Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer's disease therapy.

Here, we report a novel target of the drug memantine, ATP-sensitive K+ (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer's model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca2+ concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.

Rivastigmine improves hippocampal neurogenesis and depression-like behaviors via 5-HT1A receptor stimulation in olfactory bulbectomized mice.

Rivastigmine is a non-competitive inhibitor of both acetylcholinesterase (AChE) and butylcholinesterase (BuChE) used to treat mild to moderate dementia in Alzheimer's disease (AD) patients. Although rivastigmine reportedly ameliorates cognitive dysfunction in these patients, its ability to improve Behavioral and Psychological Symptoms of Dementia (BPSD) remains unclear. To determine whether rivastigmine treatment antagonizes depression-like behaviors, we chronically administered rivastigmine (0.1-1.0mg/kg) to olfactory bulbectomized (OBX) mice once a day for 2weeks, starting 2weeks after bulbectomy. Chronic treatment at 0.3 or 1.0mg/kg dose dependently and significantly improved depression-like behaviors, as assessed by tail suspension (TST), forced swim (FST), locomotion and novelty-suppressed feeding (NSFT) tests. Importantly, co-administration with WAY-100635 (1.0mg/kg), a 5-HT1A receptor antagonist, but not ketanserin (1.0mg/kg,), a 5-HT2A receptor antagonist, completely blocked rivastigmine-induced anti-depressive effects, suggesting that 5-HT1A receptor stimulation mediates this activity. Consistent with this observation, rivastigmine treatment significantly rescued impaired neurogenesis observed in OBX mice in a 5-HT1A receptor-dependent manner. Furthermore, enhanced protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) phosphorylation seen following rivastigmine treatment was closely associated with improved neurogenesis. These effects were blocked by WAY-100635 but not ketanserin treatment. Finally, we confirmed that 5-HT1A but not 5-HT2A receptor stimulation by specific agonists mimicked rivastigmine-induced anti-depression activity and promoted hippocampal neurogenesis. We conclude that, in addition to enhancing the cholinergic system, rivastigmine treatment restores normal function of the hippocampal serotonergic system, an activity that likely ameliorates depressive behaviors in AD patients.

Comparison of the quality of temporal subtraction images obtained with manual and automated methods of digital chest radiography.

The authors have been developing a fully automated temporal subtraction scheme to assist radiologists in the detection of interval changes in digital chest radiographs. The temporal subtraction image is obtained by subtraction of a previous image from a current image. The authors' automated method includes not only image shift and rotation techniques but also a nonlinear geometric warping technique for reduction of misregistration artifacts in the subtraction image. However, a manual subtraction method that can be carried out only with image shift and rotation has been employed as a common clinical technique in angiography, and it might be clinically acceptable for detection of interval changes on chest radiographs as well. Therefore, the authors applied both the manual and automated temporal subtraction techniques to 181 digital chest radiographs, and compared the quality of the subtraction images obtained with the two methods. The numbers of clinically acceptable subtraction images were 147 (81.2%) and 176 (97.2%) for the manual and automated subtraction methods, respectively. The image quality of 148 (81.8%) subtraction images was improved by use of the automated method in comparison with the subtraction images obtained with the manual method. These results indicate that the automated method with the nonlinear warping technique can significantly reduce misregistration artifacts in comparison with the manual method. Therefore, the authors believe that the automated subtraction method is more useful for the detection of interval changes in digital chest radiographs.

[A case of local recurrence of rectal cancer responding to local intraarterial infusion therapy].

A 42-year-old male developed pain in the right gluteal region due to local recurrence after curative resection of advanced lower rectal cancer. Radiotherapy (60 Gy) was performed, but satisfactory results were not obtained. Therefore, a reservoir was placed lowing cannulation of the internal iliac artery. The chemotherapy, in addition to intravenous administration of low dose CDDP (20 mg), included local intraarterial infusion therapy with 5-FU (1,500 mg/5 hour) once per week. After 10 courses of this chemotherapy (total dose: CDDP, 200 mg; 5-FU, 15,000 mg), the pain decreased, and the tumor size was reduced without side effects, improving the patient's QOL. At present, multidisciplinary treatments including such chemotherapy and radiotherapy is performed for local recurrence of rectal cancer, but adequate results are often not obtained. Local intraarterial infusion chemotherapy via the internal iliac artery accompanied by changes in blood flow can be safely performed on an outpatient basis, and appears to be effective for local recurrence of rectal cancer.

Reduced metastatic potential and c-myc overexpression of colon adenocarcinoma cells (Colon 26 line) transfected with nm23-R2/rat nucleoside diphosphate kinase alpha isoform.

Increased expression of nm23/nucleoside diphosphate kinase (NDP kinase) has been reported to be associated with both reduced metastatic potential in breast carcinoma and tumor progression in colon adenocarcinoma and lung adenocarcinoma. We examined effects of expression of nm23-R2 rat NDP kinase alpha isoform on mouse adenocarcinoma cells (Colon 26 line) and found a significant reduction of metastatic potential along with overexpression of c-myc. We also found that the proliferation rate of the transformed cells was the same as that of the control cells in culture. These results indicate that the cell growth potential in vitro is irrelevant to metastatic potential of the cells in vivo.

Suppression of electrophoretic anomaly of bent DNA segments by the structural property that causes rapid migration.

Intrinsic DNA curvature is speculated to be a common feature of all satellite DNA sequences and may aid in the tight winding of DNA in constitutive heterochromatin. Several satellite DNAs, however, show unusually rapid migration in non-denaturing polyacrylamide gels, which is just the opposite behavior of that shown by curved DNA structures. Employing bovine satellite I DNA monomer, we attempted to understand the molecular mechanism of 'rapid migration'. The phenomenon of rapid migration was temperature-dependent and to a small extent polyacrylamide-concentration-dependent. Physiological or near-physiological concentrations of Mg2+and Ca2+ions bent the rapid migrating DNA segment. Predominance of purine-purine base stacking over purine-pyrimidine in nucleotide sequence was strongly indicated to be the cause of the rapid migration. Furthermore, they seemed to be implicated in the formation of induced DNA bend. We also found that the satellite I monomer contains an intrinsic DNA curvature as do many other satellites. Heretofore, the rapid migration property has concealed the presence of curvature.

Epidermal growth factor receptor in human hepatocellular carcinoma.

To determine whether a relationship exists between expression of epidermal growth factor receptor (EGFR) and clinicopathological characteristics of hepatocellular carcinoma (HCC), EGFR was examined in 25 patients surgically treated for HCC using [125I]1-labeled EGF binding assay. Six cases were classified as stage I, 12 as stage II, 4 as stage III, and 3 as stage IV. Partial hepatectomy was performed in 11 cases, segmentectomy in 6, and lobectomy in 8. The level of EGFR in HCC was 8.9 (14.6) fmol/mg protein in HCC and 11.4 (9.2) fmol/mg protein in the adjacent noncancerous diseased liver tissues, and EGFR level in HCC was significantly lower than that of noncancerous liver tissues. HCC stage I + II had significantly lower levels of EGFR compared with stage III + IV (p < 0.05). Our study showed no obvious correlation between EGFR levels and other pathologic characteristics, such as tumor diameter, Edmondson's grade, extracapsular invasion, and vascular invasion. There was no significant difference in EGFR level between DNA diploid and aneuploid tumors. These results suggest that EGFR is not a relevant oncogenic factor for HCC.

Characterization of DNA fragments that show anomalously rapid migration in non-denaturing polyacrylamide gels.

Several DNA fragments that show anomalously rapid migration in nondenaturing polyacrylamide gels have been cloned from digests of bovine genomic DNA. Electrophoretic behaviors of these fragments were analyzed in detail under various conditions. The results indicated that these fragments may adopt non-B DNA conformation.

Kinetic studies of cytochrome P-45017 alpha, lyase dependent androstenedione formation from progesterone.

The reaction mechanism of androstenedione formation from progesterone was analyzed in a membrane reconstituted system consisting of P-45017 alpha, lyase and NADPH-cytochrome P-450 reductase using a rapid quenching device at 10 degrees C. In these rapid quenching experiments, only the metabolites of [3H]progesterone bound to P-45017 alpha, lyase at the initial stage were detectable during the limited cycles of the P-45017 alpha, lyase reactions (1-120 s). The level of 17 alpha-hydroxy[3H]progesterone increased rapidly in a short period (1-5 s) and then decreased to about half. That of [3H]androstenedione increased gradually from 2 s, which exactly corresponded to the decrease in 17 alpha-hydroxy[3H]progesterone. 17 alpha-Hydroxyprogesterone was conclusively the actual intermediate steroid which did not dissociate from P-45017 alpha, lyase during the successive hydroxylation reaction into androstenedione. A kinetic model can clearly describe the successive reaction catalyzed by P-450 17 alpha, lyase, in which progesterone is converted successively into androstenedione via 17 alpha-hydroxyprogesterone, some of which dissociates from the active site of P-450 17 alpha, lyase and is never metabolized into androstenedione. We analyzed the effects of pH and the amount of NADPH-cytochrome P-450 reductase on the successive reaction and proved that the reaction was regulated by the rate of electron transfer for the conversion of the bound 17 alpha-hydroxyprogesterone to androstenedione. Furthermore, we found that the product dissociation from P-450 17 alpha, lyase is the rate-limiting process in the steady-state metabolism of progesterone by P-450 17 alpha, lyase.

Changes in IL-6 and IL-8 after hepatectomy in patients with liver cirrhosis.

Changes in tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and interleukin-8 (IL-8) were investigated before and after hepatectomy in patients with or without liver cirrhosis (5 cases without liver cirrhosis and 14 cases with liver cirrhosis). Both the IL-6 and IL-8 values of the cirrhotic patients were significantly higher on the first postoperative day (POD) as compared with the non-cirrhotic patients. Overall, no significant correlation was found between peak values of IL-6 or IL-8 and blood loss or operating time. In the case of the cirrhotic patients, correlation of both IL-6 and IL-8 with operating time was significant at p < 0.05, gamma = 0.534 and 0.586, respectively. No correlation was found between blood loss and the peak value of IL-6, but significant correlation (gamma = 0.647, p < 0.05) was found between them in cirrhotic patients. There was no consistent increase in TNF-alpha and IL-1 beta following hepatectomy. These findings indicate that procedures undertaken to reduce the excessive production of these cytokines may be useful for preventing complications after hepatectomy in cirrhotic patients.

Membrane topology of bovine adrenocortical cytochrome P-450C21: structural studies by trypsin digestion in vesicle membranes.

Purified adrenocortical microsomal P-450C21 was incorporated into vesicle membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1. Trypsinolysis of the incorporated P-450C21 resulted in the formation of 30-, 25-, and 20-kDa fragments. Similar fragment formation was observed by trypsinolysis of bovine adrenocortical microsomes with Western blotting using anti-P-450C21 IgG. In the detergent-solubilized state, trypsin cleaved P-450C21 into very small peptides. Washing of the trypsin-treated vesicles with 500 mM Na2CO3 failed to cause these fragments to separate from membranes. N-Terminal amino acid sequencing of these fragments showed that trypsin cleaved the 267 Arg-268Val and 332Arg-333Val bonds of P-450C21. The time course of fragment formation indicated that trypsin cleaved the 267Arg-268Val bond first to produce 30- and 25-kDa fragments and subsequently the 332Arg-333Val bond in the 25-kDa fragment to produce the 20-kDa fragment. Neither 21-hydroxylase activity, the reduced CO difference spectrum, nor the EPR spectrum of digested P-450C21 differed from those of undigested P-450C21. Heat treatment at 50 degrees C for 20 min did not cause any decrease in activity of digested P-450C21, when the substrate progesterone was present. This high stability toward heat treatment was not observed in the solubilized state. Rotational diffusion experiments on P-450C21 showed that the size of the molecule holding the heme was not changed significantly after digestion. On the basis of these results, P-450C21 is concluded to be deeply embedded in the vesicle membranes.

Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study.

Purified adrenocortical microsomal cytochromes P-45017 alpha,lyase and P-450C21 were reconstituted with and without NADPH-cytochrome P-450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P-450 ratio of 35 (w/w) by cholate dialysis procedures. Trypsinolysis revealed that a considerable part of each P-450 molecule is deeply embedded in the lipid bilayer, on the basis of the observation of no detectable digestion for P-45017 alpha,lyase and the proteolysis-resistant membrane-bound heavy fragments for P-450C21. Rotational diffusion was measured in proteoliposomes and adrenocortical microsomes by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1-2 ms to a time-independent value r3. Coexistence of a mobile population with an average rotational relaxation time phi of 138-577 microseconds and immobile (phi > or = 20 ms) populations of cytochrome P-450 was observed in both phospholipid vesicles and microsomes. Different tilt angles of the heme plane from the membrane plane were determined in proteoliposomes to be either 47 degrees or 63 degrees for P-45017 alpha,lyase from [r3/r(0)]min = 0.04 and either 38 degrees or 78 degrees for P-450C21 from [r3/r(0)]min = 0.19, when these P-450s were completely mobilized by incubation with 730 mM NaCl. Very different interactions with the reductase have been observed for the two P-450s in proteoliposomes. In the presence of the reductase, the mobile population of cytochrome P-450C21 was increased significantly from 79% to 96% due to dissociation of P-450 oligomers.(ABSTRACT TRUNCATED AT 250 WORDS)