Effects of bone remodelling on calcium mass transfer during haemodialysis

Background. During haemodialysis, calcium balance can affect, or be affected by, mineral metabolism. However, when dialysate calcium concentration (d[Ca]) is chosen or kinetic models are employed to calculate calcium balance, bone remodelling is rarely considered. In this study, we examined whether bone remodelling affects calcium mass transfer during haemodialysis. Methods. We dialysed 23 patients using a d[Ca] of 1.0, 1.25, 1.5 or 1.75 mmol/L. Calcium mass transfer was measured and associated with remodelling bone factors. Results. Calcium balance varied widely depending on the d[Ca]. Calcium removal was -578 +/- 389, -468 +/- 563, +46 +/- 400 and +405 +/- 413 mg when a d[Ca] of 1.0, 1.25, 1.5 or 1.75 mmol/L was used, respectively (1.0 and 1.25 VS 1.5 and 1.75 mmol/L, P<0.001; 1.5 vs 1.75 mmol/L, P<0.05). Univariate analysis showed that calcium balance correlated with calcium gradient, parathyroid hormone (PTH), osteocalcin and dialysis vintage. Multivariate analysis revealed that calcium balance was dependent on calcium gradient, PTH and osteocalcin. Conclusions. These results suggest that bone remodelling could affect calcium mass transfer during haemodialysis.

Obstructive sleep apnea and dyslipidemia: implications for atherosclerosis

Purpose of review The aim of this review is to summarize current evidence about the impact of obstructive sleep apnea (OSA) and intermittent hypoxia on dyslipidemia and provide future perspectives in this area. Recent findings Intermittent hypoxia, a hallmark of OSA, induces hyperlipidemia in lean mice. Hyperlipidemia of intermittent hypoxia occurs, at least in part, due to activation of the transcription factor sterol regulatory element-binding protein-1 (SREBP-1) and an important downstream enzyme of triglyceride and phospholipid biosynthesis, stearoyl-CoA desaturase-1. Furthermore, intermittent hypoxia may regulate SREBP-1 and stearoyl-CoA desaturase-1 via the transcription factor hypoxia-inducible factor 1. In contrast, key genes involved in cholesterol biosynthesis, SREBP-2 and 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA) reductase, are unaffected by intermittent hypoxia. In humans, there is no definitive evidence regarding the effect of OSA on dyslipidemia. Several cross-sectional studies suggest that OSA is independently associated with increased levels of total cholesterol, low-density lipoprotein and triglycerides, whereas others report no such relationship. Some nonrandomized and randomized studies show that OSA treatment with continuous positive airway pressure may have a beneficial effect on lipid profile. Summary There is increasing evidence that intermittent hypoxia is independently associated with dyslipidemia. However, the role of OSA in causality of dyslipidemia remains to be established.

TRANSFER OF A FASCICLE FROM THE POSTERIOR CORD TO THE SUPRASCAPULAR NERVE AFTER INJURY OF THE UPPER ROOTS OF THE BRACHIAL PLEXUS: TECHNICAL CASE REPORT

OBJECTIVE: A new nerve transfer technique using a healthy fascicle of the posterior cord for suprascapular nerve reconstruction is presented. This technique was used in a patient with posttraumatic brachial plexopathy resulting in upper trunk injury with proximal root stumps that were unavailable for grafting associated with multiple nerve dysfunction. CLINICAL PRESENTATION: A 45-year-old man sustained a right brachial plexus injury after a bicycle accident. Clinical evaluation and electromyography indicated upper trunk involvement. Trapezius muscle function and triceps strength were normal on physical examination. INTERVENTION: The patient underwent a combined supra- and infraclavicular approach to the brachial plexus. A neuroma-in-continuity of the upper trunk and fibrotic C5 and C6 roots were identified. Electrical stimulation of the phrenic and spinal accessory nerves produced no response. The suprascapular nerve was dissected from the upper trunk, transected, and rerouted to the infraclavicular fossa. A healthy fascicle of the posterior cord to the triceps muscle was transferred to the suprascapular nerve. At the time of the 1-year follow-up evaluation, arm abduction against gravity and external rotation reached 40 and 34 degrees, respectively. CONCLUSION: The posterior cord can be used as a source of donor fascicle to the suprascapular nerve after its infraclavicular relocation. This new intraplexal nerve transfer could be applied in patients with isolated injury of the upper trunk and concomitant lesion of the extraplexal nerve donors usually used for reinnervation of the suprascapular nerve.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

The potential role of C-reactive protein in distinguishing cytomegalovirus from tuberculosis and bacterial infections in renal transplant recipients

Introduction: The delay in the diagnosis of infections can be deleterious in renal transplant recipients. Thus, laboratory tests leading to an earlier diagnosis are very useful for these patients. Purpose: To assess the behavior of C-reactive protein (CRP) in renal transplant recipients with a diagnosis of cytomegalovirus (CMV) infection, tuberculosis (TB) and bacterial infection (BI). Methods: A retrospective analysis of 129 patients admitted at our hospital, from 2006 to 2008 because of CMV, TB or BI, was carried out. Appropriate statistical analysis was done and values were expressed as medians, range. Results: When CRP levels were compared among the groups with CMV disease, TB or BI, the group with CMV disease presented lower levels of CRP (18.4 mg/L, 0.28-44 mg/L) than the TB and BI (p < 0.05) groups. The area under the receiver-operating characteristics curve, distinguishing CMV disease from TB/BI, was 0.96 (p < 0.0001), resulting in 100% sensitivity and 90.63% specificity to detect CMV disease when CRP < 44.5 mg/L. The subgroup analysis of CMV infection showed increasing levels of CRP (0.28, 16 and 29.5 mg/L) in the asymptomatic, symptomatic and invasive disease subgroups, respectively (p < 0.05). Conclusion: The measurement of CRP levels may be a useful tool for differentiating CMV infection from the other types (bacterial or TB) of infection in kidney transplant recipients.

Protein disulfide isomerase overexpression in vascular smooth muscle cells induces spontaneous preemptive NADPH oxidase activation and Nox1 mRNA expression: Effects of nitrosothiol exposure

Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). Here, we further investigated PDI effects on vascular NADPH oxidase. VSMC transfected with wild-type PDI (wt-PDI) OF PDI mutated in all four redox cysteines (mut-PDI) enhanced (2.5-fold) basal cellular ROS production and membrane NADPH oxidase activity, with 3-fold increase in Nox1, but not Nox4 mRNA. However, further ROS production, NADPH oxidase activity and Nox1 mRNA increase triggered by angiotensin-II (AngII) were totally lost with PDI overexpression, suggesting preemptive Nox1 activation in such cells. PDI overexpression increased Nox4 mRNA after AngII stimulus, although without parallel ROS increase. We also show that Nox inhibition by the nitric oxide donor GSNO is independent of PDI. PDI silencing decreased specifically Nox1 mRNA and protein, confirming that PDI may regulate Nox1 at transcriptional level in VSMC. Such data further strengthen the role of PDI as novel NADPH oxidase regulator. (C) 2009 Elsevier Inc. All rights reserved.

Influence of Single-Nucleotide Polymorphisms on C-Reactive Protein Levels in Chronic Kidney Disease Before and After Kidney Transplantation

Introduction. We sought to evaluate 2 sing] e-nucleotide polymorphisms (SNPs) in the C-reactive protein (CRP) gene promoter region for their effects on CRP levels in chronic kidney disease (CKD) patients before and after a successful kidney transplantation. Methods. Fifty CKD patients were evaluated before and at the first and second years after the graft. Two SNPs were studied, a bi-allelic (G -> A) at the -409 and a tri-allelic (C -> T -> A) variation at the -390 position in the CRP gene. Results. All patients presented the -409GG genotype. At the -390 position, the ""A"" allele was not found; there were 15 ""CC"" patients, 11 ""TT"" patients, and 24 ""CT"" patients. CRP levels were different among patients with various genotypes (P < .019). Also the presence of the allele ""T"" was sufficient to determine differences in CRP levels both in pretransplantation (P = .045) and at 1 year posttransplantation (P = .011), but not at the second year (P = .448). Conclusion. SNPs at the -390 position of the CRP gene promoter region influence CRP basal levels in such a way that the ""C"" allele correlated with the lowest and the ""T"" with the highest. We did not observe this influence in our patients at the second year posttransplantation.

New SMS mutation leads to a striking reduction in spermine synthase protein function and a severe form of Snyder-Robinson X-linked recessive mental retardation syndrome

We report the identification of a novel mutation at a highly conserved residue within the N-terminal region of spermine synthase (SMS) in a second family with Snyder-Robinson X-linked mental retardation syndrome ( OMIM 309583). This missense mutation, p.G56S, greatly reduces SMS activity and leads to severe epilepsy and cognitive impairment. Our findings contribute to a better delineation and expansion of the clinical spectrum of Snyder-Robinson syndrome, support the important role of the N-terminus in the function of the SMS protein, and provide further evidence for the importance of SMS activity in the development of intellectual processing and other aspects of human development.

Effects of exercise on leukocyte death: prevention by hydrolyzed whey protein enriched with glutamine dipeptide

Lymphocyte and neutrophil death induced by exercise and the role of hydrolyzed whey protein enriched with glutamine dipeptide (Gln) supplementation was investigated. Nine triathletes performed two exhaustive exercise trials with a 1-week interval in a randomized, double blind, crossover protocol. Thirty minutes before treadmill exhaustive exercise at variable speeds in an inclination of 1% the subjects ingested 50 g of maltodextrin (placebo) or 50 g of maltodextrin plus 4 tablets of 700 mg of hydrolyzed whey protein enriched with 175 mg of glutamine dipeptide dissolved in 250 mL water. Cell viability, DNA fragmentation, mitochondrial transmembrane potential and production of reactive oxygen species (ROS) were determined in lymphocytes and neutrophils. Exhaustive exercise decreased viable lymphocytes but had no effect on neutrophils. A 2.2-fold increase in the proportion of lymphocytes and neutrophils with depolarized mitochondria was observed after exhaustive exercise. Supplementation of maltodextrin plus Gln (MGln) prevented the loss of lymphocyte membrane integrity and the mitochondrial membrane depolarization induced by exercise. Exercise caused an increase in ROS production by neutrophils, whereas supplementation of MGln had no additional effect. MGln supplementation partially prevented lymphocyte apoptosis induced by exhaustive exercise possibly by a protective effect on mitochondrial function.

Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C). (C) 2009 Elsevier GmbH. All rights reserved.

Does Trochanteric Transfer Eliminate the Trendelenburg Sign in Adults?

Premature closure of the proximal femoral growth plate results in coxa brevis, which usually is associated with insufficiency of the hip abductors. Distal and lateral transfer of the greater trochanter sometimes is recommended to correct this problem. Most of what is known arises from studies of children and adolescents. We asked whether this procedure in adults with coxa brevis would eliminate hip abductor insufficiency and would improve their hip function based on the Harris hip score (HHS). We prospectively followed 11 patients, aged 19 to 55 years (mean, 40 years) who had distal and lateral trochanteric transfer. All patients had pain and a positive Trendelenburg test before surgery. This test was performed at the latest followup by three observers and the interobserver reliability was determined by the kappa coefficient. The HHS was obtained before surgery and at the latest followup. The minimum followup was 25 months (mean, 52 months; range, 25-77 months). Insufficiency of the hip abductors was eliminated in seven (according to two observers) and eight (according to one observer) of the 11 patients after surgery; the kappa coefficient ranged from 0.79 to 1.0. The mean HHS improved from 64 points preoperatively to 76 points at the final followup. The two patients with preexisting severe osteoarthritis of the hip had the worst final scores and persisted with a positive Trendelenburg test at the final followup. Distal and lateral transfer of the greater trochanter can eliminate insufficiency of the hip abductors and improve joint function in adult patients with coxa brevis and we believe should be considered for patients without severe osteoarthritis of the hip. Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

A low-protein, high-carbohydrate diet increases the adipose lipid content without increasing the glycerol-3-phosphate or fatty acid content in growing rats

Le taux de triacylglycerol (TAG) qui s`accumule dans le tissu adipeux depend de 2 mecanismes opposes : la lipogenese et la lipolyse. Nous avons montre anterieurement que le poids des lipides du tissu adipeux de l`epididyme (EPI) de meme que leur taux augmentent chez les rats en croissance soumis a une diete hypoproteique hyperglucidique (HPHG) pendant 15 jours. La presente etude a eu pour but d`examiner les voies impliquees dans la lipogenese et la lipolyse qui regulent l`accumulation des lipides dans le tissu. On a evalue in vivo la synthese de novo des acides gras, qui s`est revelee similaire chez les rats soumis a la diete HPHG ou a une diete temoin; toutefois, chez les rats soumis a la diete HPHG, une diminution de l`activite de la lipoproteine lipase dans le tissus adipeux de l`EPI a ete observee, ce qui laisse croire a une diminution de la capture des acides gras des lipoproteines circulantes. La diete HPHG n`a eu aucun effet sur la synthese du glycerol-3-phosphate (G3P) par la glycolyse ou la glyceroneogenese. L`activite de la glycerokinase, c.-a-d. la phosphorylation du glycerol issu de l`hydrolyse du TAG endogene pour former le GP3, n`a pas ete modifiee non plus par la diete HPHG. A l`oppose, les adipocytes des rats HPHG stimules par la norepinephrine ont eu une plus faible reponse lipolytique, meme si le taux lipolytique basal des adipocytes a ete similaire chez les 2 groupes. Ainsi, les resultats donnent a penser que la diminution de l`activite lipolytique stimulee par la norepinephrine joue un role essentiel dans l`augmentation du TAG observee dans le tissu adipeux de l`EPI des animaux HPHG, probablement en perturbant le processus d`activation de la lipolyse.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection

Of the hundreds of new tuberculosis ( TB) vaccine candidates some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65 + CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by > 60%, but also the level of myosin heavy chains (MHCs) by > 40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.