Right and left inverted lobar lung transplantation.

Adult recipients frequently withdraw from living-donor lobar lung transplantation because of the small size of donor grafts. The right lower lobe is 120% larger than the left lower lobe. We developed a novel surgical technique in which an inverted right lower lobe graft can be transplanted into the left thorax. The first patient was a 43-year-old woman with end-stage idiopathic interstitial pneumonia. Her husband was the only eligible donor for living-donor lobar lung transplantation. His right lower lobe was estimated to provide 45% of the recipient's predicted forced vital capacity, which would provide the borderline function required for living-donor lobar lung transplantation. Since lung perfusion scintigraphy of the recipient showed a right-to-left ratio of 64:36, transplanting the right lower lobe graft into the left thorax and sparing the native right lung was considered the only treatment option. We simulated this procedure using three-dimensional models produced by a three-dimensional printer. In living-donor lobar lung transplantation, all anastomoses were performed smoothly as planned preoperatively. Because of the initial success, this procedure was performed successfully in two additional patients. This procedure enables larger grafts to be transplanted, potentially solving critical size matching problems in living-donor lobar lung transplantation.

Adenosine 3',5'-monophosphate-dependent protein kinase from brain.

Adenosine 3',5'-monophosphate at a concentration of 5 x 10(-7) mole per liter causes a 400 percent increase in the rate of phosphorylation of histone catalyzed by a partially purified enzyme preparation from rabbit brain. The data provide the first direct evidence of a biochemical action of adenosine 3',5'-monophosphate in the brain.

Reliability of Japanese clinical trials estimated from GCP audit findings.

To describe the reliability of Japanese clinical trials, we compared the results of a Good Clinical Practice (GCP) audit conducted between April 1997 and March 2000 (fiscal year (FY) 1997 - 1999) with those from April 2004 - March 2005 (FY2004). The number and proportion of various types of deficiencies described in GCP audit reports were compared between the 2 periods. The audit findings in the former period were based on official audits that covered 331 hospitals and 775 trials. The audits in the latter period targeted 114 hospitals and 189 trials. The inspection of former period was undertaken by the Organization for Pharmaceuticals Safety and Research (OPSR). On the other hand, the latter period was undertaken by the Pharmaceuticals and Medical Devices Agency (PMDA). The total number of deficiencies detected in GCP audits was 1,529 in the former 3-year period (FY1997 - 1999) and 819 in the latter period (FY2004). The total number of deficiencies detected and reported was more than 1.5-fold on an annual basis in the latter period. By category of deficiencies, the proportion of protocol deviations increased from 14.7 (225/1,529) to 45.7% (374/819), while the proportion of errors in case report forms (CRFs) decreased from 43.6 (666/ 1,529) to 27.1% (222/819). There were two remarkable changes in audit findings between FY1997 - 1999 and FY2004; the increase in the proportion of protocol deviations and the decrease in the proportion of CRF-related deficiencies. We think that in Japan the improvement of research environments is needed to provide reliable clinical data responsible for the regulatory standard of GCP.

Distribution of 10 periodontal bacterial species in children and adolescents over a 7-year period.

OBJECTIVE: There is scant information available regarding the distribution of periodontal bacterial species in children and adolescents over an extended period. The purpose of this study was to compare bacterial profiles in the same individuals over a period of 7 years. SUBJECT AND METHODS: Twenty-six children and adolescents from whom dental plaque and saliva specimens were obtained during both the first (1999-2000) and second (2006-2007) periods, were analyzed. Bacterial DNA was extracted from each specimen and the presence of 10 periodontal bacterial species was determined using a PCR method, with a focus on the red complex species of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. RESULTS: Subjects with red complex species in saliva specimens obtained during the second collection possessed a significantly higher number of total bacterial species than those without. The detection rate of the red complex species in the second collection period samples was significantly greater in subjects who had two or more species detected in samples taken during the first collection compared with the other subjects. CONCLUSION: Subjects possessing red complex species may be at possible risk for infection with a high number of periodontal bacterial species during adolescent and younger adult years.

Changes in Ca(2+)/calmodulin-dependent protein kinase II activity and its relation to performance in passive avoidance response and long-term potentiation formation in mice prenatally exposed to diethylstilbestrol.

We investigated the effects of prenatal exposure to diethylstilbestrol (DES), an endocrine disrupter on learning behavior and synaptic functions. Specifically, we determined the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and related kinases that play an essential role in long-term potentiation (LTP) in the hippocampus in mice that were prenatally exposed to DES. Treatment with DES resulted in increased CaMKII autophosphorylation and Ca(2+)-independent activity in the hippocampus and cortex of male mice. Impaired passive avoidance correlated with this increased CaMKII autophosphorylation, as did the enhanced early phase of LTP (E-LTP) in hippocampus. These data suggest that prenatal exposure to DES induces deficits in passive avoidance responses as a result of increased CaMKII activity and hippocampal LTP.

Potential role of calcineurin for brain ischemia and traumatic injury.

Calcineurin belongs to the family of Ca2+/calmodulin-dependent protein phosphatase, protein phosphatase 2B. Calcineurin is the only protein phosphatase which is regulated by a second messenger, Ca2+. Furthermore, calcineurin is highly localized in the central nervous system, especially in those neurons vulnerable to ischemic and traumatic insults. For these reasons, calcineurin is considered to play important roles in neuron-specific functions. Recently, on the basis of the finding that FK506 and cyclosporin A serve as calcineurin-specific inhibitors, this enzyme has become the subject of much study. It is clear that calcineurin is involved in many neuronal (or non-neuronal) functions such as neurotransmitter release, regulation of receptor functions, signal transduction systems, neurite outgrowth, gene expression and neuronal cell death. In this review, we describe the calcineurin functions, functions of the substrates, and the pathogenesis of traumatic and ischemic insults, and we discuss the potential role of calcineurin. There are many similarities in traumatic and ischemic pathogenesis of the brain in which the release of excessive glutamate is followed by an intracellular Ca2+ increase. However, the intracellular cascade which leads to neuronal cell death after the release of excess Ca2+ is unclear. Although calcineurin is thought to be a key toxic enzyme on the basis of studies using immunosuppressants (FK506 or cyclosporin A), many of the functions of the substrates for calcineurin protect against neuronal cell death. We concluded that calcineurin is a bi-directional enzyme for neuronal cell death, having protective and toxic actions, and the balance of the bi-directional effects may be important in ischemic and traumatic pathogenesis.

Differential roles of Ca(2+)/calmodulin-dependent protein kinase II and mitogen-activated protein kinase activation in hippocampal long-term potentiation.

The roles of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) and mitogen-activated protein kinase (MAPK) in long-term potentiation (LTP) were investigated in the CA1 area of hippocampal slices, using electrophysiological and biochemical approaches. A brief high-frequency stimulation, but not low-frequency stimulation, delivered to Schaffer collateral/commissural afferents produced a stable LTP and activated both CaM kinase II and 42 kDa MAPK. Different from the activity of CaM kinase II, the increase in MAPK activity was transient. At a concentration of 50 microM, but not of 30 microM, PD098059, a potent inhibitor of MAPK kinase, markedly inhibited the induction of LTP. Although the two concentrations had similar inhibitory effects on MAPK activity, only 50 microM PD098059 suppressed the activation of CaM kinase II. Application of calmidazolium, an antagonist of calmodulin, blocked both CaM kinase II activation and the LTP induction without affecting the increase in 42 kDa MAPK activity. Application of neurotrophin brain-derived neurotrophic factor (BDNF) promoted the induction of LTP, with concomitant activation of CaM kinase II. Under the same conditions, BDNF failed to activate MAPK in hippocampal slices. These results indicate that, although the LTP induction is accompanied by increases in two kinase activities, only CaM kinase II activation is required for this event.

Phosphoprotein phosphatases for myelin basic protein in myelin and cytosol fractions of brain.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) activity for myelin basic protein was found to be present in the myelin fraction of rat brain. The enzyme activity was in a latent form and solubilized by 0.2% Triton X-100 treatment with about 50% increase of activity. The cytosol fraction from bovine brain also had phosphoprotein phosphatase activity for myelin basic protein, which was resolved into at least two peaks of activity on DEAE-cellulose column chromatography. Myelin basic protein was the best substrate for both the solubilized myelin fraction and the cytosol enzymes among the substrate proteins tested. The Km values of the solubilized myelin fraction were 4.2 muM for myelin basic protein, 7.4 muM for arginine-rich histone, 8.0 muM for histone mixture and 14.3 muM for protamine, respectively.

Regulation by human chorionic gonadotropin of protein kinases in rabbit endometrium.

The effect of human chorionic gonadotropin (CG) on protein kinase levels has been studied in rabbit endometrium. A good correlation was observed between alterations of total protein kinase activity and DNA content in the tissue. The level of type I cyclic AMP-dependent protein kinase had slightly increased by 6 h after human CG treatment, and then decreased later. In contrast, the type II enzyme gradually increased after 3 days. The increase of the activity at 7 days was mainly due to that of type II enzyme, and the ratio of type II to type I enzyme increased. The activity pattern at the later stage closely resembled that of treatment with progesterone.

A site phosphorylated in bovine cardiac troponin T by cardiac CaM kinase II.

Ca2+/Calmodulin-dependent protein kinase II isolated from heart phosphorylates bovine cardiac troponin T present in the holotroponin complex. Thr-190 has been determined as the main phosphorylation site.

Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase.

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.

Calcineurin inhibitor attenuates left ventricular hypertrophy, leading to prevention of heart failure in hypertensive rats.

BACKGROUND: There is controversy regarding the contribution of calcineurin activation to the development of pressure-overload left ventricular (LV) hypertrophy and heart failure. The aim of this study was to explore whether the inhibition of calcineurin may prevent the transition to heart failure in hypertensive rats and, if so, to clarify in which developmental stage of LV hypertrophy calcineurin plays a key role. METHODS AND RESULTS: Dahl salt-sensitive rats placed on an 8% NaCl diet from the age of 7 weeks (hypertensive rats) were randomized to no treatment (n=6) or treatment with the calcineurin inhibitor FK506 (1 mg x kg(-1) x d(-1)) from 8 weeks (FKE, n=7) or from 17 weeks (FKL, n=7). Rats placed on a 0.3% NaCl diet were defined as control rats (n=6). The administration of FK506 from 8 weeks attenuated, although it did not block, LV hypertrophy observed in the untreated rats and prevented the transition to heart failure. The development of LV fibrosis, however, was not attenuated by the administration of FK506 from 8 weeks. The administration of FK506 from 17 weeks brought no benefit for cardiac remodeling or LV function and failed to prevent heart failure. CONCLUSIONS: Calcineurin inhibition, if started from the initial stage of pressure overload, attenuated the development of LV hypertrophy without any effect on LV fibrosis and prevented the transition to heart failure. The activation of calcineurin is involved in the development of LV hypertrophy but not of LV fibrosis, and this involvement may be crucial at the initial stage.

Increased angiotensin converting enzyme activity in left ventricular aneurysm of patients after myocardial infarction.

OBJECTIVE: Angiotensin converting enzyme (ACE) inhibitors have been shown to improve left ventricular dysfunction and survival in patients with chronic myocardial infarction. The aim of this study was to examine the ACE activity in infarcted tissues in such patients in comparison with non-diseased tissues from control subjects obtained at necropsy. METHODS: ACE activity was measured in the left ventricles and right atrial auricles of patients (n = 9) with chronic myocardial infarction obtained at left ventricular aneurysmectomy, and in the hearts of control subjects at necropsy (n = 10). RESULTS: In non-diseased hearts, the ACE activity was highest in right atria and auricles [2.4(SEM 0.2), 2.2(0.3) nmol.mg-1 protein.min-1, NS, respectively], followed by left atria [1.7(0.2)], left auricles [1.5(0.1)], right ventricles [1.0(0.2)], and left ventricles [0.5(0.1)]. The ACE activity was significantly increased in aneurysmal tissues of patients with chronic myocardial infarction relative to left ventricles of control subjects [4.2(0.4) v 0.5(0.1) nmol.mg-1 protein.min-1, P < 0.01]. There was, however, no difference in the ACE activity of right atrial auricles between patients with chronic myocardial infarction and control subjects [2.8(0.5) v 2.2(0.3), NS]. In patients with chronic myocardial infarction, the ACE activity was higher in left ventricles than in right auricles (P < 0.01). The ACE activities in the infarcted and control ventricles were negatively correlated with the membrane protein content (r = -0.77, P < 0.01). CONCLUSIONS: In non-diseased human hearts, the ACE activity is higher in atria than in ventricles and higher in the right than in the left ventricle. Furthermore, the ACE activity in aneurysmal left ventricular tissue after myocardial infarction is higher than in non-diseased left ventricular myocardium. These results suggest that the local ACE in the human heart may play an important role in the pathophysiological state after myocardial infarction.

Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-kappa B in human endothelial cells.

OBJECTIVE: Nuclear factor-kappa B (NF-kappa B) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF-kappa B, the signaling pathway of oxygen free radical-induced NF-kappa B activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-kappa B activation in human umbilical vein endothelial cells (HUVECs). METHODS: Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a kappa B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against I kappa B alpha, phosphorylated by I kappa B alpha kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-kappa B luciferase reporter gene assay was also performed. RESULTS: The treatment of the cells with superoxide anion for 60 min increased the NF-kappa B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of I kappa B alpha within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of I kappa B alpha. Pretreatment of the cells with calphostin C (100-400 nmol/l) and chelerythrine chloride (5-10 mumol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-kappa B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-kappa B activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). CONCLUSION: These results suggest that PKC is involved in the activation of NF-kappa B by superoxide anion in human endothelial cells.

Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM-dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP-dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I-like protein.

Cyclosporin A stimulation of glucose-induced insulin secretion in MIN6 cells.

Effects of the immunosuppressant cyclosporin A (CsA), a specific inhibitor of Ca2+/calmodulin-dependent protein phosphatase (PP2B), were examined with regard to the induction of insulin secretion from MIN6 cells, a glucose-responsive cell line derived from mouse insulinoma. CsA had no effect on basal insulin secretion from MIN6 cells, but did increase glucose-, tolbutamide-, and KCl-induced insulin secretion. Treatment of the cells with CsA resulted in a dose-dependent increase in insulin secretion, which was maximal at 3 microM. CsA inhibited PP2B activity in a dose-dependent manner, and the increase in insulin secretion correlated with the decrease in PP2B activity. In 32P-labeled cells, treatment with CsA for 30 min increased phosphorylation of synapsin I-like protein by 50 +/- 5.7%. As revealed by one-dimensional phosphopeptide mapping of 32P-labeled synapsin I-like protein, treatment with CsA for 30 min increased phosphorylation of site II of synapsin I-like protein by 59 +/- 8%, which is phosphorylated by calmodulin kinase II. Messenger RNAs, which hybridize with complementary DNAs of calcineurin A and B subunits from rat brain, were detected in MIN6 cells. Western blot analysis showed a 61-kDa band, which interacts with rat brain calcineurin A antibody. Similar increases in secretagogue-induced insulin secretion with CsA were observed for HIT-T15 cells. These results suggest that CsA stimulates glucose-induced insulin secretion by inhibiting the activity of PP2B, an event that may be involved in mechanisms governing glucose-induced insulin secretion via dephosphorylation of synapsin I-like protein in MIN6 cells.

Involvement of mitogen-activated protein kinase in cyclic adenosine 3',5'-monophosphate-induced hormone gene expression in rat pituitary GH(3) cells.

We examined whether mitogen-activated protein (MAP) kinase was activated by stimulation of the cAMP pathway and whether MAP kinase activation was involved in synthesis of PRL and GH in GH(3) cells. Treatment of the cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP (CPT-cAMP), activated MAP kinase and increased PRL at both the protein and messenger RNA levels. The protein and messenger RNA of GH were decreased by the treatment. We constructed the luciferase reporter genes after the promoters of PRL and GH and found the activation of both promoters by the CPT-cAMP treatment. We confirmed that overexpression of the catalytic subunit of cAMP-dependent protein kinase had essentially the same effects on MAP kinase activation and synthesis of PRL and GH as the CPT-cAMP treatment. Furthermore, treatment of the cells with pituitary adenylate cyclase-activating polypeptide 27 activated MAP kinase. The activation of PRL promoter by CPT-cAMP and pituitary adenylate cyclase-activating polypeptide 27 was abolished by pretreatment with PD098059 and H89. Although the increase in PRL and GH secretion by CPT-cAMP was inhibited by H89, PD098059 had no effect on secretion. These results suggest that cAMP-induced MAP kinase activation is essential for PRL gene expression, but not for secretion of PRL and GH.

Regulation of insulin secretion by overexpression of Ca2+/calmodulin-dependent protein kinase II in insulinoma MIN6 cells.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in Ca2+-induced insulin secretion. We have previously reported that treatment of insulinoma MIN6 cells with secretagogues activated CaM kinase II and increased the phosphorylation of synapsin I, followed by insulin secretion. Here, we identified isoforms of CaM kinase II in MIN6 cells and rat islets. Immunoblot analysis suggested that the major isoforms of CaM kinase II were beta'e and delta2 at the protein level in MIN6 cells. Only the beta'e isoform was detected in rat islets by both RT-PCR and immunoblot analysis. We transiently overexpressed beta'e and delta2 isoforms in MIN6 cells and confirmed that treatment of cells with tolbutamide and glucose activated the isoforms. Immunoblot analysis with an antibody against synapsin I phosphorylated by CaM kinase II demonstrated that treatment with tolbutamide and glucose rapidly increased phosphorylation of synapsin I and that phosphorylation was potentiated by overexpression of the isoforms. The secretagogue-induced insulin secretion was potentiated by overexpression of the isoforms. Our results further support our conclusion that activation of CaM kinase II and the concomitant phosphorylation of synapsin I contribute to insulin secretion from pancreatic beta-cells.

Caudoputamen is damaged by hypocapnia during mechanical ventilation in a rat model of chronic cerebral hypoperfusion.

BACKGROUND AND PURPOSE: Postoperative brain dysfunction, such as delirium, is a common complication of anesthesia and is sometimes prolonged, especially in patients with cerebrovascular disease. In the present study we investigated the effect of hypocapnia during anesthesia on neuronal damage using a rat model of chronic cerebral hypoperfusion. METHODS: Chronic cerebral hypoperfusion was induced by clipping the bilateral common carotid arteries in male Wistar rats. Fourteen days after the operation, these animals were mechanically ventilated for 2 hours and then kept in suitable conditions for an additional 14 days. Twenty-four rats were assigned to 4 groups: those with chronic cerebral hypoperfusion with either hypocapnia or normocapnia during anesthesia, and those given sham operation with either hypocapnia or normocapnia. White matter lesions in the brain sections were evaluated with Klüver-Barrera staining. Proliferation of glial cells was estimated with the use of immunohistochemistry of glial fibrillary acidic protein, a marker for astroglia, and CD11b, a marker for microglia. Computer-assisted morphometry was applied to the immunohistochemical results of microtubule-associated protein 2 to evaluate the loss of neurons. RESULTS: The histological damage was localized almost exclusively in the white matter in the rats subjected to chronic cerebral hypoperfusion but without hypocapnia. Neuronal damage and astroglial proliferation occurred with aggravated white matter lesions in the caudoputamen in the rats with chronic cerebral hypoperfusion and hypocapnia. No lesions were observed in sham-operated rats with either hypocapnia or normocapnia. CONCLUSIONS: These results indicate that hypocapnia during anesthesia causes tissue damage in the caudoputamen, which may be responsible for long-lasting postoperative delirium in patients with stroke and/or dementia.

Neuroprotective effect of sodium orthovanadate on delayed neuronal death after transient forebrain ischemia in gerbil hippocampus.

In transient forebrain ischemia, sodium orthovanadate as well as insulinlike growth factor-1 (IGF-1) rescued cells from delayed neuronal death in the hippocampal CA1 region. Adult Mongolian gerbils were subjected to 5-minute forebrain ischemia. Immunoblotting analysis with anti-phospho-Akt/PKB (Akt) antibody showed that phosphorylation of Akt at serine-473 (Akt-Ser-473) in the CA1 region decreased immediately after reperfusion, and in turn transiently increased 6 hours after reperfusion. The decreased phosphorylation of Akt-Ser-473 was not observed in the CA3 region. The authors then tested effects of intraventricular injection of orthovanadate and IGF-1, which are known to activate Akt. Treatment with orthovanadate or IGF-1 30 minutes before ischemia blocked delayed neuronal death in the CA1 region. The neuroprotective effects of orthovanadate and IGF-1 were associated with preventing decreased Akt-Ser-473 phosphorylation in the CA1 region observed immediately after reperfusion. Immunohistochemical studies with the anti-phospho-Akt-Ser-473 antibody also demonstrated that Akt was predominantly in the nucleus and was moderately activated in the cell bodies and dendrites of pyramidal neurons after orthovanadate treatment. The orthovanadate treatment also prevented the decrease in phosphorylation of mitogen-activated protein kinase (MAPK). Pretreatment with combined blockade of phosphatidylinositol 3-kinase and MAPK pathways totally abolished the orthovanadate-induced neuroprotective effect. These results suggest that the activation of both Akt and MAPK activities underlie the neuroprotective effects of orthovanadate on the delayed neuronal death in the CA1 region after transient forebrain ischemia.