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Aim: To assess the effects of probiotic Medilac-S ® as adjunctive therapy for the induction of remission of ulcerative colitis (UC) in a Chinese population through a systematic review and meta-analysis., Methods: A systematic literature search was conducted to find randomized, controlled trials in a Chinese population with at least two study arms - a control arm which receives a conventional, oral aminosalicylate drug, and a treatment arm, which administers the same conventional drug in conjunction with the probiotic Medilac-S ® per os . Both English and Chinese databases were searched, including PubMed, EMBASE, Google Scholar, Chinese National Knowledge Infrastructure, Wanfang Data, and VIP Search, and study data was extracted onto standardized abstraction sheets. Meta-analyses were conducted for primary and secondary outcomes of interest using a fixed or random effects model. The primary outcome was the induction of clinical remission and the secondary outcomes included changes in Sutherland index, endoscopic and histological scores, proportion of reported clinical symptoms and adverse events (AEs). For outcomes with sufficient data, the type of conventional drug therapy was also assessed to determine if the effects of combination therapy with Medilac-S ® was influenced by drug type. All tests were conducted using a type I error rate of 0.05 and all confidence intervals (CI) were based on a 95% confidence level. Review protocol was uploaded to PROSPERO (CRD42018085658 upon completion)., Results: Fifty-three clinical trials with a total of 3984 participants were identified and included in the review. Medilac-S ® adjunctive therapy significantly improved induction of clinical remission (RR = 1.21; 95%CI: 1.18-1.24; P < 0.0001) with the estimated likelihood of effective treatment, on average, 21% higher for those consuming the probiotic. Sutherland index scores showed the control mean was on average 3.10 (CI: 2.41-3.78; P = 0.0428) units greater than the treatment mean, thereby demonstrating significant improvement in participants taking the probiotic. Similarly, a significant difference was seen between the overall reduction of endoscopic and histological scores of control and treatment arm participants, with score decreases in the control groups 0.71 (CI: 0.3537-1.0742) and 1.1 (CI: 0.9189-1.2300) units smaller than treatment group score decreases. The proportion of participants reporting clinical symptoms, (abdominal pain, tenesmus, blood and mucous in stool, and diarrhea) was significantly reduced after combination therapy with Medilac-S ® ( P < 0.0001) and estimated to be on average 44% (RR = 0.44, CI: 0.32-0.59), 53% (RR = 0.53, CI: 0.38-74), 40% (RR = 0.40, CI: 0.28-0.58) and 47% (RR = 0.47 CI: 0.36-0.42) respectively, of the proportion of individuals reporting the aforementioned symptoms after conventional therapy alone. The risk of AEs was also significantly reduced with adjunctive Medilac-S ® therapy. The proportion of individuals in the treatment groups reporting AEs was an estimated 72% of the proportion of individuals in the control groups reporting AEs (RR = 0.72, CI: 0.55-0.94, P = 0.0175). Upon comparing effect means for different drug types in conjunction with Medilac-S ® , evidence of significant variability ( P < 0.0001) was observed, and sulfasalazine was found to be the most effective drug in both primary and secondary outcomes., Conclusion: Evidence suggests Medilac-S ® adjunctive therapy should be considered standard care for UC in a Chinese population because it aids in the induction of clinical remission, improves symptoms of the gastrointestinal tract and reduces risk of AEs., Competing Interests: Conflict-of-interest statement: Sohail G, Xu X and Tompkins TA declare that they are paid employees of Lallemand Health Solutions Inc. (Montreal, QC), a company that studies, manufactures and sells probiotics globally, business-to-business, but not to consumers.

Clinical effects of antimicrobials and probiotics in combination have been reported, however, little is known about their impact on gut microbiota and its resistome. In this study 16S rRNA gene amplicon, shotgun metagenomics sequencing and antibiotic resistance (ABR) microarray were used on fecal samples of 70 healthy participants, taken at four time points in probiotic (Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052) and placebo groups to profile the gut bacterial microbiota and its resistome following administration of amoxicillin-clavulanic acid for one week. Significant shifts in microbiota family composition caused by the antimicrobial in both groups that included decreases in the proportion of Lachnospiraceae, Coriobacteriaceae and unidentified Clostridiales; and notable increases for the proportion of Enterobacteriaceae, Bacteroidaceae and Porphyromonadaceae compared to baseline levels. Resistome showed a corresponding enrichment of ABR genes compared to baseline from such classes as aminoglycosides and beta-lactams that were linked, by in silico inference, to the enrichment of the family Enterobacteriaceae. Despite perturbations caused by short-term antibiotic treatment, both gut microbiota and resistome showed prompt recovery to baseline levels one week after cessation of the antimicrobial. This rapid recovery may be explained by the hypothesis of community resilience.

Aims: Classical microbiology techniques are the gold standard for probiotic enumeration. However, these techniques are limited by parameters of time, specificity and incapacity to detect viable but nonculturable (VBNC) micro-organisms and nonviable cells. The aim of the study was to evaluate flow cytometry as a novel method for the specific quantification of viable and nonviable probiotics in multistrain products., Methods and Results: Custom polyclonal antibodies were produced against five probiotic strains from different species (Bifidobacterium bifidum R0071, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium longum ssp. longum R0175, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011). Evaluation of specificity confirmed that all antibodies were specific at least at the subspecies level. A flow cytometry method combining specific antibodies and viability assessment with SYTO ® 24 and propidium iodide was applied to quantify these strains in three commercial products. Analyses were conducted on two flow cytometry instruments by two operators and compared with classical microbiology using selective media. Results indicated that flow cytometry provides higher cell counts than classical microbiology (P < 0·05) in 73% of cases highlighting the possible presence of VBNC. Equivalent performances (repeatability and reproducibility) were obtained for both methods., Conclusions: This study showed that flow cytometry methods can be applied to probiotic enumeration and viability assessment. Combination with polyclonal antibodies can achieve sufficient specificity to differentiate closely related strains., Significance and Impact of the Study: Flow cytometry provides absolute and specific quantification of viable and nonviable probiotic strains in a very short time (<2 h) compared with classical techniques (>48 h), bringing efficient tools for research and development and quality control., (© 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.)

Background/aims: Visceral pain and hypothalamic-pituitary-adrenal axis (HPA) dysregulation is a common characteristic in irritable bowel syndrome (IBS) patients. Previously, we reported that a probiotic formulation ( Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) prevents chronic stress-mediated brain function abnormalities by attenuating the HPA axis response. Here, we compared the effect between different probiotic treatments on the perception of visceral pain during colorectal distension (CRD) following a chronic stress and the consequences to the activity of the HPA axis., Methods: After a 2-week treatment with a combined probiotic formulation, or L. helveticus or B. longum alone in stressed mice, the visceral pain in response to CRD was recorded. The expression of glucocorticoid receptors was determined in the different brain areas involved in the stress response (hypothalamus, hippocampus, and prefrontal cortex). The plasma levels of stress hormones were also measured., Results: A pretreatment using the combination of probiotic formulation significantly reduces the chronic stress-induced visceral hypersensitivity respectively at 0.06, 0.08, and 0.10 mL CRD volume. However, a single probiotic ( B. longum or L. helveticus ) administration is less effective in reducing visceral pain in stressed mice. Moreover, the expression of the glucocorticoid receptor mRNA was consistently up-regulated in several brain areas after pretreatment with a combined probiotic, which correlated with the normalization of stress response compared to the inconsistent effects of a single probiotic., Conclusion: The combination of L. helveticus and B. longum is more effective in regulating glucocorticoid negative feedback on the HPA axis than probiotic alone and subsequently in treating stress-induced visceral pain.

Genome-wide transcriptional analysis in intestinal epithelial cells (IEC) can aid in elucidating the impact of single versus multi-strain probiotic combinations on immunological and cellular mechanisms of action. In this study we used human expression microarray chips in an in vitro intestinal epithelial cell model to investigate the impact of three probiotic bacteria, Lactobacillus helveticus R0052 (Lh-R0052), Bifidobacterium longum subsp. infantis R0033 (Bl-R0033) and Bifidobacterium bifidum R0071 (Bb-R0071) individually and in combination, and of a surface-layer protein (SLP) purified from Lh-R0052, on HT-29 cells' transcriptional profile to poly(I:C)-induced inflammation. Hierarchical heat map clustering, Set Distiller and String analyses revealed that the effects of Lh-R0052 and Bb-R0071 diverged from those of Bl-R0033 and Lh-R0052-SLP. It was evident from the global analyses with respect to the immune, cellular and homeostasis related pathways that the co-challenge with probiotic combination (PC) vastly differed in its effect from the single strains and Lh-R0052-SLP treatments. The multi-strain PC resulted in a greater reduction of modulated genes, found through functional connections between immune and cellular pathways. Cytokine and chemokine analyses based on specific outcomes from the TNF-α and NF-κB signaling pathways revealed single, multi-strain and Lh-R0052-SLP specific attenuation of the majority of proteins measured (TNF-α, IL-8, CXCL1, CXCL2 and CXCL10), indicating potentially different mechanisms. These findings indicate a synergistic effect of the bacterial combinations relative to the single strain and Lh-R0052-SLP treatments in resolving toll-like receptor 3 (TLR3)-induced inflammation in IEC and maintaining cellular homeostasis, reinforcing the rationale for using multi-strain formulations as a probiotic., Competing Interests: All the authors (CM, OM and PB) declare they are paid employees in the Research and Development sector of Lallemand Health Solutions Inc. PS is a paid consultant to Lallemand Health Solutions Inc. Lallemand Health Solutions Inc. is a privately-owned company that researches, manufactures and sells probiotic microbes business-to-business, not directly to the consumer. This commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing our data and materials.

Broad-spectrum antibiotic use can disrupt the gastrointestinal microbiota resulting in diarrhoea. Probiotics may be beneficial in managing this type of diarrhoea. The aim of this 10-week randomised, double-blind, placebo-controlled, parallel study was to investigate the effect of Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011 supplementation on antibiotic-associated diarrhoea in healthy adults. Subjects were randomised to receive 1 week of amoxicillin-clavulanic acid (875 mg/125 mg) once per day, plus a daily dose of 8×109 colony-forming units of a multi-strain probiotic (n 80) or placebo (n 80). The probiotic or placebo intervention was maintained for 1 week after completion of the antibiotic. Primary study outcomes of consistency and frequency of bowel movements were not significantly different between the probiotic and placebo groups. The secondary outcomes of diarrhoea-like defecations, Gastrointestinal Symptoms Rating Scale scores, safety parameters and adverse events were not significantly different between the probiotic intervention and the placebo. A post hoc analysis on the duration of diarrhoea-like defecations showed that probiotic intervention reduced the length of these events by 1 full day (probiotic, 2·70 (sem 0·36) d; placebo, 3·71 (sem 0·36) d; P=0·037; effect size=0·52). In conclusion, this study provides novel evidence that L. helveticus R0052 and L. rhamnosus R0011 supplementation significantly reduced the duration of diarrhoea-like defecations in healthy adults receiving antibiotics.

Background: Recurrent Clostridium difficile infection (RCDI) is associated with repeated antibiotic treatment and the enhanced growth of antibiotic-resistant microbes. This study tested the hypothesis that patients with RCDI would harbor large numbers of antibiotic-resistant microbes and that fecal microbiota transplantation (FMT) would reduce the number of antibiotic-resistant genes., Methods: In a single center study, patients with RCDI (n = 20) received FMT from universal donors via colonoscopy. Stool samples were collected from donors (n = 3) and patients prior to and following FMT. DNA was extracted and shotgun metagenomics performed. Results as well as assembled libraries from a healthy cohort (n = 87) obtained from the Human Microbiome Project were aligned against the NCBI bacterial taxonomy database and the Comprehensive Antibiotic Resistance Database. Results were corroborated through a DNA microarray containing 354 antibiotic resistance (ABR) genes., Results: RCDI patients had a greater number and diversity of ABR genes compared with donors and healthy controls. Beta-lactam, multidrug efflux pumps, fluoroquinolone, and antibiotic inactivation ABR genes were increased in RCDI patients, although donors primarily had tetracycline resistance. RCDI patients were dominated by Proteobacteria with Escherichia coli and Klebsiella most prevalent. FMT resulted in a resolution of symptoms that correlated directly with a decreased number and diversity of ABR genes and increased Bacteroidetes and Firmicutes with reduced Proteobacteria. ABR gene profiles were maintained in recipients for up to a year following FMT., Conclusions: RCDI patients have increased numbers of antibiotic-resistant organisms. FMT is effective in the eradication of pathogenic antibiotic-resistant organisms and elimination of ABR genes., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

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.

Background: Myocardial infarction (MI) is associated with apoptosis in the amygdala and, ultimately, with clinical signs of depression. Different treatments have proven to be beneficial in preventing depression, including combination of the probiotics Lactobacillus helveticus and Bifidobacterium longum for prophylaxis. We have speculated previously that the benefit of these probiotics is due to their anti-inflammatory properties, and evidence suggests that an intact vagus nerve is important for this effect to occur. This study was designed to ascertain vagus nerve involvement in the beneficial influence of probiotics on caspase activities in our post-MI animal model of depression., Methods: Probiotics and/or vehicle were administered daily to male adult rats, 14 days before MI and until euthanasia. Vagotomy was performed in subgroups of rats 40 min before MI. They were sacrificed after 3 days of reperfusion, and MI size was assessed along with caspase-3 and -8 activities in the amygdala., Key Results: Probiotics had no effect on infarct size but vagotomy increased it. Caspase-3 and caspase-8 activities in the amygdala were higher in MI than in sham-operated rats, and this outcome was reversed by probiotics. The beneficial influence of probiotics was abolished by vagotomy., Conclusions & Inferences: Our data indicate that the effect of probiotics on caspase activities in the amygdala after MI depends on an intact vagus nerve., (© 2015 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

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.

Background: Several probiotic strains have been shown to enhance human resistance to infectious disease. It is speculated that these strains may impose this effect by excretion of anti-microbial components, by competing with pathogens for intestinal nutrients and/or mucosal adhesion sites or modulating the immune system., Objective: A parallel, double-blind, placebo-controlled 4-week intervention was performed in healthy males, to study the effect of a blend of probiotic bacteria (Lactobacillus helveticus Rosell-52, Lactobacillus rhamnosus Rosell-11, Bifidobacterium longum ssp. longum Rosell-175) and a probiotic yeast (Saccharomyces cerevisiae var boulardii CNCM I-1079) on enterotoxigenic Escherichia coli (ETEC) challenge. Primary outcomes studied were fecal ETEC excretion and total fecal output per day., Subjects/methods: Subjects were randomized to the probiotic (5 × 10(9) colony-forming units (CFUs); twice daily; n=30) or placebo group (twice daily; n=30). After 2 weeks, subjects were orally challenged with a live attenuated ETEC (3 × 10(9) CFU), previously demonstrated to induce mild, short-lived symptoms of a foodborne infection. Before and after ETEC challenge, subjects collected 24 h fecal samples. Compliance to study guidelines, stool consistency (Bristol Stool Score), stool frequency, and frequency and severity of gastrointestinal (GI) complaints were recorded by the subjects on a Daily Record Questionnaire., Results: ETEC challenge induced a significant increase in fecal ETEC excretion in both groups. However, a statistically significant increase in fecal output was only observed in the probiotic group. ETEC challenge resulted in a decrease in the percentage of fecal dry weight, and an increase in reported Bristol Stool Score, stool frequency and GI complaints. Dietary probiotics significantly decreased the percentage of fecal dry weight. In addition, ETEC increased C-reactive protein, total secretory Immunoglobulin A (IgA) and Immunoglobulin G Colonization Factor Antigen II., Conclusion: Dietary probiotics did not increase resistance to oral attenuated ETEC challenge in human subjects.

A commercially available product containing three probiotic bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033, and Bifidobacterium bifidum R0071) was previously shown in animal trials to modulate both TH1 and TH2 immune responses. Clinical studies on this combination of bacteria have also shown positive health effects against seasonal winter diseases and rotavirus infection. The goal of this study was to use a well-established in vitro intestinal epithelial (HT-29) cell model that has been shown to constitutively express double-stranded RNA (dsRNA) sensors (Toll-like receptor 3 [TLR3], retinoic acid-inducible gene I, melanoma differentiation-associated gene 5, and dsRNA-activated protein kinase). By using the HT-29 cell model, we wanted to evaluate whether or not this combination of three bacteria had the capacity to immune modulate the host cell response to a dsRNA ligand, poly(I·C). Using a custom-designed, two-color expression microarray targeting genes of the human immune system, we investigated the response of HT-29 cells challenged with poly(I·C) both in the presence and in the absence of the three probiotic bacteria. We observed that the combination of the three bacteria had a major impact on attenuating the expression of genes connected to proinflammatory TH1 and antiviral innate immune responses compared to that obtained by the poly(I·C)-only challenge. Major pathways through which the multistrain combination may be eliciting its immune-modulatory effect include the TLR3 domain-containing adapter-inducing beta interferon (TRIF), mitogen-activated protein kinase, and NF-κB signaling pathways. Such a model may be useful for selecting potential biomarkers for the design of future clinical trials.

Lactobacillus helveticus is a lactic acid bacterium widely used in the manufacture of cheese and for production of bioactive peptides from milk proteins. We present the complete genome sequence for L. helveticus CNRZ 32, a strain particularly recognized for its ability to reduce bitterness and accelerate flavor development in cheese.

Enterohaemorrhagic Escherichia coli O157:H7 and adherent-invasive Escherichia coli are two groups of enteric bacterial pathogens associated with haemorrhagic colitis and Crohn's Disease, respectively. Bacterial contact with host epithelial cells stimulates an immediate innate immune response designed to combat infection. In this study, immune responses of human epithelial cells to pathogens, either alone or in combination with probiotic bacteria were studied. Industrially prepared Lactobacillus helveticus strain R0052 was first examined by microarray analysis and then compared to broth-grown strains of R0052 and Lactobacillus rhamnosus strain GG using quantitative realt-time polymerase chain reaction. Results showed host immune activation responses increased following pathogen exposure, which were differentially ameliorated using probiotics depending on both the preparation of probiotics employed and conditions of exposure. These findings provide additional support for the concept that specific probiotic strains serve as a promising option for use in preventing the risk of enteric bacterial infections.

Proinflammatory cytokines play a central role in depression-like behaviour and apoptosis in the limbic system after myocardial infarction (MI). A PUFA n-3 diet or the combination of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 probiotics, when given before the ischaemic period, reduce circulating proinflammatory cytokines as well as apoptosis in the limbic system. The present study was designed to determine if the same nutritional interventions maintain their beneficial effects when started after the onset of the reperfusion period and attenuate depression-like behaviour observed after MI. MI was induced by the occlusion of the left anterior descending coronary artery for 40 min in rats. After the onset of reperfusion, animals were fed with a high- or low-PUFA n-3 diet, combined or not with one billion live bacteria of L. helveticus and B. longum. At 3 d post-MI, caspase-3 enzymatic activities and terminal 2'-deoxyuridine, 5'-triphosphate (dUTP) nick-end labelling (TUNEL)-positive cells were decreased in the CA1, dentate gyrus (DG) and amygdala with the high-PUFA n-3 diet, as compared to the three other diets. Probiotics attenuated caspase-3 activity and TUNEL-positive cells in the DG and the medial amygdala. At 2 weeks post-MI, depression-like behaviour was observed in the low-PUFA n-3 diet without probiotics-group, and this behaviour was attenuated with the high-PUFA n-3 diet or/and probiotics. These results indicate that a high-PUFA n-3 diet or the administration of probiotics, starting after the onset of reperfusion, are beneficial to attenuate apoptosis in the limbic system and post-MI depression in the rat.

In order to understand the appropriate use of potentially probiotic Gram-positive microbes through their introduction in the gut microbiome, it is necessary to understand the influence of individual bacteria on the host-response system at a cellular level. In the present study, we have shown that lipopolysaccharides, flagellated Gram-negative bacteria, potentially probiotic Gram-positive bacteria and yeast interact differently with human intestinal epithelial cells with a custom-designed expression microarray evaluating 17 specific host-response pathways. Only lipopolysaccharides and flagellated Gram-negative bacteria induced inflammatory response, while a subset of Gram-positive microbes had anti-inflammatory potential. The main outcome from the study was the differential regulation of the central mitogen-activated protein kinase signalling pathway by these Gram-positive microbes versus commensal/pathogenic Gram-negative bacteria. The microarray was efficient to highlight the impact of individual bacteria on the response of intestinal epithelial cells, but quantitative real-time polymerase chain reaction validation demonstrated some underestimation for down-regulated genes by the microarray. This immune array will allow us to better understand the mechanisms underlying microbe-induced host immune responses.

Lactobacillus helveticus R0052 is a commercially available strain that is widely used in probiotic preparations. The genome sequence consisted of 2,129,425 bases. Comparative analysis showed that it was unique among L. helveticus strains in that it contained genes encoding mucus-binding proteins similar to those found in Lactobacillus acidophilus.

Myocardial infarction (MI) in rats is accompanied by apoptosis in the limbic system and a behavioural syndrome similar to models of depression. We have already shown that probiotics can reduce post-MI apoptosis and designed the present study to determine if probiotics can also prevent post-MI depressive behaviour. We also tested the hypothesis that probiotics achieve their central effects through changes in the intestinal barrier. MI was induced in anaesthetised rats via 40-min transient occlusion of the left anterior coronary artery. Sham rats underwent the same surgical procedure without actual coronary occlusion. For 7 d before MI and between the seventh post-MI day and euthanasia, half the MI and sham rats were given one billion live bacterial cells of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 per d dissolved in water, while the remaining animals received only the vehicle (maltodextrin). Depressive behaviour was evaluated 2 weeks post-MI in social interaction, forced swimming and passive avoidance step-down tests. Intestinal permeability was evaluated by oral administration with fluorescein isothiocyanate-dextran, 4 h before euthanasia. MI rats displayed less social interaction and impaired performance in the forced swimming and passive avoidance step-down tests compared to the sham controls (P < 0·05). Probiotics reversed the behavioural effects of MI (P < 0·05), but did not alter the behaviour of sham rats. Intestinal permeability was increased in MI rats and reversed by probiotics. In conclusion, L. helveticus R0052 and B. longum R0175 combination interferes with the development of post-MI depressive behaviour and restores intestinal barrier integrity in MI rats.

Lactobacillus rhamnosus R0011 is a commercially available probiotic that is widely used in human dietary supplements and pharmaceutical products. We prepared a draft genome sequence consisting of 10 contigs totaling 2,900,620 bases and a G+C content of 46.7% for this strain.

Pediococcus acidilactici MA18/5M is a commercially available probiotic that is widely used in swine, poultry, aquaculture feeds, and human dietary supplements. We prepared a genome sequence for this strain consisting of 2 scaffolds totaling 1,992,928 bases including gaps for a total of 3,346 bases and a G+C content of 42%.

The probiotic preparation Lacidofil® has been commercially available in Europe, Asia and North America since 1995. This product is a combination of two strains, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011. The strains have been evaluated for safety, identity and mechanisms of probiotic action in vitro, in animal models and human clinical trials. The strains adhered to human epithelial cells, helped to maintain the barrier function and blocked the adhesion of a number of pathogens, allowing them to be cleared from the intestine. The strains also elicited an anti-inflammatory response by down-regulating IL-1β, IL-8 and TNF-α. In various stress models, the probiotic combination facilitated better coping and outcomes which may be through the maintenance of barrier function and suppressing inflammation. Overall, pre-clinical studies suggest a potential anti-infectious role for the strains and the combination. Clinical studies, primarily in children, have identified Lacidofil as an effective supplement for various gastrointestinal diseases such as antibiotic-associated diarrhoea and acute gastroenteritis. Recent research has also indicated that Lacidofil may be beneficial for individuals with atopic dermatitis or vaginal dysbacteriosis.

Commercial literature on various probiotic products suggests that they can be taken before meals, during meals or after meals or even without meals. This has led to serious confusion for the industry and the consumer. The objective of our study was to examine the impact of the time of administration with respect to mealtime and the impact of the buffering capacity of the food on the survival of probiotic microbes during gastrointestinal transit. We used an in vitro Digestive System (IViDiS) model of the upper gastrointestinal tract to examine the survival of a commercial multi-strain probiotic, ProtecFlor®. This product, in a capsule form, contains four different microbes: two lactobacilli (Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011), Bifidobacterium longum R0175 and Saccharomyces cerevisiae boulardii. Enumeration during and after transit of the stomach and duodenal models showed that survival of all the bacteria in the product was best when given with a meal or 30 minutes before a meal (cooked oatmeal with milk). Probiotics given 30 minutes after the meal did not survive in high numbers. Survival in milk with 1% milk fat and oatmeal-milk gruel were significantly better than apple juice or spring water. S. boulardii was not affected by time of meal or the buffering capacity of the meal. The protein content of the meal was probably not as important for the survival of the bacteria as the fat content. We conclude that ideally, non-enteric coated bacterial probiotic products should be taken with or just prior to a meal containing some fats.

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

An assessment of various methods to determine viable counts (CFU) in freeze-dried and dried microencapsulated (ME) probiotic cultures was carried out. Microencapsulation was done by spray-coating of dried Lactobacillus rhamnosus R0011 or Bifidobacterium longum ATCC 15708 cultures with fat. Rehydration of the ME powders was incomplete when they were added to water and gently agitated. As a result analytical methods based on vortexing of rehydrated ME cultures and which did not incorporate a high-shear homogenization (HSH) step underestimated the viable counts. The CFU of ME cultures were identical when methods using either blender or generator probes high-shear homogenization (HSH) were carried out. Furthermore HSH reduced the variability of the CFU results of both free-cell and ME cultures by a factor of three. The addition of an emulsifier (Tween 80) in the rehydrating medium to dissolve fat did not improve CFU counts when generator probes were used for HSH. The presence of fat in the ME product, or when added to the rehydration medium, improved CFU counts of B. longum but not of L. rhamnosus., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Enhancement of nutritional or vitamin content of foods is commonly touted as a major benefit of probiotics. In this paper, we examined the ability of three probiotic bacteria either alone or in combination to enhance nutritional content. Pure and mixed cultures of Streptococcus thermophilus ST5 and Lactobacillus helveticus R0052 or Bifidobacterium longum R0175 were used to prepare fermented soy beverages. The effects of strain, acidification and mixed cultures on the deconjugation of soy isoflavones were examined. Acidification to pH 4.7 alone resulted in a 7% drop in isoflavone levels. Deconjugation levels varied between the different glucosides. Fermentation by L. helveticus R0052 resulted in the 50% reduction in total glucosides with O-malonyl glucosides being reduced 64%. Fermentation by S. thermophilus ST5 or B. longum R0175 had no significant effect on isoflavone levels. Combining a S. thermophilus strain with a L. helveticus culture reduced the effectiveness of the latter. Fermentation did not significantly modify vitamin B1 or B6 levels., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

Background and Methods: The immunomodulatory activity of a synbiotic combination containing three bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033 and Bifidobacterium bifidum R0071) and short-chain fructooligosaccharide was examined in two distinct infectious rat models. In the T(h)1 model, Wistar rats were administered the synbiotic combination for 2 weeks prior to challenge with a single oral dose of enterotoxigenic Escherichia coli or vehicle. In the T(h)2 model, pretreated rats were challenged with a single subcutaneous dose of hook worm, Nippostrongylus brasiliensis. Blood samples were collected 3 hours or 4 days postchallenge and serum levels of pro- and anti-inflammatory cytokines were measured., Results: Significant reductions in pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6, and tumour necrosis factor (TNF)-α were observed in both models suggesting a single, unifying mode of action on an upstream regulator. The N. brasiliensis study also compared the effect of the individual strains to synbiotic. For most of cytokines the combination appeared to average the effect of the individual strains with the exception of IL-4 and IL-10 where there was apparent synergy for the combination. Furthermore, the cytokine response varied by strain., Conclusions: It was concluded that this synbiotic combination of these three microbes could be beneficial in both T(h)1 and T(h)2 diseases.

Lactobacillus helveticus R0052 is a bacterium used in commercial probiotic preparations. R0052 contains a small, cryptic plasmid comprised of eight open reading frames, four of which encode proteins of unknown function. Based on the sequence of the replication initiation protein RepA, pIR52-1 is a member of the recently described RepA&#95;N family of Gram-positive theta-replicating plasmids. The repA gene of pIR52-1 is the minimal origin of replication for L. helveticus and other Lactobacillus hosts. Additionally, pIR52-1 belongs to a subgroup of the RepA&#95;N plasmid family which have RepA proteins of high amino acid identity and a conserved, non-coding element upstream of repA which, in pIR52-1, is responsible for the control of plasmid copy number and contributes to plasmid maintenance.

Probiotics as dietary supplements have been readily accepted by Asian populations. Use of certain probiotic preparations is widespread and the number of clinical trials undertaken with such products is unparalleled in western scientific literature. One such preparation, containing a combination of Enterococcus faecium R0026 and Bacillus subtilis R0179, has 23 publications on post-market clinical studies involving over 1,800 adults. The majority of these publications are printed in Chinese and Korean journals. This review examines the clinical findings with this probiotic combination. As mono-therapy, it has been used to overcome symptoms associated with chronic diarrhoea and irritable bowel syndrome. It has been used as co-adjuvant therapy with sulfasalazine and mesalazine to improve remission times in mild to moderate Ulcerative Colitis and to improve compliance with conventional triple therapy for Helicobacter pylori eradication. While the much of the data is preliminary and the study designs require refinement, the contribution of these trials should not be ignored. The information derived in this review will provide practitioners with practical information on appropriate applications for probiotic supplements, expected outcomes, dosing regimes, safety and reported adverse events. Furthermore, identification of problems in these trials should help researchers design better clinical trials when investigating probiotic products.

The role of chronic infections, such as Helicobacter pylori (Hp), to produce sustained changes in host physiology remains controversial. In this study, we investigate whether the antigenic or bacterial content of the gut, after Hp eradication, influences the changes in gut function induced by chronic Hp infection. Mice were infected with Hp for 4 mo and then treated with antibiotics or placebo for 2 wk. Gastric emptying was measured using videofluoroscopy, feeding behavior using a 24-h feeding system, and intestinal permeability using an isolated jejunal segment arterially perfused with an artificial oxygen carrier, hemoglobin vesicles. Immune responses were assessed by CD3(+) cell counts and anti-Hp antibodies using ELISA. To determine the role of luminal factors in host physiology posteradication, groups of mice received the probiotics containing Lactobacillus rhamnosus R0011 and L. helveticus R0052 or placebo for 2 wk or crude Hp antigen weekly for 2 mo. Chronic Hp infection was associated with delayed gastric emptying, increased intestinal permeability, and increased gastric CD3(+) cell counts. Hp-induced altered feeding patterns did not reverse after eradication. Probiotics accelerated the recovery of paracellular permeability and delayed gastric emptying, improved the CD3(+) cell counts, and normalized altered feeding patterns posteradication. Hp antigen resulted in increased anti-Hp antibodies and increased CD3(+) cell counts in the stomach and delayed recovery of gastric function. Our results suggest that the bacterial content of the gut, as well as the presence of relevant antigens, influences the rate of recovery of host pathophysiology induced by chronic Hp infection. These changes do not seem to occur in association with modulation of intestinal permeability.

Probiotics, known for their prophylactic and therapeutic properties, are routinely used by the medical community in various regions of the world. In some Asian countries, these products are controlled as pharmaceutical substances and must adhere to strict regulatory guidelines. However, outside of Europe where the European Food Safety Authority has recently adopted a Qualified Presumption of Safety approach for probiotics used in food and feed, current safety requirements do not necessitate screening for the presence of virulence and other risk factors, which may result in the inadvertent use of probiotic strains harboring harmful genes. A safety evaluation was conducted on Enterococcus faecium R0026 and Bacillus subtilis R0179 used in several commercial probiotic products marketed in Asia. Molecular techniques were used to verify the identity of each strain and antibiotic resistance profiles were determined towards clinically relevant antibiotics. Strains were subsequently screened for the presence of enterotoxins and virulence factors and were subjected to 28 days of repeated high-dose oral toxicity testing in rats. No risk factors or aberrant activities were identified using such a detailed approach. Thus, both microbes were deemed to pose low risk to the consumer and, therefore, safe for use as probiotics.

Zinc (Zn)-enriched yeast and gluconate are considered two of the more biologically available supplements. However, there have been few reports comparing the bioavailability of these supplements. The objective of this study was to demonstrate whether Zn was absorbed better by healthy male volunteers when given supplements where the mineral is found organically bound in yeast or as a salt gluconate form. The trial used a randomized, two-way crossover design. Urine, blood, and fecal samples were collected and analyzed over a 48-h period after a single dose of supplement. The net Zn balance and the relative bioavailability were calculated. No differences were observed in urine excretion of the two supplements. Zinc gluconate gave higher Zn concentrations in the blood in the first 6 h but also showed greater losses in the feces. Zinc yeast also increased in blood with time but showed significantly less loss in the feces. Thus, the net Zn balance after 48 h for Zn yeast was 9.46 but for Zn gluconate it was -2.00, indicating that Zn gluconate supplementation contributed to a net loss of Zn. It was concluded that organic Zn yeast supplements are more biologically available than Zn gluconate salts.

Selenium has a central role in antioxidant pathways as a cofactor to glutathione peroxidase. The present study evaluated the effects of four different preparations of inactivated yeast containing various concentrations of selenium and glutathione on a combined atherosclerosis and diabetes hamster model. The hamsters were supplemented with the yeast products for three months. The enriched yeast with the highest selenium and glutathione levels reduced the weight loss induced by diabetes, inhibited an increase in plasma cholesterol and triglyceride caused by a high-cholesterol and high-fat diet, increased the time taken for oxidation of lower density lipoproteins (lag time), and inhibited the formation of atherosclerosis better than low selenium/glutathione yeast supplementation. It was concluded that the yeast prepared to provide high selenium and high glutathione was the best for effecting beneficial changes in glutathione, cholesterol, atherosclerosis, and for demonstrating an antioxidant effect. The high selenium and low glutathione yeast was the best for improving selenium and glucose levels.

There are many forms of mineral supplements currently available. Among these mineral-enriched gluconates and yeast are considered two of the more biologically available supplements. The purpose of this study was to use zinc (Zn)- or copper (Cu)-deficient rats to determine whether the organically bound mineral in yeast or the salt gluconate form was more bioavailable, i.e., is absorbed and found in a greater concentration in liver. It was demonstrated that Zn-enriched yeast was 3.7 times more bioavailable than the Zn gluconate and that Cu-enriched yeast was 1.4 times more bioavailable than the Cu gluconate.

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.

Strain R0052, isolated from a North American dairy starter culture, was initially identified as Lactobacillus acidophilus based on phenotypic analyses. However, upon sequencing the 16S rRNA gene, it became clear that the isolate was very highly related to Lactobacillus suntoryeus, Lactobacillus helveticus and Lactobacillus gallinarum, as similarities ranging from 99.3 to 99.8 % were observed. As an initial screening test to investigate the relatedness of strain R0052 and reference strains of L. suntoryeus, L. helveticus and L. gallinarum, the partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), the translational elongation factor Tu (tuf), a surface-layer protein (slp) and the Hsp60 chaperonins (groEL) were determined and they revealed high relatedness between all of the strains. The determination of the 16S-23S rRNA internally transcribed spacer (ITS) sequences revealed 98.3-100% similarity between L. suntoryeus and L. helveticus strains. SDS-PAGE of whole-cell proteins did not distinguish between these species. Fluorescent amplified fragment length polymorphism (FAFLP) could distinguish between these taxa, but they still constituted a single cluster within the L. acidophilus group. Finally, DNA-DNA hybridization experiments between strain R0052 and the type strains of L. helveticus and L. suntoryeus yielded reassociation values above 70% and confirmed that these names are synonyms.

The aim of this study was to determine if probiotics reduce epithelial injury following exposure to Escherichia coli O157:H7 and E. coli O127:H6. The pretreatment of intestinal (T84) cells with lactic acid-producing bacteria reduced the pathogen-induced drop in transepithelial electrical resistance. These findings demonstrate that probiotics prevent epithelial injury induced by attaching-effacing bacteria.

The natural resistance-associated macrophage protein (Nramp) family is functionally conserved in bacteria and eukarya; Nramp homologues function as proton-dependent membrane transporters of divalent metals. Sequence analyses indicate that five phylogenetic groups comprise the Nramp family, three bacterial and two eukaryotic, which are distinct from a more distantly related group of microbial sequences (Nramp outgroup). The Nramp family and outgroup share many conserved residues, suggesting they derived from a common ancestor and raising the possibility that the residues invariant in the Nramp family that correspond to residues which are different but also conserved in the outgroup represent candidate sites of functional divergence of the Nramp family. Four Nramp family-specific residues were identified within transmembrane domains 1, 6, and 11, and replaced by the corresponding invariant outgroup residues in the Escherichia coli Nramp ortholog (the proton-dependent manganese transporter, MntH of group A, EcoliA). The resulting mutants (Asp(34)Gly, Asn(37)Thr, His(211)Tyr, and Asn(401)Gly) were tested for both divalent metal uptake and proton transport; quasi-simultaneous analyses of uptake of metals and protons revealed for the first time protons and metals cotransport by a bacterial Nramp homologue. Additional mutations were studied for comparison (Asp(34)Asn, Asn(37)Asp and Asn(37)Val, Asn(401)Thr, His(211)Ala, His(216)Ala, and His(216)Arg). EcoliA activity was impaired after each of the Nramp/outgroup substitutions, as well as after more conservative replacements, showing that the tested sites are all important for metal uptake and metal-dependent H(+) transport. It is proposed that co-occurrence of these four Nramp-specific transmembrane residues may have contributed to the emergence of this family of metal and proton cotransporters.

The value of exogenously supplied live bacteria for the maintenance of health in humans has been recognized both scientifically in the published literature and commercially in the availability of probiotic products. Although many bacteria characterized as probiotics are strains of Lactobacillus or Bifidobacterium, sporeforming bacteria, primarily of the genus Bacillus and related genera, have also been studied and commercialized as probiotics. This article reviews the characterization, efficacy, and safety of sporeformers used as probiotics.

The selenium determination in biological materials by the classical fluorometric method (FM) is time-consuming and also hazardous, as it requires the destruction of the organic matrix samples with hot HNO3/HClO4 mixtures prior to analysis. Accordingly, commercial analytical laboratories are increasingly using faster instrumental methods; for sample digestion, avoid using HClO4. Because of these procedural changes, the results obtained by commercial laboratories may be unreliable, especially for samples containing Se in organic forms. One such "difficult" substrate is Se yeast, which contains most of its Se as selenomethionine. To establish which methods for Se analysis and sample digestion are applicable, samples of Se yeast and of selenomethionine standards were sent to laboratories employing either flame atomic absorption spectrometry (FAAS), inductively coupled plasma-mass spectrometry (ICP-MS), or hydride generation atomic absorption spectrometry (HGAAS). The results were compared with those obtained by FM and non-destructive instrumental neutron activation analysis (INAA). ICP-MS, after microwave digestion of sample with HNO3/H2O2, produced results within 5% of the expected values, as did those obtained by FM and INAA. With FAAS, acceptable results were obtained after digestion with HNO3/HCl. With HGAAS, sample digestion with HNO3/H2O2 produced values that were systematically elevated by about 10% and exhibited standard deviations of > or = 10%. Thus, current methods of sample digestion are applicable for Se yeast analysis by ICP-MS and FAAS, but not by HGAAS.

A computational model of myelin basic protein (MBP) has been constructed based on the premise of a phylogenetically conserved beta-sheet backbone and on electron microscopical three-dimensional reconstructions. Many residues subject to post-translational modification (phosphorylation, methylation, or conversion of arginines to citrullines) were located in loop regions and thus accessible to modifying enzymes. The triproline segment (residues 99-101) is fully exposed on the back surface of the protein in a long crossover connection between two parallel beta-strands. The proximity of this region to the underlying beta-sheet suggests that post-translational modifications here might have potential synergistic effects on the entire structure. Post-translational modifications that lead to a reduced surface charge could result first in a weakened attachment to the myelin membrane rather than in a gross conformational change of the protein itself. Such mechanisms could be operative in demyelinating diseases such as multiple sclerosis.

Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as negatively stained single particles adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were determined using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concentration, the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resolution of 4 nm, had an overall "C" shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resolution of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromolecule, which plays a key role in the development of multiple sclerosis.

The modulation of a brain phosphoinositide-specific phospholipase C-alpha activity was studied using a variety of compounds of different charge. Detergents such as sodium deoxycholate and cetyltrimethylammonium bromide stimulated the phospholipase C activity when used alone but when used together the effects were not additive. Spermine was an effective inhibitor of the enzyme activity while the cationic peptide, Melittin, had no effect. The inositol phosphates produced by hydrolysis with phosphoinositide-specific phospholipase C were inhibitory while diacylglycerol and inositol did not affect the phospholipase activity. Myelin basic protein, which was previously shown to stimulate phospholipase C activity by 2.5-fold, did not interact with the anionic inositol phosphatases to any significant extent. Thus we concluded that the mechanism of stimulation was not due to relief of product inhibition. Crosslinking studies with the photoactivatable reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid, showed that peptide 24-33 of myelin basic protein, which stimulated the activity almost as much as the native protein, interacted specifically with the phospholipase C. Thus the mechanism by which myelin basic protein stimulated the enzyme appeared to be through specific protein-protein interaction.

We reported previously a highly purified phosphatidylinositol-specific phospholipase C (PI-PLC) from bovine brain and from human myelin which was stimulated by myelin basic protein. In this paper we report that the stimulation of the PI-PLC activity by myelin basic protein (MBP) requires arginine residues in peptide linkage. MBP and poly-L-arginine were able to stimulate the PI-PLC activity by 250% while other basic poly amino acids were unable to stimulate the PI-PLC activity. Neither free arginine nor benzoyl-arginine ethylester was able to stimulate the activity of the enzyme. These results suggested a requirement for the guanidino group of arginine and arginine in peptidyl linkage. The arginyl residues of MBP were modified chemically with 1,2-cyclohexanedione, or enzymatically by cholera toxin which ADP-ribosylated arginyl groups, or by peptidylarginine deiminase which converted the guanidino group of arginine to the ureido group of citrulline. ADP-ribosylation did not affect the stimulation while the 1,2-cyclohexanedione modified MBP and the peptidylarginine deiminase-treated MBP showed a reduced ability to stimulate the PI-PLC activity which correlated with the number of arginyl residues modified. Sequence analysis of the peptidylarginine deiminase-treated MBP established that specific arginyl residues had been converted to citrulline to a greater extent than others. When 70% of Arg 25 and Arg31 were converted to citrulline little stimulatory activity remained, whereas the conversion of 100% of Arg 170 did not affect the ability of C1 to stimulate the enzyme. A role for "active" arginine in this MBP peptide is suggested by our data.

A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.