Physiological assay of liposome-mediated transport of a drug across Xenopus intestine: cell-liposome interaction

(1) The antagonistic effect of atropine methyl bromide entrapped in liposomes on contraction of Xenopus intestine in vitro induced by acetylcholine was studied. The results provided some insight into cell-liposome interaction. (2) Acetylcholine (0.1 mM) was added to the medium in the bath (serosal solution), while liposomes containing atropine methyl bromide in their internal and external phases were added on the mucosal side of the intestine. Large multilamellar liposomes were prepared from egg lecithin (phosphatidylcholine, PC) and cholesterol in various molar ratios. Atropine methyl bromide had most effect in liposomes composed of PC and cholesterol in a ratio of 7:3, less in those with a ratio of 4:5, and none in those with a ratio of 9:1. These effects were parallel with the sizes of these liposomes, determined by quasi-elastic light-scattering; that is, the larger the liposomes, the greater was their effect. Addition (to the liposomes) of phosphatidic acid, the negative charge of which increases the distance between the lamellar layers, increased the effect, indicating that atropine methyl bromide in the space between lamellar layers was effective. Another type of liposomes in which atropine methyl bromide was present only in the external phase of liposomes was as effective as liposomes in which atropine methyl bromide was present in both the internal and external phases. (3) From these results the following new model for liposome-mediated stimulation of transport of atropine methyl bromide is proposed. Large multilamellar liposomes have structural defects in their external lamellae through which atropine methyl bromide in the mucosal solution can penetrate into the space between the external lamellar layers and move into intestinal cells through regions of fusion between the outermost layers of the liposomes and the cell membrane.     

Effect of Clostridium perfringens Beta Toxin on Blood Pressure of Rats

Guanethidine treatment or adrenal medullectomy significantly inhibited the elevation in blood pressure induced by Clostridium perfringens beta toxin, and the combination of the two drastically reduced the pressure rise, to less than 19% of that in control rats. When rats were pretreated with tetrodotoxin or hexamethonium, the toxin-evoked rise was significantly inhibited. Elevation in blood pressure induced by the toxin in spinal rats tended to be less than that in control rats. When investigated by a microscopical technique, arteriolar constriction in the mesenteric vasculature was observed after the blood pressure elevation induced by the toxin reached a maximum. Blood flow in the skin decreased with an increase in blood pressure following intravenous injection of the toxin. It is concluded that beta toxin acts on the autonomic nervous system and produces arterial constriction.     

Congenital absence of flexor pollicis longus without hypoplasia of thenar muscles

A 7-year-old girl who was unable to flex her left thumb at the interphalangeal joint proved to have an extremely hypoplastic flexor pollicis longus with normal thenar muscles, which is very rare. The flexor digitorum superficialis of the ring finger was transferred to the inserting portion of the flexor pollicis longus tendon with good results. The patient's cooperation seems to be a factor determining the prognosis.     

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.     

Developmental regulation of calmodulin-dependent adenylate cyclase activity in an insect endocrine gland.

The insect prothoracic gland produces ecdysteroids that elicit molting and metamorphosis, and neurohormone stimulation of steroidogenesis by this gland involves both Ca2+ and cyclic adenosine monophosphate second messengers. Prothoracic gland adenylate cyclase exhibits a complex Ca2+/calmodulin (CaM) dependence, a component of which requires an activated Gs alpha for expression. A developmental switch in this system has been identified that correlates with a change in both regulation and function of the gland and involves the loss of sensitivity to extracellular Ca2+ at a time approximately concurrent with the loss of Ca2+/CaM sensitivity by the adenylate cyclase. The extent of cholera toxin activation of gland Gs alpha is lowered before this developmental switch. However, no alterations in Gs alpha levels or mobility are detected, suggesting that Gs alpha interaction with another component in the signaling pathway, perhaps adenylate cyclase itself, produces the apparent Ca2+/CaM dependence and influences the ability of toxin to modify Gs alpha.     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20-mer, and its PCR product was a 741-bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20-mer, and its PCR product was a 629-bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double-producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double-producers. However, positive results were obtained for 5 of the 10 non-ET-producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.     

Growth Regulation of Dark-grown Dwarf Barley Coleoptile by the Endogenous IAA Content

Growth curves of dark-grown coleoptiles of 11 isogenic coleoptilardwarf strains of barley (Hordeum vulagare L. cv. Akashinriki:uzu, 5, 77, 97, 105, 125, 131, 133, 136, 145 and 148) were simulatedwith a logistic equation and the endogenous IAA contents ofthe barley strains were determined. Growth analysis of the dwarfbarley coleoptiles revealed that the final coleoptile lengthwas correlated with the growth rate on the 2nd day after germination(r=0.897), when the growth rate was about maximum. The endogenousIAA Content of the barley strains, measured fluorometrically,indicated that on the 2nd day, the dwarf strains contained lessendogenous IAA than the normal Strain. The IAA content on the2nd day was correlated to the growth rate on the 2nd day (r=0.907,except for Strain 145) and the final coleoptile length (r=0.933,except for strains 77 and 145). The correlation, however, wasnot significant on the 3rd day. These results suggested thatthe dwarfism of the dark-grown coleoptiles of the barley Strainsexamined is primarily controlled by the endogenous IAA content. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University, Osaka 558, Japan. (Received February 1, 1982; Accepted April 13, 1982)     

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.