This review describes a new outcome of photoaffinity labeling methods for the analysis of functional sites within receptor molecules. Diazirine-based photoaffinity labeling was extensively investigated to improve azide-based conventional methods. First of all, a series of simple methods for modifying diazirines bearing an aromatic ring has been accomplished. This first versatile approach involving direct substitution on the aromatic ring of diazirines has been achieved by means of the aromatic thallation of alkoxyphenyldiazirines. Introduction of the thallium moiety was successfully followed by nitration, iodination, or palladiumcatalyzed carbonylation to give a family of substituted aryl diazirines useful for photolabeling. The methoxyphenyldiazirines were also found to be stable under certain demethylation conditions, and a tether to link diazirines with ligands or radioactive markers were readily introduced to the resulting phenols. These new methods of derivatization provide a practical approach to simplify the timeconsuming processes currently used for diazirine synthesis. Secondly, the diazirine-based photoaffinity labeling was systematically compared with a conventional aryl-azide method in the course of photoaffinity labeling of the eel sodium channels. A tetrodotoxin derivative carrying an aryl-diazirine was specifically photoincorporated to toxin-binding region within the sodium channel polypeptide. Labeled sites were successfully identified by probing protease-digested labeled fragments with several sequence-directed antipeptide-antibodies. The results suggest that the diazirine moiety on the toxin orients to the region between S5 and S6 in domain III and IV of the channel. The corresponding azide derivative gave no positive results probably due to their chemical limitations : their photogenerated intermediates are less reactive and the crosslinks induced between ligands and channels are less stable.
Living organisms including humans drive circadian rhythm, and this rhythm influences the social structure and daily life of human beings. The suprachiasmatic nucleus (SCN) has been established as a pacemaker for mammalian circadian rhythm. There are many kinds of neurotransmitters and neuromodulators in this nucleus. The circadian systems are involved in oscillation, input (entrainment) and output (overt rhythm). Therefore in this review, pharmacological characteristics of circadian systems in relation to the SCN are discussed by focussing especially upon the roles of various neurotransmitters and modulators.
Gangliosides are sialic acid containing glycosphingolipids found in highest concentrations in central nervous tissues (CNS). More than 100 species of gangliosides including extremely minor components exist in CNS tissues. We have developed a new method using Q-Sepharose to efficiently purify them from complex mixtures of bovine brain gangliosides. Among the isolated gangliosides a unique new series of gangliosides was shown to exist in the polysialoganglioside fraction. They possessed N-acetylneuraminic acid attached to N-acetylgalactosamine in α2-6 linkage. The immunohistochemical staining of human and rat brains with specific monoclonal antibody to the gangliosides showed that they are stained intensely the neuropile of dorsal horn on spinal cord, being presumably expressed on the cholinergic nerve terminals. On the basis of these observations, extremely minor species may play significant roles in CNS. The present data also indicated that brain tissues contain a new type of α2-6 sialytransferase to act on GalNAc in the gangliotetraose structure.
Recent studies on the three new photometric detection in the liquid chromatographic methods have been reviewed. The detector and systems based on these detection theories and their applications to several fields have been also described. Differential photometric detection using UV-visible absorbing eluents enabled us to examine the retention and detection mechanism of sample and eluent ions in ion exchange chromatography. Photometric detection in ion chromatography determined transparent ionic compounds using indirect photometric detection mode of this theory. This method was useful for many samples such as environmental water and foods. Polarized photometric detection method has been developed by introducing two polarizers on either side of the UV-visible absorbance detector flow cell. The monitor determined optically active compounds as the change in absorbance. This method was applied for determining food additives such as sugars and organic compounds. A HPLC system with a chemiluminescence detector has been developed for the determination of trace levels of mutagenic nitroarenes such as 1, 3-, 1, 6-, 1, 8-dinitropyrenes and 1-nitropyrene. Utilizing this system, their exhaust from vehicles and behavior in air were examined in detail. This system was also used for determining methamphetamines in abuser urine and hair.
A sensitive quantitative analysis by thin layer chromatography was developed for the determination of platelet activating factor (PAF) and other phospholipids in human saliva. The saliva sample (0.6ml) was pretreated by diatomite column extraction with chloroform-methanol (95 : 5, v/v). The extract (20μl) was spotted on a TLC plate. The mobile phase was chloroform-methanol-water (65 : 35 : 7, v/v). The development proceeded until the mobile phase front reached 8 cm from the spotted point, this process usually required 30 min. After development, phospholipase C and alkaline phosphatase solutions were sprayed on the TLC plate at 45°C to hydrolyze phospholipids. By spraying a mixture of ammonium molybdate and Malachite Green, the produced phosphate was changed to molybdophosphate-Malachite Green aggregate, which gave a blue green spot. The colored spots were scanned at 620 nm by chromatoscanner. A linear relationship was obtained between peak area and PAF concentration in the range from 2 to 100 pmol/spot with a relative standard deviation of 2% (n=7). By this procedure, lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in human saliva were also determined sensitively. PAF levels in the range from 40 to 300 ng/ml were found in normal human salivas. Although differences in the total amounts of phospholipids in saliva were found for each healthy volunteer and sampling time, the composition of phospholipids was proved to be virtually constant.
A simple and precise method was established for the determination of synephrine in oriental pharmaceutical decoctions containing Aurantii Fructus Immaturus using high-performance liquid chromatography with sodium dodecyl sulfate (SDS) as an ion-pair reagent. Synephrine was eluted within 32 min without interference from co-existing components using an ODS column and a mixture of water-acetonitrile-SDS-phosphoric acid (75 : 25 : 0.5 : 0.1 ; v/v/w/v, pH=2.6) as a mobile phase.
A pre-column derivatization method for the high-performance liquid chromatographic determination of taurine (1), L-glutamine (2), vitamin U (3) and L-aspartic acid (4) in pharmaceuticals has been developed. The optimum requirements for the derivatization conditions and the stability of resulting derivatives were discussed. The compounds were converted into DNT derivatives through the amino group by reaction with sodium 2, 6-dinitro-4-trifluoromethylbenzenesulfonate (DNTS) in 50% sodium borate at 60°C for 30 min (1), at 60°C for 90 min (2), at 60°C for 80 min (3) and at 80°C for 90 min (4). After the reaction mixtures were acidified with dil. HCl, the derivatives were separated on a Cosmosil 3C18 (4.6mm i.d.×50mm) column using 1% acetic acid-methanol (13 : 7) containing 2mM sodium 1-heptanesulfonate as mobile phase with a ultra violet detector at 280nm. The precisions of the analytical values expressed as the coefficient of variation were below 2.0%. The recoveries of 1-4added to various commercial samples were in the range of 97.8-100.6%.
Moisture sorption properties of gelatin powder and collagen fibers were investigated on the basis of the moisture sorption isotherm, the differential heat of moisture sorption, the decrease in entropy of moisture sorption and the parameter constants of the applicable isotherm equation. The amount of moisture sorbed on collagen fibers was larger than that on gelatin powder. The water molecules were absorbed on gelatin itself rather than on the active sites of gelatin. They were adsorbed on the polar groups of constitutive amino acids at amounts of moisture sorbed up to one or two monolayers and then were absorbed into collagen fibers at higher monolayers. They were kept loosely in gelatin and tightly in collagen. The structural stability of collagen to moisture was higher than that of gelatin.