Effects of mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium channels, on N-type calcium channel currents of rat dorsal root ganglion culture neurons.

Mirogabalin, which is a novel ligand for the α2δ subunit of voltage-gated calcium channels, is being developed for treating neuropathic pain including diabetic peripheral neuropathy and postherpetic neuralgia. Mirogabalin possesses unique α2δ subunit binding characteristics and has potent and long-lasting analgesic effects in neuropathic pain models. In the present study, we investigated the effects of mirogabalin on N-type calcium channel currents of the rat dorsal root ganglion (DRG) culture neurons using the whole-cell patch clamp technique. Small or medium DRG neurons were isolated from Sprague-Dawley rats and were incubated for 20 to 24 h with mirogabalin or pregabalin. The DRG neurons were depolarised from a holding potential of -40 mV to +40 mV in steps of 10 mV for 220 ms, and elicited N-type calcium channel currents were recorded. The N-type calcium channel currents were verified by sensitivity to ω-conotoxin GVIA, a selective N-type calcium channel blocker. Mirogabalin inhibited the calcium channel currents of rat DRG neurons at 50 µM, and pregabalin inhibited them at 200 µM. Mirogabalin and pregabalin showed significant differences in the peak current densities at depolarisation to -20 and -10 mV when compared with that shown by the vehicle control. In conclusion, mirogabalin inhibits N-type calcium channel currents in rat DRG culture neurons. The potent and long-lasting analgesic effects of mirogabalin are thought to be associated with its potent and selective binding to α2δ-1 subunits and following functional inhibition of calcium channel currents.

Analgesic effects of the novel α2δ ligand mirogabalin in a rat model of spinal cord injury.

Mirogabalin, which is a novel ligand for the α2δ subunit of voltage-gated calcium channels, is under development for the treatment of pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. Mirogabalin possesses unique binding characteristics to α2δ subunits and potent and long-lasting analgesic effects in peripheral neuropathic pain models. In the present study, we investigated the analgesic effects of mirogabalin in a rat model of spinal cord injury as an experimental animal model for central neuropathic pain. The spinal cord injury model was established by acute compression of the spinal cord at the T6/7 level with a microvascular clip in male rats. Twenty-eight days after spinal cord injury, the animals received the test compound orally, and the paw withdrawal threshold to mechanical stimulation was determined using the von Frey test at 0 (before administration), 2, 4, 6, 8, and 24 h after administration. The area under the curve of the paw withdrawal threshold (paw withdrawal threshold AUC) was also calculated. In rats subjected to spinal cord injury, mechanical allodynia was demonstrated by a decreased paw withdrawal threshold. A single oral administration of mirogabalin (2.5, 5, or 10 mg/kg) significantly increased the paw withdrawal threshold. The effects of mirogabalin were still significant 6 or 8 h after administration. The paw withdrawal threshold AUC was significantly higher in the treated animals than in the control group. In conclusion, mirogabalin showed potent and long-lasting analgesic effects in a rat model of spinal cord injury and may therefore provide effective pain relief for patients with central neuropathic pain.

Re-evaluation of stomach position as a simple prognostic factor in fetal left congenital diaphragmatic hernia: a multicenter survey in Japan.

OBJECTIVES: To document outcome and to explore prognostic factors in fetal left congenital diaphragmatic hernia (CDH). METHODS: This was a multicenter retrospective study of 109 patients with prenatally diagnosed isolated left CDH born between 2002 and 2007. The primary outcome was intact discharge, defined as discharge from hospital without major morbidities, such as a need for respiratory support including oxygen supplementation, tube feeding, parenteral nutrition or vasodilators. All patients were managed at perinatal centers with immediate resuscitation, gentle ventilation (mostly with high-frequency oscillatory ventilation) and surgery after stabilization. Prenatal data collected included liver and stomach position, lung-to-head ratio, gestational age at diagnosis and presence or absence of polyhydramnios. Stomach position was classified into four grades: Grade 0, abdominal; Grade 1, left thoracic; Grade 2, less than half of the stomach herniated into the right chest; and Grade 3, more than half of the stomach herniated into the right chest. RESULTS: Overall intact discharge and 90-day survival rates were 65.1% and 79.8%, respectively. Stomach herniation was classified as Grade 0 in 19.3% of cases, Grade 1 in 45.9%, Grade 2 in 13.8% and Grade 3 in 21.1%. Multivariate analysis revealed that liver position was the strongest prognostic variable for intact discharge, followed by stomach position. Based on our results, we divided patients into three groups according to liver (up vs. down) and stomach (Grade 0-2 vs. Grade 3) position. Intact discharge rates declined significantly from liver-down (Group I), to liver-up with stomach Grade 0-2 (Group II), to liver-up with stomach Grade 3 (Group III) (87.0%, 47.4% and 9.5% of cases, respectively). CONCLUSION: Current status and outcomes of prenatally diagnosed left CDH in Japan were surveyed. Stomach herniation into the right chest was not uncommon and its grade correlated with outcome. The combination of liver and stomach positions was useful to stratify patients into three groups (Group I-III) with different prognoses.

Multicentre study of perioperative versus adjuvant chemotherapy for resectable colorectal liver metastases.

Background: It is not known whether perioperative chemotherapy, compared with adjuvant chemotherapy alone, improves disease-free survival (DFS) in patients with upfront resectable colorectal liver metastases (CLM). The aim of this study was to estimate the impact of neoadjuvant 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX) on DFS in patients with upfront resectable CLM. Methods: Consecutive patients who presented with up to five resectable CLM at two Japanese and two French centres in 2008-2015 were included in the study. Both French institutions favoured perioperative FOLFOX, whereas the two Japanese groups systematically preferred upfront surgery plus adjuvant chemotherapy. Inverse probability of treatment weighting (IPTW) and Cox regression multivariable models were used to adjust for confounding. The primary outcome was DFS. Results: Some 300 patients were included: 151 received perioperative chemotherapy and 149 had upfront surgery plus adjuvant chemotherapy. The weighted 3-year DFS rate was 33·5 per cent after perioperative chemotherapy compared with 27·1 per cent after upfront surgery plus adjuvant chemotherapy (hazard ratio (HR) 0·85, 95 per cent c.i. 0·62 to 1·16; P = 0·318). For the subgroup of 165 patients who received adjuvant FOLFOX successfully (for at least 3 months), the adjusted effect of neoadjuvant chemotherapy was not significant (HR 1·19, 0·74 to 1·90; P = 0·476). No significant effect of neoadjuvant chemotherapy was observed in multivariable regression analysis. Conclusion: Compared with adjuvant chemotherapy, perioperative FOLFOX does not improve DFS in patients with resectable CLM, provided adjuvant chemotherapy is given successfully.

Antecedentes: Se desconoce si la quimioterapia perioperatoria en comparación con la quimioterapia adyuvante sola mejora la supervivencia libre de enfermedad (disease­free survival, DFS) en pacientes con metástasis hepáticas de origen colorrectal (colorectal liver metastases, CLM) resecables de inicio. El objetivo de este estudio fue estimar el impacto de la neoadyuvancia con 5­fluorouracilo, leucovorina y oxaliplatino (FOLFOX) sobre la DFS en pacientes con CLM resecables desde el principio. Métodos: Se incluyeron pacientes consecutivos que presentaban hasta cinco CLM resecables en dos centros japoneses y dos centros franceses entre 2008 a 2015. Ambas instituciones francesas favorecían FOLFOX perioperatorio, mientras que los dos grupos japoneses utilizaban sistemáticamente la cirugía de entrada y quimioterapia adyuvante. Se utilizaron la probabilidad inversa del tratamiento ponderado (Inverse Probability of Treatment Weighting, IPTW) y el modelo multivariable de regresión de Cox para ajustar por factores de confusión. El resultado primario fue la DFS. Resultados: Se incluyeron 300 pacientes (grupo de quimioterapia perioperatoria n = 151 y grupo de cirugía de entrada más quimioterapia adyuvante n = 149). La DFS a los 3 años ponderada fue del 33% después de quimioterapia perioperatoria versus 27% tras cirugía de entrada (cociente de riesgos instantáneos, hazard ratio HR: 0,85; i.c. del 95% (0,62­1,16); P = 0,32). Cuando se consideró el subgrupo de pacientes que (n = 165) de manera efectiva (al menos 3 meses) recibieron FOLFOX adyuvante, el efecto ajustado de la quimioterapia neoadyuvante no fue significativo (HR: 1,19 (0,74­1,90); P = 0,48). No se observó un efecto significativo de la quimioterapia neoadyuvante en el análisis de regresión multivariable. Conclusión: En comparación con la quimioterapia adyuvante, el FOLFOX perioperatorio no mejora la DFS en CLM resecables siempre y cuando la quimioterapia adyuvante se administre de forma efectiva.

Isocyano compounds as non-toxic antifoulants.

The marine sponge Acanthella cavernosa and nudibranchs of the family Phyllidiidae contain isocyanoterpenoids and their related compounds that show potent antifouling activity against cypris larvae of the barnacle Balanus amphitrite, while their toxicity to cyprids is weak. To develop non-toxic antifoulants based on isocyanoterpenoids, especially 3-isocyanotheonellin, a total of 36 isocyano compounds have been synthesized. They were evaluated by both antifouling activity and toxicity toward B. amphitrite cyprids, which led some insight into the structure-activity relationships. Since linear alkyl isocyanides showed antifouling activity at nontoxic concentrations, a large amount of 1,1-dimethyl-10-undecyl isocyanide was synthesized, incorporated into paints, and tested for antifouling activity in the field with promising results. Therefore, isocyano compounds were considered as candidate non-toxic antifouling agents.

Combined fibrolamellar carcinoma and cholangiocarcinoma exhibiting biphenotypic antigen expression: a case report.

Fibrolamellar carcinoma (FLC), a variant of hepatocellular carcinoma (HCC), very rarely occurs in association with cholangiocarcinoma (CC). This report describes the first case of FLC coexisting with CC (FLC-CC) from Japan. Although the major part of the tumour located in the right lobe of the liver showed the typical features of FLC, CC was admixed with the FLC, not only in the primary hepatic tumour, but also in the lymph node metastases. Immunohistochemical analysis revealed that, although carcinoembryonic antigen (CEA), which can be detected with monoclonal antibodies in the cytoplasm and the cell surface of CC cells but not HCC cells, was expressed in only the CC cells in the primary tumour, it was expressed extensively in the cytoplasm of both CC and FLC cells in the metastatic and recurrent tumours. Furthermore, Hep Par 1, a hepatocyte specific antigen, was also expressed in both the FLC and CC cells. These findings suggest that, in this case, both FLC and CC were possibly derived from the same cancer stem cell with the capacity to differentiate into both hepatocytes and bile duct epithelium, and that both the cellular components, therefore, exhibited biphenotypic antigen expression.

Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells.

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.

Gene expression of bone matrix proteins and endothelin receptors in endothelin-1-deficient mice revealed by in situ hybridization.

Endothelin-1 (ET-1) was first found as a vasoconstrictor protein excreted by vascular endothelial cells, but recently ET-1 has been considered to have widespread functions that include regulation of osteochondrogenic metabolism. We analyzed sections of head regions in ET-1 knockout mice that are known to have abnormalities in pharyngeal arch-derived tissues and found that there was severe hypoplasia in facial bones. The hypoplasia suggests that the matrix mineralization system of facial bones is disrupted in ET-1-/- homozygous mice. To elucidate whether osteogenic cells in facial bones are the targets for ET-1 and whether expression of bone matrix genes are modulated by ET-1, we examined gene expression of ET-1 receptors, ETA and ETB, and that of the bone matrix proteins, osteonectin (ON) and osteopontin (OP), both in the head regions of ET-1+/- heterozygous and ET-1-/- homozygous mice by means of in situ hybridization. Different patterns of expression between ETA and ETB mRNAs were observed in both groups. In 18.5 days post coitus fetuses, ETA mRNA was most strongly expressed in osteogenic cells along craniofacial bones, but ETB mRNA was most strongly expressed in trunks of trigeminal nerve. This finding suggests that ET-1 may modulate osteogenic cells through ETA receptor but not through ETB receptor. The expression patterns of ETA, OP, and ON mRNAs were distinct between the two groups. In the lower jaw of ET-1+/- heterozygous mice, the ETA, ON, and OP mRNA positive cells were scattered in the inner and outer regions of the thick bone matrix, but in ET-1-/- homozygous mice, cells containing those mRNAs were located close to each other at the surface of thin bone matrix. However, cellular expression of ON and OP mRNAs in osteogenic cells of ET-1-/- homozygous mice was not suppressed as compared with ET-1+/- heterozygous mice. We conclude that ET-1 may regulate proliferation and migration of osteogenic cells in the maxillofacial region, rather than modulating the expression level of ON and OP mRNAs.

Modulation of picryl chloride-induced contact hypersensitivity reaction in mice by nitric oxide.

We have studied the possible involvement of nitric oxide (NO) in the contact hypersensitivity reaction. A biphasic response of ear swelling was observed at 2 h (early phase) and 24 h (late phase) after application of the antigen to picryl chloride (PC1)-sensitized CBA/J mice. Intravenous injection of NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), at the time of PC1 challenge, inhibited in a concentration-dependent fashion the antigen-induced contact hypersensitivity reaction. Low-dose (1 mg/kg) L-NAME inhibited the early-phase reaction but not the late-phase reaction. High-dose (250 mg/kg) L-NAME inhibited both early- and late-phase reactions. D-NAME (enantiomer of L-NAME) did not inhibit the antigen-induced ear swelling. High-dose (250 mg/kg) L-arginine increased both early and late phase reactions. D-Arginine (enantiomer of L-arginine) did no increase the antigen-induced ear swelling. L-NAME injection, however, did not suppress phenol-induce irritant inflammation. Treatment of mice undergoing PC1-induced contact hypersensitivity reaction with L-NAME reduced the production of interleukin-2 and interferon-gamma by draining lymph node cells. Treatment with L-arginine, on the other hand increased the production of interleukin-2 and interferon-gamma. These results suggest that NO plays a modulating role in contact hypersensitivity reaction.

Effects of cholera toxin on proliferation of cultured human keratinocytes in relation to intracellular cyclic AMP levels.

In the culture of epidermal keratinocytes, the cellular growth rate is reported to be accelerated by cholera toxin. The mechanism by which cholera toxin exerts biological effects is thought to result from changes in intracellular cyclic AMP concentrations. But in many reports cyclic AMP elevating agents appeared to inhibit growth of keratinocytes in culture. This study was done to clarify the discrepancy of this problem. Determination of cyclic AMP revealed that cholera toxin over a range of 10-14-10-8 M increased the intracellular concentration of cyclic AMP of cultured keratinocytes about 100-fold over the controls after incubation for 6 hr. When a small number (10(5)) of cells were inoculated in a 60 X 15 mm culture dish, cholera toxin strongly stimulated colony growth. When a relatively larger number (8 X 10(5)) of cells were inoculated in a dish, cholera toxin moderately accelerated cell division, and increased DNA and protein levels of the culture during early days of cultivation. But after about 20 days, of cultivation when the culture reached confluence, cholera toxin decreased both DNA and protein content in a culture dish. The cultures were pulse labeled with 3H-thyrmidine at 12 and 24 hr after the addition of 10-10 M cholera toxin, and its uptake into DNA was determined. In the early days of cultivation the uptake of 3H-thymidine increased after treatment with cholera toxin. But in the late days of cultivation, cholera toxin decreased the rate of 3H-thymidine incorporation into DNA. These results indicated that cholera toxin-cyclic AMP has effects on the proliferation of keratinocytes in culture biphasically according to cellular concentrations in culture.

Expression of pemphigoid antigen by SV40-transformed human keratinocytes.

The expression and properties of pemphigoid antigen of SV40-transformed human keratinocytes were studied. By indirect immunofluorescence, SV40-transformed keratinocytes in passage 80-85 expressed the pemphigoid antigen as coarsely granular perinuclear fluorescence. To characterize this antigen, NP40 extracts of cells labeled with [14C]amino acids were immunoprecipitated using sera of 8 patients: bullous pemphigoid (6 patients), chronic localized pemphigoid (1 patient), and drug-induced lichen planus pemphigoides (1 patient). These immunoprecipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then fluorographed. All 8 sera precipitated a protein of Mr 240K, while normal human sera did not precipitate this protein. These results indicate that SV40-transformed human keratinocytes synthesize pemphigoid antigen, and that autoantibodies in the sera of pemphigoid patients with different clinical features identify the same antigen of Mr 240K in these cells.

SSA/Ro antigen expression in simian virus 40-transformed human keratinocytes.

SSA/Ro antigen is a soluble cellular component to which antibodies are frequently produced in patients with Sjögren's syndrome and systemic lupus erythematosus. Its exact location within the cell has yet to be determined. In this study we report the expression of SSA/Ro antigen in simian virus 40 (SV40)-transformed keratinocytes. The locations of SSA/Ro, U1RNP, and DNA antigens were studied by indirect immunofluorescence using monospecific antibodies. SSA/Ro antigen was detected in both the nucleus and cytoplasm of SV40-transformed keratinocytes tested with three monospecific sera. Primary cultured keratinocytes derived from adult human skin showed localized immunofluorescent staining within the nucleus. When Ca++ concentration of the medium was switched to 0.05 mM, these cells expressed cytoplasmic SSA/Ro antigens within 48 h. Depletion of the antibody activity with insolubilized human spleen extract abolished the staining. Surface expression of this antigen could not be detected in either primary or transformed cells. Localization of U1RNP and DNA was not altered. These results indicate that expression of SSA/Ro antigen in human keratinocytes is modulated by SV40 infection and that this antigen is expressed to a greater degree in cells that are less differentiated, transformed, or proliferating.

Expression of proliferating cell nuclear antigen/cyclin in human keratinocytes.

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, in human keratinocytes was examined by using the serum obtained from a SLE patient and a murine monoclonal antibody against PCNA/cyclin. In the normal epidermis, few of the nuclei were labeled with anti-PCNA/cyclin. This was in contrast to the positive nuclear staining seen in active lesions of psoriasis. In a primary culture of human keratinocytes growing as a monolayer, 20%-30% of cells expressed PCNA/cyclin. SV40-transformed human keratinocytes showed positive nuclear staining in about 40% of the cell population. In stratified keratinocytes cultured in a high Ca++ medium, PCNA/cyclin expression was decreased and only the cells in the basal and suprabasal layers showed positive staining. These results indicate that the expression of PCNA/cyclin correlates with the proliferating state in human keratinocytes and may not be associated with the mechanism of differentiation in keratinocytes.

Dual role of tropomyosin on chemically modified actin filaments from skeletal muscle.

Actin filaments copolymerized with both intact and chemically modified actin monomers restored their sliding activity when they were supplemented with tropomyosin extracted from skeletal muscle. In contrast, the ATPase activation of the copolymers was decreased when supplemented with tropomyosin. The results indicate that tropomyosin along with actin monomers may facilitate sliding activity of the entire actin filament but suppress ATPase activation of intact actin monomers themselves. Accordingly, tropomyosin molecules could be viewed as playing a dual role of both mechanical and chemical regulation of actin monomers.

Characterization of low-density lipoproteins from patients with recessive X-linked ichthyosis.

We investigated lipoprotein metabolism in 14 patients with recessive X-linked ichthyosis (RXLI), a metabolic disease characterized by scaly skin, corneal opacity and steroid sulfatase deficiency. Plasma total cholesterol (TC) levels ranged from normal to slightly low (mean +/- SD: 156 +/- 28 mg/dl). Four patients showed a mild or moderate elevation of plasma triglyceride (TG) levels ranging from 150 to 365 mg/dl. The apoprotein B (apo B) to TC ratio was higher than in normal controls (0.63 +/- 0.11 vs. 0.52 +/- 0.07, P less than 0.01), while plasma apoB levels were within the normal range (99 +/- 17 mg/dl). Polyacrylamide gel electrophoretic mobility of low-density lipoprotein (LDL) was markedly increased in all patients, and further analyses showed that this finding was not due to a change in the particle size of the LDL but to an increased content of cholesterol sulfate (1.0-2.3% of the LDL-cholesterol content). In addition to the alteration of electrophoretic mobility, marked changes in the lipid and apoprotein compositions of the LDL fraction were observed; cholesterol ester content in LDL (LDL-CE) was significantly lower than that of control subjects (37 +/- 4% vs. 41 +/- 2% of total lipids, P less than 0.01), while the triglyceride content (LDL-TG) and apo B to cholesterol ratios in LDL were significantly higher than those of controls (18 +/- 7 vs. 10 +/- 2, P less than 0.001; 1.21 +/- 0.19 vs. 0.73 +/- 0.05, P less than 0.001, respectively). This anionized LDL, in which cholesterol sulfate was increased, was shown to bind to the LDL receptor of fibroblasts to much the same extent as normal LDL. In conclusion, the increase in cholesterol sulfate in LDL fraction not only alters the electrophoretic moiety but also the relative contents of apoB, cholesterol, and triglyceride in the lipoprotein. It does not change the affinity of LDL for the LDL receptor.

Effective reduction of plasma LDL levels by LDL apheresis in familial defective apolipoprotein B-100.

The clinical response to long-term reduction of the plasma LDL cholesterol concentration was studied in a man with severe coronary artery disease associated with familial defective apolipoprotein B-100 (FDB). Plasma exchange repeated at 2-week intervals, combined with lipid-lowering drugs, led to remission of angina and improved exercise test performance. A similar clinical response was achieved after LDL apheresis with dextran sulphate columns repeated once every 2 weeks in combination with drug treatment. The reduction in plasma LDL cholesterol level brought about by LDL apheresis was at least as marked in the FDB patient as in 5 patients with familial hypercholesterolaemia. We conclude that FDB patients with coronary artery disease may derive clinical benefit from prolonged reduction of their plasma cholesterol levels and that LDL containing apo B-100 in which arginine at position 3500 is replaced by glutamine is removed from plasma by dextran sulphate columns as efficiently as is normal LDL.

Involvement of central opioid systems in human interferon-alpha induced immobility in the mouse forced swimming test.

1. We investigated the mechanism by which human interferon-alpha (IFN-alpha) increases the immobility time in a forced swimming test, an animal model of depression. 2. Central administration of IFN-alpha (0.05 - 50 IU per mouse, i.cist.) increased the immobility time in the forced swimming test in mice in a dose-dependent manner. 3. Neither IFN-beta nor -gamma possessed any effect under the same experimental conditions. 4. Pre-treatment with an opioid receptor antagonist, naloxone (1 mg kg(-1), s.c.) inhibited the prolonged immobility time induced by IFN-alpha (60 KIU kg(-1), i.v. or 50 IU per mouse. i.cist. ). 5. Peripheral administration of naloxone methiodide (1 mg kg(-1), s. c.), which does not pass the blood - brain barrier, failed to block the effect of IFN-alpha, while intracisternal administration of naloxone methiodide (1 nmol per mouse) completely blocked. 6. The effect of IFN-alpha was inhibited by a mu(1)-specific opioid receptor antagonist, naloxonazine (35 mg kg(-1), s.c.) and a mu(1)/mu(2) receptor antagonist, beta-FNA (40 mg kg(-1), s.c.). A selective delta-opioid receptor antagonist, naltrindole (3 mg kg(-1), s.c.) and a kappa-opioid receptor antagonist, nor-binaltorphimine (20 mg kg(-1), s.c.), both failed to inhibit the increasing effect of IFN-alpha. 7. These results suggest that the activator of the central opioid receptors of the mu(1)-subtype might be related to the prolonged immobility time of IFN-alpha, but delta and kappa-opioid receptors most likely are not involved.

High frequency of tetraploidy detected in malignant melanoma of Japanese patients by fluorescence in situ hybridization.

Aneuploidy and hyperploidy are often detected in malignant melanoma by cytogenetic analysis and flow cytometric analysis of DNA content. To determine the ploidy of cells in surgical specimens of melanin-producing tumors of Japanese patients, we performed fluorescence in situ hybridization (FISH) using touch smear technique to count the number of chromosomes 18 and X + Y in interphase nuclei using alpha-satellite DNA probes, D18Z1, DXZ1 and DYZ3. A normal melanocyte strain showed two D18Z1 and two [DXZ1+DYZ3] signals per nucleus, indicating 2N, and a malignant melanoma cell line showed 4 per nucleus, indicating 4N, consistent with results of cytogenetic and flow cytometric analyses. Therefore we employed this FISH method to analyze ploidy of surgical specimens. Specimens obtained from 8 patients with nevus cell nevus showed 2 FISH signals per nucleus. On the other hand, in all specimens obtained from 8 patients with malignant melanoma (6 primary and 2 metastatic melanoma), 65-90% of cells exhibited 4 signals per nucleus, indicating 4N. Histopathologically, 50-70% of cells were identified as malignant melanoma cells, indicating that our FISH method is effective to detect melanoma cells in tissue. We also analyzed allelic loss of the p53 gene by FISH with a p53 locus-specific probe and mutation of the p53 gene by immunostaining since mutation and deletion of the p53 gene may cause hyperploidy. All specimens except one obtained from a case with young-onset metastatic melanoma exhibited no allelic losses or negative p53 staining, showing the p53 gene was intact. These results indicate that tetraploidy, not caused by p53 mutation or deletion, is commonly found in malignant melanoma of Japanese patients. It is also suggested that there is no positive relationship between tetraploidy and poorer prognosis, and mutation and allelic loss of the p53 gene might be markers of aggressive form of malignant melanoma.