Ribozyme-mediated reduction of the GABA(A) receptor alpha1 subunit.

As an approach to understanding the role of the alpha1 subunit of the GABA(A) receptor, ribozymes were designed to reduce expression of this subunit protein by hydrolysis of alpha1 subunit message and antisense inactivation. The ribozyme cleavage sites were selected through homology comparison of all known murine GABA(A) receptor subunits at the amino acid and nucleotide sequence level. Two ribozymes were designed and synthesized: one against the extracellular domain and the other against the cytoplasmic domain. These ribozymes were cloned in a mammalian expression plasmid, pZeoSV2 (+). Cleavage of both extracellular and cytoplasmic domain transcripts by the respective ribozymes was observed when each ribozyme was tested against in vitro transcribed mRNA. The stable cell line, 122, expressing recombinant human GABA(A) alpha1, beta2 and gamma2S subunits of receptor was stably transfected with the cytoplasmic domain ribozyme (cy) alone and with both the cytoplasmic (cy) and extracellular domain (ex) ribozyme expression plasmids. Northern analysis showed a 55-60% reduction of alpha1 mRNA in clones of cells transfected with either the single ribozyme (Cy) or with both ribozymes (EC). The alpha1 protein level was reduced 75% in a stable Cy clone and more than 90% in a stable EC clone when compared with alpha1 expression in 122 cells and the vector transfected (Zeo) cells. Electrophysiological analysis revealed that the GABA(A) receptor properties were very similar in 122 cells and in stable clones in which the subunit protein expression had been greatly reduced. No significant difference was detected in the potentiation of the receptor response by either bretazenil or zolpidem. These data demonstrate the efficacy of the ribozyme approach in dramatically reducing GABA(A) subunit protein levels in transfected cells and identify those elements that will be important to the application of similar ribozymes to knock-down transmitter receptor subunit proteins under inducible promoters in transgenic mice.

Glycosuria and diabetes mellitus in children and adolescents in south India.

In Madras city (India) 10,513 school students between 3 and 20 yr of age were investigated for glycosuria and its causes. While no previously known cases of diabetes mellitus of any type were encountered, four students (0.038%) in the survey population were found to have glycosuria. One (0.009%) had renal glycosuria, two (0.019%) were possibly NIDDY (MODY) and one (0.009%) had transient glycosuria while receiving anti-tuberculous chemotherapy. It is therefore concluded that neither diabetes mellitus nor glycosuria of non-diabetic causes is a crucial health problem in Indian children and adolescents. While the reasons for this are not known, further research in this field could be of global interest.

Prevalence of diabetes in the young in south India.

Students from nine schools and one college in Madras city, were screened for diabetes by oral glucose tolerance test. The criteria recommended by the World Health Organization was adopted to classify glucose tolerance. Among 3,515 students, between 5 and 19 years of age, participated in this survey, 1982 (56.4%) were males and 1.533 were (43.6%) females. Family history of diabetes was positive in 302 (8.6%) students. There was no overt case of diabetes of any type. Three (0.09%) males had renal glycosuria. It is therefore concluded that insulin-dependent diabetes, non-insulin dependent diabetes or any other type of diabetes in the young is rare in South India.

Carbon microelectromechanical systems as a substratum for cell growth.

The study of the biocompatible properties of carbon microelectromechanical systems (carbon-MEMS) shows that this new microfabrication technique is a promising approach to create novel platforms for the study of cell physiology. Four different types of substrates were tested, namely, carbon-MEMS on silicon and quartz wafers, indium tin oxide (ITO) coated glass and oxygen-plasma-treated carbon thin films. Two cell lines, murine dermal fibroblasts and neuroblastoma spinal cord hybrid cells (NSC-34) were plated onto the substrates. Both cell lines showed preferential adhesion to the selectively plasma-treated regions in carbon films. Atomic force microscopy and Fourier transform infrared spectroscopy analyses demonstrated that the oxygen-plasma treatment modifies the physical and chemical properties of carbon, thereby enhancing the adsorption of extracellular matrix-forming proteins on its surface. This accounts for the differential adhesion of cells on the plasma-treated areas. As compared to the methods reported to date, this technique achieves alignment of the cells on the carbon electrodes without relying on direct patterning of surface molecules. The results will be used in the future design of novel biochemical sensors, drug screening systems and basic cell physiology research devices.

Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase.

Recent studies in Saccharomyces cerevisiae suggest that the delivery of copper to Cu/Zn superoxide dismutase (SOD1) is mediated by a cytosolic protein termed the copper chaperone for superoxide dismutase (CCS). To determine the role of CCS in mammalian copper homeostasis, we generated mice with targeted disruption of CCS alleles (CCS(-/-) mice). Although CCS(-/-) mice are viable and possess normal levels of SOD1 protein, they reveal marked reductions in SOD1 activity when compared with control littermates. Metabolic labeling with (64)Cu demonstrated that the reduction of SOD1 activity in CCS(-/-) mice is the direct result of impaired Cu incorporation into SOD1 and that this effect was specific because no abnormalities were observed in Cu uptake, distribution, or incorporation into other cuproenzymes. Consistent with this loss of SOD1 activity, CCS(-/-) mice showed increased sensitivity to paraquat and reduced female fertility, phenotypes that are characteristic of SOD1-deficient mice. These results demonstrate the essential role of any mammalian copper chaperone and have important implications for the development of novel therapeutic strategies in familial amyotrophic lateral sclerosis.