DEFB1 gene 5' untranslated region (UTR) polymorphisms are marginally involved in inflammatory bowel disease in south Brazilians.

The possible association of three DEFB1 gene polymorphisms with susceptibility to develop ulcerative colitis (UC) and Crohn's disease (CD) was investigated in Brazilian patients and controls. Although a clear and strong association between functional 5'-UTR DEFB1 SNPs and susceptibility/protection to IBDs cannot be drawn, our results suggest a possible involvement of DEFB1 gene in inflammatory bowel diseases, especially with the colonic localization of Crohn's disease.

Analysis of KIR gene frequencies and HLA class I genotypes in prostate cancer and control group.

Prostate cancer is the second most common cancer in men, with a significant increase in incidence and mortality in men over 50 years of age. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study is to evaluate the association between the KIR genes and HLA alleles in patients with prostate cancer and healthy controls. Two hundred patients with prostate cancer and 185 healthy controls were typed for HLA class I and KIR genes by PCR-SSP. When both groups were compared, no significant differences were found for HLA-C group 1 and group 2, HLA-Bw4, HLA-A3 and A11. No difference was seen either in KIR frequency between patients with prostate cancer and controls. In conclusion, our data suggest no potential role for the KIR gene system in prostate cancer.

Killer cell immunoglobulin-like receptor gene diversity in a Caucasian population of southern Brazil.

Killer immunoglobulin-like receptors (KIR) regulate the activity of natural killer and T cells through an interaction with specific human leucocyte antigen (HLA) class I molecules on target cells. Diversity in KIR gene content, KIR allelic and haplotype polymorphism has been observed between different ethnic groups. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania and Africa remain poorly studied. The aim of this study was to analyse the variability of KIR genes in 200 healthy nonrelated individuals from the Southern Brazilian population. KIR genes and HLA-A, -B and -Cw were genotyped using polymerase chain reaction-sequence-specific primers. Southern Brazilian population demonstrated several similarities to states that are closer geographically and distinct differences with Northern Brazil in the frequency of genes KIR2DS1, 2DS2, 2DS3, 2DS5, 3DL1, 3DS1, 2DL1 and 2DL2. The activating gene KIR2DS5 was the least frequent locus found in our group. Interaction of KIR/HLA was more common in the 2DS1-/2DL1+/C2+ association. This study demonstrated the diversity of KIR genes and of KIR/HLA association in a Caucasian group of Southern Brazil, establishing differences and similarities to other different populations.

A study of the killer cell immunoglobulin-like receptor gene KIR2DS1 in a Caucasoid Brazilian population with psoriasis vulgaris.

Psoriasis is a chronic inflammatory skin disease whose pathogenesis and genetic background remain unclear. Considering that previous studies have suggested an association of psoriasis vulgaris (PV) and killer cell immunoglobulin-like receptors (KIRs), we typed 15 KIR genes and human leukocyte antigen (HLA)-Cw in 79 Brazilian Caucasoid patients with PV and 110 healthy controls by polymerase chain reaction (PCR) using sequence-specific oligonucleotides and sequence-specific primers. We did not observe a relevant increase in the frequency of the activating KIR2DS1 gene in the PV group [KIR2DS1, 46 of 79 cases (58.2%) vs 40 of 110 controls (36.4%)]. However, an association of KIR2DS1 with Cw*0602+ in 26.5% of PV patients was observed, while it was present in only 5.4% of controls. These results suggest that activating KIR2DS1 gene may not confer susceptibility to PV, and an association of KIR2DS1 gene with the HLA-Cw*0602+ was observed in these patients.

A single 60-min bout of peristaltic pulse external pneumatic compression transiently upregulates phosphorylated ribosomal protein s6.

We investigated whether a single 60-min bout of whole leg, peristaltic pulse external pneumatic compression (EPC) altered select growth factor-related mRNAs and/or various phospho(p)-proteins related to cell growth, proliferation, inflammation and apoptosis signalling (e.g. Akt-mTOR, Jak-Stat). Ten participants (8 males, 2 females; aged 22·2 ± 0·4 years) reported to the laboratory 4 h post-prandial, and vastus lateralis muscle biopsies were obtained prior to (PRE), 1 h and 4 h post-EPC treatment. mRNA expression was analysed using real-time RT-PCR and phosphophorylated and cleaved proteins were analysed using an antibody array. No changes in selected growth factor-related mRNAs were observed following EPC. All p-proteins significantly altered by EPC decreased, except for p-rps6 (Ser235/236) which increased 31% 1 h post-EPC compared to PRE levels (P = 0·016). Notable decreases also included p-BAD (Ser112; -28%, P = 0·004) at 4 h post-EPC compared to PRE levels. In summary, an acute bout of EPC transiently upregulates p-rps6 as well as affecting other markers in the Akt-mTOR signalling cascade. Future research should characterize whether chronic EPC application promotes alterations in lower-limb musculature and/or enhances exercise-induced training adaptations.

Fluorescence resonance energy transfer as a structural tool for nucleic acids.

Fluorescence resonance energy transfer is a spectroscopic method that provides distance information on macromolecules in solution in the range 20-80 A. It is particularly suited to the analysis of the global structure of nucleic acids because the long-range distance information provides constraints when modelling these important structures. The application of fluorescence resonance energy transfer to nucleic acid structure has seen a resurgence of interest in the past decade, which continues to increase. An especially exciting development is the recent extension to single-molecule studies.

Ligand-induced self-association of the Escherichia coli regulatory protein TyrR.

Analyses of the sedimentation properties of the Escherichia coli regulatory protein TyrR indicated that it undergoes a ligand-induced hexamerization. This phenomenon was observed at protein concentrations approximating to those found in vivo. In the absence of added ligands, TyrR sedimented as a single molecular species with a sedimentation coefficient of 5.3 S and a relative molecular mass of 113,000. Given a subunit relative molecular mass of 57,640 for TyrR, it was concluded that this species is a dimer. Similar sedimentation properties were observed when TyrR was sedimented in the presence of either tyrosine, phenylalanine, ATP or ATP gamma S, a non-hydrolysable analogue of ATP. However, in the presence of saturating ATP gamma S and 500 microM tyrosine or 25 mM phenylalanine the sedimentation behaviour of TyrR yielded relative molecular masses of 340,000 and 310,000, respectively, indicative of hexamer formation. The sedimentation data obtained across a range of TyrR concentrations fitted equally well to dimer-hexamer and dimer-tetramer-hexamer models. For the latter model, the predicted overall association constant was 3.2 x 10(13) M-2 at saturating tyrosine, while the relative values of the association constants for the two individual steps indicated a concerted mechanism with the tetramer a minor component. There was no indication of dimer dissociation when dilute TyrR solutions (100 nM) were sedimented. A model to explain the role of hexamerization in tyrosine-mediated repression of transcription by TyrR is proposed. It is suggested that the hexameric form of TyrR is the active repressing species, interacting with two or three specific sequences (TyrR boxes) in the targeted regulatory DNA. The hexamerization reaction that takes place when the tyrosine concentration rises is envisaged as occurring in situ on the DNA, with a TyrR dimer that permanently occupies one of the TyrR boxes acting as a nucleation site for the development of the hexamer-DNA complex.

The A730 loop is an important component of the active site of the VS ribozyme.

The core of the VS ribozyme comprises five helices, that act either in cis or in trans on a stem-loop substrate to catalyse site-specific cleavage. The structure of the 2-3-6 helical junction indicates that a cleft is formed between helices II and VI that is likely to serve as a receptor for the substrate. Detailed analysis of sequence variants suggests that the base bulges of helices II and VI play an architectural role. By contrast, the identity of the nucleotides in the A730 loop is very important for ribozyme activity. The base of A756 is particularly vital, and substitution by any other nucleotide or ablation of the base leads to a major reduction in cleavage rate. However, variants of A756 bind substrate efficiently, and are not defective in global folding. These results suggest that the A730 loop is an important component of the active site of the ribozyme, and that A756 could play a key role in catalysis.

A gene for superoxide dismutase from Xanthomonas campestris pv. campestris and its expression during bacterial-plant interactions.

A recombinant plasmid selected from a library of Xanthomonas campestris pv. campestris genomic DNA by functional complementation of a superoxide dismutase (SOD)-deficient strain of Escherichia coli contained a gene encoding the major SOD activity of X. campestris pv. campestris. Inhibition and renaturation studies suggested that manganese was the metal cofactor for this SOD. Examination of the nucleotide sequence of an active subclone revealed a 612-bp open reading frame that encodes a protein with high amino acid sequence homology to a range of SOD enzymes. The sod gene was mutagenized with Tn5-lacZ. None of the insertions that abolished SOD-conferring activity were in the correct orientation for lacZ expression. Repeated attempts to introduce these insertions into the chromosome of X. campestris pv. campestris were unsuccessful and it was concluded that the sod gene may be essential for viability. In order to monitor the expression of the sod gene, a sod-gus transcriptional fusion was constructed. Expression of the sod gene varied according to the growth stage of the organism in culture. In planta, the sod gene was induced within 3 to 4 h of inoculation, with similar kinetics during compatible and incompatible interactions with turnip and pepper, respectively.

A locus determining pathogenicity of Xanthomonas campestris is involved in lipopolysaccharide biosynthesis.

A pathogenicity locus in Xanthomonas campestris pv. campestris has been shown to comprise two genes which mediate biosynthesis of the bacterial lipopolysaccharide (LPS) but not extracellular polysaccharide. Mutants with Tn5 insertions in either gene showed alterations in the electrophoretic patterns of both water-soluble and phenol-soluble LPS forms, which suggested defects in the biosynthesis of the core oligosaccharide component. On gel chromatography, core oligosaccharides of the mutants were of apparently lower molecular weight than those from the wild type. Furthermore, the content of mannose and glucose, sugars characteristic of the core oligosaccharide, were significantly lower in the water-soluble LPS of the mutants. Because of their role in LPS core biosynthesis, the two genes were called rfaX and rfaY. rfaX mutants show altered behavior in a range of host and non-host plants such that the number of recoverable bacteria drop within the first 24 h after inoculation. In contrast, the behavior of rfaY mutants only differed from the wild type in Datura, a non-host plant in which the growth of the wild type is severely attenuated. The predicted protein RfaY showed significant sequence homology to a sub-family of RNA polymerase sigma factors which are involved in extracytoplasmic functions.

Cloning and characterization of the rpfC gene of Xanthomonas oryzae pv. oryzae: involvement in exopolysaccharide production and virulence to rice.

rpfC is one of a cluster of genes which coordinately regulate the synthesis of extracellular enzymes and exopolysaccharide and pathogenicity in Xanthomonas campestris pv. campestris, the black rot pathogen of brassicas. An rpfC homolog which could functionally complement an rpfC mutant of X. campestris pv. campestris was identified in Xanthomonas oryzae pv. oryzae and the gene was characterized. Mutation of this gene in X. oryzae pv. oryzae had no effect on extracellular enzymes, but exopolysaccharide synthesis and virulence to rice were substantially reduced.

The effect of infectious bursal disease virus on B lymphocytes and bursal stromal components in specific pathogen-free (SPF) White Leghorn chickens.

The effect of infectious bursal disease virus (IBDV) was studied on adult specific pathogen-free (SPF) white Leghorn chickens through analysis of peripheral blood cell suspensions and histological staining patterns on various tissue types, with specific mAbs. A rapid, progressive loss of B lymphocytes was observed in the bursal cortex and medulla, peripheral blood and thymic medulla. There was, however, a resistant population of MUI-36+ cells at the bursal cortico-medullary junction and scattered around splenic periellipsoidal sheaths. These resistant cells were suggested to be a subpopulation of macrophages which expressed the MUI-36 marker; alternatively these may have phagocytosed virally infected B cells or their remnants. Throughout the period of infection, T lymphocytes appeared nonsusceptible. Further, while the distribution of stromal cell antigens within the bursal cortex remained unaltered, particular epitopes on the surface epithelium and in the medulla were lost as a consequence of viral infection. The data presented therefore suggests that immunodepression of chickens post-IBDV infection, may arise as a direct consequence of infection of B lymphocytes; additionally, it is possible that the elimination of certain crucial elements within the bursal microenvironment may contribute to this state.

Analysis of a ptsH homologue from Streptomyces coelicolor A3(2).

A ptsH homologue of Streptomyces coelicolor A3(2) was identified in the emerging genome sequence, cloned in Escherichia coli and the S. coelicolor HPr over-produced and purified. The protein was phosphorylated in vitro in a phosphoenolpyruvate (PEP)-dependent manner by purified enzyme I (EI) from Bacillus subtilis, and much less efficiently in an ATP-dependent manner by purified HPr kinase, also from B. subtilis. There was no indication of ATP-dependent phosphorylation of the purified protein by cell extracts of either S. coelicolor or Streptomyces lividans. Deletion of the ptsH homologue from the S. coelicolor and S. lividans chromosomes had no effect on growth when fructose was supplied as sole carbon source, and in S. coelicolor it had no effect on glucose repression of agarase and galactokinase synthesis, suggesting that the HPr encoded by this gene does not play an essential role in fructose transport nor a general role in carbon catabolite repression.

Relationship between platelet count and cardiotomy suction return.

A simple, inexpensive, accurate method of measuring the amount of blood returned by the cardiotomy suction system was devised and calibrated. Preoperative and postoperative platelet counts were obtained in 76 patients with congenital heart disease in whom the amount of cardiotomy suction return was measured. The mean percentage of total perfusate returned by the cardiotomy suction system was 8.9%. Both postoperative platelet count and the percentage change between preoperative and postoperative platelet counts correlated with the amount of blood returned by the cardiotomy suction system, time on bypass, and the percentage of total perfusate aspirated by the system.

Decreased natural killer cell activity in endometriosis patients: relationship to disease pathogenesis.

A number of reports have measured NK cell activity in patients with endometriosis with varied results. Therefore we have examined the NK activity of PBL from 44 gynecological patients undergoing laparoscopy. This analysis has demonstrated a significant reduction in NK activity only in more severe stages of endometriosis (stages III and IV) relative to patients with milder disease and controls. These data indicate that decreased NK activity is unlikely to be a primary etiological factor in the development of endometriosis but may indicate that decreased NK activity is related to the development of the more frequent and/or larger lesions characteristic of severe endometriosis. These data could indicate potential for immunotherapy of patients with advanced endometriosis by the upregulation of NK activity in vivo.

Elevated immunoglobulin E (IgE) levels in children with exposure to environmental lead.

Lead has been reported to be an immunosuppressive agent in animal systems at levels far below those recognized as overtly toxic. Little data exist on lead's effects on the human immune system, especially in young children who are at greatest risk for exposure to this environmental hazard. The effects of environmental lead exposure on the human immune system were examined in a population of young children, age 9 months-6 years, from the urban population of Springfield-Greene County, Missouri. Reported here are data from 279 children with blood lead levels ranging from 1 to 45 microg/dl. White blood cell populations have been enumerated and examined for cell surface expression of activation markers. Serum has been analyzed for IgE, specific titers to Rubella vaccine, sCD25 (the soluble form of the IL2 receptor), sCD27 (the soluble form of the lymphocyte specific member of the tumor necrosis factor receptor family), and IL4 (the cytokine interleukin 4). Variation of these assays with age of the child was considered in statistical analysis of data. A statistically significant relationship of IgE and blood lead level was found in this population; as blood lead (PbB) level increases, IgE level increases. No other statistically significant differences between risk categories or other associations with blood lead level were found. The exact mechanism for this apparent stimulus of IgE-producing B cells remains to be elucidated. The development of allergic symptoms is often preceded by an increase in IgE. These data indicate that ingested lead could play a role in this process by stimulating IgE production.

Insights from a new analytical electrophoresis apparatus.

Charge is a fundamental property of macromolecules that is inextricably linked to their structure, solubility, stability, and interactions. Progress has been made on the theoretical and structural aspects of these relationships. However, for several reasons, charge is difficult to measure in solution. Consequently, there is a lack of experimental data that, independent of other macro-ion properties, determines the effective charge. To overcome this problem, novel instrumentation and methods are being developed in our laboratory. Described here is an analytical electrophoresis apparatus that permits both the measurement of electrophoretic mobilities and the determination of steady-state electrophoresis concentration distributions. The latter provides a different-perspective on the processes that influence macro-ion behavior in an electric field. In addition, the apparatus permits the determination of diffusion coefficients either from boundary spreading during transport or from the decay of a concentration gradient. All of these determinations can be made with a single, 8-microL sample in a variety of solvents, thus providing unique insights into the charge properties of a macro-ion. Presented here is a progress report about this emerging technology, including the description of a prototype apparatus and examples of its use with a DNA oligonucleotide.

Large-volume blood patch to multiple sites in the epidural space through a single-catheter access site for treatment of spontaneous intracranial hypotension.

BACKGROUND AND PURPOSE: Spontaneous intracranial hypotension can be a therapeutic challenge to the treating physician. In this study, we present our experience with the administration of a large-volume blood patch to multiple sites in the epidural space through a single-catheter access site. MATERIALS AND METHODS: A retrospective review was conducted of patients with spontaneous intracranial hypotension who underwent a large-volume blood patch to multiple sites in the epidural space through a single-catheter access site from 2010 to 2012. Patient demographic data, clinical charts, indications for treatment, radiographic images, procedure notes, and postprocedure hospital course were reviewed. RESULTS: Overall, 9 patients were identified who underwent 20 blood patch procedures. Patients were selected to undergo the large-volume procedure either because they had a failed site-directed epidural blood patch or if imaging demonstrated multiple possible leak sites. There were 6 women and 3 men, with an average age of 33.5 years. The mean volume of blood injected per procedure was 54.1 mL (median=55 mL; range=38-70 mL). All patients had an orthostatic headache as one of their presenting symptoms; 22% also presented with neurocognitive decline and behavioral changes; 89% of patients had improvement or resolution of their symptoms; and 80% of patients who had a previously failed site-directed epidural blood or fibrin glue patch improved with a large volume catheter-directed blood patch. CONCLUSIONS: Our experience supports the use of a large-volume blood patch to multiple sites in the epidural space through a single-catheter access site for the treatment of spontaneous intracranial hypotension. Additionally, our results indicate a role for this procedure in refractory cases of spontaneous intracranial hypotension.