Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Synthesis of AZT analogues: 7-(3-azido-2-hydroxypropyl)-, 7-(3-amino-2-hydroxypropyl)-,7-(3-triazolyl-2-hydroxypropyl)theophylline

Nucleophilic displacement of the tosyloxy group in 7-(2-hydroxy-3-p-toluenesulfonyloxypropyl)theophylline (1) with azide anion afforded 7-(3-azido-2-hydroxypropyl)theophylline (2). Reduction of the 3-azido group in 2 with Ph3P/Py/NH4OH afforded the 3-amino derivative 4, alternatively obtained by regioselective amination of 7-(2,3-epoxypropyl)theophylline (3). Selective acetylation of 4 gave the N-acetyl derivative 5. 1,3-Dipolar cycloaddition of the azide group in 2 with N1-propargyl thymine (6) afforded the regioisomeric triazole 7.     

Analysis of the type IV secretion system-dependent cell motility of Helicobacter pylori-infected epithelial cells

The pathogenesis of Helicobacter pylori-associated disorders is strongly dependent on a specialized type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). Cytotoxin-associated gene A (CagA) is the only known H. pylori protein translocated into the host cell followed by tyrosine phosphorylation through host protein kinases. H. pylori induces cellular processes which are either PAI- or CagA-dependent (e.g., cell motility), PAI-dependent, but CagA-independent (e.g., interleukin-8 release), or PAI- and CagA-independent (e.g., cyclooxygenase-2 release). Here, we investigated H. pylori strains mutated in single PAI genes of the wild type strain Hp26695 and their effects on cell motility. We found 17 gene products out of 27 PAI genes playing a superordinated role and five PAI-encoded proteins exhibiting a clearly critical role in motogenic host cell responses, whereas the remaining five PAI gene products had no significant influence on the motogenic response in reaction to H. pylori infection. This study clearly demonstrated that H. pylori-induced cell motility and invasive growth involve type IV secretion system-dependent signalling as well as translocated and phosphorylated CagA. These findings reveal a deeper insight in to the meaning of the T4SS of H. pylori for host cell motility.     

Human iodine requirements determined by the saturation kinetics model

Iodine plays a decisive role in metabolism and the process of early growth and development of most organs, especially of the brain. Effects of iodine deficiency include goiter, stillbirth and miscarriage, neonatal and juvenile thyroid deficiency, dwarfism, mental defects, deaf mutism, spastic weakness and paralysis. In this study, the application of a mathematical model (derived from Machaelis-Menten enzyme kinetics) to iodine measured in urine samples from a randomly selected group derived from the Egyptian village of West El-Mawhoub in the Dakhlah Oasis resulted in the conclusion that iodine excretion parameters can be used to characterize iodine utilization and accurately predict the level of salt iodination required to maintain proper physiological functions. The four parameter saturation kinetics model analysis indicated that a salt iodination level of 63 mg/kg reduced the severity of IDD, with 83% of the studied subjects having urinary excretion levels of 1.18 micromol/L. This gives a convenient mechanism for providing adequate dietary iodine with a non-invasive index for the avoidance of IDD. Commercially available salt was analyzed using standard iodiometric titration methods to determine iodination levels. Analysis revealed that only 20% of the commercially available salt complied with the manufacturer's label and revealed the presence of large individual variability between batches amounting to -95 to +150% of the claimed iodine level. Therefore, salt iodination requires careful supervision to ensure that promised iodine levels are being delivered and consumed.     

Differential recognition of resveratrol isomers by the human estrogen receptor-alpha: molecular dynamics evidence for stereoselective ligand binding

Resveratrol (RSVL) is a phytoestrogen that occurs naturally in two forms (trans- (E) and cis- (Z)). We have conducted molecular dynamics (MD) studies to differentially characterize the estrogen receptor-alpha (ER-alpha) binding profiles of RSVL stereoisomers. Favorable orientations for RSVL isomers at the ER-alpha pocket were first inferred from (1) alignment with pharmacophoric elements of the pure ER-alpha agonists estradiol (E2) and (2) assessment of ligand recognition by the ER-alpha binding domain. Subsequently, these orientations for RSVL isomers were subjected to MD analyses versus E2. A 100-picosecond MD simulation revealed that E2 contributed four stable hydrogen bonds with the key ER-alpha pocket residue: Arg394, Glu353, His524, and Leu525. Further, E2 displayed favorable binding energy, conformational energy change (DeltaE), and movement of the binding pocket residues (RMSd). Compared to E2, (E)-RSVL lacked a hydrogen bond (HB) with His524 but formed three additional bonds with Gly521, Phe404, and Met343 of the ER-alpha pocket. Further, (E)-RSVL conferred more favorable energy of interaction, less favorable DeltaE, but comparable RMSd values. In contrast, (Z)-RSVL orientations missed hydrogen bonding (HB) with His524 and Leu525, two essential ligand binding residues, and/or produced considerably less favorable-binding energy, -DeltaE, and -RMSd values than did (E)-RSVL. In conclusion, the present study demonstrates the utility of this MD model in distinguishing between RSVL stereoisomers. The weak binding of (Z)-RSVL by the human ER-alpha binding is congruent with its inferior ligand profiles in ER-endowed biological systems. Further, evidence is provided for a considerable variation in the mode of recognition of the mixed agonist/antagonist (E)-RSVL, and the pure agonist E2.     

Polymicrobial infections in children with diarrhoea in a rural area of Jordan

Polymicrobial infections associated with diarrhoea are common in developing countries. Stool specimens were collected from 220 patient children and 100 controls. Potential pathogenic agents isolated from 143 (65%) children were identified by molecular and standard microbiological methods. Co-infections with two or more agents were detected in 50 (35%) cases. Escherichia coli, Shigella dysenteriae, Giardia and Entamoeba histolytica were found to be predominant. The etiologic agents could not be determined in 77 (35%) cases. The most significant risk factors were the age, the education level of the mother and the use of non-chlorinated water. The high infection rate of diarrhoeal diseases is a strong indication that these pathogens circulate easily through the population.     

Congestive heart failure in copper-deficient mice

Copper Deficiency (CuD) leads to hypertrophic cardiomyopathy in various experimental models. The morphological, electrophysiological, and molecular aspects of this hypertrophy have been under investigation for a long time. However the transition from compensated hypertrophy to decompensated heart failure has not been investigated in the study of CuD. We set out to investigate the contractile and hemodynamic parameters of the CuD mouse heart and to determine whether heart failure follows hypertrophy in the CuD heart. Dams of FVB mice were fed CuD or copper-adequate (CuA) diet starting from the third day post delivery and the weanling pups were fed the same diet for a total period of 5 weeks (pre- and postweanling). At week 4, the functional parameters of the heart were analyzed using a surgical technique for catheterizing the left ventricle. A significant decrease in left ventricle systolic pressure was observed with no significant change in heart rate, and more importantly contractility as measured by the maximal rate of left ventricular pressure rise (+dP/dt) and decline (-dP/dt) were significantly depressed in the CuD mice. However, left ventricle end diastolic pressure was elevated, and relaxation was impaired in the CuD animals; the duration of relaxation was prolonged. In addition to significant changes in the basal level of cardiac function, CuD hearts had a blunted response to the stimulation of the beta-adrenergic agonist isoproterenol. Furthermore, morphological analysis revealed increased collagen accumulation in the CuD hearts along with lipid deposition. This study shows that CuD leads to systolic and diastolic dysfunction in association with histopathological changes, which are indices commonly used to diagnose congestive heart failure.