Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

Canopy photosynthetic production in a Japanese larch forest. II. Estimation of the canopy photosynthetic production

Seasonal changes and yearly gross canopy photosynthetic production were estimated for an 18 year old Japanese larch (Larix leptolepis) forest between 1982 and 1984. A canopy photosynthesis model was applied for the estimation, which took into account the effect of light interception by the non-photosynthetic organs. Seasonal changes in photosynthetic ability, amount of canopy leaf area and light environment within the canopy were also taken into account. Amount of leaf area was estimated by the leaf area growth of a single leaf. The change of light environment within the canopy during the growing season was estimated with a light penetration model and the leaf increment within the canopy. Canopy respiration and surplus production were calculated as seasonal and yearly values for the three years studied. Mean yearly estimates of canopy photosynthesis, canopy respiration and surplus production were 37, 13 and 23 tCO₂ ha⁻¹ year⁻¹, respectively. Vertical trend, seasonal changes and yearly values of the estimates were analyzed in relation to environmental and stand factors.     

Construction of integrated system for selection of fused plant protoplasts

Summary An integrated system has been constructed to instantly identify and efficiently sort the heterokaryons formed by plant protoplast fusion. The system is composed of the following functions: a) a transport system, b) an electro-manipulator, c) a cell harvester, d) a flow cytometer/cell sorter, and e) a control device. The conditions for an efficient and reproducible enrichment of the heterokaryons have been investigated by this system using the fluorescein isothiocyanate stained protoplasts preparing from Glycyrrhiza glabra cell cultures and unstained protoplasts of Abrus precatorius cell cultures which contain a large quantity of chlorophyll.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2, 4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscissic acid - FITC fluorescein isothiocyanate This paper is part 96 in the series Studies on Plant Tissue Cultures. For part 95 see Orinara Y., Noguchi T. and Furuya T. (1993) submitted for publication.     

N-Glycan structures of squid rhodopsin.

To determine the glycoforms of squid rhodopsin, N-glycans were released by glycoamidase A digestion, reductively aminated with 2-aminopyridine, and then subjected to 2D HPLC analysis [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem.226, 139-146]. The major glycans of squid rhodopsin were shown to possess the alpha1-3 and alpha1-6 difucosylated innermost GlcNAc residue found in glycoproteins produced by insects and helminths. By combined use of 2D HPLC, electrospray ionization-mass spectrometry and permethylation and gas chromatography-electron ionization mass spectrometry analyses, it was revealed that most (85%) of the N-glycans exhibit the novel structure Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Galbeta1-4Fucalpha1-6)(Fucalpha1-3)GlcNAc.     

Determination of naringin and naringenin in human plasma by high-performance liquid chromatography

An HPLC method for determining a flavonoid, naringin, and its metabolite, naringenin, in human plasma is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using genistin (for naringin) or daidzein (for naringenin) as an internal standard and solid-phase extraction using a Sep-Pak t C₁₈ cartridge. For the determination, HPLC was carried out using an Inertsil ODS-2 column (250x4.6 m I.D., 5 μm particle size). The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v; pH 7.1) for naringin and acetonitrile-0.1 M ammonium acetate solution-acetic acid (30:69:1, v/v; pH 4.9) for naringenin. The flow-rate was 1 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 280 nm for naringin and at 292 nm for naringenin. The detection limits on-column were about 0.2 ng for the two flavonoids.     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.