Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Multiple genetic alterations in malignant metastatic insulinomas

Proto-oncogenes, growth factors/receptors, and tumour suppressor genes were analysed in malignant metastatic insulinomas. Normal pancreas showed only a moderate immunoreaction for c-myc proto-oncogene and a strong reaction for insulin. Benign insulinomas were slightly or moderately positive for transforming growth factor a (TGFα), weakly positive for epidermal growth factor receptor (EGF-R), and strongly positive for c-myc and insulin. In malignant insulinomas, besides a strong immunoreaction for c-myc and TGFα, activation of c-K-ras and overexpression of p53 protein were found. Insulin reaction was moderate or strong. Three out of six malignant insulinomas displayed a c-K-ras point mutation at codon 12. All mutations were guanine to cytosine transversion, resulting in amino acid substitution, glycine to arginine. Mutations were present in metastatic insulinomas only. Patients with mutated c-K-ras oncogene had overexpression of p53 protein as well as c-myc and TGFα overexpression. Our results support the view that malignant progression is a consequence of more than one genetic lesion and suggest that activation of myc, TGFα, and ras genesα plays a role in a multistep process of tumour progression, perhaps serving as an initiating event.     

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector

Summary. We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4µg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.Authors contributed equally to the results presented in this report.     

Threshold effect of urinary glycosaminoglycans and the walk test as indicators of disease progression in a survey of subjects with Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome)

A cross-sectional survey in individuals affected with the lysosomal storage disease Mucopolysaccharidosis VI (MPS VI) was conducted to establish demographics, urinary glycosaminoglycan (GAG) levels, and clinical progression of the disease. The survey evaluated 121 bona fide MPS VI-affected individuals over the age of 4 years from 15 countries across the Americas, Europe, and Australasia representing greater than 10% of the estimated world prevalence of the disease. A medical history, complete physical exam, urinary GAG determination, and assessment of several clinical measures related to physical endurance, pulmonary function, joint range of motion, strength, and quality of life were completed for each participant. Although a wide variation in clinical presentation was observed, several general findings were obtained reflecting progression of the disease. Impaired physical endurance, as measured by the distance achieved in a 6-min walk, could be demonstrated across all age groups of MPS VI-affected individuals. High urinary GAG values (>200 mug/mg creatinine) were associated with an accelerated clinical course comprised of age-adjusted short stature and low body weight, impaired endurance, compromised pulmonary function, and reduced joint range of motion. An unexpected result was the predominance of urinary GAG values <100 mug/mg creatinine for those participants over the age of 20 years. Pending the collection of longitudinal data, these results suggest that urinary GAG levels predict clinical morbidity, and longer-term survival is associated with urinary GAG levels below a threshold of 100 mug/mg creatinine.     

Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.     

Early detection of COPD by high risk population spirometric screening

COPD is the most frequent chronic lung disease in Poland. The disease is however under-diagnosed, especially at the early stages. The aim of the study was to assess the efficacy of spirometric screening for COPD in middle aged smokers. Informations on causes and symptoms of COPD were disseminated in mass media in 14 large cities. Subject aged over 39 and with smoking history of > 10 packyears were invited for a free spirometry in local chest clinic. However, everyone attending had the spirometry performed. Spirometry was performed according to ATS recommendations. Airway obstruction (AO) was diagnosed when FEV1/FVC < 85% of N and categorised as mild (FEV1 > 70% of N), moderate (FEV1 50-69% of N) or severe (FEV1 < 50% of N). Spirometry was accompanied by an antismoking advice. RESULTS: 12.781 subjects were screened (mean age 52 +/- 12 years, 57% males). In 8.269 subjects who complied with inclusion criteria AO was diagnosed in 29.8% (mild in 10.9%, moderate in 12% and severe in 6.9%). In smokers < 40 years of age and a history of < 10 packyears AO was found in 8.8% (mild in 6.0%, moderate in 1.8% and severe in 1.0%). CONCLUSION: Mass spirometry is an effective and easy method for early detection of COPD.     

Analysis of chromosome 6q in Basque families with type 1 diabetes. GEPV-N. Basque-Navarre Endocrinology and Paediatric Group

Over the last few years several studies of linkage between non-HLA loci and type 1 diabetes mellitus have mapped several putative susceptibility genes on chromosome 6q; in fact, positive evidence of linkage and/or association of IDDM5 (6q25), IDDM8 (6q27) and IDDM15 (6q21) with type 1 diabetes has been reported. We have studied these loci in diabetic families of Basque origin, a genetically homogeneous population, to avoid artifactual association results due to admixture within the sample analysed. Statistical analyses of linkage were performed using a transmission disequilibrium test (TDT). We could not confirm linkage for IDDM5, IDDM8 and IDDM15 in our population, possibly due to population-specific differences in genetic susceptibility and/or environmental triggering factors to type 1 diabetes.