Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes

Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.      

Assessing treatment progress of individual patients using expected treatment response models

The dosage model provides a normative estimate of the overall pattern of patient improvement in psychotherapy. The phase model further specifies patterns of change in the domains of subjective well-being, symptom remediation, and functioning. The expected treatment response (ETR) approach uses patient characteristics to predict an expected path of progress for each patient. With repeated measures of mental health status, the treatment progress of an individual patient can be assessed against the patient's ETR to support decisions that would enhance the quality of a clinical service while it is being delivered.     

Prevalence of the chemokine receptor CCR5-Delta32 gene mutation in periodontal disease

A 32-base-pair deletion in the CCR5 gene was previously shown to influence the susceptibility for several infectious diseases. The present study compared the frequency of the CCR5-Delta32 mutation among subjects with periodontal disease and healthy control individuals. The prevalence of the CCR5-Delta32 mutation was determined in 81 patients with generalized periodontitis and 121 healthy controls. Standardized clinical and radiographic criteria were used for the diagnosis of periodontitis for each subject. The CCR5-Delta32 mutation was identified by PCR amplification and subsequent agarose gel electrophoresis. Genotype and allele frequencies among both study groups were compared using Fisher's exact test at a level of significance of 5% (P<0.05). The frequency of the CCR5-Delta32 allele was 9.9% (16/162) for periodontitis patients and 10.7% (26/216) for the healthy controls. The allele frequencies between periodontitis patients and the control group for the CCR5-Delta32 mutation were not significantly different (P=0.801). The present study revealed no association between the CCR5-Delta32 mutation and susceptibility to periodontal disease.     

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.     

Quantitative analysis of muscle fibre type and myosin heavy chain Distribution in the frog hindlimb: implications for locomotory design

To investigate the design of the frog muscular system for jumping, fibre type distribution and myosin heavy chain (MHC) isoform composition were quantified in the hindlimb muscles of Rana pipiens. Muscles were divided into two groups: five large extensor muscles which were predicted to shorten and produce mechanical power during jumping (JP), and four much smaller muscles commonly used in muscle physiology studies, but that do not shorten or produce power during jumping (NJP). Fibres were classified as one of four different types (type 1, 2, 3 or tonic) or an intermediate type (type 1–2) based ontheir relative myosin-ATPase reactivity and MHC immunoreactivity in muscle cross-sections according to previous nomenclature established for amphibian skeletal muscle. Type 1 fibres correspond to the fastest and most powerful of the twitch fibres, and type 3 fibres are the slowest and least powerful. Myosin-ATPase histochemistry revealed that the JP muscles were co mposed primarily of type 1 fibres (89%) with a small percentage of type 2 (7%) and intermediate type 1–2 fibres (4%). The fibre type composition of NJP muscles was more evenly distributed between type 1 (29%), type 2 (46%) and type 1–2 (24%) fibres. Tonic fibres comprised less than 2% of the muscle cross-section in both JP and NJP groups. Similarly, MHC composition determined by quantitative SDS–PAGE revealed that JP muscles were composed predominantly of type 1 MHC (86%), with a balance of type 2 MHC (14%). The opposite pattern was found for MHC composition in the NJP muscles: type 1 (28%), type 2 (66%) and type 3 (6%). These results demonstrate that the large extensor muscles that produce the power required for jumping have a fibre type distribution that enables them to generate high levels of mechanical power, with the type 1 isoform accounting for 85–90% of the total M HC content.     

Effect of increasing dosage of an inhaled beta-2 specific agonist on pulmonary function, tremor, and cardiovascular indices.

In this investigation we evaluated the effect of increasing dosage, using an inhaled beta-2 specific agonist, pirbuterol, administered by a metered dose inhaler on pulmonary function, tremor, and cardiovascular parameters in nonacute adult asthmatic patients. This study was conducted with a randomized crossover study design in which each individual was administered a single dose of pirbuterol (0.4, 0.8 and 1.2 mg) [corrected] on separate days. Measurements of pulmonary function, tremor (by accelerometer readings), electrocardiogram, blood pressure and pulse were obtained at 0, 15, 30, 60, 90, 120, and 180 minutes following test drug administration where appropriate. These measurements were performed until there had been no change from baseline in tremor by 60 minutes or return of tremor measurements to within 15% of baseline for up to six hours. The data demonstrated that the onset of bronchodilator effectiveness had occurred by 15 minutes after test drug administration (the first testing time). The peak percent change from baseline for the FEV1 occurred at 60 minutes after administration for all three test doses. The duration of activity was never truly established as there was no significant difference between any of the potential side effects for any of the three test drugs at any testing time period. This study demonstrated that with the beta-2 specific agent pirbuterol, administered as a metered dose inhaler, there is little risk of development of skeletal muscle or cardiovascular toxicity when as much as three times the recommended dose is used in a single usage, and that there is no direct correlation between the onset, peak, and/or duration of tremor with the onset and peak of bronchodilator efficacy with this agent.     

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia

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Hypo- and hypertetraploidy in a case of erythroleukemia analyzed by fluorochrome banding techniques

Chromosome studies of a case of erythroleukemia in a 57-year-old female patient were made from bone marrow aspirates using the fluorescent primary stain/counterstain methodology. The chromosome number ranged from 42 to 110. There was a high proportion of hypotetraploid cells and a few hypertetraploid and hypooctaploid ones. Structurally normal chromosomes varied in number from cell to cell, ranging from one to seven in the polyploid cells. A number of marker chromosomes were observed, some of which occurred repeatedly in two copies per hypotetraploid cell. The chromosomes involved in aberrations were tentatively identified as #3, #5, #7, #12, #13, #15, #16, #18, #19, and #21. In the abnormal chromosome #16, which was missing a normal short arm, a new kind of heterochromatin was demonstrated by sequential staining with DA-DAPI and DAPI-AMD, suggesting de novo amplification of an A-T-rich satellite DNA sequence.     

Modulating parameters of excitability during and after transcranial direct current stimulation of the human motor cortex

Weak transcranial direct current stimulation (tDCS) of the human motor cortex results in excitability shifts which occur during and after stimulation. These excitability shifts are polarity-specific with anodal tDCS enhancing excitability, and cathodal reducing it. To explore the origin of this excitability modulation in more detail, we measured the input–output curve and motor thresholds as global parameters of cortico-spinal excitability, and determined intracortical inhibition and facilitation, as well as facilitatory indirect wave (I-wave) interactions. Measurements were performed during short-term tDCS, which elicits no after-effects, and during other tDCS protocols which do elicit short- and long-lasting after-effects. Resting and active motor thresholds remained stable during and after tDCS. The slope of the input–output curve was increased by anodal tDCS and decreased by cathodal tDCS. Anodal tDCS of the primary motor cortex reduced intracortical inhibition and enhanced facilitation after tDCS but not during tDCS. Cathodal tDCS reduced facilitation during, and additionally increased inhibition after its administration. During tDCS, I-wave facilitation was not influenced but, for the after-effects, anodal tDCS increased I-wave facilitation, while cathodal tDCS had only minor effects. These results suggest that the effect of tDCS on cortico-spinal excitability during a short period of stimulation (which does not induce after-effects) primarily depends on subthreshold resting membrane potential changes, which are able to modulate the input-output curve, but not motor thresholds. In contrast, the after-effects of tDCS are due to shifts in intracortical inhibition and facilitation, and at least partly also to facilitatory I-wave interaction, which is controlled by synaptic activity.