Morphological analysis of cat masseteric motoneurons after intracellular staining with horseradish peroxidase.

Intracellular injection of horseradish peroxidase (HRP) into 58 masseteric motoneurons identified by antidromic activation was performed in cats under pentobarbital anesthesia. Monosynaptic EPSPs were evoked by masseteric nerve stimuli in 52 cells, and were absent in the remaining six cells. The antidromic nature of the evoked spikes was confirmed by IS-SD separation observed at high frequency (50 Hz) stimulation. Motoneurons with monosynaptic excitation from masseter afferents showed IPSPs following stimulation of lingual and inferior alveolar nerves. Motoneurons which did not show monosynaptic excitation from masseter afferents showed no IPSPs from the above nerves. There were no differences in cell size or the number of stem dendrites between motoneurons with and without monosynaptic EPSPs. No recurrent collaterals were observed in any motor axons. Motoneurons with monosynaptic EPSPs were located at all rostrocaudal levels throughout the trigeminal motor nucleus, whereas motoneurons without such EPSPs were encountered only at the middle level. Dendrites of motoneurons with monosynaptic EPSPs did not extend into the medial portion of the nucleus where motoneurons innervating the anterior belly of the digastric muscle were located. In contrast, motoneurons without monosynaptic EPSPs had dendrite branches extending well into the medial part. The results show that there are two subpopulations of masseteric motoneurons that differ in peripheral inputs as well as dendritic morphology.

High fructose intake significantly reduces kidney copper concentrations in diabetic, islet transplanted rats.

Trace element status is known to be altered in the diabetic state, although the factors affecting trace element homeostasis in this condition are not well understood. The authors examined the effects of a high fructose diet (40% wt:wt) vs a control diet on the copper (Cu), zinc (Zn), and iron (Fe) concentrations in the kidney, plasma, and red blood cells of islet transplanted (TX) and sham-operated (SHAM) rats. Male, Wistar Furth rats made diabetic by streptozotocin injection (55 mg/kg, iv) were given an intraportal islet transplant (1000 islets); control animals were sham-injected, sham-operated (SHAM). Rats within TX and SHAM groups were assigned to either a high fructose diet (40% fructose, 25% cornstarch, FR) or a purified control diet (33% cornstarch, 33% dextrose, CNTL) containing identical amounts of mineral mixture for a period of 6 wk. Kidney Cu concentration was significantly elevated among hyperglycemic TX-CNTL rats (224+/-25 nmol/g wet wt), but was markedly reduced in hyperglycemic TX-FR rats (109+/-14 nmol/g) relative to normoglycemic controls. This occurred in spite of similar levels of glucose, insulin (fed and fasted), insulin secretory capacity, body weight, and food intake in the TX-CNTL and TX-FR groups. Among the subgroup of rats with normal glucose levels post-TX, kidney Cu levels normalized and were unaffected by dietary treatment (normoglycemic TX-CNTL = 60+/-5 nmol/g; normoglycemic TX-FR = 40+/-2 nmol/g). Kidney Cu concentrations also were unaffected by fructose feeding in SHAM animals (CNTL, 60+/-4 nmol/g and FR, 51+/-5 nmol/g). Kidney Zn and Fe concentrations were similar among the treatment groups. Plasma and red blood cell (RBC) Cu, Zn, and Fe concentrations were also similar among the groups. Since fructose feeding led to a substantial reduction of kidney Cu concentrations in the presence of hyperglycemia, the authors suggest that this model can be useful in examining effects of altered kidney Cu accumulation in the diabetic animal.

Active and passive exposure status to tobacco smoke of department store employees measured by cotinine ELISA.

Quantitation of urinary cotinine, a major metabolite of nicotine, by an enzyme-linked immunosorbent assay (ELISA), was performed in parallel with questionnaires containing items on smoking status, such as active and/or passive smokers, the number of cigarettes smoked, and the presence or absence of active smokers in the surroundings in a department store (517 employees). The cotinine values corrected by creatinine (cotinine-creatinine ratios, CCRs) approximately conformed to the extent of self-recognition of their exposure status to tobacco-smoke, and were low in the order of active smokers, passive smokers and non-smokers who felt they were not exposed to tobacco-smoke. Occupational differences of the CCRs were not found in the employees.In the active smokers, the CCRs were increasing according to the number of cigarettes per day they smoked, and the values were nearly proportional to nicotine contents of cigarette in the moderate smokers who smoked 11-20 cigarettes per day. The CCRs of males were higher than those of females in the active smokers, which also agreed well with the numbers of cigarettes they smoked per day. In the passive smokers, the CCRs were remarkably and significantly higher in subjects who felt they were exposed to tobacco-smoke both in their workplaces and homes.Urinary CCRs measured by ELISA are thus found to be a reliable and excellent objective indicator of both active and passive exposure-status to tobacco-smoke.

Copper status of young men consuming a low-copper diet.

A study was conducted in 11 young men to evaluate the effect of a low-copper diet on indexes of copper status and to define an amount of dietary copper at which adequate copper status could not be maintained. The young men were confined to a metabolic research unit for 90 d. The study was divided into three periods, with dietary copper as the only variable. Dietary copper was 0.66 mg/d for 24 d, 0.38 mg/d for 42 d, and 2.49 mg/d for 24 d. Plasma copper, ceruloplasmin activity, ceruloplasmin concentration, and erythrocyte superoxide dismutase (SOD) were measured at selected time points during each dietary copper period. Urine was collected throughout the study. Plasma copper, ceruloplasmin concentration and activity, and urinary copper declined significantly during the lowest dietary copper period. Plasma copper, ceruloplasmin concentration, and urinary copper increased in response to repletion. The average erythrocyte SOD concentration was lower during the depletion period than in the periods before or after depletion, but it did not decline significantly over time in the depletion period. The results suggest that these indexes are sensitive to copper depletion; that 0.38 mg Cu/d is not sufficient to maintain copper status in normal, healthy young men; and that the minimum dietary copper requirement is between 0.4 and 0.8 mg/d.

Influence of dietary folic acid on the developmental toxicity of methanol and the frequency of chromosomal breakage in the CD-1 mouse.

The proposed increased use of methanol (MeOH)-based fuels raises the concern for an increased risk for MeOH toxicity. MeOH, which is detoxified in part via a folate-dependent pathway, is known to be teratogenic in rodents. Previous observations have implicated maternal folate status as a critical modulator for the developmental toxicity of MeOH. The current study extends these findings, examining the effect of maternal dietary folate intake on fetal folate stores, as well as identifying a possible marker for the prediction of the developmental toxicity of MeOH. Virgin female CD-1 mice were assigned to diets containing either 400 (marginal) or 1200 (control) nmol folic acid (FA)/kg, and and 1% succinylsulfathiazole for 5 weeks prior to mating and throughout breeding and gestation. From gestation day (GD) 6 through 10 dams were given by gavage deionized, distilled water (dH2O) or MeOH at 2.5 g/kg body weight, twice daily. On GD 18, mice were weighed and killed and the liver, kidneys, and gravid uteri removed and weighed. Implantation sites, live and dead fetuses, and resorptions were counted; fetuses were weighed individually and examined for cleft palate and exencephaly. The marginal FA dietary treatment resulted in low maternal liver (50% reduction) and red cell folate (30% reduction) concentrations, as well as low fetal tissue folate concentrations (60 to 70% reduction) relative to the adequate FA dietary groups. Marginal FA treatment alone resulted in cleft palate in 13% of the litters; there were no litters affected with cleft palate in the adequate FA-control group. Marginal FA-MeOH treatment resulted in a further increase in the litters affected by cleft palate (72% of litters affected). The percent of litters affected by exencephaly was highest in the marginal FA-MeOH group. The frequency of micronuclei in maternal and fetal reticulocytes, a marker for chromosomal abnormalities, was not influenced by either the marginal FA diet or by MeOH treatment. These results show that marginal folate deficiency in pregnant dams significantly increases the teratogenicity of MeOH.

Influence of maternal folate status on the developmental toxicity of methanol in the CD-1 mouse.

Methanol, which is detoxified via a folic acid-dependent pathway, has been shown to be teratogenic in mice. Given recent observations that the level of dietary folic acid intake may be inversely related to the occurrence of select birth defects in humans, we tested the hypothesis that dietary folic acid intake would influence the developmental toxicity of methanol. Virgin female mice were fed one of three diets containing 400 (low), 600 (marginal), or 1,200 (adequate) nmol folic acid/kg diet for 5 weeks prior to and following mating. On gestation days (GD) 6-15, dams were administered by gavage either vehicle (distilled, deionized water) or methanol at 2.0 or 2.5 g/kg body weight, twice daily. On GD 18, mice were weighed and killed and the liver, kidneys, and gravid uteri removed and weighed. Implantation sites, live and dead fetuses, and resorptions were counted; fetuses were weighed individually and examined for cleft palate and exencephaly. One third of the fetuses in each litter were examined for skeletal morphology. Maternal liver folate concentrations were approximately 40-50% lower in the low dietary folic acid groups than in the marginal and adequate groups; methanol did not affect maternal liver folate concentration at term. Maternal net gestational weight gain was lowest at the lowest dietary folate level but was not affected by methanol. Gravid uterus weights were lowest in the low dietary folic acid groups exposed to the high methanol dose and the number of live fetuses per litter was lowest in the low folic acid groups. Fetal body weights were lowest in the low folic acid groups and significantly lower in the methanol groups relative to vehicle-treated animals. Fetal crown-rump lengths were shorter in the methanol-treated groups; this parameter was not affected by folic acid treatment. Both methanol and low dietary folic acid increased the incidence of cleft palate, with the highest number of affected litters in the low dietary folic acid group. These results support the concept that maternal folate status can modulate the developmental toxicity of methanol.

Effects of transfer of hybridomas producing various isotypes of immunoglobulins on the C1q metabolism in mice.

In order to elucidate possible effects of immunoglobulin on C1q metabolism at the anabolic steps, serum C1q levels and C1q mRNA of peritoneal exudate cells (PEC) and spleen cells were measured in female BALB/c mice implanted intraperitoneally with complement-(C)-fixing IgG2b- or non-C-fixing class IgG3-producing hybridomas and/or with immunoglobulin-non-productive myeloma cells (p3x63-Ag.8.653)(myeloma 653)(2 x 10(6)/0.2 ml) or without any treatment as controls. In the IgG2b-hybridoma-treated mice, the serum C1q levels and C1q mRNA in PEC increased conspicuously as compared with those in the controls, but C1q mRNA in spleen cells was almost equal to that in the control mice. On the other hand, in the IgG3-hybridoma-treated mice, the serum C1q levels decreased significantly, but the extent of such decrease and the level of C1q mRNA in their PEC were almost equivalent to those in the myeloma 653-implanted mice. The serum C1q levels and C1q mRNA in PEC fluctuated similarly in mice injected intraperitoneally with highly purified IgG2b and/or IgG3 preparations. These results suggest some anabolic interaction, as well as catabolic interaction, between the C-fixing class of immunoglobulin and C1q.

A deletion of mitochondrial DNA in murine doxorubicin-induced cardiotoxicity.

A deletion of about 4 kb has been determined in the mutated mitochondrial DNA (mtDNA) in cardiomyocytes with chronic doxorubicin (DOX)-induced cardiotoxicity in mouse. The incidence of the mtDNA deletion increased with the dosage and with the duration of the DOX administration. Coenzyme Q10 administration prevented the mtDNA deletion and decreased the thiobarbituric acid reactive substance content in the heart mitochondria, suggesting some free radical involvement in this mtDNA deletion. This mtDNA deletion may be involved in cardiomyopathy, which is known to be dosage-dependently induced by DOX administration.

Vitamin E deficiency has a pathological role in myocytolysis in cardiomyopathic Syrian hamster (BIO14.6).

This study revealed the occurrence of vitamin E deficiency in the myocardium of 60-day-old Syrian cardiomyopathic hamsters (BIO14.6), and that this deficiency might be related to the increase in lipid peroxide. Vitamin E administration for ten days effectively restored creatininekinase activity and decreased the lipid peroxide content in the myocardium, returning these to normal control levels (F1b). These results indicate that vitamin E deficiency, possibly combined with oxidative stress in the early cardiomyopathic stage plays an important role in initiating the pathogenesis of myocardial lesions.

[Quantitative analysis of myocardial fibrosis in myocardial biopsies--a comparison between computer analysis and point counting method].

To select an appropriate method to analyze quantitatively myocardial fibrosis in myocardial biopsies, two methods, the computer analysis and the point-counting method observed at magnifications of x200 and x400, were compared. Our targeted points of examination were the accuracy and reproducibility of these methods. Twenty patients (10 with dilated cardiomyopathy and 10 with hypertrophic cardiomyopathy) were randomly selected, and the percent area of myocardial fibrosis in myocardial biopsies obtained from the right ventricular septum was measured by both the computer analysis and the point-counting method. Two observers measured the same area twice on the different days, independently. In the area of analysis, the endocardium was excluded to avoid the observer's bias. By comparing the data obtained from two observers, it was shown that the point-counting method tended to give a larger mean value and standard deviation than the computer analysis method, but that the latter indicated better reproducibility than the former. Our result showed that the degree of myocardial fibrosis varied according to methods of analysis and observer's experiences. It is recommended that the comparison of myocardial fibrosis should be made only when the methods of analysis of fibrosis are identical.

Effect of UV-B irradiation on release of arachidonic acid from B-16 melanoma cells.

B-16 melanoma cells in culture were prelabeled with (3H)-arachidonate, and exposed to UV radiation. Immediately after irradiation the cells released labeled materials. This UV-stimulated release was inhibited by mepacrine (20 microM) and calmodulin inhibitor W7 (0.5 microM). To determine the influence of extracellular Ca2+ on the UV-stimulated release, experiments were made with media containing various concentrations of Ca2+. The release decreased significantly at lower Ca2+ concentrations. These results suggest that Ca2+-calmodulin-dependent phospholipase A2 was involved in UV-stimulated release of radiolabeled materials, possibly arachidonic acid and its metabolites, from the cells.

Membrane responses of B-16 melanoma cells to single exposure to ultraviolet light.

Electron spin resonance spectroscopy using the spin probe (5-, 12- and 16-deoxylstearic acid) was employed to analyze the changes in membrane fluidity in B-16 melanoma cells following UV-B exposure. The UV exposure resulted in the immediate accumulation of lipid peroxide, being accompanied by a change in membrane fluidity. The 12-DSA is the most sensitive to the changes in membrane organization caused by UV light. Na+,K+-ATPase activity was regulated by a change in membrane fluidity. Following UV exposure, the release of the prelabeled arachidonic acid from the cells was observed immediately. Ca2+-dependent calmodulin-dependent phospholipase A2-like activity was involved in the UV-stimulated arachidonic acid release from phospholipid.

Effect of dietary restriction during lactation on cardiac output, organ blood flow and organ weights of rats.

Cardiac output, organ blood flow and organ weights were examined in rats assigned at d 0 of lactation to a control (C) group fed ad libitum or an acutely restricted (AR) group fed 50% of the intake of C dams. Dams in each group were assigned to subgroups for measurement of milk yield or cardiac output, blood flow and organ weights. At d 14 of lactation, cardiac output and blood flow were measured with radiolabeled microspheres and milk yield with the tritiated water method. In AR dams cardiac output was 55% of that of C dams, but cardiac output relative to body weight did not differ between groups. Mammary gland blood flow and weight were reduced in AR dams. The weight of the kidneys, gastrointestinal tract and liver of the AR dams was less than that of C dams; however, relative blood flow to these organs did not differ between groups. Milk yield was reduced by 58% in AR dams compared to C dams. We conclude that dietary restriction during lactation negatively affects absolute cardiac output, blood flow to the mammary glands and milk yield, and that the reduced milk yield is associated with the decrease in mammary gland weight and blood flow.

Spin labeling study on membrane fluidity of epidermal cell (cow snout epidermis).

Cow snout epidermal cells digested by 0.25% trypsin were separated into three regions of keratinocytes by Percoll density gradient centrifugation. The membrane fluidity of keratinocytes in each region was measured by electron spin resonance using 5-doxyl stearic acid (5-DSA) as a labeling agent. The order parameter(s) showed higher values as the depth of the epidermis decreased: lower region of epidermis, 0.632; upper region, 0.645; and horny cells, 0.680. These data indicated that membrane fluidity of epidermal cells decreased as cells approached the surface.

Membrane fluidity in Ehrlich ascites tumor cells treated with adriamycin.

The incubation of Ehrlich ascites tumor cells with adriamycin resulted in an increase in lipid peroxide content and a decrease in membrane fluidity as measured by electron spin resonance using the paramagnetic probe 5-doxylstearic acid. Coincidently, the incorporation of [3H]thymidine into tumor cells was progressively inhibited as the concentration of adriamycin was increased. The results indicate that adriamycin induces changes in the plasma membrane of Ehrlich ascites tumor cells after exposure to a low, but cytotoxic, level of this agent.

Delayed alteration of membrane fluidity in intact cultured B-16 melanoma cells affected by ultraviolet irradiation.

Lipid peroxidation in the plasma membrane has been reported to decrease membrane fluidity. We examined membrane fluidity in relation to lipid peroxidation processes after UV-B exposure of cultured B-16 melanoma cells. UV exposure promptly increased TBA-positive material(s), but alteration of membrane fluidity was delayed. Plasma membrane fluidity increased significantly 6 hours after exposure when the TBA-value(s) had become under the control level. To examine the direct effect of lipid peroxides on the fluidity, tert-butyl hydroperoxide was added to B-16 melanoma cells. Similar results were obtained with respect to membrane fluidity. These results suggest that lipid peroxidation at UV doses maintaining cell viability does not directly induce a significant alteration of membrane fluidity, but may influence the fluidity either during metabolizing processes of UV-induced lipid peroxides or during repair processes following oxidative cell membrane damage.