Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae.

Protein expression by Haemophilus influenzae under iron-limiting growth conditions was examined. The five type b strains and four nontypeable strains studied all expressed a new protein of about 40 kDa when deprived of iron during growth. Most strains also expressed a protein of about 31 kDa under the same growth conditions. Both the 40- and 31-kDa proteins were not expressed by cells grown in iron-replete medium. The 40- and 31-kDa proteins were not expressed in iron-deficient medium to which an excess of ferric nitrate had been added, and therefore it was concluded that their expression was iron regulated. These iron-repressed proteins were localized to the periplasmic space. The amino-terminal sequences of both proteins were determined. The N-terminal sequence of the 40-kDa protein had 81% similarity to the N terminus of Fbp, the major iron-binding protein of Neisseria gonorrhoeae and N. meningitidis. The 31-kDa protein sequence showed no homology with any known protein sequence. As no plasmids were found in the strains, it was concluded that these proteins were chromosomally encoded.     

Regulation of D-alanine carboxypeptidase and peptidoglycan cross-linkage in amino acid-deprived Escherichia coli.

The effect of amino acid deprivation on the activities of D-alanine carboxypeptidase (CPase) and peptidoglycan transpeptidase in Escherichia coli was determined. Enzymes were assayed in ether-treated bacteria (ETB) which were permeable to peptidoglycan nucleotide precursors. ETB were prepared at intervals from cultures grown in the presence and absence of a required amino acid. The specific activity of CPase in ETB decreased 50 to 85% during amino acid deprivation. This was paralleled by a 60 to 70% decrease in the specific activity of peptidoglycan transpeptidase. Both enzymes reached their lowest level of activity about 40 min after the onset of amino acid deprivation. The decrease in CPase activity apparently was not due to degradation of the enzyme, since full activity was restored after disruption of ETB by sonication. A decrease in CPase activity was associated with an enhancement of transpeptidation. The peptidoglycan synthesized in vitro by amino acid-deprived ETB was 1.7 times more cross-linked than the peptidoglycan synthesized by control ETB These results support the proposal that CPase may be involved in regulating transpeptidation in E. coli.     

Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in the loggerhead (Caretta caretta) and the Green Sea Turtle (Chelonia mydas) during development.

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in the erythrocytes (RBC) of the 68-day loggerhead turtle embryo and 44-day green sea turtle embryo at levels of 7.4 and 5.5 μmoles/ml of RBC, representing the major organic phosphate during the latter period of embryonic development. (2) Inositol pentaphosphate (IPP) is absent in the red blood cells of the embryos of both the loggerhead and green sea turtle. (3) Near equimolar amounts of 2,3-DPG and IPP are present in the erythrocytes of the adult loggerhead and green sea turtle. The total concentration of these two organic phosphates is approximately 0.75 μmoles/ml of RBC in the adult of both species. (4) There is a switch from embryonic to adult hemoglobin during development of these two species of turtles; the two embryonic bands have identical electrophoretic mobilities, whereas the two adult bands migrate differently on cellulose acetate at pH 8.6. (5) The whole blood oxygen affinity of the adult loggerhead and green sea turtle is 60.3 and 32.6 Torr, respectively. (6) The stripped adult hemoglobins in these two species of turtles show no change in oxygen affinity upon addition of 2,3-DPG, ATP, or IPP. (7) It therefore appears unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in these species of turtles.     

Somatoform disorders among patients attending walk-in clinics in Trinidad: prevalence and association with depression and anxiety

Objectives Somatoform disorders are common in international primary care settings, but have been little studied in the developing world. The objective of this study was to determine the prevalence of severe undifferentiated somatoform disorder, and its relationship to depression and anxiety, among patients attending walk-in clinics in Trinidad.Methods The study participants, who were all aged 18 years or older and attending walk-in clinics at 16 randomly selected health centres, were surveyed between May and August 2007 using the PRIME-MD questionnaire.Results There were 594 participants (the response rate was 92%), of whom 72.7% were female. Their ages ranged from 18 to 93 years, and 54.5% were over 50 years of age. In total, 37.2% were married and 25.9% were single. Indo-Trinidadians represented 43.1% and Afro-Trinidadians represented 36% of the study sample; 56.5% of the participants reported that their income was less than US$ 400 per month, and 65.7% were unemployed. At walk-in clinics in Trinidad, the estimated prevalence of severe undifferentiated somatoform disorder was 10.3% (95% CI: 7.86–12.74), that of hypochondriasis was 28.5% (95% CI: 24.9–32.1), and that of body dysmorphic disorder was 15.8% (95% CI: 11.9–18.7). Severe undifferentiated somatoform disorder was statistically significantly associated with gender and ethnicity but not with age, level of education, employment status or income. Chi-square testing found significant associations between the presence of severe undifferentiated somatoform disorder and both depression and anxiety (P < 0.05), between hypochondriasis and both anxiety and depression (P < 0.05), and between body dysmorphic disorder and depression (P < 0.05) but not anxiety. Regression analysis suggested that the demographic features that predicted severe undifferentiated somatoform disorder were being female or Indo-Trinidadian.Conclusions Walk-in clinics in Trinidad that serve older patients on a lower income have a high proportion of patients with somatoform disorders as measured by the PRIME-MD scale. These patients exhibit many features of anxiety and depression. These findings have implications for medical training and service delivery.     

Predicted molecular structure of the mammalian cell entry protein Mce1A of Mycobacterium tuberculosis

The proposed role of the mammalian cell entry protein 1A (Mce1A) of Mycobacterium tuberculosis is to facilitate invasion of host cells. The structure of Mce1A was modelled on the basis of the crystal structure of Colicin N of Escherichia coli by fold prediction and threading. Mce1A, as the model predicts, is an alpha/beta protein consisting of two major (alpha and beta) domains, connected by a long alpha helix. The model further revealed that the protein contains 12 helices, 9 strands, and 1 turn. The final model of Mce1A was verified through the program VERIFY 3D and more than 90% of the residues were in the favourable region. A mouse monoclonal antibody, TB1-5 76C, is directed to an epitope within a 60-mer peptide that has been shown to promote uptake of bacteria in mammalian cells. We show here that the epitope could be narrowed down to a core of 4 amino acids, TPKD. Upstream flanking residues, KRR also contributed to binding. Mce2A does not promote uptake in mammalian cells and sequence comparison of Mce1A and Mce2A indicates that the epitope mediates uptake. The epitope was located at the surface of the Mce1A model at the distal beta strand-loop region in the beta domain. The localization of this epitope in the model confirms its potential role in promoting uptake of M. tuberculosis in host cells.     

Germinal vesicle material is essential for nucleus remodeling after nuclear transfer

Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the MII stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an MII spindle was present in both pro-MI and MII cytoplasm 1 h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.