Detection of Helicobacter species DNA by quantitative PCR in the gastrointestinal tract of healthy individuals and of patients with inflammatory bowel disease

In many animal species different intestinal Helicobacter species have been described and a few species are associated with intestinal infection. In humans, the only member of the Helicobacter family which is well described in literature is Helicobacter pylori. No other Helicobacter-associated diseases have definitely been shown in humans. We developed a sensitive quantitative PCR to investigate whether Helicobacter species DNA can be detected in the human gastrointestinal tract. We tested gastric biopsies (including biopsies from H. pylori positive persons), intestinal mucosal biopsies and fecal samples from healthy persons, and intestinal mucosal biopsies from patients with inflammatory bowel disease (IBD) for the presence of Helicobacter species. All gastric biopsies, positive for H. pylori by culture, were also positive in our newly developed PCR. No Helicobacter species were found in the mucosal biopsies from patients with IBD (n = 50) nor from healthy controls (n = 25). All fecal samples were negative. Our study suggests that Helicobacter species, other than H. pylori, are not present in the normal human gastrointestinal flora and our results do not support a role of Helicobacter species in IBD.     

Chemically modified porcine hemoglobins and their biological properties

Hemoglobin cross-linked with small molecular modifiers turns out to be more stable. Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge the molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions. In the present study, the optimal conditions for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl) fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb (DBBF-pHb) showed improved oxygen affinity and the ability to resist the dissociation of the alpha2beta2 tetramer compared with the natural protein. DBBF-pHb was then bound to the activated PEG. The results indicated that the pHb modified with DBBF and PEG had more stable tetrameric conformation with a molecular weight of 107000. Their oxygen half-saturation pressure (P50) is around 3.33 kPa, which approximates the physiological P50 of human red blood cells. Both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by enzyme-linked immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the treatment of hemorrhagic shock where modified hemoglobin solutions were used as resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model.     

Double-strand hydrolysis of DNA by a magnesium(II) complex with diethylenetriamine

The development of artificial nucleases that hydrolyze DNA or RNA is of great interest in molecular biology, biotechnology, and medicine. We now report that a magnesium(II) complex of diethylenetriamine (Mg-dien) can effectively promote the double-stranded cleavage of plasmid DNA and the dideoxynucleotide dApdA under physiological conditions of pH and temperature. Experiments performed in the presence of hydrogen peroxide, radical scavengers, or under rigorously anaerobic conditions indicate that DNA cleavage mediated by Mg-dien occurs via a hydrolytic path. Mg-dien efficiently hydrolyzes supercoiled pBR322 DNA and the pseudo-first-order rate constant at 37 degrees C and pH 8.0 is estimated to be 1.60 h(-1). The dinucleotide dApdA hydrolysis, with Mg-dien at 170 microM, shows a rate enhancement factor of ca. 5 x 10(8). 1H and 31P(1H) NMR studies show that Mg-dien effectively hydrolyzes 5'-dAMP to give deoxyadenosine and inorganic phosphate. While Mg2+ has been found at the catalytic sites of many natural nucleases, Mg-dien appears to be the first synthetic Mg2+-containing system capable of hydrolyzing dideoxynucleotides and DNA and thus may provide a simple model system to assist mechanistic studies of naturally occurring nucleases.     

Detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.     

Lysophospholipids increase ICAM-1 expression in HUVEC through a Gi- and NF-kappaB-dependent mechanism

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that are recognized by the Edg family of G protein-coupled receptors. In endothelial cells, these two ligands activate Edg receptors, resulting in cell proliferation and cell migration. The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily. This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs). This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent. Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of G_i, ammonium pyrrolidinedithiocarbamate and BAY 11–7082, inhibitors of the nuclear factor (NF)-B pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a G_i-, NF-B-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes. lysophosphatidic acid; sphingosine 1-phosphate; inflammation; intercellular adhesion molecule-1; nuclear factor-B; human umbilical cord vein endothelial cells     

Signaling from Nucleotide Receptors to Protein Kinase Cascades in Astrocytes

In the CNS, extracellular ATP can function as an excitatory neurotransmitter as well as a trophic factor. These short-term and long-term actions are mediated by nucleotide receptors. Extracellular ATP can also act as a co-mitogen in conjunction with polypeptide growth factors such as basic fibroblast growth factor (FGF2). Cellular proliferation, differentiation and survival are regulated by signaling cascades composed of protein kinases, including extracellular signal regulated protein kinase (ERK) and protein kinase B (also called Akt). Here we summarize recent studies on nucleotide receptor signaling to ERK and Akt in astrocytes and the role of protein kinase cascades in mediating the trophic actions of extracellular ATP, alone or together with FGF2. Because extracellular ATP and FGF2 contribute to the hyperplastic and hypertrophic response of astrocytes to CNS injuries, an understanding of their protein kinase signaling mechanisms may lead to novel therapeutic approaches for neurological conditions that involve gliosis and the generation of reactive astrocytes, such as trauma, stroke, seizure and neurodegenerative and demyelinating disorders.Special issue dedicated to Lawrence F. Eng.     

Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.     

Polyphenols from peanut skins and their free radical-scavenging effects

Separation of the water-soluble fraction of peanut skins led to the isolation of five proanthocyanidins. Based on the spectroscopic investigation and partial acid catalyzed degradation, their structures were determined to be epicatechin-(2beta-->O -->7, 4beta -->6)-[epicatechin-(4beta-->8)]-catechin (1), epicatechin-(2beta-->O -->7, 4beta-->8) epicatechin-(4beta-->8)-catechin-(4alpha-->8)-epicatechin (2), and procyanidins B2 (3), B3 (4) and B4 (5). The absolute configuration of the new compounds was determined from their circular dichroism curves and the (1)H NMR spectra of analysis of flavan-3-ols formed by thiolytic degradation of 1 and 2 in the presence of a chiral dirhodium complex (dirhodium tetra-(R)-(trifluoromethyl) phenyl acetate).     

Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.     

Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants

To study the role of the hinge region in nisin and to obtain mutants that exhibit altered or new biological activities and functional properties, we changed certain amino acids in the hinge region by performing site-directed mutagenesis with the nisinZ structural gene (nisZ). The results showed that the nisinZ mutants had decreased antimicrobial activities against Micrococcus flavus NCIB8166 and Streptococcus thermophilus. Interestingly, compared with wild nisinZ, mutant N20K nisinZ and M21K nisinZ displayed antimicrobial activity against gram-negative Shigella, Pseudomonas and Salmonella; and they had a higher solubility than wild-type nisinZ. At pH 8, the solubilities of N20K nisinZ and M21K nisinZ were, respectively, three-fold higher and five-fold higher than that of nisinZ. Mutant N20Q nisinZ and M21G nisinZ were considerably more stable than nisinZ at higher temperatures and neutral or alkaline pH. These mutants provided information that the central hinge region in nisinZ plays an important role in providing the conformational flexibility required for the antimicrobial activity on the membrane. Our finding documented that it may well be worth considering the construction of the new nisin mutants with changed inhibitory activity against a wide range of gram-negative bacteria and the improvement of functional properties by site-directed mutagenesis.