Isomyosin redistribution in chronic pressure overload: comparison between peptide mapping and electrophoresis under non-denaturing conditions

Summary Chronic pressure overload induces a redistribution in myosin isoenzymes as demonstrated by Ca⁺⁺-activated ATPase activity, electrophoresis under non-denaturing conditions and immunohistochemistry.We compared, in two groups of renal hypertensive rats and control rats, the isoenzymic patterns obtained by electrophoresis under non-denaturing conditions with those observed after heavy chains digestion with S. Aureus V8 protease.In the hypertensive animals in which a shift towards the slow V₂ and V₃ isomyosins was evident, peptide mapping always gave origin to a band which was not present in the controls.Since we consider this peptide as a marker of the redistribution towards the slow isoforms, peptide mapping according to Cleveland appears to be a simple and useful method to assess differences in isomyosin composition, at least between hypertrophic pressure-overloaded and normal rat ventricles. Moreover, in our experience this technique is simple, the patterns obtained from highly purified substrates are very reproducible and the digestion allows easy and clear comparisons.     

Biochemical characterization of ventricular myosin from spontaneously hypertensive turkeys

Several stimuli are able to alter the synthesis of cardiac myosin isoenzymes. Particularly in the rat a shift toward a low-ATPase isomyosin is generally observed during development and in cardiac hypertrophy due to pressure overload. On the contrary in spontaneously hypertensive turkeys both ageing and the increasing degree of cardiac hypertrophy are accompanied by a different behaviour of ventricular myosin. In fact in a previous study we have shown that the Ca2+-activated ATPase activity of ventricular myosin increases about three folds from young normotensive to old hypertensive animals. Accordingly the peptide pattern obtained after chymotryptic digestion of myosin showed that some peptides, which are not evident or barely discernible in young animals, are present in the adult ones. In this study we compare the ventricular myosin from young normotensive and adult hypertensive turkeys with atrial myosin. The results obtained suggest that in the ventricles of hypertensive turkeys the synthesis of an isomyosin with biochemical properties close to those of atrial myosin occurs.     

Enzyme Replacement Therapy Improves Respiratory Outcomes in Patients with Late-Onset Type II Glycogenosis and High Ventilator Dependency

Purpose

Type II glycogenosis (GSDII) is a rare and often fatal neuromuscular disorder caused by acid alpha-glucosidase deficiency. Although alglucosidase alfa enzyme replacement therapy (ERT) significantly improves outcomes in subjects with the infantile form, its efficacy in patients with the late-onset one is not entirely clear. The long-term efficacy of ERT in late-onset GSGII complicated by severe pulmonary impairment causing high mechanical ventilation dependency was investigated in this study.

Methods

The long-term clinical efficacy of ERT was assessed in eight late-onset GSDII patients using home mechanical ventilation (HMV) by comparing their outcomes with those of six historical control patients (GSDII patients) who had received HMV alone. The number of hospitalizations due to pulmonary exacerbations and of hours of daily use of HMV were considered the study’s primary efficacy endpoints.

Results

The treatment group showed an increased tendency toward shorter follow-up compared to the control group (35.8 ± 29.2 vs. 52.6 ± 8.55 months; p = 0.04). At the end of the study period, the daily use of HMV (12.5 ± 7.6 vs. 19 ± 14.3 h; p = 0.004) and the hospitalization rate [incidence rate ratio = 0.43 (95 % confidence interval 0.18–0.93); p = 0.03] were significantly lower in the patients receiving ERT. The differences in the forced vital capacity absolute value and percentage change from baseline were not significantly different in the two groups.

Conclusions

ERT reduces ventilator dependency in late-onset GSDII patients and the need for hospitalization due to respiratory exacerbations.     

A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for monocarboxylate transporters as metabolic targets for therapy

Metabolic adaptation is considered an emerging hallmark of cancer, whereby cancer cells exhibit high rates of glucose consumption with consequent lactate production. To ensure rapid efflux of lactate, most cancer cells express high levels of monocarboxylate transporters (MCTs), which therefore may constitute suitable therapeutic targets. The impact of MCT inhibition, along with the clinical impact of altered cellular metabolism during prostate cancer (PCa) initiation and progression, has not been described. Using a large cohort of human prostate tissues of different grades, in silico data, in vitro and ex vivo studies, we demonstrate the metabolic heterogeneity of PCa and its clinical relevance. We show an increased glycolytic phenotype in advanced stages of PCa and its correlation with poor prognosis. Finally, we present evidence supporting MCTs as suitable targets in PCa, affecting not only cancer cell proliferation and survival but also the expression of a number of hypoxia‐inducible factor target genes associated with poor prognosis. Herein, we suggest that patients with highly glycolytic tumours have poorer outcome, supporting the notion of targeting glycolytic tumour cells in prostate cancer through the use of MCT inhibitors. © 2015 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Micronuclei frequencies in hospital workers occupationally exposed to low levels of ionizing radiation: influence of smoking status and other factors

In the context of a medical surveillance program aimed at preventing cancer risk from exposure to ionizing radiation, we investigated chromosomal damage in peripheral lymphocytes from 37 hospital workers exposed to low levels of ionizing radiation and 37 controls. The micronuleus (MN) assay was used as a biomarker of genetic damage. The influence of confounding factors like smoking status, age and gender was investigated by multiple regression analysis. The results indicated that, overall, MN frequency was higher in exposed workers than in controls, although the difference was not statistically significant. Interestingly, smoking status significantly raised MN frequency among the exposed workers but not among controls. This suggests that smoking can influence chromosomal damage induced in humans by ionizing radiation. Among both exposed workers and controls, MN frequency was found to increase with age. Female gender influenced the increase in MN frequency in the exposed group. Our results suggest that the effect of cigarette smoking should be carefully factored into genetic monitoring studies assessing the risks associated with low level radiation exposure.     

McArdle disease: the mutation spectrum of PYGM in a large Italian cohort

Deficiency of the muscle isozyme of glycogen phosphorylase is causative of McArdle disease or Glycogen storage disease type V (GSD-V), the most common autosomal recessive disorder of glycogen metabolism. The typical clinical presentation is characterized by exercise intolerance with cramps, and recurrent myoglobinuria. To date, 46 mutations in the PYGM gene have been detected in GSD-V patients. We report the mutational spectrum in 68 Italian patients. We identified 30 different mutations in the PYGM gene, including 19 mutations that have not been reported previously. The novel mutations include: eight missense mutations (c.475G>A, p.G159R; c.689C>G, p.P230R; c.1094C>T, p.A365E; c.1151C>A, p.A384D; c.1182C>T, p.R428C; c.1471C>T, p.R491C; c.2444A>C, p.D815A; c.2477G>C, p.W826S), two nonsense mutations (c.1475G>A, p.W492X; c.1627A>T, p.K543X), five splice site mutations (c.855 +1G>C; c.1092 +1G>A; c. 1093-1G>T; c.1239 +1G>A; c.2380 +1G>A), and four deletions (c.715_717delGTC, p.V239del; c.304delA, p.N102DfsX4; c.1970_2177del, p.V657_G726; c.2113_2114delGG, p.G705RfsX16). Whereas we confirmed lack of direct correlation between the clinical phenotype and the genotype, we also found that the so-called 'common mutation' (p.R50X) accounted for about 43% of alleles in our cohort and that no population-related mutations are clearly identified in Italian patients.     

Comparison of different commercial FFDM units by means of physical characterization and contrast-detail analysis

The purpose of this study was to perform a complete evaluation of three pieces of clinical digital mammography equipment. Image quality was assessed by performing physical characterization and contrast-detail (CD) analysis. We considered three different FFDM systems: a computed radiography unit (Fuji "FCR 5000 MA") and two flat-panel units, the indirect conversion a-Si based GE "Senographe 2000D" and the direct conversion a-Si based IMS "Giotto Image MD." The physical characterization was estimated by measuring the MTF, NNPS, and DQE of the detectors with no antiscatter grid and over the clinical range of exposures. The CD analysis was performed using a CDMAM 3.4 phantom and custom software designed for automatic computation of the contrast-detail curves. The physical characterization of the three digital systems confirms the excellent MTF properties of the direct conversion flat-panel detector (FPD). We performed a relative standard deviation (RSD) analysis, for investigating the different components of the noise presented by the three systems. It turned out that the two FPDs show a significant additive component, whereas for the CR system the statistical noise is dominant. The multiplicative factor is a minor constituent for all the systems. The two FPDs demonstrate better DQE, with respect to the CR system, for exposures higher than 70 microGy. The CD analysis indicated that the three systems are not statistically different for detail objects with a diameter greater than 0.3 mm. However, the IMS system showed a statistically significant different response for details smaller than 0.3 mm. In this case, the poor response of the a-Se detector could be attributed to its high-frequency noise characteristics, since its MTF, NEQ, and DQE are not inferior to those of the other systems. The CD results were independent of exposure level, within the investigated clinical range. We observed slight variations in the CD results, due to the changes in the visualization parameters (window/level and magnification factor). This suggests that radiologists would benefit from viewing images using varied window/level and magnification.     

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.