Mosaicism in the expression of tumor-associated carbohydrate antigens in human colonic and gastric cancers.

Serial sequential sections from a single tumor were examined by immunohistological staining with several monoclonal antibodies directed, respectively, to different tumor-associated carbohydrate epitopes. Staining patterns were compared with those of conventional staining with hematoxylin-eosin or periodate/Schiff's reagent. Each tumor showed different areas of staining with different antibodies, and the combined staining map shows a clear mosaicism of antigen expression within the same tumor. For example, some areas of a given tumor were stained by FH4 (defining dimeric Le(x)), while other complementary areas were strongly stained, in a mutually exclusive manner, by SH1 (defining Le(x)), AH6 (defining Le(y)), FH6 (defining sialosyl dimeric (Le(x)), or TKH2 (defining sialosyl-Tn). Some areas were stained by two or three of these antibodies. Comparisons of the mosaic-staining patterns with cytohistological properties of tumor cells within specific areas suggested that the pattern of antigen expression is correlated with degree of differentiation; e.g., poorly-differentiated cells with severe dysplasia did not express high levels of Le(x) or Le(y) but did express sialyl-Le(x) or dimeric Le(x); on the other hand, moderately or well-differentiated tumor cells in some areas expressed high levels of Le(x) or Le(y) but lower levels of sialyl-Le(x). Areas showing strong expression of sialyl-Tn in their secretions were consistently correlated with presence of well-differentiated tumor cells, whereas secretions from normal mucosae were consistently characterized by lack of sialyl-Tn expression. It is postulated that the original in situ tumors (which had homogeneous glycosylation patterns) evolved into several spatially discrete cell populations displaying different degrees of glycosylation, reflecting stages of tumor cell differentiation and progression.

A new compact and cell dense continuous culture system.

A new compact and cell dense continuous culture system was developed. The system consisted of a culture bag, which had two compartments separated by a semipermeable membrane, and an external rotator. Cells were cultured in the inner compartment with medium containing serum or the necessary growth factors. The outer compartment was filled with medium without serum or growth factors. The concentration of cells in culture was normally in the order of 10(7). Due to the large number of cells, sufficient carbon dioxide was produced so that use of a CO2 incubator was unnecessary. The medium in the outer part was frequently changed, but no medium changes were necessary in the inner bag. Using this system, a concentrated yield of antibody as well as a large number of cells could be obtained.

Donor peripheral blood stem cell infusions in recipients of living-related liver allografts.

BACKGROUND: In this pilot study, donor peripheral blood stem cell (DPBSC) infusions were performed in three recipients of living-related liver transplants (LRLT). METHODS: DPBSCs were obtained by leukapheresis after mobilization with granulocyte-colony-stimulating factor (Filgrastim). Donor leukapheresis was performed on the 5th postoperative day, and half of the DPBSCs were infused into the recipient on the day of collection. The second half of the pheresed product was cryopreserved for delayed administration. RESULTS: Results from preliminary studies of chimerism in LRLT recipients, at 20 weeks posttransplant, suggested that the levels of donor cells detected in LRLT recipients treated with DPBSC infusions may be higher than those observed for recipients of cadaver donor liver allografts and vertebral body marrow infusions. CONCLUSIONS: The results of this pilot study indicate that administration of mobilized DPBSC to recipients of LRLT is a feasible procedure for both donor and recipient.

Profile of amino acid synthesis in rat hippocampus during push-pull perfusion of ethanol or morphine.

In the unanesthetized rat, the effect of ethanol or morphine on the new synthesis of six amino acids, gamma-aminobutyrate, glutamate, alanine, aspartate, glycine and glutamine, was determined at circumscribed sites in the hippocampus. In the fasted animal, 5.0 microCi of [U-14C]glucose was microinjected in a volume of 1.0 microliter into a discrete region of the hippocampus through a permanently implanted guide tube. After 10-15 min, the tip of a concentric push-pull cannula assembly was lowered through the guide tube to the labelled site, and an isotonic artificial cerebrospinal fluid was continuously perfused at a rate of 25 microliters/min. Samples of perfusate were collected every 5.0 min in a total volume of 125 microliters. During the fourth perfusion, either ethanol (94 or 471 mM) or morphine (0.1 or 1.0 microgram/microliter) was added to the push-pull perfusate. Amino acids and glucose contained in each sample of perfusate were separated by means of a two-dimensional, thin-layer chromatographic procedure. The results show that the 94 mM dose of ethanol acted only to significantly suppress the new synthesis of alanine. Conversely, the higher 471 mM dose of ethanol significantly increased the new synthesis of both glycine and glutamine from the glucose precursor. The effect of the 0.1 microgram/microliter morphine dose on amino acid synthesis paralleled that of the lower dose of ethanol since it evoked a similar decrease in the synthesis of alanine; however, the higher dose of 1.0 microgram/microliter morphine produced no effect. None of the other amino acids in the hippocampus were affected by either of the CNS depressants. Thus, whereas ethanol seems to exert significant stimulating activity on hippocampal synthesis of glycine and glutamine, morphine and ethanol possess the same inhibitory effect on alanine synthesis in this subcortical structure.

Changes in Ca2+ ion activity within unrestrained rat's hippocampus perfused with alcohol or acetaldehyde.

In the freely moving rat, the kinetics of Ca2+ ion activity were determined at circumscribed sites in the hippocampus, which was perfused with ethanol, tertiary-butyl alcohol or acetaldehyde. Initially, a region in CA1 or other cell field of the dorsal hippocampus was prelabelled by microinjection of 45Ca2+ through a permanently implanted guide tube. Then the tip of a concentric push-pull cannula assembly was lowered through the guide tube to the labelled site, and an isotonic artificial cerebrospinal fluid was repeatedly perfused at a rate of 25 microliter/min. Each perfusion was timed for 5.0 min with a 5.0 min interval between each. Once the washout curve of 45Ca2+ activity had begun to approach its asymptote, ordinarily in the midpoint of a series of perfusions, an isotonic solution of ethanol (188-942 mM), tertiary-butyl alcohol (12-580 mM) or acetaldehyde (10-98 mM) was added to the fourth perfusate. Thereafter, the hippocampal site was again perfused with the normal cerebrospinal fluid for the remainder of the experiment. Although the lowest concentration of ethanol exerted no effect on 45Ca2+ ion activity, an intermediate concentration caused mixed effects in either enhancing or suppressing the efflux into the perfusate of this cation. The highest concentration of ethanol produced in most experiments an initial suppression in Ca2+ ion efflux which was followed frequently by an elevation in the release of 45Ca2+. Similar changes in Ca2+ ion activity were produced by tertiary-butyl alcohol, but the magnitude of its effect was generally less than that of ethanol, suggesting that its effect on brain tissue differs from that of ethanol. Acetaldehyde evoked an intense and concentration-dependent enhancement of Ca2+ ion efflux from the perfused tissue at all of the sites in the hippocampus examined. These results suggest that in the unrestrained rat ethanol could unbind Ca2+ ions from hippocampal membranes or retard their uptake into cells of the hippocampus. The dual excitatory and inhibitory effect of ethanol on Ca2+ ion activity corresponds to the electrophysiological effects of this alcohol and could alter neurotransmitter release from neurons in this subcortical structure. The mechanism of action of acetaldehyde is envisaged to be due to its affinity to membrane sulfhydryl groups which alters protein conformation and thus interferes with both Ca2+ channels and Ca2+ binding properties.

Myocardial distribution of indium-111-antimyosin Fab in acute inferior and right ventricular infarction: comparison with technetium-99m-pyrophosphate imaging and histologic examination.

In a postmortem study of a 69-yr-old female patient who had suffered 2 yr previously a non-Q-wave anterior infarction and who had sustained just seven days earlier a left inferior and right ventricular infarction, the distribution of 111In-antimyosin Fab was compared to the results of 99mTc-pyrophosphate imaging and histologic examination. Indium-111-antimyosin Fab imaging could not be performed because of cardiogenic shock. However, postmortem gamma scintillation counting revealed increased activities of antimyosin Fab in the inferoapical and right ventricular infarcted regions in which 99mTc-pyrophosphate positive imagings were observed; in contrast, a histologically confirmed old subendocardial anterior infarction had no definite activity. Thus, the myocardial distribution of 111In-antimyosin Fab corresponded well to the results of 99mTc scintigrams and histologic examinations in a human heart, suggesting that this technique could be useful in vivo for detecting several-day-old myocardial infarction of the right ventricle as well as the left ventricle. Tissue from the 2-yr-old infarction was not identified by this technique.

Hypothermia caused by antipsychotic drugs in a schizophrenic patient.

In a schizophrenic patient, hypothermia was caused by combined treatment with zotepine, biperiden, and fluphenazine, although combined treatment with zotepine and biperiden had caused no side effects. Other side effects closely resembled those in neuroleptic malignant syndrome.

THE EFFECTS OF EMBRYONIC HEART RNA AND ACTINOMYCIN D BOTH ON THE ISOLATED TISSUE-PIECES OF THE CHICK BLASTODERM AND ON THE ISOLATED PRIMITIVE TUBULAR HEART OF THE EARLY CHICK EMBRYO.

The effects of embryonic heart RNA extracted from 18-day chick embryos were studied both on the isolated parts of the chick blastoderm and on the isolated primitive tubular heart of the early chick embryo. The embryonic heart RNA accelerated in vitro the beating of the heart-forming area and the pulsing of the primordial heart organ. In addition, a phenomenon of cardiac transformation was observed.

Positive direct antiglobulin tests with antilymphocyte globulin.

Patients receiving antilymphocyte globulin (ALG) of equine origin, for prophylactic immunosuppression following renal transplantation or for rejection episodes, develop a positive direct antiglobulin test (DAT), mainly of the IgG type. This finding may be observed as early as the first day following the administration of ALG. The cause for the positive DAT is due to the presence of antihorse globulin in the reagent antiglobulin serum reacting with ALG antigenic material coating the patients' red blood cells. The serum of some of these patients also may result in incompatible cross-matches. These serums and all of the eluates react with donor and reagent red blood cells demonstrating no particular blood group specificity.

Urinary metabolites of polyamines in rats.

Urinary metabolites of polyamines in rats were studied systematically by the intraperitoneal injection of radioactive polyamines. Urinary metabolites were fractionated into 4 fractions containing non-polar and acidic compounds, acidic and neutral ampholytes, basic ampholytes and polyamines. A large amount of radioactivity was detected in the fractions containing non-polar and acidic compounds and polyamines of urine of rats injected with radioactive putrescine, while in the case of the injection of radioactive spermidine or spermine a relatively large amount of radioactivity was found in the basic ampholyte fraction as well as in the polyamine fraction. Analysis of these fractions indicated that gamma-aminobutyric acid, N-monoacetylputrescine, 2(3)-hydroxyputrescine, putreanine, N-(3-aminopropyl)-4-aminobutyric acid (isoputreanine), spermic acid, N-(3-aminopropyl), N'-(2-carboxyethyl)-1,4-diaminobutane, and N-monoacetylspermidine A and B were excreted as urinary metabolites of the polyamines in addition to putrescine, spermidine and spermine.

A study on the metabolism of spermidine in mammals: purification and identification of a newly identified metabolite, 2-oxo-1-pyrrolidinepropionic acid, in rat urine.

In order to study the metabolism of spermidine in mammals, radioactive spermidine was injected intraperitoneally into a rat and urine was collected for analysis. Incorporation of radioactivity into putreanine, isoputreanine, spermidic acid, and N-aminopropylpyrrolidin-2-one was confirmed by ion-exchange chromatography, thin layer chromatography, and paper electrophoresis, the highest radioactivity being observed in the non-polar and acidic fraction of the collected urine. A radioactive compound was purified from the non-polar and acidic fraction, and identified as 2-oxo-1-pyrrolidinepropionic acid by comparison of its behavior on ion-exchange chromatography and thin layer chromatography with that of authentic 2-oxo-1-pyrrolidinepropionic acid, and recrystallization with the authentic compound. Acid hydrolysis of the radioactive compound produced radioactive spermidic acid, confirming the identification. To examine the interconversion between isoputreanine and N-aminopropylpyrrolidin-2-one, these compounds were deuterated and then intraperitoneally injected into a rat. Analysis of 24-h urine by gas-chromatography-mass-spectrometry indicated no interconversion between the two metabolites of spermidine under these conditions. An intracerebroventricular injection of radioactive spermidine into a rat showed that radioactivity was also incorporated into the metabolites of spermidine in the brain, and oxidative deamination of the aminopropyl moiety of spermidine was thought to be dominant in the central nervous system and vice versa in peripheral organs.

Biorhythm of arginine- vasopressin in the paraventricular, supraoptic and suprachiasmatic nuclei of rats.

The diurnal variations in content of arginine-vasopressin in the supraoptic nucleus, the paraventricular nucleus and the suprachiasmatic nucleus of rats were determined using radioimmunoassay. In the supraoptic nucleus and the paraventricular nucleus the arginine-vasopressin level was relatively constant during the light phase (the inactive phase). When it became dark, the level of arginine-vasopressin lowered during the early and middle dark phase and then increased to the highest level during the late dark phase. In the suprachiasmatic nucleus the level was stable during the light phase, while in the early and the late dark phase it was significantly higher than that in the middle dark phase.

Effects of prostaglandin E2 and D2 on the release of vasopressin and oxytocin.

The effects of prostaglandin (PG) E2 and D2 on the plasma levels of arginine-vasopressin (AVP) and oxytocin (OXT) in rabbits, and on the release of the both hormones from the isolated posterior pituitary of rats were examined. An intraventricular administration of PGE2 to a rabbit raised the plasma levels of the both hormones. An intraventricular injection of PGD2 also increased the plasma level of OXT but not that of AVP. The release of AVP and OXT from fragments of the posterior pituitary of a rat was not influenced by perfusion with Eagle MEM medium containing 10(-6) or 10(-5) M PGE2 and D2.

A study on the release mechanism of vasopressin and oxytocin.

The changes in plasma levels of arginine-vasopressin (AVP) and oxytocin (OXT) of rabbits by intraventricular administration of various drugs and their effects on the release of both hormones from the isolated posterior pituitary of rats were examined. An intraventricular injection of hypertonic saline, carbachol, angiotensin II, prostaglandin E2 or histamine to a rabbit increased the concentrations of plasma AVP and OXT, whereas serotonin decreased their plasma levels. Noradrenaline increased the concentration of OXT, but not that of AVP. Dopamine did not significantly affect the plasma level of either hormone. The release of AVP and OXT from the posterior pituitary fragments of rats was stimulated by changing the osmolality of the perfusion medium in vitro. Perfusion with medium containing dopamine suppressed the release of both hormones. However, the other bioactive amines and the drugs mentioned above did not affect the release of AVP and OXT.

Role of adenosine and P2 receptors in the penile tumescence in anesthetized dogs.

We studied the role of adenosine and P2 receptors in the pelvic nerve stimulation-induced penile tumescence in anesthetized dogs. A local intracavernous injection of adenosine induced the tumescence, which was abolished by intracavernous 8-(p-sulfophenyl)theophylline (8-SPT), an unspecific adenosine receptor antagonist, and by 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol (ZM241385), an adenosine A(2A) receptor antagonist. ATP also induced the tumescence, which was diminished by 8-SPT, but not by reactive blue-2, a P2 receptor antagonist. Neither intracavernous beta, gamma-meATP nor ADP(beta)S, P2X and P2Y receptor agonists, induced tumescence. N(G)-nitro-L-arginine (L-NAME), a nitric oxide synthase inhibitor, and T-1032, a phosphodiesterase type V inhibitor, had no effects on the tumescence induced by adenosine. 8-SPT and reactive blue-2 had no effects on the tumescence induced by pelvic nerve stimulation. These results show that although exogenous adenosine and ATP induce tumescence, neither the adenosine nor the P2 receptor is involved in the tumescence induced by pelvic nerve stimulation in anesthetized dogs.

T-1032, a novel specific phosphodiesterase type 5 inhibitor, increases venous compliance in anesthetized rats.

Nitric oxide (NO) donors including organic nitrates dilate capacitance vessels. As inhibition of phosphodiesterase type 5 results in the accumulation of guanosine 3'5'-cyclic monophosphate (cGMP), specific phosphodiesterase type 5 inhibitors are expected to have a vasodilator property similar to that of NO donors. To test this hypothesis, we examined the effect of methyl2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel specific phosphodiesterase type 5 inhibitor, on mean arterial pressure and mean circulatory filling pressure (an index of venodilation) compared with that of nitroglycerin and diltiazem in mecamylamine- and noradrenaline-treated anesthetized rats. Intravenous infusion of T-1032 (0.1, 1, 10 microg/kg/min) dose-dependently decreased mean arterial pressure (-3.8+/-0.3%, -9.1+/-0.8%, -16.8+/-1.5% at doses of 0.1, 1 and 10 microg/kg/min, respectively) and mean circulatory filling pressure (-6.1+/-0.9%, -12.5+/-0.7%, -18.6+/-3.0% at doses of 0.1, 1 and 10 microg/kg/min, respectively). The mean circulatory filling pressure-mean arterial pressure relationship revealed that T-1032 had a selective action on the mean circulatory filling pressure compared with diltiazem (10, 100 microg/kg/min) and a similar or more selective effect than nitroglycerin (0.3, 3 and 30 microg/kg/min). In the next study, we calculated venous compliance and unstressed volume from the mean circulatory filling pressure-volume relationship. Intravenous infusion of T-1032 (3 microg/kg/min) increased venous compliance (3.35+/-0.40 in T-1032 vs. 2.31+/-0.15 ml/kg/mm Hg in vehicle, P<0.05) without changing the unstressed volume (37.2+/-2.80 in T-1032 vs. 42.6+/-2.37 ml/kg in vehicle, P>0.05). It was concluded that T-1032 increased venous capacitance by increasing venous compliance, and that this selective phosphodiesterase type 5 inhibitor appeared to have a different vasodilator action from that of an NO donor and a Ca(2+) channel antagonist in that it had a selective action on the mean circulatory filling pressure.

Pharmacological profile of T-1032, a novel specific phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum.

This study was designed to examine the pharmacological properties of T-1032 (methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate), a novel phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum. T-1032 (3x10(-11) to 3x10(-7) M) caused an endothelium-dependent relaxation in the isolated rat aorta precontracted with phenylephrine, and the relaxation was accompanied by an increase in cGMP but not cAMP levels. The T-1032-induced relaxation was attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME) (10(-3) M), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) (10(-5) M), a guanylyl cyclase inhibitor. T-1032 (10(-9), 10(-8) M) produced a potentiation of the relaxation induced by sodium nitroprusside, but not of the relaxation induced by isoproterenol. In the isolated rabbit corpus cavernosum precontracted with phenylephrine, the electrical field stimulation-induced relaxation was attenuated by treatment with tetrodotoxin (10(-6) M) as well as L-NAME (10(-4) M). The L-NAME-inhibited relaxation was restored by treatment with L-arginine (5x10(-4) M). T-1032 (10(-9) to 10(-6) M) and sildenafil (10(-9) to 10(-6) M) produced a potentiation of the electrical field stimulation-induced relaxation as well as a decrease in basal tension in a concentration-dependent manner. It was concluded that T-1032 had potentiating effects on the NO/cGMP signaling pathway in isolated tissues, probably through specific blockade of phosphodiesterase type 5. T-1032 would be a useful compound to examine the physiologic functions of phosphodiesterase type 5 in mammalian tissues.

Quantification of antimyosin uptake and infarct size at various stages of myocardial infarction.

We studied with quantitative techniques the clinical efficacy of indium-111 antimyosin at a later stage of myocardial infarction in 18 patients at various stages after infarction. Antimyosin accumulation was detected irrespective of infarct age and size and quantified as an infarct weight with a tomographic technique. Higher intensities in a planar image were observed in anterior Q wave infarct group (36 +/- 5 g) but not in inferior and non-Q wave anterior infarct groups because of the smaller infarct weights (8 +/- 3 g, 13 +/- 6 g, respectively). Infarct area calculated from thallium-201 tomography significantly correlated with left ventricular ejection fraction in both recent (less than 2 weeks) and older (2-week- to 6-month-old) infarct groups (r = -0.969, P less than 0.001; r = -0.860, P less than 0.001, respectively), whereas there was a significant negative correlation between infarct weight and left ventricular ejection fraction in the recent infarct group (r = -0.731, P less than 0.05) but not in the older infarct group. Thus, antimyosin tomography can detect myocardial necrosis with a high sensitivity regardless of infarct age, size, and location. However, the accumulation might be affected by infarct age and correspond to necrotic mass but not necessarily to infarct volume itself at a later stage probably because of the presence of necrosed and scarred tissues in infarcted myocardium.

Formation of GABOB from 2-hydroxyputrescine and its anticonvulsant effect.

To investigate the formation of gamma-amino-beta-hydroxybutyric acid from 2-hydroxyputrescine in mammalian organs, the radioactive diamine was synthesized and was injected into rats intraperitoneally or intraventricularly. After intraperitoneal injection, the radioactive amino acid was detected in various organs, but formation of the stereoisomer of the amino acid (gamma-amino-alpha-hydroxybutyric acid) was not demonstrated. Intraventricular injection of the radioactive diamine also resulted in the formation of gamma-amino-beta-hydroxybutyric acid in the rat brain. In vivo experiments using monoamine oxidase or diamine oxidase inhibitors suggested the participation of both enzymes in the formation of the amino acid from the diamine in rat organs other than the brain, where diamine oxidase appeared to play the major role. To investigate the anticonvulsant effect of 2-hydroxyputrescine, the threshold of pentylenetetrazol-induced generalized convulsions was measured in rats after the intraventricular injection of 2-hydroxyputrescine. Both R(-)- and S(+)-2-hydroxyputrescine had an anticonvulsant effect, with a greater elevation of the threshold being observed after injection of the R(-) form. Time course experiments suggested that this anticonvulsant effect depended on the formation of gamma-amino-beta-hydroxybutyric acid from 2-hydroxyputrescine in the rat brain. The anticonvulsant action of gamma-amino-beta-hydroxybutyric acid was also examined, and the stimulation of Cl- influx plus the inhibition of GABA uptake into brain membrane vesicles were indicated to be involved.

Effect of DL-erythro-dihydroxyphenylserine on the locomotor activity of the mouse.

Effect of dihydroxyphenylserine (DOPS) on the locomotor activity of mice pretreated with beta-phenylisopropylhydrazine was studied using an Animex activity meter. An intraperitoneal injection of DL-erythro-DOPS (200 mg/kg) suppressed significantly the locomotor stimulation by the MAO inhibitor, while DL-threo-DOPS (200 mg/kg) had no effect. Only slight suppression was observed after the administration of 100 mg/kg of DL-erythro-DOPS. Effect of DOPS on the concentrations of brain catecholamines and serotonin of mice pretreated with the MAO inhibitor was also analysed. The administration of DL-erythro-DOPS significantly increased the concentration of noradrenaline, while DL-threo-DOPS did not affect the contents of brain amines in the experimental condition. The suppressive effect of DL-erythro-DOPS on the locomotor stimulation by the MAO inhibitor was confirmed by a simultaneous administration of the amino acid and d-phenylisopropylmethylamine to mice. Based on these findings, the neural mechanisms of the locomotor activity and a clinical application of DL-erythro-DOPS to the manic syndrome were discussed.