Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator

In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N- terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.      

Malaria epidemiology in the Pakaanóva (Wari') Indians, Brazilian Amazon

This paper reports the results of a longitudinal study of malaria incidence (1998-2002) among the Pakaanóva (Wari') Indians, Brazilian southwest Amazon region, based on data routinely gathered by Brazilian National Health Foundation outposts network in conjunction with the Indian health service. Malaria is present yearlong in the Pakaanóva. Statistically significant differences between seasons or months were not noticed. A total of 1933 cases of malaria were diagnosed in the Pakaanóva during this period. The P. vivax / P. falciparum ratio was 3.4. P. vivax accounted for 76.5% of the cases. Infections with P. malariae were not recorded. Incidence rates did not differ by sex. Most malaria cases were reported in children < 10 years old (45%). About one fourth of all cases were diagnosed on women 10-40 years old. An entomological survey carried out at two Pakaanóva villages yielded a total of 3.232 specimens of anophelines. Anopheles darlingi predominated (94.4%). Most specimens were captured outdoors and peak activity hours were noted at early evening and just before sunrise. It was observed that Pakaanóva cultural practices may facilitate outdoor exposure of individuals of both sexes and all age groups during peak hours of mosquito activities (e.g., coming to the river early in the morning for bathing or to draw water, fishing, engaging in hunting camps, etc). In a context in which anophelines are ubiquitous and predominantly exophilic, and humans of both sexes and all ages are prone to outdoor activities during peak mosquito activity hours, malaria is likely to remain endemic in the Pakaanóva, thus requiring the development of alternative control strategies that are culturally and ecologically sensitive.     

Amplification and overexpression of HER-2/neu in parotid gland salivary duct carcinoma. Immunohistochemical study and fluorescence in situ hybridization

Salivary duct carcinoma (SDC) is highly malignant salivary gland tumour with aggressive clinical behaviour, characterised by its histological resemblance to invasive ductal carcinoma of the breast. Amplification of gene HER-2/neu and overexpression of its gene product have been shown to have both prognostic and treatment implications in breast cancer. The reports concerning the expression of c-erbB2/HER-2/neu in salivary gland tumours are few and controversial. Thus, eleven cases of SDC were evaluated for HER-2/neu status using immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). To the best of our knowledge, this is the first molecular genetic analysis of SDCs using FISH. HER-2/neu overexpression, identified as strong membrane staining, was observed in all but one case of SDC in majority of neoplastic cells while only four tumours, of nine cases analysed, revealed HER-2/neu gene amplification by means of FISH analysis. SDCs were associated with poor clinical outcome, 6 patients (55%) died of disseminated carcinoma within 4 to 44 months after therapy. There was no difference in outcome of patients with IHC positive-nonamplified and IHC positive-amplified tumours.     

Influence of D-glucose on lipid solid support membrane system as attempt for biosensing of medically relevant molecules

The influence of D-glucose on a lipid solid support system with the aid of impedance spectrocopy as a preliminary attempt for the biosensing of medical relevant molecules was studied. In spite of some shortcomings, s-BLM's proved to be an appropriate model for the study of lipid membrane-D-glucose interactions. The shortcomings were the roughness of the metal support, and the lack of homogeneity in the monolayer or multilayer lipid structures.     

Cardiac troponins following repeated administration of an iron chelator--salicylaldehyde isonicotinoyl hydrazone (SIH)--in rabbits

Both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are considered to be reliable biomarkers with sufficient sensitivity and specificity for cardiac injury in the majority of laboratory animals. The aim of our study was to compare the diagnostic performance of cTnT and cTnI in three groups of rabbits: 1) control (saline 1 ml/kg i.v.); 2) Salicylaldehyde Isonicotinoyl Hydrazone--SIH (50 mg/kg, once weekly, i.p.; partially dissolved in 10% Cremophor solution); 3) 10% Cremophor solution in water (2 ml/kg i.v.). The drugs were given once a week, 10 administrations. The concentration of cTnT was measured using Elecsys Troponin T STAT Immunoassay (Roche). The concentration of cTnI was measured using AxSYM Troponin I (Abbott). The linear regression model was applied to see if there is a dependence between cTnT and cTnI. The coefficient of determination was not acceptable in all groups. The highest value of R2 was found in the control group (R2 = 0.424). We may conclude that in rabbits meaningful dependence between cTnT and cTnI was not found. According to our long-term experiences cTnT seems to be more suitable cardiomarker in rabbits in comparison with cTnI where the data are characterized by the large scatter.     

Separation and mapping of multiple genes that control IgE level in Leishmania major infected mice

The strain BALB/cHeA (BALB/c) is a high producer, and STS/A (STS) a low producer of IgE after Leishmania major infection. We analyzed this strain difference using 20 recombinant congenic (RC) BALB/c-c-STS/Dem (CcS/Dem) strains that carry different random subsets of 12.5% of genes of the strain STS on the BALB/c background. Strains CcS-16 and -20 exhibit a high and a low IgE level, respectively. In their F(2) hybrids with BALB/c we mapped nine Leishmania major response (Lmr) loci. Two of them we previously found to influence IgE level in CcS-5. IgE production in CcS-16 is controlled by loci on chromosomes 2, 10, 16 and 18 and in CcS-20 by loci on chromosomes 1, 3, 4, 5 and 8. The STS alleles of loci on chromosomes 1, 4, 5, 8 and 10 were associated with a low, whereas the STS alleles on chromosomes 16 and 18 with a high IgE production. The loci on chromosomes 2 and 3 have no apparent individual effect, but interact with the loci on chromosomes 10 and 1, respectively. The loci on chromosomes 10 and 18 were mapped in the regions homologous with the human regions containing genes that control total serum IgE and intensity of infection by Schistosoma mansoni, suggesting that some Lmr loci may participate in the pathways influencing atopic reactions and responses to several parasites. The definition of genes controlling anti-parasite responses will permit a better understanding of pathways and genetic diversity underlying the disease phenotypes.     

HLA-DPB1 typing by polymerase chain reaction amplification with sequence-specific primers

DPB1 is the second most polymorphic class II locus with currently 84 recognized alleles, i.e. DPB1*0101 to DPB1*8101. Most of the alleles have been described during the last few years using oligonucleotide and sequencing techniques and relatively little is known about the role and importance of the polymorphic residues as regards to the function of DP molecules. In the present study, polymerase chain reaction (PCR) primers were designed for identification of all the phenotypically different DPB1 alleles by PCR amplification with sequence-specific primers. Forty-eight standard genomic PCR reactions per sample were performed in order to achieve this resolution. Unique amplification patterns were obtained in 2983 of 3160 (94.4%) possible genotypes. The primers were combined so that only very rare genotypes gave rise to ambiguous patterns. Sixty-four Histocompatibility Workshop cell lines and 150 DNAs provided by the UCLA DNA exchange were investigated by the DPB1 primer set. All typing results were conclusive. Analysis of the distribution of DPB1 alleles was performed in 200 Caucasian samples, 100 African samples and 40 Oriental samples. The population study by the DPB1 PCR-SSP method showed a characteristic distribution of HLA-DPB1 alleles. Each ethnic group had one, or two, frequent DPB1 allele(s) and the frequency of homozygotes was high, suggesting that balancing selection does not appear to be affecting the evolution of the DPB1 locus.     

The epidemic wave of meningococcal disease in Spain in 1996-1997: probably a consequence of strain displacement

During 1996 and 1997 an epidemic wave of meningococcal disease took place in Spain. Initial studies described the antigenic expression of the epidemic strain as C:2b:P1.2,5 and proposed that it was a variant of the previously identified Spanish C:2b:non-subtypable epidemic strain. To clarify this hypothesis, 1036 C:2b:P1.2(5) and 76 C:2b:NST isolates obtained during 1992-1999 were analysed by pulsed-field gel electrophoresis. The majority of the C:2b:P1.2,5 and C:2b:P1.2 isolates showed one of two very closely related profiles. During the epidemic period, 80% of the C:2b:NST strains showed these two pulsotypes. However, before the epidemic wave, most of these C:2b:NST strains (60%) showed a profile that was found infrequently among C:2b:P1.2,5 and C:2b:P1.2 isolates. A similar evolution was observed in C:2b:P1.5 isolates. Thirty-four C:2b:P1.2(5) and 10 C:2b:NST isolates, exhibiting representative pulsotypes, were subjected to multi-locus sequence typing. Isolates belonging to both A4 and ET-37 lineages were identified. These data point to the possibility that the A4 cluster has displaced the ET-37 complex among serogroup C meningococci in Spain.     

A comparison of the potency of the oxime HLö-7 and currently used oximes (HI-6, pralidoxime, obidoxime) to reactivate nerve agent-inhibited rat brain acetylcholinesterase by in vitro methods

(1) The efficacy of the oxime HL?7 and currently used oximes (pralidoxime, obidoxime, HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. (2) Both H oximes (HL?-7, HI-6) were found to be more efficacious reactivators of sarin and VX-inhibited acetylcholinesterase than pralidoxime and obidoxime. On the other hand, their potency to reactivate tabun-inhibited acetylcholinesterase is very low and does not reach the reactivating efficacy of obidoxime. In the case of cyclosarin, the oxime HI-6 was only found to be able to sufficiently reactivate cyclosarin-inhibited acetylcholinesterase in vitro. (3) Thus, the oxime HL?-7 does not seem to be more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than HI-6 according to in vitro evaluation of their reactivation potency and, therefore, it is not more suitable to be introduced for antidotal treatment of nerve agent-exposed people than HI-6.     

Phantoms for texture analysis of MR images. Long-term and multi-center study

The application of texture analysis (TA) in magnetic resonance imaging (MRI) requires the availability of texture phantoms for use in the standardization of in vivo measurements. The aims of our study were (a) to develop a new type of phantoms suitable for MRI and TA and test their long-term stability; (b) to optimize the choice of texture parameters describing the phantoms; (c) to compare different MR imagers according to texture parameters in a multi-center study. A long-term study performed at 4.7 T proved that the developed phantom based on polystyrene spheres and an agar gel solution is stable at least 12 months. This phantom, with nodular patterns, was found useful for the modeling of structural differences. The comparison of TA parameters at 4.7 and 7 T proved that the same parameters can be used for the separation of structures. The proposed algorithm of the selection of TA parameters shows that there exists a part of texture parameters which can be measured with high reproducibility (1-3%); on the other hand, their absolute values can differ by more than 30% if the textures differ. Results obtained from the multi-center study of whole body MR imagers show the wide variation in the misclassification rates at the different sites and point out the importance of the set up of MR sequences.