[Validity and reliability of the Spanish version of the oral assessment guide (OAG) in cancer patients]. / Validez y fiabilidad de la versión española de la guía de valoración oral (OAG) en pacientes con cáncer.

BACKGROUND: The Oral Assessment Guide (OAG) is a widely used tool designed for evaluating problems of oral mucous in cancer patients, but it has not been validated in Spanish. The aim of this work is to translate and validate into Spanish the scale of the OAG designed by Eilers. METHOD: The translation process was carried out using the method of back-translation by bilingual translators. The study was carried out with cancer patients, both outpatient and inpatients, of the Hematology/Oncology Department and with oncology nurses. The following psychometric properties of the OAG were evaluated: internal consistency, concurrent validity with WHO's mucositis scale, interjudge agreement between two different nurses. The perception of patients and nurses on the use of the OAG was also assessed. RESULTS: An adequate Spanish version of the OAG was obtained. All the participants (n=40) completed the study. Internal consistency measured by Cronbach's alpha was 0.71 and interjudge agreement obtained a moderate to good Kappa index in the majority of items (k=0.4-0.81), except in "tongue and gums" (k=0.33-0.37). Concurrent validity with WHO mucositis scale was acceptable (r=0.458). All the nurses (n=6) considered that the scale was easy to understand and useful in clinical practice. The patients said that oral evaluation with the scale did not cause them discomfort. CONCLUSIONS: The Spanish version of the OAG is a valid and reliable instrument in cancer patients. It is a scale that is easy to use in clinical practice and is well accepted by patients.

Expression of the pituitary transcription factor GHF-1/PIT-1 in cell types of the adult porcine adenohypophysis.

We describe the expression of the transcription factor GHF-1/PIT-1 in adult porcine adenohypophysis by a nonradioactive in situ hybridization (ISH) method using a digoxigenin-labeled cDNA probe corresponding to the entire coding region of rat GHF-1. GHF-1 transcripts were found in 71.7% of adenohypophyseal cells. We also report the simultaneous detection of GHF-1 mRNA and pituitary hormones by combined ISH and immunocytochemistry (IC) in dispersed adenohypophyseal cells, detected with an alkaline phosphatase-NBT/BCIP technique and with an immunoperoxidase-3-amino-9-ethylcarbazole (AEC) method, respectively. The combination of the two techniques neither abolished nor diminished their sensitivity or specificity. GHF-1 is expressed in all five of the cell types in the adult porcine adenohypophysis, showing that this method is suitable for simultaneous detection of transcripts and proteins at the single-cell level.

Response of the L-arabinose forward mutation assay of Salmonella typhimurium to frameshift-type mutagens.

The present study was designed to evaluate the capacity of the L-arabinose resistance test of Salmonella typhimurium in the detection of frameshift-type mutagens. To this end the response of the Ara test was examined with respect to 15 chemicals which had been previously described as able to revert the Ames tester strain TA97. The mutagenicity of each compound was determined by the liquid test under experimental conditions which optimize the mutagenic response of the Ara test with the tester strain BA9. Strain TA97 was used simultaneously with BA9. The Ara forward-mutation assay efficiently detected the mutagenic activity of 14 out of the 15 chemicals assayed. PR toxin was the only compound which gave a weak dose response without doubling the spontaneous mutant level. In comparison with the Ara test, a total of 3 chemicals (HZ, PE and PR toxin) were not found to be mutagenic with strain TA97. In most cases (11/15) the mutagenic response of the Ara test was comparatively greater than that of strain TA97. Three chemicals (DEO, PRF and 9-AA) were detected with quite similar degrees of sensitivity by both mutation assays. ICR-191, which seems highly specific in reverting frameshift mutations with added cytosines in a run of cytosines, was the only chemical with a lower mutagenic activity in the Ara test than in strain TA97. The results enhance the interest of the L-arabinose forward-mutation assay as an alternative to the set of specific tester strains used by the histidine reverse-mutation assay in massive, general and primary screening for genotoxic agents.

Use of an infectious Simian virus 40-based shuttle vector to analyse UV-induced mutagenesis in monkey cells.

SV40 based shuttle vectors able to be packaged as pseudovirions have been used either as naked DNA or as pseudovirus to analyse the mutation frequency and the UV-induced mutation spectra obtained after transfection or infection of COS7 monkey cells. The frequency of supF spontaneous mutants was similar whatever the state of the vector, indicating that the transfection step is not responsible for the high spontaneous mutation frequency when using shuttle vectors. Nevertheless the UV-induced mutation frequency of the supF gene was higher when transfected DNA was replicated into COS7 cells than when pseudovirus infection was performed. The UV induced mutation spectra was basically similar in both situations but a new hot-spot at nucleotide 110 was obtained after pseudovirus infection. UV-pretreated and control COS7 cells were infected with untreated or UV-damaged pi SVPC7 shuttle virus and the survival and the supF mutation frequency were analysed in the progeny. The survival of UV-damaged pseudovirus replicated in 10 J/m2 UV-pretreated cells was 2-fold higher than in untreated cells. This increase in the survival was accompanied by a slight enhancement in the number of supF mutants.

Role of cell wall-degrading enzymes in pathogenicity of Fusarium oxysporum.

Fusarium oxysporum invades its host plants through the roots and colonizes the vascular system. It produces a great variety of cell-wall degrading enzymes (CWDE), such as cellulases, xylanases, pectinases and proteases. Our group has purified and characterized an endopolygalacturonase (PG1), two exopolygalacturonases (PG2 and PG3), an endoxylanase (XYL1) and an endo pectatelyase (PL1). We have isolated the following CWDE-encoding genes: pg1, pgx4, pg5, xyl2, xyl3, prt1 and pl1. Gene expression in different culture conditions has been determined by Northern analysis. The occurrence of these genes in different formae speciales has been analyzed by Southern analysis and PCR. All these genes are expressed during different stages of the interaction with the host plant indicating a possible role in pathogenesis. At present, targeted gene disruption is being carried out, in order to determine the role of each gene in the pathogenicity process.

Draft Genome of a Type 4 Pilus Defective Myxococcus xanthus Strain, DZF1.

Myxococcus xanthus is a member of the Myxococcales order within the deltaproteobacterial subdivision. Here, we report the whole-genome shotgun sequence of the type IV pilus (T4P) defective strain DZF1, which includes many genes found in strain DZ2 but absent from strain DK1622.

Draft Genome Sequence of Myxococcus xanthus Wild-Type Strain DZ2, a Model Organism for Predation and Development.

Myxococcus xanthus is a member of the Myxococcales order within the Deltaproteobacteria subdivision. The myxobacteria reside in soil, have relatively large genomes, and display complex life cycles. Here, we report the whole-genome shotgun sequence of strain DZ2, which includes unique genes not found previously in strain DK1622.

FrzZ, a dual CheY-like response regulator, functions as an output for the Frz chemosensory pathway of Myxococcus xanthus.

Myxococcus xanthus utilizes two distinct motility systems for movement (gliding) on solid surfaces: adventurous motility (A-motility) and social motility (S-motility). Both systems are regulated by the Frz signal transduction pathway, which controls cell reversals required for directed motility and fruiting body formation. The Frz chemosensory system, unlike the Escherichia coli chemotaxis system, contains proteins with multiple response regulator domains: FrzE, a CheA-CheY hybrid protein, and FrzZ, a CheY-CheY hybrid protein. Previously, the CheY domain of FrzE was hypothesized to act as the response regulator output of the Frz system. In this study, using a genetic suppressor screen, we identified FrzZ and showed FrzZ is epistatic to FrzE, demonstrating that FrzZ is the principal output component of the pathway. We constructed M. xanthus point mutations in the phosphoaccepting aspartate residues of FrzZ and demonstrated the respective roles of these residues in group and single cell motility. We also performed in vitro assays and showed rapid phosphotransfer between the CheA domain of FrzE and each of the CheY domains of FrzZ. These experiments showed that FrzZ plays a direct role as an output of the Frz chemosensory pathway and that both CheY domains of FrzZ are functional.

Conditions for the optimal use of the L-arabinose-resistance mutagenesis test with Salmonella typhimurium.

Different conditions of mutagenesis have been compared in order to optimize the use of the L-arabinose resistance test with Salmonella typhimurium. The mutagenesis protocols compared were the plate-incorporation, the pre-incubation and the liquid tests. Fourteen chemicals were used in the comparison: six direct-acting mutagens and eight pre-mutagens. Five concentrations of S9 (3, 7.5, 10, 15 and 33% v/v) were compared in the liquid test with pre-mutagens, and three densities of bacteria (ranging from 10(8) to 10(6) cells) were used in the comparison between the plate-incorporation and the pre-incubation mutagenesis test. In general, the liquid test proved the most sensitive mutagenesis protocol. When carrying out this test in a mass screening of mutagens, we propose to select the L-arabinose-resistant mutants in plates supplemented with 0.5 mg of D-glucose, and to express the mutagenic response as the absolute number of induced mutants. The plate-incorporation and the pre-incubation mutagenesis protocols could be considered as alternative procedures in the case of previous negative results with the liquid test. Two recommendations can be finally made in order to avoid false negative results: (a) a large population of bacteria (ranging from 10(8) to 10(7) cells) must be exposed to the mutagens in both the plate-incorporation and the pre-incubation mutagenesis tests, and (b) ideally, several S9 concentrations (ranging from 3 to 30% v/v) would be employed for testing in liquid samples of unknown mutagenicity.

Comparison of a forward and a reverse mutation assay in Salmonella typhimurium measuring L-arabinose resistance and histidine prototrophy.

A forward and a reverse mutation assay designed to detect environmental mutagens have been compared in Salmonella typhimurium. The forward mutation assay scored resistance to L-arabinose and the reverse assay, reversion of histidine auxotrophy. Eighteen chemicals of different structural groups, all known to be mutagenic in the histidine reverse assay, were applied to strains carrying the genetic markers needed to perform both mutation assays. The mutagenicity of each chemical was determined by both plate and liquid tests. The plate test counted absolute numbers of surviving mutants and the liquid test separately measured survival and frequency of mutants among the survivors. All the chemicals used were found to be mutagenic in both mutation assays. The response of the L-arabinose assay was equal to or larger than the response of the histidine assay in the case of 16 chemicals. The two other compounds, 2-nitrofluorene and sodium azide, were detected more efficiently by the histidine assay. Sodium azide, a non-carcinogenic compound, is a potent mutagen in the histidine assay, but very weak in the L-arabinose assay.

Molecular characterization of a novel endo-beta-1,4-xylanase gene from the vascular wilt fungus Fusarium oxysporum.

A gene, xyl5, was identified from the tomato vascular wilt fungus Fusarium oxysporum f.sp. lycopersici, whose predicted amino acid sequence shows significant homology with family 11 xylanases. Expression of xyl5 was detected during growth both on xylan and cellulose substrates as carbon sources and on tomato vascular tissue. RT-PCR analysis revealed the presence of two different transcript sizes, resulting from differential splicing of the third intron. The 3'-untranslated region of the xyl5 transcript contained a region of homology to cellulose-binding domains, suggesting that such a domain may have been part of an ancestral XYL5 version. As shown by RT-PCR, xyl5 is expressed by F. oxysporum exclusively during the initial stages of infection in tomato roots. Targeted inactivation of xyl5 had no detectable effect on virulence.

Effect of ogt expression on mutation induction by methyl-, ethyl- and propylmethanesulphonate in Escherichia coli K12 strains.

We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O6-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kanr disruption mutation was introduced into the parental ogt+ bacteria, and the ogt::kanr mutation was then eliminated by cotransduction of ogt+ with the closely linked Tetr marker (zcj::Tn10). The delta(ogt-fnr) deletion or ogt::kanr disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to L-arabinose resistance (Arar). Furthermore, the number of Arar mutants increased linearly with dose, unlike the case in ogt+ bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt- mutant strains and also methylmethanesulphonate mutagenesis in ada- bacteria. A sample of AB1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.

Folyt1, a new member of the hAT family, is active in the genome of the plant pathogen Fusarium oxysporum.

An active transposable element, Folyt1, has been isolated from the tomato pathogen Fusarium oxysporum f. sp. lycopersici as an insertion sequence within the coding region of the nitrate reductase gene (nit 1) in two independent mutants (CO66 and CO108). Folyt1 was 2615 bp in length and contained 9-bp imperfect inverted terminal repeats (ITRs) and 8 bp duplicated at the target site upon insertion. The element contained a long open reading frame interrupted by a single putative intron. The predicted amino acid sequence showed similarity to conserved domains of transposases from hobo, Ac, and Tam3 elements, which belong to the hAT family. The excision frequency of Folyt1 was determined to be less than 10(-5) in both mutants. These events restored the nit 1 wild-type allele without leaving footprints in all the revertants of strain CO66. Nevertheless, some revertants of strain CO108 showed a point mutation footprint at the target sequence. Expression of the Folyt1 transposase was detected by Northern analysis as a 2.1-kb transcript. The element exists in about 10 copies per genome in F. oxysporum f. sp. lycopersici and appears to be widely distributed among different formae speciales of F. oxysporum.

A method for selection of forward mutations in supF gene carried by shuttle-vector plasmids.

The supF gene of Escherichia coli has been widely used as a mutagenic target in several shuttle-vector plasmids. Mutations in this gene are usually screened by a colony colour assay based on the suppression of a lacZ amber mutation in an appropriate bacterial indicator strain. This screening method cannot measure the low mutation frequencies usually detected in prokaryotes, and therefore precludes the use of supF gene for studying mutational spectra in bacteria. In this paper we report the development of a simple method for the selection of supF forward mutations in shuttle-vector plasmids. The method has implied the construction of an araD- araC(Am) mutant strain (MBL50) of E.coli. The L-arabinose sensitivity caused by the accumulation of a toxic intermediate in araD- mutants is abolished in MBL50 because the araC(Am) mutation blocks the L-arabinose catabolic pathway. Strain MBL50 becomes sensitive to L-arabinose when transformed with a supF+ plasmid but remains resistant upon transformation with a supF- mutant. This new L-arabinose resistance selection method was able to detect supF- mutant fractions up to three orders of magnitude below those determined with the colony colour screening assay. The method was further validated by carrying out in vivo mutagenesis experiments with N-methyl-N-nitrosourea (MNU) and a shuttle-vector-bearing strain (UC2109) completely defective in O6-methylguanine (O6meG) alkyltransferase repair capacity. The DNA sequence alterations of 22 independent supF- mutants induced by MNU were determined. All mutations were G:C-->A:T transitions in agreement with the predicted significance of the mispairing potential of the O6meG lesion. A preference for the sequence 5'-GG-3' was detected, revealing a 5'-flanking base influence. The accumulation of all 22 MNU-induced mutations in three sites of the supF genes might be related to the lack of O6meG alkyltransferase repair capacity of strain UC2109. The L-arabinose resistance method described in this paper allows rapid scoring and sequencing of forward mutations in the supF gene on shuttle-vectors, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria. Since shuttle-vectors replicate both in bacteria and mammalian cells, this method makes it possible to compare prokaryotic and eukaryotic mutational spectra.

The Che4 pathway of Myxococcus xanthus regulates type IV pilus-mediated motility.

Myxococcus xanthus co-ordinates cell movement during its complex life cycle using multiple chemotaxis-like signal transduction pathways. These pathways regulate both type IV pilus-mediated social (S) motility and adventurous (A) motility. During a search for new chemoreceptors, we identified the che4 operon, which encodes homologues to a MCP (methyl-accepting chemotaxis protein), two CheWs, a hybrid CheA-CheY, a response regulator and a CheR. Deletion of the che4 operon did not cause swarming or developmental defects in either the wild-type (A(+)S(+)) strain or in a strain sustaining only A motility (A(+)S(-)). However, in a strain displaying only S motility (A(-)S(+)), deletion of the che4 operon or the gene encoding the response regulator, cheY4, caused enhanced vegetative swarming and prevented aggregation and sporulation. In contrast, deletion of mcp4 caused reduced vegetative swarming and enhanced development compared with the parent strain. Single-cell analysis of the motility of the A(-)S(+) parent strain revealed a previously unknown inverse correlation between velocity and reversal frequency. Thus, cells that moved at higher velocities showed a reduced reversal frequency. This co-ordination of reversal frequency and velocity was lost in the mcp4 and cheY4 mutants. The structural components of the S motility apparatus were unaffected in the che4 mutants, suggesting that the Che4 system affects reversal frequency of cells by modulating the function of the type IV pilus.

Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein-barr-virus-derived vector.

The hepatitis B virus surface antigen (HBsAg) gene, under control of the inducible mouse metallothionein I gene promoter, was inserted in an expression vector based on the Epstein-Barr virus (EBV). This vector was introduced into human cells by DNA transfection and clones were selected for their resistance to hygromycin B. The recombinant EBV vector replicates efficiently as an episome in human cells and approximately six copies per cell were found in one clone of hygromycin-B-resistant cells. These cells produce high levels of HBsAg in the presence of metals. The protein is mainly found in the cell medium, suggesting that the HBsAg is secreted from the cells.

An association between mutagenicity of the Ara test of Salmonella typhimurium and carcinogenicity in rodents for 16 halogenated aliphatic hydrocarbons.

Sixteen halogenated aliphatic hydrocarbons were assayed for genotoxicity using the Ara mutagenicity assay with Salmonella typhimurium. Seven substances (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide, hexachloro-1,3-butadiene, iodoform and vinilydene chloride) were mutagenic at non-lethal doses. Comparatively, nine compounds (chloroform, carbon tetrachloride, 1,1,1-trichloroethane, 1,1,2-trichloroethane, tetrachloroethylene, trichloroethylene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane and hexachloroethane) were non-mutagenic after being assayed both in the presence and absence of metabolic activation with a rat liver microsomal fraction (S9). All negative compounds (except hexachloroethane) gave a lethal response, which could be an indication that bacteria were adequately exposed. The concordance between mutagenicity in the Ara test and carcinogenicity in rodents for this group of halogenated hydrocarbons was (31%) significantly lower than the concordance (72%) previously found in the Ara test with respect to a wider range of chemical classes. This result is in agreement with data reported for other genotoxicity assays. The presence of non-genotoxic carcinogens versus genotoxic non-carcinogens is discussed as a possible explanation. Five positive compounds (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide and hexachloro-1,3-butadiene) were analyzed for a quantitative relationship between carcinogenic potency in rats and the potency of response in the Ara mutagenicity test. This was possible because the Ara test, for volatile compounds (such as vinyl bromide), did not require the use of special vaporization techniques, which are difficult to evaluate quantitatively for mutagenic activity. A highly significant correlation was found between the mutagenic efficiencies of the five compounds in the Ara test and their carcinogenic potencies in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Role in pathogenesis of two endo-beta-1,4-xylanase genes from the vascular wilt fungus Fusarium oxysporum.

A gene, xyl4, whose predicted amino acid sequence shows significant homology with family 11 xylanases, was identified from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Expression of xyl4 is induced on oat spelt xylan as the carbon source, subject to carbon catabolite repression and preferentially expressed at alkaline ambient pH. Transcript levels of xyl4 on an inducing carbon source are differentially regulated by the nature and concentration of the nitrogen source. As shown by RT-PCR, xyl4 is expressed by F. oxysporum during the entire cycle of infection on tomato plants. Targeted inactivation of xyl4 and of xyl3, a previously identified gene of F. oxysporum f. sp. lycopersici encoding a family 10 xylanase, had no detectable effect on virulence on tomato plants, demonstrating that both genes are not essential for pathogenicity.

Females of four mole species of genus Talpa (insectivora, mammalia) are true hermaphrodites with ovotestes.

We studied the anatomical, histological, and genetic features of the sexual tract in four European mole species of the genus Talpa (Insectivora, mammalia): T. occidentalis, T. europaea, T. romana, and T. stankovici. All XY individuals had a normal male phenotype, whereas all XX individuals in all four species had features that identified them as intersexes. These individuals were nonetheless presumed to be functionally fertile females. Intersexuality was manifested mainly as gonadal hermaphroditism, with all females possessing bilateral ovotestes. The gonads were composed of a small portion of histologically normal ovarian tissue and a variably sized, generally large mass of disgenetic testicular tissue, accompanied by a small, rudimentary epididymis. The rest of the sexual tract was typically female, including oviducts, uterus, and vagina of normal appearance. Polymerase chain reaction (PCR) and Southern blotting analyses showed that the mammalian testis-determining gene SRY is present in males but not in females. Part of the conserved sequence of the mole SRY gene was cloned and sequenced after PCR amplification in two of the four mole species (T. occidentalis from Spain and T. romana from Italy). Sequences were identical in these two species and were very similar to those of the human and mouse SRY gene. Our findings constitute the first evidence of the existence of a genus-specific case of true hermaphroditism, probably due to a very ancient mutation that fixed in populations of the ancestral species from which contemporary moles evolved. The possible nature of this mutation is discussed with regard to the cytologic, histologic, and genetic features of the gonads in Talpa females.