Transglutaminase 2 and its role in pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a deadly progressive disease with few treatment options. Transglutaminase 2 (TG2) is a multifunctional protein, but its function in pulmonary fibrosis is unknown. OBJECTIVES: To determine the role of TG2 in pulmonary fibrosis. METHODS: The fibrotic response to bleomycin was compared between wild-type and TG2 knockout mice. Transglutaminase and transglutaminase-catalyzed isopeptide bond expression was examined in formalin-fixed human lung biopsy sections by immunohistochemistry from patients with IPF. In addition, primary human lung fibroblasts were used to study TG2 function in vitro. MEASUREMENTS AND MAIN RESULTS: TG2 knockout mice developed significantly reduced fibrosis compared with wild-type mice as determined by hydroxyproline content and histologic fibrosis score (P < 0.05). TG2 expression and activity are increased in lung biopsy sections in humans with IPF compared with normal control subjects. In vitro overexpression of TG2 led to increased fibronectin deposition, whereas transglutaminase knockdown led to defects in contraction and adhesion. The profibrotic cytokine transforming growth factor-ß causes an increase in membrane-localized TG2, increasing its enzymatic activity. CONCLUSIONS: TG2 is involved in pulmonary fibrosis in a mouse model and in human disease and is important in normal fibroblast function. With continued research on TG2, it may offer a new therapeutic target.

The cannabinoid receptor 1 gene (CNR1) and multiple sclerosis: an association study in two case-control groups from Spain.

Different studies point to the implication of the endocannabinoid system in multiple sclerosis (MS) and animal models of MS. The purpose of this study was to evaluate a possible association of MS with polymorphic markers at the CNR1 gene, encoding the cannabinoid 1 (CB(1)) receptor. We have performed a genetic analysis of an AAT repeat microsatellite localized in the downstream region of the CNR1 gene, in two case-control groups of MS patients and healthy controls (HC) from Spain (Madrid and Bilbao). MS patients with primary progressive MS (PPMS) had more commonly long ((AAT) > or = (13)) alleles and genotypes with a significant difference for genotype 7/8 in Madrid (p = 0.043) and in the sum of both groups (p = 0.016); short alleles were less frequently found in PPMS with a significant difference for allele 5 in the analysis of both groups together (p = 0.039). In patients with relapsing MS, no consistent differences in allele and genotype distribution were found. Disease severity and progression was unrelated to AAT repeat variations. In conclusion, long (AAT) > or = (13) CNR1 genotypes could behave as risk factors for PPMS.

Nox1-derived oxidative stress as a common pathogenic link between obesity and hyperoxaluria-related kidney injury.

Specific relationships among reactive oxygen species, activation pathways, and inflammatory mechanisms involved in kidney injury were assessed in a combined model of obesity and hyperoxaluria. Male Wistar rats were divided into four groups: Control, HFD (high fat diet), OX (0.75% ethylene glycol), and HFD + OX (combined model) Changes in basal O2- levels were evaluated by chemiluminescence in renal interlobar arteries and renal cortex. Furthermore, the effect of different inhibitors on NADPH-stimulated O2- generation was assessed in renal cortex. Oxidative stress sources, and local inflammatory mediators, were also determined, in parallel, by RT-PCR, and correlated with measures of renal function, urinary biochemistry, and renal structure. Rats from the HFD group developed overweight without lipid profile alteration. Tubular deposits of crystals were seen in OX and severely enhanced in HFD + OX groups along with a significantly higher impairment of renal function. Basal oxidative stress was increased in renal cortex of OX rats and in renal arteries of HFD rats, while animals from the combined HFD + OX group exhibited the highest levels of oxidative stress in renal cortex, derived from xanthine oxidase and COX-2. NADPH oxidase-dependent O2- generation was elevated in renal cortex of the OX group and markedly enhanced in the HFD + OX rats, and associated to an up-regulation of Nox1 and a down-regulation of Nox4 expression. High levels of oxidative stress in the kidney, of OX and HFD + OX groups were also associated to an inflammatory response mediated by an elevation of TNFα, COX-2, NFκB1 MCP-1, and OPN. Oxidative stress is a key pathogenic factor in renal disease associated to hyperoxaluria and a common link underlying the exacerbated inflammatory response and kidney injury found under conditions of both obesity and hyperoxaluria. Nox1 pathway must be considered as a potential therapeutic target.

Multiple sclerosis associates with LILRA3 deletion in Spanish patients.

The genetic susceptibility to multiple sclerosis (MS) is only partially explained, and it shows geographic variations. We analyse here two series of Spanish patients and healthy controls and show that relapsing MS (R-MS) is associated with a gene deletion affecting the hypothetically soluble leukocyte immunoglobulin (Ig)-like receptor A3 (LILRA3, 19q13.4), in agreement with an earlier finding in German patients. Our study points to a gene-dose-dependent, protective role for LILRA3, the deletion of which synergizes with HLA-DRB1(*)1501 to increase the risk of R-MS. We also investigated whether the risk of suffering R-MS might be influenced by the genotypic diversity of killer-cell Ig-like receptors (KIRs), located only approximately 400 kb telomeric to LILRA3, and implicated in autoimmunity and defence against viruses. The relationship of LILRA3 deletion with R-MS is not secondary to linkage disequilibrium with a KIR gene, but we cannot exclude some contributions of KIR to the genetic susceptibility to R-MS.

Metabolic syndrome contributes to renal injury mediated by hyperoxaluria in a murine model of nephrolithiasis.

Metabolic syndrome (MS) individuals have a higher risk of developing chronic kidney disease through unclear pathogenic mechanisms. MS has been also related with higher nephrolithiasis prevalence. To establish the influence of MS on renal function, we designed a murine model of combined metabolic syndrome and hyperoxaluria. Four groups of male Sprague-Dawley rats were established: (1) control group (n = 10) fed with standard chow; (2) stone former group (SF) (n = 10) fed with standard chow plus 0.75% ethylene glycol administered in the drinking water; (3) metabolic syndrome group (MS) (n = 10), fed with 60% fructose diet; (4) metabolic syndrome + stone former group (MS + SF) (n = 10), 60% fructose diet and 0.75% EG in the drinking water. MS group showed a significant injury to renal function when hyperoxaluria was induced. It was demonstrated by a significant decrease of creatinine clearance (p < 0.001), with higher tubular damage (34.3%, CI 95% 23.9-44.7, p < 0.001), produced by deposition of crystals, and increased tubular synthesis of osteopontin as a response to tubular damage. Induction of hyperoxaluria in rats with MS causes severe morphological alterations with a significant impairment of renal function. This impairment is not produced in rats without MS. Therefore, this model can be useful for the study of the influence of MS in stone formation.

Codon usage in Kluyveromyces lactis and in yeast cytochrome c-encoding genes.

Codon usage (CU) in Kluyveromyces lactis has been studied. Comparison of CU in highly and lowly expressed genes reveals the existence of 21 optimal codons; 18 of them are also optimal in other yeasts like Saccharomyces cerevisiae or Candida albicans. Codon bias index (CBI) values have been recalculated with reference to the assignment of optimal codons in K. lactis and compared to those previously reported in the literature taking as reference the optimal codons from S. cerevisiae. A new index, the intrinsic codon deviation index (ICDI), is proposed to estimate codon bias of genes from species in which optimal codons are not known; its correlation with other index values, like CBI or effective number of codons (Nc), is high. A comparative analysis of CU in six cytochrome-c-encoding genes (CYC) from five yeasts is also presented and the differences found in the codon bias of these genes are discussed in relation to the metabolic type to which the corresponding yeasts belong. Codon bias in the CYC from K. lactis and S. cerevisiae is correlated to mRNA levels.

A novel anti-inflammatory and pro-resolving role for resolvin D1 in acute cigarette smoke-induced lung inflammation.

INTRODUCTION: Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation. METHODS: Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1ß or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed. RESULTS: RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure. CONCLUSIONS: RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.

Electrophilic PPARγ Ligands Attenuate IL-1ß and Silica-Induced Inflammatory Mediator Production in Human Lung Fibroblasts via a PPARγ-Independent Mechanism.

Acute and chronic lung inflammation is associated with numerous important disease pathologies including asthma, chronic obstructive pulmonary disease and silicosis. Lung fibroblasts are a novel and important target of anti-inflammatory therapy, as they orchestrate, respond to, and amplify inflammatory cascades and are the key cell in the pathogenesis of lung fibrosis. Peroxisome proliferator-activated receptor gamma (PPARγ) ligands are small molecules that induce anti-inflammatory responses in a variety of tissues. Here, we report for the first time that PPARγ ligands have potent anti-inflammatory effects on human lung fibroblasts. 2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid (CDDO) and 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibit production of the inflammatory mediators interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), COX-2, and prostaglandin (PG)E(2) in primary human lung fibroblasts stimulated with either IL-1ß or silica. The anti-inflammatory properties of these molecules are not blocked by the PPARγ antagonist GW9662 and thus are largely PPARγ independent. However, they are dependent on the presence of an electrophilic carbon. CDDO and 15d-PGJ(2), but not rosiglitazone, inhibited NF-κB activity. These results demonstrate that CDDO and 15d-PGJ(2) are potent attenuators of proinflammatory responses in lung fibroblasts and suggest that these molecules should be explored as the basis for novel, targeted anti-inflammatory therapies in the lung and other organs.

Utility of oligoclonal IgG band detection for MS diagnosis in daily clinical practice.

An early and accurate diagnosis of multiple sclerosis (MS) is very important, since it allows early treatment initiation, which reduces the activity of the disease. Oligoclonal IgG band (OCGB) detection is a good ancillary tool for MS diagnosis. However, it was argued that its usefulness was limited by the high interlaboratory variability. In the last years, different techniques for OCGB detection have appeared. We performed a blinded aleatorized multicenter study in 19 Spanish hospitals to assess the accuracy and reproducibility of OCGB detection in this new scenario. We studied cerebrospinal fluid (CSF) and serum samples from 114 neurological patients. Every hospital contributed to the study with triplicated pairs of CSF and serum samples of six patients and analyzed 18 different samples. Global analysis rendered a sensitivity of 92.1%, a specificity of 95.1% and a Kappa value of 0.81. This shows that current techniques for OCGB detection have good accuracy and a high interlaboratory reproducibility and thus, represent a good tool for MS diagnosis. When we analyzed separately the different techniques used for OCGB detection, the highest concordance was observed in western blot with alkaline phosphatase detection (kappa=0.91). This indicates that high sensitivity techniques improve the reproducibility of this assay.

Characterization of promoter regions involved in high expression of KlCYC1.

Functional analysis of the KlCYC1 promoter reveals that sequences located upstream to those already published [Freire-Picos, M. A., Rodríguez-Torres, A. M., Ramil, E., Cerdán, M. E., Breuning, K. D., Hollenberg, C. P. & Zitomer, R. S. (1993) Sequence of a cytochrome c from Kluyveromyces lactis and its upstream region, Yeast 9, 201-204] and extending from positions -780 to -371 are important for maintaining high levels of expression, although this region contains both negative and positive elements. A consensus sequence for interaction with KlCpf1p is present at position -492, into the negative site, and specific protein binding to KlCpf1p has been demonstrated. Deletion of the sequences from positions -413 to -338 diminishes KlCYC1 transcription; protein binding to two sequences included in this activator region is detected and several points of evidence indicate that the complex observed is different from the Hap2/3/4/5p complex. Binding of KlCpf1p and the activator complex to the promoter is constitutive in different carbon sources. Although the promoter contains CCAAT boxes, directed mutagenesis has revealed that they are not related to the moderate de-repression observed in glycerol media.

Heme-mediated transcriptional control in Kluyveromyces lactis.

Heme is of great importance in oxygen-dependent biological functions, since it serves as a prosthetic group for many proteins related to oxygen-binding, oxidative damage prevention and electron transport. It also regulates gene expression through the action of specific transcriptional regulatory factors. In this paper, we present an analysis of heme-dependent transcriptional regulation of several respiration-related genes in an aerobic respiratory yeast, Kluyveromyces lactis. We also report that the KlHEM13 gene, encoding the heme biosynthetic enzyme coproporphyrinogen oxidase, is under heme and oxygen transcriptional regulation, thereby controlling the synthesis of the effector, heme. KlHEM13 is induced during hypoxia, which represents the first report of a transcriptionally regulated gene with this behaviour in K. lactis.

Regulation of the CYB2 gene expression: transcriptional co-ordination by the Hap1p, Hap2/3/4/5p and Adr1p transcription factors.

Expression of the Saccharomyces cerevisiae nuclear gene CYB2 encoding the mitochondrial enzyme L-(+)-lactate-cytochrome c oxidoreductase (EC 1.2.2.3) is subject to several strict metabolic controls at the transcriptional level: repression due to glucose fermentation, derepression by ethanol, induction by lactate and inhibition under anaerobic conditions or in response to deficiency of haem biosynthesis. In this respect, the data obtained from the transcriptional analysis of the CYB2 gene contribute to a better understanding of the control of mitochondrial biogenesis. In this study, we show that Hap1p is the main transcriptional activator involved in the control of CYB2 transcription. We found that Hap1p activity, known to be oxygen dependent, is effected by DNA-protein interaction with two binding sites present in the CYB2 promoter. Control is moreover dependent on carbon sources. This regulation by the carbon substrates is subordinate to the activity of the complex Hap2/3/4/5p, which counteracts the negative effect of the URS1 element. Finally, our results suggest that the Adr1p transcriptional activator is also required in CYB2 transcription control. This work provides new data which allows a better understanding of the molecular mechanisms implicated in the co-regulation at the transcriptional level of the genes encoding proteins involved in various aspects of oxidative metabolism.

The KlCYC1 gene, a downstream region for two differentially regulated transcripts.

KlCYC1 encodes for cytochrome c in the yeast Kluyveromyces lactis and is transcribed in two mRNAs with different 3'-processing points. This is an uncommon transcription mechanism in yeast mRNAs. The 3' sequence encompassing the whole region that is needed to produce both mRNAs is analysed. We have determined identical processing points in K.lactis and in Saccharomyces cerevisiae cells transformed with KlCYC1; positions 698 and 1092 (with respect to the TAA) are the major polyadenylation points. This shows that the cis-elements present in the KlCYC1 3'-untranslated region (3'-UTR) direct a processing mechanism that has been conserved in yeast. In K. lactis there is a high predominance of the shorter transcript (1.14 kb) only at the initial logarithmic growth phase. Interestingly, this growth phase-dependent regulation of 3'-UTR processing is lost when the gene is expressed in S. cerevisiae.

Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region.

The complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory-related yeast genes.

Sequence analysis of a 10 kb DNA fragment from yeast chromosome VII reveals a novel member of the DnaJ family.

We report the sequence analysis of a 10 kb DNA fragment of Saccharomyces cerevisiae chromosome VII. This sequence contains four complete open reading frames (ORFs) of greater than 100 amino acids. There are also two incomplete ORFs flanking the extremes: one of these, G2868, is the 5' part of the SCS3 gene (Hosaka et al., 1994). ORFs G2853 and G2856 correspond to the genes CEG1, coding for the alfa subunit of the mRNA guanylyl transferase and a 3' gene of unknown function previously sequenced (Shibagaki et al., 1992). G2864 is identical to SOH1 also reported (Fan and Klein, 1994).

Respirofermentative metabolism in Kluyveromyces lactis: Insights and perspectives.

Yeasts do not form a homogeneous group as far as energy-yielding metabolism is concerned and the fate of pyruvate, a glycolytic intermediate, determines the type of energy metabolism. Kluyveromyces lactis has become an alternative to the traditional yeast Saccharomyces cerevisiae owing to its industrial applications as well as to studies on mitochondrial respiration. In this review we summarize the current knowdeledge about the K. lactis respirofermentative metabolism, taking into account the respiratory capacity of this yeast and the molecular mechanisms controlling its regulation, giving an up-to-date picture.

Descompresión nasogástrica postoperatoria / Postoperative nasogastric decompression

En el presente trabajo se estudia en forma prospectiva el uso de sonda nasogástrica profiláctica en cirugía abdominal. En la comparación entre 100 pacientes en los que se usó dicho procedimiento y 100 en los que no se usó, se observó en este segundo grupo una disminución de los días de internación y de la necesidad de reposición hidroelectrolítica, como así también una considerable mejoría en el bienestar postoperatorio. La falta de la aspiración gástrica rutinaria no ocasionó inconvenientes, aun en operaciones que incluían anastomosis intestinales. Se concluye que el procedimiento puede ser eliminado sin inconvenientes