Primary structure of the alpha-subunit of vacuolar-type Na(+)-ATPase in Enterococcus hirae. Amplification of a 1000-bp fragment by polymerase chain reaction.

A 1000-bp fragment of Enterococcus hirae genomic DNA was amplified by the polymerase chain reaction method, using the oligonucleotide primers designed from amino acid sequences of both amino-terminal and a tryptic fragment of the Na(+)-ATPase alpha-subunit in this organism. DNA sequencing of this product revealed that the amino acid sequence of Na(+)-ATPase alpha-subunit is highly homologous to the corresponding sequences of large (alpha) subunits of vacuolar (archaebacterial) type H(+)-ATPases, supporting our proposal [Kakinuma, Y. and Igarashi, K. (1990) FEBS Lett. 271, 97-101] that the Na(+)-ATPase of this organism belongs to the vacuolar-type ATPase.     

The biology of beta-adrenergic receptors: analysis in human epidermoid carcinoma A431 cells.

1. G-protein-linked transmembrane signaling has emerged as a major pathway for information transduction across the cell membrane. 2. In addition to photopigments that propagate the signal from light, cell-surface receptors for hormones, neurotransmitters, and autacoids propagate signals from ligand binding to membrane-bound effector units via G-proteins. 3. Biochemical and molecular features of one prominent member of these receptors, the beta-adrenergic receptor, will be highlighted in the present article. 4. The role of the human epidermoid carcinoma A431 cells as a model for the study of the structure and biology of beta-adrenergic receptors will be emphasized. 5. A model for receptor regulation, gleaned from recent advances in the biochemistry, cell and molecular biology of beta-adrenergic receptors, is discussed.     

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.     

Crystallization and preliminary X-ray investigation of a sarcoplasmic calcium-binding protein from amphioxus

Crystals of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group C222(1), with unit cell axes a = 59.6(1) A, b = 81.3(1) A and c = 82.4(1) A. There is one molecule in the asymmetric unit. The crystals diffract beyond 2.5 A and show less than 20% decline in diffraction intensities after a three day exposure to X-rays from a laboratory rotating anode source.     

Analysis of protein main-chain solvation as a function of secondary structure

We have analysed the hydration of main-chain carbonyl and amide groups in 24 high-resolution well-refined protein structures as a function of the secondary structure in which these polar groups occur. We find that main-chain atoms in beta-sheets are as hydrated as those in alpha-helices, with most interactions involving "free" amide and carbonyl groups that do not participate in secondary structure hydrogen bonds. The distributions of water molecules around these non-bonded carbonyl groups reflect specific steric interactions due to the local secondary structure. Approximately 20% and 4%, respectively of bonded carbonyl and amide groups interact with solvent. These include interactions with carbonyl groups on the exposed faces of alpha-helices that have been correlated previously with bending of the helix. Water molecules interacting with alpha-helices occur mainly at the amino and carbonyl termini of the helices, in which case the solvent sites maintain the hydrogen bonding by bridging between residues i and i-3 or i-4 at the amino terminus and between i and i+3 or i+4 at the carbonyl terminus. We also see a number of solvent-mediated Ncap and Ccap interactions. The water molecules interacting with beta-sheets occur mainly at the edges, in which case they extend the sheet structure, or at the ends of strands, in which case they extend the beta-ladder. In summary, the solvent networks appear to extend the hydrogen-bonding structure of the secondary structures. In beta-turns, which usually occur at the surface of a protein, exposed amide and carbonyl groups are often hydrated, especially close to glycine residues. Occasionally water molecules form a bridge between residues i and i+3 in the turn and this may provide extra stabilization.     

Epidemiology of diaphragmatic hernia in Languedoc-Roussillon.

The frequency of diaphragmatic hernia (DH) varies, according to the studies, between 1/2000 and 1/7000. In the Languedoc-Roussillon (South of France), due to the presence of a Regional Foeto-Pathology Department and Medico Surgical Paediatric Department, it was possible to itemize all of the DH over a 24 month period (June 1989----May 1991). 20 children presenting DH (10 foetuses and 10 liveborns) were examined for a total population of 49.350 foetuses and liveborns (frequency of DH: 0.40/1000). 10 DH were associated with extra-pulmonary malformations (50%). 4 chromosome abnormalities were found (20%). Prenatal chromosome analysis in cases of ultrasound malformation detection has increased the number of karyotype abnormalities diagnosed.     

Fast atom bombardment tandem mass spectrometric analysis of N-carbamoylamino acids.

Using fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), we examined 12 synthetic N-carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives. In FAB mass spectrometry and FAB MS/MS, spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. The daughter scan spectrum of the parent ion indicated that it was useful for structural elucidation and differentiation of structural isomers of CAA. We have also identified each CAA separately in a mixture using a neutral loss scan for characteristic ions. In addition, we demonstrated that CAA in urine samples from patients with ornithine carbamoyl transferase deficiency gave collision-induced dissociation (CID) spectra which correspond well with CID spectra obtained using synthetically prepared CAA.     

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.