Primary cultures of adult rat hepatocytes were treated in the presence or absence of extracellular calcium with ten different membrane-active toxins. In all cases more than half the cells were killed in 1 to 6 hours in the presence but not in the absence of extracellular calcium. An effect of calcium on the primary mechanism of membrane injury by any of the agents cannot be implicated. Viability, as determined by trypan blue exclusion correlated well with other indices of viability such as plating efficiency and the hydrolysis of fluorescein diacetate. It is concluded that the cells are killed by processes that involve at least two steps. In each type of injury, disruption of the integrity of the plasma membrane by widely differing mechanisms is followed by a common functional consequence involving extracellular calcium, and most likely representing an influx of calcium across the damaged plasma membrane and down a steep concentration gradient. This later step represents, or at least initiates, a final common pathway for the toxic death of these cells.

During cell division and during the induction of tubulin synthesis that accompanies flagellar regeneration in Chlamydomonas reinhardi, four tubulin mRNAs of discrete molecular sizes are produced. During induction two beta tubulin mRNAs (2.47 kb and 2.34 kb) and two alpha tubulin mRNAs (2.26 kb and 2.13 kb) are synthesized in high abundance and in a closely coordinated fashion. Combined data from restriction enzyme mapping (i.e., Southern analysis) of genomic DNA and of Charon 30 recombinant clones bearing inserts of Chlamydomonas tubulin genes provide direct evidence for four distinct tubulin genes in this organism. Dot-blot analysis of the level of hybridization of a 32p nick-translated beta tubulin cDNA to genomic DNA from gametic cells and to a clone containing the beta 1 tubulin gene indicate that each beta 1 tubulin gene is present in one copy per cell. Additional hybridization experiments employing fragments of cDNA clones which selectively anneal to either the 3' or 5' portions of the two alpha tubulin genes or to one or both of the two beta tubulin genes suggest that each tubulin gene is actively transcribed to give rise to one of the four tubulin mRNAs. These observations further suggest that at most four basic types of tubulin subunits are produced by Chlamydomonas and that the heterogeneity of tubulin subunits reported to exist in the flagellar axoneme must arise as a result of post-translational modification.

No abstract

The role of Ca2+ in toxic liver cell death was studied with primary cultures of adult rat hepatocytes. Within

IntroductionThe Congenital Cranial Dysinnervation Disorders are congenital, non-progressive disorders that result from developmental abnormalities of one or more cranial nerves/nuclei with primary or secondary dysinnervation and include Duane's syndrome, Möbius syndrome, Congenital Fibrosis of the External Ocular Muscles (CFEOM) and Congenital ptosis. Cases are obtained for phenotype/genotype research via a number of reporting mechanisms including the British Neurological Surveillance Unit. Recently mutations in the β-tubulin isotype 3 (TUBB3) gene have been reported to produce several phenotypes depending on the particular mutation.MethodsSequence analysis of the TUBB3 gene was undertaken in Congenital Cranial Dysinnervation Disorder families with CFEOM phenotypes for which a genetic cause had not previously been determined.ResultsFour affected individuals in two families were identified to have a previously reported heterozygous missense TUBB3 mutation, E410K. The phenotype in all individuals consisted of CFEOM with some variability but preserved eye abduction bilaterally and striking facial weakness. Other findings included developmental delay, corpus callosum hypoplasia and an adult with sensory axonal neuropathy.ConclusionThere are dramatic genotype/phenotype correlations depending on the particular TUBB3 mutation. Although there was striking facial weakness in our cases with ocular involvement they did not fulfil the criteria for Möbius syndrome as abduction was preserved.