Toru Tsumita

A simple method to measure anti-glycolipid antibody by using complement-mediated immune lysis of fluorescent dye-trapped liposomes

A simple, reproducible, and micro quantity method is described to measure the antibody against glycolipid antigens. The multilamellar liposomes containing carboxyfluorescein (CF), which is self-quenched at high concentration, are prepared by vortexing the dried lipid films consisting of egg lecithin, cholesterol, phosphatidic acid and Forssman glycolipid antigen. On addition of anti-glycolipid serum plus active complement, liposome lysis occurs, and trapped CF is released. The dilution of CF in the external volume abolishes the quenching, resulting in a high fluorescence signal. Experimental conditions to measure anti-glycolipid antibody is established in this paper.

Liposome immune lysis assay (LILA): a simple method to measure anti-protein antibody using protein antigen-bearing liposomes

A new simple immunoassay technique using immune lysis of liposomes was developed to measure antibody against protein antigens. Multilamellar liposomes were composed of dipalmitoylphosphatidylcholine, cholesterol and phosphatidylethanolamine substituted with the hetero-bifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). The protein antigen (human IgG) was coupled to these liposomes after treatment with SPDP and mild reduction. As a release marker, carboxyfluorescein (CF) was entrapped in the liposomes. The CF release was specific to anti-human IgG antibody and depended on the presence of complement. This technique could detect 10(-15) mol of anti-human IgG antibody or human IgG. The liposomes were stable over 8 months at 4 degrees C under nitrogen gas.

Antiglycolipid antibodies in normal and pathologic human sera and synovial fluids.

Antiglycolipid antibodies were measured in normal and pathologic sera and synovial fluids by means of a modified microplate method of complement‐mediated immune lysis of fluorescent dye‐trapped liposomes. All sera of normal subjects had antibodies against globopentaosylceramide (IV3 GalNAcGbOse4Cer), ganglioside GMI, gangliotriaosylceramide, gangliotetraosylceramide, and galactosylneolactotetraosylceramide antigens. Most sera of normal subjects had antibodies against lactotriaosylceramide, N‐glycolylneuraminosyl‐neolactotetraosylceramide (NeuGcnLcOse4Cer), GM3 ganglioside with N‐glycolylneuraminic acid (NeuGcGM3) and GDIa antigens. Differences of titers against IV3GalNAcGbOse4Cer, neolactotetraosylceramide, NeuGcGM3 and NeuGcnLcOse4Cer antigens were observed between sera of normal subjects and pathologic sera from cases of leukemias, lymphomas, several autoimmune diseases and liver diseases.

myo-Inositol binding and transport in brush border membranes of rat kidney

Using hypotonically treated brush border membranes, binding and transport of myo-inositol were examined. By hypotonic treatment, both total and non-specific uptake decreased significantly, but specific uptake was not affected. myo-Inositol release from membranes preloaded by incubation for 2 min was very rapid and about 98% of preloaded myo-inositol was released in 5 min of incubation. However, myo-inositol release from membranes preloaded by incubation for 20 min was fairly slow and 50% of myo-inositol remained in the membranes even after 10 min of incubation. Uptake of myo-inositol decreased by the increase of osmolarity in the medium. However, effect of osmolarity on the uptake was less significant when myo-inositol concentration was lower. Under conditions in which mainly binding occurred, myo-inositol binding to the membranes was measured. Two binding systems were demonstrated and high affinity site could bind 22 pmol/mg protein at most and the apparent Km value was 8.3 muM. Both binding and transport processes were dependent on Na+ and enhanced by Na+-gradient.

myo-Inositol transport in plasma membrane of rat kidney

Abstract The myo -inositol transport system in kidney plasma mambrane preparation was investigated. myo -Inisitol uptake was more rapid than that due to non-specific uptake. Specific myo -inisitol uptake was temperature dependent and pH sensitive; the optimum was at pH 7.4. Specific myo -insitol uptake was inhibited by scyllitol and inosose-2 but not(+)-inositol, d -glucose, d -galactose or mannitol. Inhibition of myo -inositol uptake by scyllitol was of the competitive type. It showed that the transport system is stereospecific and that myo -inositol shares the transport system with scyllitol. Moreover, the specific myo -inositol uptake was inhibited by phlorizin. Counter transport of myo -inositol was demonstrated. The results indicate that myo -inositol uptake by the membrane preparation represents the entry into the intravesicular spaces rather than binding to the membrane. It was concluded that the plasma membrane of rat kidney has a cyclitol carrier system specific to myo -inositol and scyllitol.

Elemental analysis of cataractous lenses by PIXE

Abstract Elemental analysis of the lenses of hereditary and galactose cataracts were carried out by the particle induced X-ray emission technique (PIXE) using 28 MeV α-particle beam from a cyclotron accelerator. The lenses of the hereditarily cataractous mice (ca mouse) showed marked increases with age in the concentrations of Cl and Ca. The spatial distributions of elements in the lenses of guinea pigs, galactose-fed and normal diet-fed were measured by scanning with a small beam spot of 200 μm diameter. It was commonly found that K and Cl were concentrated in the polar regions and S was predominantly located in the central region. In opaque lenses tremendous increases in the concentrations of Cl and Ca accompanied by a decrease in that of K were generally observed.

Ageing and compositional changes of rat lens

In order to elucidate chemical changes in the lenses of aged animals, carbohydrate and fatty acid compositions were studied in 36 healthy male and female Fischer 344 rats from 3 weeks to 32 months of age. Senile cataract was observed on six lenses of 12 rats aged 28-32 months. The carbohydrate content increased rapidly within 7 months of age and remained constant until 29 months. But the myoinositol content showed a maximum at 7 months of age and afterwards a decreasing trend was observed. In cataractous lenses, the myoinositol content decreased rapidly; sorbitol and fructose showed similar changes although the rates were much lower than that of myoinositol. Lens fatty acids increased steadily during the life span and the ratio of unsaturated to saturated fatty acids was maintained in a narrow range (1.20-1.30). However, the value was significantly altered in cataractous lenses. An age-dependent change was found with nervonic acid, which increased markedly from 1.8% of fatty acids at 3 weeks of age to 6.8% at 29 months. In cataractous lenses, the predominant changes noticed were a rapid decrease of arachidonic acid and a high content of nervonic acid.

Studies on lens proteins of mice with hereditary cataract. I. Comparative studies on the chemical and immunochemical properties of the soluble proteins of cataractous and normal mouse lenses

Total soluble and insoluble proteins of the lens were similar in normal and hereditary cataractous mice up to 1 week of age. Thereafter, the normal mouse lens showed a continued increase in weight and protein content until 500 days of age. In cataractous mice, while the total protein content increased up to 60 days and reached a plateau, the soluble protein content declined dramatically from day 22 to day 60, and then the rate of decrease remained constant up to 500 days. At different ages, the soluble proteins were separated by gel filtration into the high molecular weight proteins, alpha-, beta- and gamma-crystallin fractions. All of these showed an age-related increase in the normal lens, and the relative values of alpha- and beta-crystallins increased for a 410-day period. On the other hand, in the cataractous process, the high molecular weight protein increased, and alpha-, beta- and gamma-crystallins decreased: the degree was especially marked in gamma-crystallin. Immunochemical studies indicated that the aggregation of beta-crystallin occurred much earlier in the cataractous lens than in the normal. Analysis of the amino acid composition and ultraviolet absorption spectra revealed no significant chemical differences between the crystallins of the normal and the cataractous lens.

Properties of scyllitol transport in rat kidney slices

Abstract The transport of scyllitol in rat kidney slices was examined. Scyllitol was actively transported as was myoinositol but (+)-inositol was not. Scyllitol uptake was competitively inhibited by myoinositol and apparent influx Km of scyllitol and Ki by myoinositol were 1.7 · 10−5 and 8.4 · 10−5 M, respectively. Scyllitol uptake was also inhibited by inosose-3, dinitrophenol, ouabain and phlorizin. No effect was found with d -glucose, d galactose, mannitol and (+)-inositol. These results suggest that scyllitol shares the transport system with myoinositol, and that the transport system has stereospecificity with regard to OH orientation.

PIXE and microprobe analysis of normal and cataractous lenses

Abstract The external beam PIXE analysis was applied to twenty-two human lenses with senile cataract and ten normal human lenses to investigate the changes of the elements associated with opacification. The lenses with senile cataract were found to be clearly classified into two types by the amounts of potassium and calcium. One type contained the normal level of potassium and calcium and the other was characterized by the lowered potassium and the significantly raised calcium. Then, the distributions of sulfur, chlorine, potassium and calcium in the sagittal planes of the lyophilized lenses of the two types of senile cataract were compared using an external 27 MeV α-particle microprobe with resolution of around 100 μm. In both lenses, relatively uniform distributions were obtained for all elements measured. Elemental distributions were also obtained in the rat lenses with ionophore cataract induced by valinomycin in vitro . Changes in the distributions of potassium and rubidium were observed in the valinomycin treated lenses; however, the distribution of chlorine was almost unchanged.