Characteristics of a human cell line successively transformed by Rous sarcoma virus and simian virus 40.

Human enbryo cells were successively transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) and simian virus 40 (SV40) in vitro, and the double transformant HuE 13 RS was established. From this cell line the two clonal cell lines RSa and RSb were isolated. In both, presence of SV40 T antigens was demonstrated by the fluorescent antibody technique, and the presence of RSV genomes was verified in one RSb clone by focus formation after fusion with chick embryo cells. Growth of these cells was affected by dibutyryl cAMP without marked morphologic changes. Cells were extremely sensitive to the anticellular action of human leukocyte interferon.

Purification and characterization of a novel metal-containing nonheme bromoperoxidase from Pseudomonas putida.

A novel bromoperoxidase was purified to homogeneity from the bacterium Pseudomonas putida IF-3 strain, which produces the antibiotic pyrrolnitrin. The enzyme had a molecular mass of 68,000 and was composed of two identical subunits (33,000). It was specific for I- and Br- and inactive toward Cl- and F- in the monochlorodimedone assay system. The optimum pH of the enzyme was around 4.2 and it rapidly lost its activity below 3.5, but it was stable over of range pH of 4 to 11. The purified enzyme was activated several fold by incubation with only cobalt ions, and did not contain an organic prosthetic group such as heme, flavin and cobalamin. Analyses of prosthetic metal compounds in the enzyme using plasma atomic emission spectroscopy (ICP-AES) combined with mass-spectroscopy (MS) and trace metal determination by high performance liquid chromatography (HPLC)-spectrometry, revealed that the enzyme contained 0.35 +/- 0.1 mol of cobalt ions, 1.0 +/- 0.2 mol of nickel ions, 0.8 +/- 0.2 mol of zinc ions and 2.0 +/- 0.2 mol of ferric iron per mol of enzyme, assuming the molecular weight of 68,000. There was no trace of vanadium in the enzyme, unlike in some nonheme haloperoxidases. Thus the bromoperoxidase of P. putida is a novel nonheme metal-containing bromoperoxidase.

A variety of catalases and bromoperoxidases in genus Pseudomonas and their characterization.

A survey of bromoperoxidase in some Pseudomonas strains revealed that they contain different types of bromoperoxidase, catalase-bromoperoxidase and catalase. Although all Pseudomonas strains exhibited catalase activity, the enzyme isolated from P. pyrrolnitrica was named catalase-bromoperoxidase, because it catalyzed not only a catalase reaction, but also the bromination of monochlorodimedone. Except heme-type catalase-bromoperoxidase, this strain contained four isoenzymes of general nonheme bromoperoxidase, and their molecular weights were about 73,000. On the other hand, P. putida and P. aeruginosa had the usual heme-type catalase, but they differed in molecular weight and pI value. Both strains also had a nonheme bromoperoxidase which catalyzed the bromination of monochlorodimedone and aniline, and the molecular weight of each enzyme was 68,000 for P. putida and 86,000 for P. aeruginosa. Considering the results reported by Van Pée et al. [1] and Weisner et al. [2], regarding the haloperoxidases of Pseudomonas, the genus was revealed to contain a wide variety of bromoperoxidase and catalase.

Activities of enzymes in some types of peripheral leucocytes may reflect the differences in nutrient metabolism between dogs and cats.

Plasma metabolites and immunoreactive insulin (IRI) concentrations and enzyme activities of some types of peripheral leucocytes were measured to clarify one aspect of the differences in nutrient metabolism between dogs and cats. There were no significant differences in plasma concentrations of glucose, triglyceride, free fatty acids and IRI between dogs and cats. Higher total cholesterol and lower HDL cholesterol concentrations were observed in feline plasma, and H/T ratio (HDL/total cholesterol concentrations) was significantly lower than that in canine plasma. The cytosolic activities of fructokinase (FK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) were significantly higher and the activities of cytosolic malate dehydrogenase (MDH) and mitochondrial glutamate dehydrogenase (GLDH) were significantly lower in feline leucocytes than those in canine leucocytes. Higher activities of FK, PK and G6PD, which regulate the rate of biosynthesis of fatty acids, may reflect the different characteristics in nutrient metabolism in feline tissues from canine tissues.

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studies using [gamma-32P] ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppressed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.

Differentiation of HL-60 cells is promoted by H-toxin of Clostridium septicum.

H-toxin of Clostridium septicum potentiated dimethyl sulfoxide (DMSO)-induced differentiation of human promyelocytic leukemia HL-60 cells which were monitored by nuclear morphology and production of oxidative radicals. But, H-toxin did not induce differentiation of HL-60 cells in the absence of DMSO. These phenomena were not observed by staphylococcal leukocidin, a cytotoxin affecting to HL-60 cells. In HL-60 cells, ADP-ribosylation of 118, 93, 75 and 58 kDa membrane proteins was observed, but the ADP-ribosylation was not detected either in differentiated HL-60 cells by DMSO or in normal polymorphonuclear leukocytes of human. When the membranes of HL-60 cells were incubated with H-toxin, ADP-ribosylation of the membrane proteins was inhibited. Such suppressive effects on ADP-ribosylation were not observed by DMSO or staphylococcal leukocidin. These data suggest that inhibition of the ADP-ribosylation by H-toxin may play an important role in potentiation of DMSO-induced differentiation of HL-60 cells.

CTP-dependent endogenous ADP-ribosylation of a 38 kDa protein in HL-60 cell membranes.

Incubation of membranes of human promyelocytic leukemia HL-60 cells with [32P]NAD led to ADP-ribosylation of several proteins including a 38 kDa protein by endogenous ADP-ribosyltransferases. The ADP-ribosylation of the 38 kDa protein was distinctly different from others on the basis of pH dependency and heat stability at 50 degrees C, suggesting that there are at least two endogenous ADP-ribosyltransferases. It was enhanced by CTP, but not affected by ATP, GTP and UTP, whereas it was inhibited by GTP gamma S. [alpha-32P]CTP bound to the 38 kDa protein immobilized on a nitrocellulose sheet, indicating that the 38 kDa protein which bound CTP is strongly ADP-ribosylated by an endogenous ADP-ribosyltransferase.

Animal model of para-aortic lymph node metastasis.

The purpose of this study was to establish a model of experimental lymph node metastasis by intra-rectal implantation of human cancer cells in nude mice. Four types of human cancer cell lines (TE-1, MKN-45, HT-29, and MIAPaca-2) were investigated. Tumor cells suspended in Matrigel were injected into the submucosal layer of the rectum. All cancer cell lines produced locally aggressive rectal tumors and, subsequently, para-aortic lymph node metastasis. We were unable to produce other distant metastases in the dying state in such locations as the liver, spleen, lung, and peritoneum. However, using this method, we were able to evaluate the effect of the anti-cancer agent uracil/tegafur (UFT) on primary tumor growth and lymph node metastasis. Oral intake of UFT significantly suppressed implanted tumor volume and inhibited lymph node metastasis. We expect that the process of lymph node metastasis shown in this model will be studied as an experimental model of lymph node metastasis simulating human cancers.

A novel model for invasion of cancer cells using the submucosal layer of the human stomach.

An in vitro model for studying the invasion mechanism of cancer cells was established using the submucosal layer of the human stomach. Submucosa prepared from a surgical specimen was maintained in an organ culture. The cytoarchitecture of the cultured submucosa remained well preserved; viability remained for over 2 weeks. When human gastric cancer cell lines MKN45, MKN74, and Kato III were seeded onto the submucosal slices, cancer cells of MKN45 and KATO III invaded the submucosa 3 days after inoculation. However, MKN74 cells were not seen in the submucosal slices. Our invasion model, which mimics the in vivo conditions of the submucosa of human stomach, may make it possible to analyze actual events of human gastrointestinal malignant cell invasion in normal submucosa in vitro. The usefulness of our invasion model lies in the choice of the submucosal layer of the human stomach as the host tissue. The histarchitecture of the submucosal slices indicates that the model has potential for studies of the mechanism of interactions between carcinoma cells and host tissue similar to interactions that may occur in vivo. Moreover, this method allows the continuous microscopic observation of cells within the living submucosa. Using this model, a novel approach to controlling the local invasion of tumor cells may lead to a promising, radical cure for these intractable neoplasms. Our model system is an in vitro model that is facile, inexpensive, and experimentally manipulative.

Molecular analysis of the h-warts/LATS1 gene in human breast cancer.

Loss of heterozygosity (LOH) on chromosome 6q is often observed in breast cancer, suggesting the existence of a putative tumor suppressor. Recently, a human homolog of the Drosophila warts tumor suppressor gene, h-warts/LATS1, was identified and mapped at chromosome 6q24-25.1. Mutation analysis of the h-warts/LATS1 was performed using 25 breast cancer tissues by RT-PCR SSCP analysis. Although LOH of the h-warts/LATS1 was found in one patient, no mutations were found. Two polymorphisms were found, but neither of them caused amino acid substitutions. Further investigations are necessary to elucidate the role of the h-warts/LATS1 gene in the carcinogenesis of breast cancer.

Survival for 145 days with a total artificial heart.

A calf into which a biolized, total artificial heart (TAH) had been implanted survived for 145 days. All measured physiological parameters except central venous pressure (CVP) were back to normal one month after implantation, and thereafter the animal's physiological development was similar to that of a normal calf. The intimal weight, which was 96 kilograms at implantation, reached 190 kilogram at the end of experiment, with a daily gain rate of 0.9 kilogram per day. After the nineteenth postoperative week, signs of congestive heart failure appeared, such as high venous pressure, ascites, and enlarged liver although the calf outwardly appeared well. On postoperative day 146, the animal started foaming at the mouth, and a convulsion occurred; then, the experiment was terminated after 3,494 hours of pumping. At autopsy, there were acute bilateral bronchopneumonia involving mostly both upper lobes, pulmonary edema, slight chronic pneumonitis, and hepatomegaly. There were no serious thrombotic deposits inside the cardiac prosthesis.

Effects of concanavalin A on the antiviral and cell growth inhibitory action of human interferons.

Sensitivities of human transformed cell line RSa and its variant cell line IFr to the cytotoxicity of Con A were compared. IFr cells were more resistant than RSa to Con A. Con A-resistant cell lines, Con Ar-1 and Con Ar-3, were isolated from RSa, and they were slightly more sensitive than RSa cells to the cell growth-inhibitory actions of interferons. Agglutinability of RSa, IFr, and Con Ar cells by Con A was compared and found to be almost equal. The combined effects of Con A and interferon upon growth and viability of these cell lines were tested. When RSa and IFr cells were treated simultaneously with Con A and Le-IF, growth of the cells was suppressed more markedly than when treatment was with Con A or Le-IF alone. To clarify the mechanism of this phenomenon, binding of 125I-labeled Con A was examined. Though ther wee some differences, both leukocyte and fibroblast interferon enhanced the binding of Con A to RSa cells and also in Con Ar cells but, in interferon-resistant IFr and HEC cells, enhancement of Con A binding was low or not observed. Therefore, the combined effect of Con A and interferon on the inhibition of cell growth is not considered to be merely due to the enhanced binding of Con A by interferon action. Successive treatment of RSa or Con Ar cells with Con A and interferon did not enhance the antiviral action of interferon at all. On the contrary, simultaneous treatment with Con A and interferon suppressed the antiviral action of interferon, depending on the concentration of Con A used. Thus, the effect of Con A on the antiviral and cell growth-inhibitory actin of interferon seems rather different.

The contribution of endogenous mono-ADP-ribosylation to kindling-induced epileptogenesis.

We examined the alteration of endogenous mono ADP-ribosylation in the hippocampus of amygdaloid kindled rats to clarify the neurochemical basis of epilepsy. A significant increase of the ADP-ribosylation on the 38 kDa protein was observed in the hippocampal membrane of the kindled rat. Several antiepileptics (phenytoin, phenobarbital, carbamazepine, sodium valproate) significantly decreased the ADP-ribosylation on the 38 kDa protein and effaced the increase in the kindled group. The ADP-ribosylation was largely increased by sodium nitroprusside, a nitric oxide generating compound, in both the kindled and control groups. Carbamazepine could not affect the ADP-ribosylation in the presence of sodium nitroprusside. Twenty amino acids from the N-terminus of the ADP-ribosylated 38 kDa protein were determined by sequential analysis. The sequence was completely identical to that of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that the endogenous mono-ADP-ribosylation which increased in the kindled group and decreased by the antiepileptics might be a specific reaction associated with the mechanisms of epileptogenesis.

The binding of Vibrio parahaemolyticus 125I-labeled thermostable directhemolysin to erythrocytes.

Thermostable direct hemolysin (TDH) produced by Vibrio parahaemolyticus was iodinated using chloramine T. The 125I-labeled TDH retained up to 80% of the activity of intact toxin. The binding of 125I-TDH to rabbit erythrocytes was inhibited by addition of nonlabeled TDH. The binding of 125I-TDH to rabbit erythrocytes was completed in the 1st or 2nd min of incubation at 37 degrees C in contrast to that at 4 degrees C. 125I-TDH, which cannot lyse horse erythrocytes as does intact TDH, bound to horse erythrocytes as to those of rabbit. The dissociation constants (KD) derived Scatchard plots were 2.85, 4.39, 4.33 and 5.35 x 10-8M for rabbit, horse, human and sheep erythrocytes, respectively. The lytic sensitivity of various erythrocytes to TDH showed no relationship to the binding capacity.

Absence of mutatins in the analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic humangastric cancer using genomic DNA and intron primers.

Mutations in the transforming growth factor beta type II receptor (TGFbetaRII) gene have been detected in several human cancers that represent the phenotype of genomic instability. To establish a basis for diagnosis of cancer patients, we previously determined the exon-intron organization of the TGFbetaRII gene. The results indicated that TGFbetaRII protein is encoded by 567 codons in 7 exons. In this study, we further determined the nucleotide sequences surrounding these 7 exons and designed 8 sets of intron-based primers to examine the entire coding region of the TGFbetaRII gene. By using these primers, we screened for mutations of the TGFbetaRII gene in DNAs of 32 sporadic gastric cancer patients in whom one case showed MI+ (3.1%) at two loci. We found no mutations, and these data support other recent evidence that TGFbetaRII mutations rarely occur except in colon and gastric tumors with MI.

Efficacy of immunochemotherapy with Ftorafur and Krestin in rats.

The effectiveness of combined administration of Ftorafur (FT) and Krestin (PSK) on experimentally-induced liver cancer has not been established. This study was undertaken to elucidate the effect of combined administration of these drugs on tumor growth and temporal changes in the immuno-endocrine system under this immunochemotherapy. Male inbred WKA/H strain rats were used. The drugs used were FT and PSK, each dissolved in water and fed orally. The drugs were administered separately but concomitantly in standardized cycles to the tumor-bearing animals. KDH-8 ascitic liver cancer cells were subcutaneously transplanted into WKA rats. The tumor growth inhibition rate of FT and PSK was then determined. Twenty-one days after subcutaneous transplantation, tumor growth in the combined administration transplantation, tumor growth in the combined administration group was significantly inhibited, compared to the control group (p < 0.001). At fourteen days, plasma ACTH levels of the FT + PSK combined group were significantly lower than those of the control group (p < 0.001).

[Fifty-two-week oral chronic toxicity study of propiverine hydrochloride in rats].

An oral chronic toxicity study of propiverine hydrochloride (P-4), a new anti-pollakiuria agent, was carried out at dose levels of 0 (control), 0.5, 5 and 50 mg/kg/day using male and female rats. They were treated for 52 weeks, followed by 5 weeks recovery period. The results obtained from the present study were as follows. 1. There were no deaths related to P-4. Mydriasis, transitory salivation were observed in both sexes receiving 50 mg/kg/day, and soil of the abdomen was also noted in females receiving 50 mg/kg/day. 2. Body weight gain was suppressed from initiation of administration in both sexes receiving 50 mg/kg/day. 3. There were no significant or remarkable changes in food consumption, hematology and ophthalmology. 4. Urinary findings in animals receiving 50 mg/kg/day showed increases of urine and potassium excretion volumes and decrease of urine osmotic pressure in both sexes, negativity of urine protein and decrease of urobilinogen value in females. 5. Biochemical findings in animals receiving 50 mg/kg/day showed increase of urea-nitrogen (Urea N) in both sexes and decrease of triglyceride (TG), total cholesterol (T. cho), free cholesterol (F. cho), non-esterified fatty acids (NEFA) and phospholipid (PL) in males. 6. The absolute and/or relative weights of the liver increased in animals receiving 50 mg/kg/day. Histopathological examination in animals receiving 50 mg/kg/day revealed intranuclear eosinophilic inclusions and cytoplasmic eosinophilic substance in renal proximal tubular epithelium and midzonal lipid droplets in liver. Histochemical examination in animals receiving 50 mg/kg/day revealed the slight increase of gamma-GTP positive area in peripheral zone of liver. Electron-microscopic examination in animals receiving 50 mg/kg/day revealed intranuclear and intracellular large and homogeneous spherical-like structure with low electron density in renal proximal tubular epithelium, and slight hyperplasia of smooth endoplasmic reticulum with dilatation of cisternae and deposition of large lipid droplets in hepatocytes, but there was no difference of VLDL and its distributions in hepatocytes among groups.(ABSTRACT TRUNCATED AT 400 WORDS)

[A case report of long-term post-thoracotomy pain management with intrapleural bupivacaine].

A 50-year old woman with right post-thoracotomy pain was referred to us for assistance with pain control. She required pentazocine 60-150 mg per day before our treatment. First, we treated her with intercostal nerve block or oral morphine sulfate. But the result was not satisfactory after five months. Then we tried intrapleural bupivacaine. An epidural catheter was inserted into the pleural space from eight intercostal space at the anterior axillary line and 10 ml of 0.5% bupivacaine was instilled. The treatment was effective for about 4-5 hours. We continued this method for 42 days with 10 ml of 0.25% or 0.5% bupivacaine once or twice a day. She felt so good from the intrapleural analgesia and could be discharged. There was no hypotension, respiratory depression, urinary retention except burning thoracic sensation. We think it is possible to use this intrapleural bupivacaine to treat a certain kind of unilateral chronic pain.

[Utilization of D-histidine by the derivative strain TA100 of Salmonella typhimurium LT 2].

In general, wild-type gram-negative enteric bacteria are not able to utilize D-amino acids as the precursors of respective L-amino acids. We found, however, that an L-histidine auxotroph mutant, TA100, derived from Salmonella typhimurium strain LT 2 and used in the Ames test, showed a biphasic growth curve in the presence of both L- and D-histidine at concentrations of 5 micrograms/ml and 100 micrograms/ml, respectively. L-histidine may be utilized preferentially and then, after a short lag, D-histidine may be utilized. The short lag time is therefore considered to be the time required for induction of such an enzyme that converts D-histidine to L-histidine and for uptake of D-histidine by the bacterial cells.