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P Cortes

Publications and source records attributed to P Cortes.

At least 19 recordsLinked to original sources
PubMed2006

Otitis interna (labyrinthitis) associated with Salmonella enterica arizonae in turkey poults.

Otitis interna was diagnosed in five 9-to-21-day-old turkey poults with clinical signs of paralysis, opisthotonus, torticollis, blindness, and increased mortality. Gross and microscopic lesions in the poults included omphalitis, typhlitis, hepatitis, meningoencephalitis, ophthalmitis, neuritis and ganglionitis of the vestibulocochlear nerve, and otitis interna. Salmonella enterica arizonae was isolated from the brains, eyes, intestines, yolk sacs, and livers of poults. Birds with otitis interna also had meningoencephalitis. It is most likely that the S. enterica arizonae infection spread from the brain to the internal ears through the vestibulocochlear nerve. This is the first documentation of otitis interna caused by bacteria in an avian species.

Animals↗
PubMed2005

Summer and winter sensitivity of leaves and xylem to minimum freezing temperatures: a comparison of co-occurring Mediterranean oaks that differ in leaf lifespan.

Freezing sensitivity of leaves and xylem was examined in four co-occurring Mediterranean oaks (Quercus spp.) grown in a common garden to determine whether freezing responses of leaves and xylem were coordinated and could be predicted by leaf lifespan. Freezing-induced embolism and loss of photosynthetic function were measured after overnight exposure to a range of subzero temperatures in both summer and winter. Both measures were found to be dependent on minimum freezing temperature and were correlated with leaf lifespan and vessel diameter. The dependence of xylem embolism on minimum freezing temperature may result from the decline in water potential with ice temperature that influences the redistribution of water during freezing and leads to an increase in xylem tension. Winter acclimatization had a relatively small effect on the vulnerability to freezing-induced embolism, although leaf photosynthetic function showed a strong acclimatization response, particularly in the two evergreen species. Quercus ilex, the species with the longest leaf lifespan and narrowest vessel diameters, showed the highest freezing tolerance. This helps explain its ability to inhabit a broad range throughout the Mediterranean region. By contrast, the inability of the deciduous oaks to maintain photosynthetic and vascular function throughout the winter indicates a competitive disadvantage that may prevent them from expanding their ranges.

Climate↗
PubMed2001

V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.

Alleles↗
PubMed2001

Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis.

Animals↗
PubMed2000

N-terminal RAG1 frameshift mutations in Omenn's syndrome: internal methionine usage leads to partial V(D)J recombination activity and reveals a fundamental role in vivo for the N-terminal domains.

Omenn's syndrome is an autosomal recessive primary immunodeficiency characterized by variable numbers of T lymphocytes of limited clonality, hypereosinophilia, and high IgE levels with a paradoxical absence of circulating B lymphocytes. We have previously attributed this disorder to missense mutations that render the RAG1/RAG2 recombinase only partially active. Here we report seven Omenn's patients with a novel class of genetic lesions: frameshift mutations within the 5' coding region of RAG1. Interestingly, we demonstrate in transient expression experiments that these frameshift deletion alleles remain partially functional for both deletional and inversional recombination and can hence explain the partial rearrangement phenotype observed in these patients. The rearrangement activity is mediated by truncated RAG1 proteins that are generated by alternative ATG usage 3' to the frameshift deletion and that demonstrate improper cellular localization. Taken together, our results suggest a novel mechanism for the development of immunodeficiency in a subset of Omenn's syndrome patients.

Alleles↗
PubMed2000

The genetic and biochemical basis of Omenn syndrome.

Omenn syndrome (OS) is a peculiar, autosomal recessive severe combined immunodeficiency (SCID) associated with early-onset, generalized, exudative erythrodermia; lymphoadenopathy; hepato- and splenomegaly; hypereosinophilia; elevated serum IgE; and normal to high activated, yet non-functional, oligoclonal T cells. Recent investigations have shown that the primum movens of all these puzzling features lies in a defect of the lymphoid-specific V(D)J recombination process. Abnormalities in both alleles of either Rag-1 or -2 genes are found in all OS patients. At variance with T B- SCID, whose Rag mutations represent null alleles, OS mutations maintain a residual recombination activity, allowing limited T-cell receptor gene rearrangements to occur in the thymus. The gene rearrangements are subsequently expanded in the periphery after environmental antigen exposure. Missense mutations detected in OS have been examined in a number of biochemical assays and have contributed to dissect the various functional domains of both Rag-1 and Rag-2 proteins. The examination of a set of mutations occurring in the Rag-1 N-terminal portion has demonstrated that this region plays a fundamental role in vivo. The elucidation of the molecular basis of OS has allowed us to perform early prenatal diagnosis and could be the basis for trials of in utero bone marrow transplantation or gene therapy approaches.

Animals↗
PubMed2000

F-actin fiber distribution in glomerular cells: structural and functional implications.

BACKGROUND: Glomerular distention is associated with cellular mechanical strain. In addition, glomerular distention/contraction is assumed to influence the filtration rate through changes in filtration surface area. A contractile cytoskeleton in podocytes and mesangial cells, formed by F-actin-containing stress fibers, maintains structural integrity and modulates glomerular expansion. In this study, the glomerular cell distribution of F-actin and vimentin filaments was studied in normal control and nine-month streptozotocin-diabetic rats. Results in situ were compared with observations in tissue culture. METHODS: Microdissected rat glomeruli were perfused to obtain a physiological 25% glomerular expansion over the basal value. Fixation was done without change in glomerular volume. Dual fluorescent labeling of F-actin and vimentin was carried out, and samples were examined by confocal laser scanning microscopy. RESULTS: The podocyte cell bodies and their cytoplasmic projections, including the foot processes, contained bundles of vimentin-containing fibers. Except for a thin layer at the base of foot processes, they did not demonstrate F-actin. While mesangial cells in culture had F-actin as long stress fibers resembling tense cables, mesangial cells stretched in situ contained a maze of short tortuous F-actin fibers organized in bundles that often encircled vascular spaces. This fibrillar organization was disrupted in diabetic glomeruli. CONCLUSION: Mesangial cells, but not podocytes, contain a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Together with previous observations, the distribution of this cytoskeleton suggests that mesangial cell contraction may be involved in the redistribution of glomerular capillary blood flow, but not substantially in the modulation of glomerular distention. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes.

Actins↗
PubMed2000

Modelling the effects of vascular stress in mesangial cells.

It has recently been shown that mesangial cells are subjected to multiple forms of mechanical strain (fluid shear, hydrostatic pressure, and triaxial stretch) as a result of forces exerted by the vasculature. Nevertheless, the exact nature and the relative response to these stimuli have not been clarified. Although it is now well established that cyclic stretching of mesangial cells in culture results in the overproduction of extracellular matrix, indicating how intraglomerular hypertension may lead to glomerular scar formation, the contribution of different intracellular signalling mechanisms and extracellular mediators of the response are only now being identified. Recent studies point to a role for high glucose concentrations, transforming growth factor beta and its receptors, vascular endothelial growth factor, and connective tissue growth factor as important mediators, or modifiers of the response to mechanical strain. Although evidence exists for a role for protein kinase C, recent studies also implicate the mitogen-activated protein kinases along with enhanced DNA-binding activity of AP-1 as part of the signalling cascade altering matrix synthesis and cell proliferation in response to stretch. Finally, recent studies examining the effects of oscillating hyperbaric pressure demonstrate similarities, as well as differences, in comparison to those of cyclic stretch.

Animals↗
PubMed2000

Mutations in conserved regions of the predicted RAG2 kelch repeats block initiation of V(D)J recombination and result in primary immunodeficiencies.

The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed beta-propeller structure, with the second beta-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and DeltaI273 within the predicted second beta-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second beta-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.

Amino Acid Sequence↗
PubMed1999

The RAG1/RAG2 complex constitutes a 3' flap endonuclease: implications for junctional diversity in V(D)J and transpositional recombination.

During V(D)J recombination, processing of branched coding end intermediates is essential for generating junctional diversity. Here, we report that the RAG1/ RAG2 recombinase is a 3' flap endonuclease. Substrates of this nuclease activity include various coding end intermediates, suggesting a direct role for RAG1/ RAG2 in generating junctional diversity during V(D)J recombination. Evidence is also provided indicating that site-specific RSS nicking involves RAG1/RAG2-mediated processing of a localized flap-like structure, implying 3' flap nicking in multiple DNA processing reactions. We have also demonstrated that the bacterial transposase Tn10 contains a 3' flap endonuclease activity, suggesting a mechanistic parallel between RAG1/RAG2 and other transposases. Based on these data, we propose that numerous transposases may facilitate genomic evolution by removing single-stranded extensions during the processing of excision site junctions.

Animals↗
PubMed1999

Characterization of the rat mesangial cell type 2 sulfonylurea receptor.

BACKGROUND: Sulfonylurea receptors are classified as either high-affinity type 1 (SUR1) or low-affinity type 2 receptors (SUR2), and the gene expression of SURs has recently been demonstrated in kidney. However, functional data regarding a renal SUR are lacking. We previously demonstrated that mesangial cell (MC) gene and protein expression of extracellular matrix components were up-regulated by the sulfonylurea, tolazamide. After noting this biological response, we next sought to investigate the presence of a sulfonylurea receptor in rat MCs. METHODS: Equilibrium binding studies employing [3H]glibenclamide as a ligand were performed on crude MC membrane preparations. Gene expression for SUR was explored by Northern analysis of cultured MCs and whole kidney tissue. The effect of sulfonylurea on intracellular Ca2+ in MCs was assayed by spectrofluorometry, and glibenclamide-induced changes in the contractility of MCs were assessed. RESULTS: MCs bound [3H]glibenclamide with a KD of 2.6 microM and a Bmax of 30.4 pmol/mg protein as determined by Scatchard analysis. Three SUR2 transcripts were detected in MCs. A major transcript was detected at 5.5 kb and minor transcripts at 7.5 and 8.6 kb. Following sulfonylurea treatment of MCs, real-time videomicroscopy revealed intense MC contraction, coinciding with oscillatory increments of intracellular Ca2+ concentration. Further evidence of sulfonylurea-induced MC contraction was demonstrated by glibenclamide-induced deformation of a silicone rubber substrate. CONCLUSIONS: These results demonstrate that SUR2 resides on MCs. Functional activation of this receptor by sulfonylurea induces Ca2+ transients that result in MC contraction.

ATP-Binding Cassette Transporters↗
PubMed1999

TGF-beta receptor expression and binding in rat mesangial cells: modulation by glucose and cyclic mechanical strain.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured mesangial cells (MCs) exposed to high glucose concentrations and cyclic mechanical strain. This change is associated with increased levels of TGF-beta, but may also involve alterations in receptor expression and binding. METHODS: Rat MCs cultured in media containing either 8 or 35 mM glucose were seeded into culture plates with elastin-coated flexible bottoms. Thereafter, they were subjected to cyclic stretch or static conditions and then examined for 125I-TGF-beta1 binding and expression of TGF-beta receptors at the gene and protein levels. RESULTS: Kinetic studies showed that MCs bound TGF-beta1 in a time- and concentration-dependent manner, expressing 6800 high-affinity receptors per cell, with an apparent dissociation constant (Kd) of 15.4 pM, while cross-linking analysis identified three TGF-beta receptors (betaR) corresponding to betaRI, betaRII, and betaRIII of 54, 73, and 200 kDa, respectively. Immunocytochemical studies of betaRI and betaRII protein revealed MC expression in a homogeneous, punctate distribution, whereas Northern analysis demonstrated the presence of the corresponding mRNAs. Exposure to cyclic stretching significantly increased (10%) the overall number of TGF-beta receptors, whereas ligands associated with betaRs I, II, and III also increased (25 to 50%). The finding of increased (30 to 40%) betaRI and betaRII transcript levels and immunoreactive protein (163 and 59%, respectively) in the absence of significant changes in the apparent Kd indicated that stretch-induced binding was the result of increased receptor synthesis and expression and not due to a change in binding affinity. In a similar, but more dramatic fashion, exposure to high glucose also elevated (50%) the receptor number, as well as the amount of ligands associated with betaRs I, II, and III (100 to 250%). This same treatment also increased the levels of betaRI and betaRII mRNA (30 to 40%) and the immunoreactive protein (82 and 82%, respectively), without significantly altering the binding affinity of the receptor. A concerted or synergistic effect of both stimuli was not evidenced. CONCLUSION: These results suggest that the modulation of TGF-beta receptors may be an additional control point in mediating the glucose- and mechanical force-induced increase in ECM deposition by MCs.

Activin Receptors, Type I↗
PubMed1999

Mechanical strain of glomerular mesangial cells in the pathogenesis of glomerulosclerosis: clinical implications.

Due to their elasticity, glomeruli will undergo excessive expansion and repetitive cycles of distension contraction under conditions of impaired glomerular pressure autoregulation and systemic arterial hypertension. These alterations in glomerular volume are associated with mesangial cell stretch which in turn stimulates the synthesis and deposition of ECM with eventual mesangial expansion and glomerulosclerosis. Hyperactivity of growth factors with prosclerotic activity is an important component in the translation of cellular mechanical strain into the abnormal metabolism of ECM components. Although mesangial cell mechanical strain is expected to occur in both remnant glomeruli and in glomeruli of diabetic kidneys, quantitatively different factors will determine the resultant metabolic consequences. In remnant glomeruli, the mechanical stretch is intense, being accounted for largely by the marked glomerular hypertrophy and increased glomerular compliance. In diabetic glomeruli, however, the mechanical stretch is less prominent but its effect on ECM synthesis is markedly aggravated by the presence of hyperglycaemia. There are presently no methods clinically available to diminish the prosclerotic action of growth factors at the glomerular level. In addition, there are no effective means to specifically improve glomerular pressure autoregulation. Therefore, current therapies must be aimed at decreasing systemic arterial pressure, blocking angiotensin II action and reducing glomerular hypertrophy. While there are effective drugs for the treatment of hypertension and for angiotensin II inhibition, protein restriction is the only measure available to diminish glomerular hypertrophy. Finally, in diabetes correction of systemic and glomerular hypertension should be coupled with strict glycaemic control to correct both glomerular autoregulation and increased ECM deposition.

Biomechanical Phenomena↗
PubMed1998

Hairpin coding end opening is mediated by RAG1 and RAG2 proteins.

Despite the importance of hairpin opening in antigen receptor gene assembly, the molecular machinery that mediates this reaction has not been defined. Here, we show that RAG1 plus RAG2 can open DNA hairpins. Hairpin opening by RAGs is not sequence specific, but in Mg2+, hairpin opening occurs only in the context of a regulated cleavage complex. The chemical mechanism of hairpin opening by RAGs resembles RSS cleavage and 3' end processing by HIV integrase and Mu transposase in that these reactions can proceed through alcoholysis. Mutations in either RAG1 or RAG2 that interfere with RSS cleavage also interfere with hairpin opening, suggesting that RAGs have a single active site that catalyzes several distinct DNA cleavage reactions.

Alcohols↗
PubMed1998

Effects of oral antihyperglycemic agents on extracellular matrix synthesis by mesangial cells.

BACKGROUND: Increased expression of the glucose transporter GLUT1 in mesangial cells (MCs) markedly stimulates glucose transport and the formation of extracellular matrix (ECM), even when ambient glucose concentrations are low. Certain antihyperglycemic agents cause GLUT1 overexpression and increase glucose transport in various tissues. However, their effects on the kidney are unknown. Because diabetic glomerulosclerosis is characterized by the accumulation of mesangial matrix, was studied the effects of antihyperglycemic agents on matrix metabolism in MCs cultured either in 8 or 20 mM glucose. METHODS: Membrane-associated GLUT1 was measured by immunoblotting. The initial rate of glucose transport was determined according to the 2-deoxy-D[14C(U)]glucose uptake. Collagen metabolism was studied by metabolic radiolabeling with [14C]-proline. Fibronectin in the medium was measured by ELISA. GLUT1 mRNA was estimated by Northern analysis. RESULTS: The sulfonylurea tolazamide increased GLUT1 protein expression by 107 and 69% in 8 and 20 mM glucose-grown cells, respectively. However, GLUT1 mRNA levels remained unchanged. Transporter-dependent deoxyglucose uptake was increased by tolazamide up to 184% in a dose-dependent fashion and was evident at both glucose concentrations after three or five days of exposure to the drug. Tolazamide significantly stimulated transforming growth factor-beta 1 (TGF-beta 1) secretion and the total synthesis of collagen and collagen and fibronectin accumulation in the medium of MCs maintained in high or low glucose concentrations. The biguanide metformin did not alter GLUT1 expression, glucose transport, fibronectin formation, or collagen metabolism, except at high concentrations. CONCLUSION: Tolazamide markedly enhances ECM synthesis and accumulation in MCs probably by stimulating GLUT1 expression, glucose transport and TGF-beta 1 secretion, irrespective of the ambient glucose concentration. This effect was dose-dependent and minimally inducible by metformin.

Administration, Oral↗
PubMed1998

Mechanical strain- and high glucose-induced alterations in mesangial cell collagen metabolism: role of TGF-beta.

Cultured mesangial cells (MC) exposed to cyclic mechanical strain or high glucose levels increase their secretion of transforming growth factor-beta1 (TGF-beta1) and collagen, suggesting possible mechanisms for the development of diabetic renal sclerosis resulting from intraglomerular hypertension and/or hyperglycemia. This study examines whether glucose interacts with mechanical strain to influence collagen metabolism and whether this change is mediated by TGF-beta. Accordingly, rat MC were grown on flexible-bottom plates in 8 or 35 mM glucose media, subjected to 2 to 5 d of cyclic stretching, and assayed for TGF-beta1 mRNA, TGF-beta1 secretion, and the incorporation of 14C-proline into free or protein-associated hydroxyproline to assess the dynamics of collagen metabolism. Stretching or high glucose exposure increased TGF-beta1 secretion twofold and TGF-beta1 mRNA levels by 30 and 45%, respectively. However, the combination of these stimuli increased secretion greater than fivefold without further elevating mRNA. In 8 mM glucose medium, stretching significantly increased MC collagen synthesis and breakdown, but did not alter accumulation, whereas those stretched in 35 mM glucose markedly increased collagen accumulation. TGF-beta neutralization significantly reduced baseline collagen synthesis, breakdown, and accumulation in low glucose, but had no significant effect on the changes induced by stretch. In contrast, the same treatment of MC in high glucose medium greatly reduced stretch-induced synthesis and breakdown of collagen and totally abolished the increase in collagen accumulation. These results indicate that TGF-beta plays a positive regulatory role in MC collagen synthesis, breakdown, and accumulation. However, in low glucose there is no stretch-induced collagen accumulation, and the effect of TGF-beta is limited to basal collagen turnover. In high glucose media, TGF-beta is a critical mediator of stretch-induced collagen synthesis and catabolism, and, most importantly, its net accumulation. These data have important implications for the pathogenesis and treatment of diabetic glomerulosclerosis.

Animals↗
PubMed1997

V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA-bending proteins.

Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.

DNA↗