Peroxisome biogenesis occurs in late dorsal-anterior structures in the development of Xenopus laevis.

Metabolism and development are two important processes not often examined in the same context. The focus of the present study is the expression of specific peroxisomal genes, the subsequent biogenesis of peroxisomes, and their potential role in the metabolism associated with the development of Xenopus laevis embryos. The temporal and expression patterns of six peroxisomal genes (PEX5, ACO, PEX19, PMP70, PEX16, and catalase) were elucidated using RT-PCR. Functionally related peroxisomal genes exhibited similar expression patterns with their RNA levels elevated relatively late during embryogenesis. Using immunohistochemistry PMP70 and catalase protein was localized largely to dorsal-anterior structures. Peroxisomal function was assayed with peroxisomal targeted-GFP, which when microinjected, revealed peroxisomes in dorsal-anterior structures at stage 45. A requirement for peroxisomal function appears to be present only late in development as organogenesis is finishing, yolk stores are depleted, and ingestion commences.     

Vascular calcification and atherosclerosis in hemodialysis patients: what can we learn from the routine clinical practice?

BACKGROUND: Hemodialysis (HD) patients are at increased risk for arterial intimal (AIC) and medial calcification (AMC). METHODS: In a cross-sectional study on 153 HD patients we evaluated the presence of AIC and AMC using plain radiography of the pelvis and the presence of atherosclerotic lesions using high-resolution B-mode ultrasonography of the common carotid arteries (CCA). RESULTS: The radiography of the pelvis confirmed the frequent presence of AIC (35.3%) and AMC (35.9%) in our HD patients. Arterial calcification was absent (non calcified-NC) in a minority of patients (28.8%). Patients with AIC had significantly higher prevalence of atherosclerotic plaques on CCA (78.6%) compared with both other groups and a higher number of documented atherosclerotic complications, such as cardiovascular (85.2%), cerebrovascular (33.3%) and peripheral arterial disease (38.9%) in comparison with the NC patients. According to the 1-year calculated data from patient records, there were no significant differences in the specific HD risks, such as the dose of prescribed calcium carbonate and vitamin D3, serum levels of calcium, phosphate, calcium-phosphate product and intact parathyroid hormone. All four bone metabolism markers within the range proposed by K/DOQI guidelines were achieved in 9.3%, 14.5% and 20.4% in the AIC, AMC and NC group, respectively. CONCLUSIONS: Patients with AIC and AMC are frequently found in the HD population. Screening for arterial calcifications in chronic kidney disease patients is suggested even in the early pre-dialysis period. The highest proportion of patients within the guidelines proposed range for bone and mineral metabolism markers was observed in the NC group. A longer period of data analysis is required in order to evaluate the possible role of some traditional and HD-specific risk factors for the development of arterial calcifications. The achievement of the K/DOQI guidelines is an important issue in the prevention of those conditions.     

Overexpression of matrix metalloproteinases leads to lethality in transgenic Xenopus laevis: implications for tissue-dependent functions of matrix metalloproteinases during late embryonic development.

The extracellular matrix (ECM) functions as the structural support of cells and as a medium for cell-cell interactions. It is understood to play critical roles in development. ECM remodeling is mediated largely through the action of matrix metalloproteinases (MMPs), a family of Zn2+-dependent proteases capable of degrading various proteinaceous components of the ECM. MMPs are expressed in many developmental and pathologic processes. However, few studies have been carried out to investigate the function of MMPs during embryogenesis and postembryonic organogenesis. By using Xenopus development as a model system, we have previously shown that several MMP genes are expressed from neurulation to the completion of embryogenesis in distinct tissues/organs, suggesting that ECM remodeling during mid- to late embryogenesis occurs in an organ-specific manner. By using the recently developed transgenic technology for Xenopus laevis, we overexpressed Xenopus MMPs stromelysin-3 (ST3) and collagenase-4 (Col4) under the control of a ubiquitous promoter and observed that embryos with overexpressed ST3 or Col4, but not the control green fluorescent protein (GFP), died in a dose-dependent manner during late embryogenesis. The specificity of this embryonic lethal phenotype was confirmed by the failure of a catalytically inactive mutant of ST3 to affect development. Finally, overexpression of a mammalian membrane type-MMP also led to late embryonic lethality in Xenopus embryos, suggesting that membrane type-MMPs have functions in vivo for ECM remodeling, in addition to being activators of other pro-MMPs. These data together with the developmental expression of several MMPs during Xenopus development, suggest that MMPs play important roles during mid- to late embryogenesis and that proper regulation of MMP genes is critical for tissue morphogenesis and organogenesis.     

Kinetic effects of carbon monoxide inhalation on tissue protection in ventilator-induced lung injury

Mechanical ventilation causes ventilator-induced lung injury (VILI), and contributes to acute lung injury/acute respiratory distress syndrome (ALI/ARDS), a disease with high morbidity and mortality among critically ill patients. Carbon monoxide (CO) can confer lung protective effects during mechanical ventilation. This study investigates the time dependency of CO therapy with respect to lung protection in animals subjected to mechanical ventilation. For this purpose, mice were ventilated with a tidal volume of 12 ml/kg body weight for 6 h with air in the absence or presence of CO (250 parts per million). Histological analysis of lung tissue sections was used to determine alveolar wall thickening and the degree of lung damage by VILI score. Bronchoalveolar lavage fluid was analyzed for total cellular influx, neutrophil accumulation, and interleukin-1β release. As the main results, mechanical ventilation induced pulmonary edema, cytokine release, and neutrophil recruitment. In contrast, application of CO for 6 h prevented VILI. Although CO application for 3 h followed by 3-h air ventilation failed to prevent lung injury, a further reduction of CO application time to 1 h in this setting provided sufficient protection. Pre-treatment of animals with inhaled CO for 1 h before ventilation showed no beneficial effect. Delayed application of CO beginning at 3 or 5 h after initiation of ventilation, reduced lung damage, total cell influx, and neutrophil accumulation. In conclusion, administration of CO for 6 h protected against VILI. Identical protective effects were achieved by limiting the administration of CO to the first hour of ventilation. Pre-treatment with CO had no impact on VILI. In contrast, delayed application of CO led to anti-inflammatory effects with time-dependent reduction in tissue protection.     

Association of methylenetetrahydrofolate reductase (MTHFR-677 and MTHFR-1298) genetic polymorphisms with occlusive artery disease and deep venous thrombosis in Macedonians

Aim

To analyze the association of methylenetetrahydrofolate reductase polymorphisms (MTHFR-677 and MTHFR-1298) with occlusive artery disease and deep venous thrombosis in Macedonians.

Methods

We examined 83 healthy respondents, 76 patients with occlusive artery disease, and 67 patients with deep venous thrombosis. Blood samples were collected and DNA was isolated from peripheral blood leukocytes. Identification of MTHFR mutations was done with CVD StripAssay (ViennaLab, Labordiagnostika GmbH, Vienna, Austria) and the population genetics analysis package, PyPop, was used for the analysis. Pearson P values, crude odds ratio, and Wald’s 95% confidence intervals were calculated.

Results

The frequency of C alleles of MTHFR-677 was 0.575 in patients with deep venous thrombosis, 0.612 in patients with occlusive artery disease, and 0.645 in healthy participants. The frequency of T allele of MTHFR-677 was lower in healthy participants (0.355) than in patients with occlusive artery disease (0.388) and deep venous thrombosis (0.425). The frequency of A allele for MTHFR-1298 was 0.729 in healthy participants, 0.770 in patients with occlusive artery disease, and 0.746 in patients with deep venous thrombosis. The frequency of C allele of MTHFR-1298 was 0.271 in healthy participants, 0.230 in patients with occlusive artery disease, and 0.425 in patients with deep venous thrombosis. No association of MTHFR-677 and MTHFR-1289 polymorphisms with occlusive artery disease and deep venous thrombosis was found, except for the protective effect of MTHFR/CA:CC diplotype for occlusive artery disease.

Conclusion

We could not confirm a significant association of MTHFR-677 and MTHFR-1289 polymorphisms with occlusive artery disease or deep venous thrombosis in Macedonians, except for the protective effect of MTHFR/CA:CC diplotype against occlusive artery disease.Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) catalyzes the conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine. The human MTHFR gene (MIM *607093) has been localized at chromosome 1p36.3 (1) and is composed of 11 exons (2).Twelve alleles of human MTHRF gene (0001-0012) have been identified so far (3). MTHFR thermolabile polymorphisms (MTHFR, 677C-T, ALA222VAL and MTHFR, 1298A-C, GLU429ALA) were investigated in several diseases. The mutation in the heterozygous or homozygous state correlated with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of the mutagenized cDNA containing the mutation confirmed its effect on the thermolability of MTHFR. Individuals homozygous for the mutation had significantly elevated plasma homocysteine levels. Thus, the 677C-T mutation may represent an important genetic risk factor for vascular disease (4-6). There are many articles connecting MTHFR mutations, mostly MTHFR 677C-T, with plasma homocysteine levels. Several meta-analyses showed a positive association of MTHFR mutations with vascular diseases (7-9), although several did not (10-12).Since both mutations (677C-T and 1298A-C MTHFR), when homozygous, were associated with a decreased DNA methylation status (although the effect was slightly less pronounced for the 1298A-C transversion), it was suggested that 1298CC MTHFR genotype, independently of folate availability, and 677TT MTHFR genotype with concomitant low folate levels, might be potential risk factors for diseases associated with decreased DNA methylation status (13).We believe that MTHFR mutations influence the homocysteine metabolism, but are in weak association with vascular diseases and could be analyzed in combination with other candidate genes for vascular diseases. There are no data on MTHFR-677 and MTHFR-1298 polymorphisms in Macedonian population and their possible associations with different diseases. The aim of this study was to analyze the association of methylenetetrahydrofolate reductase polymorphisms (MTHFR-677 and MTHFR-1298) with occlusive artery disease and deep venous thrombosis in order to investigate the role of MTHFR mutations as candidate genes in different vascular diseases in Macedonians.