Characterization of parvalbumin isotypes in white muscle from the barbel and expression during development

Parvalbumin isotypes PA II, PA III, PA IVa, and PA IVb were isolated by chromatography from trunk white muscle of barbel and physicochemically characterized. Electrospray ionization mass spectroscopy revealed that PA II has a lower molecular weight than the other isotypes and that PA IVa and PA IVb each consist of two subforms. Isotype distribution was studied by polyacrylamide gel electrophoresis. In adult fish, the total parvalbumin titre decreased and the isotype distribution varied from the anterior to the posterior myotomes. In the course of barbel development, the total parvalbumin titre increased rapidly as fish standard length increased from 1·3 to 5 cm; then sloped down gently as the length increased to 60 cm. At least six parvalbumin isotypes were identified, three of which are different forms (a, b, and c) of PA II. These three forms were present together at the larval stage, but PA IIc and chiefly PA IIb appeared as early isotypes, contrary to PA IIa which was present until the adult period. Later PA IVb accounted for up to 90% of the total parvalbumin content; PA III and PA IVa are minor adult isotypes. Temporal and spatial variations in the total parvalbumin titre and in the differential expression of barbel parvalbumin isotypes very likely reflected the functional requirements of the fish axial musculature according to fish size and myotome location. Physiologically, the larval isotypes could promote faster relaxation of fast fibres than the adult isotypes, and hence favour shorter contraction times.     

The role of the cell wall in the mechanism of lead and zinc tolerance in Anthoxanthum odoratum L

The respiration rate and viability of cultured cells and protoplasts isolated from two clones of Anthoxanthum odoratum tolerant to both zinc and lead were unaffected by the presence of zinc. Although intact cells were largely unaffected by the presence of lead, protoplasts isolated from cultured cells were susceptible, showing a reduced respiration rate and a high mortality. In contrast cultured cells and protoplasts of non-tolerant clones of A. odoratum were susceptible to both zinc and lead. The results provide direct evidence that in A. odoratum the cell wall is part of the mechanism of tolerance to lead, but not to zinc.     

Hatching plasticity in the tropical gastropod Nerita scabricosta

Hatching plasticity has been documented in diverse terrestrial and freshwater taxa, but in few marine invertebrates. Anecdotal observations over the last 80 years have suggested that intertidal neritid snails may produce encapsulated embryos able to significantly delay hatching. The cause for delays and the cues that trigger hatching are unknown, but temperature, salinity, and wave action have been suggested to play a role. We followed individual egg capsules of Nerita scabricosta in 16 tide pools to document the variation in natural time to hatching and to determine if large delays in hatching occur in the field. Hatching occurred after about 30 d and varied significantly among tide pools in the field. Average time to hatching in each pool was not correlated with presence of potential predators, temperature, salinity, or pool size. We also compared hatching time between egg capsules in the field to those kept in the laboratory at a constant temperature in motionless water, and to those kept in the laboratory with sudden daily water motion and temperature changes. There was no significant difference in the hatching rate between the two laboratory treatments, but capsules took, on average, twice as long to hatch in the laboratory as in the field. Observations of developing embryos showed that embryos in the field develop slowly and continuously until hatching, but embryos in the laboratory reach the hatching stage during the first month of development and remain in stasis after that. Instances of hatching plasticity in benthic marine invertebrates, like the one in N. scabricosta, could greatly enhance our ability to investigate the costs and benefits of benthic versus planktonic development, a long‐standing area of interest for invertebrate larval biologists.     

Incidence and Risk Factors for Severe Bacterial Infections in People Living with HIV. ANRS CO3 Aquitaine Cohort, 2000–2012

Severe non-AIDS bacterial infections (SBI) are the leading cause of hospital admissions among people living with HIV (PLHIV) in industrialized countries. We aimed to estimate the incidence of SBI and their risk factors in a large prospective cohort of PLHIV patients over a 13-year period in France. Patients followed up in the ANRS CO3 Aquitaine cohort between 2000 and 2012 were eligible; SBI was defined as a clinical diagnosis associated with hospitalization of ≥48 hours or death. Survival analysis was conducted to identify risk factors for SBI.Total follow-up duration was 39,256 person-years [PY] (31,370 PY on antiretroviral treatment [ART]). The incidence of SBI decreased from 26.7/1000 PY [95% CI: 22.9–30.5] over the period 2000–2002 to 11.9/1000 PY [10.1–13.8] in 2009–2012 (p <0.0001). Factors independently associated to increased risk of SBI were: plasma HIVRNA>50 copies/mL (Hazard Ratio [HR] = 5.1, 95% Confidence Interval: 4.2–6.2), CD4 count <500 cells/mm³ and CD4/CD8 ratio <0.8 (with a dose-response relationship for both markers), history of cancer (HR = 1.4 [1.0–1.9]), AIDS stage (HR = 1.7 [1.3–2.1]) and HCV coinfection (HR = 1.4, [1.1–1.6]). HIV-positive patients with diabetes were more prone to SBI (HR = 1.6 [0.9–2.6]). Incidence of SBI decreased over a 13-year period due to the improvement in the virological and immune status of PLHIV on ART. Risk factors for SBI include low CD4 count and detectable HIV RNA, but also CD4/CD8 ratio, HCV coinfection, history of cancer and diabetes, comorbid conditions that have been frequent among PLHIV in recent years.     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

Métabolisme des indolamines dans l'organe pinéal de Lacerta (Reptiles,Lacertiliens)

Résumé Les sites d'incorporation du 5-HTP-³H (5-hydroxytryptophane-³H) et de rétention de ses dérivés ont été recherchés dans l'organe pinéal de Lacerta vivipara (J.).Les animaux reçoivent au cours de l'après-midi (aux environs de 15h, le 5/VII, T°=21 à 28°C) une injection intrapéritonéale de 5-HTP-³H. Ils sont sacrifiés après 2, 10, 15, 21, 30 minutes, 1, 2, 3, 4, 5, 10, 15 heures et 1, 2, 3, 4, 5, 6, 7, jours.De l'étude qualitative et quantitative, il ressort que des réactions radioautographiques spécifiques apparaissent dans les photorécepteurs rudimentaires sécrétoires (=PRS) entre 2 et 10 minutes. La concentration de radioactivité atteint un maximum au niveau des PRS, après 5 heures. Puis l'intensité du marquage diminue pour disparaître entre 2 et 3 jours. La radioactivité des PRS est toujours concentrée dans les régions de grains denses de sécrétion protéique (500 à 3400 Å) d'origine golgienne. Les autres compartiments cellulaires sont nettement moins radioactifs.Les autres cellules (interstitielles de type épendymaire; nerveuses sensorielles et probablement noradrénergiques) de l'organe pinéal et les régions voisines du cerveau ne retiennent pas significativement les composés radioactifs.Ces résultats et ceux antérieurs, cytophysiologiques et biochimiques, montrent le rôle important des PRS dans la biosynthèse et le stockage de la sérotonine. D'autres conclusions concernant la biosynthèse et le métabolisme des indolamines ne seront exposées que dans une étude ultérieure où l'incorporation du 5-HTP-³H est envisagée dans les conditions expérimentales. Le turnover des indolamines semble plus lent chez Lacerta que chez les Mammifères.

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Biosynthesis and metabolism of indolamines in the pineal organ of Lacerta (Reptiles, Lacertilians)I. Selective incorporation of ³H-5-HTP and retention of its derivatives in the secretory rudimentary photoreceptor cells

Summary The sequence of incorporation and utilization of ³H-5-hydroxytryptophan (³H-5-HTP) has been examined in the pineal organ of adult lizard (Lacerta vivipara J.).Each animal was given ³H-5-HTP in the afternoon (about 15.00 h in July, T°=21–28°C). The lizards were sacrificed 2, 10, 15, 21, 30 minutes, 1, 2, 3, 4, 5, 10, 15 hours and 1, 2, 3, 4, 5, 6, 7 days after administration.The cellular distribution of radioactivity was studied by qualitative and quantitative radioautography. The radioautographs show selective labelling, appearing in the SRP (secretory rudimentary photoreceptors) after 2–10 minutes. The labelling reaches a maximum over the SRP within 5 hours and subsequently disappears between 2 and 3 days. These radioautographic reactions are always most concentrated in the regions of proteinaceous secretory granules (500–3400 Å) originating from the Golgi complex. The other components of the SRP account only for a minor fraction of the labelling. Supporting and nervous (sensory and probably noradrenergic) cells of the pineal organ, as well as neighbouring brain structures do not retain significantly the radioactive compounds.These results, correlated with previous cytophysiological and biochemical studies, are consistant with an important role of SRP in the biosynthesis and storage of serotonin. Conclusions concerning the biosynthesis and metabolism of indolamines are presented in a subsequent paper where the incorporation of ³H-5-HTP is studied under experimental conditions. The turnover of indolamines seems to be slower in Lacerta than in mammals.

Les auteurs remercient vivement M. Robert Meiniel pour sa collaboration efficace dans la collecte des Lézards, ce qui nous a permis de travailler sur des lots homogènes.     

Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase

Summary The URA7 gene of Saccharomyces cerevisiae encodes CTP synthetase (EC 6.3.4.2) which catalyses the conversion of uridine 5-triphosphate to cytidine 5-triphosphate, the last step of the pyrimidine biosynthetic pathway. We have cloned and sequenced the URA 7 gene. The coding region is 1710 by long and the deduced protein sequence shows a strong degree of homology with bacterial and human CTP synthetases. Gene disruption shows that URA7 is not an essential gene: the level of the intracellular CTP pool is roughly the same in the deleted and the wild-type strains, suggesting that an alternative pathway for CTP synthesis exists in yeast. This could involve either a divergent duplicated gene or a different route beginning with the amination of uridine mono- or diphosphate.     

Distribution of arrestin-like protein and beta-subunit of GTP-binding proteins in quail choroid plexuses.

Monoclonal antibodies (Mabs) directed against retinal arrestin (S-antigen) were used to detect and characterize this protein in choroid plexus (CP) of quails maintained during eight days, either under long-day photoperiods or in constant darkness. Immunocytochemistry and Western blotting confirmed the presence and the distribution of an arrestin-like protein in quail CP. Arrestin-like immunoreactivities in CP were compared with those obtained with Mabs to beta 36-subunit of G proteins (G beta), alpha-subunit of transducin and rhodopsin. Rhodopsin-like and transducin-like proteins could not be detected in choroidal cells, whereas intense positive reactions were observed with anti-G beta and anti-arrestin Mabs. The strongest immunoreactivities were found in choroidal ependymocytes of the lateral and IIIrd ventricles. In CP epithelial cells lining the IVth ventricle, very weak or no immunoreactivity could be detected with Mabs to arrestin, while Mab against G beta subunit always provided a positive reaction. In quails maintained in constant darkness, arrestin- and G beta-immunoreactivities of CP epithelial cells displayed changes in cellular distribution and intensity (decrease or disappearance of the immunoreactions). The strong arrestin-like immunoreaction located in the apical region of ependymocytes suggests the preferential association of the protein with choroidal microvilli and a possible role in cerebrospinal fluid production assumed by CP cells.