Protective effect of vaccination with DNA of the H. Pylori genomic library in experimentally infected mice

Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pylori-whole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomach-adapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library.     

Melatonin Increases Serotonin N-Acetyltransferase Activity and Decreases Dopamine Synthesis in Light-Exposed Chick Retina: In Vivo Evidence Supporting Melatonin-Dopamine Interaction in Retina

The administration of melatonin, either peripherally (0.01-10 mg/kg) or intraocularly (0.001-10 mumol/eye), to light-exposed chicks dose-dependently increased serotonin N-acetyltransferase (NAT) activity in retina but not in pineal gland. The effect of melatonin was slightly but significantly reduced by luzindole (2-benzyl-N-acetyltryptamine), and not affected by two other purported melatonin antagonists, N-acetyltryptamine and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23). The elevation of the enzyme activity induced by melatonin was substantially stronger than that evoked by 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, or 5-methoxytryptamine. The melatonin-evoked rise in the retinal NAT activity was counteracted by two dopamine D2 receptor agonists, quinpirole and apomorphine, and prevented by the dopamine D2 receptor blocker spiroperidol, and by an inhibitor of dopamine synthesis, alpha-methyl-p-tyrosine. Melatonin (0.1-10 mg/kg i.p.) dose-dependently decreased the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), as well as the DOPAC/dopamine ratio, in chick retina but not in forebrain. The results obtained (1) indicate that melatonin in vivo potently inhibits dopamine synthesis selectively in retina, and (2) suggest that the increase in retinal NAT activity evoked by melatonin in light-exposed chicks is an indirect action of the compound, and results from the disinhibition of the NAT induction process from the dopaminergic (inhibitory) signal. The results provide in vivo evidence supporting the idea (derived on the basis of in vitro findings) that a mutually antagonistic interaction between melatonin and dopamine operates in retinas of living animals.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.     

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: molecular dynamics simulation of the agonist-bound state in the membrane-aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.     

CXCR3+CD4+ T cells mediate innate immune function in the pathophysiology of liver ischemia/reperfusion injury

Ischemia-reperfusion injury (IRI), an innate immune-dominated inflammatory response, develops in the absence of exogenous Ags. The recently highlighted role of T cells in IRI raises a question as to how T lymphocytes interact with the innate immune system and function with no Ag stimulation. This study dissected the mechanism of innate immune-induced T cell recruitment and activation in rat syngeneic orthotopic liver transplantation (OLT) model. Liver IRI was induced after cold storage (24-36 h) at 4 degrees C in University of Wisconsin solution. Gene products contributing to IRI were identified by cDNA microarray at 4-h posttransplant. IRI triggered increased intrahepatic expression of CXCL10, along with CXCL9 and 11. The significance of CXCR3 ligand induction was documented by the ability of neutralizing anti-CXCR3 Ab treatment to ameliorate hepatocellular damage and improve 14-day survival of 30-h cold-stored OLTs (95 vs 40% in controls; p < 0.01). Immunohistology analysis confirmed reduced CXCR3+ and CD4+ T cell infiltration in OLTs after treatment. Interestingly, anti-CXCR3 Ab did not suppress innate immune activation in the liver, as evidenced by increased levels of IL-1beta, IL-6, inducible NO synthase, and multiple neutrophil/monokine-targeted chemokine programs. In conclusion, this study demonstrates a novel mechanism of T cell recruitment and function in the absence of exogenous Ag stimulation. By documenting that the execution of innate immune function requires CXCR3+CD4+ T cells, it highlights the critical role of CXCR3 chemokine biology for the continuum of innate to adaptive immunity in the pathophysiology of liver IRI.     

Complexity of phenotypes and symbiotic behaviour of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> exopolysaccharide mutants

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 -lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of -lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l^–1). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of -lactamase was observed only in strain EHA101.Abbreviations CFU Colony-forming unit - MBC Minimum bactericidal concentration - MIC Minimum inhibitory concentration - PBP Penicillin-binding protein     

Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L.

Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (1979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases, callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiole-derived callus tissue have so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be non-regenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - KN kinetin - NAA -naphthaleneacetic acid     

Genetic polymorphism of the iron-regulatory protein-1 and -2 genes in age-related macular degeneration

Iron can be involved in the pathogenesis of AMD through the oxidative stress because it may catalyze the Haber–Weiss and Fenton reactions converting hydrogen peroxide to free radicals, which can induce cellular damage. We hypothesized that genetic polymorphism in genes related to iron metabolism may predispose individuals to the development of AMD and therefore we checked for an association between the g.32373708 G>A polymorphism (rs867469) of the IRP1 gene and the g.49520870 G>A (rs17483548) polymorphism of the IRP2 gene and AMD risk as well as the modulation of this association by some environmental and life-style factors. Genotypes were determined in DNA from blood of 269 AMD patients and 116 controls by the allele-specific oligonucleotide-restriction fragment length polymorphism and the polymerase chain reaction-restriction fragment length polymorphism. An association between AMD, dry and wet forms of AMD and the G/G genotype of the g.32373708 G>A-IRP1 polymorphism was found (OR 3.40, 4.15, and 2.75). On the other hand, the G/A genotype reduced the risk of AMD as well as its dry or wet form (OR 0.23, 0.21, 0.26). Moreover, the G allele of the g.49520870 G>A-IRP2 polymorphism increased the risk of the dry form of the disease (OR 1.51) and the A/A genotype and the A allele decreased such risk (OR 0.43 and 0.66). Our data suggest that the g.32373708 G>A-IRP1 and g.49520870 G>A-IRP2 polymorphisms may be associated with increased risk for AMD.     

Analysis of the Behavior of Mitochondria in the Ovaries of the Earthworm Dendrobaena veneta Rosa 1839

We examined six types of cells that form the ovary of the earthworm Dendrobena veneta ogonia, prooocytes, vitellogenic oocytes, trophocytes, fully grown postvitellogenic oocytes and somatic cells of the gonad. The quantitative stereological method revealed a much higher “volume density” of mitochondria in all of the types of germ-line cells except for the somatic cells. Fluorescent vital stain JC-1, however, showed a much higher oxidative activity of mitochondria in the somatic cells than in the germ-line cells. The distribution of active and inactive mitochondria within the studied cells was assessed using the computer program ImageJ. The analysis showed a higher luminosity of inactive mitochondria in all of the types of germ-line cells and a higher luminosity of active mitochondria in somatic cells. The OXPHOS activity was found in somatic cells mitochondria and in the peripheral mitochondria of the vitellogenic oocytes. The detection of reactive oxygen species (ROS) revealed a differentiated distribution of ROS in the different cell types. The amount of ROS substances was lower in somatic cells than in younger germ-line cells. The ROS level was also low in the cytoplasm of fully grown postwitellogenic oocytes. The distribution of the MnSOD enzyme that protects mitochondria against destructive role of ROS substances was high in the oogonia and in prooocytes and it was very high in vitellogenic and postvitellogenic oocytes. However, a much lower level of this protective enzyme was observed in the trophocytes and the lowest level was found in the cytoplasm of somatic cells. The lower mitochondrial activity and higher level of MnSOD activity in germ-line cells when compared to somatic cells testifies to the necessity of the organisms to protect the mitochondria of oocytes against the destructive role of the ROS that are produced during oxidative phosphorylation. The protection of the mitochondria in oocytes is essential for the transfer of healthy organelles to the next generation.