Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.     

New therapeutic prospects for the glycosphingolipid lysosomal storage diseases

The Differential Straight Arch technique, more commonly known as "Tip-Edge" (TP Orthodontics Inc.), uses a modified edgewise type bracket to allow differential tooth movement. As bracketed teeth incline toward their finishing positions, the geometry of the arch wire slot causes the vertical slot dimension available to the arch wire to increase. Advantage has been taken of this facility, to evolve an entirely new torquing system for final finishing, during the root correction phase. Instead of increasing rectangular arch wire size to fit the arch wire slot, as with conventional edgewise brackets, the Tip-Edge method reduces the vertical arch wire slot dimension to achieve a precise, three-dimensional relationship to a full-sized rectangular arch wire. The active component is provided by auxiliary springs. The method, which awaits mathematical analysis, is described and illustrated with two case reports.     

Gene structure of the human MET proto-oncogene

By direct sequencing of cosmids using primers designed from the known cDNA sequence, we identified 19 exons in the human MET proto-oncogene, and sequenced the corresponding 5' and 3' exon-intron junctions. By homology search in the database of the Washington University Genome Sequence Center (GSC), we identified one additional exon. These 20 exons, together with a previously reported exon, bring the total exon number of MET to 21. Oligonucleotide primers were designed to amplify each exon and adjacent intronic sequences to permit examination of each exon for mutations. By restriction mapping, we assembled a 110 kb genomic contig that covered almost the entire MET proto-oncogene. This information is relevant for the screening of recently reported mutations of the MET gene which cause hereditary papillary renal carcinomas and for the search for additional mutations of the same gene which may play a role in the pathogenesis of common human carcinomas including carcinomas of the breast, ovary and pancreas.     

Comparative performance of disposable soft contact lenses.

A short-term clinical study was undertaken to compare and evaluate the fit and clinical behaviour of eight disposable hydrogel contact lenses. Thirty subjects were enrolled in a 1-day, randomised, double-masked study. The lenses compared were Vistakon Acuvue (8.80), Vistakon Surevue (8.80), Aspect Frequency-55, Bausch & Lomb Medalist (SeeQuence 2), Ciba Focus (8.90), Lunelle Rythmic, Pilkington Barnes Hind Precision-Wand Wesley Jessen Freshlook. Three of the eight designs, Acuvue (86.7%), Precision-UV (86.7%) and Frequency-55 (90%), achieved relatively high levels of fitting success. The Rythmic lenses achieved the lowest success rate (70%) showing significantly greater downward decentration than the other designs. Although all the lenses were designed to fit a wide variety of eyes, they exhibited widely varying fitting characteristics. Some were more successful than others. The study shows that it is necessary for practitioners to utilise a range of lens designs in order to fit satisfactorily the typical contact lens population.     

Pulp reactions to restoration of experimentally induced crown fractures

OBJECTIVES: Reattachment of the avulsed enamel-dentine coronal fragment to the remaining tooth structure has become an accepted clinical alternative to a resin composite build-up for the restoration of crown fractured teeth. Since little knowledge exists as to the pulpal response to this procedure, this study was designed to observe the condition of the pulp following experimentally induced crown fracture and restoration in monkeys. METHODS: Experiments were conducted in eight young green Vervet monkeys (Cercopithecus aethiops). In all, 64 fractured incisors were investigated. Light microscopic examination of pulp tissue specimens was carried out after 3 months of observation. RESULTS: The evaluation was restricted to specimens having a fracture plane within 2 mm of the pulp and no pulpal exposure. In general, pulp tissue was well preserved irrespective of the restorative procedure. Even if the restoration or the bonded tooth fragment had been lost during the follow-up period, the pulp generally remained in good condition. Inflammatory infiltrates where seen in only a few specimens and then as clusters of mononuclear leukocytes. Hard tissue repair was frequently observed and displayed various configurations from isolated hard tissue deposits to areas of extensive hard tissue repair in the coronal portion of the pulp. Pronounced hard tissue repair and occurrence of inflammatory cell infiltrates correlated with the presence of stainable bacteria on the fractured dentine surface. CONCLUSIONS: In the absence of direct exposure, reparative dentine is a frequent feature of the pulp's response to crown fracture and restoration with composite or reattachment of the crown fragment with dentine bonding. These restorative procedures appear to ensure continued function of the underlying pulp.     

Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis

The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)