Expressed sequence enrichment for candidate gene analysis of citrus tristeza virus resistance.

Several studies have reported markers linked to a putative resistance gene from Poncirus trifoliata ( Ctv-R) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (CTV). To be successful in both marker-assisted selection and transformation experiments, its accurate mapping is needed. Several factors may affect its localization, among them two are considered here: the definition of resistance and the genetic background of progeny. Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange ( Citrus aurantium), a citrus rootstock well-adapted to arid and semi-arid areas, were used for linkage group-4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. The new one A(7+3+4) is now saturated with 48 markers including expressed sequences. Surprisingly, sour orange was as resistant to the CTV isolate tested as was P. trifoliata, and three hybrids that carry Ctv-R, as deduced from its flanking markers, are susceptible to CTV. The new linkage maps were used to map Ctv-R under the hypothesis of monogenic inheritance. Its position on linkage group 4 of P. trifoliata differs from the location previously reported in other progenies. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction, which is not compatible with the hypothesis of a single gene controlling resistance. Two major issues are discussed: another type of genetic analysis of CTV resistance is needed to avoid the assumption of monogenic inheritance, and transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees.

The diversification of Citrus clementina Hort. ex Tan., a vegetatively propagated crop species.

Clementines, due to their high quality, are one of the most important cultivated citrus mandarins. As in the case of sweet orange and satsuma mandarins, genetic variability within this species is minimal when analyzed by molecular markers, because the existing varieties have not been obtained through hybridization, but through the selection of spontaneous mutations affecting traits of agronomic interest. This would explain, at least in part, the greater diversity for agronomic traits when compared to the variability for molecular markers. Another possible (nonexclusive) reason is that the types of molecular marker used are not focused on the kind of molecular change mainly involved in the origination of new clementine cultivars; i.e., are all sources of variation equally involved in the diversification of these plants? To answer this question, different kinds of markers based on primers of random sequence, simple sequence repeats, and retrotransposon sequences that may reveal point mutations, and somatic recombination and transposon activity, respectively, were used to compare the level of variability among 24 clementine varieties. Their ISSR, RAPD, and AFLP analysis provided only two polymorphic bands, distinguishing just two varieties. No variability was found by SSRs, i.e., no new allele arising through somatic recombination was detected. Instead, the amplification of sequences adjacent to retrotransposons yielded a higher number of polymorphisms (14.6 vs 2.4% for the previous mentioned marker types). Two geographical distant groups, one from North Africa and the other from Spain, have evolved in agreement with polymorphisms based on IRAP markers anchored to, at least, two different Copia-like retrotransposon sequences. Therefore, this study suggests that the DNA of this type of mobile elements is evolving faster than the DNA of other markers in this clonal lineage.

Hyperlipidemia in liver transplant recipients: prevalence and risk factors.

Hyperlipidemia is common in transplant patients. Although a causal relationship to the use of cyclosporine is accepted, additional risk factors are as yet unidentified. Eighty-five liver transplant recipients treated with standard triple immunosuppression with a survival of at least 6 months were evaluated. Pretransplantation and posttransplantation variables were analyzed as predictive factors of posttransplantation hyperlipidemia. Serum cholesterol and triglyceride levels were considered elevated if they were > 250 mg/dL and > 150 mg/dL, respectively. Before and after transplantation, hyperlipidemia occurred in 8% (95% confidence interval [CI], 3% to 16%) and 66% (95% CI, 55% to 76%), respectively. After transplantation, 47% (95% CI, 36% to 58%) of the patients had isolated high triglyceride levels, 12% (95% CI, 6% to 21%) had both elevated cholesterol and triglyceride levels, and 7% (95% CI, 3% to 15%) had isolated elevated cholesterol levels. Hypertriglyceridemia occurred early after transplantation (67% by first month) and persisted nearly unchanged throughout the first year. In contrast, cholesterol levels increased with time (5%, 13%, and 27% at 1, 3, and 6 months, respectively). In univariate analysis, factors predictive of hypercholesterolemia included female sex, pretransplantation cholestatic liver disease, pretransplantation cholesterol levels > 141 mg/dL, and > 3 methylprednisolone "boluses." In multivariate analysis, only a pretransplantation cholesterol level of > 141 mg/dL (odds ratio [OR], 5.5; 95% CI, 1.4 to 21) was an independent risk factor. Risk factors associated with hypertriglyceridemia included pretransplantation hepatocellular liver disease (OR, 6.8; 95% CI, 1.2 to 40) and posttransplantation renal dysfunction (OR, 5.4; 95% CI, 1.9 to 15.4). Hyperlipidemia is a frequent finding in liver transplant recipients, and hypertriglyceridemia is the most common abnormality. Hypertriglyceridemia can be predicted on the basis of pretransplant hepatocellular disease and posttransplant renal dysfunction. Pretransplant serum cholesterol level is an independent risk factor for posttransplant hypercholesterolemia.

[Changes in lipoprotein(a) after heart transplantation]. / Alteraciones de la lipoproteína (a) tras el trasplante cardíaco.

AIM: The purpose of this study was to analyze the evolutionary changes of lipoprotein (a) levels occurring in heart transplant and to evaluate the possible relationship between the plasma concentration of this lipoprotein and the immunosuppressor drugs normally used in this type of transplant. METHOD: 17 patients undergoing heart transplant and with no history of dyslipemia or dysglucemia were studied. Patients with metabolic alterations after the transplant were excluded (except when these alterations occurred during the first week), as well as those who showed intercurrent processes near to the determinations. These were performed before the transplant, and 1, 2, 4 and 6 months later. RESULTS: An increase of lipoprotein (a) was observed after the transplant, with a subsequent progressive decrease. Significant differences were found between the levels prior to the transplant (9.18 +/- 8.66) and 6 months later (7.53 +/- 8.86), with no differences found between the previous concentrations and the determinations after one month (10.29 +/- 7.58), two months (8.06 +/- 7.90) and four months (8.82 +/- 7.84). Differences were also observed between the values of the first month in relation to the subsequent months, as well as between the 4th and the 6th month. No relationship was noticed between the levels of this lipoprotein and those of cyclosporin (r = 0.10), azatioprine (r = 0.17) and deflazacort (r = 0.19). CONCLUSIONS: The lipoprotein (a) levels increase after heart transplant, with a subsequent gradual decrease even below the previous figures. These levels bear no relationship with the dose of immunosupressors normally used in heart transplant.

Salt tolerance in Lycopersicon species. III. Detection of quantitative trait loci by means of molecular markers.

A segregating population derived from a cross between L. esculentum cv Madrigal and a line of L. pimpinellifolium was used to identify genetic markers linked to QTLs involved in salinity tolerance in terms of yield, under a conductivity of 15 dS/m (171.1 mM NaCl). Six markers resulted, associated with QTLs affecting average fruit weight, fruit number and total weight under salinity. One of them, Aco-1, behaves reversely to the expectation from parental means; this and other features make it a promising target to obtain salt-tolerant tomatoes. Epistatic interactions were also found, thus affecting the criteria for marker-assisted selection. Although only 41% of the loci assayed were polymorphic, a high efficiency in identifying QTLs was achieved, since 43% of the marker loci are linked to QTLs for the trait under study.

Genetic variability in Lycopersicon species and their genetic relationships.

Genetic diversity has to be described and measured in order to establish breeding strategies and manage genetic resources. It is also fundamental to develop a comparative intraspecific study before attempting to discuss and conclude any phylogenetic relationship. The genetic variability of Lycopersicon species was studied using starch gel electrophoresis of 11 enzymatic systems in a hierarchical fashion. The species with the greatest genetic variability are L. chilense, L. peruvianum and L. pennellii, mainly due to the within-line component. L. chmielewskii, L. parviflorum and L. pimpinellifolium show an intermediate total variability and their between-component clearly predominates over the within-component. The least variable species are L. cheesmanii and L. esculentum. Cluster analysis resulted in three main groups: one formed by the cultigen, L. pimpinellifolium, L. cheesmanii and L. peruvianum;another by two species with self-incompatibility systems, L. pennelli and L. chilense; and another by two autogamous species L. chmielewskii and L. parviflorum. With respect to L. esculentum the farthest related species is Solanum rickii and the closest, L. pimpinellifolium.

Salt tolerance in Lycopersicon species. I. Character definition and changes in gene expression.

Salt tolerance defined in terms of fruit yield under different NaCl concentrations (171.1 and 325.1 mM) is analyzed in 11 lines belonging to: Lycopersicon esculentum, L. cheesmanii, L. chmielewski, L. peruvianum and L. pimpinellifolium. Four L. pimpinellifolium lines and two L. cheesmanii lines tolerated the 171.1mM treatment; the latter species even tolerates 325.1 mM of NaCl. Changes in gene expression induced by salt treatment were also investigated by studying anther and leaf zymograms for L. esculentum and one salt-tolerant L. pimpinellifolium line, and leaf proteinograms for all lines. Changes in leaf PRX and MDH enzymatic systems were detected, mainly in the salt-sensitive genotype (L. esculentum). Four saltrelated peptides from 14 500 to 40 000 daltons were found. A polyclonal antibody raised against one of these peptides (number 2), also binds another peptide, named 2', of much higher molecular weight, present both in control and salt-tolerant L. cheesmanii lines at the end of 171.1 mM treatment. The xero-halophyte shrub Atriplex halimus also showed a likely 2'-homologous peptide with this treatment, while its counterpart C3 species A. triangularis did not.

Salt tolerance in Lycopersicon species. II. Genetic effects and a search for associated traits.

Eleven quantitative traits, mostly related to tomato plant growth and fruit set, and their association with salt tolerance in terms of fruit yield under a 171.1 mM NaCl treatment have been investigated in 206 progeny derived from an interspecific hybrid, L. esculentum x L. pimpinellifolium, by self-pollination. None of the traits were highly correlated phenotypically to salt tolerance; however, the immunologically-detected presence of peptide 2' was significantly associated with high total fruit weight (TW) and number (FN) under saline treatment. Broad-sense heritability was estimated for these two salt-tolerance components as 53.44 and 72.59 %, respectively. Non-additive gene effects, which have to be considered in a breeding program for salt tolerance, have been detected in TW, FN and in average fruit weight (FW). Given that different types of gene action have been found depending on the presence or absence of a high NaCl concentration in the nutrient solution, a different set of genes, or genes, differently regulated, must be involved in the expression of TW, FN and other fruit-related characters depending on this environmental condition.

Euglobulin clot lysis induced by tissue-type plasminogen activator is reduced in subjects with increased levels of lipoprotein (a).

Several reports have evaluated the in vitro effect of lipoprotein(a) [Lp(a)] levels on the fibrinolytic system, suggesting that high Lp(a) levels may inhibit fibrinolysis by competing for plasminogen binding in different systems. We have studied plasminogen activation induced by tissue-type plasminogen activator (t-PA), as well as other fibrinolytic parameters, in 25 subjects with Lp(a) levels greater than 30 mg/dl and the results were compared with those found in 23 subjects with Lp(a) less than 30 mg/dl. Both groups were similar in age, sex distribution, living habits and lipid pattern. Plasminogen activation, when measured by t-PA-induced euglobulin clot lysis, was significantly decreased in the group with elevated Lp(a) levels (lysis time, 16.7 +/- 3.3 min) compared with the group with low Lp(a) levels (11.8 +/- 2.0 min), although 8 of the 25 subjects with high Lp(a) levels showed plasminogen activation within the range of the control group. A positive significant correlation between Lp(a) levels and t-PA-induced euglobulin clot lysis time was found. No statistical differences were demonstrated between groups for the other fibrinolytic parameters studied. Addition of purified Lp(a) to the euglobulin fraction or to plasma resulted in a decrease in euglobulin clot lysis. The present study shows that t-PA induced plasminogen activation is decreased in individuals with high circulating levels of Lp(a) supporting the hypothesis that Lp(a) may interfere with the physiological functions of plasminogen.

[Combined therapy with chenodeoxycholic acid and ursodeoxycholic acid of radiolucent biliary lithiasis]. / Tratamiento combinado con los ácidos quenodesoxicólico (AQDC) y ursodesoxicólico (AUDC) en la litiasis biliar radiotransparente.

27 patients with radiolucent biliary stones in normofunctioning gall bladder were treated with a combination of CDCA and UDCA (CDCA doses = 7.7 mg/kg/day; UDCA--6.5 mg/kg/day); only 23 continued in the study for at least one year. The reasons for leaving were in one case the necessity of a cholecystectomy and treatment interruption in 3 cases. The rate of complete dissolution was of 39%, being of 17% the partial dissolution rate. The only biochemical alteration was the increase of HDL cholesterol at 9 and 12 months of treatment (p less than 0.05 and p less than 0.01 respectively). Transient diarrhea was seen in 17% of the patients and it did not require treatment. The incidence of liver colic during the year before treatment was 26% and it decreased during the 1st year of treatment to 4%. The non-specific dyspepsia observed during the year before treatment was of 48%, and decreased to 17% during the first year of treatment. Only one patient developed calcification of the gallstone, 12 months after treatment.

Increase in serum total cholesterol and low-density lipoprotein cholesterol by high-dose chenodeoxycholic acid in patients with radiolucent gallstones significantly reversed during preventive low dose after gallstone dissolution.

This study examines the effects of high-dose chenodeoxycholic acid (CDCA) on serum lipids and lipoproteins in 25 patients who underwent a 12-month therapy with CDCA for the dissolution of cholesterol gallstones. All patients received a daily dose of CDCA of 15 mg/kg body weight. Complete dissolution of gallstones was achieved in 16 cases. After 6 months of therapy the mean total cholesterol levels and low-density lipoprotein cholesterol (C-LDL) significantly increased (p less than 0.001). The decrease of mean triglyceride levels was significant too (p less than 0.01), although it was greater among patients with effective gallstone dissolution than in patients with persistent gallstones. The effects of high-dose CDCA after 12 months of therapy were similar to those observed at 6 months. 12 patients of the gallstone dissolution group were treated with a continuous low dose of CDCA (250 mg/day) for preventing gallstone recurrence. 6 months after dissolution, the mean total cholesterol levels and C-LDL significantly decreased (p less than 0.01 and less than 0.05, respectively). High-dose administration of CDCA produced an increase in total cholesterol and C-LDL, but did not alter high-density lipoprotein cholesterol levels. These effects were significantly reversed when a preventive low dose of CDCA was given after gallstone dissolution.

Reflex cardiovascular response to acute myocardial ischaemia.

The usual cardiovascular response to acute myocardial ischaemia is either pressor, characterised by tachycardia and hypertension or depressor, manifested by bradycardia, hypotension and systemic vasodilatation. We studied the incidence of ectopic arrhythmias, the changes in heart rate, in left ventricular pressure and dP/dt and the changes in plasma level of free fatty acids. Acute anterior ischaemia increased the heart rate, left ventricular dP/dtmax and plasma level of free fatty acids and developed frequent ventricular ectopic arrhythmias. Previous injection of propranolol prevented the elevation of plasma free fatty acids. Acute inferior ischaemia produced a slight decrease in heart rate, no change in plasma level of free fatty acids and a significant decrease in left ventricular dP/dtmax. The experimental model allowed us to study the response to acute myocardial ischaemia in conscious dogs; the results obtained point to a preferential location of the pressor and depressor receptors respectively to the anterior and inferior surfaces of the left ventricle.

[Assessment of fetal maturity by measurement of the phospholipids in the amniotic fluid. Comparison of the results with the lecithin/sphingomyelin ratio]. / Appréciation de la maturité foetale par la mesure des phospholipides du liquide amniotique. Comparaison des résultats avec le rapport lécithine/sphingomyéline.

The authors explored the possibility of assessing fetal lung maturity by measuring the phospholipids in the amniotic fluid by the enzymatic technique of Takayama et al. [8]. The study was conducted on 41 pregnancies. The results obtained were evaluated comparatively with the lecithin/sphingomyelin ratio (L/S). The authors found that the L/S ratio can be used to determine fetal lung maturity in 93% of cases with 2/14 false maturities and 1/27 false immaturities. The assay of the phospholipids containing choline by the method of Takayama et al. [8] gives a much less precise assessment of fetal lung maturity: only 68% of the results were concordant; 5/14 false maturities and 8/27 false immaturities. These differences can be explained by the fact that the method of Takayama et al. [8] takes into account substances which are not related to process of fetal lung maturation.

The influence of several factors that intervene in hemodialysis on serum levels of triglycerides and free fatty acids.

The effect of the administration of heparin and glucose, in doses and sequence similar to those of hemodialysis, on serum free fatty acids and triglycerides, was studied in eight patients with chronic renal failure who had not undergone hemodialysis. The results are compared with the action exerted by hemodialysis on the same parameters. During hemodialysis, the values of triglycerides are not significantly different from those obtained at similar intervals after the administration of glucose and/or heparin, with the exception that the values obtained five hours after beginning hemodialysis are lower than those obtained five hours after the administration of glucose (P less than 0.005). The levels of free fatty acids obtained during hemodialysis are higher (P less than 0.005) than those found during the other tests. The possible origin of these high fatty acid values is discussed, and the possibility that they originate from the acetate in the dialysis liquid is suggested.