NAD(P)H oxidase p22phox C242T polymorphism and ischemic stroke in Japan: the Fukuoka Stroke Registry and the Hisayama study.

The C242T polymorphism of p22phox, a component of NAD(P)H oxidase, may have an impact on cardiovascular diseases; however, the association between this polymorphism and brain infarction is not fully understood. Here, we investigate the relationship between the C242T polymorphism and brain infarction in Japan. We recruited 1055 patients with brain infarction and 1055 control subjects. A chi-squared test revealed that the T-allele frequency was lower in patients with cardioembolic infarction (5.6%) than in control subjects (11.0%, P < 0.001); however, allele frequencies in patients with lacunar and atherothrombotic infarction (11.2%) were not significantly different from those in control subjects (11.0%). A multivariate-adjusted conditional logistic regression analysis also revealed no association between CT + TT genotype, and lacunar and atherothrombotic infarction (odds ratio = 0.97, 95% confidence interval: 0.72-1.32). To investigate the functional effects of the C242T polymorphism, we examined superoxide production in COS-7 cells cotransfected with Nox4 and p22phox of each genotype. The superoxide-producing activity in those cells expressing p22phox with the T allele was not significantly different from that in cells expressing p22phox with the C allele. The present results suggest that the p22phox C242T polymorphism may have a protective effect against cardioembolic infarction, but is not related to lacunar and atherothrombotic infarction in Japan.

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells.

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (alpha, beta-1, beta-2, beta-3, beta-4 and beta-5), PP2C alpha was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2C alpha was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2C alpha is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.

Mutational analysis of the domain structure of mouse protein phosphatase 2Cbeta.

The structures of five distinct isoforms of mammalian protein phosphatase 2Cbeta (PP2Cbeta-1, -2, -3, -4 and -5) have previously been found to differ only at their C-terminal regions. In the present study, we performed mutational analysis of recombinant mouse PP2Cbeta-1 to determine the functional domains of the molecule and elucidate the biochemical significance of the structural differences in the isoforms. Differences in affinity for [32P]phosphohistone but not for [32P]phosphocasein were observed among the five PP2Cbeta isoforms. Deletion of 12 amino acids from the C-terminal end, which form a unique sequence for PP2Cbeta-1, caused a 35% loss of activity against [32P]phosphohistone but no loss of activity against [32P]phosphocasein. Deletion of up to 78 amino acids from this end did not cause any further alteration in activity, whereas deletion of 100 amino acids totally eliminated the activity against both [32P]phosphohistone and [32P]phosphocasein. On the other hand, deletion of 11 amino acids from the N-terminal end caused a 97% loss of enzyme activity, and further deletions caused a total loss of activity. Substitution of any of the six specific amino acids among 16 tested in this study, which were located among the 250 N-terminal residues, caused 98-100% loss of enzyme activity. Among these amino acids, three (Glu-38, -60 and -243) have recently been reported to be essential for the binding of metal ions in the catalytic site of the PP2C molecule [Das, Helps, Cohen and Barford (1996) EMBO J. 15, 6798-6809]. These observations indicate that PP2Cbeta is composed of at least two distinct functional domains, an N-terminal catalytic domain of about 310 amino acids and the remaining C-terminal domain, which is involved in determination of substrate specificity.

Effects of high atmospheric pressure and oxygen on middle cerebral blood flow velocity in humans measured by transcranial Doppler.

BACKGROUND AND PURPOSE: There are several reports that have studied the effects of hyperbaric oxygen (HBO) on cerebral blood flow (CBF). However, most of the reports have been of animal experiments, and human studies are few so far. The aim of this study is to clarify the relationship between HBO and CBF in humans. METHODS: Middle cerebral arterial blood flow velocity (MCV) was measured using transcranial Doppler (TCD) technique in a multiplace hyperbaric chamber. The Doppler probe was fixed on the temporal region by a head belt, and the transcutaneous gas measurement apparatus (tcPO2 and tcPCO2) was fixed on the chest wall. MCV and transcutaneous gas were measured continuously in eight healthy volunteers under four various conditions: 1 atmosphere absolute (ATA) air, 1 ATA oxygen (O2), 2 ATA air, and 2 ATA O2. On the next step, the effect of environmental pressure was studied in another eight healthy volunteers, in whom the tcPO2 was kept at almost the same level under conditions of both 1 ATA and 4 ATA by inhaling oxygen at 1 ATA. RESULTS: MCV of 1 ATA O2, 2 ATA air, and 2 ATA O2 decreased, and tcPO2 increased significantly in comparison with that of 1 ATA air. A significant difference in MCV was observed between the O2 group and the air group under the same pressure circumstance. On the other hand, there were no differences in MCV or tcPO2 between 4 ATA air and 1 ATA plus O2, and the influence for the MCV of the environmental pressure was not observed. CONCLUSIONS: We conclude that hyperoxemia caused by HBO reduces the CBF, but the high atmospheric pressure per se does not influence the CBF in humans.

Differentiation-dependent enhanced expression of protein phosphatase 2Cbeta in germ cells of mouse seminiferous tubules.

The presence of five distinct isoforms of protein phosphatase 2Cbeta (PP2Cbeta-1 approximately -5) is known. In this study, we demonstrate that the mRNA levels of PP2Cbeta-3, -4 and -5 and PP2Cbeta protein level increased during the course of the first wave of spermatogenesis in neonatal mouse testis. Northern blot and in situ hybridization analyses revealed that PP2Cbeta-3, -4 and -5 were expressed predominantly in pachytene spermatocytes and in more highly differentiated germ cells. The substrate specificity of PP2Cbeta-4 determined with artificial substrates differed from those of PP2Cbeta-3 and -5, suggesting that the difference in the structure of PP2Cbeta-3, -4 and -5 reflect their unique physiological functions in testicular germ cells.

Okadaic acid suppresses neural differentiation-dependent expression of the neurofilament-L gene in P19 embryonal carcinoma cells by post-transcriptional modification.

Mouse P19 embryonal carcinoma cells in aggregation culture in the presence of 10(-6) M retinoic acid followed by monolayer culture differentiate into nerve and glial cells. In this study, we demonstrated that the neurofilament-L (NF-L) mRNA and protein levels of these cells were enhanced in accordance with their retinoic acid-induced neural differentiation. Okadaic acid (OA) treatment of the cells markedly suppressed this differentiation-dependent NF-L gene expression increase and neurite outgrowth of the cells. Similar results were obtained when tautomycin was used instead of OA, suggesting that inhibition of protein phosphatase(s) is involved in the suppression of neural differentiation. OA treatment did not affect the NF-L gene transcription level, determined by the nuclear run-on transcription assay, but it did reduce the stability of both the 3.5- and 2.3-kilobase NF-L mRNAs. The expression and activity levels of protein phosphatase 2A (PP2A) and 2B (PP2B) but not protein phosphatase 1 (PP1) in P19 cells increased in accordance with the enhanced NF-L gene expression. The presence of OA in the culture medium during the course of the neural differentiation caused a reduced PP2A activity but not PP1 and PP2B activities of the cell extracts. On the other hand, both PP1 and PP2B activities but not PP2A activity of cell extracts were suppressed by the addition of cyclosporin A or FK506 in the culture medium. However, both cyclosporin A and FK506 treatments affected neither NF-L gene expression nor neurite outgrowth. These results demonstrate that the OA treatment inhibits the differentiation-dependent increase in NF-L gene expression by destabilizing its mRNAs and suggest that PP2A plays key roles in the differentiation-dependent enhanced expression of the NF-L gene and is the point of the action of OA.

Brain ischemia following bilateral carotid occlusion during development of hypertension in young spontaneously hypertensive rats--importance of morphologic changes of the arteries of the circle of Willis.

The present study was designed to examine the effect of morphologic changes of the arteries of the circle of Willis on cerebral blood flow (CBF) and metabolism in young spontaneously hypertensive rats (SHR). CBF in the parietal cortex was measured by the hydrogen clearance method before and during a one-hour bilateral carotid artery occlusion (BCO), and supratentorial brain metabolites were determined by standard enzymatic methods at a one-hour BCO. The internal diameters of the main arteries of the circle of Willis were estimated morphologically. With increase in age, systemic arterial pressure at rest was significantly raised, while cortical CBF tended to decrease and calculated cerebral vascular resistance increased. During BCO, CBF and supratentorial metabolism (adenosine triphosphate and lactate/pyruvate ratio) tended to be better preserved in two-month-old rats as compared with those in one- or three-month-old rats. The internal diameter of the posterior communicating artery (PcomA) was significantly smaller in the one-month-old group than in the other groups, while the diameter of the internal carotid artery was significantly smaller in rats aged three months than those in rats aged one or two months. It is indicated that cortical CBF reduction and impairment of supratentorial metabolism following occlusion of carotid arteries, at least in part, depend on the morphologic changes of the arteries of the circle of Willis associated with age and development of hypertension in young SHR.

Enhanced UV sensitivity of yeast cells induced by overexpression of Mg(2+)-dependent protein phosphatase alpha (type 2C alpha).

The UV sensitivity of wild-type Saccharomyces cerevisiae cells was increased 2-fold when rat Mg(2+)-dependent protein phosphatase alpha (protein phosphatase type 2C alpha) was overexpressed in the cells. The overexpression of this enzyme rendered the rad 18 mutant (defective in postreplication repair) more UV-sensitive than was observed in the wild-type cells. However, this increase in UV sensitivity disappeared when the host cells had a rad 1 mutation (defective in excision repair). These results suggest that the Mg(2+)-dependent protein phosphatase overexpressed in the yeast cells inhibited their excision repair system.

Localization of the mouse protein serine/threonine phosphatase 2C beta gene to chromosome 17E 4-5.

Protein phosphatase 2C (PP2C) is one of four major classes of protein serine/threonine phosphatase and is considered to have a role in signal transduction of stress responses. It has two isotypes, alpha and beta, encoded by different genes. In this study, the mouse PP2C beta gene was mapped by in situ hybridization to chromosome 17E 4-5.

Molecular cloning and expression of mouse mg(2+)-dependent protein phosphatase beta-4 (type 2C beta-4).

A full-length complementary DNA (cDNA) clone (pTK-3) encoding an isoform of Mg(2+)-dependent protein phosphatase beta (MPP beta-4) was isolated for the first time from a mouse melanocyte cDNA library. It was strongly suggested that the mRNA corresponding to the pTK-3 insert was a splicing variant of a single pre-mRNA that also encodes MPP beta-1 and -2 (T. Terasawa, T. Kobayashi, T. Murakami, M. Ohnishi, S. Kato, O. Tanaka, H. Kondo, H. Yamamoto, T. Takeuchi, and S. Tamura, 1993, Arch. Biochem. Biophys. 307, 342-349). The amino acid sequence of MPP beta-4 differed from those of MPP beta-1 and -2 only at the carboxyl terminal region. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) revealed that MPP beta-4 mRNA was expressed only in testis and intestine and not in other mouse tissues tested. Specific expression of the mRNA signals of two other isoforms of MPP beta, MPP beta-3 and -5 (a novel isoform), in testis and intestine was also demonstrated by the RT-PCR. The carboxyl terminal region of MPP beta-5 was found to have a chimera structure composed of part of MPP beta-1 and part of MPP beta-3. The recombinant MPP beta-3 and -4 and the putative MPP beta-5 expressed in Escherichia coli cells exhibited Mg(2+)-dependent and okadaic acid-insensitive protein phosphatase activities. It was demonstrated that the mRNA expression levels of MPP beta-3, -4, and -5 alter according to the maturation of mouse testis. These results suggest that the complex structure of MPP beta isoforms and their tissue- and developmental stage-specific expression reflect the variety of their physiological functions.

Effects of acute superior cervical ganglionectomy on cerebral blood flow and metabolism in stroke-prone spontaneously hypertensive rats subjected to cerebral ischaemia.

1. The effects of acute bilateral superior cervical ganglionectomy on cerebral blood flow and metabolism were investigated in stroke-prone spontaneously hypertensive rats (SHRsp), before and during cerebral ischaemia. 2. The resting cerebral blood flow was comparable between the control and denervated animals. 3. There was no significant difference in cerebral blood flow or concentration of tissue energy metabolites (adenosine triphosphate [ATP], lactate and pyruvate) between the sham-operated control and denervated animals during ischaemia. 4. The results suggest that sympathetic innervation of cerebral vessels originating from superior cervical ganglia may not play a major role in the progression of cerebral ischaemia in SHRsp.

[Disturbances of fluid and electrolyte balance in patients with acute stroke].

Serum sodium and potassium concentrations were measured in 196 patients with acute cerebral infarction and 56 with cerebral hemorrhage. All patients were admitted within 7 days of onset and the data within 2 weeks of admission were recorded. The incidences of hypernatremia (serum Na greater than or equal to 149 mEq/l), hyponatremia (less than or equal to 134 mEq/l), hyperkalemia (serum K greater than or equal to 4.8 mEq/l) and hypokalemia (less than or equal to 3.2 mEq/l) were higher in patients with hemorrhage (18, 7, 13 and 14%, respectively) than infarction (4.5, 4.5, 11 and 6%, respectively). The incidences of hypernatremia and hyponatremia in infarction were higher in those who had cortical lesions than in those who had lesions in the basal ganglia or infratentorium. In cerebral hemorrhage, the incidence of hypernatremia was the highest in those with brain stem lesion. Hypernatremia was found in 27% of large sized hematoma, being significantly higher than that of those with medium (16%) or small (1%) hematoma. A similar tendency was also observed in hyponatremia and hyperkalemia. In elderly patients, electrolyte disturbances were more common than in young or middle-aged patients. Renal insufficiency and diabetes mellitus were frequent complications in stroke patients with hypernatremia (42 and 32%, respectively), of which 57% died within one month of admission.

Regional cerebral blood flow and metabolism in patients with transient global amnesia: a positron emission tomography study.

In four patients who experienced transient global amnesia (TGA), clinical features and neuroradiological findings including positron emission tomography (PET) were studied within three months of the episodes, and compared with those in seven cases with cerebral transient ischaemic attacks (TIA). None of TGA patients had a previous history or significant risk factors for the cerebrovascular diseases. Their electroencephalogram, brain CT and angiogram for the head and neck were almost normal. PET study showed better preserved cerebral blood flow and oxygen metabolism in each area of the brain in patients with TGA compared with those with TIA in whom focal reductions of flow and metabolism were evident. These observations suggest that TGA is caused by reversible circulatory and/or metabolic disturbance, of which mechanism might be different from that in TIA.

Iodide-induced thyrotoxicosis in a thyroidectomized patient with metastatic thyroid carcinoma.

An unusual case of iodide-induced thyrotoxicosis is documented in this article. The patient was a 64-year-old euthyroid man with acromegaly. He also had multiple follicular and papillary thyroid carcinomas with a metastatic lesion in the lumbar vertebrae. After a total thyroidectomy, he became slightly hypothyroid, and the lumbar lesion began to incorporate 131I by scintigraphy. When an iodine-containing contrast medium happened to be injected, a transient increase of serum thyroid hormone level was observed. After complete thyroid ablation with 83 mCi of 131I, the oral administration of 100 mg of potassium iodide for 7 days induced a prominent increase of serum thyroid hormone level. These findings indicated that the metastatic thyroid carcinoma could produce excess thyroid hormone insofar as a sufficient amount of iodide was given. Although this is the first report of such a case, iodide-induced thyrotoxicosis may not be rare in patients with thyroid carcinomas because the Wolff-Chaikoff effect is thought to be lost, and the organic iodinating activity and lysosomal protease activity are well-preserved.

Importance of bilateral sympathetic innervation on cerebral blood flow autoregulation in the thalamus.

Effects of bilateral sympathetic innervation on the regulation of cerebral blood flow to the thalamus were examined in spontaneously hypertensive rats (SHR). The superior cervical ganglion was removed on one side or bilaterally, and blood flow in the thalamus was repeatedly measured with a hydrogen clearance technique during a stepwise increase in arterial pressure. Regional blood flow in the thalamus was unchanged following acute ganglionectomy: 55 +/- 6 ml/100 g/min in the intact rats and 56 +/- 4 in the denervated rats. Sympathectomy on one side neither had effects on the pressure-flow relationship nor on the blood pressure levels of upper limits of autoregulation in the ipsilateral thalamus. In contrast, bilateral sympathetic denervation impaired the autoregulatory function in the thalamus and the upper limits were significantly lower than those in intact rats: 206 +/- 8 vs 226 +/- 10 mm Hg, respectively (P less than 0.02). It is concluded that overlapping innervation of sympathetic nerves has an important role in regulation of blood flow to the thalamus during an acute rise in arterial pressure in SHR.

Cerebrospinal fluid lactate in patients with hepatic encephalopathy.

Cerebrospinal fluid (CSF) lactate and pyruvate concentrations were determined in 16 patients with hepatic encephalopathy before and/or after treatment. CSF lactate was significantly increased to 1.92 +/- 0.11 mmol/l in hepatic encephalopathy before the treatment in comparison to 1.40 +/- 0.05 mmol/l in control subjects. In 9 of 11 patients with moderate or stage 2 encephalopathy, CSF lactate levels were below 2 mmol/l. In contrast, in 4 of 5 patients with stage 3-4 encephalopathy, CSF lactate levels were higher than 2 mmol/l. CSF lactate was decreased with the recovery of neurological symptoms by the treatment. These findings indicate that CSF lactate levels reflect the severity of metabolic impairment of the brain. Hypocapnia was frequently observed in these encephalopathic patients, and arterial PCO2 correlated inversely with CSF lactate and linearly with CSF HCO3-, suggesting that CSF lactic acidosis contributes to hyperventilation in hepatic encephalopathy. It is concluded from present results that metabolic disorder of neuronal cells might be one of the important factors for the development of hepatic encephalopathy.

Cerebral and cerebellar blood flow autoregulations in acutely induced cerebral ischemia in spontaneously hypertensive rats--transtentorial remote effect.

Autoregulation of cerebral (CBF) and cerebellar blood flow (CeBF) was studied before, during and after acutely induced cerebral ischemia in spontaneously hypertensive rats. Cerebral ischemia of the supratentorial portion was induced for one hour by bilateral carotid artery ligation (BCL). The animals were artificially ventilated and the blood flow was measured with a hydrogen clearance technique. To test the autoregulation, the blood pressure was stepwise lowered by bleeding and maintained at a new level, i.e. 15% or 30% lower than the baseline values before, during and after cerebral ischemia. At the preischemic state, CBF and CeBF were 52.1 +/- 6.2 and 58.9 +/- 4.6 ml/100 g/min (mean +/- SEM), of which autoregulations were normally preserved. Following BCL, CBF was markedly decreased to about 10% of control value while CeBF was minimally reduced to 46.9 +/- 8.6 ml/100 g/min (80%). At the ischemic state, CBF became almost zero flow during hypotension. CeBF was also reduced to 74% and further to 58% of the resting value by 15% and 30% decrease in the blood pressure, respectively, indicating impaired CeBF autoregulation. At the 30 min post-ischemic state, CBF was recovered to 48.0 +/- 4.9 and CeBF to 53.9 +/- 5.4 ml/100 g/min. Autoregulation of CBF was still abolished, whereas CeBF was kept constant by 15% fall of blood pressure and slightly reduced to 84% by 30% hypotension, indicating almost recovery of CeBF autoregulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional cerebral blood flow autoregulation in normotensive and spontaneously hypertensive rats--effects of sympathetic denervation.

The present study was designed to investigate the effect of acute sympathetic denervation on the regional cerebral blood flow (CBF) autoregulation during acute elevation of blood pressure in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). CBF to the parietal cortex and thalamus was measured by the hydrogen clearance method and, to test autoregulation, systemic arterial blood pressure was elevated by intravenous infusion of phenylephrine. Superior cervical ganglia were removed on both sides to interrupt sympathetic innervation in the deeper structures of the brain. Acute bilateral sympathetic denervation did not alter the resting blood pressure or CBF in either SHR or WKY. In innervated SHR, resting mean arterial pressure (MAP) was 165 +/- 5 mm Hg (mean +/- SEM) and the upper limit of autoregulation in the cortex was 210 +/- 3 mm Hg, which was significantly lower than that in the thalamus (229 +/- 3 mm Hg, p less than 0.02). In bilaterally denervated SHR, the upper limits were lowered to 193 +/- 4 mm Hg in the cortex (p less than 0.02 vs. innervated SHR) and to 207 +/- 5 mm Hg in the thalamus (p less than 0.02 vs. innervated). In WKY, resting MAP was approximately 55 mm Hg lower than that in SHR. Acute denervation reduced the upper limits from 142 +/- 3 mm Hg to 130 +/- 4 in the cortex (p less than 0.05) and from 158 +/- 4 to 145 +/- 4 in the thalamus (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)