Melanogenic effect of dersimelagon (MT-7117), a novel oral melanocortin 1 receptor agonist.

Background: The activation of melanocortin 1 receptor (MC1R) on melanocytes stimulates the production of eumelanin. A tridecapeptide α melanocyte-stimulating hormone (αMSH) is known to induce skin pigmentation. Objectives: We characterised the properties of a novel oral MC1R agonist dersimelagon (MT-7117) with respect to its specific binding to MC1R, downstream signalling and eumelanin production in experimental models. Methods: The competitive binding and production of intracellular cyclic adenosine 3', 5'-monophosphate in cells expressing recombinant melanocortin receptors were examined. A mouse melanoma cell line B16F1 was used for the evaluation of in vitro melanin production. The in vitro activity of MT-7117 was determined with αMSH and [Nle4, D-Phe7]-αMSH (NDP-αMSH) as reference comparators. The change of coat colour and skin pigmentation were evaluated after repeat administration of MT-7117 by oral gavage to C57BL/6J-Ay/+ mice and cynomolgus monkeys, respectively. Results: MT-7117 showed the highest affinity for human MC1R compared to the other melanocortin receptors evaluated and agonistic activity for human, cynomolgus monkey and mouse MC1R, with EC50 values in the nanomolar range. In B16F1 cells, MT-7117 increased melanin production in a concentration-dependent manner. In vivo, MT-7117 (≥0.3 mg/kg/day p.o.) significantly induced coat colour darkening in mice. MT-7117 (≥1 mg/kg/day p.o.) induced significant skin pigmentation in monkeys and complete reversibility was observed after cessation of its administration. Conclusions: MT-7117 is a novel oral MC1R agonist that induces melanogenesis in vitro and in vivo, suggesting its potential application for the prevention of phototoxic reactions in patients with photodermatoses, such as erythropoietic protoporphyria and X-linked protoporphyria.

Ca2+ release induced by myotoxin alpha, a radio-labellable probe having novel Ca2+ release properties in sarcoplasmic reticulum.

1. Myotoxin alpha (MYTX), a polypeptide toxin purified from the venom of prairie rattlesnakes (Crotalus viridis viridis) induced Ca2+ release from the heavy fraction (HSR) but not the light fraction of skeletal sarcoplasmic reticulum at concentrations higher than 1 microM, followed by spontaneous Ca2+ reuptake by measuring extravesicular Ca2+ concentrations using the Ca2+ electrode. 2. The rate of 45Ca2+ release from HSR vesicles was markedly accelerated by MYTX in a concentration-dependent manner in the range of concentrations between 30 nM and 10 microM, indicating the most potent Ca2+ releaser in HSR. 3. The Ca2+ dependency of MYTX-induced 45Ca2+ release has a bell-shaped profile but it was quite different from that of caffeine, an inducer of Ca(2+)-induced Ca2+ release. 4. 45Ca2+ release induced by MYTX was remarkable in the range of pCa between 8 and 3, whereas that by caffeine was prominent in the range of pCa, i.e., between 7 and 5.5. 5. MYTX-induced 45Ca2+ release consists of both early and late components. The early component caused by MYTX at low concentrations (30-300 nM) completed within 20 s, while the late component induced by it at higher concentrations (> 0.3 microM) was maintained for at least 1 min. 6. Both the components were almost completely inhibited by inhibitors of Ca2+ such as Mg2+, ruthenium red and spermine. 7. 45Ca2+ release induced by caffeine or beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) was completely inhibited by high concentrations of procaine. Procaine abolished the early component but not the late one, suggesting that at least the early component is mediated through Ca(2+)-induced Ca2+ release channels. 8. On the basis of these results, the character of Ca2+ release induced by MYTX was quite different from that caused by caffeine or AMP-PCP, suggesting that MYTX induces Ca2+ release having novel properties in HSR. MYTX is the first polypeptide Ca2+ inducer and has become a useful pharmacological tool for clarifying the mechanism of Ca2+ release from skeletal muscle SR.

Closed reduction of developmental dislocation of the hip by prolonged traction.

The efficacy of traction before an attempted closed reduction for patients with developmental dislocation of the hip remains controversial. We treated 55 children (62 dislocations of the hip) by preliminary, prolonged traction for a mean of eight weeks. All were followed up for at least two years in order to observe the development of any avascular changes within the femoral head. Of the 55 children, 27 (31 dislocations) were followed up until they were over six years of age. Fifty-seven of the 62 hips (92%) showed a successful closed reduction. Only one had radiological evidence of avascular necrosis of the femoral head. Of the 31 hips which were followed up to over six years of age, 15 (48%) showed residual subluxation. Our method of prolonged preliminary traction leads to a high rate of successful closed reduction, a low incidence of avascular necrosis and a reduced need for secondary operations.

[Cis-dichlorodiammineplatinum in osteosarcoma. Osteosarcoma Cooperative Study Group report].

Thirty-two patients with primary osteosarcoma and 18 patients with advanced osteosarcoma were treated by iv or ia infusion of cisplatinum at a dose of 100 mg/m2 every three weeks. The efficacy of the agent for primary osteosarcoma was mainly estimated by X-ray findings and histologic examination. One patient had a partial response, and 7 patients had a minor response. Pathologic evaluation of the extent of the primary tumor necrosis was performed on 27 resected specimens. Eight of 27 cases showed a good response (Ayala III A less than). One of 18 patients with advanced osteosarcoma had a partial response, and 2 a minor response. Nausea and vomiting (88%), liver dysfunction (42%), leukopenia (36%), nephrotoxicity (20%) and auditory disturbance (20%) were the main side effects in 50 patients. However, side effects of cisplatinum were generally reversible. The results suggest that cisplatinum is effective against osteosarcoma and may enhance the therapeutic results in osteosarcoma.

[A study on the interrelation of growth changes of upper apical base and alignment of permanent upper anterior teeth].

A comparative study was made between the crowding group and the normal aligned group to clarify the relationship between the growth of the maxilla, occlusion and the alignment of teeth. The material consisted of longitudinal dental casts and lateral cephalograms (II A, III A, III C of Hellman's dental age) taken from each of the 10 children. Differences between the two groups were investigated concerning the size of teeth dental arches and the coordinates of the landmarks. Then a factor analysis on the growth change, which the coordinates of landmarks showed, was performed. The results were as follows: 1. The differences of both the tooth size and the coordinates of the landmarks on the cephalograms were not significant. 2. From the factor analysis, it was observed that in the crowding group the migrations of the upper anterior teeth and molars were related more closely to the cranial base, the palate and the apical base, and also the dental arch migrated in unity. 3. It was suggested from the above results that although, in the normal group, the anterior teeth and molars migrated independently, stringently controlled migration of the teeth in the crowding group caused the united migration of the arch, resulting in uncompensatory accommodation of the teeth.

Effect of chronic antigen inhalation in guinea pigs.

To examine the role of airway inflammation in airway hyperresponsiveness (AHR), we examined the effect of chronic antigen inhalation in sensitized guinea pigs. Guinea pigs were actively sensitized with dinitrophenylated Ascaris suum extract (DNP-As) and repeatedly exposed to aerosolized DNP-As antigen once a day for 4 or 10 days. Twenty-four hours after the last antigen exposure, airway responsiveness to inhaled acetylcholine (ACh) and bronchoalveolar lavage (BAL) were studied. The guinea pigs receiving 4 days of exposure to antigen demonstrated an increase in airway responsiveness to inhaled ACh (p < 0.05). On the other hand, the guinea pigs receiving 10 days of exposure to antigen showed no significant change in airway responsiveness to inhaled ACh. BAL fluid analysis indicated that a significant increase in the number of eosinophils and neutrophils was observed in both groups of guinea pigs. A significant increase in the number of lymphocytes in BAL fluid was observed in guinea pigs exposed for 10 days, but not in those exposed for 4 days. We conclude that repeated exposure to antigen induced both development and suppression of AHR. Our results suggest that airway inflammation may play a role in both the development and suppression of AHR.

Determination of lycopene, alpha-carotene and beta-carotene in serum by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring.

A selected-ion monitoring (SIM) determination of serum lycopene, alpha-carotene and beta-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI-MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC-MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C18 column of mightsil ODS-5 (75 mm x 4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI-MS, lycopene, alpha-carotene and beta-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15-150 ng for lycopene, 7-70 ng for alpha-carotene and 25-50 ng for beta-carotene. The detection limit of LC-APCI-MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N = 3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, alpha-carotene and beta-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11.2%, 8.8% and 6.5% for lycopene, alpha-carotene and beta-carotene, respectively.

Vascular supply to slipped capital femoral epiphysis.

The etiology of avascular necrosis associated with slipped capital femoral epiphysis has not been well understood. The aims of this study were to clarify the blood supply to the slipped epiphysis and to examine whether this vascular supply is damaged before the reduction. Twelve patients (12 hips) underwent selective angiography of the medial circumflex femoral artery. There were seven stable slips and five unstable slips. All patients with slips underwent angiography before reduction, and one patient with an unstable slip underwent angiography both before and after reduction. The superior retinacular artery (SRA) was filled in all stable slips. This result was in accordance with the previous report that stable slips result in low rates of avascular necrosis. Of five unstable slips, the SRA was stained in two and was not filled in three. In one slip examined both before and after the manipulative reduction, the SRA was not seen before it but was well stained after it. These results have suggested that in some unstable slips the vascular injury occurs at the time of injury, before reduction, and that the reduction dose not necessarily contribute to the risk of avascular necrosis after slipped capital femoral epiphysis.

Tenotomy and postoperative brace treatment for muscular torticollis.

A postoperative corrective brace for congenital muscular torticollis is introduced and the results and indications for its use are presented. Thirty-three of 55 patients who underwent open tenotomy of the sternocleidomastoid muscle with application of the brace following surgery were evaluated in follow-up. Tenotomy was performed at the sternoclavicular origin of the muscle. The mean age at operation was six years; the mean follow-up period was seven years. The results were good in 21 patients (64%), fair in seven (21%), and poor in five (15%). Facial asymmetry remained in all patients over the age of ten who underwent operation. Alopecia, one of the complications of the brace, was found only in patients under age five. This combined treatment with tenotomy and postoperative brace is considered best indicated for patients between six and ten years of age.

Effect of a major metabolite of the antiallergic drug repirinast on phosphodiesterase and adenylate cyclase activities.

MY-1250 (5,6-dihydro-7,8-dimethyl-4,5-dioxo-4H-pyrano [3,2-c]quinoline-2-carboxylic acid, CAS 63768-47-8), a major metabolite of the antiallergic drug repirinast (MY-5116, CAS 73080-51-0), inhibits antigen-induced histamine release from rat peritoneal mast cells. MY-1250 causes a rapid increase in cyclic adenosine monophosphate (AMP) levels in rat peritoneal mast cells. MY-1250 inhibited cyclic AMP phosphodiesterase activities from rat peritoneal cells and guinea pig lung tissue in a concentration dependent manner with IC50 values of 2000 mumol/l and 1670 mumol/l, respectively. However, MY-1250 showed no effect on adenylate cyclase activity from rat peritoneal cells. These results suggest that the inhibitory effect of MY-1250 on histamine release may be partly due to the inhibition of cyclic AMP phosphodiesterase activity.

Production of recombinant granulocyte colony-stimulating factor by knocking into the active immunoglobulin heavy chain gene locus in the hybridoma cell line.

The hybridoma cell line KM50 originally produces a monoclonal antibody at a concentration of approximately 40 mg ml(-1) in ascites. To investigate the possibility to apply this expression system to the production of useful proteins, the cDNA encoding human granulocyte colony-stimulating factor was inserted by homologous recombination into just downstream of the promoter of the active immunoglobulin heavy chain gene of KM50. Site directed integration of targeting DNAs resulted in the disruption of expression of the immunoglobulin heavy chain proteins with a frequency of 1 in 10 approximately 100 G418-resistance transfectants. One of the monoclonal antibody-deficient transfectants produced25 ng ml(-1) of granulocyte colony-stimulating factor in the supernatant of its cell culture the number of molecules of which corresponds to that of the monoclonal antibody originally produced by KM50. However, when this transfectant was injected intraperitoneally, it produced only a 9 mug ml(-1) concentration of granulocyte colony-stimulating factor in ascites, which is approximately 3 orders of magnitude less than the monoclonal antibody. This method may be applicable to production of other recombinant proteins, although further optimization in the conditions of production would be needed in order to reach much higher yields.

Avascular necrosis associated with fractures of the femoral neck in children: histological evaluation of core biopsies of the femoral head.

There is no well-documented effective treatment for avascular necrosis following fractures of the femoral neck in children. Six children who suffered avascular necrosis following these fractures were treated with a long period of non-weight bearing. We tried to predict the advisable period of non-weight bearing by histological finding on core biopsy taken from the femoral head and present long-term follow-up results. The time interval for the biopsy ranged from 4 to 21 months after injury. Two specimens obtained within 1 year after injury showed total necrosis. The other four specimens taken more than 1 year after injury showed partial repair. Two specimens obtained from patients who had minimally displaced fractures also revealed necrotic tissue. Four patients were initially placed non-weight bearing for over 1 year. Two patients started weight bearing immediately after surgery, and late segmental collapse occurred within 1 year. They were then placed non-weight bearing for a further period in excess of 1 year. All patients, including those who had severely displaced fractures, avoided severe collapse of the femoral head. To avoid severe collapse of the femoral head due to avascular necrosis after pediatric femoral neck fractures, a long period of non-weight bearing of at least 1 year may be recommended treatment.

Expression cloning of a novel alpha 1,3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes.

The sialyl Lewis x (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc-NAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium or platelets. In our efforts to identify glycosyltransferases involved in the biosynthesis of those ligands, we achieved expression cloning of a novel human alpha 1,3-fucosyltransferase termed Fuc-TVII from a THP-1 cDNA library by enrichment of the Namalwa cells highly expressing that determinant with a fluorescence-activated cell sorter. Expression of the COOH-terminal catalytic domain of Fuc-TVII showed an alpha 1,3-fucosyltransferase activity for a type II oligosaccharide with a terminal alpha 2,3-linked sialic acid among various acceptors, consistent with that in vivo acceptor specificity. Alignment of the primary sequences of five alpha 1,3-fucosyltransferases and assignment of the chromosomal location of Fuc-TVII gene, together with that acceptor specificity, indicate that Fuc-TVII consists of a unique class of the alpha 1,3-fucosyltransferase family. Determination of the expression levels of these alpha 1,3-fucosyltransferases in various cells revealed that both Fuc-TVII and a myeloid fucosyltransferase Fuc-TIV were significantly expressed in myeloid lineage cells. Fuc-TVII-transfected Namalwa cells exhibited significant binding to E-selectin in contrast to little binding of the Fuc-TIV-transfected cells. These results suggest that Fuc-TVII may participate in the biosynthesis of the selectin ligands.

Small-Angle Scattering and Electron Microscopy Investigation of Nanotubules Made from a Perfluoroalkylated Glucophospholipid.

Anionic glucophospholipids were recently reported as a new family of tubule-forming lipids. We report here investigations on the structure of nanotubules made from a glucophospholipid with a mixed fluorocarbon-hydrocarbon hydrophobe, using freeze fracture and cryo-transmission electron microscopy (TEM) and X-ray and neutron small angle scattering (SAXS, SANS). The hollow and regularly shaped tubules are very thin: they have an external radius of 140 Å and an internal radius of 35 Å on the average. Their 105 Å-thick wall appears to consist in three bilayers in which the glucophospholipid molecules are probably in a tilted and/or interdigitated configuration. Upon heating these nanotubes convert reversibly into vesicles; transformation is complete at 60 degrees C. Copyright 1999 Academic Press.