No abstract

Isolated yeast nuclei were able to incorporate 3H-labeled pJDB219 DNA in vitro in the presence of ATP and Mg2+. The number of plasmid molecules incorporated into each nucleus was calculated to be 60 under the conditions we used. Enzyme-histochemical staining of the incorporated biotinylated pJDB219 with streptavidin-biotinylated-peroxidase complex indicated a uniform distribution of the incorporated plasmids within each nudeus. After intranuclear incorporation, substrate pJDB219 DNAs (open and closed circular forms) were changed to the linear form and were weakly digested over the longer incubation period (over 60 min). Facile release of the once-incorporated plasmid DNA was never observable; discharge of the incorporated [3H]pJDB219 during a 60-min incubation was less than 5%. The addition of adenylyl-imidodiphosphate, N,N'-dicyclohexylcarbodiimide (DCCD), or quercetin inhibited in vitro DNA uptake reaction. DCCD and quercetin inhibited the nuclear ATPase and apparent protein kinase, respectively; hence, the involvement of these enzymes in the nuclear DNA transport system was suggested.When eucaryotic cells are used as recipients for transfection, DNA introduced in the cytoplasm must enter the nucleus during the process of transformation. The materials entering the nucleus are known to be selected by the nuclear pore complex. Molecules larger than a critical size are excluded from the nucleus, whereas smaller ones are able to enter. The maximum size limit for entry to the nuclear compartment is consistent with a channel radius of 4.5 nm, which corresponds to a physical size of the globular protein of approximately 60,000 daltons (6). The plasmid vectors used for transformation in the yeast Saccharomyces cerevisiae are larger than 5 kilobase pairs; i.e., over 3 x 106 daltons. The mechanism for entry of DNA molecules into the nucleus cannot be explained simply by the size selection mechanism postulated for protein entrance, because the three-dimensional form of plasmid molecules introduced in the cytoplasm is not clear; i.e., supercoiled, relaxed, or bound with some other cytoplasmic molecules, etc. Since little is known about the nuclear DNA transport mechanism in yeasts, we describe in this report the establishment and characterization of an in vitro assay system for DNA transport through the nuclear membrane by using isolated yeast nuclei. MATERIALS AND METHODSYeast strain and isolation of nuclei. S. cerevisiae A364A was grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose, 50 ,ug of adenine per ml) at 28°C with shaking. The nuclei were isolated from early-log-phase cells by our previously described method (12).Preparation of 3H-or biotin-labeled pJDB219 DNA. Plasmid pJDB219 (pMB9 LEU2 2,um DNA; for the structure, see

Allergic rhinitis is an inflammatory disease of the nasal mucosa, induced by histamine, leukotrienes, and other substances released from mast cells. Fexofenadine hydrochloride, the active metabolite of terfenadine, is a novel, nonsedating antiallergic drug having H₁ receptor antagonistic activity. Fexofenadine is effective for the treatment of allergic rhinitis. However, its mechanism of action in attenuating nasal congestion has not yet been elucidated. Therefore, we first examined the effects of fexofenadine on a guinea pig model of antigen-induced rhinitis. We also evaluated the effects of mepyramine, zafirlukast and ramatroban in this model; these drugs are an H₁ receptor antagonist, a selective leukotriene antagonist and a selective thromboxane antagonist, respectively. Rhinitis was induced by ovalbumin (OVA) instillation into the nasal cavity of animals that had been sensitized by two earlier OVA injections (s.c. and i.p.). The nasal airway resistance was measured for 45 min after the challenge. Fexofenadine hydrochloride (20 mg/kg) and terfenadine (20 mg/kg) administered orally 70 min prior to the challenge significantly inhibited (fexofenadine, p < 0.001, terfenadine, p < 0.05) the increase in nasal airway resistance. Ramatroban (30 mg/kg) administered orally 60 min prior to the challenge also significantly inhibited (p < 0.05) the increase in nasal airway resistance. In contrast, mepyramine (3 mg/kg i.v.) and zafirlukast (3 mg/kg p.o.) failed to reduce the increase in nasal airway resistance. These results suggest that thromboxane may be involved in the increase in the nasal airway resistance in this model. Accordingly, fexofenadine may reduce the increase in nasal airway resistance by inhibiting the release of chemical mediators, including thromboxane, that are involved in the increase in nasal airway resistance in this model.

These results suggest that mepartricin suppresses hyperplastic growth of the DLP and SV induced by estrogen in immature castrated rats, the underlying mechanism being a reduction in the serum estrogen level, thereby suppressing stromal cell proliferation and activation.