Selective growth inhibition by suppression of F1Fo ATPase in canine malignant melanoma cell lines.

Canine malignant melanoma (CMM) is a highly aggressive and fatal neoplasm. To identify potential therapeutic compounds and/or targets, 320 compounds were screened for their growth inhibitory activity in a CMM line (CMM-1) using a chemical library known to target specific signaling pathways/cell growth-related molecules. Among the compounds screened, the F1Fo ATPase inhibitor oligomycin showed potent growth inhibitory effects in CMM-1 cells, while exhibiting less toxic effects in a non-neoplastic control cell line (MDCK cells). The growth inhibitory effect of oligomycin A was then examined using six CMM lines and MDCK cells. Three CMM lines were highly sensitive to oligomycin A, with around 3000-20 000 times lower IC50 compared with oligomycin A-resistant CMM lines and MDCK cells. Oligomycin A-sensitive CMM-1 cells exhibited much greater oligomycin A-induced decreases in cellular ATP compared to oligomycin A-resistant cell lines. Although the oligomycins are clinically unsuitable because of its in vivo toxicity, these findings implicate the potential of F1Fo ATPase as a therapeutic target in a subset of CMM.

A rare case of Lemierre`s syndrome caused by Porphyromonas asaccharolytica.

Lemierre's syndrome is only very rarely caused by Porphyromonas asaccharolytica. Here, we report the case of a 35-year-old man who developed a left peritonsillar abscess, thrombophlebitis of the left internal jugular vein, and septic embolization of both lungs. Anaerobic P. asaccharolytica was isolated in the blood cultures, and we subsequently confirmed the diagnosis as Lemierre's syndrome. Our case indicates that although P. asaccharolytica is not commonly found in oral cavities, this organism may still cause Lemierre's syndrome. Consequently, when it is detected in blood cultures, the treating physician should perform the medical examination while keeping in mind the possibility that the patient could have Lemierre's syndrome.

Oxygen reduction activity of carbon nitride supported on carbon nanotubes.

Fuel cells offer an alternative to burning fossil fuels, but use platinum as a catalyst which is expensive and scarce. Cheap, alternative catalysts could enable fuel cells to become serious contenders in the green energy sector. One promising class of catalyst for electrochemical oxygen reduction is iron-containing, nanostructured, nitrogen-doped carbon. The catalytic activity of such N-doped carbons has improved vastly over the years bringing industrial applications ever closer. Stoichiometric carbon nitride powder has only been observed in recent years. It has nitrogen content up to 57% and as such is an extremely interesting material to work with. The electrochemical activity of carbon nitride has already been explored, confirming that iron is not a necessary ingredient for 4-electron oxygen reduction. Here, we synthesize carbon nitride on a carbon nanotube support and subject it to high temperature treatment in an effort to increase the surface area and conductivity. The results lend insight into the mechanism of oxygen reduction and show the potential for carbon nanotube-supported carbon nitride to be used as a catalyst to replace platinum in fuel cells.

Oxidation and reduction of bile acid precursors by rat hepatic 3 alpha-hydroxysteroid dehydrogenase and inhibition by bile acids and indomethacin.

Enzyme kinetics of purified rat hepatic 3 alpha-hydroxysteroid dehydrogenase for bile acid precursors and effects of bile acids and indomethacin on those activities were studied. This enzyme catalyzed the oxidoreduction of the C3 position of bile acid precursors. Km for 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one (1.6 microM) was markedly lower than Km for 7 alpha-hydroxy-5 beta-cholestan-3-one (28 microM) but Vmax was similar. Km for 3 alpha, 7 alpha-dihydroxy-5 beta-cholestane (12 microM) was lower than Km for 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestane (150 microM) although Vmax/Km values were similar for both compounds. Bile acids and indomethacin inhibited the reduction of 3-oxo bile acid precursors. NADPH inhibited the binding of lithocholic acid (3 alpha-hydroxy-5 beta-cholanic acid) by 3 alpha-hydroxysteroid dehydrogenase. These data suggest that intrahepatic bile acid concentrations may affect the reduction of 3-oxo-bile acid precursors and intrahepatic redox conditions may affect intracellular bile acid transfer.

Metabolism of intravenously administered 7 alpha-hydroxycholesterol-3 beta-stearate in the hamster.

In order to investigate the metabolic fate of serum esterified 7 alpha-hydroxycholesterol, [4-14C]7 alpha-hydroxycholesterol-3 beta-stearate was synthesized from labeled cholesterol and administered to bile fistula hamsters intravenously. Bile samples were collected at every 20 min for 7 h. Radioactivity was detected in bile 40 min after the beginning of the infusion of the labeled compound and 56.5 +/- 5.7% (48.7-66.0%) of the administered radioactivity was recovered in bile during 7 h. The liver contained appreciable radioactivity (19.5 +/- 7.6% of the administered dose) at the time of sacrifice. Only a trace amount of radioactivity was detected in urine and blood. Cumulative recovery of the radioactivity was 76.3 +/- 8.6% (63.3-90.4%). Major radioactive metabolites in the bile samples were identified to be taurine- and glycine-conjugated cholic acid and chenodeoxycholic acid by radioactive thin-layer chromatographic analysis of the bile samples before and after enzymatic hydrolysis and 3 alpha-hydroxysteroid dehydrogenase treatment. The conversion was nearly complete and we could not detect neutral metabolites, such as the mother compound, free 7 alpha-hydroxycholesterol and bile alcohols, as well as glucuronidated or sulfated bile acids. It is concluded that serum esterified 7 alpha-hydroxycholesterol could be effectively taken up by the liver, hydrolyzed by cholesterol esterase and metabolized via the normal biosynthetic pathway to taurine- or glycine-conjugated primary bile acids to be excreted into bile.

Congenital myopathy with type II muscle fiber hypoplasia.

We examined a 14-month-old boy with severe muscular hypotonia and weakness. Loss of tendon reflexes were noted at age 2 months. Right-sided heart failure and dyspnea during sleep developed at age 4 months. Muscle biopsy revealed selective hypoplasia of type II fibers and normal type I fibers. Intercostal and diaphragmatic muscles showed similar changes at necropsy and probably accented the respiratory failure.

HLA and T-cell receptor gene polymorphisms in Guillain-Barré syndrome.

OBJECTIVE: We examined a possible involvement of genetic factors influencing the development of Guillain-Barré syndrome (GBS). METHODS: We studied T-cell receptor (TCR), alpha-chain constant (AC), and beta-chain variable (BV) gene polymorphisms using microsatellite markers and serologic HLA class I antigens, HLA-DRB1, and HLA-DQB1 alleles in 81 Japanese patients with GBS and 87 controls. RESULTS: There were no significant differences in these genetic markers between GBS patients and controls. Subgrouping of GBS patients according to recent Campylobacter jejuni infection, the presence of anti-GM1 antibody in the sera, or their combinations also failed to reveal significant associations with these genetic markers. There was, however, a tendency for an increased frequency of HLA-DRB1*0803 in the C. jejuni + GM1 + GBS group, when compared with controls. CONCLUSIONS: The data suggest that the roles of TCRAC, T-cell receptor beta-chain variable (TCRBV), HLA class I or class II in the development of GBS are not critical, and further research is necessary to clarify other genes encoded within the HLA region for genetic susceptibility to GBS.

Low-dose ACTH therapy for West syndrome: initial effects and long-term outcome.

BACKGROUND: Most Japanese pediatric neurologists attempt other treatments before using adrenocorticotropic hormone (ACTH) therapy for West syndrome (WS), and even then, they use only a low-dose synthetic ACTH to avoid serious adverse effects. In this multi-institutional study, the authors analyzed the initial effects, adverse effects, and long-term outcome in patients treated with low-dose synthetic ACTH in Japan. METHODS: The medical records of 138 patients with WS, who were treated with low-dose synthetic ACTH therapy for the first time at the authors' institutions between 1989 and 1998, were analyzed. RESULTS: At the end of ACTH therapy, excellent effect on seizures was noted in 106 of 138 (76%) patients, good effect in 23 (17%), and poor effect in 9 (7%). Initial effects on EEG were excellent in 53 of 138 (38%) patients, good in 76 (55%), and poor in 9 (7%). As for seizure prognosis at the time of follow-up, 51 of 99 (52%) patients were seizure-free, whereas 48 (48%) patients had seizures. Mental outcome was normal in 6 of 98 (6%) patients, mild mental retardation in 16 (16%), moderate mental retardation in 26 (27%), and severe mental retardation in 50 (51%). The initial effects of ACTH on seizures and long-term outcome were not dose dependent (daily dosage 0.005 to 0.032 mg/kg, 0.2 to 1.28 IU/kg; total dosage 0.1 to 0.87 mg/kg, 4 to 34.8 IU/kg). The severity of adverse effects correlated with total dosage of ACTH, and the severity of brain volume loss due to ACTH correlated well with the daily dosage and total dosage of ACTH. CONCLUSION: Low-dose synthetic ACTH therapy is as effective for the treatment of WS as the higher doses used in previous studies. The dosage of synthetic ACTH used in the treatment of WS can be decreased as much as possible to avoid serious adverse effects.

Antibodies to gangliosides and galactocerebroside in patients with Guillain-Barré syndrome with preceding Campylobacter jejuni and other identified infections.

The relationship between preceding infections and antibodies to glycolipids was investigated in 205 Japanese patients with Guillain-Barré syndrome (GBS). Serological evidence of recent Campylobacter jejuni (C. jejuni) infection was found in 45% of the patients, compared with 1% in healthy controls. In contrast, recent infection of cytomegalovirus (CMV), Mycoplasma pneumoniae (M. pneumoniae) and Epstein-Barr virus (EBV) was detected in only 5%, 2% and none of the patients, respectively. C. jejuni-associated GBS was more frequent in early spring than in other seasons. All stool specimens positive for C. jejuni isolation were obtained within 10 days after the onset of GBS symptoms. Of 13 C. jejuni isolates from GBS patients, 10 (77%) belonged to Penner serotype 19 (heat-stable, HS-19). Elevated titers of anti-GM1 antibody were found in 8 (80%) of 10 GBS patients whose C. jejuni isolates belonged to HS-19 and in none of those infected with non-HS-19 C. jejuni (P = 0.04), and in 49% of 92 patients with C. jejuni infection and 25% of patients without infection of C. jejuni, CMV, EBV, or M. pneumoniae (P = 0.0007). The frequencies of elevated antibody titers to GD1a, GD1b and GQ1b were also significantly higher in GBS patients associated with C. jejuni than those not associated with C. jejuni, CMV, EBV, and M. pneumoniae. GBS in Japan seems to be associated more frequently with C. jejuni and less frequently with CMV than in Europe and North America.

Cancer chemopreventive activity of phenylpropanoid esters of sucrose, vanicoside B and lapathoside A, from Polygonum lapathifolium.

To search for cancer chemopreventive agents from natural resources, many phytochemicals have been screened using the in vitro synergistic assay indicated by the inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA). Two phenylpropanoid esters of sucrose, vanicoside B and lapathoside A, were isolated from the aerial part of Polygonum lapathifolium as inhibitors on the EBV-EA induction. These compounds also exhibited significant anti-tumor-promoting effects on mouse two-stage skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA, as an initiator) and TPA as a promoter. Further, vanicoside B exhibited the remarkable inhibitory effect on two-stage carcinogenesis test of mouse skin tumors initiated with an NO donor, NOR-1.

Clinical spectrum and genetic studies of Fukuyama congenital muscular dystrophy.

The association of congenital muscular dystrophy (CMD) with type II lissencephaly and ocular anomalies is found in Fukuyama CMD (FCMD), the Walker-Warburg syndrome (WWS), and muscle-eye-brain disease (MEBD). The classification of these disorders remains controversial. Between 1972 and 1992, we performed clinical and genetic studies in 41 families of FCMD, which is particularly frequent in Japan. Nine families (22%) had multiple affected children ("familial" FCMD). The other 32 families had only one affected child ("sporadic" FCMD). Parental consanguinity was documented in 5 sporadic FCMD families and in none of the familial cases. In total, 48 patients, including 7 sib pairs, were evaluated with regard to maximum motor ability, mental and convulsion states, cranial CT or MRI findings, and EEG and ophthalmological data. A difference between the sibs in motor ability was apparent in 4 families. Mental status also showed wide variation. Two of 7 sib pairs differed in EEG findings. The familial FCMD patients showed relatively more severe motor disability than that in the sporadic FCMD patients, while in mental and convulsion states no significant difference was found in both groups. Interestingly, in one family hydrocephalus was found in only one of the sibs. In addition, this patient showed encephalocele and retinal detachment at birth. Based on these observations, we consider the clinical spectrum of FCMD to be much broader than previously described and to overlap with that of "mild" WWS and MEBD.

Effects of temperature and cholesterol on human erythrocyte membranes.

The effects of temperature and cholesterol on the membrane fluidity of human erythrocytes were studied using 5-nitroxide stearic acid (5NS), 12-nitroxide stearic acid (12NS), and 16-nitroxide stearic acid (16NS). Human erythrocytes and their lipid vesicles were treated in the range of 5--55 degrees C. In erythrocytes, ESR signals for 12NS and 16NS showed line broadening above 40 degrees C, whereas those for 5NS became sharper with increasing temperature as was the case with the signals of lipid vesicles for each label molecule. Lipid extraction from the heated sample caused no radical reduction. Only in 12NS-labeled erythrocytes did a weakly immobilized component and a strongly immobilized component appear. In the time course at 50 degrees C, the former decreased and the latter remained constant. From the ratio of both components, it was found that the interaction of the label molecules with the binding sites was determined by the physical state of the membrane. Furthermore, the dependence on temperature of the molecular motion of the labels in the cell membrane was irreversible above 40 degrees C. On addition of cholesterol to the membrane, the outer hyperfine splittings for 12NS and 16NS increased but that for 5NS decreased at C/P greater than 1, perhaps indicating a spread between the head groups of phospholipids by cholesterol.

Cloning and expression of the Ca2+ channel alpha1C and beta2a subunits from guinea pig heart.

Complimentary DNA clones encoding the alpha1C and beta2a subunits of guinea-pig cardiac L-type Ca2+ channels were isolated using the PCR method. The open reading frame encoded 2,169 amino acids for the alpha1C and 597 amino acids for the beta2a subunit. The proteins showed 94.2 and 94.8%, respectively, identity to the respective subunit of the rabbit protein. The message size of the guinea pig alpha1C and beta2a subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the alpha1C subunit is expressed exclusively in the heart, while the beta2a subunit is expressed in the heart, cerebellum, whole brain, and stomach. The alpha1C and beta2a subunits are transiently expressed in BHK (baby hamster kidney) cells, and the channel currents were studied using the whole-cell patch clamp technique in medium containing 30 mM Ba2+. In cells expressing alpha1C alone, the Ba2+ current was activated at -30 mV and more positive potentials and peaked at about 10 mV. The co-expression of beta2a with alpha1C did not affect the voltage-dependence of the current, but increased the peak current and accelerated current decay. In cells transfected with guinea pig alpha1C and rabbit beta1+alpha2/delta, a Ba2+ current comparable to those in native myocytes was observed. The Ba2+ current can be blocked completely by nifedipine and is enhanced 3-fold by Bay K 8644. On the other hand, neither forskolin nor okadaic acid affects the Ba2+ current, suggesting that cAMP-mediated modulation is not easily reproduced in transfected cells, unlike that seen in native cardiac myocytes.

A study on the reaction of human erythrocytes with hydrogen peroxide.

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.

Gadolinium pretreatment decreases survival and impairs liver regeneration after partial hepatectomy under ischemia/reperfusion in rats.

BACKGROUND: Modulation of Kupffer cell functions by treatment with gadolinium chloride protects the liver against reperfusion injury. However, its effect on liver regeneration after hepatectomy under ischemia/reperfusion has not been studied. Using a common clinical ischemia/reperfusion technique, we examined the effect of gadolinium on liver regeneration after hepatectomy in rats. METHODS: After an initial 15-minute ischemia and 15-minute reperfusion, 70% hepatectomy was performed during the second 15-minute ischemia period in gadolinium-pretreated (gadolinium group) and saline solution--pretreated (control group) rats. The 24-hour survival rate, relative liver weight, DNA synthesis rate, and hepatic adenosine triphosphate level were examined immediately after hepatectomy and on postoperative days (PODs) 1, 2, 3, and 7. Serum levels of total bilirubin, glutamic pyruvic transaminase, and endotoxin were also measured. RESULTS: The 24-hour survival rate was significantly lower in the gadolinium group (67%) than in the control group (100%). On POD 1, the relative liver weight and DNA synthesis rate were significantly lower in the gadolinium group than in the control group. On POD 1, serum total bilirubin and endotoxin levels were significantly higher in the gadolinium group than in the control group. Immediately after hepatectomy, the hepatic adenosine triphosphate level was significantly lower in the gadolinium group than in the control group. CONCLUSIONS: Under ischemia/reperfusion, gadolinium pretreatment impairs liver regeneration and energy status after hepatectomy and decreases postoperative survival.

Vascular endothelial growth factor and platelet-derived growth factor are potential angiogenic and metastatic factors in human breast cancer.

BACKGROUND: Angiogenesis is a prerequisite for tumor growth and metastasis. Tumor angiogenesis may be mediated by several angiogenic factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha, and basic fibroblast growth factor. METHODS: Differential mRNA expressions of VEGF, PDGF (A chain), transforming growth factor-alpha and basic fibroblast growth factor in 32 primary invasive breast tumors were examined by reverse transcriptase-polymerase chain reaction. We analyzed relationships between mRNA expressions of these angiogenic factors and the degree of angiogenesis, tumor size, and metastasis. Quantification of angiogenesis was achieved by the immunohistochemical staining of endothelial cells with antibody to CD31. RESULTS: VEGF and PDGF-A mRNAs were expressed more frequently in breast tumors than in nontumor breast tissues, whereas no difference was found in expression frequency of either transforming growth factor-alpha or basic fibroblast growth factor mRNA. Vascular counts in tumors correlated with each expression frequency of VEGF and PDGF-A mRNA. PDGF-A mRNA was expressed more frequently in tumors with lymph node metastasis than in those without metastasis. CONCLUSIONS: Expression of VEGF and PDGF mRNAs detected by reverse transcriptase-polymerase chain reaction in breast tumors correlates with tumor-related characteristics of angiogenesis and metastatic potential. Analysis of these mRNAs by reverse transcriptase-polymerase chain reaction may be useful for assessing the biologic behavior of a breast tumor before surgical treatment.

Current management and long-term prognosis of hepatolithiasis.

OBJECTIVE: To assess the surgical and endoscopic treatment results and long-term prognosis of hepatolithiasis. DESIGN: Retrospective case series. SETTING: University hospital. PATIENTS: Eighty-five consecutive patients with hepatolithiasis treated between 1980 and 1993. MAIN OUTCOME MEASURES: Location of calculi, associated diseases, treatment procedures, and long-term prognosis were analyzed. RESULTS: Thirty-eight percent of the 85 patients had stones only in intrahepatic ducts, while others had intrahepatic and extrahepatic calculi. Seventy-two percent of the patients had repeated biliary surgery, and 14% were associated with liver cirrhosis. After surgery combined with cholangioscopy and electrohydraulic lithotripsy, the complete stone clearance rate was 71%. The treatment-related mortality rate was 3.8%; the stone recurrence rate after complete stone removal was 11%. However, it was notable that 17 patients (20%) died of related diseases during a mean follow-up of 6 years. The reasons for death were liver failure, bleeding from esophageal varices, hepatocellular carcinoma, and cholangiocarcinoma. CONCLUSION: Improvement of stone clearance rate is evident by a systematic approach. Complete relief from calculi, bile stasis, and cholangitis at an early stage of the disease is mandatory to prevent progressive liver damage, because repeated recurrence of intrahepatic calculi and cholangitis ultimately lead to irreversible liver damage and hepatobiliary carcinomas with a high mortality.

Functional contribution of preoperative portal vein occlusion to hepatectomy. With special reference to hepatic energy charge and DNA synthesis after hepatectomy in rats.

OBJECTIVE: To examine possible functional contributions of preoperative portal branch ligation before hepatectomy (PBL-Hx). DESIGN: Rats were randomly divided into 3 groups. In the PBL-Hx group, the portal branch supplying the left lateral and median lobes of the liver was ligated and the corresponding lobes (48% of the whole liver) were excised 2 days later. In the sham groups (one 68% Hx; the other 47% [hereafter, sham-67% Hx, and sham-47% Hx]), originally ligated lobes and left lateral and caudate lobes, similar to the excised liver volume in the PBL-Hx group, respectively, were excised 2 days after sham operation without PBL. MAIN OUTCOME MEASURES: Hepatic adenine nucleotides and energy charge, which are essential for vital function of hepatocytes, and liver regeneration were assessed by the DNA synthesis rate and weight before Hx and on days 1, 2, 3, and 7 after Hx. RESULTS: The remaining liver weight was restored similarly in the PBL-Hx and sham-47% Hx groups and more rapidly than in the sham-68% Hx group. Further enhancement of DNA synthesis did not occur after Hx in the PBL-Hx group, and hepatic energy charge did not decrease. In contrast, hepatic DNA synthesis was significantly activated depending on the excised liver volume in both the sham-Hx groups and was accompanied by corresponding decreases in hepatic energy charge. CONCLUSION: Preoperative PBL has a functional advantage because the recovery of the remaining liver volume is not impaired and hepatic energy charge is preserved with no further enhancement of DNA synthesis after Hx.

Clinical pharmacology and toxicity of low daily administration of oral etoposide in advanced lung cancer patients.

In this study, pharmacokinetics, clinical effects, and toxicity of daily oral etoposide were studied in 12 patients with advanced lung cancers. Administration schedule was 25 mg/body everyday of a 4-week cycle or 50 mg (25 mg twice)/body for 14 consecutive days of a 4-week cycle. The pharmacokinetic parameters of both groups in serum were as follows: 1) mean peak plasma concentration of 0.91 +/- 0.18 micrograms/mL (25 mg) and 1.24 +/- 0.12 micrograms/mL (25 mg twice); 2) elimination half-lives of 6.48 +/- 1.09 hours (25 mg) and 3.64 +/- 0.27 hours (25 mg twice); and 3) the area under the plasma concentration curve of 7.09 +/- 1.26 micrograms.hr/mL (25 mg) and 8.67 +/- 0.77 micrograms.hr/mL (25 mg twice). There was a significant difference between those two schedules in terms of the plasma concentration and the duration at a low concentration (greater than 1 micrograms/mL) of etoposide. Low daily administration of oral etoposide is shown to be well tolerated and easy on the patient. More studies are needed to study whether prolonged schedules of lower doses of etoposide could result in improved prognosis.