Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Poly-cis carotene pathway in the Scenedesmus mutant C-6D

The mutation of Scenedesmus obliquus strain C-6D is expressed in the dark only. Under these conditions, this strain synthesizes acyclic poly-cis carotenes. Cyclic carotenoids like -carotene or xanthophylls are absent. In the light the normal cyclic carotenes and xanthophylls are synthesized in the all-trans configuration. The poly-cis carotencs present in dark cultures have been analysed and quantitated. It could be shown that these poly-cis carotenes are identical with the poly-cis carotenes synthesized by the tomato mutant Lycopersicon esculentum var. Tangella. The poly-cis pathways, however, are different regulated in the two organisms. The tomato mutant accumulates prolycopene as the major carotene, whereas the mutant C-6D accumulates mainly pro--carotene. Furthermore, the mutation in Tangella is constitutive in light in contrast to Scenedesmus C-6D. Besides that, Scenedesmus C-6D synthesizes a further cis-carotene isomer of phytofluene as well as of -carotene. The configuration of these carotenes still have to be elucidated. The occurrence of this poly-cis carotene biosynthetic pathway by a mutation of only one enzyme, the phytoene desaturase which, however, is only expressed in darkness under heterotrophic conditions, is discussed.     

Functional vasoactive intestinal polypeptide (VIP)-system in salt glands of the Pekin duck

Summary In saltwater-acclimated ducks with fully specialized supraorbital salt glands, intracarotid application of acetylcholine (5 nmoles/min/kg b.w.) or porcine vasoactive intestinal polypeptide (pVIP) (240 pmoles/min/kg b.w.) induced secretion from the salt glands at threshold conditions of secretory activity. pVIP-like immunoreactivity could be localized in fibers of the postganglionic secretory nerve ramifying throughout the glandular parenchyma. Both middle-sized arterioles and secretory tubules were innervated, and pVIP-immunoreactive varicose fibers formed peritubular baskets around the basal region of secretory tubules indicating direct innervation of the secretory tissue. pVIP-specific staining could be abolished by preabsorption of the antiserum with peptide extracts of salt-gland tissue. Synthetic pVIP and endogenous VIP from salt glands of the duck co-eluted on the HPLC system, suggesting structural similarity of the peptides. Membrane-binding studies with radioiodinated pVIP revealed the presence of high-affinity binding sites in salt-gland tissue. Affinities of unlabeled pVIP analogues to compete for these binding sites were as follows: pVIP > PHI > pVIP antagonist > secretin > pVIP (10–28) > chicken VIP (16–28). Peptide extracts of salt glands had affinities similar to pVIP. Binding sites could be localized mainly at the apical end of the radially arranged secretory tubules, as demonstrated by receptor autoradiography.It is concluded that, in addition to the classical parasympathetic transmitter acetycholine, VIP serves as neuromodulator/transmitter in cranial parasympathetic control of avian salt-gland secretion by acting on both the arteriolar network and the secretory tubules of the gland.     

Chemical characterization of rat cholecystokinin-58

Cholecystokinin-58 (CCK-58) was purified from rat intestines using an extraction method that yields large amounts of this peptide. Greater than 30% of total CCK immunoreactivity eluted before CCK-39 upon gel permeation chromatography (Sephadex G-50) if extracts were loaded onto Sep Pak cartridges before freezing. If the extracts were frozen and stored at −70°C for six weeks, only 20% of the material eluted in this region and total immunoreactivity was reduced by 50%, suggesting that proteases were active under these storage conditions. This early eluting peak was purified by reverse phase and ion-exchange HPLC to a single absorbance peak. Microsequence analysis of this peak detected AVLRPDSEP which is the amino terminus of rat CCK-58 predicted from the rat preprocholecystokinin cDNA. Because degradation of CCK-58 occurred in these extracts, it is possible that CCK-58 is the predominant molecule form in the rat small intestine.     

Fast HPLC Estimation of γ-Aminobutyric Acid in Microdialysis Perfusates: Effect of Nipecotic and 3-Mercaptopropionic Acids

A method for rapid, automated (less than 5 min), and sensitive (detection limit 50 fmol/10 microliter) determination of gamma-aminobutyric acid (GABA) is described. The method is based on precolumn derivatization with o-phthaldialdehyde/t-butylthiol reagent and separation by reverse-phase HPLC with electrochemical detection under isocratic conditions. A 100 X 4 mm Nucleosil 3 C18 column was used; the mobile phase consisted of 0.15 M sodium acetate, 1 mM EDTA (pH 5.4), and 50% acetonitrile; the flow rate was 0.8 ml/min. The potential of the glassy carbon working electrode was +0.75 V. The method allows for the monitoring of GABA levels in the extracellular fluid sampled by microdialysis as documented in the present study when 0.5 mM nipecotic acid is infused via the probe, or 3-mercaptopropionic acid is injected at a dose of 100 mg/kg i.p. There was a 15-fold increase of extracellular GABA after nipecotic acid, whereas in the second case the inhibition of GABA synthesis was followed by a 74% decrease of GABA as compared to basal levels.     

寡糖的荧光标记高效液相色谱分析

 利用氨化还原的方法把高量子产率的荧光标记物——α-萘胺,接到异麦芽糖寡糖的还原端。用硅胶薄层色谱、荧光光谱及快原子轰击质谱证实反应物的完全性及其结构。高效液相色谱用Micropak si5硅胶柱,以乙腈-水(含0.05％三乙胺)为梯度洗脱液可使含有二到九个糖残基的异麦芽糖寡糖的α-萘胺衍生物全部分离。本法对异麦芽糖二糖的荧光检测灵敏测度为2.35Pmol。     

The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine.

A stereospecific high-performance liquid chromatographic (HPLC) method was developed for the quantitation of the enantiomers of venlafaxine, an antidepressant, in dog, rat, and human plasma. The procedure involves derivatization of venlafaxine with the chiral reagent, (+)-S-naproxen chloride, and a postderivatization procedure. The method was linear in the range of 50 to 5,000 ng of each enantiomer per ml of plasma. No interference by endogenous substances or known metabolites of venlafaxine occurred. Studies to characterize the disposition of the enantiomers of venlafaxine were conducted in dog, rat, and human, following oral administration of venlafaxine. The Cmax, area under the curve (AUC) and (S)/(R) concentration ratios of the (R)- and (S)-enantiomers were compared. In rats, the mean plasma ratio of (S)-venlafaxine to that of (R)-venlafaxine over 0.5 to 6.0 h varied from 2.97 to 8.50 with a mean value of 5.51 +/- 2.45. The Cmax, AUC0-infinity, and t 1/2 values of the (R)- and (S)-enantiomers in dogs were not significantly different from one another (P greater than 0.1). The mean ratios [(S)/(R)] of enantiomers of venlafaxine in human over a 2 to 6 h interval ranged from 1.33 to 1.35 with an overall ratio of 1.34 +/- 0.26 (n = 12). These ratios of the enantiomers [(S)/(R)] were not statistically different from unity (P greater than 0.1) indicating that the disposition of venlafaxine enantiomers in humans is not stereoselective and is more similar to that in dogs than that in rats.     

4',6-Dichloroflavan (BW683C): Enantiomeric separation by hplc/dad and antirhinovirus activity

Racemic 4',6-dichloroflavan (BW683C), a highly effective inhibitor of rhinovirus serotype 1B in vitro, was resolved by high-performance liquid chromatography on a chiral stationary phase. The enantiomers were separately collected and circular dichroism curves were obtained, in order to determine the absolute configuration of the two enantiomers. The activity of the isomers was studied on human rhinovirus serotype 1B multiplication in HeLa cell cultures, by means of the plaque reduction assay. Both enantiomers were potent inhibitors of virus replication; by comparing the IC₅₀ values, the S form was 3.5 times more effective than the R form.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.