Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids.

A new member of the UDP-N-acetylglucosamine:beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3Gn-T motifs was cloned from rat and human cDNA libraries and named beta3Gn-T5 based on its position in a phylogenetic tree. We concluded that beta3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc(3)Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto- or neolacto-series carbohydrate chains on glycolipids. beta3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc(4)Cer; paragloboside), resulting in the synthesis of Lc(3)Cer and neolactopentaosylceramide (nLc(5)Cer), respectively. A marked decrease in LacCer and increase in nLc(4)Cer was detected in Namalwa cells stably expressing beta3Gn-T5. This indicated that beta3Gn-T5 exerted activity to synthesize Lc(3)Cer and decrease LacCer, followed by conversion to nLc(4)Cer via endogenous galactosylation. The following four findings further supported that beta3Gn-T5 is Lc(3)Cer synthase. 1) The beta3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc(3)Cer synthase in those cells. 2) The beta3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The beta3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in beta3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc(3)Cer synthase activity reported previously.

Preoperative diagnosis of ileal lipoma by endoscopic ultrasonography probe.

The occurrence of tumor in the small intestine is relatively rare. It has been demonstrated that lipoma of the ileum is a cause of intussusception. We report a 59-year-old man admitted to our hospital for lower abdominal pain. Diagnosis of intussusception was made by abdominal x-ray and ultrasonography. Enema contrast studies revealed ileocolic intussusception. Colonoscopy revealed a tumor with an submucosal tumor (SMT)-like head and coil-spring appearance in the ascending colon. Endoscopic ultrasonography (EUS) revealed a hyperechoic submucosal lesion with features compatible with lipoma. Subsequently, this was confirmed histopathologically after resection. To our knowledge, this is the first report of preoperative diagnosis of ileal lipoma by EUS.

Antitumorigenic effects of several food proteins in a rat model with colon cancer and their reverse correlation with plasma bile acid concentration.

In order to obtain information on the preventive effects of various food proteins against colonic cancer, six groups of azoxymethane-initiated mature Fischer rats (n = 10) were fed respective diets different in protein sources such as bovine milk casein (casein), high-molecular-weight fraction from protolytic digest of soy protein isolate (soybean HMF), hen's yolk defatted protein (yolk protein), wheat gluten and codfish meat, which had been supplemented with sodium deoxycholate (hereinafter, DCA) as a cancer promoter except for an additional DCA-unfed casein group. All of the living rats at checkpoints during the feeding period were examined by the use of a bronchus fiberscope for colonic tumor incidence at 6 wk intervals between the 10th and 34th wk, from which both blood and feces samples were taken at times of endoscopy. Tumorigenesis in the colon was perceived by endoscopy at wk 22 in the group fed DCA casein only and at wk 28 in the other groups except the DCA-unfed casein group. At wk 34, both soybean HMF and yolk protein groups ranked inferior to the DCA-unfed group in tumor incidence. When plasma steroid or lipid concentration was plotted against tumor incidence at wk 28 or 34, positive correlations were found between plasma bile acid concentration and tumor incidence at both weeks. With the exception of the DCA-unfed casein group, plasma bile acid concentration was reversely correlated to fecal bile acid excretion. Taken altogether, these results suggest that bile acids at higher concentrations in the plasma may serve as risk factors of colon tumor incidence.

Electroacupuncture potentiates the antinociceptive effect of intrathecal endomorphin-1 in the rat formalin test.

Endomorphin-1 is a novel endogenous mu-opioid peptide. In this study, we examined the effects of 2 Hz electroacupuncture in the rat tail flick test and the formalin test (a persistent noxious model). Moreover, we investigated if the electroacupuncture potentiated the effect of intrathecal endomorphin-1. The results demonstrated that electroacupuncture alone produced a significant antinociception in the tail flick test, but not in the formalin test, and that intrathecal endomorphin-1 dose-dependently suppressed the biphasic nociceptive behavior in the formalin test. Electroacupuncture enhanced the antinociceptive effect of intrathecal endomorphin-1 in the formalin test, resulting in a significant leftward shift in the dose-response curves for intrathecal endomorphin-1 antinociception. The enhanced effect was antagonized by intraperitoneal naltrexone. The study suggests that electroacupuncture may potentiate the intrathecal endomorphin-1 antinociception partially mediated by opioid receptors.

Soybean curd refuse alleviates experimental tumorigenesis in rat colon.

Adult Fischer-344 rats which underwent administration of azoxymethane were fed diets containing soybean curd refuse (SCR) or a high-molecular-weight fraction of soy protein digest (HMF), or Hammarsten casein (CAS) as a protein source over a period of 34 weeks. All the living rats of each group at 22, 28 or 34 weeks were endoscopically inspected for tumor incidence in the colon. SCR turned out to be comparable to HMF in anti-tumorigenicity, or rather better than HMF.

[An autopsy case of interstitial pneumonia probably induced by Sho-saiko-to].

A 66-year-old woman had been treated for 3 years by her local physician with Sho-saiko-to for chronic hepatitis C virus (HCV) infection and liver cirrhosis. She was admitted to our hospital because of cough, fever, and infiltrative shadows on chest x-ray films. Sho-saiko-to-induced pneumonitis was diagnosed and steroid therapy started. Though a temporary improvement was observed, interstitial pneumonitis relapsed and the patient died of respiratory failure and liver dysfunction. Autopsy findings showed diffuse alveolar damage and honeycombing. Furthermore, reverse-transcriptase polymerase chain reaction techniques detected HCV-RNA in specimens of fibrotic lung tissue. For comparison, HCV-RNA was not histologically detected in lung tissue specimens from 4 control subjects who were positive for HCV antibodies but who did not have interstitial lung disease. It was speculated that the progression of interstitial pneumonia in the present case may have been caused by HCV in combination with Sho-saiko-to-induced lung injury.

1alpha,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) hydroxylates the carbon at the end of the side chain (C-26) of the C-24-fluorinated analog of 1alpha,25-dihydroxyvitamin D3.

The sequential oxidation and cleavage of the side chain of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha,25(OH)2D3, 24, 24-difluoro-1alpha,25-dihydroxyvitamin D3 (F2-1alpha,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha,25(OH)2D3-supplemented rats were incubated with F2-1alpha,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha,25,26-trihydroxyvitamin D3 (F2-1alpha,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha,25,26(OH)3D3. Metabolism of F2-1alpha,25(OH)2D3 into F2-1alpha,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha,25(OH)2D3 into rats. The C-24 oxidation of 1alpha,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha,25(OH)2D3 but also at C-26 of F2-1alpha,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.

[Potentiation of procaine-induced local sensory block by verapamil in rats].

We have studied the effects of calcium-channel blocker, verapamil, on procaine-induced local sensory block. Standardized tail-flick (TF) test was used to investigate the duration and intensity of procaine-induced local conduction block in rats. After obtaining baseline TF latencies (mean; 3.3 sec), two 100 microliters of 0.4% procaine alone, a combination of 0.4% procaine and verapamil (100 micrograms, or 200 micrograms), or a large dose of verapamil (200 micrograms) were injected to the opposite sites of the tail base and TF test was performed every five minutes for 45 minutes. A large dose of verapamil showed no prolongation of TF latencies. The administration of 0.4% procaine alone produced a significant increase of TF latencies and the peak effect of % MPE (percent maximum possible effect) was demonstrated at 4 minutes after the drug injection (mean % MPE; 37.0%). Coadministration of 0.4% procaine and two doses of verapamil produced significant increases of % MPE in a dose-dependent fashion. It was concluded that sensory block by procaine of the peripheral nerves is potentiated by coadministration of calcium-channel blocker, verapamil.

Potentiation of local lignocaine-induced sensory block by calcium channel blockers in rats.

We have studied the effects of three different types of calcium channel blockers (verapamil, diltiazem, and nicardipine) on local lignocaine sensory block. The standardized tail flick test was used to measure the duration and degree of lignocaine-induced conduction block in rats. After obtaining baseline tail flick latencies (mean 3.2 s), two 100-microliter doses of 0.3% lignocaine alone, a combination of verapamil 25, 100 or 200 micrograms, diltiazem 25, 100 or 200 micrograms, or nicardipine 0.5, 1.0 or 2.0 micrograms, and a large dose of calcium channel blockers (verapamil 200 micrograms, diltiazem 200 micrograms or nicardipine 2.0 micrograms) were injected on opposite sites of the tail base and the tail flick test was performed every 5 min for 45 min. A large dose of the calcium channel blockers showed no prolongation of tail flick latencies. Administration of 0.3% lignocaine alone produced a significant increase in tail flick thresholds and the peak effect of the percentage maximum possible effect (% MPE) was demonstrated at 5 min after drug injection (mean % MPE 28.8%; P < 0.01 vs baseline). Co-administration of 0.3% lignocaine and three doses of verapamil produced significant increases in area under the curve (AUC) in a dose-dependent fashion. Mean AUC values for 0.3% lignocaine alone and a combination of verapamil 25, 100 or 200 micrograms were 217.5, 502.5, 529.1 and 1600.3, respectively. Almost similar patterns of augmentation in AUC values were demonstrated after addition of different doses of diltiazem or nicardipine to 0.3% lignocaine. We conclude that the use of mixtures of local anaesthetic and calcium channel blocker potentiated lignocaine sensory block at the level of the peripheral nerves.

Comparison of atherogenicity of soybean oil and peanut oil, and effect of clentiazem on diet-induced atherosclerosis in rabbits.

Rabbits were fed with two kinds of atherogenic diet, one containing 0.5% cholesterol and 3% soybean oil and the other 0.5% cholesterol and 6% peanut oil, for three months to compare the atherogenic property of the diets. The soybean oil diet seemed to be superior to the peanut oil diet for evaluation of the anti-atherogenic effect of drugs, because the former caused milder vascular lesions than the latter. Using this rabbit model for atherosclerosis, the anti-atherogenic effect of clentiazem, a new calcium antagonist, was examined. Clentiazem at an oral dose of 30 mg/kg/day significantly reduced the size of atheromatous lesion in the aortic arch and thoracic aorta, and lowered the collagen content of the aortic intima and media, although it did not decrease serum lipid levels. On the other hand, clentiazem showed no clear effect on reducing the coronary atherosclerotic lesions. These results suggest that clentiazem may inhibit the progression of diet-induced aortic atherosclerosis without normalizing the serum lipid levels.

[A case of recurrent hepatocellular carcinoma after hepatic resection surviving over five years by hepatic arterial infusion of lipiodol-anticancer drug suspension].

A 63-year-old male with four intrahepatic recurrences of surgically resected hepatocellular carcinoma was admitted to our hospital in June 1985. He underwent lateral segmentectomy of the liver in November 1983. Pathologic finding of Edmondson II with liver cirrhosis had been confirmed by the operative specimen. Sizes of four recurrent tumors were assessed by CT as 3.5 x 2.2 cm, 2.6 x 2.2 cm, 2.2 x 2.2 cm and 2.2 x 2.2 cm, respectively. During five years until July 1990, the patient was treated with hepatic arterial infusion of Lipiodol-anticancer drug suspension eight times (total 5-FU 900 mg, ADM 77 mg, MMC 73 mg, and Lipiodol 36 ml) and hepatic arterial chemoembolization of MMC microcapsules one time. In addition, two hepatic arterial infusions of CDDP (total 70 mg) were given and 5-FU (total 10 g) was administered intravenously. Partial response (PR) was obtained for 19 months. Hepatic arterial infusion of Lipiodol-anticancer drug suspension was given only once every 6 months, and he maintained a good quality of life for over four and half years. The man died in July 1990. In general, multiple intrahepatic recurrence of surgical resected hepatocellular carcinoma has a poor prognosis. Therefore it was considered that hepatic arterial infusion of this drug brought about the relatively long survival of more than five years.

Successful treatment of a hyperprolactinemic infertile woman with a pituitary microadenoma using hachimijiogan.

A hyperprolactinemic infertile woman with a pituitary microadenoma, who was resistant to bromocriptine as a method of bringing on pregnancy, succeeded in having a normal pregnancy and delivery with Hachimi-jio-gan treatment. This paper reports that the patient did not succeed in pregnancy in spite of 16 months of bromocriptine treatment, though her prolactin level gradually decreased and continued to be between 40-60 ng/ml with Hachimi-jio-gan treatment. Although the level of prolactin of the patient did not normalize, she, nevertheless, succeeded in having a normal pregnancy and delivery.

Possible mechanism of vasogenic brain oedema induced by arachidonic acid.

Free arachidonic acid was infused into normal rat brains and the effect of arachidonic acid on capillary permeability was investigated by measuring the regional uptake of 14C-aminoisobutyric acid with a quantitative autoradiographic method. Arachidonic acid increased capillary permeability in a dose-dependent manner up to 2 mM. A high dose of arachidonic acid (greater than 5 mM) produced a profound tissue destruction around the needle track and less increased capillary permeability than 2 mM arachidonic acid. Time-course study disclosed that arachidonic acid markedly increased capillary permeability within 2 hours after infusion, and continued to increase with time to 24 hours. The effect of 48 hours infusion was about a half of that at 24 hours, indicating that the effect of arachidonic acid was partially reversible. Pretreatment with dexamethasone significantly inhibited the arachidonic acid-induced increase in capillary permeability and the administration of actinomycin D 1 hour before the pretreatment with dexamethasone suppressed the inhibitory effect of dexamethasone. These results suggest that arachidonic acid, which is deposited in the extracellular space, increases brain capillary permeability by two different ways. One is the direct detergent effect of arachidonic acid, and the other is the effect of arachidonic acid that is released from the membrane by the activation of phospholipase A2.

Cytocidal effects of hematoporphyrin derivative and argon dye laser on human gynecologic tumor cells in vitro.

Antitumor effects of hematoporphyrin derivative (HpD) plus argon dye laser were examined using various human gynecologic tumor cells in vitro. Irradiation alone with argon dye laser showed no effect on the DNA synthesis of cells. Treatment of the cells with HpD inhibited the DNA synthesis depending on the concentration and the exposure time. Photoradiation by argon dye laser following treatment of the cells with HpD 40 micrograms/ml for 2 h killed more than 80% of cells when the cells were irradiated for more than 3 min. Prominent degenerative changes of the cytoplasm and the nucleus appeared within 1 h after photoradiation. These changes were confirmed by morphology and DNA histogram. There were no differences of sensitivity to photoradiation among the three histologically different kinds of cells lines used.

[The effect of an arginine-imbalanced diet on the growth of implanted MM-48 and MH-134 tumors in C3H/He mice].

The arginine-imbalanced diet used in this study was a diet of 27% casein supplemented with 8% arginine monochloride. There was no difference in the serum protein level between C3H/He mice fed a casein diet (N-mice) and mice fed on the arginine-supplemented diet (A-mice). The serum arginine level was increased in the A-mice compared with the N-mice. Significant inhibition of MM-48 tumor incidence was seen in the A-mice when the transplanted tumor was below 5 X 10(6) cells. Growth inhibitory effect against MM-48 tumor was significantly enhanced in the A-mice with i.p. injection of OK-432. Interferon production of spleen cells was increased in the A-mice compared with the N-mice. We speculate that the effect of arginine depends partially on the immune functions in the mice.

Growth physiology of red-tide microorganisms.

The occurrence of red tides, which often cause mass mortality of marine animals and/or shellfish poisoning in humans, is increasing in the estuaries and coastal zones of the world. Species of dinoflagellates are most commonly responsible, though other flagellates, and even ciliates, can also cause red tides. In recent years, understanding of the growth physiology of red-tide microalgae has grown. Each causative organism has a species-specific preference and requirement for temperature, salinity, pH, the basic nutrients, and growth factors, and the toxin formation is affected by these environmental factors.