The enhancing effect of nasal absorption of FITC-dextran 4,400 by beta-sitosterol beta-D-glucoside in rabbits.

The effect and mechanism of action of beta-sitosterol beta-D-glucoside (Sit-G) on the in vitro and in vivo nasal absorption of FITC-dextran (molecular weight, 4400; FD-4) in rabbits were studied in comparison with beta-sitosterol (Sit). The FD-4 permeation in the powder dosage form was increased by Sit-G and Sit and related to the uptake of Sit-G and Sit with no changes in the amount of cholesterol in the excised nasal mucosa. The application of Sit and Sit-G increased FD-4 permeation with and without a decrease in transepithelial resistance (TEER), respectively. These results suggested that the mechanism of the enhancement by Sit-G was different from those of Sit and sodium caprate; Sit-G may exert its effects mainly via the transcellular pathway due to perturbation of the mucosal membrane.

Effect of cod-liver oil extract on the buccal permeation of ergotamine tartrate.

Ergotamine tartrate (ET) is used clinically in the treatment of migraines. However, the bioavailability of ET is rather poor following oral administration. Therefore, we tried to improve ET delivery using buccal administration. The purpose of this study was to investigate the characteristics of the permeation of ET through the hamster cheek pouch in vitro using a two-chamber diffusion cell, and to evaluate the effect of permeation enhancers on the transbuccal delivery of ET. Cod-liver oil extract (CLOE), polyoxyethylene hydrogenated castor oil (HCO 60), sodium glycocholate (GC), and sodium caprate (CA) were selected as premeation enhancers considering their low irritancy of the mucosa. When the enhancers were added to the donor cell at a 5% concentration each, the ET permeation rate markedly increased compared with that in a control not containing enhancer. Among these enhancers, CLOE exhibited the greatest effect. Because CLOE is composed of 16 kinds of fatty acids, the enhancement action of each of the major components was separately determined. As major fatty acids, palmitic acid, oleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were selected and their enhancing effects were studied. The enhancing effect of each fatty acid was significantly lower than that of CLOE.

Distribution of glutamate- and GABA-immunoreactive neurons projecting to the cardioacceleratory center of the intermediolateral nucleus of the thoracic cord of SHR and WKY rats: a double-labeling study.

We aimed at (1) determining the distribution of glutamate (Glu)- and gamma-aminobutyric acid (GABA)-containing neurons in the brainstem with projections to the cardioacceleratory sympathetic preganglionic neurons in the intermediolateral nucleus (IML) of the upper thoracic cord and (2) determining whether such afferent projections in spontaneously hypertensive rats (SHR) differ from those of control Wistar-Kyoto (WKY) rats. We used a combination of electrophysiological methods to determine the site of HRP injection in the spinal cord and double-labeling methods for plotting the distribution of Glu- and GABA-immunoreactive neurons with projections to this site. HRP/Glu-labeled neurons (possibly glutamatergic) and HRP/GABA-labeled neurons (possibly GABAergic) were detected in 27% and 7% of the total HRP-labeled neurons of the central autonomic nuclei of 3 SHR rats and 3 WKY rats. HRP/Glu-labeled neurons were distributed predominantly ipsilaterally in 20 nuclei of the medulla oblongata, pons and hypothalamus, while HRP/GABA-labeled neurons were distributed in 7 nuclei of the medulla oblongata. No significant differences were found between the average percentages of HRP/Glu-labeled and HRP/GABA-labeled neurons in SHR and WKY rats. These findings indicate that: (1) the Glu-containing neurons represent a greater proportion than the GABA-containing neurons, (2) the proportions of these neurons appear to be similar in WKY and SHR rats and (3) generation of inbred tachycardia and hypertension in SHR rats can not be attributed to the topological and quantitative differences in the distribution of the glutamatergic and GABAergic neurons in the central autonomic nuclei.

Hypothalamic projections to the spinal segments and preganglionic regions by means of choleratoxin subunit B conjugated HRP in the chicken.

Hypothalamo-spinal projections were studied using the method of retrograde axonal transport of choleratoxin subunit B conjugated to horseradish peroxidase (CTb-HRP) in chickens. CTb-HRP was injected into the segmental and preganglionic regions of spinal segments 23-24 and segments 31-32 which are sympathetically and parasympathetically connected with the cloaca, respectively. Many labeled SP-neurons (segmental projection neurons) were found in the paraventricular nucleus (PVN), except in its lateral protrusion. The distribution of these labeled neurons continued to the more caudal areas such as the vicinity of the nucleus periventricularis hypothalami (PHN), areas medial to the stratum cellulare externum (SCE), the nucleus interstitialis (IS), and the posterior hypothalamic areas. Some labeled SP-neurons were also found in the lateral hypothalamic area. These labeled SP-neurons were distributed in almost the same areas in both cases of injection into segments 23-24 and 31-32. However, the number of labeled neurons was somewhat different rostrocaudally according to the spinal levels injected. In contrast, after microinjections into the spinal preganglionic regions, a small number of PP-neurons (preganglionic projection neurons) were found only in the ventral part of the PVN. These PP-neurons showed the rostrocaudally discernible segregation; PP-neurons in the rostral PVN and those in the caudal PVN were predominantly labeled by microinjections into segments 23-24 and segments 31-32, respectively. The present study suggests that the rostral PVN is more sympathetic and the caudal PVN is more parasympathetic in chickens.(ABSTRACT TRUNCATED AT 250 WORDS)

[Evaluation of toxic effects on yusho causal substances by chick embryo hepatic microsomal enzymes activities].

PCBs, non-ortho chlorine substituted PCBs (Co-PCBs), PCQs and (PCDFs + PCDDs), all of which contained similar compositions of those corresponding in yusho oil, were prepared from a PCB preparation used as a heat exchanger fluid. After dissolved in 1, 4-dioxane, they were applied into the air sac of white leghorn eggs incubated for 16.5 days at 37.5 degrees C. Forty eight hours after injection, the hepatic benzo(a)pyrene hydroxylase (AHH) and 7-ethoxyresorufin deethylase (EROD) activities were assayed. The average relative potencies of induction for the two microsomal drug metabolizing enzymes by (PCDFs + PCDDs), Co-PCBs, PCBs and PCQs were 100, 13.4, 0.0006 and 0.0004, respectively. The toxic effects for yusho disease by these substances were calculated from the relative enzyme induction potencies and the average concentrations in yusho oils with the production dates of February 9 and 10, 1968. Consequently, the relative toxicities of (PCDFs+PCDDs), Co-PCBs, PCBs and PCQs were 100, 13.2, 0.06 and 0.12, respectively. This result, as well as our previous investigations using rats and monkeys, insists that (PCDFs+PCDDs) are the primary causal agents for yusho disease. However, the Co-PCBs, which were recently detected in the yusho oils by us, were revealed to be fairly effective in yusho manifestation. In addition, it was cleared that the hepatic enzyme induction by the Co-PCBs fraction, which contained other several PCB isomers, was almost completely contributed by only Co-PCBs such as 3,4,3',4'-tetra- 3,4,5,3',4'-penta- and 3,4,5,3',4',5'-hexachlorinated biphenyls present in the fraction. A chemical uptake rate from the air sac by the chick embryo decreased significantly in the cases of extremely high doses of PCBs (10,000 micrograms/egg) and PCQs (3,333 and 10,000 micrograms/egg), and result the elevations of hepatic enzymes activities were depressed, indicating that the suitable chemical dose amount to be less than about 1,000 micrograms/egg.

Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia coli. Purification by high performance liquid chromatography and direct analysis by plasma desorption mass spectrometry.

Lipopolysaccharide (LPS) extracted from the deep rough mutant of Escherichia coli D31m4 was disaggregated with 0.1 M EDTA, pH 7.0, and fractionated on a diethylaminoethyl-cellulose column to yield the biphosphate form of LPS. After methylation, the derivative was purified by reverse-phase high performance liquid chromatography using a C18-bonded silica cartridge. A linear gradient of 50-100% isopropyl alcohol/water (93:7, v/v) in acetonitrile/water (93:7, v/v) was used over a period of 60 min. The derivatized LPS showed a single major peak by high performance liquid chromatography, and this hexamethyl hexaacyl LPS was recovered and subjected to chemical analysis, plasma desorption mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. Chemical analysis of the purified hexamethyl LPS quantitated certain key chemical compositions. Plasma desorption mass spectrometry showed a molecular ion (M + CH2 + Na)+ at m/z 2360, which established the molecular formula and Mr to be C116H214N2O39P2 and 2323, respectively. Thus, it contained two each of glucosamine, 2-keto-3-deoxyoctonate, and phosphate; four beta-hydroxymyristates; one laurate; and one myristate. NMR spectroscopy confirmed the locations of the four ester-linked fatty acyl groups. Based on these results and the known structure of free lipid A, the complete structure of the deep-rough chemotype LPS from E. coli can now be presented with confidence. This is the first report of a successful purification to homogeneity and the characterization of the simplest of the LPS at the intact level. This study shows that the natural distribution of the lipid A moiety of LPS from E. coli D31m4 is hexaacyl/pentaacyl in a molar ratio of greater than 90:less than 10. Acid hydrolysis of LPS causes the formation of the lower homologues of the free lipid A.

Projections of upper structure to the spinal cardioacceleratory center in cats: an HRP study using a new microinjection method.

We studied, with the horseradish peroxidase (HRP) method, the supraspinal structure projections to the cardioacceleratory center in the intermediolateral nucleus (ILN) of T3-4 segments of the cat spinal cord. A fiber-filled double-barrel coaxial electrode was devised. The inner barrel electrode, filled with 3 M NaCl solution, was used for stimulating intraspinal structures to determine the cardioacceleratory center. The outer barrel electrode with an Agar stop, filled with a 20% HRP solution, was used for injecting HRP by pulses of positive electric current. In every experiment, HRP was injected into 3 points within the cardioacceleratory center at intervals of 1 mm. Out of 36 experiments, 10 showed that the HRP injection sites were centered in and almost confined to the ILN, and the population of the HRP-labeled cells was not less than 50. Some 1146 HRP-labeled cells were thus identified. They were distributed in the medulla oblongata (72.1%), pons (10.2%), midbrain (8.5%) and hypothalamus (9.2%). They were concentrated in the medullary reticular formation (37.8%), median raphe (26.9%) and pontine reticular formation (10.2%). Contrary to expectation, the HRP-labeled cells were few in the nucleus tractus solitarii (3.7%), paraventricular hypothalamic area (3.3%) and nucleus locus coeruleus (none).