Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.     

Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system

A BacMam baculovirus was designed in our laboratory to express the reporter protein secreted alkaline phosphatase (SEAP) driven by the immediate early promoter of human cytomegalovirus promoter (CMV). In vitro tests have been carried out using this recombinant baculovirus to study the secreted protein in two cell lines and under various culture conditions. The transductions were carried out on two commonly used mammalian cell lines namely the human embryonic kidney (HEK 293A) and Chinese hamster ovary (CHO-K1). Initial studies clearly demonstrated that the transient expression of SEAP was at least 10-fold higher in the HEK 293 cells than the CHO cells under equivalent experimental conditions. Factorial design experiments were done to study the effect of different parameters such as cell density, MOI, and the histone deacetylase inhibitor, trichostatin A concentration. The multiplicity of infection (MOI) and the cell density were found to have the most impact on the process. The enhancer trichostatin A also showed some positive effect. The production of secreted protein in a batch reactor was studied using the Wave disposable bioreactor system. A semi-continuous perfusion process was developed to extend the period of gene expression in mammalian cells using a hollow fiber bioreactor system (HFBR). The growth of cells and viability in both systems was monitored by offline analyses of metabolites. The expression of recombinant protein could be maintained over an extended period of time up to 30 days in the HFBR.     

Glutathione peroxidase3 of <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis> protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 μM cadmium (Cd)-treatment, whereas an untreated gpx3Δ strain showed a drastic reduction in overall phospholipids which was further reduced with 50 μM Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3Δ cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3Δ. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3Δ cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3Δ cells during Cd-treatment.     

Influence of medium- and long-range interactions in different folding types of globular proteins

Recognition of protein fold from amino acid sequence is a challenging task. The structure and stability of proteins from different fold are mainly dictated by inter-residue interactions. In our earlier work, we have successfully used the medium- and long-range contacts for predicting the protein folding rates, discriminating globular and membrane proteins and for distinguishing protein structural classes. In this work, we analyze the role of inter-residue interactions in commonly occurring folds of globular proteins in order to understand their folding mechanisms. In the medium-range contacts, the globin fold and four-helical bundle proteins have more contacts than that of DNA-RNA fold although they all belong to all-alpha class. In long-range contacts, only the ribonuclease fold prefers 4-10 range and the other folding types prefer the range 21-30 in alpha/beta class proteins. Further, the preferred residues and residue pairs influenced by these different folds are discussed. The information about the preference of medium- and long-range contacts exhibited by the 20 amino acid residues can be effectively used to predict the folding type of each protein.     

Correlation of Trace Elemental Profiles in Blood Samples of Indian Patients with Leukoplakia and Oral Submucous Fibrosis

In order to ascertain possible correlation between alterations in trace elemental profile and the progression or regression of two most common potentially malignant disorders affecting oral cavity, namely oral submucous fibrosis and leukoplakia, blood from 60 patients from each group of patients as well from 30 healthy individuals was analyzed for elemental profiling employing EDXRF technique. Out of the 16 detected elements (K, Si, Ca, V, Cr, Ni, Mn, Fe, Cu, Zn, Se, Br, Rb, Sr, Co, and Pb), Mn, Fe, Zn, Br, and Co showed remarkable alteration in their profile in both leukoplakia and oral submucous fibrosis patients with respect to the normal healthy individuals. While Zn, Br, and Fe reflected similar changes--showing gross depletion in both the diseased groups, Mn and Co depicted inverse pattern of alterations in their concentrations in the two types of precancerous disorders when compared to the control subjects.     

Thermodynamic database for protein-nucleic acid interactions (ProNIT).

MOTIVATION: Protein-nucleic acid interactions are fundamental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein-nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data. Thus, we have developed a Thermodynamic Database for Protein-Nucleic Acid Interactions (ProNIT). RESULTS: We have collected experimentally observed binding data from the literature. ProNIT contains several important thermodynamic data for protein-nucleic acid binding, such as dissociation constant (K(d)), association constant (K(a)), Gibbs free energy change (DeltaG), enthalpy change (DeltaH), heat capacity change (DeltaC(p)), experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data are integrated into a relational database system together with structural and functional information to provide flexible searching facilities by using combinations of various terms and parameters. A www interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. AVAILABILITY: ProNIT is freely accessible at the URL http://www.rtc.riken.go.jp/jouhou/pronit/pronit.html.     

Phagocytosis and leucocyte enzymes in protein–calorie malnutrition

1. Enzymes pertinent to bactericidal activities of leucocytes were assayed in children suffering from protein-calorie malnutrition. 2. Leucocytes obtained from malnourished and control children contained similar activities for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Granule-bound NADPH oxidase activity was low in leucocytes isolated from malnourished patients and failed to show the phagocytic stimulation which is normally seen in control leucocytes. Further, leucocytes obtained from malnourished patients did not release the acid phosphatase from lysosomes during phagocytosis, unlike those from controls. 3. Treatment of the malnourishment with a diet high in calories and protein resulted in significant increase in the activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADPH oxidase and in releasing the acid phosphatase from the lysosomes into the supernatant fraction during phagocytosis. 4. The significance of these enzyme changes are discussed in relation to the increased susceptibility of these patients to infection.