A genome guided evaluation of the Lab4 probiotic consortium.

Draft genome sequences of the Lab4 probiotic consortium were deposited in Genbank: Bifidobacterium animalis subsp lactis CUL34 (PRJNA482550), Bifidobacterium bifidum CUL20 (PRJNA559984), Lactobacillus acidophilus CUL60 (PRJNA482335), Lactobacillus acidophilus CUL21 (PRJNA482434). Probiogenomic analyses confirmed existing taxonomies and identified putative gene sequences that were functionally related to the performance of each organism during in vitro assessments of bile and acid tolerability, adherence to enterocytes and susceptibility to antibiotics. Genomic stability predictions identified no significant risk of gene acquisition of both antibiotic resistance and virulence genes. These observations were supported by acute phase and repeat dose tolerability studies in Wistar rats. High doses of Lab4 did not result in mortalities, clinical/histopathological abnormalities nor systemic toxicity. Increased faecal numbers of Lab4 in supplemented rats implied survival through the gastrointestinal tract and/or impact the intestinal microbiota composition. In summary, this study provides multifaceted support for probiotic functionality and the safety of the Lab4 consortium.

A randomised controlled study shows supplementation of overweight and obese adults with lactobacilli and bifidobacteria reduces bodyweight and improves well-being.

In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years old) with BMI 25-34.9 kg/m2 received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, p < 0.0001), BMI (0.045 kg/m2, p < 0.0001), WC (0.94 cm, p < 0.0001) and WtHR (0.006, p < 0.0001) were in favour of the probiotic. Stratification identified greater body weight reductions in overweight subjects (1.88%, p < 0.0001) and in females (1.62%, p = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (-2.5%, p < 0.0001) alongside a significant between group reduction in small dense LDL-cholesterol (0.2 mmol/L, p = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, p < 0.0001) were recorded for the probiotic group. No adverse events were recorded. Six months supplementation with Lab4P probiotic resulted in significant weight reduction and improved small dense low-density lipoprotein-cholesterol (sdLDL-C) profiles, QoL and URTI incidence outcomes in overweight/obese individuals.

In vitro neuroprotective activities of two distinct probiotic consortia.

Neurodegeneration has been linked to changes in the gut microbiota and this study compares the neuroprotective capability of two bacterial consortia, known as Lab4 and Lab4b, using the established SH-SY5Y neuronal cell model. Firstly, varying total antioxidant capacities (TAC) were identified in the intact cells from each consortia and their secreted metabolites, referred to as conditioned media (CM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Crystal Violet (CV) assays of cell viability revealed that Lab4 CM and Lab4b CM could induce similar levels of proliferation in SH-SY5Y cells and, despite divergent TAC, possessed a comparable ability to protect undifferentiated and retinoic acid-differentiated cells from the cytotoxic actions of rotenone and undifferentiated cells from the cytotoxic actions of 1-methyl-4-phenylpyridinium iodide (MPP+). Lab4 CM and Lab4b CM also had the ability to attenuate rotenone-induced apoptosis and necrosis with Lab4b inducing the greater effect. Both consortia showed an analogous ability to attenuate intracellular reactive oxygen species accumulation in SH-SY5Y cells although the differential upregulation of genes encoding glutathione reductase and superoxide dismutase by Lab4 CM and Lab4b CM, respectively, implicates the involvement of consortia-specific antioxidative mechanisms of action. This study implicates Lab4 and Lab4b as potential neuroprotective agents and justifies their inclusion in further in vivo studies.

The anti-cholesterolaemic effect of a consortium of probiotics: An acute study in C57BL/6J mice.

Hypercholesterolaemia is a major risk factor for cardiovascular disease and it has been found that some probiotic bacteria possess cholesterol-lowering capabilities. In this study, the ability of the Lab4 probiotic consortium to hydrolyse bile salts, assimilate cholesterol and regulate cholesterol transport by polarised Caco-2 enterocytes was demonstrated. Furthermore, in wild-type C57BL/6J mice fed a high fat diet, 2-weeks supplementation with Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 resulted in significant reductions in plasma total cholesterol levels and suppression of diet-induced weight gain. No changes in plasma levels of very low-density lipoprotein/low-density lipoprotein, high-density lipoprotein, triglycerides, cytokines or bile acids were observed. Increased amounts of total and unconjugated bile acids in the faeces of the probiotic-fed mice, together with modulation of hepatic small heterodimer partner and cholesterol-7α-hydroxylase mRNA expression, implicates bile salt hydrolase activity as a potential mechanism of action. In summary, this study demonstrates the cholesterol-lowering efficacy of short-term feeding of the Lab4 probiotic consortium plus L. plantarum CUL66 in wild-type mice and supports further assessment in human trials.

Complement activation and expression during chronic relapsing experimental autoimmune encephalomyelitis in the Biozzi ABH mouse.

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE) in mice recapitulates many of the clinical and histopathological features of human multiple sclerosis (MS), making it a preferred model for the disease. In both, adaptive immunity and anti-myelin T cells responses are thought to be important, while in MS a role for innate immunity and complement has emerged. Here we sought to test whether complement is activated in crEAE and important for disease. Disease was induced in Biozzi ABH mice that were terminated at different stages of the disease to assess complement activation and local complement expression in the central nervous system. Complement activation products were abundant in all spinal cord areas examined in acute disease during relapse and in the progressive phase, but were absent in early disease remission, despite significant residual clinical disease. Local expression of C1q and C3 was increased at all stages of disease, while C9 expression was increased only in acute disease; expression of the complement regulators CD55, complement receptor 1-related gene/protein y (Crry) and CD59a was reduced at all stages of the disease compared to naive controls. These data show that complement is activated in the central nervous system in the model and suggest that it is a suitable candidate for exploring whether anti-complement agents might be of benefit in MS.

Deletion of Crry, the murine ortholog of the sporadic Alzheimer's disease risk gene CR1, impacts tau phosphorylation and brain CFH.

Large-scale genome-wide SNP association studies have identified an association between variants of CR1, the gene encoding complement component receptor 1, and the sporadic form of Alzheimer's disease. The role of CR1 and the complement system in Alzheimer's disease remains far from clear. In rodents the closest ortholog of CR1 is the Crry gene (Cr1-related protein Y). To begin to explore its role in Alzheimer's disease we examined hippocampal lysates from Crry(-/-) mice and age matched controls by immunoblotting. We measured complement factor H, a component of the complement system and biomarker for Alzheimer's disease progression, and tau phosphorylation at the serine 235 site, hyperphosphorylated forms of tau being a defining neuropathological hallmark of the disease. We found that levels of CFH and of tau phosphorylation at serine 235 were strongly and significantly reduced in Crry(-/-) samples. These observations provide a starting point for further attempts to determine the role of CR1 in the neuropathological process driving Alzheimer's disease.

Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular degeneration.

PURPOSE: There is evidence for complement dysfunction in age-related macular degeneration (AMD). Complement activation leads to formation of the membrane attack complex (MAC), known to assemble on retinal pigment epithelial (RPE) cells. Therefore, the effect of sub-lytic MAC on RPE cells was examined with regard to pro-inflammatory or pro-angiogenic mediators relevant in AMD. METHODS: For sub-lytic MAC induction, RPE cells were incubated with an antiserum to complement regulatory protein CD59, followed by normal human serum (NHS) to induce 5% cell death, measured by a viability assay. MAC formation was evaluated by immunofluorescence and FACS analysis. Interleukin (IL)-6, -8, monocytic chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were quantified by enzyme-linked immunosorbent assay (ELISA). Intracellular MCP-1 was analysed by immunofluorescence, vitronectin by western blotting, and gelatinolytic matrix metalloproteinases (MMPs) by zymography. RESULTS: Incubation of RPE cells with the CD59 antiserum followed by 5% NHS induced sub-lytic amounts of MAC, verified by FACS and immunofluorescence. This treatment stimulated the cells to release IL-6, -8, MCP-1, and VEGF. MCP-1 staining, production of vitronectin, and gelatinolytic MMPs were also elevated in response to sub-lytic MAC. CONCLUSIONS: MAC assembly on RPE cells increases the IL-6, -8, and MCP-1 production. Therefore, sub-lytic MAC might have a significant role in generating a pro-inflammatory microenvironment, contributing to the development of AMD. Enhanced vitronectin might be a protective mechanism against MAC deposition. In addition, the increased expression of gelatinolytic MMPs and pro-angiogenic VEGF may be associated with neovascular processes and late AMD.

Introduction to "a handbook of transcription factors".

This chapter briefly summarizes the topics in this volume.

A catalogue of eukaryotic transcription factor types, their evolutionary origin, and species distribution.

Transcription factors (TFs) play key roles in the regulation of gene expression by binding in a sequence-specific manner to genomic DNA. In eukaryotes, DNA binding is achieved by a wide range of structural forms and motifs. TFs are typically classified by their DNA-binding domain (DBD) type. In this chapter, we catalogue and survey 91 different TF DBD types in metazoa, plants, fungi, and protists. We briefly discuss well-characterized TF families representing the major DBD superclasses. We also examine the species distributions and inferred evolutionary histories of the various families, and the potential roles played by TF family expansion and dimerization.

Systemic and local anti-C5 therapy reduces the disease severity in experimental autoimmune uveoretinitis.

Activation of complement occurs during autoimmune retinal and intraocular inflammatory disease as well as neuroretinal degenerative disorders. The cleavage of C5 into fragments C5a and C5b is a critical event during the complement cascade. C5a is a potent proinflammatory anaphylatoxin capable of inducing cell migration, adhesion and cytokine release, while membrane attack complex C5b-9 causes cell lysis. Therapeutic approaches to prevent complement-induced inflammation include the use of blocking monoclonal antibodies (mAb) to prevent C5 cleavage. In these current experiments, the rat anti-mouse C5 mAb (BB5.1) was utilized to investigate the effects of inhibition of C5 cleavage on disease progression and severity in experimental autoimmune uveoretinitis (EAU), a model of organ-specific autoimmunity in the eye characterized by structural retinal damage mediated by infiltrating macrophages. Systemic treatment with BB5.1 results in significantly reduced disease scores compared with control groups, while local administration results in an earlier resolution of disease. In vitro, contemporaneous C5a and interferon-gamma signalling enhanced nitric oxide production, accompanied by down-regulation of the inhibitory myeloid CD200 receptor, contributing to cell activation. These experiments demonstrate that C5 cleavage contributes to the full expression of EAU, and that selective C5 blockade via systemic and local routes of administration can suppress disease. This presents great therapeutic potential to protect against tissue damage during autoimmune responses in the retina or inflammation-induced degenerative disease.

Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins.

BACKGROUND: Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates. RESULTS: We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG. We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific. Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway. CONCLUSIONS: The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins. Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Cdc13 both positively and negatively regulates telomere replication.

Cdc13 is a single-strand telomeric DNA-binding protein that positively regulates yeast telomere replication by recruiting telomerase to chromosome termini through a site on Cdc13 that is eliminated by the cdc13-2 mutation. Here we show that Cdc13 has a separate role in negative regulation of telomere replication, based on analysis of a new mutation, cdc13-5. Loss of this second regulatory activity results in extensive elongation of the G strand of the telomere by telomerase, accompanied by a reduced ability to coordinate synthesis of the C strand. Both the cdc13-5 mutation and DNA polymerase alpha mutations (which also exhibit elongated telomeres) are suppressed by increased expression of the Cdc13-interacting protein Stn1, indicating that Stn1 coordinates action of the lagging strand replication complex with the regulatory activity of CDC13. However, the association between Cdc13 and Stn1 is abolished by cdc13-2, the same mutation that eliminates the interaction between Cdc13 and telomerase. We propose that Cdc13 participates in two regulatory steps-first positive, then negative-as a result of successive binding of telomerase and the negative regulator Stn1 to overlapping sites on Cdc13. Thus, Cdc13 coordinates synthesis of both strands of the telomere by first recruiting telomerase and subsequently limiting G-strand synthesis by telomerase in response to C-strand replication.

DNA microarrays for expression profiling.

DNA microarrays enable the transcript levels of an entire genome to be measured simultaneously. Recent improvements in array manufacture, sample preparation, and data analysis are shifting emphasis from the technology itself to experimental design and the broader range of biological questions that can be addressed. The past year has also seen a transition from experiments involving a small number of conditions, with an emphasis on the specific genes induced or repressed, to experiments involving hundreds of conditions in which patterns of global gene expression are used to classify disease specimens and discover gene functions and drug targets. Basic research, medicine, and pharmacology are all likely to benefit from these advances.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein alpha gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1.

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPalpha genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPalpha genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPalpha gene (xC/EBPalpha). The -1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPalpha or xC/EBPss. Deletion analysis showed that the -321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPalpha promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPalpha promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPalpha expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

Differential regulation of macrophage CCAAT-enhancer binding protein isoforms by lipopolysaccharide and cytokines.

The regulation of the C/EBP family in macrophages by LPS and cytokines is of potentially crucial importance in several pathophysiological conditions. The action of LPS and three cytokines on the expression of C/EBP mRNA, protein and functional DNA binding activity in the murine J774.2 cell line was therefore studied. Exposure of the cells to LPS, IL-1, IFN-gamma and TNF-alpha produced a reduction of C/EBP alpha mRNA levels and a corresponding increase in the expression of C/EBP beta and C/EBP delta. EMSA showed time-dependent changes in the DNA binding activity of individual C/EBP isoforms and demonstrated the participation of heterodimers between the different members in DNA-protein interactions. Additionally, mediator-specific changes in the kinetics and magnitude of C/EBP mRNA expression pattern and profile of DNA-protein interactions were observed. These studies provide novel insights into the potential mechanisms that may be responsible for the mediator-specific regulation of macrophage gene expression through the C/EBP family.

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Widespread aneuploidy revealed by DNA microarray expression profiling.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

The Est3 protein is a subunit of yeast telomerase.

EST1, EST2, EST3 and TLC1 function in a single pathway for telomere replication in the yeast Saccharomyces cerevisiae [1] [2], as would be expected if these genes all encode components of the same complex. Est2p, the reverse transcriptase protein subunit, and TLC1, the templating RNA, are subunits of the catalytic core of yeast telomerase [3] [4] [5]. In contrast, mutations in EST1, EST3 or CDC13 eliminate telomere replication in vivo [1] [6] [7] [8] but are dispensable for in vitro telomerase catalytic activity [2] [9]. Est1p and Cdc13p, as components of telomerase and telomeric chromatin, respectively, cooperate to recruit telomerase to the end of the chromosome [7] [10]. However, Est3p has not yet been biochemically characterized and thus its specific role in telomere replication is unclear. We show here that Est3p is a stable component of the telomerase holoenzyme and furthermore, association of Est3p with the enzyme requires an intact catalytic core. As predicted for a telomerase subunit, fusion of Est3p to the high affinity Cdc13p telomeric DNA binding domain greatly increases access of telomerase to the telomere. Est1p is also tightly associated with telomerase; however, Est1p is capable of forming a stable TLC1-containing complex even in the absence of Est2p or Est3p. Yeast telomerase therefore contains a minimum of three Est proteins for which there is both in vivo and in vitro evidence for their role in telomere replication as subunits of the telomerase complex.

Cytokine-mediated differential regulation of macrophage activator protein-1 genes.

The regulation of macrophage activator protein-1 (AP-1) gene expression by LPS and cytokines is of potentially crucial importance in the pathogenesis of several diseases. The action of LPS and four cytokines on AP-1 gene expression in the murine macrophage J774.2 cell line was, therefore, studied. Exposure of the cells to IL-6 produced no changes in the mRNA levels of all AP-1 members studied. In contrast, the expression of JunB, c-jun and c-fos, but not JunD, was increased by LPS, TNF-alpha, IFN-gamma and IL-1, albeit with different kinetics and magnitude of induction. Electrophoretic mobility shift assays showed a close correlation between the expression of the AP-1 genes and the functional AP-1 DNA binding activity and, additionally, demonstrated the participation of heterodimeric interactions between the different members. These studies provide insights into the potential mechanisms that may be involved in the mediator-specific modulation of AP-1 regulated macrophage gene expression.