Regulation of the nitrate-reducing system enzymes in wild-type and mutant strains of Chlamydomonas reinhardii

Summary Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H-nitrate reductase and its associated activities (NAD(P)H-cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301, 102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase activity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301, 102, 104, and 307, when properly induced, exhibit an NAD(P)H-cytochrome c reductase distinguishable electrophoretically from contitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.     

Preparation of new C-nucleosides by intramolecular dehydration of 2-pentahydroxypentyl-4,5,6,7-tetrahydroindol-4-ones

Acid-catalysed dehydration of the polyhydroxyalkyl chain of 6,6-dimethyl-2-(d-gluco-pentitol-l-yl)-4,5,6,7-tetrahydroindol-4-one and of 6,6-dimethyl-2-(d-manno-pentitol-l-yl)-4,5,6,7-tetrahydroindol-4-one gave 2-α-d-arabinofuranosyl-6,6-dimethyl-4,5,6,7-tetrahydroindol-4-one (3). In a similar way, 2-β-d-lyxopyranosyl-6,6-dimethyl-4,5,6,7-tetrahydroindol-4-one (8) and 2-β-d-lyxopyranosyl-4,5,6,7-tetrahydroindol-4-one (9) were obtained by dehydration of 6,6-dimethyl-2-(d-galacto-pentitol-l-yl)-4,5,6,7-tetrahydroindol-4-one and 2-(d-galacto-pentitol-l-yl)-4,5,6,7-tetrahydroindol-4-one, respectively. The structures of the new C-nucleosides described (3, 8, and 9) were elucidated by chemical and physical methods.     

Trophic control of biogenic carbon export in Bransfield and Gerlache Straits, Antarctica

Size-fractionated chlorophyll a and photosynthetic carbon incorporation,microbial oxygen production and respiration and particulatevertical flux were measured in January 1996 at three regions,characterized by distinct hydrographic fields and planktoniccommunities, of the Antarctic Peninsula: (1) a diatom-Phaeocystissp., dominated community associated with the relatively stratifiedwaters of the Gerlache Strait, (2) a nanoplankton-Cryptomonassp. dominated assemblage at the Gerlache–Bransfield confluence;and (3) a nano- and picoplankton community in mixed waters ofthe Bransfield Strait. Despite the marked differences in bothcommunity structure and total phytoplankton biomass and primaryproduction, and against predictions from models about trophiccontrol of C export, the lowest respiration rates were measuredat Bransfield (pico- and nanoplankton), and no difference wasobserved between the Gerlache (large diatoms) and Bransfieldstations in relative vertical particle flux (6.4 vs. 5.1 % ofsuspended C; 14.9 vs. 10.4 % of net community production, respectively).Growth and loss rates of the phytoplankton population studiedfor each community indicate that microbial populations can beexplained by in situ growth, but spatial (diatom-Phaeocystissp., bloom) and temporal (diatom-Phaeocystis sp. bloom and nanoplanktoncommunities) scales of study were shown to be insufficient foraddressing the coupling between primary production and biogeniccarbon export, especially after the appreciation of the accumulationof dissolved organic carbon in the water column. This wouldexplain the unexpected results and highlights the necessityof including the mechanisms controlling accumulation and consumptionof dissolved organic matter into conceptual models about thetrophic control of C export.     

Molecular characterization of porphyrias in Italy: a diagnostic flow-chart.

The porphyrias are disorders associated with inherited or acquired enzyme deficiencies in the heme biosynthetic pathway. The differential diagnosis is often difficult since the phenotype is very similar in some forms and the biochemical tests are not commonly available. Here we provide an update on the molecular diagnosis of porphyrias in Italy and a flow-chart to facilitate the identification of mutations in heme biosynthetic genes. The molecular analysis has allowed us to identify the molecular defect underlying the disease in 66 probands with different porphyrias [acute intermittent porphyria (AIP), variegate porphyria (VP), porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP)]. No Italian patients with defects in coproporphyrinogen oxidise (CPOX) gene, responsible for hereditary coproporphyria (HCP), have been detected. The molecular characterization has been extended to 115 relatives with the identification of 55 asymptomatic mutation carriers and 60 normal subjects. We have so far identified 50 different mutations among 4 genes associated with the most common porphyrias showing a high molecular heterogeneity: 22 in the hydroxymethylbilane synthase (HMBS) gene (AIP), 7 in the protoporphyrinogen oxidase (PPOX) gene (VP), 16 in the uroporphyrinogen decarboxylase (UROD) gene (PCT) and 5 in the ferrochelatase (FECH) gene (EPP). Among the 50 molecular defects, 29 seem to be restricted to the Italian population.     

Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency.

Leigh disease associated with cytochrome c oxidase deficiency (LD[COX-]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients. Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.     

Mental retardation in heterozygotes for the fragile-X mutation: evidence in favor of an X inactivation-dependent effect.

The still debated question of whether the expression of mental retardation in heterozygous carriers of the Martin-Bell syndrome is influenced by X inactivation has been investigated in a group of phase-known double heterozygotes for the FRA-X mutant and the G6PD Mediterranean variant. In these individuals, the number of somatic cells (fibroblasts or red cells) with an active FRA-X chromosome could be assessed through the G6PD phenotype at the single-cell level. The data reported indicate a significant inverse correlation between the IQ level (as measured by the Wechsler-Bellevue test) and the percentage of fibroblast cells with an FRA-X active chromosome. In contrast, no significant correlation was found when the IQ level and red cell data were compared, thus suggesting the occurrence of somatic selection against hematopoietic stem cells with an active FRA-X chromosome.     

Assignment of the slow troponin T (TNNT1) gene to chromosome 19 using polymerase chain reaction

Summary Confirmation that the slow troponin (TNNT1) gene lies on chromosome 19 has been obtained by means of the polymerase chain reaction and somatic cell hybrids.     

Aspects of neural plasticity in the central nervous system—VI. Studies of the effects of gangliosides on brain metabolic lesions

The effects of gangliosides have been studied in two models of metabolic insult (insulin-induced hypoglycemia and transient forebrain ischemia) of the central nervous system. In the severe hypoglycemia experiments lactate extracellular fluid levels were evaluated by means of a microdialysis probe implanted in the frontoparietal cortex. Ganglioside GM1, given either peripherally (10 mg/kg, i.p.) or intracerebrally (2 × 10⁻⁴ M, via the microdialysis probe) 2 h before insulin injection, was able to reduce the decay of the perfusate levels of lactate induced by the insulin injection. In the same animal model peripheral, but not central, administration of GM1 reduced the hypoglycemia-induced increase of cerebral blood flow and increased the survival time observed after the insulin injection. In the experiments on transient forebrain ischemia, a GM1 derivative, AGF2 (5 mg/kg/day, i.p.), was administered chronically, starting 5 days before or the day after the ischemic insult. With both treatment schedules a similar protective effect was observed in a neurological test battery and in the step-through latency in a passive avoidance test.     

Aspects of neural plasticity in the central nervous system—V. Studies on a model of transient forebrain ischemia in male Sprague-Dawley rats

A morphological and functional characterization of the four-vessel occlusion model of transient (30 min) forebrain ischemia has been carried out. The rats were classified as fully ischemic when an isoelectric pattern of electroencephalographic activity was present within 5 min of the occlusion of carotid arteries. Otherwise they were considered as partially ischemic rats. The modifications of cerebral blood content during and after the ischemic insult were assessed by a histochemical method which visualizes red blood cells in cerebral vessels. The periods of increase and decrease of red blood cell content were found to correspond to previous reports of post-ischemic hyper- and hypoperfusion. Neuronal damage was assessed by a quantitative analysis of Nissl stained preparations of cingulate cortex, dorsal hippocampus and striatum. The signs of morphological damage were quantified by means of computer-assisted image analysis of Nissl preparations. The highest vulnerability to the ischemic insult was demonstrated in the pyramidal layer of the hippocampal CA1 field and in the lateral striatum. Arterial blood pressure measurements were performed during the ischemic and post-ischemic periods, demonstrating a peak increase of arterial blood pressure within 2 min after carotid artery occlusion, followed by a slow decrease towards basal levels during the ischemic period and a full recovery within 15 min of reperfusion. Ischemic rats were tested in a neurological test battery and in a passive avoidance task. While a full recovery of the relatively simple tasks of the neurological test battery was attained within 14 days of reperfusion, a highly significant impairment of passive avoidance behavior was still present 15 days after the ischemic insult. Finally, a discriminant analysis was applied to separate, on the basis of non-invasive techniques (neurological tests and hot plate), the group of completely ischemic rats from that of partially ischemic rats.