Human-mouse genome comparisons to locate regulatory sites.

Elucidating the human transcriptional regulatory network is a challenge of the post-genomic era. Technical progress so far is impressive, including detailed understanding of regulatory mechanisms for at least a few genes in multicellular organisms, rapid and precise localization of regulatory regions within extensive regions of DNA by means of cross-species comparison, and de novo determination of transcription-factor binding specificities from large-scale yeast expression data. Here we address two problems involved in extending these results to the human genome: first, it has been unclear how many model organism genomes will be needed to delineate most regulatory regions; and second, the discovery of transcription-factor binding sites (response elements) from expression data has not yet been generalized from single-celled organisms to multicellular organisms. We found that 98% (74/75) of experimentally defined sequence-specific binding sites of skeletal-muscle-specific transcription factors are confined to the 19% of human sequences that are most conserved in the orthologous rodent sequences. Also we found that in using this restriction, the binding specificities of all three major muscle-specific transcription factors (MYF, SRF and MEF2) can be computationally identified.

A new measurement for optimal antenatal care: determinants and outcomes in Cameroon.

A combined measure of optimal antenatal care can provide more information on the role it plays in maternal health. Our objectives were to investigate the determinants of a measure of optimal antenatal care and the associated pregnancy outcomes. Data on 7,557 women taken from the 2004 Demographic and Health Survey in Cameroon were used to develop a new measurement of optimal antenatal care based on four indicators: at least four visits, first visit in first trimester, last visit in third trimester and a professional provider of antenatal care. We studied the relationship of this new variable with other related variables in a multivariate analysis, taking into account the complex study design. Almost sixty six percent of the women had optimal antenatal care. Secondary or higher education (OR 1.74; 95% CI 1.28-2.36), greater wealth (OR 2.31; 95% CI 1.73-3.1), urban residence (OR 1.42; 95% CI 1.12-1.82) and parity of 3-4 (OR 0.79; 95% CI 0.62-0.99) were independently associated with optimal antenatal care. Women with optimal antenatal care were more likely to deliver in a health unit (OR 2.91; 95% CI 2.42-3.49), to be assisted by a skilled health worker during delivery (OR 1.88; 95% CI 1.49-2.37) and to have a baby with a normal birthweight (OR 1.62; 95% CI 1.11-2.38). Obtaining and using a new measure for ANC is feasible. The association of optimal antenatal care to education, wealth and residence in this study, consistent with others, highlights the role of the country's development in maternal health.

Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.

Expulsion of gastrointestinal nematodes is associated with pronounced mucosal mast cell (MMC) hyperplasia, differentiation, and activation, accompanied by the systemic release of MMC granule chymases (chymotrypsin-like serine proteases). The beta-chymase mouse mast cell protease-1 (mMCP-1) is expressed predominantly by intraepithelial MMCs, and levels in the bloodstream and intestinal lumen are maximal at the time of worm expulsion in parasitized mice. To address the in vivo functions of MMC-specific beta-chymases, we have generated transgenic mice that lack the mMCP-1 gene. They were backcrossed onto a congenic BALB/c background to investigate the response to nematode infection. The deletion of the mMCP-1 gene is associated with significantly delayed expulsion of Trichinella spiralis and increased deposition of muscle larvae in BALB/c mice despite the presence of normal and sometimes increased numbers of MMCs. Neither worm fecundity nor worm burdens were altered in Nippostrongylus-infected mMCP-1(-/)- BALB/c mice. These data demonstrate, for the first time, that the ablation of an MMC-derived effector molecule compromises the expulsion process.

Transmembrane tumor necrosis factor alpha is required for enteropathy and is sufficient to promote parasite expulsion in gastrointestinal helminth infection.

To study the specific role of transmembrane tumor necrosis factor (tmTNF) in protective and pathological responses against the gastrointestinal helminth Trichinella spiralis, we compared the immune responses of TNF-alpha/lymphotoxin alpha (LTalpha)(-/-) mice expressing noncleavable transgenic tmTNF to those of TNF-alpha/LTalpha(-/-) and wild-type mice. The susceptibility of TNF-alpha/LTalpha(-/-) mice to T. spiralis infection was associated with impaired induction of a protective Th2 response and the lack of mucosal mastocytosis. Although tmTNF-expressing transgenic (tmTNF-tg) mice also had a reduced Th2 response, the mast cell response was greater than that observed in TNF-alpha/LTalpha(-/-) mice and was sufficient to induce the expulsion of the parasite. T. spiralis infection of tmTNF-tg mice resulted in significant intestinal pathology characterized by villus atrophy and crypt hyperplasia comparable to that induced following the infection of wild-type mice, while pathology in TNF-alpha/LTalpha(-/-) mice was significantly reduced. Our data thus indicate a role for tmTNF in host defense against gastrointestinal helminths and in the accompanying enteropathy. Furthermore, they also demonstrate that TNF-alpha is required for the induction of Th2 immune responses related to infection with gastrointestinal helminth parasites.

Detecting subtle sequence signals: a Gibbs sampling strategy for multiple alignment.

A wealth of protein and DNA sequence data is being generated by genome projects and other sequencing efforts. A crucial barrier to deciphering these sequences and understanding the relations among them is the difficulty of detecting subtle local residue patterns common to multiple sequences. Such patterns frequently reflect similar molecular structures and biological properties. A mathematical definition of this "local multiple alignment" problem suitable for full computer automation has been used to develop a new and sensitive algorithm, based on the statistical method of iterative sampling. This algorithm finds an optimized local alignment model for N sequences in N-linear time, requiring only seconds on current workstations, and allows the simultaneous detection and optimization of multiple patterns and pattern repeats. The method is illustrated as applied to helix-turn-helix proteins, lipocalins, and prenyltransferases.

Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond.

Single-stranded regions in RNA secondary structure are important for RNA-RNA and RNA-protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals 'well-determined' single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit beta-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA-RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.

Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes.

Toward the goal of identifying complete sets of transcription factor (TF)-binding sites in the genomes of several gamma proteobacteria, and hence describing their transcription regulatory networks, we present a phylogenetic footprinting method for identifying these sites. Probable transcription regulatory sites upstream of Escherichia coli genes were identified by cross-species comparison using an extended Gibbs sampling algorithm. Close examination of a study set of 184 genes with documented transcription regulatory sites revealed that when orthologous data were available from at least two other gamma proteobacterial species, 81% of our predictions corresponded with the documented sites, and 67% corresponded when data from only one other species were available. That the remaining predictions included bona fide TF-binding sites was proven by affinity purification of a putative transcription factor (YijC) bound to such a site upstream of the fabA gene. Predicted regulatory sites for 2097 E.coli genes are available at http://www.wadsworth.org/resnres/bioinfo/.

Trihalomethanes in drinking water and human colorectal cancer.

The relation of trihalomethanes (THM) to colorectal cancer was evaluated. A total of 395 colorectal cancer deaths among white women teachers in New York State was compared to an equal number of deaths of teachers from noncancerous causes. Cumulative chloroform (CHCl3) exposure was estimated by the application of a statistical model to operational records from the individual water treatment facilities that served the home and work addresses of each study subject during the 20 years prior to death. The odds of exposure to a surface source containing little or no THM was no greater for cases than for controls. The odds ratio = 1.07; the 90% confidence interval = 0.79-1.43; and the P = .68. The distribution of CHCl3 exposure was not significantly different between cases and controls (rated by Wilcoxon signed rank statistic = -0.52; P = .60). No effect of cumulative CHCl3 exposure on outcome was seen in a logistic analysis controlling for average source type, population density, marital status, age, and year of death (likelihood ratio test statistic = 0.047; P = .83).

Relationship of hair dye use, benign breast disease, and breast cancer.

An epidemiologic case-control study of 118 breast cancer patients and 233 controls was conducted to test the hypothesis that hair dyes are related to breast cancer. Matched controls were selected by "random digit dialing," and all epidemiologic data were collected by telephone interviews. No overall association was detected. On a prospective basis, the interaction between hair dye exposure and six variables known to be risk factors for breast cancer then were examined: previous benign breast disease (BBD), "ever" versus "never" pregnant, age at first pregnancy, menopause induced by operation, age at menarche, and education. A statistically significant increased risk of breast cancer was found for women with a history of BBD and exposure to hair dyes as compared to women with BBD but no hair dye exposure: The relative risk (RR) was 4.5, and the 95% confidence intervals (C) were 1.20 and 15.78. A total of 24 women (19 patients and 5 controls) reported a history of BBD and hair dye use. Further analysis revealed a significant association between hair dye use and breast cancer among women 40-49 years of age (RR = 3.33; 95% CI: 1.1 and 10.85) and a highly significant (P = 0.0008) dose-response relationship among women who used hair dyes for changing their natural color as opposed to covering gray hair. The numbers of patients and controls included in this study were small and several hypotheses were tested. Additional epidemiologic studies are needed before firm conclusions can be reached concerning the nature of these associations.

Anatomy of a preferred target site for the bacterial insertion sequence IS903.

Like many transposons the bacterial insertion sequence IS903 was thought to insert randomly. However, using both genetic and statistical approaches, we have derived a target site for IS903 that is used 84% of the time. Computational and genetic analyses of multiple IS903 insertion sites predicted a preferred target consisting of a 21 bp palindromic pattern centered on the 9 bp target duplication generated during transposition. Here we show that targeting can be dissected into four components: the 5 bp flanking sequences, the most important sequences required for site-specific insertion; the 7 bp palindromic core within the target duplication; the dinucleotide pair at the transposon-target junction; and the local DNA context. Finally, using a substrate with multiple target sites we show that a target site is more likely found by a local bind-and-slide model and not by extended DNA tracking.

Contrasting effects of acute and chronic gastro-intestinal helminth infections on a heterologous immune response in a transgenic adoptive transfer model.

We have previously found that co-immunisation with ovalbumin (OVA) and the body fluid of the helminth Ascaris suum inhibited an OVA-specific delayed type hypersensitivity (DTH) response by reducing OVA-specific CD4+ T lymphocyte proliferation via an IL-4 independent mechanism. In the present study, we determined whether parasite infections themselves could induce similar changes to peripheral immunisation by examining the modulation of OVA-specific immune responses during acute and chronic helminth infections. Surprisingly, an acute infection with Trichinella spiralis, but not a chronic infection with Heligmosomoides polygyrus, inhibited the OVA-specific DTH reaction. Correspondingly, the T helper 1 (Th1) OVA-specific response was decreased in mice infected with T. spiralis, but not with H. polygyrus. Inhibition of the Th1 response may be a result of a shift in the Th1/Th2 balance as although both H. polygyrus and T. spiralis infected mice induced a Th2 OVA-specific response, that exhibited by T. spiralis was more potent. Furthermore, although IL-10 secretion upon OVA restimulation was similarly increased by both infections, production of this immunoregulatory cytokine may play a role in the suppression of immune responses observed with T. spiralis infection depending on the context of its release. Interestingly, analysis of the OVA-specific T lymphocyte division by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining revealed that gastro-intestinal infection with the acute helminth T. spiralis, but not with chronic H. polygyrus, inhibited the systemic immune response by significantly inhibiting the antigen-specific T cell proliferation during the primary response, a mechanism similar to that observed when A. suum parasite extracts were directly mixed with the OVA during immunisation in our previous studies.

Motifs and structural fold of the cofactor binding site of human glutamate decarboxylase.

The pyridoxal-P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal-P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo- and holoenzymes. PROBE identified six motifs shared by the two GADs and four proteins of known structure: bacterial ornithine decarboxylase, dialkylglycine decarboxylase, aspartate aminotransferase, and tyrosine phenol-lyase. Five of the motifs corresponded to the alpha/beta elements and loops that form most of the conserved fold of the pyridoxal-P binding cleft of the four enzymes of known structure; the sixth motif corresponded to a helical element of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal-P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functions were evaluated by examining the proteins of known structure. The key residues that interact directly with pyridoxal-P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a specific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates that the highly regulated transition between apo- and holoGAD is accomplished by modifications in this basic fold rather than through a novel folding pattern.

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles.

Effects of reagent and instrument on prothrombin times, activated partial thromboplastin times and patient/control ratios.

Activated partial thromboplastin times accumulated from two proficiency testing surveys were analyzed to determine simultaneously the effects of the method and reagent used. Prothrombin time results were reevaluated concomitantly for comparison. A robust two-way analysis of variance was applied to determine the effect of method and reagent on APTT results. The effect of the reagent and method on the ratio of abnormal to normal plasma clotting times was determined. We found a substantial difference in ratios for the PT using different reagents on the same instrument. There was an even larger effect of reagents on APTT ratios. Our finding of substantial reagent effects for the PT and APTT clearly support the need for standardization. We found standardization to be feasible only for the PT, and only if applied in a form consistent with the inherent error structure of the data. For the APTT, the present methodology and plasma samples did not achieve consistent standardization.

Prothrombin time proficiency testing: a robust grading method.

A robust two-way analysis of variance technique was applied to determine simultaneously the effects of method and thromboplastin on prothrombin time. A new approach to outlier detection for two-way analysis of variance was used. Focusing on the underlying error structure improved the uniformity of the grading procedure in the hematology proficiency testing program of the New York State Department of Health. The logarithm-transformed scale produced constancy of error variance and resulted in uniformity of the acceptable spread of data. The common variance was lower than that obtained by previous methods and allowed for a narrower acceptable range of reported prothrombin times by reducing the inflated standard deviation, thus improving the efficiency of the grading procedure. For proficiency testing, no advantage was found in the use of either a common thromboplastin or freeze-dried, coumadinized patient plasmas rather than artificially depleted commercial plasmas, except for special purposes.

Immune response profiles in responsive and non-responsive mouse strains infected with Heligmosomoides polygyrus.

The immune response to a primary infection with Heligmosomoides polygyrus was monitored in three strains of mice (SJL, BALB/c, CBA) with different degrees of susceptibility to the infection. Parameters measured included circulating leucocyte numbers, T and B cell numbers in the mesenteric lymph nodes, the mucosal mast cell response, and quantitative and qualitative antibody responses to parasite antigens. From these data it would appear that resistance was governed by the relative speed and magnitude of the immune response mounted by the host. The possible immunological mechanisms governing this process are discussed.

Differential secretion of acetylcholinesterase and proteases during the development of Heligmosomoides polygyrus.

The development of the gastrointestinal nematode Heligmosomoides polygyrus was studied in the mouse. Levels of production of acetylcholinesterase and proteases were measured in excretory/secretory products of various stages of the parasite. The production of acetylcholinesterase was found to be maximal between days 4 and 6 post-infection, corresponding to the fourth larval stage of the parasite's life-cycle. Analysis of proteolytic activity revealed both quantitative and qualitative differences between the stages. Quantitative examination showed a maximal concentration of proteolytic enzymes in the early third larval stage (L3). Qualitative analysis revealed L3-associated molecules at 96, 15 and 8 kDa, L4-associated molecules at 58 and 33 kDa and adult-associated molecules at 116, 102, 39 and 25 kDa. A number appeared to be shared by all stages (18, 16 and 13 kDa), whilst others (76 and 42 kDa) appeared to be associated with the late L4/early adult parasite. The biological and immunological implications of variation in the production of proteases and acetylcholinesterase during the development of H. polygyrus are discussed.

Immunosuppressive proteins secreted by the gastrointestinal nematode parasite Heligmosomoides polygyrus.

Experiments are described in which the conditions for the production, assay and isolation of immunomodulatory factors from the excretory-secretory (ES) products of Heligmosomoides polygyrus have been standardized. For the inhibition of an in vitro antibody response to keyhole limpet haemocyanin, immunosuppressive activity was most reproducibly produced by 10-20-day-old adult worms maintained in culture for 24 h. This activity was relatively stable at room temperature, at 50 degrees C and pH 2, but was destroyed by boiling. Immunosuppressive activity was eluted from Sephadex G-100 in fractions preceding those containing the bulk of ES proteins, and resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with molecular masses of 67, 54 and 20 kDa. The relative purity of these factors was confirmed by iso-electric focusing, where immunosuppressive activity was associated with proteins of pI values of approximately 4.2 and 4.35.

Decreased mitogen responsiveness and elevated tumor necrosis factor production in cats shortly after feline immunodeficiency virus infection.

We present the results of an investigation into the effects of feline immunodeficiency virus (FIV) infection on the response to mitogens and cytokine production in the first month of infection. We were able to demonstrate a depression of response of peripheral blood mononuclear cells to the mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen, with the response to pokeweed mitogen being most severely affected. The response of the cells of the spleen were affected by 10 days post infection and these could not be augmented by the addition of exogenous interleukin-2 (IL-2). The response of mesenteric lymph node cells was not affected until 20 days post infection and this could be partially restored by the addition of exogenous IL-2. IL-2 production was unaffected in peripheral blood mononuclear cells, slightly depressed in mesenteric lymph node cells and slightly elevated in spleen cells. Tumor necrosis factor levels were significantly elevated with respect to controls within 10 days of infection. These studies suggest that there are a number of changes in the immune response of FIV infected cats early in infection and this may determine the subsequent outcome of the infection.

Clinical and pathological findings in feline immunodeficiency virus experimental infection.

A study is described of the clinical and pathological findings in 20 specific pathogen free cats infected when 1 year old with feline immunodeficiency virus and monitored over 12 months. Cats were divided into two groups (A and B). The clinical and clinicopathological features were studied in Group A. In Group B, at 1, 2, 4, 9 and 12 months post infection two cats were necropsied. Clinically all cats developed generalised lymphadenopathy, six cats were neutropenic and five cats lymphopenic. Three cats became febrile with conjunctivitis and anterior uveitis and one of these cats ultimately developed jaundice. Postmortem examinations confirmed a generalised lymphadenopathy involving peripheral and visceral lymph nodes with concurrent stimulation of splenic white matter and mucosal lymphoid tissue of the digestive tract and conjunctiva. Within the lymph nodes there was a reactive follicular hyperplasia accompanied by a paracortical hyperplasia with an increased paracortical vascularity. Unusual features were the presence of lymphoid follicles in the bone marrow, thymus and parathyroid tissue. In addition, aggregates of lymphoid cells were found within salivary glands, kidneys, sclera and choroid of the eye. One cat developed a lymphosarcoma affecting the liver and kidneys at 36 weeks post infection. The cat with jaundice had a cholangitis with marked biliary epithelial hyperplasia.