Enhancement of cellular accumulation of cyclosporine by anti-P-glycoprotein monoclonal antibody MRK-16 and synergistic modulation of multidrug resistance.

BACKGROUND: Drug resistance is a major obstacle to successful cancer chemotherapy. P-glycoprotein, which transports certain antitumor agents out of resistant tumor cells, is known to be a major factor in some types of multidrug resistance. Studies have shown that verapamil and the immunosuppressors cyclosporine and FK-506 can reverse multidrug resistance in vitro and in vivo and that the P-glycoprotein monoclonal antibody MRK-16 increases drug toxicity in multidrug-resistant tumors. PURPOSE: The purpose of this in vitro study was to establish effective treatment modalities for overcoming multidrug resistance. We assessed the synergistic effects of verapamil, cyclosporine, or FK-506 in combination with MRK-16 and antitumor agents. METHODS: Human myelogenous leukemia K562 cells and multidrug-resistant K562/ADM cells were treated with vincristine or doxorubicin combined with MRK-16 and cyclosporine alone or together; MRK-16 and verapamil alone or together; or MRK-16 and FK-506. The effects of MRK-16 and cyclosporine or verapamil on the accumulation of vincristine and doxorubicin were examined in K562/ADM cells, and the mechanisms of action were analyzed. RESULTS: MRK-16 and cyclosporine synergistically enhanced the antitumor effects of vincristine and of doxorubicin in K562/ADM cells. However, the combined use of MRK-16 with verapamil or FK-506 did not show such synergistic effects in these cells. Studies of the effect of MRK-16 on cellular accumulation of cyclosporine and verapamil revealed that MRK-16 substantially increased accumulation of cyclosporine in K562/ADM cells, but did not increase accumulation of verapamil. CONCLUSIONS: MRK-16 and cyclosporine synergistically enhanced the antitumor effects of vincristine and doxorubicin because MRK-16 increased cellular accumulation of cyclosporine. IMPLICATIONS: These results, together with our previous finding that intravenous administration of MRK-16 induced regression of multidrug-resistant subcutaneous tumors in athymic mice, support the hypothesis that the combined use of MRK-16 and cyclosporine might increase the efficacy of antitumor agents against multidrug-resistant tumors expressing P-glycoprotein. Clinical phase I trials of MRK-16 in the treatment of multidrug-resistant tumors are under consideration.

A novel purification and some properties of rat liver mitochondrial choline dehydrogenase.

Choline dehydrogenase (choline:(acceptor) oxidoreductase, EC 1.1.99.1) was purified from rat liver mitochondria. An approx. 240-fold purification was achieved by chemically modified enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) through columns of DEAE-Sepharose CL-6B and the choline-Sepharose 4B with C3-spacer, and after the subsequent release of the thionitrobenzoate with dithiothreitol, through a second column of DEAE-Sepharose CL-6B in the presence of 0.1% Triton X-100. The purified preparation gave a specific activity of 6.3 mumol of O2 consumed/min per mg protein at 30 degrees C with phenazine methosulfate as the primary electron acceptor. After polyacrylamide gel electrophoresis in the presence of 0.5% sodium dodecyl sulfate, the enzyme showed activity in the gel. The preparation thus purified oxidized only choline and betaine aldehyde. The Km value for choline was 7.0 mM at an infinite concentration of phenazine methosulfate at pH 7.6 and 30 degrees C. 2-Dimethyl aminoethanol (Ki,app = 1.0 mM) and monoethanolamine did not work as substrates, inhibiting the enzyme competitively. The absolute requirement of any electron acceptor other than the molecular oxygen was confirmed. The Km value for phenazine methosulfate was about 1.1 mM at infinite concentration of choline. These findings suggested that coenzyme Q served as the primary electron acceptor in vivo.

Change of choline metabolism in rat liver on chronic ethionine-feeding.

On chronic ethionine-feeding (0.1% DL-ethionine in 0.5% sucrose solution) for 2, 4 or 6 months, choline metabolism in rat hepatic cells was altered considerably, although RNA contents were virtually unchanged. Choline dehydrogenase activity in the hepatic mitochondrial fraction was suppressed by about 1/2 or 1/3, compared to its normal level, whereas choline kinase activity, which existed in the cytoplasmic fraction, was elevated more than 1.5-fold. The normal value for choline-metabolizing enzymes in terms of the choline dehydrogenase/choline kinase activity ratio was estimated to be about 70 under the defined conditions, while the average value of the activity ratio drastically changed to about 10-20 on chronic ethionine-feeding. The present results suggest that an alteration of hepatic choline-flux occurred, due both to an increase in choline kinase activity and to a counteractive decrease in choline dehydrogenase activity.

A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR.

Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.

Crystallization and preliminary X-ray crystallographic studies of recombinant human leukotriene A4 hydrolase complexed with bestatin.

Recombinant human leukotriene A4 hydrolase complexed with bestatin, an inhibitor of metalloprotease, has been crystallized by the hanging drop vapor diffusion method using 0.1 M phosphate buffer (pH 6.5) and 50 to 54% saturated ammonium sulfate. The orthorhombic crystals belong to the space group I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 273.6 A, b = 261.3 A and c = 52.9 A. They diffract beyond 2.5 A resolution and a native data set up to 3 A resolution has been collected on an imaging plate Weissenberg camera using synchrotron radiation.

Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein.

Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.

The calcium-binding property of equine lysozyme.

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio-Gel P-4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metallo-protein like alpha-lactalbumin, which is a homologue of hen egg white lysozyme.

Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethyla mide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10(-7) M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10(-7) M in vitro.

Purification, properties, and molecular features of glucose oxidase from Aspergillus niger.

4. FHD (flavin-hypoxanthine dinucleotide) has coenzymatic activity equal to that of FAD.

Crystallographic studies of a calcium binding lysozyme from equine milk at 2.5 A resolution.

The crystal structure of a calcium binding equine lysozyme has been determined at 2.5 A resolution by means of molecular replacement. The energy minimized equine lysozyme as the starting model, was refined with the molecular dynamics program, X-PLOR, and the R factor of the current model was found to be 24% without any water molecules. The conformation of the calcium binding loop is similar to that of alpha-lactalbumin. The profiles of backbone atomic displacements throughout the lysozyme and alpha-lactalbumin superfamilies are comparable as well as their homologous tertiary structures.

Effects of gastrectomy on motility, perfusion pressure, and caerulein-induced relaxation of sphincter of Oddi in dogs.

The effects of subtotal-gastrectomy (gastrectomy) on the spontaneous motility and caerulein-induced relaxation of the sphincter of Oddi (SO) were investigated in the dog. The spontaneous motility and the response to caerulein of the SO were recorded using perfusion method. The basal perfusion pressure (5.1 +/- 0.5 cmH2O) and the frequency of phasic contractions (6.1 +/- 0.5 cycles/min, c/min) of the SO increased to 8.2 +/- 0.6 cmH2O (p < 0.05) and 9.3 +/- 0.4c/min (p < 0.05) after gastrectomy, respectively. They were observed one month after operation (7.8 +/- 0.5 cmH2O and 9.1 +/- 0.9 c/min, p < 0.05), but did not change by vagotomy with sympathectomy (vagosympathectomy). In the spontaneous motility of the SO, the motility index increased to 143.7 +/- 18.7% (p < 0.05) at 4 hrs and 135.0 +/- 9.1% (p < 0.05) at one month after gastrectomy, but did not increase after vagosympathectomy. Caerulein had an inhibitory effect on the SO motility in the normal animal 48.0 +/- 4.2%). Gastrectomy reversed to the excitatory effect from the inhibitory effect to caerulein at 4 hrs (127.6 +/- 5.3%, p < 0.05) and at one month (126.6 +/- 5.3%, p < 0.05) after operation, but not reversed by vagosympathectomy and sham gastrectomy. The excitatory response to caerulein after gastrectomy was not effected by vagosympathectomy. It is concluded that gastrectomy induced the SO dysfunction, an increase of the perfusion pressure and the frequency of phasic contractions of the SO, and a change of the response to caerulein of the SO. These alterations suggests that one of the mechanisms of the regulation of the SO motility exist as the reflex from the stomach and/or uppermost duodenum through intrinsic nervous pathways.

[Effects of gabexate mesilate (FOY) on the gallbladder, sphincter of Oddi and duodenum of the normal and gastrectomized dogs].

FOY induced a dose-dependent inhibitory response on the gallbladder, sphincter of Oddi and duodenum of normal and gastrectomized dogs, although it induced an excitatory response in some dogs. The inhibitory response was not reduced or terminated by pretreatment with atropine, guanethidine, hexamethonium or/and proglumide. The FOY-induced inhibitory response reversed to the excitatory response in the sphincter of Oddi and duodenum by pretreatment with tetrodotoxin, but not in the gallbladder. These results suggested that the FOY-induced inhibitory response of the sphincter of Oddi and duodenum was caused by stimulation of nonadrenergic noncholinergic inhibitory neurons, not by FOY-induced cholecystokinin secretion. The excitatory response was induced by direct stimulation of their smooth muscles. The inhibitory response of the gallbladder to FOY was induced by direct stimulation of the smooth muscles.

Metabolism of theanine, gamma-glutamylethylamide, in rats.

The metabolism of theanine, one of the major amino acid components in tea (Camellia sinensis), was studied in rats. High-performance liquid chromatography (HPLC) with fluorometric detection was used to evaluate the nature of theanine's metabolites in plasma, urine, and tissues. In the urine samples collected after administration of 100, 200, and 400 mg each of theanine, intact theanine, L-glutamic acid, and ethylamine, these compounds were detected in a dose-dependent manner. When 200 mg of theanine was orally administered to rats, the plasma concentrations of theanine and ethylamine reached their highest levels about 0.5 and 2 h after administration, respectively. It seems most likely that the enzymatic hydrolysis of theanine to glutamic acid and ethylamine was accomplished in the kidney. These results indicate that orally administered theanine is absorbed through the intestinal tract and hydrolyzed to glutamic acid and ethylamine in the rat kidney.

Isolated pancreatectomy for ductal carcinoma of the head of the pancreas.

Hepatic carcinoma recurrence in the early months after pancreatectomy for ductal carcinoma of the head of the pancreas is one of the major factors of the poor survival rate. Early hepatic recurrence might be caused by carcinoma cells entering the portal vein as a result of surgical manipulations. To deal with this problem, we devised a new operative procedure called "isolated pancreatectomy" in 1987. This method involves no-touch pancreatic resection combined with extensive retroperitoneal skeletonization. Both the feeding and draining vessels of the pancreatic carcinoma are ligated and divided prior to pancreatectomy. The confluence of the portal vein, the superior mesenteric vein, and the splenic vein is removed together with the pancreatic head. The wide surgical field thus obtained facilitates subsequent skeletonization of the retroperitoneum. We have experienced 71 cases of pancreatectomy for ductal carcinoma of the head of the pancreas among whom isolated pancreatectomy has been performed in 16 patients since 1987. In all cases, long-term survival was obtained in the patients undergoing curative and extended resection. The cumulative five-year survival rate for stages I and II was 40.7%, and that for stage III was 17.3%. After the introduction of isolated pancreatectomy, hepatic recurrence rates within six months and 12 months after pancreatectomy have dropped to 0% and 16.7%, respectively, from the 22.9% and 31.3% of the other pancreatectomy. The survival rate for the patients with stage III in isolated pancreatectomy significantly exceeded that obtained with other pancreatectomy.

Evaluation of preoperative and postoperative sodium and water loading in patients undergoing hepatectomy for liver cirrhosis complicated by hepatocellular carcinoma.

Evaluation of postoperative disturbance in the sodium and water balance was made in eight patients with compensated liver cirrhosis who had undergone segmental hepatectomy for hepatocellular carcinoma and had received unrestricted administration of sodium and water to maintain a normal urine output. On postoperative day 3, a significantly higher plasma atrial natriuretic peptide level and a significantly lower plasma aldosterone level were noted compared with postoperative day 1: the hormonal levels on postoperative day 3 were similar to the postinfusion levels obtained by the preoperative saline-loading test, which was performed to assess the physiological control of effective extracellular fluid and blood volume. Pulmonary artery and pulmonary wedge pressures were slightly, but significantly, higher on postoperative day 3 than on postoperative day 1. These results suggest that unrestricted fluid management prevents the depletion of effective extracellular fluid and blood volume on postoperative day 1, and permits their slight excess on postoperative day 3.

Interruption of hepatic arterial blood flow after resection of pancreaticobiliary carcinoma.

BACKGROUND/AIMS: We reviewed 17 cases of patients with interrupted hepatic arterial blood flow to determine the effect of such an interruption. MATERIALS AND METHODS: In 17 patients undergoing radical resection of pancreaticobiliary carcinoma with simultaneous hepatic artery excision (n=15) or intraoperative hepatic artery obstruction (n=2), morbidity and mortality were reviewed. Nine hepatic artery anastomoses were performed in 7 of these patients, and postoperative patency was obtained for 5 anastomoses. The patients were classified into the following groups: group I was 6 patients with complete interruption of hepatic arterial flow to the whole liver or the remnant liver, group II was 6 patients with interruption of hepatic arterial flow to a lobe of the liver, and group III was 5 patients with preservation of hepatic arterial flow. RESULTS: Disruption of the bilioenteric anastomosis occurred in all 6 patients from group I versus none of those in groups II and III (p<0.05). Liver abscess and liver failure developed in 1 patient each from group I, but the mortality rate in this group was not high. CONCLUSIONS: Hepatic artery reconstruction in patients with complete interruption of hepatic arterial blood flow appears to be necessary to avoid ischemic breakdown of the bilioenteric anastomosis.

Block resection of the hepatoduodenal ligament for carcinoma of the bile duct and gallbladder. Surgical technique and a report of 11 cases.

Carcinoma of the bile duct and gallbladder often infiltrates the entire hepatoduodenal ligament. Therefore radical resection should include block resection of the hepatoduodenal ligament. Over the last two years, block resection of the hepatoduodenal ligament for carcinoma of the bile duct and gallbladder was performed in 11 patients. When the carcinoma was located in the hilar bile duct, a combination of hemihepatectomy including the caudate lobe and ligamentetomy, "hepato-ligamentectomy", was performed (six cases). When the carcinoma was in the lower bile duct, a combination of pancreatico-duodenectomy and ligamentectomy, "ligamento-pancreatectomy", was performed (three cases). In two extremely advanced cases a combination of both hepatectomy and pancreatectomy with ligamentectomy, "hepato-ligamento-pancreatectomy", was performed. To accomplish these procedures safely, double catheter bypass of the portal circulation, devised by the authors in 1986, proved very effective in maintaining sufficient hepatic circulation and preventing portal congestion during block resection of the hepatoduodenal ligament. Histological evidence of invasion of the carcinoma cells into the hepatoduodenal ligament was detected in 10 cases, and in half of them the hepatic artery or portal vein was involved. As of April 1988, five cases in whom curative resection was performed are still alive, the longest survival period being 18 months. Four cases died in the early postoperative period, three of the deaths being due to sepsis and one to respirator malfunction.

Effects of insulin and glucagon on energy and carbohydrate metabolism of rat hepatocytes in primary culture.

We studied the effects of insulin and glucagon on energy and carbohydrate metabolism of rat hepatocytes in primary culture. The aim of this study is to elucidate the mechanism of the synergistic action of insulin and glucagon and to evaluate the combined effects of these hormones on liver injury. Insulin increased the level of adenosine triphosphate in hepatocytes in the presence of glucagon. Insulin increased the activities of glucokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40) type L and glucose 6-phosphate dehydrogenase (EC 1.1.1.49). Glucagon had no antagonistic effect on these increases. Glucagon increased the activity of glucose 6-phosphate (EC 3.1.3.9) (G6Pase) in the presence or absence of insulin, while insulin had no effects on the levels of G6Pase and fructose 1,6-bisphosphatase (EC 3.1.3.11) in the presence or absence of glucagon. Metabolite analysis of cultured hepatocytes indicated that insulin and glucagon have antagonistic effects on the glycolytic activity of hepatocytes. These combined effects of insulin and glucagon may partially explain the preventive effects of these hormones on liver injury.

Pancreaticogastrostomy: effect of partial gastrectomy on the pancreatic stump in rabbits.

To assess the influence of digestive juice on the pancreatic stump when pancreaticogastrostomy was performed after pancreatoduodenectomy, the pancreatic stump was anastomosed to the intact stomach (group I), the stomach after partial gastrectomy (group II), or the jejunum (group III) in rabbits, and the nature of the digestive juice at the anastomotic site as well as the histologic changes of the pancreatic tissue were investigated. The digestive juice was highly acidic in group I, slightly acidic in group II, and almost neutral in group III. Histological examination of the pancreatic stump revealed extensive coagulative necrosis and delayed replacement with granulation tissue in group I, while there was less prominent liquefactive necrosis and early replacement with granulation tissue in groups II and III. Intraperitoneal abscess formation around the anastomotic site and atrophic fibrosis of the pancreas (similar to the changes after pancreatic duct ligation) occurred in 27.8% and 46.2% of group I rabbits, respectively, but no such changes were detected in groups II and III (both P < 0.05). These results indicate that the highly acidic gastric juice had a widespread corrosive effect on the anastomosed pancreatic tissue, and that partial gastrectomy may be necessary to prevent anastomotic leakage and pancreatic duct obstruction after pancreaticogastrostomy.