Structural and functional study in the rhizosphere of Oryza sativa L. plants growing under biotic and abiotic stress.

AIMS: A structural and functional study has been carried out in the rice production area of the Guadalquivir marshes in southern Spain aiming to increase knowledge of rice rhizosphere structure and function for further application on integrated management practices. METHODS AND RESULTS: Rhizosphere bacterial structure (analysis of 16S rRNA partial sequences from total soil DNA), metabolic diversity (analysed by Biolog FF for fungal community and GN for microbial community) and a screening for putative plant growth-promoting rhizobacteria (PGPR) to identify potential isolates for development of local biofertilizers, and biodiversity of culturable micro-organisms (analysis of 16S rRNA partial sequences) from four areas differing in salinity and Magnaporthe oryzae incidence in two moments of the crop cycle were studied. Results indicate that the dominant taxon in libraries from the four areas was Proteobacteria. Metabolic diversity was higher in areas affected only by salinity or incidence of Magnaporthe than in the control or area affected by both stresses. It seems that rice plants selected, in their rhizosphere, micro-organisms able to affect plant hormonal balance under all conditions, and this activity relied in different bacterial genera depending on the environmental stress. CONCLUSIONS: Bacterial genera for each stress, as well as generalist strains, were found present in all the studied areas. Potential molecular markers and taxonomic markers (Sphingobacteria for salt and Thermococci for Magnaporthe) of the different stress situations have been highlighted, and Class Verrucomicrobiae could be a marker for nonstressed areas. In addition, putative PGPR strains isolated in this study could be used as biofertilizers. SIGNIFICANCE AND IMPACT OF THE STUDY: Rice paddies are great ecologically important ecosystems. The results are very relevant as they may be included in the process of rice production, improving crop conditions with less environmental impact.

Bacterial siderophores efficiently provide iron to iron-starved tomato plants in hydroponics culture.

Iron is one of the essential elements for a proper plant development. Providing plants with an accessible form of iron is crucial when it is scant or unavailable in soils. Chemical chelates are the only current alternative and are highly stable in soils, therefore, posing a threat to drinking water. The aim of this investigation was to quantify siderophores produced by two bacterial strains and to determine if these bacterial siderophores would palliate chlorotic symptoms of iron-starved tomato plants. For this purpose, siderophore production in MM9 medium by two selected bacterial strains was quantified, and the best was used for biological assay. Bacterial culture media free of bacteria (S) and with bacterial cells (BS), both supplemented with Fe were delivered to 12-week-old plants grown under iron starvation in hydroponic conditions; controls with full Hoagland solution, iron-free Hoagland solution and water were also conducted. Treatments were applied twice along the experiment, with a week in between. At harvest, plant yield, chlorophyll content and nutritional status in leaves were measured. Both the bacterial siderophore treatments significantly increased plant yield, chlorophyll and iron content over the positive controls with full Hoagland solution, indicating that siderophores are effective in providing Fe to the plant, either with or without the presence of bacteria. In summary, siderophores from strain Chryseobacterium C138 are effective in supplying Fe to iron-starved tomato plants by the roots, either with or without the presence of bacteria. Based on the amount of siderophores produced, an effective and economically feasible organic Fe chelator could be developed.

Russell-Silver syndrome due to paternal H19/IGF2 hypomethylation in a patient conceived using intracytoplasmic sperm injection.

Epigenetic alterations at several maternal loci have been associated with imprinting disorders in children conceived using assisted reproductive technologies. To date, epimutations at paternal loci have been observed in the spermatozoa of infertile men, but there is little evidence of paternal epimutations in babies conceived using assisted reproductive treatment. This is a report of a female infant with classic Russell-Silver Syndrome (RSS) who was conceived using intracytoplasmic injection of spermatozoa obtained from testicular aspiration. Methylation studies revealed hypomethylation of the paternally derived H19/IGF2 locus. As far as is known, this is the second assisted reproduction treatment-conceived patient with classic RSS and this epigenotype. This case provides further evidence that epimutations affecting paternal alleles might be associated with assisted reproductive treatment.

A WT1 antisense oligonucleotide inhibits proliferation and induces apoptosis in myeloid leukaemia cell lines.

The response of the CML-BC cell line, K562, the myelomonocytic cell line MM6 and the promyelocytic leukaemia cell line HL-60, to a 15 mer WT1 antisense oligonucleotide, targeted to the translation initiation site of the WT1 mRNA was examined. K562 cells exposed to 0.4 microM antisense oligonucleotide showed markedly reduced proliferation which was associated with reduced cell viability. Sense, scrambled and mutant antisense oligonucleotides had no effect on the proliferation of K562 cells. MM6 cells exposed to 0.4 microM antisense oligonucleotide also showed significantly reduced cellular proliferation which was also accompanied by loss of cell viability. In the K562 and MM6 antisense cultures that exhibited reduced cell viability, both DNA fragmentation and morphological features consistent with apoptosis could be identified. In contrast the growth of HL-60 cells was unaffected by exposure to 0.4 microM antisense oligonucleotide. In each of the cell lines examined, WT1 antisense oligonucleotide abrogated WT1 protein expression, and analysis of WT1 coding sequence in these cells showed that no oncogenic point mutations in the gene were present. We propose therefore that in some myeloid leukaemia cell lines, the expression of a normal WT1 protein is necessary for cell proliferation and that it plays a role in maintaining the viability of some leukaemia cells.

Purification and properties of mouse stomach aldehyde dehydrogenase. Evidence for a role in the oxidation of peroxidic and aromatic aldehydes.

The major isozyme of aldehyde dehydrogenase in mouse stomach, AHD-4, has been purified to homogeneity and characterized with a range of aldehyde substrates at pH 7.4. The enzyme was a dimer with a subunit size of 65 kDa. Using V/Km values as an indication of substrate efficacy, aromatic aldehydes were the preferred substrates. The enzyme used either NAD+ or NADP+ as cofactor, but showed a preference for NAD+. AHD-4 showed 'high-Km' properties with respect to acetaldehyde, but differed from the 'high-Km' liver mitochondrial enzyme (AHD-1), in that it was not a semialdehyde dehydrogenase. The enzyme was significantly active towards the peroxidic aldehyde, 4-hydroxynonenal, and may play a role in vivo in the detoxification of aromatic aldehydes and the aldehyde products of lipid peroxidation.

Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis.

The Drosophila slit gene (sli) encodes a secreted leucine-rich repeat-containing protein (slit) expressed by the midline glial cells and required for normal neural development. A putative human sli homolog, SLIT1, has previously been identified by EST database scanning. We have isolated a second human sli homolog, SLIT2, and its murine homolog Slit2. Both SLIT1 and SLIT2 proteins show approximately 40% amino acid identity to slit and 60% identity to each other. In mice, both genes are expressed during CNS development in the floor plate, roof plate and developing motor neurons. As floor plate represents the vertebrate equivalent to the midline glial cells, we predict a conservation of function for these vertebrate homologs. Each gene shows additional but distinct sites of expression outside the CNS suggesting a variety of functions for these proteins.

Mutation analysis of the WT1 gene in sporadic childhood leukaemia.

The 10 coding exons of the WT1 gene, from 39 bp upstream of the translation initiation codon to 12 bp downstream of the stop codon, were examined for point mutations in a panel of 48 sporadic childhood acute leukaemias using the single-stranded conformational polymorphism (SSCP) assay. The panel included 33 cases of acute lymphocytic leukaemia and 15 cases of acute myeloid leukaemia. This is the first study in which sporadic childhood leukaemias have been examined for WT1 point mutations across the entire coding region of the WT1 gene, however, no tumorigenic point mutations or small deletions or insertions could be identified in these patients. A previously described polymorphism in exon 7, resulting in an A to G transition in an arginine codon, was observed at a frequency of 21.5%, equivalent to that seen in the normal population. This study suggests that point mutations in the coding regions of the WT1 occur infrequently in leukaemias of childhood.

Silencing of CDKN1C (p57KIP2) is associated with hypomethylation at KvDMR1 in Beckwith-Wiedemann syndrome.

CONTEXT: Beckwith-Wiedemann syndrome (BWS) arises by several genetic and epigenetic mechanisms affecting the balance of imprinted gene expression in chromosome 11p15.5. The most frequent alteration associated with BWS is the absence of methylation at the maternal allele of KvDMR1, an intronic CpG island within the KCNQ1 gene. Targeted deletion of KvDMR1 suggests that this locus is an imprinting control region (ICR) that regulates multiple genes in 11p15.5. Cell culture based enhancer blocking assays indicate that KvDMR1 may function as a methylation modulated chromatin insulator and/or silencer. OBJECTIVE: To determine the potential consequence of loss of methylation (LOM) at KvDMR1 in the development of BWS. METHODS: The steady state levels of CDKN1C gene expression in fibroblast cells from normal individuals, and from persons with BWS who have LOM at KvDMR1, was determined by both real time quantitative polymerase chain reaction (qPCR) and ribonuclease protection assay (RPA). Methylation of the CDKN1C promoter region was assessed by Southern hybridisation using a methylation sensitive restriction endonuclease. RESULTS: Both qPCR and RPA clearly demonstrated a marked decrease (86-93%) in the expression level of the CDKN1C gene in cells derived from patients with BWS, who had LOM at KvDMR1. Southern analysis indicated that downregulation of CDKN1C in these patients was not associated with hypermethylation at the presumptive CDKN1C promoter. CONCLUSIONS: An epimutation at KvDMR1, the absence of maternal methylation, causes the aberrant silencing of CDKN1C, some 180 kb away on the maternal chromosome. Similar to mutations at this locus, this silencing may give rise to BWS.

Splicing of exon 5 in the WT1 gene is disrupted in Wilms' tumour.

Using a reverse transcriptase polymerase chain reaction to examine alternate splicing at site I (exon 5) and site II (exon 9) in the Wilms' tumour suppressor gene, WT1, we found that in seven of the 10 Wilms' tumours examined, splicing at site I was disrupted. This is predicted to result in isoform imbalance in Wilms' tumours, with an increase in isoforms in which the 17 amino acids encoded by exon 5 are missing. These observations could not be explained by mutations or rearrangements in flanking introns. Disrupted alternate splicing of exon 5 may play a role in the aetiology of Wilms' tumour.

Overexpression of murine WT1 + / + and - / - isoforms has no effect on chemoresistance but delays differentiation in the K562 leukemia cell line.

The Wilms' tumor gene (WT1) encodes a zinc-finger transcription factor that is expressed as four distinct isoforms designated as, + / +, + / -, - / + and - / -. It is expressed in leukemic cells, and is proposed to play a role in their proliferation and differentiation. In this study we have shown that cell lines of the erythroleukemia, K562, overexpressing the murine + / + and - / - WT1 isoforms grow normally and do not exhibit altered responses to the induction of apoptosis by the reagents cisplatin and adriamycin, or to serum withdrawal. However, differentiation of K562 cells with 12-O-tetradecanoylphorbol 13-acetate, modeling aspects of megakaryopoiesis, was partially inhibited by the persistent expression of both the murine + / + and - / - WT1 isoforms. This finding suggests that WT1 plays a role in the regulation of hematopoietic differentiation and is consistent with an oncogenic role for WT1 in leukemogenesis.

Allelic imbalance at chromosome 1q21 in Wilms tumor.

The Wilms tumor suppressor gene 1, WT1, located on chromosome 11p13 is mutated in only a subset of Wilms tumors. Cytogenetic studies of Wilms tumors show that the most frequent structural anomalies after those affecting chromosome 11p are rearrangements of 1q, suggesting that there is a gene involved in Wilms tumor etiology in this region. The WT1 target sequence +P5 (D1S3309E), isolated using whole-genome polymerase chain reaction (PCR), binds all WT1 isoforms in vitro and has been mapped to 1q21-22. As +P5 may mark a 1q Wilms tumor gene, constitutional and tumor DNA from 33 Wilms tumor patients (36 tumors) was screened for allele imbalance using microsatellite markers from 1p21 to 1q44. Although no gross rearrangements of the +P5 region were found, this study demonstrates allele imbalance for 1q in 12% of patients (5/36 tumors), defining a smallest region of overlap at 1q21. This finding supports a role for 1q21 in Wilms tumorigenesis.

High frequency of t(12;21) in childhood acute lymphoblastic leukemia detected by RT-PCR.

Recently, a new recurrent translocation, t(12;21)(p13;q22), has been identified in B-cell lineage acute lymphoblastic leukemia (ALL). The translocation results in the fusion of two known genes, ETV6/TEL(12p13) and AML1(21q22), both of which have been shown to be involved in other hematological malignancies. The t(12;21) is virtually undetectable by routine cytogenetics, but the chimeric transcript ETV6-AML1 has been detected in childhood ALL by molecular techniques in up to 36% of cases, making it the most common genetic abnormality in these patients. It has been shown to be associated with a B-precursor phenotype and an excellent prognosis. We tested 66 diagnostic pediatric ALL samples by reverse transcription polymerase chain reaction (RT-PCR) and found evidence of the t(12;21) in 22 (33%). None of these had previously been identified as harboring the t(12;21), although six had karyotypic abnormalities involving either 12p13 or 21q22. ETV6-AML1 expression defined a subgroup of patients characterised by an age of between two and 12 years, B-lineage immunophenotype and non-hyperdiploid DNA content. Our data further support the importance of molecular diagnostic methods in the identification of clinically distinct subgroups of patients with ALL.

Biochemical and genetic studies on mouse aldehyde dehydrogenases.

Aldehyde dehydrogenase (AHD) exists as isozymes which are differentially distributed among tissues and subcellular fractions of mouse tissues. Genetic variants for liver mitochondrial (AHD-1) and cytoplasmic (AHD-2) isozymes have been used to map the responsible loci (Ahd-1 and Ahd-2) on chromosomes 4 and 19 respectively. Evidence for a regulatory locus (Ahd-3r) controlling the inducibility of the mouse liver microsomal isozyme (AHD-3) has also been obtained. More recent studies have described genetic and biochemical evidence for three additional AHD isozymes: a stomach isozyme (AHD-4); another liver mitochondrial enzyme (AHD-5); and a testis isozyme (AHD-6). Genetic analyses have indicated that AHD-4 and AHD-6 are encoded by distinct but closely linked loci on the mouse genome (Ahd-4 and Ahd-6), which segregate independently of Ahd-1 and Ahd-2. Liver mitochondrial isozymes, AHD-1 and AHD-5, have been purified to homogeneity using affinity chromatography. The very high affinity of AHD-5 for acetaldehyde suggests that this enzyme is predominantly responsible for acetaldehyde oxidation in mouse liver mitochondria.

Microbe associated molecular patterns from rhizosphere bacteria trigger germination and Papaver somniferum metabolism under greenhouse conditions.

Ten PGPR from different backgrounds were assayed on Papaver somniferum var. Madrigal to evaluate their potential as biotic elicitors to increase alkaloid content under the rationale that some microbe associated molecular patterns (MAMPs) are able to trigger plant metabolism. First, the 10 strains and their culture media at two different concentrations were tested for their ability to trigger seed germination. Then, the best three strains were tested for their ability to increase seedling growth and alkaloid levels under greenhouse conditions. Only three strains and their culture media enhanced germination. Then, germination enhancing capacity of these best three strains, N5.18 Stenotrophomonas maltophilia, Aur9 Chryseobacterium balustinum and N21.4 Pseudomonas fluorescens was evaluated in soil. Finally, the three strains were applied on seedlings at two time points, by soil drench or by foliar spray. Photosynthesis was measured, plant height was recorded, capsules were weighted and alkaloids analyzed by HPLC. Only N5.18 delivered by foliar spray significantly increased plant height coupled to an increase in total alkaloids and a significant increase in opium poppy straw dry weight; these increases were supported by a better photosynthetic efficiency. The relative contents of morphine, thebaine, codeine and oripavine were affected by this treatment causing a significant increase in morphine coupled to a decrease in thebaine, demonstrating the effectivity of MAMPs from N5.18 in this plant species. Considering the increase in capsule biomass and alkaloids together with the acceleration of germination, strain N5.18 appears as a good candidate to elicit plant metabolism and consequently, to increase productivity of Papaver somniferum.

Liver cytosolic aldehyde dehydrogenases from "alcohol-drinking" and "alcohol-avoiding" mouse strains: purification and molecular properties.

Liver cytosolic aldehyde dehydrogenases (AHD-2) have been isolated in a highly purified state from "alcohol-drinking" (C57BL/6J) and "alcohol-avoiding" (DBA/2J) strains of mice. The purified enzymes were resolved into three major and one minor form of activity by isoelectric focusing (IEF) techniques and showed similar zymogram patterns. The enzymes had identical subunit sizes on SDS-polyacrylamide gels: 53,000. Gel exclusion chromatography, using Ultrogel AcA34, indicated that the enzymes were dimers. The enzymes exhibited biphasic kinetic characteristics and were readily distinguished from each other. The purified forms of AHD-2 from C57BL/6J and DBA/2J mice exhibited two apparent Km values in each case: 10 microM/100 microM and 30 microM/330 microM respectively. AHD-2 exhibited a broad pH optimum in the range 7.0-9.0 and was very sensitive towards disulphuram inhibition, with 50% inhibition occurring at 0.17 microM. The kinetic results support proposals that AHD-2 may be the primary enzyme for oxidizing acetaldehyde during ethanol oxidation in vivo.

Mouse mitochondrial aldehyde dehydrogenase isozymes: purification and molecular properties.

Aldehyde dehydrogenase isozymes (AHD-1 and AHD-5) have been isolated in a highly purified state from extracts of mouse liver mitochondria. The enzymes have distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: AHD-1, 63,000; AHD-5, 49,000. Gel exclusion chromatography, using sephadex G-200, indicated that both isozymes are dimers, although AHD-1 may also exist as a monomeric form as well. The enzymes exhibited widely divergent kinetic characteristics. The purified allelic forms of AHD-1, AHD-1A (C57BL/6J mice) and AHD-1B (CBA/H mice), exhibited high Km values with acetaldehyde as substrate, 1.4 mM and 0.78 mM respectively, whereas AHD-5 exhibited a low Km value with acetaldehyde of 0.2 microM. In addition, the isozymes exhibited distinct pH optima for catalysis (AHD-1, pH range 6.5-7.5; AHD-5, pH range 8.5-10.0), and were differentially sensitive towards disulphuram inhibition, with 50% inhibition occurring 13 and 0.1 microM for the AHD-1 and AHD-5 isozyme respectively. Based upon the kinetic characteristics, it is suggested that AHD-5 may be the primary enzyme for oxidizing mitochondrial acetaldehyde during ethanol oxidation in vivo.