Hydration structure of strongly bound water on the sulfonic acid group in a Nafion membrane studied by infrared spectroscopy and quantum chemical calculation.

The hydration structure of the 'strongly bound water' around the sulfonic acid (SA) groups in Nafion, which has recently been revealed by (1)H NMR spectroscopy (Anal. Chem., 2013, 85, 7581), is studied using infrared spectroscopy with the aid of quantum chemical (QC) calculations. During a heated drying process, bulky water is firstly dehydrated, which is followed by the disappearance of the hydronium ion and the appearance of bands that have been assigned to the fully dehydrated species at 140 °C. However, a spectral simulation based on QC reveals that the spectrum at 140 °C comes from the SA group associated with a single-water molecule via two H-bonds. This implies that a thoroughly dried membrane is unavailable even at 140 °C, and the involved water corresponds to the 'strongly bound water.' The QC-analytical results are experimentally confirmed by evolved gas analysis mass spectrometry (EGA-MS). At ca. 300 °C, which is the temperature where the SA group is selectively decomposed, the molecular fragment of SO2 is observed accompanying water molecules as expected. This confirms that the last single-water molecule can remain on the SA group until the thermal decomposition.

Cloning of the RNase H genes from a metagenomic DNA library: identification of a new type 1 RNase H without a typical active-site motif.

AIMS: The study aimed to combine a metagenomics approach with complementary genetics to identify novel bacterial genes with orthologous functions, with the identification of novel RNase H genes as a test case. METHODS AND RESULTS: A metagenomic DNA library was prepared from leaf-and-branch compost and used to screen for the RNase H genes by their abilities to complement the temperature-sensitive growth phenotype of the rnhA mutant Escherichia coli strain MIC3001. Determination of the nucleotide sequences of the cloned DNA fragments allowed us to identify 12 different genes encoding type 1 RNases H. Eleven of them encode novel RNases H, which show 40-72% amino acid sequence identities to those available from database. One of them lacks a typical DEDD/E active-site motif, which is almost fully conserved in various RNases H. CONCLUSIONS: Functional screening of environmental DNA without cultivation of microbes is a useful procedure to isolate novel RNase H genes. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the identified RNase H genes had no sequence similarity to a previously assumed conserved motif, suggesting multiple catalytic mechanisms exist. This test case illustrates that metagenomics combined with complementary genetics can identify novel genes that are orthologous without sequence similarity to those from cultivated bacteria.

Identification of a novel gene, MASL1, within an amplicon at 8p23.1 detected in malignant fibrous histiocytomas by comparative genomic hybridization.

We used comparative genomic hybridization to study malignant fibrous histiocytomas (MFHs) from 19 patients to detect changes in the copy number of DNA sequences, along entire chromosomes. Together with losses and gains in various chromosomal regions, distinct high-level amplifications were found at six loci (4q12-21, 8p21-pter, 8q24.1-qter, 9q12-13, 12p11.2-pter, and 15q11.2-15), suggesting that those regions may contain unknown (proto) oncogenes. We focused on the 8p amplicon, where detailed characterization allowed us to determine that the minimal common amplified region lay between markers D8S1819 and D8S550 at 8p23.1. A novel gene designated MASL1 (MFH-amplified sequences with leucine-rich tandem repeats 1) was isolated from within this narrowly defined region. Expression of the MASL1 gene was enhanced significantly in MFH tumors bearing the 8p amplicon. The primary structure of its deduced product revealed an ATP/GTP-binding site, three leucine zipper domains, and a leucine-rich tandem repeat, all of which are important structural or functional elements for interactions among proteins related to the cell cycle. These features suggest that overexpression of MASL1 might well be oncogenic with respect to MFH.

Nicardipine increases cerebral blood flow but does not improve neurologic recovery in a canine model of complete cerebral ischemia.

The effects of the calcium entry blocker nicardipine on CBF, CMRO2, and neurologic outcome following 10 min of complete cerebral ischemia were examined in dogs. In CBF and CMRO2 studies, the CBF in the untreated group (seven dogs) and the nicardipine group (seven dogs; 20 micrograms kg-1 at 30 min postischemia and a subsequent infusion of 2 micrograms kg-1 min-1 for 90 min) initially increased to 300-400% and then returned to preischemic values at 30 min postischemia. Thereafter the CBF in the untreated group significantly decreased to 50% of preischemic values for the following 90-min period (hypoperfusion), while the CBF in the nicardipine group did not differ from preischemic values. The CMRO2 in both groups decreased to approximately 50-80% of preischemic values after 15 min postischemia and did not differ between the groups throughout the study. In neurologic outcome studies, 18 dogs were divided into three groups (of six dogs each): untreated; saline infusion only, posttreated; nicardipine as in CBF and CMRO2 studies, pretreated; nicardipine 20 micrograms kg-1 at 2 min preischemia and a subsequent infusion of 2 micrograms kg-1 min-1 from immediately postischemia to 120 min postischemia. Nicardipine treatment initiated either before or after ischemia failed to improve neurologic outcome at 48 h postischemia. Thus, the increase of postischemic global CBF by nicardipine is not accompanied by neurologic recovery in a canine model of complete cerebral ischemia.

Characterization of spinal amino acid release and touch-evoked allodynia produced by spinal glycine or GABA(A) receptor antagonist.

Intrathecal strychnine (glycine antagonist) or bicuculline (GABA(A) antagonist) yields a touch-evoked agitation that is blocked by N-methyl-D-aspartate receptor antagonism. We examined the effects of intrathecal strychnine and bicuculline on touch-evoked agitation and the spinal release of amino acids. Fifty-two Sprague-Dawley rats were prepared under halothane anesthesia with a lumbar intrathecal catheter and a loop dialysis catheter. Four days after implantation, rats were randomized to receive an intrathecal injection of N-methyl-D-aspartate (3 microg), strychnine (3 microg) or bicuculline (10 microg), or a combination of N-methyl-D-aspartate with bicuculline or strychnine. The agitation produced by brief light tactile stroking of the flank (tactile allodynia), and the spontaneous spinal release of glutamate, taurine and serine was measured. Intrathecal N-methyl-D-aspartate, strychnine and bicuculline produced similar touch-evoked allodynia. Intrathecal bicuculline and N-methyl-D-aspartate alone evoked a transient spinal release of glutamate and taurine, but not serine, in the 0- 10 min sample, while strychnine did not affect spinal transmitter release at any time. As GABA(A) but not glycine receptor inhibition at equi-allodynic doses increases glutamate release, while the allodynia of both is blocked by N-methyl-D-aspartate receptor antagonism, we hypothesize that GABA(A) sites regulate presynaptic glutamate release, while glycine regulates the excitability of neurons postsynaptic to glutamatergic terminals.

Intoxication with sulindac, tiaramide hydrochloride and diclofenac sodium.

Overdosage intoxication of sulindac, tiaramide and diclofenac caused excitability of central nervous system, followed by unconsciousness. The case was treated with ordinary therapies and direct hemoperfusion (DHP). Serum concentrations of these drugs and their metabolites were correlated well with the clinical symptoms. DHP may be effective to eliminate these drugs and their metabolites.

Intracranial pressure following cardiopulmonary resuscitation.

Intracranial pressure (ICP) was measured in six patients following cardiopulmonary resuscitation (CPR). The causes of cardiac arrest were respiratory or circulatory problems and the primary intracranial pathology was not detected. The measurement of ICP started 3 to 10 h following CPR except one patient in whom it started on the day 7. Duration of ICP measurement ranged from 2 to 7 days. In five out of six patients, ICP persistently remained below 20 mmHg. In the remaining one patient, ICP elevation associated with seizure activity was observed and ICP ultimately increased to 57 mmHg. Among these, four patients died and two remained in a persistent vegetative state. These results suggest that ICP following CPR does not necessarily increase if the patient has no primary intracranial pathology or seizures.

Glutamate release and neuronal injury after intrathecal injection of local anesthetics.

High concentrations of local anesthetics are neurotoxic, but the mechanism for this neurotoxicity is obscure. Here, we report increased concentrations of glutamate in the cerebrospinal fluid after intrathecal injections of high concentrations of tetracaine (a local anesthetic). The peak concentrations of glutamate after administration of 1%, 2%, and 4% tetracaine were 4-fold, 6-fold, and 10-fold higher than baseline values, respectively. Animals in the 1% group were all neurologically normal one week after tetracaine injection. In the group receiving 4%, no animal was able to hop and vacuolation of the white matter and/or central chromatolysis of the motor neurons were observed. Because high concentrations of glutamate are known to be neurotoxic, our results may provide some insight into the mechanisms for neurotoxicity of intrathecal local anesthetics.

The hydroxyl radical scavenger Nicaraven inhibits glutamate release after spinal injury in rats.

Neuronal degeneration after trauma is mediated in part by release of excitatory amino acids (EAAs) and oxygen free radicals (OFR). We evaluated the effect of i.v. treatment with a hydroxyl radical scavenger ((+/-)-N,N'-propylenedinicotinamide; AVS) and spinal hypothermia (33 degrees C) on spinal CSF glutamate release after spinal trauma. In a control group, spinal compression evoked at 10 min a significant increase (5-fold) in glutamate which declined over 4 h (2.1-fold). AVS treatment attenuated glutamate release but had no additive effect. These data suggest that this compound can be effective in modulating spinal excitotoxicity resulting from increased OFR synthesis and corresponding potentiation of EAA release.

Cerebral blood flow and oxygen consumption in the rat brain after lesions of the noradrenergic locus coeruleus system.

The effect of lesions of the locus coeruleus neuron system on cerebral metabolic rate for oxygen (CMRO2) and blood flow (CBF) was evaluated in paralyzed and mechanically ventilated rats, using a 133xenon modification of the Kety-Schmidt inert gas technique. Bilateral electrothermic lesions of its ascending bundle caused no significant change in CBF or CMRO2. The 6-hydroxydopamine lesions did not influence the CBF and CMRO2 responses to hypercapnia and hypoxia. It is concluded that the locus coeruleus does not exert any resting tone on CBF and CMRO2 and that no influence on the CBF and CMRO2 responses to hypercapnia and hypoxia is mediated via its ascending projections.

Thiopentone inhibits endothelium-dependent relaxations of rat aortas regulated by endothelial Ca2+-dependent K+ channels.

The present study was designed to examine the mechanisms of inhibitory effect of barbiturates on endothelial function by determining whether thiopentone and phenobarbitone reduce relaxations to acetylcholine mediated by endothelial Ca2+-dependent K+ channels in rat aortas. Cumulative applications (10(-9) to 10(-5) M) of acetylcholine induced endothelium-dependent relaxations, which are abolished by inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester, 10(-4) M) and of soluble guanylate cyclase (1H-[1,2,4]oxadiazolo [4,3,-a]quinoxaline-1-one; ODQ, 5 x 10(-6) M). Selective inhibitors of large-conductance Ca2+-dependent K+ channels (iberiotoxin, 5 x 10(-8) M), but not of those with small-conductance (apamin, 5 x 10(-8) M), significantly reduced the acetylcholine-induced vasorelaxation. ODQ, but neither iberiotoxin nor apamin, blocked the relaxations of arteries without endothelium induced by nitric oxide donors, sodium nitroprusside (10(-9) to 10(-5) M) and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7; 10(-10) to 10(-5) M). Thiopentone (10(-4) and 3 x 10(-4) M) but not phenobarbitone (3 x 10(-4) M) significantly impaired relaxations to acetylcholine, whereas thiopentone did not alter relaxations to sodium nitroprusside. Thiopentone (3 x 10(-4) M) did not affect relaxations to acetylcholine in arteries treated with iberiotoxin (5 x 10(-8) M), whereas it reduced these relaxations in arteries treated with apamin (5 x 10(-8) M). These results suggest that in rat aortas, large-conductance, but not small-conductance, Ca2+-dependent K+ channels in endothelial cells, play a role in endothelium-dependent relaxations to acetylcholine, and that thiopentone, but not phenobarbitone, impairs relaxations to acetylcholine mediated by these channels.

A saponin conjugated with 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one from Dolichos lablab.

A new 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated saponin, lablab saponin I, was isolated from the hypocotyl of hyacinth bean (Dolichos lablab). The structure was elucidated by 1H NMR and 13C NMR spectroscopy and chemical techniques as 3-O-[alpha-L-rhamnopyranosyl- (1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D-glucuronopyranosyl(1 -->)]-22-O- [2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one(2'-->)]-3 beta, 22 beta, 24-trihy-droxyolean-12-en-28-al. SOD (superoxide dismutase)-like activity depended upon the DDMP group and the aldehyde group (C-28) of the aglycone was observed in the order of lablab saponin I > glutathione > soyasaponin beta g > maltol. The soybean saponin Bb, lacking the DDMP moiety was found not to exhibit the SOD-like activity.

Establishment and characterization of a cisplatin-resistant human neuroblastoma cell line.

BACKGROUND: To investigate the mechanisms of cisplatin (CDDP)-resistance in neuroblastoma(NB), we established a CDDP-resistant human NB cell line, BM1R2. MATERIALS AND METHODS: We characterized BM1R2 in terms of the susceptibilities to other anticancer agents, MDR1 and MRP expression, MYCN amplification, intracellular gultathione-S-transferase(GST-pi), metallothionein(MT) and gultathione(GSH) levels, and immunocytochemical and cytogenetic features. RESULTS: When compared to parent BM1 line, BM1R2 exhibited a 17.0-fold resistance to CDDP and cross-resistance to other agents. MRP expression was only observed in BM1R2, whereas MDR1 was expressed in both lines. Notably higher intracellular GST-pi and MT levels were observed in BM1R2 cells. MYCN amplifications were 50 and 6 copies in BM1 and BM1R2, respectively, and additional aberrations were observed in chromosome 1 and 2 in BM1R2. CONCLUSION: It was suggested that GST-pi and MT could exert crucial roles on CDDP-resistance in our system. BM1R2 is of great interest for investigating the mechanisms of CDDP-resistance in NB.

Prognostic factors of colorectal cancer. Results of multivariate analysis of curative resection cases with or without adjuvant chemotherapy.

One thousand two hundred fifty-four cases (610 colonic and 644 rectal cancers resected during 2 years from 1984 and followed up for more than 5 years) were entered from 140 institutions in Japan and analyzed by means of Cox's proportional hazards model. The analyzed pathologic variables were the size and depth of invasion, Dukes' stage, venous invasion, lymphatic permeation, and other clinical features, such as the sex and age of the patient and location of the tumor. The extent of dissection, serum carcinoembryonic antigen (CEA) level, and the presence or absence of adjuvant chemotherapy were also analyzed. Adjuvant chemotherapy consisted of three arms for both colonic and rectal cancers. For colonic cancer, arm I was a combination of i.p. (intraportal) and i.v. mitomycin C (MMC) + p.o. 5-fluorouracil (5-FU); arm II was i.v. MMC + p.o. 5-FU; and arm III was surgery only. For rectal cancer, arm IV was a combination of i.a. (inferior mesenteric artery) and i.v. MMC + p.o. 5-FU; arm V was i.v. MMC + p.o. 5-FU; and arm VI was surgery only. As for the factors affecting the disease-free survival of the patient, multivariate analysis disclosed nodal involvement, venous invasion, an elevated CEA level, and the lower part of the rectum. The effect of adjuvant chemotherapy on the patient's survival was proven for rectal cancer but not for colonic cancer. We conclude that these factors should be considered in setting the stage of tumor pre- and postoperatively.

Modulation of cerebrospinal metabolic responses to peripheral stimulation by enflurane anesthesia in rats.

Enflurane-induced modulation of cerebrospinal metabolic responses to peripheral nerve stimulation was examined in 30 rats. Local glucose utilization in the brain and lumbar spinal cord was measured using the autoradiographic 2-[C]deoxyglucose method at three anesthetic concentrations (0,5, 2, and 4%) either with or without electrical stimulation (5 mA, 0.5 ms, 10 Hz) of the unilateral sciatic nerve. Stimulation produced a 71 to 111% increase in glucose utilization in the ipsilateral dorsal horn of the spinal cord at all anesthetic concentrations examined. Stimulation also produced a 32 to 48% increase in glucose utilization in the hindlimb projectionarea of the contralateral somatosensory cortex at the two lowest concentrations (0.5 and 2%), while at 4% no stimulus-induced increase in glucose utilization was observed. The results show that there is a threshold at which enflurane suppresses the metabolic responses to peripheral stimulation in the somatosensory cortex but not in the spinal cord. If electrical stimulation of a peripheral nerve is regarded as analogous to surgical stimulation, considerable increase in the spinal cord metabolism may occur during surgery even in a deeply anesthetized subject.

Levels of interleukin (IL)-6, IL-8, and IL-1 receptor antagonist in the hepatic vein following liver surgery.

BACKGROUND/AIMS: The liver produces various cytokines, but local changes in the concentrations of these reaction products after liver surgery are unknown. We investigated the local changes of interleukin-6, interleukin-8 and interleukin-1 receptor antagonist after liver surgery. METHODOLOGY: We determined levels of interleukin-6, interleukin-8, and interleukin-1 receptor antagonist in the hepatic vein and radial artery after liver resection in 13 patients. These cytokine levels in the portal vein were also measured in 6 patients. RESULTS: Interleukin-6, interleukin-8, and interleukin-1 receptor antagonist levels were significantly increased during liver surgery (P < 0.05). The level of interleukin-6 was significantly lower in the hepatic vein than in the radial artery as well as in the portal vein at the end of the operation (P < 0.05, < 0.03). The level of interleukin-8 and interleukin-1 receptor antagonist was significantly higher in the hepatic vein than in the artery (P < 0.05). CONCLUSIONS: Interleukin-6 may be taken up by the liver after liver surgery, and the difference between hepatic venous and peripheral arterial interleukin-6 levels may be an indicator of liver regeneration after liver resection. Interleukin-8 and interleukin-1 receptor antagonist appear to be produced in the remaining liver.

Decreased hepatic nitric oxide synthesis during liver surgery.

BACKGROUND/AIMS: Liver cells can produce nitric oxide from L-arginine through the action of nitric oxide synthase, but local changes in the concentrations of nitric oxide-related metabolites during liver surgery are not well characterized. We investigated such changes during and after liver surgery. METHODOLOGY: We determined nitrite plus nitrate, L-arginine and L-citrulline concentrations in radial arterial and hepatic venous blood during and after liver surgery in 17 patients. Portal venous blood concentrations were also measured at the end of surgery in 7 patients. RESULTS: Both arterial and hepatic venous nitrite plus nitrate concentrations were significantly decreased during surgery and remained low compared to pre-operative values until post-operative day 2. Arterial and hepatic venous nitrite plus nitrate concentrations were not significantly different. L-arginine concentrations in both arterial and hepatic venous blood were significantly decreased during surgery, but returned to pre-operative levels on post-operative day 1. L-arginine concentrations in hepatic venous blood were significantly lower than in arterial blood. L-citrulline concentrations in both arterial and hepatic venous blood were significantly decreased during surgery compared to pre-operative values, and tend to be decreased until post-operative day 2. L-citrulline concentrations were significantly higher in hepatic venous blood than in arterial blood. CONCLUSIONS: Hepatic nitric oxide production was decreased peri-operatively during liver surgery. The decreases in L-arginine concentrations and in nitric oxide synthase activity may account for the decrease in nitric oxide production.

[Comparative clinical study of aspoxicillin and sulbenicillin in postoperative wound infections].

A well-controlled comparative study was performed to evaluate efficacy, safety and utility of aspoxicillin (ASPC) as compared with sulbenicillin (SBPC) in the treatment of postoperative wound infections. Either 2 g of ASPC or 2 g of SBPC was administered to patients by intravenous drip infusion twice a day for 7 days. The following results were obtained: Overall clinical effectiveness rates were 82.5% (66/80) in ASPC group and 77.0% (57/74) in SBPC group, with no statistically significant difference between 2 groups. Final overall clinical improvement rates were 83.8% (67/80) in ASPC group and 81.1% (60/74) in SBPC group, with no statistically significant difference between 2 groups. As to bacteriological effectiveness, eradication rates of clinical isolates were 70.4% (38/54) in ASPC group and 74.4% (32/43) in SBPC group. There was no statistically significant difference in 2 groups. Side effects and abnormal laboratory findings were observed in 6 cases (6.7%) and 11 cases (12.4%) in ASPC group (89 cases) respectively, and 4 cases (4.4%) and 7 cases (7.8%) in SBPC group (90 cases) respectively. Especially severe adverse reactions were not observed, and there was no significant difference in the incidences of side effects and abnormal laboratory findings between 2 groups. As to overall clinical utility, utility rates were 77.5% (62/80) in ASPC group and 70.3% (52/74) in SBPC group. There was no statistically significant difference between 2 groups. These results may be indicated that ASPC is as useful as SBPC in the treatment of postoperative wound infections.

[Microbiological laboratories for sepsis in the 21st century].

A new concept, "SIRS (systemic inflammatory response syndrome)", introduced for the use of innovative therapies in sepsis, indicates the importance of clinically satisfying services in the laboratories. Appropriate laboratory management of the host, parasite and environment, is needed for the diagnosis, treatment and prevention of sepsis. Improvement of environmental conditions is expected to contribute to the prevention of sepsis by nosocomial infections. Another important key in the management is quality assurance in blood cultures, which involves three issues that must be improved. Concerning the first two issues, high sensitivity and low contamination, balanced procedures must be performed to ensure cost-effectiveness. Regarding the third issue, we proposed a quick return system for positive blood cultures. We need to reconsider the traditional concept of "time" in microbiological tests and employ practical measures for sepsis in clinical laboratories.

[Effects of isosorbide dinitrate on regional cerebral blood flow and intracranial pressure in cats].

The effects of isosorbide dinitrate (ISDN) on regional cerebral blood flow (rCBF) and intracranial pressure (ICP) were examined in cats. A low dose of ISDN (2.5 micrograms/kg/min) infusion did not show any changes in cerebral hemodynamics. During high dose of ISDN (5.0 micrograms/kg/min) or NTP (5.0 micrograms/kg/min) infusion, mean blood pressure (mBP) decreased by 10 to 20% accompanied by decreased cerebral perfusion pressure (CPP: mBP-ICP), however, rCBF or ICP did not change. It is concluded that intravenous administrations of ISDN in a dose of 2.5-5.0 micrograms/kg/min that produce slight decrease in blood pressure did not influence on cerebral hemodynamics.