‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity

The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ_m) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca^2 + uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100 nM FCCP for 30 min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ_m, that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial ‘uncoupling’ stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ_m and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5 min of the uncoupling stimulus and elevated up to 1 h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.     

Determination of ovulation time in bitches based on teasing, vaginal cytology, and elisa for progesterone

The estrous cycle of 16 mature mongrel female dogs was monitored to evaluate the accuracy of teasing, vaginal cytology and quantitative ELISA progesterone assay to determine ovulation. The dogs were presented to male, and blood samples and vaginal swabs were taken daily during proestrus and estrus. Selected serum samples collected during estrus were assayed for endogenous LH by radioimmunoassay (RIA). Plasma samples collected during proestrus and estrus were assayed for progesterone with a commercially avialable ELISA kit. Ovulation was considered to take place 48 h after the preovulatory LH peak. Vaginal cytology smears were stained with Wright's stain and evaluated for the percentage of superficial squamous cells. Day 1 of diestrus (Day 1) was defined as a drop of 20% or more in the total number of superficial cells. Two standard curves (linear and best fitted curves) commonly used with ELISA were compared together and with the RIA progesterone assay. Ovulation was estimated to occur when progesterone concentration was 4.9 +/- 1.0ng/ml (mean +/- SD, n = 15), with a range of 3.4 to 6.6 ng/ml. Based on vaginal cytology, ovulation took place 6.9 +/- 1.6 d (n = 15) after 80% of the squamous cells were superficial and 6.8 +/- 1.4 d (n = 16) before Day 1. Ovulation took place 2.1 +/- 3.9 d (n=11) after the first day of standing estrus and 8.8 +/- 1.5 d (n = 10) before the last day of receptivity. The two standard curves were found parallel to each other and to the RIA progesterone assay. Based on the results of the present study, ELISA progesterone assay and determination of the first day of estrus by vaginal cytology are reliable methods for predicting ovulation, whereas the last day of receptivity as determined by teasing and Day 1 as determined by vaginal cytology are reliable methods to retrospectively estimate ovulation time.     

A high frequency of distinct ATM gene mutations in ataxia-telangiectasia.

The clinical features of the autosomal recessive disorder ataxia-telangiectasia (AT) include a progressive cerebellar ataxia, hypersensitivity to ionizing radiation, and an increased susceptibility to malignancies. Epidemiological studies have suggested that AT heterozygotes may also be at increased risk for malignancy, possibly as a consequence of radiation exposure. A gene mutated in AT patients (ATM) has recently been isolated, making mutation screening in both patients and the general population possible. Because of the relatively large size of the ATM gene, the design of screening programs will depend on the types and distribution of mutations in the general population. In this report, we describe 30 mutations identified in a panel of unrelated AT patients and controls. Twenty-five of the 30 were distinct, and most patients were compound heterozygotes. The most frequently detected mutation was found in three different families and had previously been reported in five others. This corresponds to a frequency of 8% of all reported ATM mutations. Twenty-two of the alterations observed would be predicted to lead to protein truncation at sites scattered throughout the molecule. Two fibroblast cell lines, which displayed normal responses to ionizing radiation, also proved to be heterozygous for truncation mutations of ATM. These observations suggest that the carrier frequency of ATM mutations may be sufficiently high to make population screening practical. However, such screening may need to be done prospectively, that is, by searching for new mutations rather than by screening for just those already identified in AT families.     

Human T-cell receptor V beta gene polymorphism and multiple sclerosis.

Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the beta chain of the T-cell receptor for antigen (TCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of approximately 110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sublocalization of an Ataxia-Telangiectasia Gene Distal to D11S384 by Ancestral Haplotyping In Costa Rican Families

In an effort to localize a gene for ataxia-telangiectasia (A-T), we have genotyped 27 affected Costa Rican families, with 13 markers, in the chromosome 11q22-23 region. Significant linkage disequilibrium was detected for 9/13 markers between D11S1816 and D11S1391. Recombination events observed in these pedigrees places A-T between D11S1819 and D11S1960. One ancestral haplotype is common to 24/54 affected chromosomes and roughly two-thirds of the families. Inferred (ancestral) recombination events involving this common haplotype in earlier generations suggest that A-T is distal to D11S384 and proximal to D11S1960. Several other common haplotypes were identified, consistent with multiple mutations in a single gene. When considered together with all other evidence, this study further sublocalizes the major A-T locus to ≈200 kb, between markers S384 and S535.     

Variability in T cell receptor V beta gene usage in human peripheral blood lymphocytes. Studies of identical twins, siblings, and insulin-dependent diabetes mellitus patients.

Recent studies focused on the diversity and molecular organization of the human TCR-beta complex have begun to establish the genetic basis for the potential repertoire of V beta specificities in T cells. The scope and variability of the actual repertoire derived from this potential repertoire, however, remains to be clarified. In this study, V beta usage by human peripheral T cells derived from serial samples of the same individual, identical twins, and the members of three nuclear families that include four members with insulin-dependent diabetes mellitus (IDDM) was assessed by both quantitative polymerase chain reaction and Northern blotting with V beta subfamily-specific probes. Samples taken from the same individual over a period of 21 months and analyzed in separate experiments indicated stability in the peripheral repertoire, whereas the similarity in peripheral V beta usage in a pair of identical twins suggested a strong role for genetics in shaping the peripheral T cell repertoire. In contrast, V beta usage in siblings and in unrelated individuals was observed to differ substantially. In particular, peripheral expression of V beta 3 and V beta 20 differed by more than sixfold among members of two different families. Segregation analysis of TCR and HLA haplotypes in these families suggested that variation in V beta 20 expression was TCR haplotype specific. Subsequent nucleotide sequence analysis of the V beta 20 gene segment in multiple members of these families revealed the presence of a null allele for V beta 20 expression. No consistent significant differences in V beta usage were observed in IDDM patients relative to their siblings or between identical twins discordant for IDDM. These results suggest that the repertoire of peripheral T cell specificities present in different individuals in human populations varies dramatically because of the effects of multiple factors, including TCR germ-line polymorphism.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.