Mitochondrial and ribosomal DNA variation among members of the Anopheles quadrimaculatus (Diptera: Culicidae) species complex.

The extent of intra- and inter-specific variation in mitochondrial DNA and nuclear ribosomal RNA gene restriction sites was determined for the four sibling species of the Anopheles quadrimaculatus complex. Individual mosquitoes were identified by allozyme analysis according to previously published keys, and the total genomic DNA of these same individuals was then cleaved with restriction enzymes. Restriction maps of mitochondrial DNA, including the positions of variable sites, were constructed for each species. No evidence for interspecific hybridization was found in the populations surveyed. There was little variation in restriction patterns within any given species, but differences occurred among the four. Three restriction enzymes (AvaI, HindIII, and PvuII) yielded species-specific DNA restriction patterns for the mitochondrial DNA, while AvaI and HindIII produced diagnostic patterns for the ribosomal DNA. Thus, restriction patterns were very useful for detecting cryptic species but less appropriate than isozymes for studying genetic structure of populations within species.     

Synthesis of a human insulin gene V. Enzymatic assembly,cloning and characterization of the human proinsulin DNA

To form a 258-bp sequence coding for human proinsulin, 41 synthetic deoxyribo-oligonucleotide fragments of 11 to 15 nucleotides in length were assembled by enzymatic methods. The coding sequence is preceded by ATG and followed by TGA for translation start and stop signals, and terminated in an EcoRI and a BamHI recognition sequence. The complete synthetic sequence was ligated to a plasmid and cloned in Escherichia coli. The cloned DNA was shown to have the correct human proinsulin coding sequence.     

Decrease in N-ethylmaleimide-sensitive ATPase activity in collecting duct by metabolic alkalosis

Changes in systemic acid-base balance are known to influence acidification in the collecting duct. The H+ secretion in the collecting duct has been shown to be an electrogenic process and it has been suggested that an H-ATPase sensitive to inhibition by N-ethylmaleimide (NEM) is responsible for H+ secretion. This study was designed to determine the effect of metabolic alkalosis on NEM-sensitive ATPase activity in the microdissected segments of the distal nephron. Metabolic alkalosis was produced by giving NaHCO3 to normal rats for 7 days. The plasma total CO2 concentration in the experimental group was 31.5 +/- 1.8 mM compared with 23.4 +/- 1.0 mM in the control group. NEM-sensitive ATPase activity was significantly lower in the cortical collecting duct and in the outer and inner medullary collecting ducts of alkali-loaded rats than those of control rats. There was no significant difference in the enzyme activity between the two groups of animals in the other nephron segments examined. Our results suggest that NEM-sensitive H-APTase activity in all three segments of the collecting duct is modulated by the acid-base status of the animal.     

Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.

The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.     

Voltammetric detection of anti-HIV replication drug based on novel nanocomposite gold-nanoparticle–CaCO<Subscript>3</Subscript> hybrid material

A novel bionanocomposite, horse radish peroxidase- gold-nanoparticle–Calcium carbonate (HRP-AuNPs-CaCO₃), hybrid material was encapsulated by silica sol on a glassy carbon electrode (GCE). The fabricated modified electrode was used as a novel voltammetric sensor for electrochemical sensing of anti-HIV replication drug i.e. deferiprone. The surface morphology of the modified electrode was characterized by scanning electron microscopy (SEM). Results obtained from the voltammetric measurements show that HRP-AuNPs-CaCO₃ modified GCE offers a selective and sensitive electrochemical sensor for the determination of deferiprone. Under experimental conditions, the proposed voltammetric sensor has a linear response range from 0.01 to 10,000 μM with a detection limit of 0.01 μM. Furthermore, the fabricated sensor was successfully applied to determine deferiprone level in spiked urine and serum samples.