Characterizing the phenotypic manifestations of MFN2 R104W mutation in Charcot-Marie-Tooth type 2

Mutations of the mitofusin 2 (MFN2) gene have been reported to be the most common cause of the axonal form of Charcot Marie Tooth disease (CMT). The aim of this study was to describe a de novo MFN2 p.R104W mutation and characterize the associated phenotype. We screened the entire coding region of MFN2 gene and characterized its clinical phenotype, nerve conduction studies and sural nerve biopsy. Neuropsychological tests and brain MRI were also performed. A de nova mutation was found in exon 4 (c.310C > T; p.R104W). In addition to a severe and early onset axonal neuropathy, the patient presented learning problems, obesity, glucose intolerance, leukoencephalopathy, brain atrophy and evidence of myelin involvement and mitochondrial structural changes on sural nerve biopsy. These results suggest that MFN2 p.R104W mutation is as a hot-spot for MFN2 gene associated to a large and complex range of phenotypes. (C) 2011 Elsevier B.V. All rights reserved.

Masticatory muscle activity in children with a skeletal or dentoalveolar open bite

Forty-five children (31 boys and 14 girls), aged 6-11 years, were included in the study, 15 with a skeletal anterior open bite (SAOB), 15 with a dentoalveolar anterior open bite (DAOB), and 15 with a normal occlusion (CG), defined by clinical evaluation and lateral cephalograms. EMG recordings of the temporal and masseter muscles were performed under maximal voluntary clenching and during chewing. Analysis of variance was used for inter-group analysis, followed by the Tukey post hoc test. A Student`s t-test for paired data was used for intra-group analysis. There were statistically significant differences among the three groups (P < 0.05), with the mean EMG being highest in the CG and lowest in children with a SAOB. The percentage EMG activity during chewing in relation to that during maximal voluntary clenching was more than 100 per cent in the SAOB group. The CG and DAOB groups presented higher EMG activity during clenching compared with chewing (P < 0.001), as well as a greater difference between tasks. In the SAOB group, the neuromuscular system appeared to have a lower capacity to produce EMG activity according to the task, while that in the DAOB group suggests that their functional capacity during growth should also be carefully observed.

Activated satellite cells in medial rectus muscles of patients with strabismus

PURPOSE. The goal of this study was to determine whether the medial rectus muscles of patients with a history of medial rectus underaction or overaction show alterations in the process of satellite cell activation when compared with normal age-matched control muscles. METHODS. Medial rectus muscles were obtained with consent from adult patients undergoing surgical resection due to medial rectus underaction or overaction and were prepared for histologic examination by fixation and paraffin embedding. Control muscles were obtained from cornea donor eyes of adults who had no history of strabismus or neuromuscular disease. Cross sections were obtained and stained immunohistochemically for the presence of activated satellite cells, as identified by MyoD immunoreactivity, and the presence of the total satellite cell population, as identified by Pax7 immunoreactivity. The percentages of MyoD- and Pax7-positive satellite cells per 100 myofibers in cross section were calculated. RESULTS. As predicted from results in the literature, MyoD-positive satellite cells, indicative of activation, were present in both the control and resected muscles. In the underacting medial rectus muscles, the percentages of MyoD- and Pax7-positive satellite cells, based on the number of myofibers, were approximately twofold higher than the percentages in the control muscles. In the overacting medial rectus muscles, the percentage of MyoD- positive satellite cells was twofold less than in the control muscles, whereas the percentage of Pax7-positive satellite cells significantly increased compared with that in the control specimens. CONCLUSIONS. The presence of an increased number of activated satellite cells in the resected underacting medial rectus muscles and the decreased numbers of activated satellite cells in the overacting muscles was unexpected. The upregulation in the number of MyoD- positive satellite cells in underacting muscles suggests that there is potential for successful upregulation of size in these muscles, as the cellular machinery for muscle repair and regeneration, the satellite cells, is retained and active in patients with medial rectus underaction. The decreased number of activated satellite cells in overacting MR muscle suggests that factors as yet unknown in these overacting muscles are able to affect the number of satellite cells and/or their responsiveness compared with normal age-matched control muscles. These hypotheses are currently being tested.

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as `guacatonga`, has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright (C) 2008 John Wiley & Sons, Ltd.

Protective effects of carvedilol on systemic vascular damage induced by angiotensin II: Organ-specific effects independent of antihypertensive effects

Background: The protective effect of carvedilol on multiple organ damage induced by angiotensin II (Ang II) remains unclear. The aim of this study was to evaluate the protective effect of carvedilol on the heart, liver, and kidney in rats infused with Ang II. Material/Methods: Wistar rats were randomly distributed into three groups: control (no treatment), continuously infused with Ang II (150 eta g/min for 72 hr), and treated with Ang II + carvedilol (90 mg/kg/d). Histological sections of the myocardium, kidney, and liver were analyzed for the presence of necrosis. Results: Ang II induced arterial hypertension which was not affected by carvedilol treatment (tail-cuff blood pressures, control: 125 +/- 13.6, Ang II: 163 +/- 27.3, Ang II + CV: 178 +/- 39.8 mmHg, p<0.05). Also, there were perivascular inflammation and necrosis in the myocardium, kidney, and hepatocytes necrosis around the terminal vein. Carvedilol treatment fully prevented damage to the heart and kidney and attenuated liver lesions induced by the Ang II infusion. Conclusions: The protective effect of carvedilol on perivascular damage induced by Ang II infusion depended on the target organ. The prevention of heart damage occurred independently of the antihypertensive effects of carvedilol.

Bispectral monitoring in dogs subjected to ovariohysterectomy and anesthetized with halothane, isoflurane or sevoflurane

Objective To assess the effect of halothane (H), isoflurane (I) or sevoflurane (S) on the bispectral index (BIS), and the effect of the addition of meperidine in dogs subjected to ovariohysterectomy. Study design Prospective, randomized, blinded, clinical trial. Animals Forty-eight female mixed-breed dogs, with weights varying from 10 to 25 kg. Methods All dogs were premedicated with acepromazine (A) (0.1 mg kg(-1) IM) or A and meperidine (M) (3 mg kg(-1) IM) and they were divided into six groups of eight animals (AH, AMH, AI, AMI, AS, and AMS). Fifteen minutes after premedication they were anesthetized with propofol (5 mg kg(-1) IV) and then orotracheally intubated. Anesthesia was maintained with halothane, isoflurane or sevoflurane, respectively. The BIS, E`(anest) variables were recorded at 15 minutes after administering pre-anesthetic medication (T0); 10 minutes of anesthesia maintenance (T1); right ovarian pedicle ligation (T2); muscle suturing (T3); skin suture (T4) and 10 minutes after terminating the inhalant anesthetic (T5), respectively. Results BIS values were decreased at all times when compared to the baseline values in all groups (p < 0.05). In the comparative assessment between groups, the values obtained at T0 and T1 were similar for all groups. At T2, the values in AMH were lower than those obtained in AI, AMI and AS (p < 0.05). At the same time significantly higher values were found for AI when compared to AMS (p < 0.01). There was a correlation between the bispectral index and the expired anesthetic fraction in all groups. Conclusions and clinical relevance Within groups given the same inhalant anesthetic the bispectral index was a good indicator for the degree of hypnosis in dogs, indicating a good correlation with the amount of anesthetic and the nociceptive stimulation. BIS was a less reliable indicator of relative anesthetic depth when comparing equipotent end-tidal concentrations between the three inhalants.

Ultrastructure of Motor Nerve Terminals in the Anterior Third of Wistar Rat Tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4 degrees C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi`s apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech. 72:464-470, 2009. (C) 2009 Wiley-Liss. Inc.

Gingival Overgrowth Among Patients Medicated With Cyclosporin A and Tacrolimus Undergoing Renal Transplantation: A Prospective Study

Background: The aim of this study is to make a longitudinal evaluation of the incidence and severity of gingival overgrowth (GO) induced by immunosuppressive agents, such as tacrolimus (Tcr) and cyclosporin A (CsA), in the absence of calcium channel blockers in patients undergoing renal transplantation (RT). Methods: This longitudinal study is conducted in 49 patients with RT who were divided into a CsA group (n = 25) and Tcr group (n = 24). The individuals were assessed at four time intervals: before transplant and 30, 90, and 180 days after RTs. Demographic data and periodontal clinical parameters (plaque index, cemento-enamel junction to the gingival margin, probing depth, clinical attachment level, bleeding on probing [BOP], and GO) were collected at all time intervals. Results: The mean GO index was significantly lower in the Tcr group compared to the CsA group after 30 (P = 0.03), 90 (P = 0.004), and 180 (P = 0.01) days of immunosuppressive therapy. One hundred eighty days after RTs, a clinically significant GO was observed in 20.0% of individuals in the CsA group and 8.3% of individuals in the Tcr group. However, this difference was not statistically significant (P = 0.41). There was a reduction in periodontal clinical parameters regarding the time of immunosuppressive therapy for PI and BOP (P<0.001) in both groups. Conclusion: Although there was no statistical difference in the incidences of clinically significant GO after 180 days of immunosuppressive therapy, it was observed that GO occurred later in the Tcr group, and the severity of GO in this group was lower than in patients who used CsA. J Periodontol 2011;82:251-258.

Comparative study on antioxidant effects and vascular matrix metalloproteinase-2 downregulation by dihydropyridines in renovascular hypertension

The vascular remodeling associated with hypertension involves oxidative stress and enhanced matrix metalloproteinases (MMPs) expression/activity, especially MMP-2. While previous work showed that lercanidipine, a third-generation dihydropyridine calcium channel blocker (CCB), attenuated the oxidative stress and increased MMP-2 expression/activity in two-kidney, one-clip (2K1C) hypertension, no previous study has examined whether first- or second-generation dihydropyridines produce similar effects. We compared the effects of nifedipine, nimodipine, and amlodipine on 2K1C hypertension-induced changes in systolic blood pressure (SBP), vascular remodeling, oxidative stress, and MMPs levels/activity. Sham-operated and 2K1C rats were treated with water, nifedipine 10 mg/kg/day, nimodipine 15 mg/kg/day, or amlodipine 10 mg/kg/day by gavage, starting 3 weeks after hypertension was induced. SBP was monitored weekly. After 6 weeks of treatment, quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin-stained sections. Aortic and systemic reactive oxygen species levels were measured by using dihydroethidine and thiobarbituric acid-reactive substances (TBARs), respectively. Aortic MMP-2 levels and activity were determined by gelatin zymography, in situ zymography, and immunofluorescence. Nifedipine, nimodipine, or amlodipine attenuated the increases in SBP in hypertensive rats by approximately 17% (P<0.05) and prevented vascular hypertrophy (P<0.05). These CCBs blunted 2K1C-induced increases in vascular oxidative stress and plasma TBARs concentrations (P<0.05). All dihydropyridines attenuated the increases in aortic MMP-2 levels and activity associated with 2K1C hypertension. These findings suggest lack of superiority of one particular dihydropyridine, at least with respect to antioxidant effects, MMPs downregulation, and inhibition of vascular remodeling in hypertension.

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress. we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201 +/- 5 vs. 163 +/- 7 mm Hg in diabetic rats untreated and treated with lercaniclipine, respectively; P < 0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P < 0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P < 0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P < 0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2MMP-2 expression. (C) 2008 Published by Elsevier B.V.

Post-polio syndrome: epidemiologic and prognostic aspects in Brazil

Objectives - To describe the clinical and epidemiological aspects of post-polio syndrome (PPS) and identify predictors of its severity. Materials and methods - 132 patients with PPS were selected at the Neuromuscular Disease Outpatient Clinic of the Federal University of Sao Paulo. Descriptive analysis was carried out and predictors of PPS severe forms were investigated using an unconditional logistic regression. Results - The average age at onset was 39.4 years. The most common symptoms were fatigue (87.1%), muscle pain (82.4%) and joint pain (72.0%); 50.4% of the cases were severe. The following were associated with PPS severity: a < 4-year period of neurological recovery (OR 2.8), permanent damage in two limbs (OR 3.6) and residence at the time of acute polio in a city with more advanced medical assistance (OR 2.5). Conclusions - Health professionals should carefully evaluate polio survivors for PPS and be aware of the implications of muscle overuse in the neurological recovery period.

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation

Objectives: Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes. Study design: Immature oocytes obtained from cows slaughtered in a slaughterhouse (n = 840) were divided into the following groups: (1) control (n = 325), submitted only to IVM in TCM199 for 24 h; (2) BLI 18 h (n = 208) submitted to meiotic blockage with 100 mu M BLI for 24 h (Prematuration) and then induction of IVM in TCM199 for 18 h; and (3) BLI 24 h (n = 307), pre-matured with 100 mu m BLI for 24 h followed by 24 h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated. Results: Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18 h and BLI 24 h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18 h and BLI 24 h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18 h and BLI 24 h, respectively). Conclusion: Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Downregulation of VAPB expression in motor neurons derived from induced pluripotent stem cells of ALS8 patients

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope, since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but, in contrast to over-expression systems, cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8, in agreement with the observed reduction of VAPB in sporadic ALS.

Stem cell Researches in Brazil: Present and Future Challenges

A bill allowing researches with human embryonic stem cells has been approved by the Brazilian Congress, originally in 2005 and definitively by the Supreme Court in 2008. However, several years before, investigations in Brazil with adult stem cells in vitro in animal models as well as clinical trials, were started and are currently underway. Here, we will summarize the main findings and the challenges of going from bench to bed, focusing on heart, diabetes, cancer, craniofacial, and neuromuscular disorders. We also call attention to the importance of publishing negative results on experimental trials in scientific journals and websites. They are of great value to investigators in the field and may avoid the repeating of unsuccessful experiments. In addition, they could be referred to patients seeking information, aiming to protect them against financial and psychological harm.