Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products.

Protein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.

Effects of rapid rewarming on cerebral nitric oxide production and cerebral hemodynamics after hypothermia therapy for kainic acid-induced seizures in immature rabbits.

BACKGROUND: The aim of the present study was to investigate whether rapid rewarming after hypothermia therapy during seizures alters the endogenous nitric oxide (NO) production in and around hippocampus, cortical cerebral blood flow (cCBF), and mean arterial blood pressure (MABP) in immature rabbits. METHODS: The hypothermic rabbits (rectal temperatures, 33 degrees C) were given kainic acid (KA; 12 mg/kg, i.v; at 0 min), followed by cooling (33 degrees C) for 60 min (at 60 min), then either rewarming (RW; 33-37 degrees C) was started (KA[+]RW[+] group, n = 7) or cooling was continued (KA[+]RW[-] group, n = 7) for another 60 min (at the end 120 min). In the KA(-)RW(+) group (n = 5), 0.5 mL normal saline was given (at time 0 min), followed by cooling (33 degrees C) for 60 min (at 60 min), then rewarming to 37 degrees C was started with observation for another 60 min (at the end 120 min). NO production in and around hippocampus was continuously measured by an NO-selective electrode, cCBF by laser Doppler flowmetry, cortical electroencephalogram (EEG), rectal and cerebral temperatures, and MABP during the experiment. Comparisons were made of these parameters between the values at 60 min and 120 min after the KA administrations. RESULTS: KA administration induced abnormal discharges in both KA(+)RW(+) and KA(+)RW(-) groups at the same degree. The KA(+)RW(+) group had a significant increase in %NO, and significant decreases in %cCBF and MABP after rapid rewarming, compared with before rewarming. In the KA(+)RW(-) group, there were no significant changes in %NO, %cCBF, and MABP between values at 60 and 120 min. These changes after rapid rewarming in the KA(+)RW(+) group were different from those with only elevation in brain temperature from 33 to 37 degrees C without seizures (KA[-]RW[+] group). CONCLUSIONS: These results suggest that rapid rewarming after hypothermia therapy induces an increase in the NO production in and around hippocampus and the decreases in cCBF and MABP during seizures in immature rabbits.

Hypothermia during kainic acid-induced seizures reduces hippocampal lesions and cerebral nitric oxide production in immature rabbits.

We investigated (1) whether cerebral hypothermia during kainic acid (KA)-induced seizures was neuroprotective; and (2) whether nitric oxide (NO) production in the brain during seizures was altered by cerebral hypothermia in immature rabbits. Twelve female rabbits, aged 2 weeks, were anesthetized, paralyzed and mechanically ventilated. We continuously measured NO production in the brain by NO-selective electrode, cortical electroencephalogram (EEG), regional cerebral blood flow (rCBF) by laser Doppler flowmetry, rectal and cerebral temperatures and mean arterial blood pressure (MABP) during KA (12 mg/kg, i.v.)-induced seizures in the hypothermic group (n = 6; rectal temperature, 33 degrees C), and in the normothermic group (n = 6; rectal temperature, 37 degrees C). The normothermic group showed a gradual increase in NO generation in the brain, which was significantly inhibited in the hypothermic group. There were no significant differences in the increases in rCBF, MABP, arterial blood gases, blood glucose, or EEG abnormalities between the two groups. Neuronal damages in the hippocampus (CA3) were significantly lower in hypothermia than in normothermia. These results suggest that hypothermia attenuates NO production during drug-induced seizures and decreases hippocampal brain lesions in the immature rabbit brain. These results may help to explain the neuroprotective effects of hypothermia.

Nitric oxide donor increases cerebral blood flow and oxygenation during kainic acid-induced seizures in newborn rabbits.

BACKGROUND: We investigated the hypothesis that sodium nitroprusside (SNP), a nitric oxide (NO) donor, increased the cerebral blood flow and oxygenation during kainic acid (KA)-induced seizures in newborn rabbits. METHODS: After KA administration (i.v. 12 mg/kg)to induce seizures, either 1.2 mg/kg SNP (SNP group, i.v., n = 6) or 1 mL normal saline (vehicle group, i.v., n = 6)was given. Regional cerebral blood flow (rCBF), cerebral oxyhemoglobin(oxy-Hb), deoxyhemoglobin (deoxy-Hb), total hemoglobin (t-Hb), mean arterial blood pressure (MABP), heart rate (HR) and electroencephalography(EEG) were continuously monitored throughout the experiment, lasting at least 60 min after the KA administration. RESULTS: The value for rCBF was greatly increased during seizures in the SNP group than in the vehicle group. The values for oxy-Hb and t-Hb were significantly increased, and deoxy-Hb was significantly decreased. There were ameliorations of cerebral oxygenation in the SNP group during the acute phase of seizures in the neonatal animals, compared with the vehicle group. There were no significant differences in the MABP, HR, arterial blood gases, rectal and brain temperatures, blood hemoglobin concentrations,blood glucose levels, the latencies to first abnormal discharges in EEG, the total sum of the duration of abnormal discharges in EEG and the incidences of subclinical electric status epileptics between the two groups. CONCLUSIONS: These results suggest that the treatment with SNP contributed to the increases in cerebral blood flow and oxygenation, and that EEG abnormalities were unchanged by the treatment with SNP during neonatal seizures.