A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

Identification and characterization of the miniature pig Huntington's disease gene homolog: evidence for conservation and polymorphism in the CAG triplet repeat.

Huntington's disease (HD) is associated with a significant expansion of a CAG trinucleotide repeat, which results in a lengthened polyglutamine tract in the single gene product, huntingtin, on human 4p16.3. We isolated cDNA clones that encompassed the entire coding sequence of the miniature pig HD gene (Sus HD) from two porcine testis cDNA libraries. The cDNA contig revealed a 12,749-nucleotide transcript coding for a 345-kDa protein (3139 amino acid residues), which exhibited 96% peptide sequence homology to human huntingtin. Northern blot analysis revealed that the Sus HD gene was ubiquitously expressed as two large transcripts of approximately 11 and 13 kb in size in all the tested tissues, much like the human HD gene. The CAG trinucleotide repeat was found to be interrupted by CAA triplets and to encode 17 or 18 consecutive glutamine residues. In our laboratory stock of miniature pig, three allotypes in the triplet repeat sequence were found. Thus, the Sus HD gene closely resembles its human counterpart in terms of sequence and expression pattern. In particular, human-miniature pig similarities in the normal length of the CAG triplet repeat as well as its repeat-number polymorphism may indicate that miniature pig would provide a good animal model for Huntington's disease.

Cyclin-dependent kinases as a therapeutic target for stroke.

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4/cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.

Neuronal apoptosis inhibitory protein (NAIP) may enhance the survival of granulosa cells thus indirectly affecting oocyte survival.

In mammals, the postnatal loss of more than 99% of female germ cells is due mainly to the process of ovarian follicular atresia. Atresia is known to be mediated by apoptotic granulosa cell-death. Here we show the involvement of neuronal apoptosis inhibitory protein (NAIP) in ovarian folliculogenesis in which it prevents granulosa cell-death. NAIP has been isolated in association with a neurodegenerative disorder, spinal muscular atrophy (SMA), in which it has been shown to suppress mammalian cell-death induced by a variety of stimuli (Liston et al., 1996, Nature 379:349-353). In an in situ hybridization analysis with mouse ovaries, active expression of NAIP mRNA was localized in the granulosa cells of developing follicles from the primary stage to the Graafian stages, whereas the NAIP gene was not expressed or was weakly expressed in follicles that might be undergoing atresia. Gonadotropin, which is known to inhibit apoptosis in ovarian follicles, caused a 2.4-fold increase in NAIP gene expression in the ovary. To study the role of ovarian NAIP, antisense NAIP oligonucleotides were locally delivered into the ovarian bursa. Suppression of ovarian NAIP expression with antisense oligonucleotides evoked a decrease in the number of morphologically normal ovulated oocytes, implying an indirect involvement of NAIP in germ cell development by enhancing the survival of granulosa cells. These findings suggest that gonadotropin-inducible NAIP may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa cell-death during folliculogenesis.

In vivo uptake of [3H]nimodipine into brain during cortical spreading depression.

We report autoradiographic measurements of the in vivo uptake of [3H]nimodipine during the nonischemic depolarization of cortical spreading depression (CSD) in rat brain. [3H]Nimodipine uptake in brain was determined regionally in rats undergoing CSD (n = 8) and was significantly increased in cortex (14 +/- 7%) and hippocampus (10 +/- 6%) on the stimulated side relative to the contralateral hemisphere when compared with the same measurements in a control group (n = 8). A similar measurement using the physiologically inert radiotracer [14C]iodoantipyrine to control for potential effects of CSD on radioligand distribution showed a minimal increase (2.4 +/- 0.7%) of radiotracer uptake in cortex after CSD. This increase was significantly less than that observed in the [3H]nimodipine uptake studies. We hypothesize that increased in vivo [3H]nimodipine uptake in CSD identifies regions of depolarization and thus infers activation of the L-type voltage sensitive calcium channels.

Relationship between extracellular glutamate concentration and voltage-sensitive calcium channel function in focal cerebral ischemia in the rat.

Ischemic cell death occurs when extracellular glutamate levels increase, causing tissue depolarization and an excessive rise in intracellular calcium concentrations. The relative occurrence of the depolarization events and the changes in glutamate concentration in ischemia have not been studied. In a model of focal cerebral ischemia in the rat, three measurements were made simultaneously in vivo: cerebral blood flow (CBF) by the H2-clearance method, extracellular glutamate concentration by microdialysis, and activation of the voltage-sensitive calcium channel (VSCC) by its binding to [3H]nimodipine. Effects of probe implantation on these measurements were accounted for. The CBF to control ratio obtained during the experiments spanned the range of 1.08 to 0.07. Binding to [3H]nimodipine became significantly activated when CBF fell to approximately 0.49 of its control value while extracellular glutamate concentrations increased significantly only at a CBF ratio of < 0.33. Activation of the VSCC at this high CBF ratio may be due to ischemic depolarization, which has been shown to activate the binding to [3H]nimodipine. It may be useful to define a CBF threshold of 50% of normal in focal ischemia for opening of the VSCC. The same threshold has been linked to an overall depression of protein synthesis and to activation of a number of molecular responses.

Relevance of interstitial glutamate to selective vulnerability in focal cerebral ischemia.

Glutamate concentrations in striatum and cortex were measured by means of in vivo cerebral microdialysis before and for 4 h after middle cerebral and ipsilateral common carotid artery occlusion in rats. The peak glutamate concentration reached 7.28 +/- 3.60 microM in dialysate from striatum and 5.64 +/- 2.24 microM in that from cortex. An index of exposure of each region to glutamate was calculated by integrating glutamate concentrations after occlusion. During ischemia the striatum was exposed to statistically higher cumulative concentrations of glutamate than the cortex (p < 0.01). The difference in vulnerability between striatum and cortex may arise from the additional time needed for the cortex to be exposed to cumulative threshold levels of glutamate.

Hepatic portoenterostomy for congenital biliary atresia - method of dissection of porta hepatis, evaluation of revision, re-boring and supplementary operations.

A few guidelines for surgical dissection at the porta hepatis in hepatic portoenterostomy for congenital biliary atresia as evolved from our experiences on 52 cases are suggested. Indication for revision is also shown. Supplementary procedures in case of complications, such as ascending cholangitis and the onset of portal hypertension, are also described. An early primary operation as well as a positive revision and supplementary procedures for ascending cholangitis and oesophageal varices have resulted in an improvement of the therapeutic results.