The development of a radioimmunoassay for formoterol

The development of a radioimmunoassay (RIA) for the beta 2-stimulant formoterol is described. The sensitivity of the method is 0.1 ng/ml in plasma and urine, when a 1-ml sample is used. The cross-reactivity of the antiserum with formoterol glucuronide was 30%. Since formoterol is metabolized extensively to formoterol glucuronide in rats, dogs and humans, extraction with ethyl ether prior to the radioimmunoassay was carried out. Satisfactory agreement was obtained for levels of formoterol in plasma and urine when they were determined by RIA and gas chromatography-mass spectrometry. The concentration of formoterol was determined in dog plasma and human urine after oral administration of formoterol fumarate to dogs (61 mcg/kg) and humans (40 mcg).     

Evidence for the involvement of multiple forms of cytochrome P-450 in the occurrence of sex-related differences of drug metabolism in the rat

Multiple forms of cytochrome P-450 in liver microsomes of untreated male and female rats could be divided into several fractions by the use of ω-amino-n-octyl Seph. 4B and DE-52 columns. Male cytochrome P-450 fractions (I-b - I-e) differed from female fractions (I-b - I-e) with respect to absorption peaks in carbon monoxide difference spectra and 7-prop-oxycoumarin O-depropylation activities. Although male and female I-a fractions showed quite similar protein bands on SDS-polyacrylamide gel electrophoresis, some protein bands in other male fractions (I-b - I-e) were absent in corresponding female fractions. Immunochemical examinations using immunoglobulin G raised to cytochrome P-450 purified from untreated male rats also showed that liver microsomes from male and female rats contain different forms of cytochrome P-450. Based on these results, we propose that sex-related differences of drug metabolizing activities in liver microsomes are caused by multiple forms of cytochrome P-450.     

Development of clonidine-tolerance in the rat vas deferens: Cross tolerance to other presynaptic inhibitory agents

Possibility of the development of clonidine-tolerance in the peripheral nervous tissue was examined using vas deferens isolated from rats chronically treated with clonidine. Rats were treated with clonidine for 10 days by adding the drug to drinking water (10 μg/ml). For the control rats, drug-free tap water was provided. Electrically evoked twitch response of vas deferens was suppressed by adenosine, β-endorphine and α₂-adrenergic agonists, such as clonidine and B-HT 933, both in control and clonidine-treated groups. Vas deferens isolated from clonidine-treated rats showed significantly lower responsiveness to the inhibitory effects of clonidine and B-HT 933 compared to those from control rats. Vas deferens from clonidine-treated rats also was less responsive to adenosine and β-endorphin, both of which interact with presynaptic inhibitory receptors other than α₂-adrenergic and muscarinic cholinergic stimulation responsiveness of the postsynaptic smooth muscle to both α-adrenergic and muscarinic cholinergic stimulation did not change after 10 days of treatment with clonidine. These results suggest that clonidinetolerance can be induced in the peripheral nervous system by chronic treatment of this drug and that the tolerance is not specific to α₂-adrenergic agonists. Some common pathway in the inhibitory mechanisms of various agents or possible interactions between the different types of presynaptic inhibitory receptors may be involved in this phenomenon.     

Triglyceride turnover: a comparison of simultaneous determinations using the radioglyceride and the lipolytic rate procedures.

Two different methods of examining triglyceride turnover are described. One measures the clearance of endogenously labeled triglyceride glycerol, the radioglyceride method. The other assesses the hydrolysis of endogenous very low density lipoproteins after a primed infusion of maximal doses of heparin, the lipolytic rate. Both depend on widely different assumptions. A comparison of data obtained by both methods at the same time in single individuals with widely differing triglyceride concentrations revealed them to agree quite closely. This correspondence indirectly suggests the validity of the procedures. The procedures have been applied to show that fat-free high-carbohydrate diets accelerate triglyceride production in some humans.     

Distribution of Corynebacterium renale among healthy bulls with special reference to inhabitation of type III in the prepuce.

Distribution of Corynebacterium renale among apparently healthy bulls reared in Hokkaido was investigated. The organism was detected from 46 (39.3%) of 117 specimens of preputial cavity washing and from 60 (51.7%) of 116 specimens of semen. The isolates studied in this survey belonged to type III, except a few which belonged to type II. No type I strain was isolated from any bull. C. renale type III was isolated from the prepuce in six of seven bulls slaughtered and from urethra in three, but not at all from any other organ. In the seven bulls, no macroscopic changes were seen, but a slight infiltration of lymphocytes and formation of lymph nodules were noticed in the prepuce. No other microscopical changes could be demonstrated in any other organ. No serum antibody response was detected. To ascertain the virulence of C. renale isolated from the bulls, a strain of type II was inoculated into the urinary bladder of a healthy cow. The cow exhibited fever and hematuria on and after the 10th day. Typical cystitis was proved when the cow was necropsied on the 14th day after inoculation. From these result it is conceived that C. renale type II organisms inhabit the prepuce of apparently healthy bulls at a high rate, without inducing any disturbance.     

Enhancement of binding of N-hydroxy-Trp-P-2 to DNA by seryl-tRNA synthetase

Covalent binding to DNA of a mutagenic metabolite of Trp-P-2, N-hydroxy-Trp-P-2, was examined in the presence of seryl-tRNA synthetase. Both ATP and L-serine were essential requirements for this binding. In the absence of seryl-tRNA synthetase, the binding was reduced to about 14% of the complete system. These results indicate that seryl-tRNA synthetase which is widely distributed in tissues of experimental animals might act as the activating enzyme of N-hydroxy-Trp-P-2.     

Resolution of the enantiomers of aldoses by liquid chromatography of diastereoisomeric 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates

Acyclic diastereoisomers, namely, 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates are readily obtained by reductive amination of aldoses with chiral α-methylbenzylamine (MBA) in the presence of sodium cyanoborohydride, and may be separated by reversed-phase 1.c. and, more effectively, by adsorption 1.c. According to this procedure, enantiomers of the common, neutral aldoses are resolved. In adsorption 1.c., l-l_* [defined as an adduct of an l-aldose and l-(-)-MBA] is eluted before d-l_* for erythrose, xylose, ribose, glucose, 4-O-methylglucose, galactose, and fucose, and the elution order is the reverse for arabinose, lyxose, mannose, rhamnose, and glyceraldehyde. This behavior is probably related to the configuration of C-2 of the aldoses.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Unequal first cleavage in the Tubifex egg: involvement of a monastral mitotic apparatus

The first cleavage in the freshwater oligochaete Tubifex hattai is unequal and meridional, and produces a smaller cell AB and a larger cell CD. This study traces the process of furrow formation, reorganization of cortical F-actin and the assembly of a mitotic apparatus during this unequal division. Cleavage furrow formation consists of two stages: (i) when eggs are viewed from the animal pole, meridionally running furrows emerge at two points of the egg's equator that are 90° apart from each other and approach the egg axis as they deepen; and (ii) at the midpoint between the equator and the egg center, the bottoms of these furrows link to each other on the animal and vegetal surfaces of the egg and form a continuous ring of constriction in a plane parallel to the egg axis. Egg cortices, isolated during the first step and stained with rhodamine-phalloidin, show that the bottoms of recently formed furrows are underlaid by a belt of tightly packed actin bundles (i.e. a contractile arc). The transition to the second stage of furrow formation coincides with the conversion of these actin belts into a continuous ring of F-actin. Whole-mount immunocytochemistry of microtubules reveals that the first cleavage in Tubifex involves an asymmetric mitotic spindle, which initially possesses an aster at one pole but not the other. This ‘monastral’ spindle is located at the egg's center and orients itself perpendicular to the egg axis. During anaphase, astral rays elongate to reach the cell surface, so that the array of astral microtubules in the plane of the egg's equator covers a sector of 270–300°. In contrast, it is not until the transition to telophase that microtubules emanating from the anastral spindle pole approach the cell margin. If eggs are compressed along the egg axis or forced to elongate, they form monastral spindles and divide unequally. In living compressed eggs, mitotic spindles, which are recognizable as bright streaks at the egg's center, appear not to shift their position along the spindle axis during division, suggesting that without eccentric migration of spindles Tubifex eggs are able to divide unequally. These results suggest that mechanisms that translocate the mitotic spindle eccentrically do not operate in Tubifex eggs during the first cell cycle. The mechanisms that generate asymmetry in spindle organization are discussed in the light of the present results.