Comparison of genomic structures and antigenic reactivities of orthologous 29-kilodalton outer membrane proteins of Helicobacter pylori

We purified a 29-kDa Helicobacter pylori outer membrane protein (Omp29 protein) and cloned the gene encoding the protein from H. pylori strain ATCC 43504. The Omp29 gene corresponded to the reported JHP73 and the HP78-79 genes of H. pylori strains. A corresponding nucleotide fragment was detected in all 150 tested H. pylori clinical isolates by PCR or Southern blotting. The amplified Omp29-corresponding fragments were categorized into a ca. 770-bp-long group and a larger-fragment group. Sequence analysis indicated that the larger fragments were likely synthesized from the 770-bp fragments by insertion of an irrelevant fragment via 17-bp-long repeat sequences. Immunoblot analysis implies that the ca. 770-bp fragment is responsible for the protein homologous to Omp29, whereas the larger fragments are not responsible for those proteins or encoding antigenically distinct proteins. We postulate that the H. pylori outer membrane protein Omp29 can alter its antigenicity through gene modifications mediated by nucleotide transfer.     

Laryngeal Injury and Pneumomediastinum Due to Minor Blunt Neck Trauma: Case Report

Background

Serious isolated laryngeal injuries are uncommon in children.

Pigment Epithelium-Derived Factor Inhibits Lysosomal Degradation of Bcl-xL and Apoptosis in HepG2 cells

Pigment epithelium-derived factor (PEDF) has several biological actions on tumor cells, but its effects are cell-type dependent. The aim of this study was to examine the pathophysiological role of PEDF in hepatocellular carcinoma (HCC). PEDF expression was examined in various hepatoma cell lines and human HCC tissues, and was seen in various hepatoma cell lines including HepG2 cells. In human HCC tissues, PEDF expression was higher than in adjacent non-HCC tissues. In addition, serum PEDF levels were higher in HCC patients than in non-HCC patients, and curative treatment of HCC caused significant reductions in serum PEDF levels compared with pretreatment levels. In vitro experiments, camptothecin (CPT) was used to induce apoptosis and the effect of PEDF was investigated by knockdown of the PEDF gene in CPT-treated HepG2 cells. Knockdown of the PEDF gene enhanced CPT-induced apoptosis, simultaneously down-regulating Bcl-xL expression in HepG2 cells. Expression of apoptosis-related molecules and effects of bafilomycin A1 on CPT-induced apoptosis were also examined in PEDF gene knockdown HepG2 cells. Treatment with bafilomycin A1 suppressed CPT-induced decreases in Bcl-xL expression and increases in apoptosis in PEDF gene knockdown HepG2 cells. PEDF may, therefore, exert anti-apoptotic effects through inhibition of lysosomal degradation of Bcl-xL in CPT-treated HepG2 cells.Hepatocellular carcinoma (HCC) is a common cancer that causes nearly 1 million deaths a year worldwide.¹ The incidence of HCC is predicted to continue to increase over the next 30 years.² To develop new therapeutic strategies, it is important to elucidate molecular mechanisms underlying hepatocarcinogenesis.Pigment epithelium-derived factor (PEDF) is a 50-kDa glycoprotein initially isolated from fetal human retinal pigment epithelial cells.³ PEDF exerts a range of biological effects depending on the type of the target cell. PEDF induces apoptosis of endothelial cells and results in inhibition of neovascularization.⁴ Overexpression of PEDF causes a reduction in tumor microvessel density and subsequent anti-tumor effects in pancreatic adenocarcinoma and melanoma cells.^5,6 In contrast to its effects in endothelial cells, PEDF causes the opposite effect in other types of cells. PEDF protects granule cells against both natural and potassium-induced apoptosis through activation of prosurvival genes.⁷ In cultured retinal pericytes, PEDF inhibits oxidative stress-induced apoptosis through an increased ratio of B-cell leukemia/lymphoma 2 (Bcl-2)-associated X protein (bax) to bcl-2 mRNA levels with subsequent activation of caspase-3.^8,9 PEDF also inhibits light-induced apoptotic processes in photoreceptor cells in vivo.^10,11HCC is a hypervascular solid tumor, in which neovascularization plays an important role in disease progression and prognosis. However, changes in PEDF expression in human HCC have never been investigated and therefore, it is unclear whether PEDF is a useful target for therapeutic strategies in patients with HCC. Dysregulation of apoptosis is also deeply involved in hepatocarcinogenesis. Although HCC is known to be resistant to apoptosis,¹² it remains unknown whether PEDF has anti-apoptotic effects in HCC.Thus, PEDF is a multifunctional protein with opposing activities, apoptotic and anti-apoptotic activities. These disparate effects depend on cell type. The aims of the present study were to investigate changes in PEDF expression and the role of PEDF in HCC.     

Evolutionary changes in the structures of the cox2 and atp6 loci in the mitochondrial genome of soybean involving recombination across small interspersed sequences

Mitochondrial DNA (mtDNA) fragments that contained cox2 or atp6 loci were cloned from three accessions of wild soybean (Glycine soja) in order to understand the evolutionary changes of mitochondrial genomes in the genus Glycine subgenus Soja. Cox2 was cloned as a single configuration, while atp6 was cloned as either one or two configurations from each accession. Structural variations were detected in the 5′ upstream region of cox2 and in both the 5′ upstream and 3′ downstream regions of atp6. These variations appeared to be the results of recombination events. A comparison of the mtDNA fragments previously cloned from a cultivated soybean (G. max) and a wild soybean revealed various sites of recombination, as well as various combinations of the 5′ and 3′ regions, at the cox2 and atp6 loci. Some of the cloned fragments were found to contain a set of repeated sequences, namely 299-bp and 23-bp repeats in the 5′ region of cox2 or atp6, which were interspersed in the mitochondrial genome in the subgenus Soja. Recombination events involving the 299-bp or 23-bp repeated sequences were shown to account for the generation of structural variations in the 5′ regions of these loci. Received: 21 March / 4 August 1998     

Independent association of antibodies against human papillomavirus type 16 and E7 proteins with cervical cancer

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac- and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti- antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti- and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.     

Isothermal and Nonisothermal Kinetics in the Stability Prediction of Vitamin A Preparations

A nonisothermal method was applied to shelf-life estimation of commercial vitamin A preparations. The usefulness and limitations of the nonisothermal method, as predicted by computer simulation, were validated. Degradation of vitamin A palmitate followed zero-order kinetics, and non-Arrhenius behavior was suggested for the rate constant. The nonisothermal method predicted a higher shelf-life value than the isothermal method for a syrup preparation, and vice versa, for an injection preparation. Analysis of the results suggested that the nonisothermal method provides better estimates of shelf lives than the isothermal method when drug degradation does not follow the Arrhenius equation near ambient conditions.     

Aggregates Formed During Storage of β-Galactosidase in Solution and in the Freeze-Dried State

Aggregates formed during storage of freeze-dried -galactosidase were compared with those formed in solutions. Freeze-dried -galactosidase aggregated during storage in the presence of moisture, producing a protein precipitate which was soluble in guanidine hydrochloride solution but not in buffer solution. The protein precipitate dissolved in guanidine solution exhibited a large molecular size by high-performance size exclusion chromatography and converted to proteins of original size in the presence of dithiothreitol. It is suggested that the aggregation involves chemical interaction via covalent disulfide bonding. In contrast, -galactosidase in aqueous solution aggregated without formation of protein precipitates. Soluble aggregates were converted to proteins of original size in guanidine solution without dithiothreitol, suggesting noncovalent bonding. The difference in aggregation behavior may be ascribed to the difference in the water:protein ratio. We propose that inactivation of -galactosidase is due to formation of thermally denatured (unfolded) protein, which aggregates dependent on the water:protein ratio, either via noncovalent interactions at a high water:protein ratio in solution or via covalent interaction at a low water:protein ratio in the freeze-dried state.     

A Quantitative Assessment of the Significance of Molecular Mobility as a Determinant for the Stability of Lyophilized Insulin Formulations

Purpose The purpose was to explore a method for quantitatively assessing the contribution of molecular mobility to the chemical reactivity of amorphous solids. Degradation of insulin in lyophilized formulations containing trehalose and poly(vinylpyrrolidone)(PVP) was chosen as a model system, and the temperature- and glass transition temperature (T_g)-dependence of the degradation rate was analyzed to obtain the relative contributions of molecular mobility and that of the chemical activational barrier reflected in the energy of activation.Methods Insulin degradation and dimerization in lyophilized trehalose and PVP formulations were monitored at various relative humidities (6–60% RH) and temperatures (10–60°C) by reverse-phase high-performance liquid chromatography (HPLC) and high-performance size-exclusion chromatography (HP-SEC), respectively. The T_g and fragility parameter of the lyophilized insulin formulations were determined by differential scanning calorimetry (DSC).Results Insulin degradation in the initial stage was describable with first-order kinetics for both of the trehalose and PVP formulations. The temperature- and T_g-dependence of the degradation rate indicated that the reactivity of insulin in the trehalose formulation is affected by molecular mobility at low humidity (12% RH), such that the ratio of the observed rate constant (k′) to the rate constant governed only by the activational barrier (k) was 0.051 at the T_g. At higher humidities, in contrast, the value of k′/k was much higher (0.914, 0.978, and 0.994 for 23% RH, 33% RH, and 43% RH, respectively), indicating that insulin degradation rate is determined predominantly by the activational barrier. For insulin degradation in the PVP formulation at temperatures below T_g, the contribution of molecular mobility to the degradation rate appeared to be negligible, as the extrapolated value of t₉₀ at the T_g exhibited a large difference between the formulations with differing T_g values (because of differing water contents).Conclusions The reactivity of insulin in the trehalose and PVP formulations can be described by an equation including factors reflecting the activational barrier (activation energy and frequency coefficient) and factors reflecting the molecular mobility (T_g, fragility parameter and a constant representing the relationship between the molecular mobility and the reaction rate). Thus, analysis of temperature dependence based on the proposed equation allows quantitative assessment of the significance of molecular mobility as a factor affecting chemical reactivity.