Laboratory investigation of microelectronics-based stimulators for large-scale suprachoroidal transretinal stimulation (STS).

This paper describes the technological developments underlying the realization of a reliable and reproducible microchip-based stimulator with a large number of stimulus electrodes. A microchip-based stimulator with over 500 electrodes for suprachoroidal transretinal stimulation (STS) is proposed in this paper, and an example is presented. To enhance reliability and reproducibility for such a large array, we introduce a flip-chip bonding technique and place microchips on the reverse side of a substrate. A square microchip of size 600 microm was fabricated using 0.35 microm standard CMOS process technology. Twelve microchips were flip-chip bonded on a polyimide substrate through Au bumps. To evaluate the feasibility of the proposed device, we successfully fabricated a stimulator with 12 microchips and 118 electrodes made of Pt/Au bumps, and demonstrated their operation in a saline solution for 2 weeks. Also, to evaluate the device operation in vivo, a stimulator with one active IrO(x) electrode was implanted into the scleral pocket of a rabbit and electrical evoked potential (EEP) signals with a threshold of 100 microA were obtained. We also fabricated a simulator with 64 microchips that has 576 electrodes (9 electrodes in a microchip times 64 microchips).

Gene expression and in situ localization of diacylglycerol kinase isozymes in normal and infarcted rat hearts: effects of captopril treatment.

Diacylglycerol (DG) kinase (DGK) terminates signaling from DG, which serves as an activator of protein kinase C (PKC), by converting DG to phosphatidic acid. DGK is thus regarded as an attenuator of the PKC activity. In rats, five DGK isozymes have been cloned, but little is known about their role in the heart. In this study, the spatiotemporal expression of DGK isozymes was investigated in rat hearts under a normal condition and after myocardial infarction (MI) by in situ hybridization histochemistry and immunohistochemistry. In normal left ventricular myocardium, DGKalpha, DGKepsilon, and DGKzeta mRNAs were expressed evenly throughout the myocardium, although the DGKalpha expression was very low. In infarcted hearts, the expression of DGKzeta was enhanced in the peripheral zone of the necrotic area and at the border zone 3 and 7 days after MI, and to a lesser extent in the middle layer of the granulation tissue 21 days after MI. The enhanced DGKzeta expression in the infarcted and border areas could be attributed to granulocytes and macrophages. In contrast, the expression of DGKepsilon in the infarcted and border areas was lower than that in the viable left ventricle (LV) throughout the postoperation period. Furthermore, DGKepsilon expression in the viable myocardium 21 days after MI decreased significantly compared with left ventricular myocardium in the sham-operated rats and was completely restored by treatment with captopril. Our results demonstrate that three DGK isozymes are expressed in the heart and that each isozyme might have different functional characteristics in the healing and LV remodeling after MI.

Expression of UDP-glucose: poriferasterol glucosyltransferase in the process of differentiation of a true slime mold, Physarum polycephalum.

An expression of UDP-glucose:poriferasterol glucosyltransferase activity associated with differentiation of a true slime mold, Physarum polycephalum, from haploid myxoamoebae to diploid plasmodia was demonstrated. In the haploid cells, this enzyme activity was not detected, but after conjugation of the myxoamoebae, the enzyme activity was expressed and increased definitely. In the plasmodial stage, high enzyme activity was maintained constantly. The enzyme was partially purified (35-fold purification, and 28% yield), and molecular weight of 72,000, pH optimum of 7.0, and some characteristics were demonstrated.

A new metabolite of propargylglycine, gamma-glutamylpropargylglycylglycine, in liver of D,L-propargylglycine-administered rats.

A new metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) was isolated from liver of rats intraperitoneally administered D,L-propargylglycine with ion-exchange chromatography, and identified as a glutathione analogue, N-[N-gamma-glutamyl(propargylglycyl)]glycine (gamma-Glu-PPG-Gly), by fast-atom-bombardment-mass spectrometry and reactions of the compound including acid hydrolysis, carboxypeptidase reaction, and gamma-glutamyltranspeptidase reaction. The content of gamma-Glu-PPG-Gly in rat liver increased dose-dependently with the increase of D,L-propargylglycine. When the dose of D,L-propargylglycine was 50 mg/kg of body weight, the increase of gamma-Glu-PPG-Gly was proportional to the time after the administration of D,L-propargylglycine, up to 8 h, and then gradually decreased to about 50% of the maximum at 24 h, where the maximum level of gamma-Glu-PPG-Gly at 8 h was 1.15 +/- 0.08 micromol/g of liver. The propargylglycine moiety of gamma-Glu-PPG-Gly in rat liver at 14 h after the administration of D,L-propargylglycine corresponded to 2-7% of the propargylglycine administered when the dose of D,L-propargylglycine was 3.125-200 mg/kg of body weight. The present results indicate that gamma-Glu-PPG-Gly is a major intermediate of propargylglycine metabolism in rat liver. The structural resemblance between glutathione and gamma-Glu-PPG-Gly suggests a possible involvement of propargylglycine and gamma-Glu-PPG-Gly as cysteine and glutathione analogues, respectively, in sulfur amino-acid metabolism.

Characterization of hydrogen peroxide removal activities in mouse hemolysates: catalase activity and hydrogen peroxide removal activity by hemoglobin.

Hydrogen peroxide removal activities in normal and acatalasemic mouse hemolysates were examined to determine the optimal temperature of catalase. From thermal stability of the removal activities in hemolysates, the removal activities were divided into two activities. The removal activity deactivated at lower temperature was catalase, and the 50% inactivation was observed after 10 min incubation at 47.2 +/- 0.5 degrees C for normal hemolysates and 34.0 +/- 0.8 degrees C for acatalasemic ones. The removal activity deactivated at a higher temperature remained after the addition of sodium azide, and the 50% inactivation was observed at 63.5 +/- 1.4 degrees C. After separation of the removal activities by carboxymethyl-cellulose column chromatography, the removal activity deactivated at higher temperature was attributed to the activity by hemoglobin. From Lineweaver-Burk plot analysis of the removal rates by hemoglobin at 37 degrees C, the Michaelis constant for hydrogen peroxide and the maximum velocity were 201 +/- 53 microM and 5.37 +/- 1.39 micromol/s per g of Hb, respectively. Removal rates by hemoglobin in mouse hemolysates at 37 degrees C in 70 microM hydrogen peroxide were 1.32 +/- 0.12 micromol/s per g of Hb. Catalase activity (k/g Hb: rate constant related to the hemoglobin content) in normal mouse hemolysates was 104 +/- 12 at 25 degrees C and 117 +/- 10 at 37 degrees C, and that in acatalasemic hemolysates was 10.5 +/- 1.7 at 25 degrees C. These results indicate that activity of hydrogen peroxide removal by hemoglobin is substantial and the activity in acatalasemic hemolysates is predominant at low concentration of hydrogen peroxide.

DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum.

Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.

Changes in phospholipid composition and phospholipase D activity during the differentiation of Physarum polycephalum.

Changes in phospholipid composition and phospholipase D activity were observed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum. In the amoeboid stage, the main components of phospholipid fraction were phosphatidylethanolamine (PE, 43.3%), phosphatidylcholine (PC, 28.8%) and phosphatidylinositol (PI, 8.0%), but in the plasmodial stage, PC was dominant (40.7%) and other main components were PE (31.5%) and phosphatidic acid (PA, 11.0%). The specific activity of phospholipase D in the plasmodia was 5.7-times higher than that in the myxoamoebae when measured in the presence of Ca2+ at the alkaline pH. In the amoeboid stage, phospholipase A activity (A1 or A2) was detected at the alkaline pH with Ca2+. Phospholipase D activity in the plasmodia was characterized: pH optimum was 6.0; Ca2+ was required for the reaction and Ba2+ could substitute partly for Ca2+; PE was the best substrate for the hydrolytic activity and PC and PI were not appreciably hydrolyzed; and all detergents tested inhibited the enzyme activity.

Fluorescence in situ hybridization evaluation of c-erbB-2 gene amplification and chromosomal anomalies in bladder cancer.

Oncogene amplification and chromosomal anomalies are found in many solid tumors and are often associated with aggressiveness of cancer. We evaluated the frequency and the role of c-erbB-2 gene amplification, relative increase in c-erbB-2 gene copy number, and gain of chromosome 17 in bladder cancer. A total of 29 bladder cancer specimens were examined using fluorescence in situ hybridization (FISH). Dual labeling hybridization with a directly labeled centromere probe for chromosome 17 together with a probe for the c-erbB-2 locus was performed. c-erbB-2 gene amplification was found in 3.4% (1 of 29) of specimens. Relative increase in c-erbB-2 gene copy number was found in 41.4% (12 of 29) of specimens and was significantly associated with tumor grade (P = 0.044 by Fisher's exact test). Gain of chromosome 17 was identified in 65.5% (19 of 29) of specimens and was significantly associated with tumor grade (P = 0.002 by Fisher's exact test) and tumor stage (P = 0.003 by Fisher's exact test). Our results suggest that c-erbB-2 gene amplification, relative increase in c-erbB-2 gene copy number, and gain of chromosome 17 may play important roles in the development and progression of bladder cancers. Moreover, the use of c-erbB-2 amplification, relative increase in c-erbB-2 gene copy number, and gain of chromosome 17 using FISH, together with tumor grade and stage, may provide a more useful clinical indicator in bladder cancer.

Augmentation of antitumor immunity using genetically M-CSF-expressing L1210 cells.

Macrophage colony-stimulating factor (M-CSF) enhances tumoricidal activities of macrophages. We transduced human M-CSF cDNA into the mouse lymphoid cell line, L1210, and examined the antitumor effect of the locally expressed M-CSF. Mice injected with the M-CSF-producing subline showed improved survival in comparison with the mock-transfected cell line or parental cell line plus M-CSF administration (20 microg/kg for 3 days) at inoculated cell numbers of 10(2) or 5 x 10(3). The survival rate at 50 days after injection of 10(6) high M-CSF-expressing cells was 80%, significantly higher than that after injection of the mock-transfected cells, which killed all the mice by day 23. The survival rate appeared to depend on the amount of M-CSF produced. Moreover, all surviving mice after intravenous injection of the M-CSF-expressing sublines were rechallenged with 10(6) parental L1210 cells at day 50, and all survived up to day 100, demonstrating that M-CSF-expressing cells induced immune protection against the parental cells. The same improvement of survival was observed in mouse M-CSF-expressing cell lines. These observations imply that M-CSF cDNA is a candidate gene for use in gene therapy in leukemia.

Modified Fontan procedure in ninety-nine cases of atrioventricular valve regurgitation.

Between January 1985 and August 1995, among 242 patients who underwent a modified Fontan procedure, 99 had atrioventricular valve regurgitation ranging in degree from 1 to 4, for which concomitant repair of the atrioventricular valve regurgitation was done in the majority of cases. In all but 4 cases the atrioventricular valve was repaired mainly by circular annuloplasty and valve replacement was not done in any case. Although the hospital mortality rate was significantly higher in cases with atrioventricular valve regurgitation (12/99, 12%) than in cases without (4/143, 3%; p < 0.0037, chi 2 test), actuarial survival in atrioventricular valve regurgitation was 84% for years 5 through 10. The degree of atrioventricular valve regurgitation before operation was 1.6 +/- 0.7 on average: in 49 cases with higher than grade 2 regurgitation before operation there was a significant decrease to 0.4 +/- 0.49 (p < 0.0001) after operation in short-term survivors. Patients with atrioventricular valve regurgitation can be treated with reasonable risk, provided proper repair of the valve is done. Circular annuloplasty is a simple and uniformly effective method to control regurgitation even in cases of common atrioventricular valve.

Purification of a novel serpin-like protein from bovine brain.

We purified a novel serine proteinase inhibitor (serpin)-like protein from the bovine brain and named it B-43 from its molecular mass, 43 kDa. A cleaved peptide from B-43 was copurified with the native B-43. Partial amino acid sequencing of the purified B-43 showed that this protein was homologous to glia-derived nexin/protease nexin-1 (GDN/PN-1), plasminogen activator inhibitor 2, leukocyte elastase inhibitor (LEI) and placental thrombin inhibitor (PTI) among the serpins. Although B-43 had a similar amino acid composition to these serpins, the biochemical features of B-43 were different from them. B-43 did not form sodium dodecyl sulfate (SDS)-resistant serpin-proteinase complexes with thrombin, urokinase, pancreatic elastase and plasmin, suggesting that these proteinases were not the targets of B-43. In contrast to GDN/PN-1, B-43 did not have an affinity for heparin. B-43, having different biochemical properties from GDN/PN-1, appears to be an additional serpin expressed in the brain.

Origin of the left coronary artery from the right pulmonary artery.

We report the successful surgical treatment of a 12-year-old boy with a rare type of Bland-White-Garland syndrome with mitral regurgitation, in which an anomalous left coronary artery arose from the middle portion of the right pulmonary artery, employing the direct translocation of the left coronary artery and mitral valvuloplasty.

Etoposide induces programmed death in neurons cultured from the fetal rat central nervous system.

The effects of etoposide on the death of neurons cultured from the central nervous system (CNS) of fetal rats were examined. The cultured neurons died in the presence of 1-40 micrograms/ml of etoposide, which is known to induce programmed death in some kinds of cells, and this cytotoxic effect was prevented by inhibition of protein synthesis and/or RNA synthesis. Furthermore, DNA degradation, including a ladder-like pattern, became evident in these neurons 3 h after incubation with etoposide (10 micrograms/ml), whereas cell death commenced after about 6 h. These results indicate that etoposide-treated CNS neurons require new protein and RNA synthesis to undergo an active death programme, and that internucleosomal fragmentation of DNA mediates the etoposide-induced programmed cell death. This culture system of etoposide-treated CNS neurons is thought to be a useful model for the study of programmed neuronal cell death.

Gastrectomy with combined resection of other organs for carcinoma of the stomach with invasion to adjacent organs: clinical efficacy in a retrospective study.

BACKGROUND: Carcinoma of the stomach invading one or more adjacent organs raises serious concerns over en bloc dissection because en bloc resection has an associated high risk and such advanced carcinoma is frequently associated with incurable factors. Thus, it is important to understand the efficacy of gastrectomy combined with other organ resection and to refine the indications for en bloc dissection. STUDY DESIGN: Seventy-seven patients with carcinoma of the stomach directly invading adjacent organs or structures were analyzed retrospectively to investigate the efficacy of en bloc resection. Forty-one patients underwent gastrectomy combined with resection of one or more invaded organs (combined resection group), while the other 36 patients underwent gastrectomy with palliative abrasion between the primary tumor and the invasion site (noncombined resection group). RESULTS: The five-year survival rate was 23 percent in the combined resection group and 0 percent in the noncombined resection group (p < .05). The 23 curative cases and 18 noncurative cases in the combined resection group had a survival rate of 41 percent and 0 percent, respectively (p < .05). The survival rate after a single organ resection was 29 percent, and after a multiple organ resection, 0 percent (p < .05). Cases of carcinoma invading either the pancreas or mesocolon had a slightly but not significantly better prognosis. In Borrmann type IV carcinoma there was no difference in survival between the curative and noncurative operation. Cases with P1 dissemination had a better prognosis than those of P2 and P3 dissemination. CONCLUSIONS: The best indication for an en bloc combined organ resection was an invasion limited to only one other organ, not more than N2, no incurable factor, and any type except Borrmann type IV. Additionally, an en bloc combined resection would be worth trying for any type of gastric carcinoma with not more than P1 dissemination and with no other incurable factor.

Nerve growth factor and epidermal growth factor rescue PC12 cells from programmed cell death induced by etoposide: distinct modes of protection against cell death by growth factors and a protein-synthesis inhibitor.

A rat pheochromocytoma cell line (PC12 cells) died within 24 h in the presence of etoposide (1-40 micrograms/ml), an inhibitor of topoisomerase II. This cytotoxic effect was prevented by either nerve growth (NGF) or epidermal growth factor (EGF). Cycloheximide and actinomycin D also suppressed the cell death as well. Furthermore, a difference among protective modes against etoposide-induced death by growth factors and a protein-synthesis inhibitor was observed: the protective effect of either NGF or EGF remained rather constant as a function of incubation time with etoposide whereas that of cycloheximide declined. These results indicate that etoposide induces programmed death in PC12 cells and that prevention of the programmed cell death by both NGF and EGF is mainly due to inactivation of molecules involved in the death processes rather than suppression of specific protein and/or mRNA synthesis.

Clinical improvement of adrenoleukodystrophy following intravenous gammaglobulin therapy.

A patient with adrenoleukodystrophy was successfully treated by means of intravenous gammaglobulin injections. The clinical symptoms, especially visual loss, were apparently relieved and no neurological deterioration was observed during a 18-month period following the start of the gammaglobulin treatment.

Early gastric cancer in the remnant stomach.

BACKGROUND/AIMS: Early gastric cancer in the remnant stomach is rare. Periodical endoscopic examinations are mandatory for patients with partial gastrectomy for a good prognosis. Our goal is to improve the surgical management of gastric cancer in the remnant stomach. We have retrospectively investigated a total of 15 rare cases of early gastric cancer after partial gastrectomy. METHODOLOGY: From 1976 to 1994, a total of 2,102 cases of gastric cancer were resected in our Department. Among these resected cases, 845 cases were histologically diagnosed as having early gastric cancer of the stomach. Of these, 15 patients had previously undergone a partial gastric resection. The time interval between the initial partial gastrectomy and the second resection of the remnant stomach, was more than 10 years for 8 patients (Group 1) and less than 10 years for 7 patients (Group 2). Here we investigate these rare cases of remnant early gastric cancer. RESULTS: The incidence of early gastric cancer in the remnant stomach was 1.8% (15/845). The cancer location in the remnant stomach was around the stoma and suture line in 75% of Group 1 and in 28.6% of Group 2. The incidence rate of mucosal cancer (m-cancer) was 87.5% for Group 1, and 14.3% for Group 2. Total gastrectomy was selected for 37.5% of Group 1, and for 100% of Group 2. No lymph node metastasis was discovered in both groups. The postoperative mortality was zero in both groups. One patient from Group 2, later died of liver metastasis 2 years after the second total gastrectomy, while the other 9 patients continued to live for more than 5 years with no gastric cancer recurrence to date. CONCLUSIONS: The outcome for patients with gastric cancer in the remnant stomach is generally considered poor. However, the outcome of early gastric cancer in the remnant stomach was good without major postoperative complications. Therefore, to improve surgical management of remnant-stump gastric cancer, early diagnosis is most important, using periodic endoscopic follow-up examinations, especially around the stoma. When mucosal cancer around the stoma is diagnosed, subtotal gastrectomy can be selected even in gastrectomized patient for a good prognosis.

Reduction of 3-mercaptopyruvate in rat liver is catalyzed by lactate dehydrogenase.

It has been assumed that the in vivo reduction of 3-mercaptopyruvate, an intermediate of cysteine metabolism, to 3-mercaptolactate is catalyzed by lactate dehydrogenase (EC 1.1.1.27) though no definitive evidence has been presented. In order to examine this assumption, reduction of 3-mercaptopyruvate and its inhibition were studied using rat liver homogenate, lactate dehydrogenase purified from rat liver and anti-lactate dehydrogenase antiserum. Reduction of 3-mercaptopyruvate was actively catalyzed by rat liver homogenate and by the purified lactate dehydrogenase. This reducing activity was completely inhibited by anti-lactate dehydrogenase antiserum. These results indicate that the reduction of 3-mercaptopyruvate to 3-mercaptolactate in rat liver is catalyzed by lactate dehydrogenase.

Formation of gamma-glutamylpropargylglycylglycine from propargylglycine in human blood and erythrocytes.

Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.

Excretion of sulfate and taurine in rats fed with a high protein diet.

Sulfate and taurine are the main metabolites of L-cysteine in mammals and are excreted in the urine. The effect of a high protein diet on the ratio of sulfate to taurine excretion was studied in rats using synthetic 25% (standard protein diet group, group A) and 40% (high protein diet group, group B) casein diets. Average taurine and sulfate excretions (mumol/kg of body weight per day) were 280.4 +/- 93.8 and 943.2 +/- 144.8 in group A and 553.4 +/- 124.5 and 2675.0 +/- 390.9 in group B, respectively. Thus, the average taurine/sulfate ratio in group A was 0.30 +/- 0.08. By a single administration of 5 mmol of L-cysteine/kg of body weight in group A, the average taurine and sulfate excretions increased to 1127.5 +/- 120.2 and 4043.0 +/- 305.6, respectively, but the taurine/sulfate ratio changed only slightly (0.28). The average taurine/sulfate ratio in group B was 0.22 +/- 0.07, a significantly lower ratio than that in group A, which means that daily intake of a high protein diet resulted in more sulfate excretion. The taurine/sulfate ratio in group B was affected only slightly (0.19) by the cysteine administration as well. These results suggest that the ratio of taurine and sulfate production was determined by dietary protein content and that the increase in sulfate production is larger than that of taurine production when the intake of dietary protein is increased.