Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Release kinetics of high-sensitivity cardiac troponins I and T and troponin T upstream open reading frame peptide (TnTuORF) in clinically induced acute myocardial infarction

Context: Troponin T upstream open reading frame peptide (TnTuORF) may be useful as a novel biomarker in acute cardiac syndromes.

Objective: The study examined the early release kinetics of TnTuORF.

Materials and methods: We analyzed the time course of the release of cardiac troponins I and T and TnTuORF in patients (n?=?31) with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH).

Results: Fifteen minutes after TASH, the levels of both troponins increased significantly (cTnT median: 18?ng/L versus 27?ng/L; cTnI median: 15?ng/L versus 25?ng/L). TnTuORF showed no variation.

Discussion: We observed a significantly greater increase in cTnI compared with cTnT.

Conclusion: Our results demonstrate that troponin assays allow early detection of myocardial injury, whereas TnTuORF levels remain unchanged in this setting.     

Thiol-based redox metabolism of protozoan parasites

The review considers redox enzymes of Plasmodium spp., Trypanosomatida, Trichomonas, Entamoeba and Giardia, with special emphasis on their potential use as targets for drug development. Thiol-based redox systems play pivotal roles in the success and survival of these parasitic protozoa. The synthesis of cysteine, the key molecule of any thiol metabolism, has been elucidated in trypanosomatids and anaerobes. In trypanosomatids, trypanothione replaces the more common glutathione system. The enzymes of trypanothione synthesis have recently been identified. The role of trypanothione in the detoxification of reactive oxygen species is reflected in the multiplicity of trypanothione-dependent peroxidases. In Plasmodium falciparum, the crystal structures of glutathione reductase and glutamate dehydrogenase are now available; another drug target, thioredoxin reductase, has been demonstrated to be essential for the malarial parasite.     

Intravital observation of Plasmodium berghei sporozoite infection of the liver

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.     

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

Isolation of Pinus radiata Genomic DNA Suitable for RAPD Analysis

A protocol is presented for Pinus radiata genomic DNA isolation based on an alkyltrimethyl-ammonium bromide (CTAB) method described for other woody species. The method involves mortar grinding of tissue, a modified CTAB extraction employing high salt concentrations and polyvinyl pyrrolidone, a RNase A treatment and successive isoamyl alco- hol-chloroform extractions. The yield was approximately 15 g DNA per 100 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in simple sequence repeat and random-amplified-polymorphic DNA reactions. This extraction method would allow the molecular analysis of shoots from different clones within P. radiata families.     

Preclinical insights into the gut‐skeletal muscle axis in chronic gastrointestinal diseases

Muscle wasting represents a constant pathological feature of common chronic gastrointestinal diseases, including liver cirrhosis (LC), inflammatory bowel diseases (IBD), chronic pancreatitis (CP) and pancreatic cancer (PC), and is associated with increased morbidity and mortality. Recent clinical and experimental studies point to the existence of a gut‐skeletal muscle axis that is constituted by specific gut‐derived mediators which activate pro‐ and anti‐sarcopenic signalling pathways in skeletal muscle cells. A pathophysiological link between both organs is also provided by low‐grade systemic inflammation. Animal models of LC, IBD, CP and PC represent an important resource for mechanistic and preclinical studies on disease‐associated muscle wasting. They are also required to test and validate specific anti‐sarcopenic therapies prior to clinical application. In this article, we review frequently used rodent models of muscle wasting in the context of chronic gastrointestinal diseases, survey their specific advantages and limitations and discuss possibilities for further research activities in the field. We conclude that animal models of LC‐, IBD‐ and PC‐associated sarcopenia are an essential supplement to clinical studies because they may provide additional mechanistic insights and help to identify molecular targets for therapeutic interventions in humans.     

In vitro selective effect of melphalan on human T-cell populations

Summary In vitro treatment with 2 g/2×10⁶ cells melphalan (l-PAM: l-phenylalanine mustard) significantly decreased the total number of T lymphocytes from peripheral blood (PBL) of healthy human donors and of the OKT₄ population (precursor suppressor/helper/inducer) T cells as defined by monoclonal antibodies OKT₃ and OKT₄, respectively. No changes in the OKT ₈ ⁺ lymphocyte population (cytotoxic/mature suppressor cells) were observed following the same treatment. Preincubation of PBL with l-PAM at concentrations that do not affect the rate of DNA synthesis in PHA-stimulated lymphocytes inhibited the generation of T suppressor lymphocytes by ConA, as shown by their effect on PHA stimulation. Treatment of allogeneic PBL with l-PAM had no effect on mature suppressor T cells already induced by Con A, as shown by incubation of PBL with l-PAM after incubation with ConA.     

Song characteristics and morphological traits in four populations of the grasshopper Chorthippus biguttulus L.

We investigated four populations of the grasshopper Chorthippus biguttulus with respect to differences in morphological traits and characteristics of their communication signals. A special focus was laid on possible correlations between morphological and song traits of males that could be used by females to infer quality cues of potential mates. We also tested whether females exhibit preferences for males of their own population. Specific song features (onset accentuation, offset, syllable period) of males—but not of females—differed between populations. We observed size differences both in males and females from different populations, but the size ranks of the two sexes were not always correlated. Environmental factors appear to have a strong influence on different size traits, compared to genetic origin. In all populations a specific song feature, the accentuation of syllable onsets, showed a similar correlation with a morphological trait, hind leg size, but its correlation with other size indicators sometimes differed in sign. Females did not prefer songs of males from their own population. The best predictor for song attractiveness was—unexpectedly—not the onset accentuation but the offset depth.