Minori Kamaya

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.

Loss and Gain of Human Acidic Mammalian Chitinase Activity by Nonsynonymous SNPs

Acidic mammalian chitinase (AMCase) is implicated in asthma, allergic inflammation, and food processing. Little is known about genetic and evolutional regulation of chitinolytic activity of AMCase. Here, we relate human AMCase polymorphisms to the mouse AMCase, and show that the highly active variants encoded by nonsynonymous single-nucleotide polymorphisms (nsSNPs) are consistent with the mouse AMCase sequence. The chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. By creating mouse-human chimeric AMCase protein we found that the presence of the N-terminal region of human AMCase containing conserved active site residues reduced the enzymatic activity of the molecule. We were able to significantly increase the activity of human AMCase by amino acid substitutions encoded by nsSNPs (N45, D47, and R61) with those conserved in the mouse homologue (D45, N47, and M61). For abolition of the mouse AMCase activity, introduction of M61R mutation was sufficient. M61 is conserved in most of primates other than human and orangutan as well as in other mammals. Orangutan has I61 substitution, which also markedly reduced the activity of the mouse AMCase, indicating that the M61 is a crucial residue for the chitinolytic activity. Altogether, our data suggest that human AMCase has lost its chitinolytic activity by integration of nsSNPs during evolution and that the enzyme can be reactivated by introducing amino acids conserved in the mouse counterpart.

Determination of sulphate and phosphate by flow-injection analysis using a barium chloranilate packed column

SummaryA flow-injection analysis of sulphate using a reaction column packed with barium chloranilate (BaCh) is described. The chloranilate ion released by the reaction is monitored at the isosbestic point (310 nm) of chloranilate. An aqueous solution of 2-propanol (60%) is used as the carrier solution at a flow-rate of 1 ml . min−1. The linear dynamic range of the detection system is approximately 2.5×10−6∼1.0×10−3 mol/l per 10 μl sample injection. The time required for a determination is only 20 s per sample injection. The method has also been applied to the amplified determination of phosphate by reaction with molybdate resulting from the decomposition of molybdophosphate (H3PO4.MoO3) extracted.

Photometric determination of selenium with ferrocene

A sensitive spectrophotometric method for the determination of selenium has been developed. The method depends on a redox reaction between selenious acid in 8M hydrochloric acid and a chloroform solution of ferrocene. One mole of selenious acid produces 4 moles of ferricenium ions, which are then oxidized by bromine water. The resulting iron(III) is reduced to iron(II) and determined with 1,10-phenanthroline. The relative standard deviation for 20 mug of selenium was 1.1%. The apparent molar absorptivity of the final solution, referred to selenious acid, is 4.23 x 10(4) 1. mole(-1). cm(-1) at 512 nm. This method has been used to determine selenium in copper metal.

Determination of trace amounts of sulfide in human serum by high-performance liquid chromatography with fluorometric detection after derivatization with 2-amino-5-N,N-diethylaminotoluene and iron(III)

Abstract A fluorometric high-performance liquid-chromatographic method is described for the determination of sulfide in human serum. The sulfide ion had been derived into a fluorescent compound with 2-amino-5-N, N-diethylaminotoluene and Fe (III) under an acidic condition. The compound was extracted into 2-octanol as an ion-pair with sodium 1-octane sulfonate. The extract was separated by a reversed phase method (column, Inertsil ODS-2, 250mm × 4.6mm i.d.; mobile phase, 90% (V/V) acetonitrile aqueous solution containing sodium 1-octanesulfonate as a counter ion reagent; flow rate 0.5cm3· min−1 and detected fluorometrically (Ex. 640nm; Em. 675nm). The sulfide ion could be determined over the range from 4.4 × 10−10M to 4.4 × 10−7M. The coefficient of variation at 4.4 × 10−8M of sulfide was 3.2% (n=7). The range of concentration of sulfide ion in human serum were from 3.04 × 10−8M to 2.24 × 10−8M by this method.

Electrochemical gas sensors using electrolytic films of poly(ethylene oxide)/Zn, Cu, Ni trifluoromethane sulphonates for flow injection analysis of nitrogen dioxide

Abstract Au/poly(ethylene oxide)nM(CF3SO3)2/M (M = Zn, Cu, Ni) galvanic gas sensors, consisting of a disk 13 mm in diameter and 1.1 mm thick, have been developed by using a polymeric electrolyte film (0.1 mm thick). The polymeric films are made by mixing with an acetonitrile solution both M(CF3SO3)2 and poly(ethylene oxide) (m.w. ≈ 6 × 105). The working electrode (25 nm thick) is made by sputtering gold in an argon atmosphere. Of the three kinds of sensors, the zinc one (n = 55) shows the greatest sensitivity. For the zinc cell, the maximum response appears within 3 s after the sample (2.6 ml) is injected into the nitrogen carrier stream, flowing at a rate of 28 ml min−1; the response returns to the baseline within 3 min at the longest. The maximum current flowing in the external circuit is linearly related to the concentration of NO2 in the range 0.02–1 ppm at ambient temperature.

Comparative study of barium salts of 2,5-dihalo-3,6-dihydroxy-p-benzoquinone as reagents for the spectrophotometric determination of sulphate

Abstract The spectrophotometric determination of sulphate employing barium salts of fluoranilic, bromanilic or iodanilic acid was studied. The synthesis of fluoranilic and iodanilic acids and the preparation of the barium salts of the haloanilic acids as reagents are described. Suitable reaction conditions for linear calibration were established. The molar absorptivities for the reaction products of the Ba haloanilates with sulphate were between 1.87 × 10 4 and 2.78 × 10 4 1 mol −1 cm −1 at their absorption maxima. The relative standard deviation at 5 μg of sulphate was

Mouse acidic mammalian chitinase exhibits transglycosylation activity at somatic tissue pH

Mouse acidic mammalian chitinase (AMCase) degrades chitin with highest efficiency at pH 2.0 and is active up to pH 8.0. Here, we report that mouse AMCase also exhibits transglycosylation activity under neutral conditions. We incubated natural and artificial chitin substrates with mouse AMCase at pH 2.0 or 7.0 and analyzed the resulting oligomers using an improved method of fluorescence‐assisted carbohydrate electrophoresis. Mouse AMCase produces primarily dimers of N‐acetyl‐d‐glucosamine [(GlcNAc)2] under both pH conditions while generating transglycosylated (GlcNAc)3 primarily at pH 7.0 and at lower levels at pH 2.0. These results indicate that mouse AMCase catalyzes hydrolysis as well as transglycosylation and suggest that this enzyme can play a novel role under physiological conditions in peripheral tissues, such as the lungs.

Indirect spectrophotometric determination of sulfate with barium nitranilate.

An indirect spectrophotometric method for the determination of sulfate using barium nitranilate has been investigated. The analytical procedure is as follows: Add 30 mg of barium nitranilate to 5 ml of ethanolic solution (1 + 10) containing sulfate (525 μg) in a 10-ml stopped centrifuge tube. Shake for 5 min with mechanical shaker, and centrifuge the precipitate for 10 min at 300 rpm. Measure the absorbance of the supernatant liquid at 300 nm with a 1-cm quartz cell against a reagent blank. Beers law holds and the present method showed good reproducibility of relative standard deviation of 1.1 %, and the molar absorptivity for sulfate is 1.72 × 104 1mol-1 cm-1. Phosphate, thiosulfate, arsenate, chromate, sulfite, oxalate, calcium and strontium ions interfered with the determination of the analyte. The present method was applied to the determination of sulfate in tap water.

REFLECTIOMETRIC DETERMINATION OF TRACE UREA USING UREASE

Sample solution (10 μl) was mixed with the urease solution (5 μl), which contained 1800 units urease and 25 ml glycerin in 100 ml, in the tip of a micro pipette. After standing for 20 min, the mixture (15 μl) was dropped on the pH test paper, and the reflection absorbance (λ=570 nm) of the test paper was measured by spectrophotometry. The pH test paper was prepared by placing 40 μl of 0.1 % (w/v) Bromophenol Red {pH 5.2(yellow)pH 7.0(red)} aq. solution (pH 5.2) onto the filter paper (5B) and drying it in a desicator for 12 h. A linear relation was obtained between the concentration of urea (5120 ngN/10 μl) and the reflection absorbance of the test paper. The detection limit (S/N=3) was 2 ngN/10μl. Reproducibility tests (n=7) showed that the relative standard deviation of the response was 5.5% for 30 ngN/10μl.