Increase in histamine synthesis by liver macrophages in CCl4-injured mast cell-deficient W/Wv mice

This study set out to examine the possible role of liver macrophages in histamine synthesis in the injured liver. The effects of the hepatotoxins Escherichia coli lipopolysaccharide (LPS) and CCl4 on histamine synthesis in the liver of mice were evaluated. C3H/HeJ mice were resistant to LPS in including histidine decarboxylase (HDC) in the liver compared with C3H/HeN mice and mast cell-deficient W/Wv mice. However, C3H/HeJ mice did respond strongly to another hepatotoxin, CCl4, leading to a significant increase in HDC activity. CCl4 also caused a marked increase in HDC activity and histamine levels in the liver of W/Wv mice. In addition, injection of CCl4 produced a large increase in the activity of HDC in the spleen and lung of W/Wv mice. HDC activity was confined to the nonparenchymal cells, with parenchymal cells expressing essentially no HDC activity. The CCl4-induced increase in HDC activity was confined, at least in part, to the liver macrophages. These results indicate that the macrophages are responsible for the increase in HDC-dependent histamine production in the liver caused by the injection of hepatotoxins. The possible role of histamine in liver regeneration after injury is discussed.     

Histological effect of (R)-alpha-methylhistamine on ethanol damage in rat gastric mucosa: influence on mucus production

(R)-alpha-Methylhistamine, a selective agonist of histamine H3 receptors, prevents macroscopically visible gastric lesions by absolute ethanol in the rat. A further insight into its activity was the aim of our study. Rats were given saline or (R)-alpha-methylhistamine (100 mg/kg) intragastrically. After 30 min, absolute ethanol was given and gastric mucosa was sampled 60 min later. Histologic damage and intracellular and adherent mucus were quantified. Luminal surface and mucous cells were examined by scanning and transmission electron microscopy. (R)-alpha-Methylhistamine reduced the extent of lesions by ethanol from 96 to 18%. Surface mucous cells and mucous neck cells were increased in volume and number, packaging of intracellular mucus was modified, and the secretory processes were promoted by (R)-alpha-methylhistamine itself, although these modifications were mostly evident in stomachs subsequently exposed to ethanol. Adherent mucus layer thickness was increased by (R)-alpha-methylhistamine only after ethanol exposure. It is concluded that (R)-alpha-methylhistamine predisposes mucous cells to react to ethanol.     

Effects of configuration on the myocardial uptake of radioiodinated 3(R)-BMIPP and 3(S)-BMIPP in rats

Radioiodinated 3(R)-(+)- and 3(S)-(-)-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) were prepared and evaluated in rats to investigate the effects of absolute configuration of the 3(beta)-methyl group on myocardial uptake and release kinetics. METHODS: The 3(R)-(+)-BMIPP analog was synthesized by initial acylation of a thiophene template with the acid chloride of ethyl 3(R)-methylglutarate. 3(S)-(-)-BMIPP was obtained by separation from the mixture of diastereomeric amides prepared from reaction of the acid chloride of racemic BMIPP with the S-(-)-alpha-methylbenzylamine. The amide of synthetic 3(R)-BMIPP prepared from S-(-)-alpha-methylbenzylamine was identical to the chromatographically more polar isomer. Free acids were obtained by acid hydrolysis of the amides, fully characterized and then converted to the radioiodinated BMIPP isomers. RESULTS: Biodistribution studies in rats with the dual-labeled [(131)I]-3(S)-BMIPP/[(125)I]-3(R)-BMIPP mixture demonstrated greater myocardial uptake of 3(R)-BMIPP compared with the 3(S)-BMIPP isomer [60 min: 3(R)-BMIPP = 4.37 %ID/g; 3(S)-BMIPP = 3.44; p < 0.05; 180 min, 2.31 and 1.78 %ID/G, respectively, p < 0.01], although both isomers had similar myocardial washout curves (5-180 min). Percent ID/g values for other tissues which were examined (blood, lungs, thyroid) were similar. CONCLUSION: Higher myocardial uptake of the 3(R)-BMIPP isomer observed in these animal studies may suggest differences in carrier-mediated myocyte uptake of the two isomers. These studies suggest that [(123)I]-3(R)-BMIPP is a candidate for clinical evaluation and may show greater myocardial uptake than the 3(S)-BMIPP isomer and may thus require reduced injected dose.     

Effects of gestational exposure to a video display terminal-like magnetic field (20-kHz) on CBA/S mice

Possible adverse effects of magnetic fields (MFs) on reproduction have been an open question. To verify the embryo-lethal effect of pulsed MF of the type emitted by video display terminals (VDTs) reported previously in CBA/S mice, a developmental toxicity study was conducted in animals of the same origin. Mated CBA/S mice (80-86 pregnant animals per group) were exposed to a 20-kHz MF with sawtooth waveform continuously from gestational day 0-18. The flux density of the vertical MF was 15 microT peak-to-peak (150 mG). This field was previously reported to increase the number of resorptions in CBA/S mice. On gestational day 18, the dams were killed and blood and bone marrow samples were taken for hematology and micronuclei analysis, respectively. The number of corpora lutea was counted and the content of the uterus examined. There were no statistically significant differences in maternal or fetal body weights, number of corpora lutea, implantations, resorptions, dead and live fetuses, or external and skeletal malformations. MF did not alter the number of blood cells or cause micronuclei in bone marrow erythrocytes in the dams. The mean number of resorptions was slightly but not statistically significantly, higher in the MF group than in controls. The results do not indicate marked developmental, hematological, or clastogenic effects of 20-kHz MFs.     

Syntheses and beta-adrenergic binding affinities of (R)- and (S)-fluoronaphthyloxypropanolamines

Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485-fold, resulting in a specific activity for hydrogen evolution of 75.7 micromol/min/mg of protein at 25 degrees C, using reduced methyl viologen as an electron donor. The K(m) value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 55 kDa by SDS-PAGE, and 57 kDa by gel filtration. The optimum temperature and pH value for hydrogen evolution were 50 degrees C and 7.5, respectively. The partially purified hydrogenase catalyzed hydrogen evolution from ferredoxin that had been isolated from the same cells, but not from NADH or NADPH. The K(m) value for ferredoxin was 0.68 microM. The enzyme was extremely oxygen sensitive, losing over 95% of its activity upon exposure to air within minutes, even at 4 degrees C. Two peptide fragments were obtained from the hydrogenase protein digested enzymatically, and their amino acid sequences were determined. No significant homology was found to any other known sequences of hydrogenases.     

Effects of cytogenin on spontaneous arthritis in MRL/1 mice and on pristane-induced arthritis (PIA) in DBA/1J mice

Cytogenin, 8-hydroxy-3-hydroxymethyl-6-methoxyisocoumarin, has low cytotoxicity on murine and human tumour cells in vitro. It augments or suppresses phagocytosis of macrophages and lymphocyte proliferation. It has been reported that cytogenin has a potent inhibitory effect clinically on adjuvant arthritis in Lewis rats and on type II collagen-induced arthritis in DBA/1J mice. Our experimental findings provide evidence that cytogenin has beneficial effects on spontaneous polyarthritis in MRL/1 mice and pristane-induced arthritis in DBA/1J mice. The results suggest that the mode of the anti-arthritic action of cytogenin is different from those of NSAIDs; although the precise mode of action remains unclear, cytogenin may become an excellent therapeutic agent for rheumatoid arthritis.     

Effects of bacterial DNA on cytokine production by (NZB/NZW)F1 mice

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.     

Intra-individual comparison of 3(R)-BMIPP and 3(S)-BMIPP isomers in humans

The racemic 15-(p-iodophenyl)-3(R,S)-methylpentadecanoic acid (BMIPP) is currently used at several centers for myocardial metabolic imaging with SPECT. Recently, the 3(R)-BMIPP isomer showed a 20%-25% higher myocardial uptake and lower liver uptake than 3(S)-BMIPP in fasted rats. The aim of this study was to determine if these differences in myocardial and liver uptake also occur in humans. METHODS: Iodine-123-labeled 3(R)-BMIPP and 3(S)-BMIPP isomers were injected at rest, on two separate days, in six patients with stable coronary artery disease. Dual-head, whole-body scintigraphy was performed 20 min and 3 hr after injection. SPECT cardiac imaging was performed 60 min after injection. RESULTS: Myocardial activity averaged (% injected dose +/- s.d.) 3.15 +/- 0.49 versus 3.01 +/- 0.44 at 20 min (p = ns) and 2.64 +/- 0.38 versus 2.55 +/- 0.41 at 3 hr postinjection (p = ns) for the 3(R)-BMIPP and 3(S)-BMIPP isomers, respectively. Liver activity averaged 9.50 +/- 1.18 versus 9.44 +/- 0.66 at 20 min and 5.33 +/- 0.64 versus 5.43 +/- 0.66 at 3 hr, respectively (p = ns). SPECT showed no difference in the distribution of the two isomers between normal and infarcted myocardium. CONCLUSION: There is no significant difference in myocardial and liver distribution of the 3(R)-BMIPP and 3(S)-BMIPP isomers in humans.     

Reinitiation of spermatogonial mitotic differentiation in inactive old BDF1 mouse seminiferous tubules transplanted to W/Wv mouse testis

The seminiferous epithelia of old mice (33 mo of age) are composed of spermatogonia and Sertoli cells. Histochemical examination using the anti-c-kit monoclonal antibody demonstrated that the number of differentiating type A spermatogonia decreases with age. To elucidate the differential activity of old mouse spermatogonia, we transplanted extremely thin seminiferous epithelia of old BDF, mice into W/Wv mouse testes and examined whether or not they could reinitiate differentiation. Artificially cryptorchid mice were used as the control. At 2 wk after transplantation, spermatocytes and round spermatids were detected in transplanted seminiferous tubules of the control, whereas the most advanced spermatogenic cells in those of old mice were spermatocytes. At 4 wk after transplantation, although elongated spermatids were detected in transplanted tubules of the control, haploid cells (spermatids) were still undetectable in those derived from old mice. Thus, meiosis was never restored, although spermatogonia of old mice can reinitiate differentiation into spermatocytes under suitable testicular conditions. Since it has been reported in several mammalian species that age-related changes in the testicular microenvironment lead to the gerontal cessation of spermatogenesis, the present results suggest that both a defective extratubular environment and a defective intratubular environment may cause the cessation of spermatogenesis in old BDF, mice.     

Synthesis of optically active (R)- and (S)-tai-ding-an(TDA) and their anti-HSV activity evaluation]

Tai-Ding-An (3-phthalimido-2-oxo-n-butyraldehyde bisthiosemicarbazone, TDA) is an antiviral drug first synthesized in this institute. In order to clarify the difference between the two enantiomeric isomers of TDA, (R)- and (S)-TDA were synthesized from (R)- and (S)-alanine, respectively, via the following steps: fusing with phthalic anhydride gave 2-phthalimido alanine(2a or 2b). The resulting acid was reacted with thionyl chloride to offer the corresponding acid chloride(3a or 3b), which was treated with diazomethane to give the diazoketone(4a or 4b). Bromination of the ketone with hydrobromic acid gave the key intermediate 3-phthalimido-2-oxo-1-bromobutanone (5a or 5b). Compound 5a or 5b was oxidized with DMSO to give 6a or 6b, which was directly condensed with thiosemicarbazide to afford the desired (R)- or (S)-TDA. (R)-TDA, (S)-TDA and (RS)-TDA have been tested in cell culture for anti-Herpes simplex virus I (HSV-1) and HSV-2 activities by plaque reducing method. All of them showed inhibitory effects on HSV-1 and HSV-2 replication with IC50 of 0.0296 mmol.L-1, 0.0359 mmol.L-1 and 0.0418 mmol.L-1 for HSV-1 and 0.88 mmol.L-1, 1.04 mmol.L-1 and 1.06 mmol.L-1 for HSV-2. Not much difference was found among these compounds either on IC50 or on therapeutic indexes.     

Acoustic startle, prepulse inhibition, locomotion, and latent inhibition in the neuroleptic-responsive (NR) and neuroleptic-nonresponsive (NNR) lines of mice

To date, studies on the mitral flow convergence method have used rigid, circular, or slit orifices to represent the regurgitant orifice. In this study, explanted porcine mitral valves, with the entire mitral apparatus preserved, were mounted in an in vitro model to reproduce the three-dimensional regurgitant orifice geometry while permitting close control and measurement of the experimental conditions. This experimental setup permitted the evaluation of the hemispheric and hemielliptic formulas under realistic physiologic conditions. In this study, a heart rate of 70 beats/min was used with cardiac outputs between 1.5 and 6 L/min. Peak regurgitant flow rates ranged from 7 to 16 L/min (regurgitant jet velocities ranged from 2 to 5.5 m/sec); peak aortic flow rates ranged from 9 to 30 L/min. Four native mitral valves were used for these studies for a total of 28 stages. Although the hemielliptic modification has previously shown success in vitro and computationally, it has not been used clinically because of difficulty imaging the flow convergence region in three orthogonal planes. A curve-fitting algorithm was developed to extract the hemielliptic dimensions from two standard ultrasound views by rotating the transducer 90 degrees. Improved agreement was obtained between true and calculated flow rates by the hemielliptic formula (y = 1.02 x + 0.29; r = 0.91) compared with the hemispheric formula (y = 1.18 x - 2.2; r = 0.66). This method provides accurate results with a realistic three-dimensional regurgitant orifice geometry and has the capability of being incorporated as a function key on an ultrasound machine for clinical application.     

Inhibitory effect of tumor metastasis in mice by saponins, ginsenoside-Rb2, 20(R)- and 20(S)-ginsenoside-Rg3, of red ginseng

We examined the inhibitory effect of two saponin preparations from Red ginseng, 20(R)- and 20(S)-ginsenoside-Rg3, in comparison with that of ginsenoside-Rb2, on lung metastasis produced by two highly metastatic tumor cells, B16-BL6 melanoma and colon 26-M3.1 carcinoma, in syngeneic mice. In an in vitro analysis, both saponin preparations showed a significant inhibition of adhesion to fibronectin (FN) and laminin (LM) by B16-BL6 melanoma. Similarly, they significantly inhibited the invasion of B16-BL6 cells into the reconstituted basement membrane (Matrigel)/FN in a dose-dependent manner. In an experimental metastasis model using B16-BL6 melanoma, consecutive intravenous (i.v.) administrations of 100 micrograms/mouse of 20(R)- or 20(S)-ginsenoside-Rg3 1, 2, 3 and 4 d after tumor inoculation led to a significant decrease in lung metastasis. The inhibitory effect of i.v. administration of both ginseng saponins on the tumor metastasis of B16-BL6 melanoma was also recognized in a low dose of 10 micrograms/mouse. The oral administration (p.o.) of both saponins (100-1000 micrograms/mouse) induced a significant decrease in lung metastasis of B16-BL6 melanoma. Moreover, both ginseng saponins were effective in inhibiting of lung metastasis produced by colon 26-M3.1 carcinoma. When 20(R)- or 20(S)-ginsenoside-Rg3 was orally administered consecutively after tumor inoculation in a spontaneous metastasis model using B16-BL6 melanoma, both of them significantly inhibited lung metastasis. In the experiment involving neovasculization by tumor cells in vivo, both mice groups given each saponin preparation after tumor inoculation exhibited a significant decrease in the number of blood vessels oriented toward the tumor mass, with no repression of tumor size. These findings suggest that both ginseng saponins, 20(R)- and 20(S)-ginsenoside-Rg3, possess an ability to inhibit the lung metastasis of tumor cells, and the mechanism of their antimetastatic effect is related to inhibition of the adhesion and invasion of tumor cells, and also to anti-angiogenesis activity.     

Stereoselective effects of (R)- and (S)-zacopride on cognitive performance in a spatial navigation task in rats

For many disorders of the retinal pigment epithelium (RPE) for which there are no effective treatments, transplantation of RPE cells may provide a viable means of restoring function. Using a solvent casting technique, we have manufactured thin films of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) 75:25 and 50:50. Non-porous, flexible films with controlled thickness as thin as 12 +/- 3 microns and reproducible surface morphologies and flexural properties were produced. Fetal human RPE cells were found to attach to these substrates when cultured in vitro. The films made using this technique may provide a means of transplanting allogeneic RPE cells as a therapy for a number of ocular diseases related to RPE dysfunction.     

Stereochemistry of the in vitro and in vivo methylation of DNA by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and (R)- and (S)-N-nitroso-N-[2H1,3H]methyl-N-methylamine

Reaction of DNA with the carcinogens N-methyl-N-nitrosourea and N-nitroso-N,N-dimethylamine produces several methylated species including the premutagenic O6-methylguanine. The mechanism of methylation is believed to be through a methanediazonium ion. We have studied the mechanism of methylation of DNA by these carcinogens by analyzing the stereochemistry of the methyl transfer. DNA was methylated in vitro by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and in vivo by (R)- and (S)-N-[2H1,3H]methyl-N-methyl-N-nitrosamine and (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea. 7-Methylguanine, 3-methyladenine, O6-methylguanine, and the methylated phosphate backbone were isolated. The methyl groups were converted into acetic acid, and the stereochemistry was analyzed. The identity of the nucleophile did not influence the stereochemistry of the methylation reaction. It was found that the methyl group was transferred with an average of 73% inversion and 27% retention of configuration. The most likely mechanism for the retention of configuration is through multiple methylation events in which nucleophiles which initially react with the methanediazonium ion react as electrophiles with DNA.     

Prolonged release of tegafur from S/O/W multiple emulsion

To develop a prolonged and sustained release preparation, we prepared an albumin microsphere-in-oil-in-water emulsion (S/O/W) and examined sustained release from it in comparison with other control preparations such as water-in-oil (W/O) emulsions and microspheres in vitro and in vivo, respectively. Tegafur was used as a model drug. A microsphere-in-oil emulsion was prepared by adding albumin microspheres to soybean oil containing 20% Span 80. To prepare an S/O/W emulsion, the microsphere-in-oil emulsion was added into an aqueous solution of hydroxypropyl methylcellulose containing Pluronic F68. The mean particle size of the albumin microspheres was 3 microns, and the ratio of entrapment of tegafur into albumin microspheres was about 25%. In an in vitro release test, the t75 of the S/O/W emulsion was fourfold greater and in an in vivo release test the mean residence time of tegafur from the S/O/W emulsion was more than twofold that from a W/O emulsion or microsphere system. The mean residence time of 5-fluorouracil (5-FU) from an S/O/W emulsion was also greater than with other dosage forms. These results suggest the possible usefulness of an S/O/W emulsion for the sustained and prolonged release of tegafur.     

Chronic, low-level (1.0 W/kg) exposure of mice prone to mammary cancer to 2450 MHz microwaves

In a previous study (Frei et al., Bioelectromagnetics 19, 20-31, 1998), we showed that low-level (0.3 W/kg), long-term exposure of mice prone to mammary tumors to 2450 MHz radiofrequency (RF) radiation did not affect the incidence of mammary tumors, latency to tumor onset, tumor growth rate or animal survival when compared to sham-irradiated animals. In the current study, the specific absorption rate (SAR) was increased from 0.3 W/kg to 1.0 W/kg. The same biological end points were used. One hundred C3H/HeJ mice were exposed in circularly polarized waveguides for 78 weeks (20 h/day, 7 days/week) to continuous-wave, 2450 MHz RF radiation; 100 mice were sham-exposed. There was no significant difference between exposed and sham-exposed groups with respect to the incidence of palpated mammary tumors (sham-exposed = 30%; irradiated = 38%), latency to tumor onset (sham-exposed = 62.0 +/- 2.3 weeks; irradiated = 62.5 +/- 2.2 weeks) and rate of tumor growth. Histopathological evaluations revealed no significant difference in numbers of malignant, metastatic or benign neoplasms between the two groups. Thus long-term exposures of mice prone to mammary tumors to 2450 MHz RF radiation at SARs of 0.3 and 1.0 W/kg had no significant effects when compared to sham-irradiated animals.