Biosynthesis of methionine in an ethionine-resistant strain ofCandida utilis

The strainCandida utilis T 20 adapted to a high concentration of ethionine, excretes considerable amounts of methionine in a synthetic medium, about 40 times as much as the original non-adapted strain. At the same time, the amount of methionine in yeast cells incrncreased, predominantly in the pool (9 times as much as in the control). This ability to produce greater amounts of methionine in the pool or to excrete it into the medium is not permanent, since after 5 passages on agar with ut ethionine the amount of methionine was practically not increased as compared with the original non-adapted strain. An increase in free methionine and of methionine excreted into the medium was found on cultivating the strain in a molasses-containing medium, too.     

Verapamil: a novel probe of surfactant secretion from rat type II pneumocytes

Surfactant from type II pneumocytes prevents the alveolar atelectasis found in both the neonatal and adult forms of respiratory distress syndrome. We have found that verapamil, a phenylalkene with calcium channel and alpha 1-receptor binding properties, has a multiphasic concentration effect on surfactant secretion from [3H]choline-labeled rat type II pneumocytes in culture. Verapamil (10(-8) M) caused a 24% stimulation of surfactant secretion, whereas an 8% inhibition was found at 10(-6) M and a 70% stimulation was found at 10(-4) M. Lactate dehydrogenase release occurred at 5 x 10(-4) M verapamil. Verapamil (10(-4) M) also produced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) in comparison with concentrations of less than or equal to 10(-6) M, an effect that could not be blocked by propranolol (10(-4) M). Verapamil (10(-6) M) increased the total formation of inositol phosphates (IP) by 23% in comparison with IP formation in control cells. Calcium influx was inhibited 15% by 10(-8) M verapamil and 37% by 10(-4) M verapamil. Calcium efflux was stimulated 44% by 10(-5) M verapamil. In combination with 50% effective concentrations (EC50) of terbutaline, phorbol ester, and ATP, the respective effects of verapamil (10(-4) M) on surfactant secretion were approximately additive. We conclude that verapamil has a novel multiphasic concentration effect on surfactant secretion, which appears to involve several signal transduction pathways including cAMP formation, IP formation, inhibition of calcium influx, and stimulation of calcium efflux.     

Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata

Fifth (last) instar nymphs of th e tick Ornithodoros moubata convert ingested 20-hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to th e more polar conjugates API. Only small quantities of free hormone were transferred to th e hemolymph and the carcass within t h e first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [³H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of API conjugates. API obtained in second instar nymphs fed with [3H]ecdysone ([³H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of APT remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., API mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle. Apolar products (mainly AP2) that accumulated in eggs of females injected with [³H]E or [³H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to API, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs. We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Identification of phytochrome B amino acid residues mutated in three new phyB mutants of Arabidopsis thaliana

The growth and development of plants is regulated by light viathe action of photoreceptors which are responsive to the red/far-red,blue and UV regions of the spectrum. Phytochrome B (the apoproteinof which is encoded by the PHYB gene) is one of the red/far-redabsorbing photoreceptors active in this process. In this paper,the isolation and characterization of three new EMS-inducedmutations of Arabidopsis which confer phytochrome B deficiencyare described. Complementation analysis showed that these mutations(phyB-101, phyB-102 and phyB-104) were allelic with PHYB. DNAsequence analysis showed that all three mutants contain nucleotidesubstitutions in the PHYB-101 gene sequence. phyB-101 carriesa nucleotide substitution within the second exon of the PHYBgene. This G-to-A substitution is a missense mutation that convertsa glutamate residue at position 812 of the phytochrome B apoproteinto a lysine residue. phyB-102, another missense mutant, carriesa C-to-T substitution which converts a serine residue at position349 of the phytochrome B apoprotein to a phenylalanine residue.phyB-104 carries a premature stop codon as a result of a G-to-Amutation 1190 bp down-stream of the ATG start codon of the PHYBsequence. The missense mutations in phyB-101 and phyB-102 causesignificant alterations in the predicted second ary structureof their respective mutant polypeptides, and identify aminoacid residues playing crucial roles in phytochrome B function,assembly or stability. Key words: Arabidopsis thaliana, phytochromet, phyB mutants, missense mutations     

Responses of nitrate assimilation and N translocation in tomato (Lycopersicon esculentum Mill) to reduced ambient air humidity

The impact of low humidity in ambient air on water relations,nitrate uptake, and translocation of recently absorbed nitrogen,was investigated in 5-week-old tomato (Lycopersicon esculentumMill cv. Ailsa Craig) plants grown hydroponically in a completenutrient solution. Plants were subjected to dry air (relativehumidity 2–4% for 6 h. The transpiration rate increasedseveral-fold and the shoot water content decreased by almost20%, whereas root water content was unaffected. No effect onin vitro nitrate reductase (NR) activity was detected when usingan EDTA-contraining assay buffer. Replacement of EDTA with Mg²⁺revealed a significant decline in shoot NR activity, which suggestsphosphorylation of the enzyme during the stress treatment. Plantswere grown in a split-root system, in which one root half wasfed ¹⁵N-nitrate during the treatment, in order to determinenitrate uptake and translocation of recently absorbed nitrogenin the plants. Uptake of nitrate was substantially inhibited,but the proportion of absorbed ¹⁵N that was translocated tothe shoots was only slightly affected. In untreated plants,71% of the ¹⁵N recovered in roots had been retranslocated fromthe shoots, whereas in plants subjected to stress the deliveryof ¹⁵N from shoots to roots appeared to be completely inhibited.The data show that lowered humidity in air has significant effectson both uptake of nitrate as well as translocation of nitrogenwithin the plants. Some of these effects appear to be commonwith those observed in plants subjected to reduced water potentialsin the root environment and point to the possibility of theshoot water relations being highly influential on nitrogen uptakeand translocation. Key words: Air humidity, nitrate assimilation, nitrate reductase activity, nitrogen translocation, tomato, water stress     

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.The authors contributed equally to this work