Eiko Wada

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.

Retarded and distinct progress of lens opacification in congenic hereditary cataract mice, Balb/c-nct/nct

The Nakano cataract gene, nct, was introduced into Balb/c mice by repeated backcrosses to elucidate the possible effects of background genes on its expression. The resulting congenic Balb/c-nct/nct mice were characterized by retarded and sporadic cataract formation with a tendency of further retardation in males and by the different disease process of cataract as compared with Nakano mice. The age of 50% cataract incidence was 60 days in females and 90 days in males compared with 22 days in Nakano mice, and lens opacification commenced in a diffuse, mild form at the cortex in congenic but in a pin-head, intense form at the core in Nakano mice. Sex hormones seemed to be involved in the difference in cateractogenesis between male and female mice. Microphthalmia was slighter in degree in Balb/c background mice. The results indicated that the nct-dependent cataractogenesis may be influenced by background genes and some non-hereditary factors. Balb/c-nct/nct mice will provide a new type of hereditary cataract model.

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.

BIOCHEMICAL EVIDENCE FOR CONVERSION TO MILDER FORM OF HEREDITARY MOUSE CATARACT BY DIFFERENT GENETIC BACKGROUND

Congenic hereditary cataract mice, BALB/c-nct/nct, were established by introducing the nct gene from Nakano into BALB/c mice. These mice developed a milder cortical form of cataract which developed sporadically and later in life than in Nakano mice. Combined use of BALB/c and BALB/c-nct/nct mice enables biochemical comparison of normal clear lenses, congenic clear lenses which are destined to be opacified some time later, and opacified lenses in the same genetic and aging statuses. We compared the age-related changes in water content and water-soluble and -insoluble fractions among these three types of lenses. Congenic clear lenses and opaque lenses were more similar to BALB/c normal clear lenses and Nakano opaque ones, respectively, in these parameters. These results suggest, in addition to formation of aggregated crystallins and their accumulation in water-insoluble fractions, that decreased protein synthesis, increased protein degradation and augmented leakage of crystallin might have a significant role in the nct-induced lens opacification.

A cyclotron microbeam and its application to biomedical samples

Abstract The experimental setup to produce a cyclotron microbeam of 27 MeV α particles and 25 MeV protons is described. Using an ordinary doublet of quadrupole magnets with an aperture diameter of 2.5 in. as a focussing lens, a beam line with demagnification factors D CD = −3.3 and D DC = −16 was constructed. A beam spot with a diameter of less than 15 μm fwhm and an intensity of 0.06 nA was obtained for an extracted beam current of 300 nA. The experimental result is compared with the result of ray trace calculations. The microbeam of 27 MeV α particles is applied to the PIXE positional analysis of biomedical samples and that of 25 MeV protons is used to measure the density distributions in the samples based on the computed tomography (CT) principle. The experimental results of microPIXE and proton CT measurements on the eye from a mouse with a hereditary cataract are presented.

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.

r-Galactonolactone in experimental galactosemic animals

An accumulation of galactose-1,4-lactone, an oxidation product of galactose, was observed in various tissues of galactosemic guinea pigs fed a 40% galactose diet for 6 weeks. In addition, an accumulation of the two galactose metabolites varied among organs. The highest content of the lactone was observed in the liver and the content of the lactone exceeded that of the reduced counterpart. The lens gave the highest galactitol content. In the serum the level of the lactone was very low. A trace amount of the lactone was detected in the kidney while it was mostly excreted into urine within 54 h upon withdrawal of the diet. On the other hand, in the animals kept on a high galactose diet for only 2 days, urinary lactone rapidly decreased. These observations indicated that a high galactose level in the circulation was associated with the production of the lactone in various tissues and that the accumulated lactone was released into the circulation very slowly and then excreted into the urine. Suppression of galactitol production by administration of an aldose reductase inhibitor resulted in the accumulation of the lactone in the lens, the testis, and the muscle, as well as in the circulation. The lactone thus produced was excreted exclusively into the urine. This observation indicates a close relationship between the oxidative and reductive metabolisms of galactose at a toxic level.

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.

Finding of a galactose-oxidation-product in lens of galactose-fed guinea pig

From studies on polyols in lens of galactose-fed guinea pigs, r-galactono-1,4-lactone was found, which proves the presence of galactonic acid as a product of galactose oxidation, by gas liquid chromatography and mass spectrometry. The content of this component was one tenth of that of galactitol. In vitro culture of rat lens in 30 mM galactose-loaded media demonstrated the formation of the lactone. The significance of the lactone was discussed with respect to the galactose metabolism in lens.