Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.     

Effect of Z-100, an immunomodulator extracted from human type tubercle bacilli,on the pulmonary metastases of lewis lung carcinoma in attempt to regulate suppressor T cells and suppressor factor,IL-4

In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation, survival rate of these mice was increased significantly (p<0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased by 38% at 21 days, as compared with that of control mice (p<0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice, as compared with that of control mice. When splenic suppressor cells (5×10⁷ cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected 15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4.     

Interaction between histamine and vagal stimulation on tracheal smooth muscle in dogs

We examined the interaction between histamine and vagal efferent activity on airway smooth muscle reactivity in 11 anesthetized vagotomized dogs using an isolated closed segment of the intrathoracic trachea filled with Tyrode solution under an isovolumetric condition. Intratracheal pressure change was measured as an index of tracheal smooth muscle tone. The administration into the tracheal segment of histamine (0.1 or 1.0 mg/ml) in six dogs and methacholine chloride (0.001 or 0.01 mg/ml) in the other five dogs elevated intratracheal pressure by about 5 cmH2O. The electrical stimulation of the peripheral ends of both of the cut cervical vagus nerves in the presence of histamine produced significantly greater responses than the additive responses of these two stimuli applied individually (two-way analysis of variance, P less than 0.025). However, the combined effects of vagal stimulation and methacholine were not significantly different from the additive responses of these two stimuli applied individually. The average values of intratracheal pressure elevated by the combined effects of vagal stimulation and histamine were significantly higher than those obtained by the combination of vagal stimulation and methacholine (two-way analysis of variance, P less than 0.01). This suggests that histamine potentiates tracheal smooth muscle reactivity to electrical vagal stimulation, which may contribute to the hyperreactivity observed in patients with asthma.     

Ejaculations induced by p-chloroamphetamine (PCA) in rats, hamsters and mice.

The ejaculatory response induced by p-chloroamphetamine (PCA) in male rats, hamsters and mice was observed during 2 hours after the injection. The animals were treated intraperitoneally with PCA at doses ranging from 0.78125 to 160 mg/kg. The ED50 (effective dose in 50% of animals) values of PCA for the initiation of ejaculation in rats and hamsters were 1.3397 (1.0732-1.6725) and 0.1105 (0.0802-0.1522) mg/kg, respectively. On the other hand, no ejaculation was observed in any mice at any doses examined. So we concluded that there are species differences in the ejaculatory response, induced by PCA, among rats, hamsters and mice.     

A protein factor extracted from murine brains confers physiological Ca2+ sensitivity to exocytosis in sea urchin eggs.

Exocytosis in sea urchin eggs can be reconstituted in vitro using the cell ghosts (the isolated cortices). When the isolated cortices were handled in the medium primarily composed of non-chaotropic ions, exocytosis can be induced by a micromolar level of Ca2+. However, when the cortices are exposed to chaotropic anions such as Cl-, it is induced only at higher Ca2+ concentrations of 10(-5) to 10(-4) M, due to the chaotropic anionic effect, by which a specific protein(s) is dissociated from the cortex. The dissociated protein can be added back to the cortex to restore the original Ca2+ sensitivity [(1984) Dev. Biol. 101, 125-135]. A protein which has the similar effect on the isolated cortex was also found in the extract of murine brain. This protein was neither calmodulin, a G-protein or a kinase. The data suggest the general regulatory mechanism of the Ca2+ sensitivity of exocytosis by a protein factor widely distributed among cells.     

Immunological detection of propolypeptide of von Willebrand factor on platelet surface

Recently we have found that propolypeptide of von Willebrand factor (pp-vWF) obtained from platelets binds to type I collagen. It is known that pp-vWF is present in platelet alpha-granules and is secreted upon activation. In this paper, we demonstrate the two following evidences to show that it is also present on the surface of resting platelets. [1] The antibody against pp-vWF bound to the surface of platelets. [2] The antibody induced aggregation of platelets. The binding of the antibody and the antibody-induced aggregation of platelets were inhibited in a dose-dependent manner by Fab fragment of the antibody. Platelets from von Willebrand disease patients bound less of the antibody and responded weakly to the antibody.     

Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression

The nucleotide and amino acid sequences for rat type I angiotensin II receptor were deduced through molecular cloning and sequence analysis of its complementary DNAs. The rat angiotensin II receptor consists of 359 amino acid residues and has a sequence similar to G protein-coupled receptors. The expression of this receptor gene was detected in the adrenal, liver and kidney by Northern blotting. Sodium deprivation positively modulated the expression of the receptor gene in the adrenal. No detectable change was observed in the expression levels of this receptor gene between spontaneously hypertensive rats and Wistar-Kyoto rats in the tissues examined including the adrenal, brain, kidney and liver. Interestingly the expression of this receptor gene was developmentally regulated.