Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.

RNA degradation differentially affects quantitative mRNA measurements of endogenous reference genes in human placenta

It has been highlighted that RNA quality and appropriate reference gene selection is crucial for the interpretation of RT-qPCR results in human placental samples. In this context we investigated the effect of RNA degradation on the mRNA abundance of seven frequently used reference genes in 119 human placental samples. Combining RNA integrity measurements, RT-qPCR analysis and mathematical modeling we found major differences regarding the effect of RNA degradation on the measured expression levels between the different reference genes. Furthermore, we demonstrated that a modified RNA extraction method significantly improved RNA quality and consequently increased transcript levels of all reference genes.

The meanings of pasture in resource degradation negotiations: Evidence from post-socialist rural Kyrgyzstan

Pasture use in the Kyrgyz Republic has changed significantly as a result of fundamental political, economic, and societal changes following the collapse of the Soviet Union and the subsequent changes in people’s livelihoods. Government institutions criticize current land use patterns as unsustainable and the cause of degradation. But at the local level, pasture quality is rarely seen as a major problem. This article uses a qualitative approach to examine the tension between these views and addresses current land use practices and related narratives about pasture degradation in rural Kyrgyzstan. By focusing on meanings ascribed to pastures, it shows how people closely relate current practices to the experiences and value systems of the Soviet period and to changing identities emerging in the post-Soviet transformation process. It argues that proper understanding of resource degradation issues requires adequate consideration of the context of meaning constructed by local resource users when they make sense of their environment.

Bioremediation with White-Rot Fungi at Fisherville Mill: Analyses of Gene Expression and Number 6 Fuel Oil Degradation

Extracellular enzymes that white-rot fungi secrete during lignin decay have been proposed as promising agents for oxidizing pollutants. We investigated the abilities of the white-rot fungi Punctularia strigosozonata, Irpex lacteus, Trichaptum biforme, Phlebia radiata, Trametes versicolor, and Pleurotus ostreatus to degrade Number 6 fuel oil in wood sawdust cultures. Our goals are to advise bioremediation efforts at a brownfield redevelopment site on the Blackstone River in Grafton, Massachusetts and to contribute to the understanding of decay mechanisms in white-rot fungi. All species tested degraded a C10 alkane. When cultivated for 6 months, Irpex lacteus, T. biforme, P. radiata, T. versicolor and P. ostreatus also degraded a C14 alkane and the polycyclic aromatic hydrocarbon phenanthrene. Gene expression analyses of P. strigosozonata indicate differential gene expression in the presence of Number 6 oil and on pine and aspen sawdust.

Herpes-simplex virus encephalitis is characterized by an early MMP-9 increase and collagen type IV degradation

Cerebrovascular complications including cerebral edema, raised intracranial pressure and hemorrhage contribute to the high mortality and morbidity of herpes-simplex virus encephalitis (HSE). We examined changes of collagen type IV, the major constituent of the neurovascular matrix, together with expression and localization of matrix-degrading enzymes during the development of acute HSE. In an experimental model of focal HSE, we found that early, symptomatic HSE (3 days after infection) and acute, fully developed HSE (7 days after infection) are associated with significantly raised levels of matrix-metalloproteinase-9 (MMP-9) (both P<0.05). In situ zymography of brain sections revealed that the increase of MMP-9 was restricted to the cerebral vasculature in early HSE and further expanded towards the perivascular space and adjacent tissue in acute HSE. Around the cerebral vasculature, we observed that MMP-9 activity was insufficiently counterbalanced by its endogenous tissue inhibitor of MMP (TIMP) TIMP-1, resulting in loss of collagen type IV. Our findings suggest that MMP-9 is involved in the evolution of HSE by causing damage to the cerebral vasculature. The degradation of the neurovascular matrix in HSE facilitates the development of cerebrovascular complications and may represent a target for novel adjuvant treatment strategies.

In bacterial meningitis cortical brain damage is associated with changes in parenchymal MMP-9/TIMP-1 ratio and increased collagen type IV degradation

Adverse outcome in bacterial meningitis is associated with the breakdown of the blood-brain barrier (BBB). Matrix-metalloproteinases (MMPs) facilitate this process by degradation of components of the BBB. This in turn results in acute complications of bacterial meningitis including edema formation, increased intracranial pressure and subsequent ischemia. We determined the parenchymal balance of MMP-9 and TIMP-1 (tissue inhibitor of MMP) and the structural integrity of the BBB in relation to cortical damage in an infant rat model of pneumococcal meningitis. The data demonstrate that the extent of cortical damage is significantly associated with parenchymal gelatinolytic activity and collagen type IV degradation. The increased gelatinolysis was found to be associated with a brain parenchymal imbalance of MMP-9/TIMP-1. These findings provide support to the concept that MMPs mediated disruption of the BBB contributes to the pathogenesis of bacterial meningitis and that protection of the vascular unit may have neuroprotective potential.

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Quantitative magnetic resonance imaging of enzymatically induced degradation of the nucleus pulposus of intervertebral discs

STUDY DESIGN: The structural integrity of the nucleus pulposus (NP) of intervertebral discs was targeted by enzyme-specific degradations to correlate their effects to the magnetic resonance (MR) signal. OBJECTIVE: To develop quantitative MR imaging as an accurate and noninvasive diagnostic tool to better understand and treat disc degeneration. SUMMARY OF BACKGROUND DATA: Quantitative MR analysis has been previously shown to reflect not only the disc matrix composition, but also the structural integrity of the disc matrix. Further work is required to identify the contribution of the structural integrity versus the matrix composition to the MR signal. METHODS: The bovine coccygeal NPs were injected with either enzyme or buffer, incubated at 37 degrees C as static, unloaded and closed 3-disc segments, and analyzed by a 1.5-Tesla MR scanner to measure MR parameters. RESULTS: Collagenase degradation of the NP significantly decreased the relaxation times, slightly decreased the magnetization transfer ratio, and slightly increased the apparent diffusion coefficient. Targeting the proteoglycan and/or hyaluronan integrity by trypsin and hyaluronidase did not significantly affect the MR parameters, except for an increase in the apparent diffusion coefficient of the disc after trypsin treatment. CONCLUSIONS: Our results demonstrate that changes in the structural integrity of matrix proteins can be assessed by quantitative MR.