Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen.

The oxidation of human and rat erythrocyte ghost membranes by molecular oxygen has been performed in an aqueous suspension at 37 degrees C. A constant rate of oxygen uptake was observed in the presence of radical initiator. alpha-Tocopherol in the membrane suppressed the oxidation and the induction period was clearly observed. alpha-Tocopherol decreased linearly during the induction period and when it was depleted the induction period was over and a rapid oxidation started. The rate of oxidation was proportional to the square root of the rate of initial radical generation. The kinetic chain length, the ratio of the rate of propagation to that of initiation, was long, ranging from 7 to 100. These results indicate that the erythrocyte ghost membranes are oxidized by a free radical chain mechanism by molecular oxygen. Among the fatty acids of membrane lipids, polyunsaturated fatty acids were oxidized exclusively. Proteins as well as polyunsaturated fatty acids were oxidized and the formation of the high- and low-molecular-weight proteins and the decrease of protein bands were observed on gel electrophoresis.

Y chromosome haplogroup D2a1 is significantly associated with high levels of luteinizing hormone in Japanese men.

The association between the Y chromosome haplogroup D2 and risk of azoospermia and low sperm motility has been previously studied, and it was indicated that haplogroups DE (YAP lineage) are associated with prostate cancer risk in Japanese males. Our assumption had been that Y chromosome haplogroups may be associated with sex hormone levels, because sex hormones have been deemed responsible for spermatogenesis and carcinogenesis. In this study, we assessed the association between Y chromosome haplogroups and sex hormone levels, including those of testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin-B, and calculated free testosterone (cFT), in 901 young men from the general Japanese population (cohort 1) and 786 Japanese men of proven fertility (cohort 2). We found that the haplogroup D2a1 was significantly associated with high LH levels in a combined analysis involving two cohorts (ß = 0.068, SE = 0.025, p = 0.0075), following correction for multiple testing. To date, this result is the first evidence that implicates Y chromosome haplogroups in an association with sex hormone levels.

NF-kappa B and I-kappa B overexpression in articular chondrocytes with progression of type II collagen-induced arthritis in DBA/1 mouse knees.

OBJECTIVE: To investigate the roles of NF-kappa B and I-kappa B in chondrocytes in the cartilage destruction of mouse collagen-induced arthritis. METHODS: DBA/1 mice were immunized with bovine type II collagen (C II) emulsified with Freund's incomplete adjuvant. Histological abnormalities in the mouse knees were evaluated in sections stained with hematoxylin and eosin, or toluidine blue. Immunostaining using anti-NF-kappa B and anti-I-kappa B antibodies was performed to observe the expression and nuclear shift of NF-kappa B and I-kappa B in articular chondrocytes. RESULTS: Loss of metachromasia of perichondrial cartilage was found in 8 out of 10 knees of the mice at 5 weeks, and in all 10 knees at 7 weeks after C II-immunization. The mean percentage of NF-kappa B-positive chondrocytes at 3, 5 and 7 weeks and in the control was 33.6 +/- 7.6, 54.9 +/- 2.7, 75.9 +/- 1.0 and 27.9 +/- 3.8%, respectively. The mean percentage of NF-kappa B-positive nuclei was 9.1 +/- 0.7, 19.5 +/- 3.7, 47.5 +/- 3.0 and 8.6 +/- 0.1%, respectively. NF-kappa B-positive chondrocytes significantly increased from 3 to 7 weeks in a time-dependent manner (p < 0.0001). The percentage of I-kappa B-positive chondrocytes at 3, 5 and 7 weeks and in the control was 15.5 +/- 1.7, 41.1 +/- 2.5, 51.2 +/- 5.7 and 12.1 +/- 2.1%, respectively. I-kappa B-positive chondrocytes significantly increased from 3 to 5 weeks (p < 0.005). CONCLUSION: These findings suggest that nuclear shift of NF-kappa B localization causes cartilage destruction in the early stage of arthritis in DBA/1 mice immunized with C II.

The anxiolytic-like effect of MCI-225, a selective NA reuptake inhibitor with 5-HT3 receptor antagonism.

We have previously reported that MCI-225, a selective noradrenaline (NA) reuptake inhibitor with serotonin (5-HT)3 receptor antagonism, shows antidepressant-like properties in experiments using rodents. In this study, we investigated the effect of MCI-225 in anxiety models in comparison with diazepam, ondansetron, maprotiline, imipramine, and trazodone. In social interaction (SI) test in rats, MCI-225 (10 and 30 mg/kg, p.o.), diazepam (1-10 mg/kg, p.o.), and a selective 5-HT3 receptor antagonist ondansetron (1 mg/kg, p.o.) significantly increas

Effects of a novel compound MCI-225 on impaired learning and memory in rats.

Effects of MCI-225, [4-(2-fluorophenyl)-6-methyl-2-(1-piperazinyl)thieno[2,3-d]pyrimidine monohydrate hydrochloride] on experimental amnesia were studied in rats and compared with those of THA [9-amino-1,2,3,4-tetrahydroacridine]. In the Morris-type water maze task, MCI-225 (1-10 mg/kg, PO) reduced the spatial learning impairment induced by scopolamine (0.5 mg/kg, IP). In a passive avoidance (PA) task, administration of MCI-225 prior to training (1-30 mg/kg, PO) lessened the carbon dioxide (CO2)-induced amnesia in a dose-dependent manner. MCI-225 (1-100 mg/kg) did not affect gross behavior. THA (0.1-3 mg/kg, PO) reduced scopolamine-induced learning deficits in the water maze task, but the effect was not significant. THA (0.3-3 mg/kg, PO) also ameliorated the CO2-induced amnesia, although slightly, in the PA task. THA (10 mg/kg, PO) increased locomotor activity and higher dose of THA (30 mg/kg, PO) induced tremor, hypersalivation, and muscle relaxation. These results suggest that MCI-225 lessens impairments in learning and memory without causing serious behavioral abnormalities.

MCI-225, a novel thienopyrimidine analog, enhances attentional eye tracking in midpontine pretrigeminal preparation.

The effects of MCI-225, a novel psychoactive compound, and reference drugs on attention behavior were studied using visual stimulus induced vertical eye tracking movements in midpontine pretrigeminal (PTG) feline preparation. Surgery was performed under ether anesthesia and subsequently switched to nitrous oxide-fluothane which was discontinued only during experimental sessions. In addition xylocaine was locally injected. Vertical eye movements were monitored by electrooculogram (EOG) and a TV camera. To compare the effects of drugs on eye movement, numbers of spontaneous and tracking eye movements exceeding a present amplitude in EOG were counted before and during the visual stimulation, respectively. MCI-225 (1 and 3 mg/kg, i.v.) enhanced tracking movements dose-dependently without an increase in spontaneous eye movements. No or little change of the electrocorticogram (ECoG) was seen with 1 mg/kg MCI-225 and a slight increase in low voltage fast pattern was observed with 3 mg/kg, i.v.. On the other hand, tacrine (0.3 mg/kg, i.v.), physostigmine (0.03 mg/kg, i.v.) and methylphenidate (0.3 mg/kg, i.v.) enhanced both types of eye movement and induced ECoG arousal. Desipramine (3 mg/kg, i.v.) slightly increased spontaneous eye movement without affecting tracking movements. Piracetam (100 mg/kg, i.v.) decreased spontaneous eye movements only. These data clearly show that MCI-225 enhances attention to a moving object and suggest that MCI-225 could be useful in the treatment of attentional deficits and related cognitive dysfunctions in psychiatric disorders.

Effects of MCI-225 on memory and glucose utilization in basal forebrain-lesioned rats.

The effects of MCI-225 on amnesia, the cerebral glucose metabolism, and choline acetyltransferase (ChAT) activity in basal forebrain (BF)-lesioned rats were studied in comparison with those of tacrine. Bilateral BF lesions with ibotenic acid impaired the performance in passive avoidance (PA) tasks. Single administration of MCI-225 (10 mg/kg, PO) after a 2-week postoperative recovery period, increased the escape latencies in the PA task, but was not statistically significant. Repeated administration of MCI-225 (0.3 and 1 mg/kg, PO for 6 days) significantly reversed the PA failure. The BF-lesioned rat exhibited a marked decrease in the local cerebral glucose utilization (LCGU) in the frontal cortex, parietal cortex, and caudate-putamen. MCI-225 (1 mg/kg, PO for 5 days) significantly ameliorated the reduction of the LCGU in the parietal cortex. MCI-225 did not change the decrease in the cortical ChAT activity induced by the BF lesion. Repeated administration of tacrine reversed the PA failure (0.3 mg/kg, PO) but failed to prevent the decrement in the LCGU and the ChAT activity. These results suggest that MCI-225 could be effective in the treatment of senile dementia of the Alzheimer type, which is accompanied with both deficit in the BF-cortex cholinergic neuron and cerebral glucose hypometabolism.

Interferon-gamma and tumor necrosis factor-alpha levels in sera and whey of cattle with naturally occurring coliform mastitis.

Concentrations of interferon-gamma (lFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-gamma and TNF-alpha-specific ELISA. In serum and whey samples from healthy cows. IFN-gamma was almost undetectable and TNF-alpha was detected at low levels. At the onset of illness, INF-gamma in sera and whey and TNF-alpha in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-gamma and TNF-alpha in whey from mastitic cattle decreased significantly as the cows recovered.

[Study on free enzymes and proteins in the urine. Report IV: The effects of Iohexol on elderly patients with renal function disorder].

We assessed the nephrotoxicity of a non-ionic contrast medium, Iohexol, in 18 elderly patients. They were divided into two groups. Group 1 consisted of 12 patients with glomerular flow rate (GFR) above 60 ml/min/1.73 m2 (range 69-136; average 96) and group 2 consisted of 6 patients with GFR below 60 ml/min/1.73 m2 (range 38-59; average 49). Urine samples, collected before, immediately after, and on the third and sixth days after drip infused pyeloureterography (DIP) were analyzed for gamma-glutamyl transpeptidase (gamma-GTP), N-acetyl-beta-D-glucosaminidase (NAG), beta 2-microglobulin (beta 2-MG) and albumin. In group 1, immediately after DIP, the urinary excretion of albumin, gamma-GTP and NAG were significantly increased (p < 0.01, p < 0.01, p < 0.01). In group 2, immediately after DIP, the urinary excretion of gamma-GTP and beta 2-MG were significantly increased (p < 0.01, p < 0.1). The gamma-GTP and NAG values in both groups suggest that the damage of proximal tubules cells will not continue for more than 4 days. However, as for the reabsorption of beta 2-MG on proximal tubules, in group 2 the value did not return to the pre DIP level until 6 days after DIP. We believe it is important to postpone injection of non-ionic contrast medium within 6 days of its last usage.

[Clinical evaluation of renal angiomyolipoma].

Between April 1980 and December 1999, 23 kidneys in 20 patients were diagnosed as having renal angiomyolipoma at our institution. The patients were 6 males and 14 females aged 24 to 79 years, with a mean age of 55.4 years. Two patients had associated tuberous sclerosis and 3 had bilateral disease. Of all patients the main clinical symptoms were pain (45%) and palpable mass (40%); 5 patients (25%) had asymptomatic lesions. The size of the tumor ranged from 1.3 to 24 cm (mean 7.7 cm). Treatment consisted of nephrectomy in 9 patients, partial nephrectomy in 6 and selective embolization in one. Pre-operative diagnosis was renal cell carcinoma in 5 of the 9 patients who underwent nephrectomy. Six patients with 7 diseased kidneys were followed radiologically. One patient underwent percutaneous biopsy to confirm the diagnosis. We suggest that nephron-sparing surgery for patients with renal angiomyolipoma should be the first step if tumor size is 4 cm < or = or increasing rapidly. Selective embolization is also a useful method. However, we need long-term follow-up to evaluate the effectiveness of embolization.

[A case of esophageal carcinoma with multiple liver metastases successfully treated with nedaplatin (NDP) and 5-fluorouracil (5-FU) combined with radiotherapy].

A 78-year-old male patient had esophageal carcinoma with multiple liver metastases. Chemoradiotherapy was performed. The chemotherapy consisted of protracted infusion of 5-fluorouracil (5-FU), combined with infusion of nedaplatin (NDP). Radiation of the mediastinum was administered concomitantly with chemotherapy. The patient showed a complete response (CR) of the primary lesion and a partial response (PR) of the liver metastasis for 11 months. Since liver metastasis recurred after initial treatment, chemotherapy consisting of NDP infusion combined with vindesine sulfate (VDS) infusion was performed. The patient again showed PR. Grade 3 leukocytopenia occurred during treatment, but there were no major toxicities such as thrombocytopenia, nausea, renal dysfunction or esophagitis. Survival time was one year and 7 months. In conclusion, concurrent chemoradiotherapy including NDP is effective and safe for patients with esophageal carcinoma accompanied by multiple liver metastasis. This nonsurgical approach may be an option for standard care in such cases.

Microvascular free tissue transfer in oral and maxillofacial reconstruction.

Forty-two patients with head and neck cancer were submitted to microvascular reconstructive procedures. We divided patients in three groups; a first group of 30 patients, in whom the oral floor (8 patients), the part of tongue (14 patients), the lower gingiva (6 patients) and the oropharynx (2 patients) were reconstructed using various sizes of forearm flaps; a second group of 7 patients who underwent buccal mucosa reconstructions with the forearm flaps; and a third group of 5 patients who received rectus abdominis flaps for total tongue reconstruction. Three illustrative cases, one from each group, are presented in detail. Good results were obtained in 39 patients (94%), with both functional and morphological rehabilitation. There were three flap losses due to thrombosis of the microvascular anastomoses. There was no surgical mortality. The average operating time was about 10 hours in total. We concluded that there is a place for these complex procedures in the treatment of selected cases of head and neck tumors.

Effects of interferon-α-transduced tumor cell vaccines and blockade of programmed cell death-1 on the growth of established tumors.

Interferon-alpha (IFN-α) has strong antitumor effects, and IFN-α gene therapy has been used clinically against some cancers. In this study, we evaluated the efficacy of the combination of IFN-α-transduced tumor cell vaccines and programmed cell death 1 (PD-1) blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. A poorly immunogenic murine colorectal cancer cell line, MC38, was transduced to overexpress IFN-α. In a therapeutic model, parental tumor-bearing mice were inoculated with MC38-IFNα cells and an anti-PD-1 antagonistic antibody. Analyses of immunohistochemistry and tumor-specific lysis were performed. The outgrowth of the established tumors was significantly reduced in mice treated with the combination of IFN-α and anti-PD-1. Immunohistochemical analyses of the therapeutic model showed marked infiltration of CD4(+) cells and CD8(+) cells in the established MC38 tumors of mice treated with both IFN-α and anti-PD-1. Significant tumor-specific cytolysis was detected when splenocytes of mice that were treated with both IFN-α and anti-PD-1 were used as effector cells. These results suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by IFN-α. The combination of IFN-α gene-transduced tumor cell vaccines and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials.

Dendritic cell therapy with interferon-alpha synergistically suppresses outgrowth of established tumors in a murine colorectal cancer model.

Both dendritic cell (DC)-based immunotherapy and interferon (IFN)-alpha therapy have been proved to have potent long-lasting antitumor effects. In anticipation of synergistic antitumor effects, we performed combination therapy with DCs and IFN-alpha gene-transduced murine colorectal cancer MC38 cells (MC38-IFN-alpha). DCs incubated with MC38-IFN-alpha, but not neomycin-resistance gene-transduced MC38 cells (MC38-Neo), effectively enhanced proliferation of allogeneic splenocytes in vitro. In 12 of 17 mice, DCs in combination with MC38-IFN-alpha prevented the development of a parental tumor, while DCs and MC38-Neo did in only three of 17 mice (P=0.008). In a therapeutic model of an established parental tumor, inoculation of DCs and MC38-IFN-alpha suppressed the growth of the established parental tumors significantly compared with the administration of DCs with MC38-Neo or naive splenocytes with MC38-IFN-alpha (P=0.016 and 0.024, respectively). Analyses of immunohistochemistry and tumor-infiltrating mononuclear cells showed that CD8(+), CD11c(+), and NK1.1(+) cells markedly infiltrated the established tumors of mice treated with DCs and MC38-IFN-alpha. From the results of observation of parental tumor outgrowth in immune cell-depleted mice, CD8(+) cells, and asialo-GM-1(+) cells were thought to contribute to the antitumor effects induced by the combination therapy. Furthermore, MC38-specific cytolysis was detected when splenocytes of mice inoculated with DCs and MC38-IFN-alpha cells were stimulated with MC38-IFN-alpha cells in vitro. Since DC-based immunotherapy in combination with IFN-alpha-expressing tumor cells induces potent antitumor cellular immune responses, it should be considered for clinical application.

Interleukin-4 gene transduced tumor cells promote a potent tumor-specific Th1-type response in cooperation with interferon-alpha transduction.

To investigate antitumor mechanisms in interleukin (IL)-4 therapy, we established an IL-4-overexpressing MC38 murine colorectal cancer cell line (MC38-IL4). As a therapy against established tumors, MC38-IL4 cells were inoculated contralaterally 7 days after wild-type (MC38-WT) cells had been injected, significantly reducing growth of wild-type tumors (P=0.030). Immunohistochemical analysis showed numerous granulocytes infiltrating wild-type tumors of MC38-IL4-inoculated mice. Injection of MC38-IL4 cells in leukocyte-depleted mice confirmed that granulocytes were involved in IL-4-related primary antitumor effects. Inoculation of MC38-WT in leukocyte-depleted mice initially injected with MC38-IL4 suggested that T cells contributed to the antitumor effects. To investigate tumor-specific responses, we stimulated splenocytes of MC38-immune mice with MC38-IL4 cells in vitro, resulting in MC38-specific lysis (57.5+/-7.2%, effector to target ratio=20). Treatment of established wild-type tumors with MC38-IL4 in combination with interferon (IFN)-alpha-overexpressing MC38 cells (MC38-IFNalpha) significantly reduced the growth of wild-type tumors (P=0.009). In vitro IFN-gamma production by splenocytes from mice injected with both MC38-IL4 and -IFNalpha was greatly enhanced in comparison with MC38-IL4 alone, while IL-10 production was not increased. Thus, granulocytes concern early antitumor effects of IL-4 therapy. Subsequently, IL-4 induces long-lasting, tumor-specific immune responses. IL-4 appears to promote a T-helper 1-type antitumor immune response, which is enhanced in cooperation with IFN-alpha.

PCR method is essential for detecting Mycobacterium tuberculosis in oral cavity samples.

Tuberculosis is a re-emerging infectious disease, and infection by Mycobacterium tuberculosis has been increasing in immunocompromised hosts, including elderly persons. M. tuberculosis-infected persons may receive dental treatment. To evaluate the risk of M. tuberculosis infection in dental clinics, we examined the detection rates of M. tuberculosis in sample of mixed saliva, dental plaque, extracted teeth, caries lesions, and denture plaque by nested polymerase chain reaction (PCR). The detection rates by PCR in samples from mixed saliva, dental plaque, caries lesions and denture plaque obtained from tuberculosis patients were 98.0%, 92.0%, 89.0%, and 100%, respectively. The detection rates by the culture method were 17.3%, 2.0%, 0%, and 0%, respectively. M. tuberculosis also was detected from the nontuberculous mycobacteria-infected group. Strains of Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum inhibited the growth of clinical strains of M. tuberculosis, but strains of Streptococcus mutans, Streptococcus sanguinis, and Actinobacillus actinomycetemcomitans did not. The present study concludes that the PCR method is essential for detecting M. tuberculosis in oral samples.

Human simple renal cyst fluid contains a cyst formation-promoting activity for Madin-Darby canine kidney cells cultured in collagen gel.

Simple renal cysts are commonly observed in the elderly, but few examinations have been performed to study the mechanism of this disease. We have previously shown that simple renal cyst fluid contains some growth factors, including epidermal growth factor (EGF; less than 54 pg mL-1) and insulin-like growth factor I (IGF-I; 30-2070 pg mL-1) (M. Taide et al. Clin Chim Acta, 1993; 217:199-203). In this report, the biological significance of growth factors or of human simple renal cyst fluid on the formation of renal epithelial cyst was determined using Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Both EGF (200 pg mL-1 or more) and IGF-I (100 pg mL-1 or more) were found to stimulate cyst formation in MDCK cells. Additionally, 12/21 human simple renal cyst fluid samples were found to be capable of stimulating cyst formation in MDCK cells. This activity was due to a heat- and acid-labile protein and not inhibited by the anti-IGF-I neutralizing antibody. These results indicate that in some instances simple renal cyst fluid would thus appear to contain specific activity for cyst formation, which may affect cyst formation in vivo.

Relationship between ventilation and arterial potassium concentration during incremental exercise and recovery.

The purpose of this study was to compare the relationship of ventilation (VE) with pH, arterial concentrations of potassium [( K+]a), bicarbonate [( HCO3-]a), lactate [( la]a), and acid-base parameters which would affect hyperpnoea during exercise and recovery. To assess this relationship, ten healthy male subjects exercised with intensity increasing as a ramp function of 20 W.min-1 until voluntary exhaustion and they were then allowed a 5-min recovery period. Breath-by-breath gas exchange data, [HCO3-]a, pH, [la]a, [K+]a and blood gases were determined during both exercise and recovery. Using a linear regression method, the VE/[K+]a relationship was analysed during both exercise and recovery. Several interesting results were obtained: a significant relationship between [K+]a and VE was observed during recovery as well as during exercise; the VE at any given values of [K+]a was significantly higher during recovery than during exercise and out of those factors affecting exercise hyperpnoea, only [K+]a had a similar time-course to VE during recovery. Changes in [K+]a during recovery were shown to occur significantly faster than VE with an [K+]a time constant of 70.0 s, SD 16.2 as opposed to 105.5 s, SD 10.0 for VE (P less than 0.01). These results provided further evidence that [K+]a might play an important role as a substance which can stimulate exercise hyperpnoea as has been suggested by other workers. The present study also showed that during recovery [K+]a contributed significantly to the control of VE.