Ancestral genetic legacy of the extant population of Argentina as predicted by autosomal and X-chromosomal DIPs.

Aiming to determine their ancestry diagnostic potential, we selected two sets of nuclear deletion/insertion polymorphisms (DIPs), including 30 located on autosomal chromosomes and 33 on the X chromosome. We analysed over 200 unrelated Argentinean individuals living in urban areas of Argentina. As in most American countries, the extant Argentinean population is the result of tricontinental genetic admixture. The peopling process within the continent was characterised by mating bias involving Native American and enslaved African females and European males. Differential results were detected between autosomal DIPs and X-DIPs. The former showed that the European component was the largest (77.8%), followed by the Native American (17.9%) and African (4.2%) components, in good agreement with the previously published results. In contrast, X-DIPs showed that the European genetic contribution was also predominant but much smaller (52.9%) and considerably larger Native American and African contributions (39.6% and 7.5%, respectively). Genetic analysis revealed continental genetic contributions whose associated phenotypic traits have been mostly lost. The observed differences between the estimated continental genetic contribution proportions based on autosomal DIPs and X-DIPs reflect the effects of autosome and X-chromosome transmission behaviour and their different recombination patterns. This work shows the ability of the tested DIP panels to infer ancestry and confirm mating bias. To the best of our knowledge, this is the first study focusing on ancestry-informative autosomal DIP and X-DIP comparisons performed in a sample representing the entire Argentinean population.

Potential forensic use of a 33 X-InDel panel in the Argentinean population.

Polymorphic genetic markers located on the X chromosome might become a complement in particular forensic identification when the biological kinship are deficient. We analyzed forensic statistical parameters of 33 X-chromosome InDel polymorphisms in a sample of 320 individuals from Argentina. The X-chromosome InDel polymorphism (X-InDel) panel was amplified in a single multiplex PCR reaction. Hardy-Weinberg Equilibrium was determined in the female cohort, whereas the male cohort was used to calculate linkage disequilibrium (LD) tested by an extension of Fisher's exact test, D', and Chi-square values. Regarding LD, 15 markers were organized and grouped into six blocks containing two or three linked loci each, namely block I (MID356-MID357), block II (MID448804-MID3703-MID218), block III (MID3705-MID3706-MID304737), block IV (MID197147-MID3754), block V (MID3664-MID284601-MID103547), and block VI (MID3763-MID3728). The haplotype diversity was higher than 0.99 in all cases. Blocks III and VI showed the highest match probability in the studied population, whereas block II showed the lowest. The accumulated power of discrimination was 99.9999991 % in women and 99.9992925 % in men. The mean exclusion chance in trios and duos were 99.9891736 and 99.6099391 %, respectively. Since 15 markers are associated as haplotypic blocks, for a conservative treatment of the data, statistical evaluation should consider their haplotypic frequencies and the remaining 18 markers can be evaluated as independent loci.

Differential gene expression analysis provides new insights into the molecular basis of iron deficiency stress response in the citrus rootstock Poncirus trifoliata (L.) Raf.

Iron chlorosis is one of the major abiotic stresses affecting fruit trees and other crops in calcareous soils and leads to a reduction in growth and yield. Usual remediation strategies consist of amending iron to soil, which is an expensive practice, or using tolerant cultivars, which are difficult to develop when not available. To understand the mechanisms underlying the associated physiopathy better, and thus develop new strategies to overcome the problems resulting from iron deficiency, the differential gene expression induced by iron deficiency in the susceptible citrus rootstock Poncirus trifoliata (L.) Raf. have been examined. The genes identified are putatively involved in cell wall modification, in determining photosynthesis rate and chlorophyll content, and reducing oxidative stress. Additional studies on cell wall morphology, photosynthesis, and chlorophyll content, as well as peroxidase and catalase activities, support their possible functions in the response to iron deficiency in a susceptible genotype, and the results are discussed.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

[Adult living donor liver transplantation. Analysis of the first 30 cases]. / Trasplante hepático de donante vivo en adultos. Análisis de los 30 primeros casos.

INTRODUCTION: In the last 2-3 years, adult living donor liver transplantation (ALDLT) has been developed on an international scale, multiplying the number of procedures performed. Despite this, analysis of the results is still incomplete. The aim of the present study was to perform a descriptive analysis of the results after the first 3 years of the initiation of the program. MATERIAL AND METHODS: During this period, 30 ALDLT were performed. In all procedures, right lobe grafts were used. The mean age of donors and recipients was 31.8 and 52.7 years, respectively. The main indication for liver transplantation was liver cirrhosis due to hepatitis C virus (70%) and 38% of recipients were stage C in the Child-Pugh classification. A total of 46.7% of recipients had hepatocellular carcinoma. RESULTS: Donors: The mean volume of the remnant liver was 632 cc (40.5% of the previous hepatic mass). Ten donors (33%) presented complications. The most frequent complication was biliary fistula (20%) and three patients required reintervention. The mean length of hospital stay among donors was 11.7 days. Recipients: The mean weight of the graft was 775 g, with a mean difference between graft weight and that of the recipient of 1.11. Fifteen recipients (50%) presented biliary leaks and nine of these (30%) required reintervention. There were no graft losses for technical reasons. Four patients died. With a median follow-up of 14 months, actuarial survival at 18 months was 92.9%. CONCLUSION: ALDLT is an effective method for reducing the number of patients on the waiting list. The probability of survival is similar to that of cadaveric transplantation. Biliary complications in the recipient constitute a problem, the long-term repercussions of which remain to be resolved.

Coledocolitiasis no sospechada: abordaje laparoscópico durante la colecistectomía / Laparoscopic exploration of the common bile duct for unsuspected stones during cholecystectomy

Introducción. En los pacientes con colelitiasis sintomática, la colangiografía intraoperatoria transcística durante la colecistectomía laparoscópica puede mostrar la presencia de cálculos no sospechados en la vía biliar principal entre el 1 y el 9 por ciento, según las distintas series publicadas. El objetivo de este estudio es valorar la utilidad y los resultados del abordaje transcístico laparoscópico de la coledocolitiasis no sospechada, diagnosticada durante la colecistectomía laparoscópica, en una serie de pacientes con antecedentes de pancreatitis aguda biliar y coledocolitiasis tratada por colangiopancreatografía retrógrada endoscópica (CPRE).Pacientes y métodos. Desde enero de 1997 hasta abril de 2001, en 349 pacientes que fueron sometidos a colecistectomía laparoscópica (201 pacientes pospancreatitis aguda biliar, 75 pacientes post-CPRE y 73 pacientes con colelitiasis sintomática), la realización de colangiografía objetivó la existencia de coledocolitiasis en 34 pacientes (10 por ciento). De ellos, 19 eran mujeres y 15 eran varones con una edad media de 67 años. El abordaje de la vía biliar principal mediante extracción transcística fue utilizado en 15 pacientes (44 por ciento), la dilatación neumática de la papila en 12 (35 por ciento) y coledocotomía laparoscópica en tres (9 por ciento).Resultados. Todos los pacientes fueron tratados por laparoscopia de forma satisfactoria sin necesidad de conversión. El número de cálculos extraídos varió de uno a ocho, y el tamaño de 2 a 8 mm. El tiempo operatorio medio fue de 79 min (límites, 35125) para la extracción transcística, 82 min (límites, 50-105) para la dilatación neumática de la papila y de 105 min (límites, 90-120) para la coledocotomía laparoscópica. Cuatro pacientes requirieron la realización de una CPRE postoperatoria (12 por ciento) por coledocolitiasis residual. La morbilidad asociada a la cirugía fue del 15 por ciento.Conclusiones. La colangiografía transcística durante la colecistectomía laparoscópica es necesaria para el diagnóstico de la coledocolitiasis no sospechada (10 por ciento). El abordaje por vía transcística o por coledocotomía es posible y consigue eliminar la litiasis de la vía biliar principal en el 91 por ciento de nuestros pacientes. Ambas técnicas laparoscópicas son reproducibles si se dispone de la experiencia y del material adecuado, evitando un número importante de conversiones y CPRE postoperatorias. (AU)

New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

Mutants of Arabidopsis defective in a sequence-specific mRNA degradation pathway.

One of the ways a cell can rapidly and tightly regulate gene expression is to target specific mRNAs for rapid decay. A number of mRNA instability sequences that mediate rapid mRNA decay have been identified, particularly from multicellular eukaryotes, but pinpointing the cellular components that play critical roles in sequence-specific decay in vivo has been more difficult. In contrast, general pathways of mRNA degradation in yeast have been well established through the analysis of mutants affecting the general mRNA decay machinery. Strategies to isolate mutants in sequence-specific mRNA decay pathways, although extremely limited so far, have the potential to be just as powerful. In the study reported here, a selection in transgenic plants allowed the isolation of rare mutants of Arabidopsis thaliana that elevate the abundance of mRNAs that contain the plant mRNA instability sequence called DST (downstream element). This instability sequence is highly conserved in unstable small auxin up RNA (SAUR) transcripts. Genetic analysis of two dst mutants isolated via this selection showed that they are incompletely dominant and represent two independent loci. In addition to affecting DST-containing transgene mRNAs, mutations at both loci increased the abundance of the endogenous DST-containing SAUR-AC1 mRNA, but not controls lacking DST sequences. That these phenotypes are caused by deficiencies in DST-mediated mRNA decay was supported by mRNA stability measurements in transgenic plants. Isolation of the dst mutants provides a means to study sequence-specific mRNA degradation in vivo and establishes a method to isolate similar mutants from other organisms.

Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis.

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

Arginase, Arginine Decarboxylase, Ornithine Decarboxylase, and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin).

Arginase (EC 3.5.3.1) activity has been found in the ovaries and Young fruits of tomato (Lycopersicon esculentum Mill. cv Rutgers).Changes in arginase, arginine decarboxylase (EC 4.1.1.19), and ornithine decarboxylase activity (EC 4.1.1.17) and levels of free and conjugated putrescine, spermidine, and spermine were determined in unpollinated ovaries and in parthenocarpic fruits during the early stages of development induced by 2,4-dichlorophenoxyacetic acid (2,4-D) or gibberellic acid (GA3). Levels of arginase, free spermine, and conjugates of the three polyamines were constant in unpollinated ovaries and characteristic of a presenescent step. A marked decrease in arginase activity, free spermine, and polyamine conjugates was associated with the initiation of fruit growth due to cell division, and when cell expansion was initiated, the absence of arginase indicated a redirection of nitrogen metabolism to the synthesis of arginine. A transient increase in arginine decarboxylase and ornithine decarboxylase was also observed in 2,4-D-induced fruits. In general, 2,4-D treatments produced faster changes than GA3, and without treatment, unpollinated ovaries developed only slightly and senescence was hardly visible. Sensitivity to 2,4-D and GA3 treatment remained for at least 2 weeks postanthesis.

N4-Hexanoylspermidine, a New Polyamine-Related Compound That Accumulates during Ovary and Petal Senescence in Pea.

A previously unknown polyamine conjugate that accumulates in senescing ovaries of pea (Pisum sativum L.) was shown by mass spectrometry, nuclear magnetic resonance, and chemical synthesis to be N4-hexanoylspermidine (hexanoyl-spd) This structure was indicated by analysis of the dansylated polyamine using fast atom bombardment mass spectrometry, following purification by high-performance liquid chromatography. Furthermore, acid hydrolysis of the compound yielded spermidine and hexanoic acid. 1H-nuclear magnetic resonance suggested that spermidine was substituted at N4 in the conjugate. Hexanoyl-spd was synthesized, and its didansyl derivative was shown to have an identical mass spectrum and high-performance liquid chromatography retention time as the derivatized natural compound. Further confirmation of its structure was obtained by comparison of the synthetic and natural polyamines as trifluoroacetyl derivatives using gas chromatography-mass spectrometry. This new polyamine conjugate is present in pea ovaries at low levels at anthesis and its concentration remains low in developing seeded fruit or in parthenocarpic fruit that have been induced by application of growth regulators to emasculated flowers or by topping the plant. Conjugate levels are also low in parthenocarpic fruit induced naturally in the slender (la crys) mutant. However, levels of hexanoyl-spd increase progressively in senescing petals and ovaries, beginning at anthesis or 2 d later, respectively.

Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum (L.).

A cDNA coding for arginine decarboxylase (ADC, EC 4.1.1.19) has been isolated from a cDNA library of parthenocarpic young fruits of Pisum sativum (L.). The deduced aminoacid sequence is 74%, 46% and 35% identical to ADCs from tomato, oat and Escherichia coli, respectively. When the pea ADC cDNA was put under the control of the galactose inducible yeast promoter CYC1-GAL10 and introduced into Saccharomyces cerevisiae, it conferred galactose-regulated expression of the ADC activity. The ADC activity expressed in S. cerevisiae was inhibited 99% by alpha-DL-difluoromethylarginine (DFMA), a specific inhibitor of ADC activity. No activity was detected in the untransformed S. cerevisiae, nor when it was transformed with an antisense ADC construct. This provides direct evidence that the ADC cDNA from pea encoded a functional, specific ADC activity and that S. cerevisiae is able to process correctly the protein. In the pea plant, gene expression of the ADC is high in young developing tissues like shoot tips, young leaflets and flower buds. Fully expanded leaflets and roots have much lower, but still detectable, levels of the ADC transcript. In the ovary and fruit, they are developmentally regulated, showing high levels of expression during the early stages of fruit growth, which in pea is mainly due to cell expansion. The observed changes in the steady-state levels of ADC mRNA alone, however, cannot account for the differences in ADC activity suggesting that other regulatory mechanisms must be acting.

Arginine Decarboxylase and Putrescine Oxidase in Ovaries of Pisum sativum L. (Changes during Ovary Senescence and Early Stages of Fruit Development).

Enzymatic activities involved in putrescine metabolism in ovaries of Pisum sativum L. during ovary senescence and fruit set were investigated. Accumulation of putrescine was observed during incubation of extracts from gibberellic acid-treated unpollinated ovaries (young developing fruits) but not in extracts from untreated ovaries (senescent ovaries). Extracts from pea ovaries showed arginine decarboxylase (ADC) activity, but ornithine decarboxylase and arginase activity were not detected. ADC activity decreased in presenescent ovaries and increased markedly after induction of fruit set with gibberellic acid. Increases in ADC activity were also observed with application of other plant growth substances (benzy-ladenine and 2,4-dichlorophenoxyacetic acid), after pollination, and in the slender (la crys) pea mutant. By contrast, putrescine oxidase activity increased in presenescent ovaries but did not increase during early fruit development. All of these results suggest that ADC and putrescine oxidase are involved in the control of putrescine metabolism. Ovary senescence is characterized by the absence of putrescine biosynthesis enzymes and increased levels of putrescine oxidase and fruit development by an increase in ADC and a constant level of putrescine oxidase.

Short report: dispersal of Aedes aegypti in an urban area after blood feeding as demonstrated by rubidium-marked eggs.

Strategies for the control of Aedes aegypti during urban outbreaks of dengue or yellow fever assume that this species has a maximum flight range of 50-100 meters. Because Ae. aegypti distributes its eggs among several oviposition sites, we postulated that dispersal is driven by the search for oviposition sites, so an ovipositing female may have to fly much further than 50-100 meters to lay all of her eggs. We developed a method for marking Ae. aegypti eggs with a rare alkali metal (rubidium) and showed that in an urban area, oviposition activity in a single gonotrophic cycle lasts several days and covers an area at least 840 meters in diameter (55.4 hectares). We suggest that current practice for the control of dengue and yellow fever transmission by focal treatments with insecticides 50-100 meters around presumed or confirmed cases is unlikely to be effective. Moreover, source reduction (the elimination of breeding sites) may enhance dissemination of virus-infected mosquitoes by reducing the number of available oviposition sites.

Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants Infected by Citrus Exocortis Viroid or Treated with Ethephon.

We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M. Belles, J. Carbonell, V. Conejero [1991] Plant Physiol 96: 1053-1059). This decrease correlates with a decrease in ODC activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. Interference with ethylene action by silver ions prevented the decrease in ODC activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infection and ethephon treatment are regulated via ODC activity and that conjugation is not involved.

Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations.

An ovitrap containing hay infusion and a second ovitrap adjacent to it containing a 10% dilution of the infusion in tap water together yielded 8 times more Aedes aegypti eggs than single CDC ovitraps containing tap water. These "enhanced pairs" were significantly more attractive than pairs with other combinations of infusion, water or methyl propionate, and have proven useful for daily monitoring of Ae. aegypti populations. Our results shed light on the oviposition behavior of Ae. aegypti in the field.