Calcium phosphate supplementation increases faecal Lactobacillus spp. in a randomised trial of young adults.

The aim of the studies was to determine the effects of calcium carbonate and calcium phosphate supplementation on faecal Lactobacillus spp., with and without a probiotic supplement, in healthy adults. Study 1 comprised of a randomised, double-blind, crossover design; participants (n=15) received 2 capsules/d of 250 mg elemental calcium as calcium carbonate (Ca1) and calcium phosphate (Ca2) each for 2-week periods, with 2-week baseline and washout periods. Study 2 was a randomised, double-blind, crossover design; participants (n=17) received 2 capsules/d of Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011 (probiotic) alone, the probiotic with 2 capsules/d of Ca1, and probiotic with 2 capsules/d of Ca2 each for 2-week periods with 2-week baseline and washout periods. In both studies, stools were collected during the baseline, intervention and washout periods for Lactobacillus spp. quantification and qPCR analyses. Participants completed daily questionnaires of stool frequency and compliance. In Study 1, neither calcium supplement influenced viable counts of resident Lactobacillus spp., genome equivalents of lactic acid bacteria or stool frequency. In Study 2, faecal Lactobacillus spp. counts were significantly enhanced from baseline when the probiotic was administered with Ca2 (4.83±0.30, 5.79±0.31) (P=0.02), but not with Ca1 (4.98±0.31) or with the probiotic alone (5.36±0.31, 5.55±0.29) (not significant). Detection of L. helveticus R0052 and L. rhamnosus R0011 was significantly increased with all treatments, but did not differ among treatments. There were no changes in weekly stool frequency. Calcium phosphate co-administration may increase gastrointestinal survival of orally-administered Lactobacillus spp.

Evaluation of Bacillus subtilis R0179 on gastrointestinal viability and general wellness: a randomised, double-blind, placebo-controlled trial in healthy adults.

A probiotic formulation of Enterococcus faecium R0026 and Bacillus subtilis R0179 has been evaluated in previous clinical trials. However, B. subtilis R0179 has not been evaluated as a single probiotic strain or in combination with other strains at doses higher than 0.1×109 cfu. To establish oral dose-response tolerance and gastrointestinal (GI) viability of B. subtilis R0179, a randomised, double-blind, placebo-controlled trial in healthy adults (n=81; 18-50 years old) was conducted. Participants received B. subtilis R0179 at 0.1, 1.0 or 10×109 cfu/capsule/day or placebo for four weeks. General wellness was assessed using a daily questionnaire evaluating GI, cephalic, ear-nose-throat, behavioural, emetic, and epidermal symptoms. GI symptoms were further evaluated using a weekly gastrointestinal symptom rating scale (GSRS). GI transit viability of B. subtilis R0179 was assessed by plating and microbiota analysis by 16S rRNA at baseline, week 4 of the intervention and washout. General wellness and GI function were not affected by oral consumption of B. subtilis R0179 at any dose. Daily questionnaire syndrome scores were not different from baseline and did not exceed a clinically significant score of 1. GSRS syndrome scores were not different from baseline and ranged from 1.1±0.1 to 1.9±0.2. Faecal viable counts of B. subtilis R0179 demonstrated a dose response: the placebo group (1.1±0.1 log10 cfu/g) differed from 0.1×109 (4.6±0.1 log10 cfu/g), 1×109 (5.6±0.1 log10 cfu/g) and 10×109 (6.4±0.1 log10 cfu/g) (P<0.0001). No significant changes in phyla were observed, but sequence reads binned to multiple operational taxonomic units matching closest to Ruminococci increased during probiotic supplementation. B. subtilis R0179 survives passage through the human GI tract and is well tolerated by healthy adults at intakes from 0.1 to 10×109 cfu/day. The trial has been registered at www.clinicaltrials.gov under NCT01802151.

Gut microbiota correlates with energy gain from dietary fibre and appears to be associated with acute and chronic intestinal diseases.

Improvements in high-throughput sequencing technologies have spurred a large number of studies aimed at obtaining a better understanding of the composition and the dynamics in gut microbiota and its associations with various human diseases, especially those in the intestinal tract. Here we briefly summarize results from three different such studies from our group, all of which used 454 based high-throughput 16S rRNA sequence analysis combined with other microbiota profiling methods to determine faecal microbiota composition. In the first study, a controlled feeding trial, we establish that energy gain from the consumption of up to 50 g/day of a resistant maltodextrin depends on the prevalent microbiota composition. Over time, resistant maltodextrin supplementation increased the proportion of total faecal bacteria as well as potentially beneficial bifidobacteria. Thus, energy gain from resistant maltodextrin in an individual appears to vary over time and depend on the adaptation of gut microbiota. We then illustrate the power of molecular tools for identifying (i) distortions in early microbiota development in pre-term infants and the presence of potentially novel pathogens contributing to necrotizing enterocolitis and (ii) a specific microbiota signature, based on discriminant analysis of the 16S rRNA sequences, that correlates with the prevalence of an early risk marker associated with colorectal carcinogenesis, intestinal adenoma, in elderly adults.

Expression of the secreted factors noggin and bone morphogenetic proteins in the subependymal layer and olfactory bulb of the adult mouse brain.

The antagonism between noggin and the bone morphogenetic proteins (BMPs) plays a key role during CNS morphogenesis and differentiation. Recent studies indicate that these secreted factors are also widely expressed in the postnatal and adult mammalian brain in areas characterized by different types of neural plasticity. In particular, significant levels of noggin and BMP expression have been described in the rodent olfactory system. In the mammalian forebrain, the olfactory bulb (OB) and associated subependymal layer (SEL) are documented as sites of adult neurogenesis. Here, using multiple approaches, including the analysis of noggin-LacZ heterozygous mice, we report the expression of noggin and two members of the BMP family, BMP4 and BMP7, in these regions of the adult mammalian forebrain. We observe that along the full extent of the SEL, from the lateral ventricle to the olfactory bulb, noggin and BMP4 and 7 are mainly associated with the astrocytic glial compartment. In the OB, BMP4 and 7 proteins remain primarily associated with the SEL while strong noggin expression was also found in cells located in different OB layers (i.e. granule, external plexiform, glomerular layers). Taken together our data lead us to hypothesize that within the SEL the antagonism between noggin and BMPs, both produced by the glial tubes, act through autocrine/paracrine inductive mechanisms to maintain a neurogenetic environment all the way from the lateral ventricle to the olfactory bulb. In the OB, their expression patterns suggest multiple regulatory roles on the unusual neural plasticity exhibited by this region.

Molecular cloning of a putative cyclic nucleotide-gated ion channel cDNA from Limulus polyphemus.

Cyclic nucleotide-gated channels have been proposed to mediate the electrical response to light in the ventral photoreceptor cells of the horseshoe crab, Limulus polyphemus. However, a cyclic nucleotide-gated channel has not been identified from Limulus. We have cloned a putative full-length cyclic nucleotide-gated channel cDNA by screening cDNA libraries constructed from Limulus brain using a probe developed from Limulus ventral eye nerves. The putative full-length cDNA was derived from two overlapping partial cDNA clones. The open reading frame encodes 905 amino acids; the sequence shows 44% identity to that of the alpha subunit of the bovine rod cyclic GMP-gated channel over the region containing the transmembrane domains and the cyclic nucleotide binding domain. This Limulus channel has a novel C-terminal region of approximately 200 amino acids, containing three putative Src homology domain 3 binding motifs and a putative coiled-coil domain. The possibility that this cloned channel is the same as that detected previously in excised patches from the photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves.

Putative inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in Limulus photoreceptors.

Invertebrate microvillar photoreceptors utilize the phosphoinositide cascade to transduce light stimuli and inositol 1,4,5-trisphosphate is thought to be one of the messengers that triggers the electrical response by mobilizing intracellular stored calcium. To further characterize the role of the phosphoinositide signaling pathway in invertebrate phototransduction, we have examined the distribution of inositol 1,4,5-trisphosphate receptors in Limulus lateral eye and ventral nerve photoreceptors using an immunohistochemical approach combined with confocal microphotolysis of caged inositol 1,4,5-trisphosphate. We have localized the inositol 1,4,5-trisphosphate receptor using an antibody raised against a highly conserved region of the N-terminal of the protein. In lateral eye photoreceptors, the antibody intensely stains cytoplasm directly beneath the photoreceptive microvilli, containing subrhabdomeral cisternae of endoplasmic reticulum. In ventral nerve photoreceptors, the distribution of immunostaining was more homogeneous than within the lateral eye photoreceptors. Simultaneous confocal microphotolysis of caged inositol 1,4,5-trisphosphate and Ca2+ measurements using the fluorescent indicator Calcium Green 5N were performed to estimate inositol 1,4,5-trisphosphate-induced Ca2+ release in functionally distinct areas of the ventral nerve photoreceptors. This is the first direct demonstration of the localization of putative inositol 1,4,5-trisphosphate receptor in invertebrate visual cells. The inositol 1,4,5-trisphosphate receptor appears to be localized predominantly to endoplasmic reticulum and taken in conjunction with earlier physiological data from other workers, our result supports a central role for the phosphoinositide pathway in visual transduction in Limulus photoreceptors.

Digitalis-like and vasoconstrictor effects of endogenous digoxin-like factor(s) from the venom of Bufo marinus toad.

Digitalis glycoside-like properties of the Bufo marinus toad crude venom and one of its constituents, bufalin, were studied in various assay systems. In concentrations 0.3-30 micrograms/ml crude venom increased the contractility of isolated electrically driven rat atria, constricted rat aortic rings, inhibited ouabain-sensitive Na+,K(+)-ATPase in rat erythrocytes and the Na+,K(+)-pump in rat aorta, and cross-reacted with antidigoxin antibody from the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA). These effects were unaffected by adrenoceptor blockers and the 5-HT antagonist, deseril, but were blocked by antidigoxin antibody. Bufalin (10-30 microM) increased myocardial contractility and inhibited Na+,K(+)-ATPase in rat erythrocytes similarly to crude Bufo marinus venom. In rat aorta bufalin showed weak and delayed vasoconstrictor activity which was antagonized by 2 microM phentolamine, and had a biphasic effect on the Na+,K(+)-pump; 0.5-1.0 microM bufalin stimulated the pump, while higher concentrations inhibited its activity. Although the effects of bufalin were blocked by antidigoxin antibody, bufalin showed very low digoxin-like immunoreactivity in the DELFIA. These observations suggest that, in addition to bufalin, Bufo marinus venom contains at least one more digitalis-like steroid with significant intrinsic vasoconstrictor activity which, unlike bufalin, constricts the blood vessels acting directly via inhibition of the sodium pump in the vascular smooth muscle membrane.

Endogenous plasma Na,K-ATPase inhibitory activity and digoxin like immunoreactivity after acute myocardial infarction.

PURPOSE OF INVESTIGATION: The aim was to look for the presence of circulating factor(s) with Na,K-ATPase inhibitory properties and digoxin like immunoreactivity in patients after acute myocardial infarction. DESIGN: Venous blood samples were obtained when the patients were admitted and different methods were used to monitor the plasma concentrations of factor(s) with properties of digitalis. SUBJECTS - These were 26 patients of both sexes (mean age 57.7 years, range 40-72) during the first 24 h of a first transmural acute myocardial infarct, 11 male patients with unstable angina pectoris (52.5 years, 45-67), and 18 healthy male controls (25 to 50 years). MEASUREMENTS AND MAIN RESULTS: There was significant inhibition of ouabain sensitive Na,K-ATPase in intact erythrocytes in patients with myocardial infarction [1.4(SEM 0.15)mumol Pi.mg-1.h-1] compared with patients with unstable angina pectoris [3.1(0.4), p less than 0.01] and healthy controls [3.4(0.25), p less than 0.01]. In myocardial infarction complicated by ventricular fibrillation (n = 5) Na,K-ATPase activity was significantly lower than in the other 21 patients [0.95(0.2) and 1.55(0.11) mumol Pi.mg-1.h-1 respectively, p less than 0.05]. There was no change in erythrocyte Na,K-ATPase activity in myocardial infarction complicated by acute pulmonary oedema, nor was there any difference in activity in erythrocyte ghosts obtained from the patients with myocardial infarction v healthy controls, at 0.47(0.13) v 0.50(0.02) mumol Pi.mg-1.h-1. Boiled plasma supernatants obtained from the patients with myocardial infarction inhibited Na,K-ATPase in erythrocytes from healthy subjects. This inhibitory effect was antagonised by antidigoxin antibody. Plasma inhibitory potency was correlated with erythrocyte Na,K-ATPase activity in the patients with myocardial infarction (r = -0.65, p less than 0.001, n = 23). There was a 2.5-fold increase in plasma digoxin like immunoreactivity in the patients with myocardial infarction [1.65(0.5) ng.ml-1] using DELFIA fluoroimmunoassay as compared with five healthy controls [0.04(0.12), p less than 0.05] and nine patients with unstable angina [0.48(0.11), p less than 0.05]. There was no difference in plasma digoxin like immunoreactivity in myocardial infarction complicated or not by ventricular fibrillation, but there was very low digoxin like immunoreactivity in patients with myocardial infarction complicated by acute pulmonary oedema [0.26(0.08) ng.ml-1, n = 7]. There was no correlation between plasma digoxin like immunoreactivity and either plasma Na,K-ATPase inhibitory potency or erythrocyte Na,K-ATPase activity. CONCLUSIONS: The results show that plasma factor(s) with some of the properties of digitalis are increased in acute myocardial infarction.

[Glucagon receptor binding and its effect on the cAMP level in isolated rat and chicken hepatocytes]. / Sviazyvanie gliukagona retseptorami i vliianie na uroven' tsAMF v izolirovannykh gepatotsitakh krys i kur.

The binding sites to glucagon with KD 10(-9) M in rat and chicken isolated hepatocytes and with KD 10(-7) M in chicken hepatocytes only where revealed. The first, but not the second binding sites where decreased in their affinity by the adding of GTP nonhydrolyzing analogue Gpp(NH). Glucagon activated the cAMP accumulation in rat and chicken hepatocytes in dose-depended manner. The dose-response curve being plateau after maximum in rats and decreased in chicken. Glucagon inserted an inhibiting effect when occupying chicken-specific sites of binding with KD 10(-7) M. The nature of these binding sites and mechanism of glucagon inhibiting effect on the cAMP accumulation are discussed.

[The role of an endogenous digoxin-like factor in regulating blood circulation and in the origin of arrhythmia in myocardial ischemia]. / Rol' éndogennogo digoksinopodobnogo faktora v reguliatsii krovoobrashcheniia i proiskhozhdenii aritmii pri ishemii miokarda.

The history of the discovery of endogenous digoxin-like factor (EDF) is described and the role played by the substance in blood circulation regulation, in the pathogenesis of arterial hypertension is discussed. The authors provide their own data (both experimental and clinical ones) concerned with EDF participation in the pathogenesis of early ventricular fibrillations in acute myocardial ischemia. Experiments on rats demonstrated that myocardial infarction (MI) is marked by a negative linear correlation between the intensity of ventricular fibrillations and the activity of Na,K-ATPase of intact red blood cells (r = -0.84) that mirrors the content of circulating EDF. Administration to the animals of digoxin antibodies binding EDF resulted in the antiarrhythmic effect and in the recovery of the enzyme activity. The patients demonstrated, within the first day of MI, a 76-percent inhibition of the activity of Na,K-ATPase of red blood cells. A correlation was discovered between the enzyme activity and the capacity of protein-free supernatants of blood plasma for inhibiting Na,K-ATPase, which indicates the presence of circulating EDF in blood plasma.

[Glucagon binding by isolated chick hepatocytes]. / Sviazyvanie gliukagona izolirovannymi gepatotsitami kur.

Studies have been made on the binding of 125I-glucagon by isolated chick hepatocytes. It was shown that pH and temperature dependence of the binding does not differ from that in rat hepatocytes. Optimum binding was observed at pH 7.6, the rate of binding being higher at 37 degrees C as compared to that at 20 degrees C, although the binding capacity increased with the decrease in the temperature. Unlabeled glucagon was able to compete with 125I-glucagon at the binding sites. Scatchard plot was found to be curvilinear revealing two classes of the binding sites with Kd values 10(-9) and 10(-7) M at temperatures 20 and 37 degrees C correspondingly. Earlier studies revealed in rats the binding sites of a sole class with Kd value 10(-9) M. Preincubation of cells with native glucagon results in changes of labeled glucagon binding, the effect being proportional to the concentration of native glucagon. Preincubation effect was observed at 37 degrees C, being absent at 20 degrees C; the effect was due to the decrease in the number of both high and low affinity binding sites. The presence of down-regulation of glucagon receptors in chick hepatocytes is suggested.

[Insulin receptor reaction of the lamprey Lampetra fluvialis to hyperinsulinemia induced by administration of the hormone]. / Reaktsiia insulinovykh retseptorov minogi Lampetra fluvialis na giperinsulinemiiu, vyzvannuiu vvedeniem gormona.

Insulin injections to the lamprey Lampetra fluviatilis result in prolonged and significant hyperinsulinemia, but do not decrease the number of receptors in its target cells. These findings were made in studies on the specific binding to 125I-insulin by membrane fractions of the brain and cardiac muscle in migrating and winter (fasting) lampreys. It is also suggested that no regulation of the number of the receptors by hormonal level takes place in the lamprey, which is presumably due to a low rate of internalization of hormone-receptor complexes and to a total decrease in the turnover of the receptors in a pre-spawning period.