rules.     

MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression

microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.