Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation

The embryonation of nematode eggs has been shown to increase their resistance to anthelmintics when parasites are submitted to egg hatch assays. Nevertheless, no mechanism has been suggested to explain this phenomenon. Earlier observations by other authors showed that the biochemical composition of eggshells is altered during the embryonation of eggs. The functional consequences of these changes have not been identified. We studied the changes in membrane environment (eggshells) of Haemonchus contortus eggs during the embryonation by fluidity measurements and their effects on nonspecific mechanisms of resistance to anthelmintics. We previously demonstrated that these mechanisms imply P-glycoproteins (Pgp) belonging to the multi-drug resistance (MDR) system and that the Pgp activity is very susceptible to their lipidic environment. The results obtained here show that the embryonation induced a significant and gradual increase in eggshell fluidity which was associated with an increased resistance to anthelmintics. Differences were observed between H. contortus isolates with various levels of resistance which might result from their specific biology and/or membrane composition. The membrane environment changes could act both on the solubilization of anthelmintics into the eggs and on the efflux of these lipophilic molecules by Pgp.     

Characterization of cells with a high aldehyde dehydrogenase activity from cord blood and acute myeloid leukemia samples

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells, and this is thought to be important in chemoresistance. A method for the assessment of ALDH activity in viable cells recently has been developed and made commercially available in a kit format. In this study, we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Via multicolor flow cytometry of cord blood ALDH+ cells, we have expanded on their phenotypic analysis. We then assessed the incidence, morphology, phenotype, and nonobese diabetic/ severe combined immunodeficiency engraftment ability of ALDH+ cells from acute myeloid leukemia (AML) samples. AML samples had no ALDH+ cells at all, an extremely rare nonmalignant stem/progenitor cell population, or a less rare, leukemic stem cell population. Hence, in addition to identifying nonmalignant stem cells within some AML samples, a high ALDH activity also identifies some patients' CD34+/ CD38- leukemic stem cells. The incidence of normal or leukemic stem cells with an extremely high ALDH activity may have important implications for resistance to chemotherapy. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis.     

Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies

DYSF encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 34 unrelated patients from various ethnic origins. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using SSCP or dHPLC of PCR products of each of 55 exons of the gene was followed by sequencing whenever a sequence variation was detected. All together, 54 sequence variations were identified in DYSF, 50 of which predicting either a truncated protein or one amino-acid substitution and most of them (34 out of 54) being novel. In 23 patients, we identified two pathogenic mutations, while only one was identified in 11 patients. These mutations were widely spread in the coding sequence of the gene without any mutational "hotspot."     

Morphometric and immunohistochemical characterization of the intimal layer throughout the arterial system of elderly humans

The purpose of the present study was to obtain insight into the natural development of adaptive intimal thickening and atherosclerosis in the arterial tree of human species. The morphometry and composition of the intimal layer were studied in the arterial system of elderly individuals. Post mortem, a total of 703 arterial segments were dissected from 24 subjects (age 81.9 ± 9.9 years). From each subject, segments were dissected from 31 different arteries. Area stenosis [(plaque area/vessel area) × 100%] was determined in each segment. By (immuno)histochemistry, lipid content and the presence of inflammatory cells (macrophages) were assessed in the coronary, common carotid, brachial, radial and internal iliac arteries. Adaptive intimal thickening or advanced atherosclerosis was observed in all studied artery types. Area stenosis was highest in the coronary arteries (median, 30%) and lowest in the arteries supplying the brain (median, ≤ 7%). Plaques hiding a lipid‐rich core and plaques with macrophage infiltration were observed in all five selected artery types. In summary, the present observation demonstrates that intimal thickening is a systemic process involving most artery types. Within elderly humans, features of advanced atherosclerotic disease, a lipid‐rich core and macrophages, can be observed in the intimal layer of artery types that are recognised for their relation with clinical syndroms as well as artery types that remain clinically silent.     

Patients affected with Fabry disease have an increased incidence of progressive hearing loss and sudden deafness: an investigation of twenty-two hemizygous male patients

Background  

Fabry disease (FD, OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism due to the deficient activity of alpha-galactosidase A, a lysosomal enzyme. While the progressive systemic deposition of uncleaved glycosphingolipids throughout the body is known to have protean clinical manifestations, few data are available regarding the cochlear involvement.     

Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states

Sensory relay structures in the spinal cord dorsal horn are now thought to be active processing structures that function before supraspinal sensory integration. Dorsal horn neurons directly receive nociceptive (pain) signals from the periphery, express a high degree of functional plasticity and are involved in long-term sensitization and chronic pain. We show here that deep dorsal horn neurons (DHNs) in Wistar rats can switch their intrinsic firing properties from tonic to plateau or endogenous bursting patterns, depending upon the balance of control by metabotropic glutamate (mGlu) and GABA(B) receptors. We further show that this modulation acts on at least one common target, the inwardly rectifying potassium channel (Kir3). Finally, we found that these firing modes correspond to specific functional states of information transfer in which dorsal horn neurons can faithfully transmit, greatly enhance or block the transfer of nociceptive information.     

Local Suppression of Epileptiform Activity by Electrical Stimulation in Rat Hippocampus In Vitro

High frequency electrical stimulation of deep brain structures (DBS) has been effective at controlling abnormal neuronal activity in Parkinson's patients and is now being applied for the treatment of pharmacologically intractable epilepsy. The mechanisms underlying the therapeutic effects of DBS are unknown. In particular, the effect of the electrical stimulation on neuronal firing remains poorly understood. Previous reports have showed that uniform electric fields with both AC (continuous sinusoidal) or DC waveforms could suppress epileptiform activity in vitro . In the present study, we tested the effects of monopolar electrode stimulation and low-duty cycle AC stimulation protocols, which more closely approximate those used clinically, on three in vitro epilepsy models. Continuous sinusoidal stimulation, 50 % duty-cycle sinusoidal stimulation, and low (1.68 %) duty-cycle pulsed stimulation (120 μs, 140 Hz) could completely suppress spontaneous low-Ca²⁺ epileptiform activity with average thresholds of 71.11 ± 26.16 μA, 93.33 ± 12.58 μA and 300 ± 100 μA, respectively. Continuous sinusoidal stimulation could also completely suppress picrotoxin- and high-K⁺-induced epileptiform activity with either uniform or localized fields. The suppression generated by the monopolar electrode was localized to a region surrounding the stimulation electrode. Potassium concentration and transmembrane potential recordings showed that AC stimulation was associated with an increase in extracellular potassium concentration and neuronal depolarization block; AC stimulation efficacy was not orientation-selective. In contrast, DC stimulation blocked activity by membrane hyperpolarization and was orientation-selective, but had a lower threshold for suppression.     

β-Adrenergic and muscarinic receptor mRNA accumulation in the sinoatrial node area of adult and senescent rat hearts

The sinoatrial (SA) node is the cardiac pacemaker and changes in its adrenergic-muscarinic phenotype have been postulated as a determinant of age-associated modifications in heart rate variability. To address this question, right atria were microdissected, the SA node area was identified by acetylcholinesterase staining, and, using a RT-PCR method, the accumulation of mRNA molecules encoding β₁- and β₂-adrenergic (β₁- and β₂-AR) and muscarinic (M₂-R) receptor was quantified to define the proportion between β-AR and M₂-R mRNAs within the sinoatrial area of adult (3 months) and senescent (24 months) individual rat hearts. In adult hearts, the highest M₂-R/β-AR mRNA ratio was observed within the sinoatrial area compared with adjacent atrial myocardium, while in the senescent hearts, no difference was observed between sinoatrial and adjacent areas. This change was specific of the sinoatrial area since adult and senescent whole atrial or ventricular myocardium did not differ in their M₂-R/β-AR mRNA ratio, and was associated with a fragmentation of acetylcholinesterase staining of the senescent SA node. Quantitative changes in the expression of genes encoding proteins involved in heart rate regulation specifically affect the sinoatrial area of the senescent heart.     

Refined genetic mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.3 and evidence of linkage disequilibrium in European families

Chronic distal spinal muscular atrophy (Chronic DSMA, MIM (*)607088) is a rare autosomal recessive disorder characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of Chronic DSMA gene has been previously mapped to chromosome 11q13 in the 10.3 cM interval defined by loci D11S1889 and D11S1321. By linkage analysis in 12 European Chronic DSMA families, we showed that a disease gene maps to chromosome 11q13.3 (Z(max)=6.66 at theta=0.00 at the DSM4 locus) and suggested that this condition is genetically homogeneous. Recombination events allowed us to reduce the genetic interval to a 2.6 cM region, telomeric to the IGHMBP2 gene, excluding this gene as the disease causing gene in Chronic DSMA. Moreover, partial linkage disequilibrium was found between three rare alleles at loci D11S1369, DSM4 and D11S4184 and the mutant chromosome in European patients. Analysis of the markers at these loci strongly suggests that most Chronic DSMA chromosomes are derived from a single ancestor. Refinement of the Chronic DSMA locus will hopefully allow to test candidate genes and lead to identification of the disease-causing mutations.