Polyol pathway in tissues of spontaneously diabetic Chinese hamsters (Cricetulus griseus) and the effect of an aldose reductase inhibitor, ONO-2235

1. Sorbitol and fructose levels were significantly elevated in the lens, the sciatic nerve, the retina and the kidney of diabetic Chinese hamsters and inositol level was significantly decreased in the lens and sciatic nerve of diabetics. 2. The activity of an aldose reductase in the kidney was not different between normal and diabetic Chinese hamsters. 3. An aldose reductase inhibitor (ONO-2235) had no effect in sorbitol, fructose and inositol contents of all these tissues from diabetic Chinese hamsters. 4. These results suggest that diabetic Chinese hamsters produce polyol accumulation in tissues but that there is a clear species-specific difference to inhibition of aldose reductase.     

Frequency domain studies of impedance characteristics of biological cells using micropipet technique. I. Erythrocyte.

This study aims at precise measurement of the membrane capacity and its frequency dependence of small biological cells using the micropipet technique. The use of AC fields as an input signal enables the magnitude and phase angle of membrane impedance to be measured at various frequencies. The micropipet technique was applied to human erythrocyte, and passive membrane capacity and conductivity were determined between 4 Hz and 10 KHz. Membrane capacity thus determined changed from 1.05 to 0.73 microF/cm2 between 4 Hz and 10 KHz. In addition to the micropipet technique, we used suspension method between 50 KHz and 10 MHz for the purpose of supplementing the new method with the one which has been in use for many years. We obtained a membrane capacity of 0.65-0.8 microF/cm2 using this technique. These values agree with the capacitance obtained with the micropipet method. Although this paper discusses only human erythrocytes, the study has been performed with lymphocytes and various forms of cancer cells. This paper is the first of the series of reports on frequency domain studies of the impedance characteristics of various biological cells.     

Suppression of overt diabetes in NOD mice by anti-thymocyte serum or anti-Thy 1, 2 antibody

Effects of anti-thymocyte serum (ATS) and anti-Thy 1, 2 monoclonal antibody on the spontaneously occurring diabetes in NOD mice were examined. Spontaneous diabetes in female mice was markedly suppressed by intravenous injection of rabbit anti-mouse thymocyte serum diluted to 1:4 on three consecutive days during the time period from 70 to 100 days after birth; the cumulative incidence of overt diabetes upto 195 days of age was greatly reduced and the onset of diabetes was delayed. Similar effect was observed with anti-Thy 1, 2 antibody treatment. These findings suggest that T lymphocytes play a role in the production of spontaneous diabetes in this mouse strain.     

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.     

Genetic analysis for insulitis in NOD mice

Non-obese diabetic (NOD) mice spontaneously develop diabetic signs akin to those of Type I diabetes in man. Insulitis, i.e., lymphocytic infiltration around and into the pancreatic islets is one of the characteristics of such mice. It is also the etiologic pathological lesion in the development of diabetes mellitus in NOD mice. Thus, we chose insulitis as a marker for genetic analysis of the development of diabetes mellitus in NOD mice and clarified the mode of its inheritance. In breeding studies between NOD and C57BL/6J mice, insulitis was not observed in the F1 and (F1 X C57BL/6J) backcross generations, but was found with incidences of 3.9% in females and 1.4% in males in the F2 generation and 23.7% in females and 12.1% in males in the (F1 X NOD) backcross generation. These incidences in the F2 and (F1 X NOD) backcross females corresponded approximately to 1/16 and 1/4 of the incidences of 89.7% in the NOD females, respectively. A similar relationship was observed between the F2 and (F1 X NOD) backcross males and the NOD males. When the gene expressivity of both sexes for a double recessive homozygote was assumed to be the incidences of insulitis in 9-week-old NOD females and males, respectively, the expected numbers of both sexes with and without insulitis in the F2 and backcross generations agreed well with the observed ones. These observations suggest that two recessive genes on independent autosomal chromosomes are necessary for the development of insulitis in NOD mice.     

4-Hydroxyaminoquinoline 1-oxide metabolism and DNA adducts in the early stage of tumorigenesis in rats: comparison of target organ pancreas with non-target organ liver

In order to probe key early molecular events which might be responsible for the initiation of rat pancreatic tumorigenesis by 4-hydroxyaminoquinoline 1-oxide (4-HAQO), the uptake and metabolism of carcinogen and the formation and subsequent repair of DNA adducts were monitored under conditions of high and low tumorigenicity, respectively in partially pancreatectomized and non-operated animals, and in the liver, a non-target organ for this carcinogen. Although uptake of radioactively labelled 4-HAQO was higher in the liver than in the pancreas, generation of DNA adducts was 20 times greater in the latter organ. This discrepancy was probably due to a difference in the metabolic profile of 4-HAQO. The spectrum of the adducts was qualitatively similar in both organs. No qualitative or quantitative differences could be established under the high and low tumorigenicity conditions with regard to DNA adduct formation or persistence. The major difference was the presence of a relatively large extent of pancreatic DNA replication under the high tumorigenic condition. The results indicated that metabolic profile of 4-HAQO, quantity of DNA adducts and levels of DNA replication are key factors involved in initiation of tumorigenesis.     

Sarcolemmal Na+-Ca2+ exchange during the development of genetically determined cardiomyopathy

The UM-X7.1 myopathic and control hamsters at 40, 120 and 280 days of age were employed for the examination of heart sarcolemmal Ca2+-transport activities. Na+-dependent Ca2+ uptake activities were significantly depressed in myopathic animals at 120 and 280 days of age in comparison to the control values. No difference in Na+-induced Ca2+ release activities was found between control and experimental sarcolemmal vesicles. ATP-dependent Ca2+ binding and Ca2+-stimulated, Mg2+ ATPase activities were depressed in the experimental animals at 120 and 280 days of age. Similar alterations in the sarcolemmal Na+-dependent Ca2+ exchange and Ca2+-pump activities were seen upon treating the control hamsters with 40 mg/kg isoproterenol for 24 hr. It is suggested that a depression in the sarcolemmal Ca2+ transport activities may contribute to the development of intracellular Ca2+ overload in the genetically determined cardiomyopathy in hamsters and such a defect may be due to excessive amount circulating catecholamines in these animals.     

Benzodiazepines and their metabolites: relationship between binding affinity to the benzodiazepine receptor and pharmacological activity

Experiments were carried out to study the relationship between binding affinity to the benzodiazepine receptor and pharmacological activity, especially anti-anxiety activity, of clinically useful benzodiazepines. In the in vitro experiments, fludiazepam showed the highest affinity to the benzodiazepine receptor with 4 times more potency than that of diazepam, which paralleled the in vivo activity. Diazepam and nimetazepam also bound with high affinities as expected from their in vivo activities. On the contrary, medazepam and cloxazolam showed extremely low affinities and oxazolam showed no affinity, although they showed moderate in vivo activity. However, their metabolites were found to have both high affinity and in vivo activities. These results strongly suggest that in the case of medazepam, cloxazolam and oxazolam, their metabolites may bind to receptor sites in the brain and then elicit pharmacological action. This conclusion was supported by the fact that a good correlation between the binding affinity and the anti-anxiety activity of the tested compounds was observed.     

Molecular cloning of the cls gene responsible for cardiolipin synthesis in Escherichia coli and phenotypic consequences of its amplification.

The cls gene responsible for cardiolipin synthesis in Escherichia coli K-12 was cloned in a 5-kilobase-pair DNA fragment inserted in a mini-F vector, pML31, and then subcloned into a 2.0-kilobase-pair fragment inserted in pBR322. The initial selection of the gene was accomplished in a cls pss-1 double mutant that had lesions in both cardiolipin and phosphatidylserine synthases and required either the cls or the pss gene product for normal growth at 42 degrees C in a broth medium, NBY, supplemented with 200 mM sucrose. The cloned gene was identified as the cls gene by the recovery and amplification of both cardiolipin and cardiolipin synthase in a cls mutant as well as by the integration of a pBR322 derivative into its genetic locus at 27 min on the chromosome of a polA1 mutant. The maxicell analysis indicated that a protein of molecular weight 46,000 is the gene product. The cls gene is thus most likely the structural gene coding for cardiolipin synthase. Hybrid plasmids of high copy numbers containing the cls gene were growth inhibitory to pss-I mutants under the above selective conditions, whereas they inhibited neither the growth of pss-I mutants at 30 degrees C nor that of pss+ strains at any temperature. Amplification of cardiolipin synthase activity was observed, but was not proportional to the probable gene dosage (the enzyme activity was at most 10 times that in wild-type cells), and cardiolipin synthesis in vivo was at the maximum 1.5 times that in wild-type strains, implying the presence in E. coli cells of a mechanism that avoids cardiolipin overproduction, which is possibly disadvantageous to proper membrane functions.