Bifidobacterium bifidum BF-1 suppresses Helicobacter pylori-induced genes in human epithelial cells.

Helicobacter pylori infection alters gene expression in host cells. Specifically, inflammatory chemokines such as IL-8 are upregulated in the gastric mucosa during H. pylori infection. Although the mechanism by which H. pylori causes inflammation of the gastric mucosa is not yet understood, many studies have suggested that nuclear factor kappa B (NF-κB) plays a key regulatory role in host cells. We have shown that preincubation with Bifidobacterium bifidum strain BF-1, a probiotic strain known to improve H. pylori-associated gastritis, suppresses induction of IL-8 by the pathogen. To investigate how how BF-1 affects gene expression in H. pylori-infected cells, we performed microarray analysis to assess gene expression in epithelial cells, which had been preincubated with BF-1 and infected with H. pylori. We found that preincubation with BF-1 suppresses the expression of H. pylori-induced genes in human cells and that most of the affected genes are related to the NF-κB signaling pathways. These results suggest that BF-1 can affect the regulatory mechanism of the NF-κB signaling pathways.

Early detection of the Limulus amebocyte lysate reaction evoked by endotoxins.

The gelation of Limulus amebocyte lysate (LAL) evoked by bacterial endotoxins can be detected earlier than with usual methods by using laser scattering photometry to recognize the formation of small particles of clotted enzyme produced when the reaction mixture is agitated. The appearance of these small particles means that the influence of endotoxins has stimulated activation of the clotting enzyme across the LAL cascade, and the timing of their appearance is related to endotoxin concentration. This new method can be used for quick and sensitive endotoxin assay. The average endotoxin level of healthy volunteers was assayed to be 0.0738 pg/ml [0.0312-0.3445 pg/ml] (n = 11) within 70 min from the start of the assay.

Thrombopoietin primes human platelet aggregation induced by shear stress and by multiple agonists.

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine-diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

Transdominant inhibition of Moloney murine leukemia virus proliferation by defective mutants of reverse transcriptase.

To investigate the dominant-negative inhibition of Moloney murine leukemia virus (Mo-MuLV) proliferation by polymerization-defective mutants of reverse transcriptase (RT), we constructed several plasmids harboring the Mo-MuLV RT gene in which the YVDD sequence, one of the conserved sequences in RNA-dependent polymerases, was altered, and then transformed mouse NIH3T3 cells and Escherichia coli with the mutant plasmids. Mouse NIH3T3 cells expressing these mutant RT genes were highly resistant to Mo-MuLV proliferation. The mutant RT expressed in E. coli exhibited no polymerization activity, but it retained its binding activity to the template RNA and inhibited in vitro poly(dG) synthesis occurring with the wild-type RT. These results suggest that the competition for binding of the two types of enzymes to the template is responsible for the resistance to Mo-MuLV proliferation and that the YVDD sequence of Mo-MuLV may be a good target for dominant-negative inhibition of retroviral proliferation.

Detection of platelet aggregates with a particle counting method using light scattering.

A novel method to detect platelet aggregation by means of the particle counting technique using light scattering has been developed. An optical device designed to focus on a limited area of platelet-rich plasma measured the intensity of light scattered by particles passing through the area, minimizing multiple light scattering. The use of polystyrene spheres of different diameters confirmed that the light scattering intensity increases in proportion to the particle size in a suspension. Platelet activation induced by various agonists resulted in light scattering of higher intensities, which correlated well with the number and size of aggregates as observed under a microscope. These findings confirmed that the intensity of light scattering detected by the new device provides information on the number and size of aggregates in a suspension. The new method was compared with conventional platelet aggregometry using overall light scattering or changes in light transmission (optical density). The new device appeared to be particularly sensitive to small aggregates such as those formed in platelet activation induced by low concentrations of agonists. Furthermore, the new method has an advantage over the conventional aggregometry, in that it allows the aggregate size distribution and the extent of aggregation to be estimated.

Thrombin-induced calcium oscillation in human platelets and MEG-01, a megakaryoblastic leukemia cell line.

Digital imaging microscopy revealed that human platelets show periodic intracellular Ca++ elevation in response to 0.01 U/ml thrombin. MEG-01, a megakaryoblastic leukemia cell line, also responded with oscillatory intracellular Ca++ elevation (0.7-1 times/min) to thrombin (0.001-0.003U/ml). Ca++ transients appears to be fused with higher thrombin doses. With extracellular Ca++ concentrations of 0.1 mM or less, Ca++ oscillation could not be elicited, or even when present, it disappeared after a few spikings of [Ca++]i. Extracellular Ca++ concentrations of 0.3 mM or more were required to facilitate ongoing Ca++ oscillation, suggesting an important role of Ca++ influx for Ca++ oscillation.

Different usage of two polyadenylylation signals in transcription of the N-myc gene in rat tumor cells.

The complete nucleotide sequence of a rat genomic DNA fragment of 6.9 kbp containing the entire N-myc gene was determined. A unique structural feature of the rat N-myc gene is the presence of two polyadenylation signals in the exon 3 region resulting in formation of two poly(A)+ N-myc mRNAs of 2.9 and 2.2 kb length. Elevated expression of the 2.9 kb mRNA, which was not due to gene amplification, was observed in normal rat tissues such as brain, adrenal and testis, and in rat tumor cells such as ascites hepatoma AH130, AH70Btc and AH7974 cells, and pituitary tumor GH3 cells. In contrast, expression of 2.2 kb mRNA, for which transcription was terminated at the upstream polyadenylation site, was observed only in GH3 cells.

Mechanisms of the contractile effect induced by uridine 5-triphosphate in canine cerebral arteries.

This study was performed to elucidate mechanisms responsible for the contraction of isolated canine cerebral arteries induced by uridine 5'-triphosphate (UTP) and to ascertain whether UTP given intracisternally causes cerebral arterial constriction. The latter was proven arteriographically to be the case. In vitro, UTP (10(-4)M) and UDP were similar in potency, produced sustained contractions, and were more effective than other pyrimidine nucleotides or uridine. Unlike serotonin (5-HT), UTP was not antagonized by cinanserin and failed to cause constriction of mesenteric arteries. Adenosine similarly antagonized 5-HT and UTP. The Ca2+ antagonist nimodipine abolished contractions caused by high K+ but only incompletely antagonized 5-HT or UTP. On the other hand, procedures that hyperpolarize the cell membrane (low K+ followed by K+) abolished tonic contractions induced by UTP. Hyperpolarization prior to UTP (with or without nimodipine) did not, however, prevent the occurrence of a phasic contraction. Papaverine or lanthanum antagonized this phasic response. It was concluded that UTP selectively affects cerebral arteries, may initiate contraction by releasing membrane bound Ca2+, depolarizes the cell membrane to open receptor operated and potential sensitive calcium channels, but does not inhibit the electrogenic Na-pump nor specifically antagonize the vasodilator adenosine.

Vasoactive and beta-adrenoceptor blocking properties of 3,4-dihydro-8-(2-hydroxy-3-isopropylamino) propoxy-3-nitroxy-2H-1-benzopyran (K-351), a new antihypertensive agent.

Single oral administration of K-351 showed a long lasting antihypertensive action in spontaneously hypertensive rats, accompanied by slight bradycardia. K-351 reduced systolic and diastolic blood pressures almost to the same degree. K-351 showed a competitive antagonistic effect on norepinephrine-induced contraction of isolated canine blood vessels. This alpha-adrenoceptor blocking action of K-351 was about 5 times less potent than that of phentolamine. Furthermore, K-351 produced a nitroglycerin-like relaxant action on isolated blood vessels previously contracted with high K+. K-351 showed a nonselective beta-adrenoceptor blocking action in isolated guinea-pig atrium and trachea, and its action was about 2 times more potent than that of propranolol. K-351 did not show an intrinsic sympathomimetic action. In anesthethized dogs, low doses of K-351 reduced the heart rate and antagonized the positive chronotropic and hypotensive responses to isoproterenol. In higher doses, K-351 lowered the blood pressure and showed an antagonistic action on the pressor response to phenylephrine. The desnitro compound of K-351 was deprived of vasoactive properties on isolated blood vessels and hypotensive activity. Results have revealed that K-351 has both beta-adrenoceptor blocking and vasoactive properties which may result in an antihypertensive effect without reflex tachycardia in hypertensive animals.

[Effects of adenosine triphosphate on the postrotatory nystagmus and disorders of vestibular function (author's transl)].

Effects of adenosine triphosphate (ATP) on the postrotatory-induced nystagmus in rabbits and disorders of vestibular function induced by repeated administration of streptomycin in guinea pigs were studied. ATP-2Na exerted little influence on the postrotatory nystagmus in doses of 3 and 10 mg/kg, i.v., while slight suppressive effects were noted with a dose of 30 mg/kg. On the other hand, diphenidol hydrochloride remarkably suppressed the postrotatory nystagmus in a dose of 3 mg/kg i.v.. The disturbance in vestibular function following administration of streptomycin sulfate (400 mg/kg i.m., once daily x 9) was significantly alleviated when concomitant treatment with ATP-2Na (3 mg/kg i.p., 10 mg/kg i.p. and 100 mg/kg p.o., once daily x 14) or diphenidol hydrochloride (50 mg/kg p.o., once daily x 14) was given. ATP as well as diphenidol alleviated disorders in the vestibular function, while ATP had little influence on vestibular function in intact animals.

[Effects of 1, 4-bis-(3-(3, 4, 5-trimethoxy benzoyl oxy)-propyl) perhydro-1, 4-diazepine (dilazep) on the isolated smooth muscles].

The effects of a new coronary vasodilator (dilazep) on isolated intestine, taenia coli, trachea, vas deferens, uter, aorta and coronary arteries were investigated in rats, guinea pigs, rabbits and dogs. Dilazep showed relaxant effects on isolated smooth muscle in a concentration of 10(-5) 3 X 10(-4) M and non-competitive inhibition on contraction induced by agonists (pD'2: 4.405.05). In guinea pig taenia coli, dilazep had a relaxant effect on K-contracture. The effect was qualitatively similar to that of papaverine. In guinea pig taenia coli, dilazep showed a Ca++ antagonistic effect in a concentration as high as 3 X 10(-6)M. The potency was stronger than dipyridamole, NaNO2 and aminophylline and equalled that of papaverine and hexobendine. In guinea pig taenia coli, dilazip potentiated relaxant effects induced by adenosine and adenine nucleotides in a concentration as high as 10(-8)M. The potency was stronger than that of dipyridamole. From these results, it is suggested that the potentiating effect of both adenosine and adenine nucleotides and Ca++ antagonistic effect of dilazep may play an important role in producing the coronary vasodilating effect.