Charcot-Marie-Tooth neuropathy type 2 and P0 point mutations: two novel amino acid substitutions (Asp61Gly; Tyr119Cys) and a possible "hotspot" on Thr124Met

Mutations in the gene for the major protein component of peripheral nerve myelin, myelin protein zero (MPZ, P0), cause hereditary disorders of Schwann cell myelin such as Charcot-Marie-Tooth neuropathy type 1B (CMT1B), Dejerine-Sottas syndrome (DSS), and congenital hypomyelinating neuropathy (CHN). More recently, P0 mutations were identified in the axonal type of CMT neuropathy, CMT2, which is different from the demyelinating variants with respect to electroneurography and nerve pathology. We screened 49 patients with a clinical and histopathological diagnosis of CMT2 for mutations in the P0 gene. Three heterozygous single nucleotide changes were detected: two novel missense mutations, Asp61Gly and Tyr119Cys, and the known Thr124Met substitution, that has already been reported in several CMT patients from different European countries. Haplotype analysis for the P0 locus proved that our patients with the 124Met allele were not related to a cohort of patients with the same mutation, all of Belgian descent and all found to share a common ancestor. Our data suggest that P0 mutations account for a detectable proportion of CMT2 cases with virtually every patient harbouring a different mutation but recurrence of the Thr124Met amino acid substitution. The high frequency of this peculiar genotype in the European CMT population is presumably not only due to a founder effect but Thr124Met might constitute a mutation hotspot in the P0 gene as well.     

hSKCa3: no association of the polymorphic CAG repeat with bipolar affective disorder and schizophrenia

hSKCa3 is a neuronal small conductance calcium-activated potassium channel, which contains a polyglutamine tract, encoded by a polymorphic CAG repeat in the gene. Since an association between longer alleles of this CAG repeat and bipolar disorder or schizophrenia has been reported, we genotyped the polymorphic CAG repeat in 91 German family trios of patients with bipolar disorder I and used the transmission disequilibrium test (TDT) to test for association. Applying a dichotomized model (< or = 19 or > 19 CAG triplets), we found no evidence for an association of longer alleles with bipolar disorder (TDT = 0.75, P = 0.386). Regarding the whole range of alleles, there was no preference in transmitting the larger of the two observed alleles from parents to the affected offspring. In parallel we performed an independent case-control study on German patients with bipolar disorder and schizophrenia. Again we did not detect an overrepresentation of longer CAG repeats in patients. Thus, our data do not support the hypothesis that longer CAG repeats in the hSkCa3 gene contribute to the susceptibility for bipolar disorder and schizophrenia.     

Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.     

Factors related to postmenopausal muscle performance: a cross-sectional population-based study

The aim of the present study was to investigate cross-sectionally the association of postmenopausal muscle strength with simple performance tests. A random sample of 1,166 naturally postmenopausal women (born 1932–1941) was selected from the Kuopio Osteoporosis Risk Factor and Prevention (OSTPRE) study cohort. Grip and quadriceps strength were measured with strain gauge dynamometers and reported in both absolute values (KPa and kg) and per kilogram of body mass (N cm^–2 kg^–1 and N kg^–1). In addition, two performance tests, ability to stand on one foot and ability to squat down to touch the floor were carried out. A five-category self-assessment of overall health (very good, good, moderate, bad, and very bad) was obtained by postal questionnaire. The women that were able to stand on one foot and able to squat down to touch the floor had greater grip and quadriceps strength than their counterparts (P<0.001 and P<0.03 in ANOVA, respectively). In addition, self-assessed health had a strong positive association with muscle strength in the grip and leg extensor muscles in ANOVA (P<0.001 between very good and moderate or poorer state of health) and regression model (P<0.001). Adjustment for age, duration of menopause, body mass, height, physical activity level, use of HRT, and number of diseases and medications did not change any of the main effects. Also, there were no differences in results between absolute measurement values and values reported per kilogram of body mass. According to the present study, a simple performance test may be useful in the prediction of postmenopausal muscle strength. Furthermore, self-assessed state of health is strongly associated with muscle strength in postmenopausal women.     

Analysis of E-cadherin in diffuse-type gastric cancer using a mutation-specific monoclonal antibody

In-frame deletions from the E-cadherin mRNA, coding for a homophilic cell adhesion molecule, are characteristic for diffuse-type gastric carcinomas. Using immunohistochemical analysis the mutant form cannot be distinguished from normal E-cadherin, making results difficult to interpret. In this study, a rat monoclonal antibody, designated E-cad delta 9-1, was generated against a peptide spanning the fusion junction region between exons 8 and 10. This new epitope is present in an E-cadherin variant that lacks exon 9 from the mRNA due to different splice-site gene mutations. Using Western blotting and immunohistochemistry of E-cadherin-transfected cells, we demonstrate that E-cad delta 9-1 specifically reacts with E-cadherin lacking exon 9 but not with the wild-type protein. No immunoreactivity was observed in 31 nontumorous and embryonal tissues analyzed. In gastric carcinoma specimens known to express mutant E-cadherin mRNA lacking exon 9, E-cad delta 9-1 targets exclusively tumor cells in routine formalin-fixed and paraffin-embedded material from biopsies, primary tumors, and lymph node metastases. In a retrospective series of 172 diffuse-type gastric carcinomas expressing E-cadherin, E-cad delta 9-1 reacted with 22 tumors (13%). This new tumor marker-monoclonal antibody system could open novel avenues for selective diagnosis and specific therapy of a subgroup of diffuse-type gastric cancer patients.     

Oxygenation and lung morphology in a rabbit pediatric ARDS- model under high peak pressure ventilation plus nitric oxide and surfactant compared with veno-venous ECMO

The aim of the study is to investigate which of two treatment options of saline lavage induced ARDS in rabbits is better in terms of oxygenation and prevention of barotrauma: combined high peak pressure ventilation with surfactant administration and inhaled nitric oxide or veno-venous ECMO combined with low peak inspiratory pressure ventilation. MATERIALS AND METHODS: After saline lavage (10 cc/kg repeated as long as foamy retrieval was observed) two combined therapeutic strategies were examined: ventilation with high inspiratory pressures (35 cm H2O) with additional exogenous surfactant administration (100 mg/kg) and inhaled nitric oxide (10 PPM) (n=5, group 1) and low inspiratory pressure (20 cm H2O) ventilation under veno-venous ECMO support (n=5, group 2). The FiO2 was maintained at 1.0 in both groups. The paO2/FiO2 ratio was calculated in 30 minute intervals for 4 hours. After that the animals were sacrificed and the lungs examined macro- and microscopically. Aeration was described in a semiquantitative method using the alveolar expansion index. Oxygenation in group 1 was significantly better than in group 2, it increased significantly after surfactant but not after additional nitric oxide administration. However, the lungs in group 1 showed severe signs of baro/ergotrauma (Hyaline membranes, air leaks, infiltration of polymorphonuclear (PMN) granulocytes and macrophages, break down of alveolar capillary membranes) after 4 hrs of combined therapy, whereas the lungs in group 2 appeared normal. Adding surfactant and NO to a high tidal volume ventilation improved oxygenation, but did not prevent baro/ergotrauma. Ventilation with low inspiratory pressures combined with ECMO caused little baro/ergotrauma but adequate oxygenation could not be achieved, probably due to anatomical features of the rabbit which do not allow appropriate blood flow within the ECMO-circuit.     

Untersuchungen über die Bedeutung einzelner Lipid- und Proteinfraktionen des Serums als Streptolysininhibitoren

Zusammenfassung Die vorliegenden Untersuchungen haben zu folgendem Ergebnis geführt:1. Die im Serum nachweisbare Streptolysinhemmwirkung zeigt Beziehungen zum Phosphatidgehalt des-Glubulinbereiches und zu den Eiweißanteilen der Albumin-, ₂-Globulin- und-Globulinfraktion.2. Neben der durch Antikörper bedingten Strepto-lysinhemmwirkung — die wohl in der-Globulinfraktion lokalisicrt ist — sind noch weitere unspezifische Serumfaktoren nachzuweisen, die eine derartige Hemmwirkung entfalten. Sie werden von uns als Streptolysininhibitoren bezeichnet.3. Der für die unspezifische Hemmwirkung maßgebliche Lipoidanteil ist an Bentonit adsorbierbar, und zwar werden hohe wie niedrige Titer um etwa 25% reduziert.An Bentonit werden neben den Lipoiden auch gewisse Eiweißanteile adsorbiert. Von den danach im Serum verbliebenen Eiweißfraktionen zeigen die Albumin-, ₂-Globulin- und-Globulinfraktion statistisch gesicherte Beziehungen zu dem verbliebenen Resttiter.4. Die Bedeutung dieser Ergebnisse für die klinische Bewertung des Streptolysintiters wird diskutiert.