Genetic Analysis of Resistance to Leaf Rust and Yellow Rust in Spring Wheat Cultivar Kenya Kongoni.

The Kenyan wheat (Triticum aestivum L.) 'Kenya Kongoni' exhibits high levels of adult plant resistance (APR) to leaf rust (LR) and yellow rust (YR). We determined the genomic regions associated with LR and YR resistance in a population of 148 recombinant inbred lines generated from a cross between 'Avocet-YrA' and Kenya Kongoni. Field experiments to characterize APR to LR and YR were conducted in four and two Mexican or Uruguayan environments, respectively. A linkage map was constructed with 438 diversity arrays technology and 16 simple-sequence repeat markers by JoinMap 4.1 software. Genetic analyses showed that resistance to both rusts was determined by four to five APR genes, including Lr46/Yr29 and Sr2/Lr27/Yr30. Quantitative trait loci (QTL) analysis indicated that pleiotropic APR loci QYLr.cim-1BL corresponding to Lr46/Yr29 and QYLr.cim-7BL that is a putative novel QTL accounted for 5 to 57% and 12 to 35% of the phenotypic variation for resistance to LR and YR, respectively. These loci, in combination with another three LR QTL and two YR QTL, respectively, conferred high levels of resistance to both LR and YR in wheat under Mexican and Uruguayan environments. Among other detected QTL, QLr.cim-1DS, QLr.cim-2BL, and QYLr.icm-7BL may be new loci for APR to both rusts in common wheat.

Mapping and pyramiding of qualitative and quantitative resistance to stripe rust in barley.

The identification and location of sources of genetic resistance to plant diseases are important contributions to the development of resistant varieties. The combination of different sources and types of resistance in the same genotype should assist in the development of durably resistant varieties. Using a doubled haploid (DH), mapping population of barley, we mapped a qualitative resistance gene ( Rpsx) to barley stripe rust in the accession CI10587 (PI 243183) to the long arm of chromosome 1(7H). We combined the Rpsx gene, through a series of crosses, with three mapped and validated barley stripe rust resistance QTL alleles located on chromosomes 4(4H) (QTL4), 5(1H) (QTL5), and 7(5H) (QTL7). Three different barley DH populations were developed from these crosses, two combining Rpsx with QTL4 and QTL7, and the third combining Rpsx with QTL5. Disease severity testing in four environments and QTL mapping analyses confirmed the effects and locations of Rpsx, QTL4, and QTL5, thereby validating the original estimates of QTL location and effect. QTL alleles on chromosomes 4(4H) and 5(1H) were effective in decreasing disease severity in the absence of the resistance allele at Rpsx. Quantitative resistance effects were mainly additive, although magnitude interactions were detected. Our results indicate that combining qualitative and quantitative resistance in the same genotype is feasible. However, the durability of such resistance pyramids will require challenge from virulent isolates, which currently are not reported in North America.

Molecular marker mapping of leaf rust resistance gene lr46 and its association with stripe rust resistance gene yr29 in wheat.

ABSTRACT Leaf and stripe rusts, caused by Puccinia triticina and P. striiformis, respectively, are globally important fungal diseases of wheat that cause significant annual yield losses. A gene that confers slow rusting resistance to leaf rust, designated as Lr46, has recently been located on wheat chromosome 1B. The objectives of our study were to establish the precise genomic location of gene Lr46 using molecular approaches and to determine if there was an association of this locus with adult plant resistance to stripe rust. A population of 146 F(5) and F(6) lines produced from the cross of susceptible 'Avocet S' with resistant 'Pavon 76' was developed and classified for leaf rust and stripe rust severity for three seasons. Using patterns of segregation for the two diseases, we estimated that at least two genes with additive effects conferred resistance to leaf rust and three to four genes conferred resistance to stripe rust. Bulked segregant analysis and linkage mapping using amplified fragment length polymorphisms with the 'Avocet' x 'Pavon 76' population, F(3) progeny lines of a single chromosome recombinant line population from the cross 'Lalbahadur' x 'Lalbahadur (Pavon 1B)', and the International Triticeae Mapping Initiative population established the genomic location of Lr46 at the distal end of the long arm of wheat chromosome 1B. A gene that is closely linked to Lr46 and confers moderate levels of adult plant resistance to stripe rust is identified and designated as Yr29.

Chiral inversion of (R)-(-)-fenoprofen in guinea-pigs pretreated with clofibrate.

The influence of clofibrate on the stereoconversion of fenoprofen (FPF) was studied in guinea pigs. This hypolipidaemic agent has been related to some biochemical changes in the liver leading to an increase in the chiral inversion process. Two groups of animals (n = 6 per group) were pretreated with oral doses of clofibrate (280 mg/kg per day) for three days and were then given (R)- or (S)-FPF (5 mg/kg, IV). The FPF enantiomers were extracted from the guinea-pigs' plasma using a solid phase procedure and analysed by HPLC with previous derivatization with L-leucinamide. Pretreatment with clofibrate increased the chiral inversion of (R)-FPF in favour of the pharmacologically active (S)-FPF enantiomer. Before this metabolic interaction can be applied to therapy with fenoprofen, the toxic effects of (S)-(+)-FPF on the gastrointestinal and renal tracts and the interference by (R)-(-)-FPF with the metabolism of lipids should be thoroughly evaluated.

Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C.

Zonula occludens 2 (ZO-2) protein is a tight-junction phos phorylated protein that belongs to the membrane-associated guanylate kinase ('MAGUK') family. Here we study the interaction between ZO-2 and protein kinase C (PKC). We have constructed two ZO-2 fusion proteins of the middle (3PSG) and C-terminal (AP) regions of the molecule and demonstrate that they are phosphorylated by PKC isoenzymes beta, epsilon, lambda and zeta. To understand the physiological significance of the interaction between ZO-2 and PKC, we analysed the phosphorylation state of ZO-2 immunoprecipitated from monolayers with mature tight junctions or from cells that either lack them or have them disassembled through Ca(2+) chelation. We found that in the latter condition the phosphorylation level of ZO-2 is significantly higher and is due to the action of both PKC and cAMP-dependent protein kinase. These results therefore suggest that the phosphorylated state of ZO-2 restrains its capacity to operate at the junctional complex.

Correction of insulin resistance in methimazole-treated patients with Graves disease.

The effect of thyroid hormone excess on glucose tolerance as well as insulin secretion and its peripheral action has been a matter of debate for many years. Thyrotoxicosis caused by Graves' disease is associated in some patients with impaired glucose tolerance and insulin resistance. The objective of the present study was to investigate if the insulin sensitivity, assessed by the euglycemic insulin clamp technique, increase after correction of hyperthyroidism due to Graves' disease. After four months of medical treatment, patients became euthyroid and insulin sensitivity increased significantly from 3.47 to 6.39 mg/kg/min; therefore it was concluded that insulin resistance could be improved after successful treatment of hyperthyroidism. The precise mechanism underlying the effect of thyroid hormone excess on insulin sensitivity remains to be elucidated.

Tight junction proteins ZO-1, ZO-2, and occludin along isolated renal tubules.

BACKGROUND: Tight junctions play a critical role in tubular function. In mammalian kidney, the transepithelial electrical resistance and the complexity of the tight junction increase from the proximal to the collecting tubule. The differential expression of three tight junction proteins, ZO-1, ZO-2, and occludin, along isolated rabbit renal tubules is examined in this article. METHODS: Microdissected rabbit renal tubules were processed for immunofluorescence detection of ZO-1, ZO-2, and occludin. The quantitation of these proteins was done by Western blot determinations in Percoll isolated tubules. RESULTS: ZO-1 stained cell boundaries independently of the identity of the tubule. However, the amount found in distal segments was significantly higher than that expressed in proximal regions. ZO-2 in the proximal region was found diffusely distributed in the cytoplasm, with faint staining at cell borders, while a clear signal at cell perimeters was detectable from the Henle's loop to collecting tubules. Nuclear staining of ZO-2 was found along the whole nephron. The presence of occludin at the proximal region was faint and discontinuous, while its expression in the more distant portions was conspicuous. The quantity of ZO-2 and occludin present at the distal region was significantly higher compared with the proximal segment. CONCLUSIONS: The distribution of ZO-1, ZO-2, and occludin follows the increase in junction complexity encountered in renal tubules. The amount of the three proteins found in proximal and distal segments is significantly higher in the latter.

Molecular characterization of the tight junction protein ZO-1 in MDCK cells.

Most of the information on the structure and function of the tight junction (TJ) has been obtained in MDCK cells. Accordingly, we have sequenced ZO-1 in this cell type, because this protein is involved in the response of the TJ to changes in Ca2+, phosphorylation, and the cytoskeleton. ZO-1 of MDCK cells comprises 6805 bp with a predicted open reading frame of 1769 amino acids. This sequence is 92 and 87% homologous to human and mouse ZO-1, respectively. Two nuclear sorting signals located at the PDZ1 and GK domains and 17 SH3 putative binding sites at the proline-rich domain were detected. We found two new splicing regions at the proline-rich region: beta had not been reported in human and mouse counterparts, and gamma, which was previously sequenced in human and mouse ZO-1, is now identified as a splicing region. The expression of different beta and gamma isoforms varies according to the tissue tested. With the information provided by the sequence, Southern blot, and PCR experiments we can predict a single genomic copy of MDCK-ZO-1 that is at least 13.16 kb long. MDCK-ZO-1 mRNA is 7.4 kb long. Its expression is regulated by calcium, while the expression of MDCK-ZO-1 protein is not.

Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium.

The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and alpha-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.

Higher prevalence of diabetes in hypertensive subjects with upper body fat distribution.

OBJECTIVE: To analyze the association of hypertension and upper body fat distribution on the occurrence of non-insulin-dependent diabetes mellitus in Mexicans. MATERIAL AND METHODS: It was a population-based cross-sectional study in Cuajimalpa, a district of Mexico City. A total of 1066 subjects were home interviewed, and attended our clinic for fasting plasma glucose sampling, blood pressure and anthropometric measurements. Diabetes was defined according to the World Health Organization criteria, and hypertension as a blood pressure equal to or greater than 140/90. The ratio of upper to lower body skinfolds was used to estimate body fat distribution. RESULTS: The prevalence of diabetes was 12.0%. There was a significant positive trend in the age and sex adjusted prevalence of diabetes according to the magnitude of hypertension (p = 0.0006) and upper body fat distribution (p = 0.007). The age and sex adjusted prevalence in normotensive subjects with lower body fat distribution was 7.1% (95% confidence interval 5.9-8.2) whereas it was 19.9% (CI 17.0-22.8) in those with hypertension and upper body fat distribution. The prevalence of diabetes in Mexicans was high and it may be related to a genetic susceptibility for an insulin resistance syndrome. CONCLUSIONS: These results indicate that there is a dose response effect in the association of hypertension and upper body fat distribution with diabetes in Mexicans, and that there may be an interaction in the effect of hypertension and body fat distribution in this syndrome.

Antibody to hepatitis C virus in volunteer blood donors of the Hospital de Especialidades, National Medical Center, Mexico City.

The present survey was performed to assess the hepatitis C virus seroprevalence in volunteer blood donors of the National Medical Center, Hospital de Especialidades, Mexico City. Serum samples from 1100 individuals were collected. We selected second generation enzyme-linked immunosorbent assay (ELISA) (Abbott HCV EIA) test for the screening. The antibodies against hepatitis C virus (anti-HCV) in the volunteer blood donors were positive in 0.7%. The second generation ELISA test is useful as a screening test and may lead to decreasing the incidence of posttransfusional hepatitis C virus infection.

[Monoclonal antibodies against the lipopolysaccharide of Salmonella typhi 9,12,VI:d. Analysis of passive protection in a mouse model of typhoid fever]. / Anticuerpos monoclonales antilipopolisacarido de Salmonella typhi 9,12,Vi:d: análisis de protección pasiva en un modelo murino de fiebre tifoidea.

The current work was undertaken in order to assess the role of the monoclonal antibodies (Mabs) to lipopolysaccharide (LPS) of Salmonella typhi in the induction of passive protection against the challenge with the bacteria in a mice model. BALB/c mice were immunized with the whole bacteria, mice with high anti-LPS antibody titers were killed, the spleens were removed and splenocytes were fused with the mouse plasmocytoma SP2/0. Two IgM monoclonal antibodies against porins were developed. Each one of these Mabs recognized the polysaccharide region of LPS. Passive immunization with supernatant fluid of mice with one of these monoclonal antibodies did not protect against challenged with 20 LD50 and 100 LD50 of Salmonella typhi. The results suggest that LPS is not valuable immunogen for the induction of a protective status against typhoid fever.

[Elaboration of an immunosorbent for the purification of porins from Salmonella typhi 9, 12, Vi:d]. / Elaboración de un inmunoadsorbente para la purificación de porinas de Salmonella typhi 9, 12, Vi: d.

The current work was undertaken to purify porins of Salmonella typhi, which are outer membrane proteins (OMPs) that induce protection in mice against challenge with the bacteria in mucin. OMPs, isolated with a non-ionic detergent, had a 4% contamination with LPS (endotoxin) and molecular sizes ranging from 17 to 70 KDa. Porins (Mw 38-41 KDa) were isolated from OMPs preparative SDS-PAGE. Anti-porins antisera were raised in rabbits and specific IgG was purified, which was coupled to Sepharose-CNBr. This immunosorbent was used to purify LPS-free porins.