Simultaneous determination of nitrazepam and its metabolites in urine by micellar electrokinetic capillary chromatography

We applied micellar electrokinetic capillary chromatography to simultaneous separation and determination of nitrazepam and its major metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, in spiked urine. Prior to electrophoresis, the three compounds were successfully extracted from the spiked urine with commercial disposable solid-phase cartridges. The optimum running buffer for the separation was prepared by combining 85 parts of 60 mM sodium dodecyl sulphate—6 mM phosphate—borate, adjusted to pH 8.5, with 15 parts of methanol. The separation order, completed within 25 min, was 7-aminonitrazepam > 7-acetamidonitrazepam > nitrazepam, at an applied potential of 20 kV. We obtained reproducible electropherograms in successive repetitions, and few other peaks or interferences appeared in the electropherogram. The detection limits of the three compounds were 50–100 pg (0.1–0.2 μg/ml of analyte in spiked urine), and the recoveries were 78.9–100.8% for 1 μg/ml and 84.1–100.3% for 5 μg/ml. The application of this method to forensic or clinical samples is demonstrated.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

Inhibition of azoreductase activity by antibodies against cytochromes P-450 and P-448

Hepatic microsomal azoreductase activity in mice was induced with phenobarbital (PB) and 3-methylcholanthrene (3-MC). Antibodies against cytochrome P-450 inhibited azoreductase activity of PB-treated animals while antibodies against cytochrome P-448 inhibited liver azoreductase activity of 3-MC-treated animals, each by about 90%. These antibodies also inhibited microsomal 7-ethoxycoumarin-O-deethylase activity to the same extent. It is concluded that hepatic microsomal azoreductase activity is almost totally dependent on cytochromes P-450 and P-448 and the contribution, if any, of other microsomal components is negligible.     

Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.     

Biosystematic studies onDisporum (Liliaceae-Polygonatae)

Both the floral biology and morphometrics of two Japanese species of AsianDisporum (sectionEudisporum) are presented. These two species,D. sessile andD. smilacinum, represent extremes in both floral morphology and divergence in pollination within the section. The inverted flowers ofD. sessile have an elongate floral tube formed by the imbrication of the oblanceolate tepals. The tepal bases are modified into well developed, saccate nectaries. The stamens have rigid, vertical filaments which tightly encircle the ovary-style axis, and extrorse anthers located within a floral cavity which can accommodate a large pollinator (cross-pollination). The stigma is exserted and the depth of its cleft formation constant.D. smilacinum, in contrast, has an open, nodding campanulate flower with lanceolate tepals which have only shallow nectaries at their bases. The stamens have widely divergent filaments with versatile anthers that have laterally introrse dehiscence (wind and/or self-pollination). The depth of the stigma cleft is variable. For both species, the pattern of differential UV absorption and reflectance is similar. It is suggested on morphological grounds and by pollinator observation, thatD. sessile with a high energy flower requiring specialized visitors represents a more advanced condition than that observed inD. smilacinum, which is more generalized and primitive. Seasonal herbivore pressure on the tepal nectaries ofD. sessile is discussed in relation to its pollination.     

Deterrent effect of soybean meal on feeding of the silkworm, Bombyx mori

The occurrence of a factor in soybean which deters feeding of the silkworm was demonstrated. The deterrent was not extractable with ether or ethanol, but was extracted with 90% ethanol or hot water. Preliminary experiments showed that the deterrent was adsorbed on charcoal, but neither on activated alumina nor in a steam distillate. Autoclaving did not inactivate the deleterious effect. A crude extract of the deterrent showed a marked negative feeding response by including 0·4 mg/g of the dry diet.Inclusion of the crude extract in the diet retarded the growth of larvae, whereas larvae grew well when receiving the 90% ethanol-washed meal. Feeding response for various soybean meals and proteins were compared, and better larval growth has generally obtained when the feeding response was more positive. It is, however, inconclusive whether or not the deleterious principle possesses some physiologically toxic effect.The feeding behaviour of newly hatched larvae was observed in the presence of favourable and/or unfavourable diets, and a brief discussion is made concerning the feeding behaviour of this insect.     

Biosystematic studies inErythronium (Liliaceae-Tulipeae)

The floral biology ofErythronium japonicum has been studied from two approaches: a reinvestigation of its floral morphology and a pollinator case history. The perianth, differentiated into a sepal and petal cycle, has a tubular, but free arrangement basally around a slightly stipitate ovary. The two cycles of stamens with dimorphic filaments are positioned by the differently lobed auricles of the mature sepals and petals. These auricles also form a trap-lid mechanism for the inverted nectary which also has passageways. The perianth parts are highly UV absorbant due to the presence of flavonoids. This pattern contrasts strikingly with the purple trident basal guide lines so prominent in the visible spectrum. The weakly protandrous flowers also have exserted styles, thus functioning to exclude its own pollen and insure outbreeding. These floral adaptations are related specifically to the pollination behavior ofXylocopa appendiculata, and in general to the floral evolution within the genusErythronium. This work was supported in part by the U.S.-Japan Cooperative Science Program Grant GF-41367 and Grant-in-Aid No. 934053 from the Ministry of Education, Japan.     

Ultrastructure of perivascular amyloid fibrils in Alzheimer’s disease

Perivascular amyloid fibrils in the brains of patients with Alzheimer's disease have been examined by electron microscopy. The amyloid fibrils showed a hollow rod structure and consisted of globular substances. Each turn appeared to be composed of five globular subunits. These findings coincide with the ultrastructure of amyloid fibrils obtained from replicas made by a rapid freezing method.