Absence of nitric oxide synthase I despite the presence of the dystrophin complex in human striated muscle

Summary Recently, it has been shown that in human striated muscle the signalling enzyme, brain-type nitric oxide synthase I (NOS I), is associated with the sarcolemma and complexes with dystrophin and/or members of the dystrophin complex. In order to find out whether there exists a regular association between NOS I and the complex, muscle biopsies from patients with various muscle disorders were analysed by enzyme histochemistry and immunohistochemistry. In patients suffering from Duchenne muscular dystrophy, and to a lesser extent in those with Becker-type dystrophy, NOS I and dystrophin complex components were absent or drastically reduced in the sarcolemma region. In other dystrophies, as well as in metabolic and inflammatory myopathies, NOS I and dystrophin complex constituents were expressed normally, while in the case of neurogenic diseases leading to denervation atrophy and especially congenital idiopathic clubfoot, the immunohistochemical patterns of the distribution of the dystrophin complex constituents were normal, but NOS I activity and protein were deficient or dramatically diminished. The results can be interpreted as indicating that, in general, NOS I targeting to the sarcolemma is dependent on particular members of the dystrophin complex, such as ·-1 syntrophin, yet the expression and/or positioning of NOS I may be under the control of further factors, probably of neurogenic origin. NOS I-associated diaphorase may thus be a useful complementary tool in the diagnosis of muscle disorders. This revised version was published online in November 2006 with corrections to the Cover Date.     

Histochemistry of nitric oxide synthase in the nervous system

Summary Nitric oxide synthase, which generates the physiological messenger molecule nitric oxide, and its associated NADPH diaphorase (NADPHd) activity are distributed throughout selective neuronal populations of the central and peripheral nervous system. Considerable evidence has been accumulated to indicate that NADPHd activity labels cells lacking neuronal nitric oxide synthase, i.e., the specificity of the reaction has to be considered for the reliable detection of the enzyme in neuronal but also non-neuronal tissue. In the present review, critical aspects of nitric oxide synthase visualization in neurones, using its NADPHd activity, are discussed. Furthermore, the organization of the central and peripheral nitric oxide synthase-containing neuronal systems is described. Nitric oxide synthase is present in local cortical and striatal neurones, hypothalamic magnocellular neurones, mesopontine cholinergic neurones, cerebellar interneurones, preganglionic sympathetic and parasympathetic neurones, neurones in parasympathetic autonomic and enteric ganglia and primary viscero-afferent neurones. Finally, injury-related alterations in nitric oxide synthase activity are briefly outlined. In this respect, the histochemistry of nitric oxide synthase may represent a valuable marker for neurochemical, if not structural, alterations observed in neural diseases, regeneration and transplantation.     

Integrative proteomic and microRNA analysis of primary human coronary artery endothelial cells exposed to low-dose gamma radiation

High doses of ionising radiation significantly increase the risk of cardiovascular disease (CVD), the vascular endothelium representing one of the main targets. Whether radiation doses lower than 500 mGy induce cardiovascular damage is controversial. The aim of this study was to investigate radiation-induced expression changes on protein and microRNA (miRNA) level in primary human coronary artery endothelial cells after a single 200 mGy radiation dose (Co-60). Using a multiplex gel-based proteomics technology (2D-DIGE), we identified 28 deregulated proteins showing more than ±1.5-fold expression change in comparison with non-exposed cells. A great majority of the proteins showed up-regulation. Bioinformatics analysis indicated “cellular assembly and organisation, cellular function and maintenance and molecular transport” as the most significant radiation-responsive network. Caspase-3, a central regulator of this network, was confirmed to be up-regulated using immunoblotting. We also analysed radiation-induced alterations in the level of six miRNAs known to play a role either in CVD or in radiation response. The expression of miR-21 and miR-146b showed significant radiation-induced deregulation. Using miRNA target prediction, three proteins found differentially expressed in this study were identified as putative candidates for miR-21 regulation. A negative correlation was observed between miR-21 levels and the predicted target proteins, desmoglein 1, phosphoglucomutase and target of Myb protein. This study shows for the first time that a low-dose exposure has a significant impact on miRNA expression that is directly related to protein expression alterations. The data presented here may facilitate the discovery of low-dose biomarkers of radiation-induced cardiovascular damage.     

Synthesis, physicochemical and spectroscopic characterization of copper(II)-polysaccharide pullulan complexes by UV-vis, ATR-FTIR, and EPR

Bioactive copper(II) complexes with polysaccharides, like pullulan and dextran, are important in both veterinary and human medicine for the treatment of hypochromic microcitary anemia and hypocupremia. In aqueous alkaline solutions, Cu(II) ion forms complexes with the exopolysaccharide pullulan and its reduced low-molecular derivative. The metal content and the solution composition depend on pH, temperature, and time of the reaction. The complexing process begins in a weak alkali solution (pH >7) and involves OH groups of pullulan monomer (glucopyranose) units. Complexes of Cu(II) ion with reduced low-molecular pullulan (RLMP, M_w 6000 g mol⁻¹) were synthesized in water solutions, at the boiling temperature and at different pH values ranging from 7.5 to 12. The Cu(II) complex formation with RLMP was analyzed by UV–vis spectrophotometry and other physicochemical methods. Spectroscopic characterizations (ATR-FTIR, FT-IRIS, and EPR) and spectra–structure correlation of Cu(II)–RLMP complexes were also carried out.     

Low-dose irradiation causes rapid alterations to the proteome of the human endothelial cell line EA.hy926

High doses of ionising radiation damage the heart by an as yet unknown mechanism. A concern for radiological protection is the recent epidemiological data indicating that doses as low as 100-500 mGy may induce cardiac damage. The aim of this study was to identify potential molecular targets and/or mechanisms involved in the pathogenesis of low-dose radiation-induced cardiovascular disease. The vascular endothelium plays a pivotal role in the regulation of cardiac function and is therefore a potential target tissue. We report here that low-dose radiation induced rapid and time-dependent changes in the cytoplasmic proteome of the human endothelial cell line EA.hy926. The proteomes were investigated at 4 and 24 h after irradiation at two different dose rates (Co-60 gamma ray total dose 200 mGy; 20 mGy/min and 190 mGy/min) using 2D-DIGE technology. Differentially expressed proteins were identified, after in-gel trypsin digestion, by MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. We identified 15 significantly differentially expressed proteins, of which 10 were up-regulated and 5 down-regulated, with more than ±1.5-fold difference compared with unexposed cells. Pathways influenced by the low-dose exposures included the Ran and RhoA pathways, fatty acid metabolism and stress response.     

Isolated splenic metastasis from colorectal carcinoma in a high risk patient: a case report

Isolated splenic metastasis arising from a colorectal carcinoma is a rare finding. We report a case of 74-year-old man with a medical history of diabetes type II and paroxysmal atrial fibrillation, who underwent a right hemicolectomy for an adenocarcinoma of caecum in August 2004. In June 2007 the patient was diagnosed with high grade aortic valve stenosis as well as long segment stenosis of the first obtuse marginal branch of left coronary artery. He was suggested aortic valve replacement with coronary artery bypass grafting but he refused the surgery. In October 2007 the patient underwent alpha 18FDG - PET scanning, due to increasing values of CEA serum level, which showed a 5 cm big isolated hypermetabolic lesion in the spleen. Due to operative risk, splenectomy was refused by surgeons. The patient underwent a chemotherapy with capecitabine in total of 8 cycles before his CEA level began to rise and MSCT showed a progression in size of splenic metastasis. The patients condition was reevaluated by a team of experts and splenectomy was performed in September 2008. In May 2009 during the postoperative follow up, MSCT scanning revealed enlarged lymph nodes in celiac region and hepatic lesion suspicious of metastasis and the patient was admitted for further chemotherapy treatment. There is still no standardized treatment for this condition due to small number of cases reported in literature. Splenectomy followed by chemotherapy seems to be an optimal treatment but still no final conclusions can be made.     

Proteomic analysis by SILAC and 2D-DIGE reveals radiation-induced endothelial response: four key pathways

Epidemiological data show that ionising radiation increases the risk of cardiovascular disease. The endothelium is one of the main targets of radiation-induced damage. Rapid radiation-induced alterations in the biological processes were investigated after exposure to a clinically relevant radiation dose (2.5 Gy gamma radiation). The changes in protein expression were determined using the human endothelial cell line EA.hy926 as a model. Two complementary proteomic approaches, SILAC (Stable Isotope Labelling with Amino acids in Cell culture) and 2D-DIGE (Two Dimensional Difference-in-Gel-Electrophoresis) were used. The proteomes of the endothelial cells were analysed 4h and 24h after irradiation. Differentially expressed proteins were identified and quantified by MALDI-TOF/TOF and LTQ Orbitrap tandem mass spectrometry. The deregulated proteins were mainly categorised in four key pathways: (i) glycolysis/gluconeogenesis and synthesis/degradation of ketone bodies, (ii) oxidative phosphorylation, (iii) Rho-mediated cell motility and (iv) non-homologous end joining. We suggest that these alterations facilitate the repair processes needed to overcome the stress caused by irradiation and are indicative of the vascular damage leading to radiation-induced cardio- and cerebrovascular impairment.     

A Novel Reading Scheme for Assessing the Extent of Radiographic Abnormalities and Its Association with Disease Severity in Sputum Smear-Positive Tuberculosis: An Observational Study in Hyderabad/India

Background

Existing reading schemes for chest X-ray (CXR) used to grade the extent of disease severity at diagnosis in patients with pulmonary tuberculosis (PTB) are often based on numerical scores that summate specific radiographic features. However, since PTB is known to exhibit a wide heterogeneity in pathology, certain features might be differentially associated with clinical parameters of disease severity.

Objective

We aimed to grade disease severity in PTB patients at diagnosis and after completion of DOTS treatment by developing a reading scheme based on five different radiographic manifestations and analyze their association with the clinical parameters of systemic involvement and infectivity.

Methods

141 HIV-negative adults with newly diagnosed sputum smear-positive PTB were enrolled in a prospective observational study in Hyderabad, India. The presence and extent on CXRs of five radiographic manifestations, i.e., lung involvement, alveolar infiltration, cavitation, lymphadenopathy and pleural effusion, were classified using the new reading scheme by using a four-quadrant approach. We evaluated the inter-reader reliability of each manifestation, and its association with BMI and sputum smear positivity at diagnosis. The presence and extent of these radiographic manifestations were further compared with CXRs on completion of DOTS treatment.

Results

At diagnosis, an average lung area of 51.7% +/- 23.3% was affected by radiographic abnormalities. 94% of the patients had alveolar infiltrates, with 89.4% located in the upper quadrants, suggesting post primary PTB and in 34.8% of patients cavities were found. We further showed that the extent of affected lung area was a negative predictor of BMI (β value -0.035, p 0.019). No significant association of BMI with any of the other CXR features was found. The extent of alveolar infiltrates, along with the presence of cavitation, were strongly associated with sputum smear positivity. The microbiological cure rate in our cohort after 6 months of DOTS treatment was 95%. The extent of the affected lung area in these patients decreased from 56.0% +/- 21.5% to 31.0 +/- 20% and a decrease was also observed in the extent of alveolar infiltrates from 98.4% to 25.8% in at least one quadrant, presence of cavities from 34.8% to 1.6%, lymphadenopathy from 46.8% to 16.1%, and pleural effusion from 19.4% to 6.5%.

Conclusions

We established a new assessment scheme for grading disease severity in PTB by specifically considering five radiographic manifestations which were differently associated with the BMI and sputum smear positivity, changed to a different extent after 6 months of treatment and exhibited an excellent agreement between radiologists. Our results suggest that this reading scheme might contribute to the estimation of disease severity with respect to differences in disease pathology. Further studies are needed to determine a correlation with short and long-term pulmonary function impairment and whether there would be any benefit in lengthening or modulating therapy based on this CXR severity assessment.