Analysis of expressed sequence tags from Uromyces appendiculatus hyphae and haustoria and their comparison to sequences from other rust fungi.

Hyphae, 2 to 8 days postinoculation (dpi), and haustoria, 5 dpi, were isolated from Uromyces appendiculatus infected bean leaves (Phaseolus vulgaris cv. Pinto 111) and a separate cDNA library prepared for each fungal preparation. Approximately 10,000 hyphae and 2,700 haustoria clones were sequenced from both the 5' and 3' ends. Assembly of all of the fungal sequences yielded 3,359 contigs and 927 singletons. The U. appendiculatus sequences were compared with sequence data for other rust fungi, Phakopsora pachyrhizi, Uromyces fabae, and Puccinia graminis. The U. appendiculatus haustoria library included a large number of genes with unknown cellular function; however, summation of sequences of known cellular function suggested that haustoria at 5 dpi had fewer transcripts linked to protein synthesis in favor of energy metabolism and nutrient uptake. In addition, open reading frames in the U. appendiculatus data set with an N-terminal signal peptide were identified and compared with other proteins putatively secreted from rust fungi. In this regard, a small family of putatively secreted RTP1-like proteins was identified in U. appendiculatus and P. graminis.

[Oligodendrogliomas: historical background of classifications]. / Oligodendrogliomes: historique des classifications.

The story of the classifications for gliomas is related to the development of the techniques used for cytological and histological examination of brain parenchyma. After a review of these techniques and the progressive discovery of the central nervous system cell types, the main classifications are presented. The first classification is due to Bailey and Cushing in 1926. It was based on histoembryogenetic theory. Then Kernohan introduced, in 1938, the concept of anaplasia. The WHO classification was published in 1979, then revised in 1993 and 2000. It took into account some data from both previous systems and introduced gradually the notion of histological criteria of malignancy. More recently; molecular genetics data and clinical evolution were retained. The Sainte-Anne classification for oligodendrogliomas is based on both histological and imaging data. It includes the notion of spatial histological structure of oligodendrogliomas. Contrast enhancement is closely related to endotheliocapillary hyperplasia. Gliomas classifications are changing and confusions can be made because of lack of reproductibility and misinterpretations of samples.

Molecular Characterization of Arginine Kinases in the Soybean Cyst Nematode (Heterodera glycines).

Arginine kinase (AK) is a phosphagen kinase that plays a key role in energy mobilization in invertebrates. Alignment of expressed sequence tags (ESTs) for soybean cyst nematode (SCN) (Heterodera glycines) produced two separate contiguous sequences (contigs) and three singletons encoding peptides with high similarity to AKs. One contig, Hg-AK1, had 244 ESTs in the alignment whereas the other, Hg-AK2, had only three; nonetheless, the consensus sequence for Hg-AK1 was missing much of the 5' end. Polymerase chain reaction (PCR) was used to prepare clones that were then sequenced to obtain full-length sequences for both Hg-AK1 and Hg-AK2. Hg-AK1 has an open reading frame of 1080 nucleotides (nt) encoding a protein of 360 amino acids (aa) with a predicted molecular weight of 40 kDa. The open reading frame for Hg-AK2 is 1221 nt, 407 aa, and 46 kDa with a 71% aa identity with Hg-AK1. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that Hg-AK1 and Hg-AK2 are expressed constitutively throughout the SCN life cycle. Phylogenetic analysis of peptide sequences for near full-length nematode contigs and other AKs in the Swisspro database indicates that the nematode AKs evolved from a single gene after divergence of insects and nematodes.

Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma.

The tomato (Lycopersicon esculentum cv Ailsa Craig) polygalacturonase genes TAPG1 (LYCes;Pga1;2) and TAPG4 (LYCes;Pga1;5) are abundantly expressed in both abscission zones and the pistils of mature flowers. To further investigate the spatial and temporal expression patterns for these genes, the TAPG gene promoters were ligated to beta-glucuronidase (GUS) reporter genes and transformed into tomato. GUS expression with both constructs was similar and entirely consistent with the expression patterns of the native gene transcripts. GUS activity was observed in the weakening abscission zones of the leaf petiole, flower and fruit pedicel, flower corolla, and fruit calyx. In leaf petiole and flower pedicel zones this activity was enhanced by ethylene and inhibited by indole-3-acetic acid. On induction of abscission with ethylene, GUS accumulation was much earlier in TAPG4:GUS than in TAPG1:GUS transformants. Moreover, TAPG4:GUS staining appeared to predominate in the vascular bundles relative to surrounding cortex cells whereas TAPG1:GUS was more evenly distributed across the separation layer. Like the native genes, GUS was also expressed in the stigma. Activity was not apparent in pistils until the flowers had opened and was confined to the stigma and style immediately proximal to it. A minimal promoter construct consisting of a 247-bp 5'-upstream element from TAPG1 was found to be sufficient to direct GUS expression in both abscission zones and the stigma.

High level expression of "male specific" pheromone binding proteins (PBPs) in the antennae of female noctuiid moths.

Pheromone Binding Proteins (PBPs) are one branch of a multigene family of lepidopteran Odorant Binding Proteins (OBPs) that are known for their relatively high levels of expression in male antennae. However, PBP expression has been observed at low levels in female antennae of the Saturniidae, Bombycidae and Lymantriidae, and at relatively high levels in members of the Noctuiidae. The function of female PBP expression is unclear, as female lepidoptera are consistently noted for their failure to respond physiologically or behaviorally to sex-pheromone. In this study, the sexual dimorphism of PBP expression was examined in the noctuiid moths Helicoverpa zea, Heliothis virescens and Spodoptera frugiperda. A PBP cDNA clone was isolated from female H. zea, PBP-Hzea(f). Northern blot analysis indicated relatively high levels of PBP-Hzea(f) expression in both male and female antennae, though females consistently expressed about 50% that of males. Western blot analysis of male and female PBP expression supported these relative differences. Immunocytochemical analysis indicates discrete expression localized beneath olfactory sensilla of both male and female antennae. These results suggest female noctuiids possess the biochemistry to detect at least components of their sex-pheromone. Alternatively, these results may suggest that PBPs have a more general function in noctuiids, possibly reflecting behavioral and life history differences that distinguish this the Noctuiidae from other Lepidopteran families.

Molecular characterization of a tomato polygalacturonase gene abundantly expressed in the upper third of pistils from opened and unopened flowers.

A polygalacturonase (PG) gene, TPG7 (Lyces;Pga1;8), has been cloned from tomato (Lycopersicon esculentum Mill., cv. Rutgers). RNA blot analysis reveals that TPG7 is highly expressed in pistils (ovary removed) from unopened and fully open flowers. Dissection of mature pistils demonstrated that TPG7 expression is limited to the top third (stigmatic region) of the pistils. This is contrasted with another tomato PG, TAPG4, which is also expressed in the same region of the pistil but only in mature pistils from fully open flowers. Hybridization of the TPG7 probe to anther RNA was nil to none and was barely detectable in RNA from leaf and flower abscission zones. The TPG7 polypeptide shares 39% sequence identity with the tomato fruit PG and between 63% and 73% sequence identities with six other tomato PGs.

Molecular characterization of a tomato endo-beta-1,4-glucanase gene expressed in mature pistils, abscission zones and fruit.

A cDNA (TAC1) and genomic clone (cel5) encoding an endo-beta-1,4-glucanase (EGase) were identified from tomato (Lycopersicon esculentum Mill., cv. Rutgers). The cel5 gene is expressed in pistils, flower pedicel and leaf abscission zones, and ripening fruit. The genomic sequence includes a 22 bp 5' upstream sequence that is conserved in a closely related peach EGase gene, ppEG1.

Genomic organization of six tomato polygalacturonases and 5' upstream sequence identity with tap1 and win2 genes.

Recently, three polygalacturonase (PG) cDNAs (TAPG1, TAPG2, and TAPG4) were identified in a library prepared from tomato (Lycopersicon esculentum cv. Rutgers) leaf abscission zones. Genomic clones encoding these three cDNAs have been identified. Moreover, the genomic clones include three additional PG genes, TPG3, TAPG5 and TPG6, which have not been previously reported. A transcript for TAPG5 was detected in the RNA from leaf and flower abscission zones; however, transcripts for TPG3 and TPG6 were not. DNA sequence analysis revealed that TAPG1, TAPG2, and TPG3 are linked in a close tandem array. TAPG4, TAPG5 and TPG6 are also closely linked to each other but in divergent and inverted orientations and are not closely linked to TAPG1, TAPG2, or TPG3. TAPG4, TAPG5 and TPG6 map to the middle of chromosome 12. TPG6 contains two introns. The other five PG genes include four exons and three introns. The relative positions of introns 1 and 2 are shared by all six PG genes. The position of intron 3 is conserved in the other five. The structure of the tomato fruit PG gene, which contains 8 introns, is compared with that of the six PG genes described above. Of interest is an approximately 300 bp inverted repeat found in TAPG1, TAPG2 and TAPG4 that shares significant sequence identity with sequence in the first intron of the tomato anionic peroxidase gene, tap1. RNA blot analysis indicates that the transcript for an anionic peroxidase increases during abscission. In addition, a 250 bp sequence found in TPG3 shares high sequence identity with a 5' upstream region in a wound-induced win2 gene from potato. Potential sites of transcriptional regulation in these genes are discussed.

Oligodendrogliomas. Part I: Patterns of growth, histological diagnosis, clinical and imaging correlations: a study of 153 cases.

The present study has attempted to demonstrate that the morphological spectrum of oligodendrogliomas includes tumors which are traditionally misinterpreted as 'diffuse fibrillary astrocytoma'. We have shown that these tumors are in fact made of isolated neoplastic oligodendrocytes which are entrapped in a fibrillary background composed of axons and fibrillary reactive gliosis. Analysis in a series of 153 'pure' supratentorial oligodendrogliomas composed of 'classical' or pseudo 'diffuse fibrillary oligodendrogliomas' diagnosed by imaging-based serial stereotactic biopsies showed that 2/3 of the tumors were exclusively made of isolated tumor cells (ITCs) (structure type III) and that only 1/3 of them exhibited both ITCs and solid tumor tissue components (structure type II). The tumor tissue destroys the brain parenchyma and contains new formed microblood vessels whereas ITCs do not destroy the parenchyma and are not associated with microangiogenesis. These fundamentally opposite morphological characteristics were reflected by the following findings: 1) contrast enhancement was observed in 64% of structure type II but was never seen in structure type III oligodendrogliomas. 2) a neurological deficit occurred in 57% of structure type II but in only 8% of structure type III oligodendrogliomas. 3) using the new grading system described in the companion paper to this study, we found that the biological behavior of oligodendrogliomas was also closely related to the patterns of tumor growth. From a synthesis of data gathered in this study it is suggested that emergence of microangiogenesis within a tumor which at first grows slowly with a structure type III pattern is a crucial event toward more aggressive behavior.

Oligodendrogliomas. Part II: A new grading system based on morphological and imaging criteria.

This second part of our study of 'pure' oligodendrogliomas focuses on survival data analysis. In order to identify potentially useful prognostic factors and to assess the effectiveness of a new grading system, the 79 patients in the previously analyzed series for whom adequate follow-up could be obtained (52%) were entered in the present analysis. Statistical analysis demonstrated that contrast enhancement and endothelial hyperplasia had powerful and similar influence on survival. Median survival with and without contrast enhancement were: 3 versus 11 years, and with or without endothelial hyperplasia were: 3.5 versus 11 years. Conversely, the degree of nuclear atypia and presence or absence of mitosis or necrosis were not correlated with survival. These findings allowed us to devise a simple grading system which discriminates two malignancy grades as follows: absence of endothelial hyperplasia and of contrast enhancement = Grade A, presence of endothelial hyperplasia and/or of contrast enhancement = Grade B. Of the 79 oligodendrogliomas in this study, 59 tumors were categorized as grade A and 20 as grade B. Median survival were: 11 years in grade A and 3.5 years in grade B. Five-year and 8-year survival rates were: 89% and 60% in grade A and: 33% and 15% in grade B. Double blind grading between two independent observers was concordant in 96% of the cases. Application of this simple efficient and reproducible grading scheme should permit reliable comparison of retrospective or prospective therapeutic data emanating from various institutions.

Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern.

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76-93% identity) yet different from the those of tomato fruit PG (38-41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.

Cloning and characterization of a rhamnogalacturonan hydrolase gene from Botrytis cinerea.

Rhamnogalacturonan hydrolase (Rgase A) cleaves alpha 1--2 linkages between rhamnosyl and galacturonosyl residues in pectin. A 1.9 kb RGase A cDNA clone (BCRHGA) was isolated from a B. cinerea cDNA library using a PCR-amplified Aspergillus aculeatus RGase A probe. It's 1.7 kb open reading frame had 62% identity at the amino acid level with A. aculeatus RGase A. Northern blots of B. cinerea total RNA probed with BCRHGA revealed a 2 kb band, suggesting the cDNA clone is full or nearly-full length. To determine mRNA expression of the gene, B. cinerea was grown in media containing 0.5% apple pectin, 0.5% rhamnogalacturonan-I and 1% glucose carbon sources. Northern analysis revealed the BCRHGA gene was expressed on all carbon sources, but with different patterns of expression. B. cinerea RGase A appeared to be coded for by a single or low copy number gene based on Southern analysis.

The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase.

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues.

Dominant mutations in the Arabidopsis ETR1 gene block the ethylene signal transduction pathway. The ETR1 gene has been cloned and sequenced. Using the ETR1 cDNA as a probe, we identified a cDNA homologue (eTAE1) from tomato. eTAE1 contains an open reading frame encoding a polypeptide of 754 amino acid residues. The nucleic acid sequence for the coding sequence in eTAE1 is 74% identical to that for ETR1, and the deduced amino acid sequence is 81% identical and 90% similar. Genomic Southern blot analysis indicates that three or more ETR1 homologues exist in tomato. RNA blots show that eTAE1 mRNA is constitutively expressed in all the tissues examined, and its accumulation in leaf abscission zones was unaffected by ethylene, silver ions (an inhibitor of ethylene action) or auxin.

The preclinical toxicological evaluation of sumatriptan.

1 Sumatriptan is a potent and selective 5-HT1 receptor agonist marketed for the treatment of migraine by both oral and subcutaneous routes. An extensive toxicological programme employing high doses of sumatriptan was carried out in a range of animal species. The studies evaluated both the local and systemic tolerance to single and repeated dosing, effects on all stages of reproduction, as well as the genotoxic and oncogenic potential of sumatriptan. 2 The administration of relatively high single and repeated doses of sumatriptan was well tolerated by both rodents and dogs by the oral, subcutaneous and intravenous routes. Behavioural effects, suggestive of involvement of the central nervous system, were the most obvious result of such doses and were generally more pronounced in dogs than rodents. The reason for this may be related to the higher plasma concentrations of the drug achievable in dogs. Additional observations restricted to dogs, were transient, and included tachycardia, facial oedema and breaks in the continuity of secretion films on the corneal surface. A tendency for an increase in weight gain was seen for rats, while a slight decrease was usually seen for dogs. The only pathological changes related to treatment with high concentrations of sumatriptan consisted of local reactions at the site of subcutaneous administration. 3 Sumatriptan is an indole; the structures of this chemical class show varying propensities for nitrosation. However, appropriate testing with sumatriptan failed to identify any mutagenic nitroso compounds. 4 Sumatriptan was neither genotoxic nor oncogenic. 5 Reproductive studies demonstrated that sumatriptan was not teratogenic and had no effect on peri- and postnatal development. Some embryotoxicity was observed, but only at maternally toxic doses. A slight decrease in the success of insemination was also noted at high oral doses in rats. 6 Results of the toxicological programme performed in support of migraine therapy with sumatriptan provide good assurance of safety for subcutaneous and oral use.

Cloning of a tomato polygalacturonase expressed in abscission.

Abscission, organ separation, is accompanied by cell wall breakdown in separation layer cells. In tomato (Lycopersicon esculentum), ethylene-induced abscission is correlated with an increase in polygalacturonase (PG) and endo-beta-1,4-D-glucanase (cellulase) activity. We have identified a putative, abscission-specific cDNA clone for PG, pTAPG1. The TAPG1 cDNA has 43% identity at the amino acid level with the tomato fruit PG. Genomic blot analysis suggests that the gene for TAPG1 is a member of a small subfamily of PG genes that is distinct from the tomato fruit PG. The TAPG1 cDNA hybridizes to mRNA expressed during the course of ethylene-induced leaf and flower abscission. A high level of PG transcript accumulation coincides with the occurrence of abscission. Auxin, an abscission inhibitor, and silver thiosulfate, an ethylene action inhibitor, suppressed accumulation of mRNA in leaf abscission zones complementary to the TAPG1 cDNA. Expression of TAPG1 transcripts is several-fold higher in flower abscission zones than in leaf abscission zones. The identification of cDNAs that encode abscission-specific PG provide and additional tool to study the regulation of abscission and cell wall dissolution in separation layer cells.

Comparative study of cellulases associated with adventitious root initiation, apical buds, and leaf, flower, and pod abscission zones in soybean.

Cellulase activity was measured in soybean (Glycine max) leaf abscission zones, flower abscission zones, pod abscission zones, apical buds, and adventitious rooting hypocotyls. Immunoprecipitation data showed that a cellulase immunologically similar to the bean abscission cellulase (isoelectric point 9.5) is present in soybean leaf, flower, and pod abscission zones, but is not present in soybean apical buds or rooting hypocotyls. cDNA and genomic clones for two different soybean genes were identified and show sequence similarity with the bean abscission cellulase clone pBAC10. The cDNA clone pSAC1, isolated from a soybean abscission cDNA library, hybridized to transcripts in soybean leaf, flower, and pod abscission zones. Although ethylene has been shown to play a role in the increase in cellulase activity associated with both abscission and adventitious root initiation, no signal was seen for hybridization of the soybean abscission cellulase clone, pSAC1, to RNA from soybean adventitious rooting hypocotyls. In addition, no soybean abscission cellulase transcripts were detected in apical buds. Transcripts for a second soybean cellulase gene (SC2) were not detected in any of the tissues surveyed.