Physiology of a choline-requiring strain of Torulopsis pintolopesii

Summary A choline-requiring strain of Torulopsis pintolopesii when growing on choline-methyl-¹⁴C or choline-methyl-³H excretes the radioactivity incorporated in the first 24–48 h of incubation up to ca. 85–95% of the radioactivity added at the beginning of the incubation. The addition of non-radioactive methionine did not interfere with the uptake and excretion of radioactivity from choline-Me-¹⁴C. Radioactive methyl group of methionine previously incorporated by growing cells of T. pintolopesii on varying concentrations of choline was not excreted in appreciable amounts. No evidence was obtained for the oxidation of choline to betaine, degradation to trimethylamine, or net incorporation of labelled choline into lecithin. The occurrence of a new pathway for the utilization of choline in yeasts is suggested. The requirement of choline by T. pintolopesii is explained tentatively by the formation and excretion of a compound containing the carbon and hydrogen atoms from the methyl groups of choline and whose chemical structure still under study, may comprise a heteroside containing mannitol as the polyhydroxylated moiety.     

The relationship of conceptus number and fetal sex to maternal serum testosterone concentration in the rat

On Day 8 of pregnancy, the number of implantation sites in pregnant rats was adjusted to 1, 2, 4, 6, or greater than 10. Blood was collected on Days 11, 12, 15, 18, and 20 for the determination of serum testosterone, progesterone, and androstenedione. Serum testosterone levels exhibited a direct linear relationship with implantation number, increasing from 1 through greater than 10 implants. This linear relationship was particularly evident at Days 12, 15, and 18 of pregnancy. Serum progesterone levels increased from one to four conceptuses and plateaued above this number. There was no apparent relationship between the number of conceptuses and serum androstenedione levels, which may reflect the multiple origins of this steroid in the pregnant rat. In a separate group of rats in which the number of conceptuses was adjusted to three on Day 8 of pregnancy, blood was collected on Days 11, 12, 15, 18, and 20. Fetal sex was determined between the last bleeding and the day of parturition. Serum testosterone was determined and results were examined with regard to the number of male/female fetuses in the litter of three. There was no relationship between maternal serum testosterone levels and the number of male fetuses, indicating that the fetal testis does not make a significant contribution to circulating maternal testosterone levels.     

Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes.

Transposon Tn21 codes for a site-specific integration system, which is probably a novel recombination mechanism, responsible for the acquisition of resistance genes in this widespread family of transposons. Using insertion and deletion mutagenesis we have identified the genetic loci of the various recombination hot-spots (RHS) and of the gene product (the integrase) that catalyses the reaction. The site of recombination has been localized in two of the RHSs to the DNA sequence GTTAG, which is present at the 3' termini of a loosely conserved palindromic sequence of approximately 59 bp. This 59 bp sequence, which flanks the inserted genes in a number of naturally occurring transposons, is the only element required in cis for the recombination reaction.     

Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration originally described in families of Portuguese-Azorean ancestry. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised. To test this possibility we have conducted a linkage disequilibrium study of markers segregating with the MJD1 locus in a total of 64 unrelated families of different geographical origins. Significant association was detected between the MJD1 locus and marker alleles at loci D14S280, D14S1050 and D14S81. All affected individuals, except one Chinese family, had allele 3 (237 bp) at D14S280. This finding is consistent with a founder effect in our MJD population. However, distinct haplotypes were observed in patients originating from the two Azorean islands showing the highest disease prevalence; therefore, the possible existence of more than one founder mutation can not be excluded with the markers currently available. Received: 27 February 1996 / Revised: 4 June 1996     

Neural control of the expression of a Ca2+-activated K+ channel involved in the induction of myotonic-like characteristics

Summary 1. Expression of the apamin-sensitive K⁺ channel (SK⁺) in rat skeletal muscle is neurally regulated. The regulatory effect of the nerve over the expression of some muscle ion channels has been attributed to the electrical activity triggered by the nerve and/or to a trophic effect of some molecules transported from the soma to the axonal endings. 2. SK⁺ channels apparently are involved in myotonic dystrophy (MD), therefore understanding the factors that regulate their expression may ultimately have important clinical relevance. 3. To establish if axoplasmic transport is involved in this process, we used two experimental approaches in adult rats: (a) Both sciatic nerves were severed, leaving a short or a long nerve stump attached to the anterior tibialis (AT). (b) Colchicine or vinblastine (VBL), two axonal transport blockers of different potencies, was applied on one leg to the sciatic nerve. To determine whether electrical activity affects the expression of SK⁺ channels, denervated AT were directly stimulated. The corresponding contralateral muscles were used as controls. 4. With these experimental conditions we measured (a) apamin binding to muscle membranes, (b) muscle contractile characteristics, and (c) electromyographic activity. 5. In the short- and long-nerve stump experiments, 5 days after denervation¹²⁵I-apamin binding to AT membranes was 2.0 times higher in the short-stump side. This difference disappeared at longer times. The delayed expression of SK⁺ channels in the muscle left with a longer nerve stump can be attributed to the extra axoplasm contained in the longer stump, which maintains a normally repressive signal for a longer period of time. Ten to 15 days after application of axonal transport blockers we found that the muscle half-relaxation time increased in the drug-treated side and apamin partially reverted the prolonged relaxation. Myotonic-like discharges specifically blockable by apamin were always present in the drug-treated leg.¹²⁵I-Apamin binding, which is undetectable in a microsomal preparation from hind leg control muscles, was increased in the drug-treated preparations. Apamin binding to denervated and stimulated AT muscles was lower than in the contralateral unstimulated muscles [3.3±1.0 vs 6.8±0.8 (n=4) fmol/mg protein]. 6. Our results demonstrate that electrical activity and axoplasmic transport are involved in the control of expression of SK⁺ in rat skeletal muscle. However, the increased expression of this channel induces myotonic-like characteristics that are reversed by apamin. This myotonic activity could be a model for MD.     

Isolation and promoter characterization of barley geneItr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutantlys3a endosperm

The geneItr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library ofHordeum vulgare L. The gene has no introns and presents in its 5-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the copia-like retro-transposon Bare-1. Functional analysis of theItr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system theItr1 promoter retains its endosperm specifity and thetrans-regulation mediated by theLys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer fullGUS expression and for endosperm specifity. In protoplasts derived from thelys3a mutant, Risø 1508,GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.Equal authours     

Examining the relationship between secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus

Summary The conformation of a peptide that represents antigenic site A of foot-and-mouth disease virus strain C-S8c1 (residues 136–156 of VP1; YTASARGDLAHLTTTHARHLP) has been studied by circular dichroism and compared with three analogs that reproduce amino acid substitutions at position 146 (HisArg, Gln or Asp) which affect antibody recognition. Four other peptides, incorporating replacements at position 147 predicted to maintain (LeuIle, Nle and Ala) or disrupt (LeuGly) helical structure at this site, have also been studied. In aqueous solution or in 4 M urea, the spectra of all eight peptides were typical of aperiodic conformation and independent of concentration or pH. However, upon addition of solvents such as methanol or hexafluoroisopropanol, spectral patterns evidenced significant levels (ca. 50%) of helical structure. The single residue substitutions at positions 146 and 147 caused minor to significant variations in the calculated amount of -helix of the peptides. An attempt to relate these changes in helical content to the antigenic behaviour of the peptides towards five monoclonal antibodies elicited with virus and mapping at site A could not find any straightforward correspondence between the two sets of results. The parent peptide and its His¹⁴⁶Arg analog were also analyzed by circular dichroism in the presence of the Fab fragment of SD6, a monoclonal antibody mapping at site A and much less reactive with viruses carrying the referred mutation. Although a peptide-antibody interaction was evident from spectral changes, careful inspection of the difference spectra (peptide-Fab minus Fab) of both peptides failed to detect any significant distinction between them that could be attributed to their different immunoreactivity. While these findings do not necessarily conflict with previous reports that the interaction of antigenic site A with antibodies is mediated to some extent by the adoption of a helix structure, they suggest that, at least for C-serotype viruses, other structural features in addition to a helical conformation are critically involved in antigenic recognition.