Penicillin-binding proteins 4 and 5 of Haemophilus influenzae are involved in cell wall incorporation

Abstract Permeabilized cells of Haemophilus influenzae incorporate wall precursors into murein material in an ampicillin-sensitive reaction. In resistant transformants that contain the low antibiotic affinity penicillin-binding proteins (PBPs) 4 and 5, the sensitivity of this incorporation reaction to ampicillin is proportionally lower, suggesting a catalytic role for these proteins in wall synthesis. We conclude that, analogous to the reaction in Escherichia coli , PBPs 4 and 5 of H. influenzae have transpeptidase activity.     

Biotransformation of penicillin V to 6-aminopenicillanic acid using immobilized whole cells of E. coli expressing a highly active penicillin V acylase

The production of 6-aminopenicillanic acid (6-APA) is a key step in the manufacture of semisynthetic antibiotics in the pharmaceutical industry. The penicillin G acylase from Escherichia coli has long been utilized for this purpose. However, the use of penicillin V acylases (PVA) presents some advantages including better stability and higher conversion rates. The industrial application of PVAs has so far been limited due to the nonavailability of suitable bacterial strains and cost issues. In this study, whole-cell immobilization of a recombinant PVA enzyme from Pectobacterium atrosepticum expressed in E. coli was performed. Membrane permeabilization with detergent was used to enhance the cell-bound PVA activity, and the cells were encapsulated in calcium alginate beads and cross-linked with glutaraldehyde. Optimization of parameters for the biotransformation by immobilized cells showed that full conversion of pen V to 6-APA could be achieved within 1?hr at pH 5.0 and 35°C, till 4% (w/v) concentration of the substrate. The beads could be stored for 28 days at 4°C with minimal loss in activity and were reusable up to 10?cycles with 1-hr hardening in CaCl₂ between each cycle. The high enzyme productivity of the PVA enzyme system makes a promising case for its application for 6-APA production in the industry.     

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)-IAM and GPR17(−)-IAM stationary phases. Frontal chromatography experiments on both GPR17(+)-IAM and GPR17(−)-IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)-IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (K_d) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y₁₂/P2Y₁₃ antagonist), MRS2179 (a P2Y₁ receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K_d values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)-IAM in ranking affinity studies for the selection of new potential candidates.     

Induction of tyrosine phosphorylated proteins in THP-1 cells by Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae porins

The effect of porins purified from Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae on induction of tyrosine phosphorylation in THP-1 cells and C3H/HeJ macrophage was investigated. Incubation of porins at concentration of 1.0-5.0 microg ml(-1) with either THP-1 or macrophage from C3H/HeJ mice resulted in tyrosine phosphorylation of specific host cell proteins. After porin stimulation a pattern of tyrosine phosphorylated proteins appeared in the soluble cytoplasmic fraction, in the membrane fraction and in the insoluble protein fraction. The observed effects were dependent on the porin concentrations; they reached a maximal expression at 3 min and declined at 60 min. Porin and lipopolysaccharide treatments induce a similar phosphorylation pattern in all of the three cellular fractions studied. A difference can be observed in the cytoplasmic fraction bands of 50-60 kDa, which are more evident after treatment with lipopolysaccharide, and in the insoluble fraction band of 80 kDa and the cytoplasmic fraction band of 250 kDa, which are more evident after treatment with porins. The events of tyrosine protein phosphorylation were present in macrophage from lipopolysaccaride-hyporesponsive C3H/HeJ mice stimulated with porins, while they were markedly reduced when the cells were stimulated with lipopolysaccharide. Staurosporine, genistein and cytochalasin D induced in the three cellular fractions a different inhibition pattern of tyrosine protein phosphorylation in porin stimulated cells. Porins extracted from the three bacterial species investigated behave in a similar way as stimuli more or less potent; Hib porin seems to be the most powerful stimulator and, moreover, it specifically induces phosphorylation of a 55 kDa band.     

On the use of cells or membranes for receptor binding: Growth hormone secretagogues

Receptor binding techniques have been widely used in different biochemical applications, with isolated membranes being the most used receptor preparation in this type of assays. In this study, intact cells were compared with isolated membranes as receptor support for radioligand receptor binding assay. The growth hormone secretagogue receptor 1a (GHSR-1a) expressed in human embryonic kidney 293 (HEK293) cells was used as a model of G-protein-coupled receptors. Differences between using intact cells in suspension and using isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation, and competition using different agonists. Intact cells are more prone to variability. Although under optimized settings both preparations were equivalent, the K_d value for intact cells was three times higher than that using isolated membranes. However, no significant differences were observed in competition assays obtaining practically identical K_i values for all ligands tested. For the GHSR-1a, isolated membranes are the better choice if particular incubation conditions are required (less variability), whereas intact cells yield easy, fast, and physiological conditions for receptor binding assays.     

Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication

Tetraploidy has been proposed as an intermediate state in neoplastic transformation due to the intrinsic chromosome instability of tetraploid cells. Despite the identification of p53 as a major factor in growth arrest of tetraploid cells, it is still unclear whether the p53-dependent mechanism for proliferation restriction is intrinsic to the tetraploid status or dependent on the origin of tetraploidy. Substrate adherence is fundamental for cytokinesis completion in adherent untransformed cells. Here we show that untransformed fibroblast cells undergoing mitosis in suspension produce binucleated tetraploid cells due to defective cleavage furrow constriction that leads to incomplete cell abscission. Binucleated cells obtained after loss of substrate adhesion maintain an inactive p53 status and are able to progress into G1 and S phase. However, binucleated cells arrest in G2, accumulate p53 and are not able to enter mitosis as no tetraploid metaphases were recorded after one cell cycle time. In contrast, tetraploid metaphases were found following pharmacological inhibition of Chk1 kinase, suggesting the involvement of the ATR/Chk1 pathway in the G2 arrest of binucleated cells. Interestingly, after persistence in the G2 phase of the cell cycle, a large fraction of binucleated cells become senescent. These findings identify a new pathway of proliferation restriction for tetraploid untransformed cells that seems to be specific for loss of adhesion-dependent cytokinesis failure. This involves Chk1 and p53 activation during G2. Inhibition of growth and entrance into senescence after cytokinesis in suspension may represent an important mechanism to control tumor growth. In fact, anchorage independent growth is a hallmark of cancer and it has been demonstrated that binucleated transformed cells can enter a cycle of anchorage independent growth.     

RecJ, ExoI and RecG are required for genome maintenance but not for generation of genetic diversity by repeat-mediated phase variation in Haemophilus influenzae

High levels of genetic diversity are generated in Haemophilus influenzae populations through DNA repeat-mediated phase variation and recombination with DNA fragments acquired by uptake from the external milieu. Conversely, multiple pathways for maintenance of the genome sequence are encoded in H. influenzae genomes. In Escherichia coli, mutations in single-stranded-DNA exonucleases destabilise tandem DNA repeats whilst inactivation of recG can stabilise repeat tracts. These enzymes also have varying effects on recombination. Deletion mutations were constructed in H. influenzae genes encoding homologs of ExoI, RecJ and RecG whilst ExoVII was refractory to mutation. Inactivation of RecJ and RecG, but not ExoI, increased sensitivity to irradiation with ultraviolet light. An increase in spontaneous mutation rate was not observed in single mutants but only when both RecJ and ExoI were mutated. None of the single- or double-mutations increased or decreased the rates of slippage in tetranucleotide repeat tracts. Furthermore, the exonuclease mutants did not exhibit significant defects in horizontal gene transfer. We conclude that RecJ, ExoI and RecG are required for maintenance of the H. influenzae genome but none of these enzymes influence the generation of genetic diversity through mutations in the tetranucleotide repeat tracts of this species.     

Two populations of TSPO binding sites in oral cancer SCC-15 cells

Oral cancer mortality and morbidity rates remain high. The main inducer of oral cancer is cigarette smoke (CS). Translocator protein 18 kDa (TSPO) was shown to play a role in carcinogenesis. We characterized TSPO binding sites in human oral cancer cell line SCC-15 and examined effect of CS on TSPO binding. We exposed SCC-15 human squamous cells to cigarette smoke. [³H]PK 11195 binding results were assessed in cells confluent for one day. To characterize the number of population sites, a custom written Matlab program compared Pearson linear correlation coefficients between all points in the Scatchard plot. Using [³H]PK 11195 as a radio ligand, we found that TSPO binding sites are not uniform, but separated into two sub-populations, one with high affinity (respective Kd and Bmax values of 1.40±0.08 nM and 1586±48 fmol/mg protein), another with lower affinity (respective Kd and Bmax values of 61±5 nM and 26260±1050 fmol/mg protein). We demonstrate rapid decrease in TSPO binding to the high affinity site induced by exposure to CS; specifically, significant 36% decrease in binding after 30 min CS exposure (p<0.05), and 69% decrease after 2 h CS exposure (p<0.05). Association between TSPO and CS exposure may contribute to understanding the underlying mechanism of oral carcinogenesis.     

Citric acid extracts a specific set of proteins from isolated cell nuclei

The treatment of isolated cell nuclei with citric acid was described as a method for separating inner and outer nuclear membrane. Using cell nuclei from bovine cerebral cortex, we can show that citric acid does not cause a separation of the two nuclear membranes, but extracts a specific set of proteins from the nuclei. The extraction of proteins is not just an effect of damaging the nuclear membrane or destructing the cytoskeleton, but rather a specific effect of citric acid treatment. One of the extracted proteins, chosen as a marker for the putative outer nuclear membrane fraction, has an apparent molecular weight of 145 kDa and is located in the nucleoplasm as shown by immunofluorescence microscopy. By sequencing tryptic peptides it was identified as RNA helicase A, an abundant nuclear protein assumed to participate in the processing of mRNA. © 1995 Wiley-Liss, Inc.     

Ionic modulation of electrically induced fusion of mammalian cells

Summary During the last few years, a new technique has been developed for the electrofusion of mammalian cells. No previous treatment of the culture is needed, for the contact between cells is spontaneous. Short DC electric pulses are applied directly to a culture growing in monolayers on a culture dish. When the cell density is high enough, contacts occur between cells giving the so-called contact inhibition. In the present study, a systematic investigation of the modulation of the extent of the fusion by the ionic content of the bathing medium during the pulsation is described. An increase in the content in monovalent ions decreases the fusion yield. But this decrease is modulated by the nature of the ion; Li⁺, a potent water structure maker, is less effective than Na⁺ or K⁺. Ca²⁺, when present in the millimolar range, leads to the lysis of the cells. Mg²⁺, when present at concentrations smaller than 4mm, promotes the fusion but prevents it at larger concentrations. Microelectrophoresis measurements show that the electric surface charge is not strongly affected by these changes in ionic content. Our observations are relevant of a modulation of the cell-cell interactions by the ionic content of the bathing medium.     

The fibronectin binding proteins of Staphylococcus aureus are required for adhesion to and invasion of bovine mammary gland cells

We recently described adhesion to and invasion of bovine mammary gland cells by Staphylococcus aureus in vitro. Here, we show that the levels of adhesion and invasion are dependent on the bacterial growth phase and are controlled by the agr locus. Incubation of exponential growth phase cells of S. aureus with mammary gland cells resulted in bacterial cell clumping. Strains of S. aureus deficient in expression of the fibronectin binding proteins (FnBPA and FnBPB) failed to clump and their ability to adhere to and to invade the bovine mammary gland cells is strongly reduced. This indicates that the fibronectin binding proteins are essential for S. aureus adhesion to and invasion of bovine mammary gland cells.     

Changes in the electric characteristics of the inner surface of Escherichia coli cytoplasmic membrane after the binding of microdoses of copper to its outer surface

The electric characteristics of the surface structures of intact Escherichia coli spheroplasts and the spheroplasts with the outer membrane modified by microdoses of copper were studied by electron spin resonance using the positively charged spin probes of KAT_n type and the newly synthesized KAT₁₅. The experimental results were analyzed using the Gouy-Chapman formalism. It was found that microdoses of copper ions bound to the outer surface of the cytoplasmic membrane influenced the charge density on the inner surface of the membrane.     

Two proteins regulate the cell wall extensibility and the yield threshold in glycerinated hollow cylinders of cowpea hypocotyl

Glycerinated hollow cylinders of hypocotyl segments excised from the elongation region of cowpea seedlings were heated for 15s in 50% glycerol at 70, 80 or 90°C. Their in vitro yield threshold tension (y) and extensibility (φ) were determined by stress-strain experiments under the perfusion of solutions of pH 4·0 or 6·2. The decrement in y and the increment in φ with acidification were extinguished at 80 and 90°C, respectively. Moreover, such changes in φ and y with acidification were prevented by proteinase treatment for 6 and 10 h, respectively. These results suggest that these two cell wall mechanical properties are controlled, respectively, by two functional proteins activated by acid.     

Multicenter phase 1/2 application of adenovirus-specific T cells in high-risk pediatric patients after allogeneic stem cell transplantation

Background

Adenovirus (ADV) reactivation can cause significant morbidity and mortality in children after allogeneic stem cell transplantation. Antiviral drugs can control viremia, but viral clearance requires recovery of cell-mediated immunity.

Heat shock proteins and other components of cellular machinery for protein synthesis are up-regulated in vascular endothelial cell growth factor-activated human endothelial cells

Proteome analysis of human umbilical endothelial cells was performed to identify proteins that are modified during vascular endothelial cell growth factor (VEGF)-induced transition from the quiescent into the proliferating-migrative phenotype. Subtractive analysis of two-dimensional gel patterns of human endothelial cells, before and after stimulation with VEGF(165), revealed differences in 85 protein spots. All proteins were identified by peptide sequencing and peptide mass fingerprinting using an electrospray spectrometer. The proteins identified were members of specific families including Ca(2+)-binding proteins, fatty-acid binding proteins, structural proteins, and chaperones. Remarkably, there was a massive activation of cellular machinery for both protein synthesis and protein degradation. Thus, among up-regulated proteins there were members of all groups of heat shock proteins (HSPs; HSP 27, HSP 60, HSP 70p5, HSP 70p8, HSP 90, and HSP 96) and some other proteins showing either chaperone activity or which participate in assembly of multimolecular structures (TCP-1, desmoplakins, junction plakoglobin, GRP 94, thioredoxin related protein, and peptidylprolyl isomerase). The increased expression of HSPs was confirmed at the mRNA level at different stages of treatment with VEGF. Similarly, components of the proteolytic machinery for the degradation of misfolded proteins (ER-60, cathepsin D, proteasome subunits, and protease inhibitor 6) were also up-regulated. On the other hand, changes in the expression of structural proteins (T-plastin, vimentin, alpha tubulin, actin, and myosin) could account, at least in part, for the different morphologies displayed by migrating endothelial cells. In summary, our data show that VEGF levels similar to those during physiological stresses induce a number of genes and multiple endogenous pathways seem to be engaged in restoring cellular homeostasis. To ensure cell survival, the molecular chaperones (the heat shock family of stress proteins) are highly up-regulated providing protein-folding machinery to repair or degrade misfolded proteins.