Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.     

Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l -[³H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and ²²Na⁺ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O₂/79% N₂ (control) or 100% N₂ (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the K_D under hypoxic conditions was twice as large as that under control conditions, whereas the B _max was unchanged. When the specific [³H]nicotine binding was kinetically analyzed, the association constant ( k ₁) but not the dissociation constant ( k ₋₁) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine-evoked ²²Na⁺ flux into the cells was suppressed by hypoxia. In contrast, specific [³H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.     

The role of polypyrrole as charge transfer mediator and immobilization matrix for d-fructose dehydrogenase in a fructose sensor

A fructose dehydrogenase (FDH) modified electrode is produced by the electroadsorption of a layer of FDH on a platinum electrode followed by the electropolymerization of a polypyrrole (PPy) film around and over the enzyme. This immobilizes and stabilizes the enzyme as well as providing an electron transfer pathway to the electrode. The amperometric response to fructose and the enzymatic activity are measured as a function of PPy film thickness. The electrode is shown to have a maximum response at a PPy thickness of approximately the thickness of the enzyme layer. A measure of the electrode efficiency is also obtained, this is the amperometric response to fructose as a percentage of that expected on the basis of the enzyme activity. The functioning of the electrode is also dependent on the counter-ion used for PPy polymerization. This is shown to be mainly related to the nucleation and growth of the PPy film in the interfacial region.     

Influence of hydraulic retention time on anaerobic digestion of pretreated sludge

Continuous anaerobic digestion of waste activated sludge pretreated at low temperatures below 100°C increased methane generation by 30%. pH values of the digestion mixture increased, approximately from 0.3 to 0.55 by pretreatment, although its volatile fatty acids concentration was greater than the control. An abrupt increase in propionate : acetate ratio in digestion stage (e.g. from less than 1:1 to over 3.5 :1), provided a reliable indicator for impending failure.This revised version was published online in October 2005 with corrections to the Cover Date.     

Cell-free synthesis of ornithine decarboxylase. Changes in mRNA activity in the liver of thioacetamide-treated rats

Ornithine decarboxylase (ODC)mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell-free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC-mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC-mRNA but also to a translational and/or posttranslational control.