Large scale purification of myo-inositol monophosphate phosphatase from porcine brains

myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol . min(-1) . mg(-1). The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 +/- 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent K(m) value of the phosphatase for the utilization of inositol-1-phosphate and beta-glycerol phosphate are 3.20 x 10(-4) and 8 x 10(-3) M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K(1) was determined to be 6.38 x 10(-4) M and the inhibition is uncompetitive. (c) 1995 John Wiley & Sons, Inc.     

Interactions of pyridoxal kinase and aspartate aminotransferase emission anisotropy and compartmentation studies

Physical interactions between pyridoxal kinase and aspartate aminotransferase were detected by means of emission anisotropy and affinity chromatography techniques. Binding of aspartate aminotransferase (apoenzymes) to pyridoxal kinase tagged with a fluorescent probe was detected by emission anisotropy measurements at pH 6.8 (150 mM KCl). Upon saturation of the kinase with the aminotransferase, the emission anisotropy increases 22%. The protein complex is characterized by a dissociation constant of 3 microM. Time-dependent emission anisotropy measurements conducted with the mixture 5-naphthylamine-1-sulfonic acid-kinase aspartate aminotransferase (apoenzyme), revealed the presence of two rotational correlation times of phi 1 = 36 and phi 2 = 62 ns. The longer correlation time is attributed to the stable protein complex. By immobilizing one enzyme (pyridoxal kinase) through interactions with pyridoxal-Sepharose, it was possible to demonstrate that aspartate aminotransferase releases pyridoxal kinase. A test of compartmentation of pyridoxal-5-phosphate within the protein complex using alkaline phosphatase as trapping agent, indicates that the cofactor generated by the catalytic action of the kinase is channeled to the apotransaminase. The main function of the stable complex formed by the kinase and the aminotransferase is to hinder the release of free pyridoxal-5-phosphate into the bulk solvent.     

Papillomavirus-specific CD4+ T cells exhibit reduced STAT-5 signaling and altered cytokine profiles in patients with recurrent respiratory papillomatosis

Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.     

Kinetic FP-TDI assay for SNP allele frequency determination

Strategies for identifying genetic risk factors in complex diseases by association studies require the comparison of allele frequencies of numerous SNPs between affected and control populations. Theoretically, hundreds of thousands of SNP markers across the genome will have to be genotyped in these studies. Genotyping SNPs one sample at a time is extremely costly and time consuming. To streamline whole genome association studies, some have proposed to screen SNPs by pooling the DNA samples initially for allele frequency determination and perform individual genotyping only when there is a significant discrepancy in allele frequencies between the affected and control populations. Here we describe a new method for determining the allele frequency of SNPs in pooled DNA samples using a two-color primer extension assay with real-time monitoring of fluorescence polarization (named kinetic FP-TDI assay). By comparing the ratio of the rate of incorporation of the two allele-specific dye-terminators, one can calculate the relative amounts of each allele in the pooled sample. The accuracy of allele frequency determination with pooled samples is within 3.3 +/- 0.8% of that determined by genotyping individual samples that make up the pool.     

A comparison of the inhibitory effects of somatostatin-14, -25, and -28 on motility of the guinea pig ileum

A number of studies have suggested that somatostatin-14 (SS-14) and somatostatin-28 (SS-28) exhibit a similar spectrum of biological activities but have different potencies. In the present study the effects of SS-14, SS-28, and somatostatin-25 on electrically induced contractions of the guinea pig ileum have been compared. All three peptides exhibited equipotent inhibitory effects. Inhibition was obtained at a threshold concentration less than 10(-10) M, with maximal inhibition at 10(-7) M and IC50 values of 6.0-6.5 X 10(-10) M. The N-terminal 14 amino acid fragment of SS-28 had no effect either on motility, when added alone, or on the actions of SS-28, suggesting that this region of the molecule is not critical for biological activity.     

Retroviral-mediated gene transfer of the leukocyte integrin CD18 subunit

Children with leukocyte adherence deficiency (LAD) exhibit heterogeneous defects in the leukocyte integrin CD18 subunit that prevent surface expression of functional CD11/CD18 leukocyte integrin adherence complexes. We used a retroviral vector, designated LCD18SN, to transfer the CD18 cDNA into K562 human myeloid leukemia cells and into EBV B-cells from a child with LAD. Transfer of the LCD18SN retroviral construct, which expresses the CD18 cDNA from the Moloney Murine leukemia virus (MoMLV) long terminal repeat (LTR), into K562 cells resulted in relatively high levels of CD18 mRNA and intracellular protein. Retroviral-mediated gene transfer of CD18 into LAD EBV B-cells resulted in low, but readily measurable, levels of surface expression of the CD11a/CD18 complex in these previously deficient lymphocytes. The reconstitution of surface expression of the CD11a/CD18 complex by gene transfer of the CD18 cDNA into LAD EBV B-cells indicates that this syndrome represents a candidate disorder for gene therapy.     

BMI, body composition, and physical functioning in older adults

Objective: Recent studies have emphasized the importance of muscle and fat mass in relation to age‐related decline in physical function. Our objective was to determine whether BMI, as a surrogate measurement of fat mass, may be used as a measure of risk factor for physical functioning in older adults and whether body composition measurements confer any advantage over BMI. Research Methods and Procedures: Four thousand men and women ≥65 years of age living in the community, stratified by age and sex, underwent the following measurements: body composition by DXA; grip strength; and timed 6‐m walk. Subjects were grouped into five categories of BMI using Asian criteria for health‐related risks, and between‐group differences in physical performance measures and body composition were analyzed using analysis of covariance adjusting for age, physical activity level, and presence of chronic disease. Results: Subjects in the two obese categories had a significantly greater number of instrumental activities of daily living (IADL) impairments compared with the underweight and normal‐weight groups. Those with BMI ≥30 kg/m² had the worst walking performance, and the groups with BMI in the normal and overweight range had optimal performance. Fat mass, but not appendicular muscle mass, was associated with walking speed after adjusting for BMI. Discussion: Fat mass seems to be a more important factor than appendicular muscle mass in determining walking speed in community‐living older adults, even after adjusting for BMI.     

Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-alpha

Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth and suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-alpha for 24 hours, the mRNA level of glucose transporter 1 (GLUT 1), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dose-dependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNF-alpha inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells.     

Genomic regions associated with microdeletion/microduplication syndromes exhibit extreme diversity of structural variation

Segmental duplications (SDs) are a class of long, repetitive DNA elements whose paralogs share a high level of sequence similarity with each other. SDs mediate chromosomal rearrangements that lead to structural variation in the general population as well as genomic disorders associated with multiple congenital anomalies, including the 7q11.23 (Williams–Beuren Syndrome, WBS), 15q13.3, and 16p12.2 microdeletion syndromes. Population-level characterization of SDs has generally been lacking because most techniques used for analyzing these complex regions are both labor and cost intensive. In this study, we have used a high-throughput technique to genotype complex structural variation with a single molecule, long-range optical mapping approach. We characterized SDs and identified novel structural variants (SVs) at 7q11.23, 15q13.3, and 16p12.2 using optical mapping data from 154 phenotypically normal individuals from 26 populations comprising five super-populations. We detected several novel SVs for each locus, some of which had significantly different prevalence between populations. Additionally, we localized the microdeletion breakpoints to specific paralogous duplicons located within complex SDs in two patients with WBS, one patient with 15q13.3, and one patient with 16p12.2 microdeletion syndromes. The population-level data presented here highlights the extreme diversity of large and complex SVs within SD-containing regions. The approach we outline will greatly facilitate the investigation of the role of inter-SD structural variation as a driver of chromosomal rearrangements and genomic disorders.