Identification of PEX10, the gene defective in complementation group 7 of the peroxisome-biogenesis disorders.

The peroxisome-biogenesis disorders (PBDs) are a group of genetically heterogeneous, lethal diseases that are characterized by neuronal, hepatic, and renal abnormalities; severe mental retardation; and, in their most severe form, death within the 1st year of life. Cells from all PBD patients exhibit decreased import of one or more classes of peroxisome matrix proteins, a phenotype shared by yeast pex mutants. We identified the human orthologue of yeast PEX10 and observed that its expression rescues peroxisomal matrix-protein import in PBD patients'' fibroblasts from complementation group 7 (CG7). In addition, we detected mutations on both copies of PEX10 in two unrelated CG7 patients. A Zellweger syndrome patient, PBD100, was homozygous for a splice donor-site mutation that results in exon skipping and loss of 407 bp from the PEX10 open reading frame. A more mildly affected neonatal adrenoleukodystrophy patient was a compound heterozygote for a missense mutation in the PEX10 zinc-binding domain, H290Q, and for a nonsense mutation, R125ter. Although all three mutations attenuate PEX10 activity, the two alleles detected in the mildly affected patient, PBD052, encode partially functional PEX10 proteins. PEX10-deficient PBD100 cells contain many peroxisomes and import peroxisomal membrane proteins but do not import peroxisomal matrix proteins, indicating that loss of PEX10 has its most pronounced effect on peroxisomal matrix-protein import.     

PCR for Detection of Shigella spp. in Mayonnaise

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.     

Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes in Escherichia coli

Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF), were grown on minimal media in batch fermentations. The strain lacking both PKs showed a 28% decrease on its specific growth rate when compared to the wild type. However, protein and CO2 yields did not change. Using radioactive 1-C14 glucose and collecting the CO2 produced by the cultures, it was found that the mutant lacking both pyruvate kinases, metabolized glucose mainly through the pentose pathway (PP). The increased participation of the PP in glucose metabolism in this strain, was also reflected on the levels of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases.Copyright 1998 John Wiley & Sons, Inc.     

Mitochondrial Membrane Potential and DNA Stainability in Human Sperm Cells: A Flow Cytometry Analysis with Implications for Male Infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.     

Free Amino Acid Composition of Phloem Sap and Growing Fruit of Lycopersicon esculentum

Pure phloem sap of tomato leaves was collected by stylectomy.Glutamine and glutamate were the predominant free amino acidstranslocated by the phloem stream. In developing fruits glutaminecontent increased significantly, reaching 35% of the total freeamino acids. Comparison in the amino acid composition betweenthe two tissues are discussed. (Received October 6, 1997; Accepted January 27, 1998)     

Evolution of the Structure and Chromosomal Distribution of Histidine Biosynthetic Genes

A database of more than 100 histidine biosynthetic genes from different organisms belonging to the three primary domains has been analyzed, including those found in the now completely sequenced genomes of Haemophilus influenzae, Mycoplasma genitalium, Synechocystis sp., Methanococcus jannaschii, and Saccharomyces cerevisiae. The ubiquity of his genes suggests that it is a highly conserved pathway that was probably already present in the last common ancestor of all extant life. The chromosomal distribution of the his genes shows that the enterobacterial histidine operon structure is not the only possible organization, and that there is a diversity of gene arrays for the his pathway. Analysis of the available sequences shows that gene fusions (like those involved in the origin of the Escherichia coli and Salmonella typhimurium hisIE and hisB gene structures) are not universal. In contrast, the elongation event that led to the extant hisA gene from two homologous ancestral modules, as well as the subsequent paralogous duplication that originated hisF, appear to be irreversible and are conserved in all known organisms. The available evidence supports the hypothesis that histidine biosynthesis was assembled by a gene recruitment process.     

Long- and short-term phosphate deprivation in bean roots: plasma membrane lipid alterations and transient stimulation of phospholipases

In a long-term experiment bean (Phaseolus vulgaris L.) seedlings were grown for 18 days in hydroponics in either phosphate-sufficient (+P) or phosphate-deficient (-P) nutrient solutions. Phosphate deprivation halved the phosphorous content of roots. In plasma membrane (PM) fractions isolated from -P roots the phospholipid (PL) level was reduced from 35 to 21 mol%, while PL composition and degree of unsaturation were hardly altered. Digalactosyldiacylglycerol (DGDG) accumulated up to 26% of total PM lipids, replacing PL to a large extent. Molecular species and fatty acid compositions of DGDG in root PM were different compared to DGDG present in the -P plastids. In a short-term study, bean seedlings were grown for 18 days in hydroponics with a complete nutrient solution containing phosphate and then incubated in a -P medium for increasing time ranging from 1 up to 96 h. At the end of the starvation period phosphorous content of -P roots was reduced by 30% compared to +P ones. An activation of phospholipase D and phospholipase C was observed after 1 and 2h of phosphate deprivation, respectively. Maximal phosphatidic acid accumulation was detected after 4h of phosphate deprivation, when also DGDG started to accumulate in PM of bean roots. The fatty acid composition of PLD-derived phosphatidylbutanol resembled that of phosphatidylcholine.     

Seed bank of seasonally flooded grassland: experimental simulation of flood and post-flood

Wetland seed banks comprise the propagules of plant species that have species-specific germination requirements for germination in either flooded or dry conditions. At the community level, wetland structure and succession during and after a seasonal flooding event depends upon the early life-history requirements of species, including germination under flooded and dry conditions. We examined the effects of simulated flood and post-flood scenarios on seedling emergence from a seed bank of seasonally flooded grassland in the Pantanal, Brazil. Field samplings were conducted in both wet and dry seasons, both of which were subject to flood and post-flood conditions. A total of 70 species emerged from the seed bank, dominated by Poaceae and Cyperaceae. Sixteen species were exclusive to the wet and one exclusive to the dry season. The richness of perennial species was higher under flood conditions, while the richness of annuals was greater post-flood. In general, the aquatic and amphibious species exhibited a significant germination response to flooding. Terrestrial species only germinated in post-flood conditions, with higher richness in the dry season. Four species had high seedling abundance in both treatments. The capacity of regeneration by seeds is high in these grasslands and can be increased by seasonal flooding and drawdown. In these seasonally flooded grasslands, we observed three main germination strategies: under flooded conditions, aquatic and amphibious species; post-flood conditions, an explosion of annual amphibious and terrestrial species; and in moist soil, perennial terrestrial species. The differential responses to flooding versus post-flood conditions help to maintain the structure and species richness in the community over time.     

Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photo-oxidative damage

Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.