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.

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.

Detection and serotype distribution of Actinobacillus actinomycetemcomitans in cardiovascular specimens from Japanese patients.

Actinobacillus actinomycetemcomitans, an important pathogen in periodontitis, has also been detected in cardiovascular tissues. Sixty heart valves were collected during valve replacement surgery from 60 patients (one from each), 10 were from patients with infective endocarditis (IE group) and 50 were from patients with other valvular diseases (non-IE group). In addition, 46 samples of aneurysmal tissue were taken from 46 patients with a thoracic or abdominal aneurysm (Aneurysm group, one from each). Dental plaque samples were taken from 54 of the patients, 31 in the IE and non-IE groups and 23 in the aneurysm group. First, the distribution of A. actinomycetemcomitans in all specimens was analysed using a polymerase chain reaction method, which resulted in a positive reaction in 33 (31.1%) of the cardiovascular specimens and 25 (46.3%) of the dental plaque samples. Next, using serotype-specific sets of primers, the serotype distribution of A. actinomycetemcomitans in the cardiovascular specimens and dental plaque samples was found to be significantly different compared to dental plaque samples from Japanese subjects reported previously.

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.

Inhibitory effect of propofol on ketamine-induced c-Fos expression in the rat posterior cingulate and retrosplenial cortices is mediated by GABAA receptor activation.

BACKGROUND: Non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, including ketamine, have psychotomimetic activities and cause neuronal damage in the posterior cingulate and retrosplenial cortices (PC/RS), which are suggested to be the brain regions responsible for their psychotomimetic activities. We previously demonstrated that ketamine induced marked c-Fos (c-fos protein) expression in the rat PC/RS, which was inhibited by propofol, and the expression was closely related to ketamine-induced abnormal behavior. In the present study, we investigated whether the inhibition by propofol was mediated by GABAA receptor receptor activation. METHODS: Using Wistar rats, propofol alone, propofol with bicuculline or propofol with flumazenil was injected intravenously and then continuously infused. Fifteen minutes later, 100 mg kg-1 of ketamine or normal saline was injected intraperitoneally. Two hours after the ketamine or saline injection, the brain was extracted and brain sections were prepared, and c-Fos expression was detected using immunohistochemical methods. RESULTS: Ketamine induced marked c-Fos expression in the PC/RS (171 +/- 9/0.4 mm2), which was significantly inhibited by propofol (5 +/- 5/0.4 mm2). The inhibition by propofol was disinhibited dose-dependently by both bicuculline (0.5 and 1.0 mg kg-1 bicuculline groups: 46 +/- 15 and 143 +/- 16, respectively) and flumazenil (0.1 and 1.0 mg kg-1 flumazenil groups: 79 +/- 6 and 130 +/- 15, respectively). CONCLUSION: These results demonstrate that the inhibitory effect of propofol on ketamine-induced c-Fos expression in the PC/RS is mediated by GABAA receptor activation, and suggests that ketamine-induced psychoneuronal adverse effects may be suppressed by propofol via the activation of GABAA receptors.

[Calcium signaling and brain functions].

Calcium signaling plays a critical role in various cell types by activation of receptors and Ca2+ channels in response to neurotransmitters, hormones, growth, factors etc. Although a variety of functions of intracellular Ca2+ are reported, Ca2+/calmodulin-dependent protein kinases (CaMK) are involved in their mediation. We have been studying on CaMK I, II, III, IV and K in the dynamic regulation in the cells in relation to functions. In this study, we elucidated the structures of the isoforms of CaMKII subunits with nuclear translocation signal (NTS). NTS is included in the variable domain following the regulatory domain with a sequence of KKRK. The isoforms of CaMK subunits such as alpha B, gamma A, gamma A.B, delta 3 and delta 7 contain NTS in the sequences of the structures. Transfection of the isoforms with NTS into NG108-15 cells stimulated the expression of brain-derived neurotrophic factor in the cytoplasm. Activation of CaMKII and IV and mitogen-activated protein kinase (MAPK) was observed during long-term potentiation (LTP) induction in the CA1 area of hippocampus. The activation of CaMKII was sustained for a long period, whereas that of CaMKIV and MAPK was transient. The results suggest that CaMKII is involved in LTP induction, while CaMKIV and MAPK are rather involved in LTP maintenance. We present and discuss our recent studies on regulation of CaMKs in neuronal functions.

A case of paraneoplastic syndrome accompanied by two types of cancer.

A case of paraneoplastic syndrome accompanied by two types of cancer is reported. The patient was a 62 year old man who progressively developed cerebellar ataxia, especially an abnormal gait. The anti-Hu antibody titre was high. A small tumour was detected in the middle lobe of the right lung and was surgically treated. The histology was adenocarcinoma. After lobectomy, however, the ataxia deteriorated, and plasma exchange, 250 ml/kg/day, was conducted for 6 days. After plasma exchange, the anti-Hu antibody titre decreased and the ataxia temporarily ceased to progress. A week after the last plasma exchange, a mass appeared in the anterior cervical region and rapidly increased in size. The biopsy of the neck tumour disclosed a small cell carcinoma. Five months later small cell carcinoma appeared in the left lung. This case shows the importance of searching for small cell carcinoma when anti-Hu antibodies are detected. It is assumed that plasma exchange removed not only a pathogenic factor of ataxia but also a factor which inhibited the growth of the small cell carcinoma. It is recommended that plasmapheresis should be performed with caution in paraneoplastic syndrome when the origin of a tumour is obscure.

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.

Inactive corrinoid-compound significantly decreases in Spirulina platensis grown in a cobalt-deficient medium.

Spirulina platensis NIES-39 was grown under open culture system in the presence or absence of CoSO(4) (12 microg/L) and/or vitamin B(12) (10 microg/L) to confirm whether CoSO(4) and/or vitamin B(12) stimulate or are essential for growth of the algal cells and for accumulation of vitamin B(12). The addition of CoSO(4) and/or vitamin B(12) could not affect both cell growth and cell yield of the alga. The amount of corrinoid-compound was increased significantly by the addition of CoSO(4) but not by vitamin B(12). A C18 reversed-phase HPLC pattern of the Spirulina corrinoid-compound increased by the addition of CoSO(4) was identical to that of authentic pseudovitamin B(12), which is inactive for human. These results indicate that the algal cells grown in the absence of CoSO(4) are suitable for use of human health foods because the inactive corrinoid-compound can be reduced significantly.

An anxiolytic agent, dihydrohonokiol-B, inhibits ammonia-induced increases in the intracellular Cl(-) of cultured rat hippocampal neurons via GABA(c) receptors.

The effects of an anxiolytic honokiol derivative, dihydrohonokiol-B (DHH-B) [3'-(2-propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diaol], on ammonia-induced increases in the intracellular Cl(-) concentration ([Cl(-)](i)) were examined using primary cultured rat hippocampal neurons. DHH-B (1-100 ng/ml), but not an inactive isomer of honokiol, magnolol (100 ng/ml), dose-dependently inhibited the ammonia-induced increases in [Cl(-)](i) without any changes in the control [Cl(-)](i). Such an effect of DHH-B was blocked by a gamma-aminobutylic acid A (GABA(A)) and GABA(C) Cl(-) channel blocker, 100 microM picrotoxin, and a GABA(C) receptor blocker, 10 microM (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid, but not by a GABA(A) receptor blocker, 10 microM bicuculline. Further, a GABA(C) receptor agonist, 200 microM cis-4-aminocrotonic acid, but not a GABA(A) receptor agonist, 10 microM muscimol, mimicked the effect of DHH-B. Thus, DHH-B appears to protect neurons from the ammonia-induced increases in [Cl(-)](i) through GABA(C) receptor stimulation.

Microglial signaling by amyloid beta protein through mitogen-activated protein kinase mediating phosphorylation of MARCKS.

Myristoylated alanine-rich C kinase substrate (MARCKS), an acidic protein associated with cell motility and phagocytosis, is activated upon phosphorylation by protein kinase C (PKC) and proline-directed protein kinases. In Alzheimer disease (AD), activated microglia expressing MARCKS migrates around senile plaques. We reported that amyloid beta protein (A beta), a major component of senile plaques, activated MARCKS through a tyrosine kinase and PKC-delta. We have now identified another A beta signaling pathway through a mitogen-activated protein kinase (MAPK) involved in the phosphorylation of MARCKS and analysed cross-talk between PKC and MAPK pathways in primary cultured rat microglia. A selective inhibitor for MAPK kinase, PD098059, significantly inhibited the phosphorylation of MARCKS induced by A beta. Extracellulary regulated kinases, the activities of which were induced by A beta, directly phosphorylated a recombinant MARCKS in vitro. The MAPK pathway was sensitive to wortmannin, but not to a PKC inhibitor or to tyrosine kinase inhibitors. The activation of PKC by A beta was not sensitive to wortmannin. Our findings suggest involvement of the MAPK pathway through phosphoinositol 3-kinase in the phosphorylation of MARCKS in rat cultured microglia, an event may be associated with mechanisms activating microglia in AD.

Intravenously injected FK506 failed to inhibit hippocampal calcineurin.

FK506 (tacrolimus) is known as an inhibitor for calcineurin and is used in numerous research fields. It is not clear whether intravenously injected FK506 inhibits neuronal calcineurin. We measured the calcineurin activities of normal and postischemic rat hippocampi after intravenous injection of FK506 (3 mg/kg). Intravenously injected FK506 had no inhibitory effect on calcineurin activity in the hippocampi of normal and postischemic rats, whereas FK506 inhibited the calcineurin in vitro (purified enzyme, hippocampal homogenate, and hippocampal slice culture homogenate). Thus, it is considered that intravenously injected FK506 does not act on intraneuronal calcineurin and that several effects of FK506 are not due to the inhibition of neuronal calcineurin.

Characterization of a vitamin B12 compound from unicellular coccolithophorid alga (Pleurochrysis carterae).

A unicellular coccolithophorid alga, Pleurochrysis carterae, contained 125.4 +/- 1.2 microg of vitamin B12 per 100 g dry cell weight of the lyophilized algal cells. A vitamin B12 compound was purified from the lyophilized algal cells and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B12, but not those of vitamin B12 analogues inactive for humans. When 22-week-old B12-deficient rats which excreted substantial amounts of methylmalonic acid (75.5 +/- 12.3 mg/day) in urine were fed the P. carterae (10 g per kg diet)-supplemented diet for 12 d, urinary methylmalonic acid excretion (as an index of vitamin B12 deficiency) of the rats became undetectable and hepatic vitamin B12 level of the rats was significantly increased.

Characterization of vitamin B12 compounds from edible shellfish, clam, oyster, and mussel.

Vitamin B12 content of various edible shellfish was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor-chemiluminescence methods. The values determined by the microbiological method were 1.2-19.8 (M/C ratio) fold greater in the shellfish than the values determined by the chemiluminescence method. Vitamin B12 compounds were purified from most eaten shellfish, oyster (M/C, 1.5), mussel (M/C, 1.2), and short-necked clam (M/C, 2.7), and partially characterized. TLC and HPLC patterns of each red-colored vitamin B12 compound (M/C, 1.0-1.2) purified from these shellfish were identical to those of authentic vitamin B12. Although the higher values in the determination of vitamin B12 by the microbiological method may be due to the occurrence of vitamin B12-substitutive compounds, the edible shellfish would be excellent vitamin B12 sources judging from the values (> or = 6 micrograms/100 g) determined by the chemiluminescence method.

Feeding dried purple laver (nori) to vitamin B12-deficient rats significantly improves vitamin B12 status.

To clarify the bioavailability of vitamin B12 in lyophylized purple laver (nori; Porphyra yezoensis), total vitamin B12 and vitamin B12 analogue contents in the laver were determined, and the effects of feeding the laver to vitamin B12-deficient rats were investigated. The amount of total vitamin B12 in the dried purple laver was estimated to be 54.5 and 58.6 (se 5.3 and 7.5 respectively) microg/100 g dry weight by Lactobacillus bioassay and chemiluminescent assay with hog intrinsic factor respectively. The purple laver contained five types of biologically active vitamin B12 compounds (cyano-, hydroxo-, sulfito-, adenosyl- and methylcobalamin), in which the vitamin B12 coezymes (adenosyl- and methylcobalamin) comprised about 60 % of the total vitamin B12. When 9-week-old vitamin B12-deficient rats, which excreted substantial amounts of methylmalonic acid (71.7(se 20.2) micromol/d) in urine, were fed the diet supplemented with dried purple laver (10 microg/kg diet) for 20 d, urinary methylmalonic acid excretion (as an index of vitamin B12 deficiency) became undetectable and hepatic vitamin B12 (especially adenosylcobalamin) levels were significantly increased. These results indicate that vitamin B12 in dried purple laver is bioavailable to rats.

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.

Activation of calcium/calmodulin-dependent protein kinase IV in long term potentiation in the rat hippocampal CA1 region.

The importance of well characterized calcium/calmodulin-dependent protein kinase (CaMK) II in hippocampal long term potentiation (LTP) is widely well established; however, several CaMKs other than CaMKII are not yet clearly characterized and understood. Here we report the activation of CaMKIV, which is phosphorylated by CaMK kinase and localized predominantly in neuronal nuclei, and its functional role as a cyclic AMP-responsive element-binding protein (CREB) kinase in high frequency stimulation (HFS)-induced LTP in the rat hippocampal CA1 region. CaMKIV was transiently activated in neuronal nuclei after HFS, and the activation returned to the basal level within 30 min. Phosphorylation of CREB, which is a CaMKIV substrate, and expression of c-Fos protein, which is regulated by CREB, increased during LTP. This increase was inhibited mainly by CaMK inhibitors and also by an inhibitor for mitogen-activated protein kinase cascade, although to a lesser extent. Our results suggest that CaMKIV functions as a CREB kinase and controls CREB-regulated gene expression during HFS-induced LTP in the rat hippocampal CA1 region.