Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. INTRODUCTION: Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. MATERIALS AND METHODS: Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. RESULTS: In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. CONCLUSIONS: We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.     

Neurilemmoma of tongue

Neurilemmoma is usually soimry, benign tumour derived from schwan cells of the Sheaths of peripheral cranial or autonomie nerves. In thehead and neck region it occurs most commonly in association with acoustic nerve within the skuil and is rely fottnd in the oral cavity (1,2). We report here two cases of the iongue diagnosed on histopathohgy.     

Matrix Metalloproteinases

Drugs & Aging - Matrix metalloproteinases (MMPs), or matrixins, are a family of zinc endopeptidases that play a key role in both physiological and pathological tissue degradation. Normally,...     

Anterior eye focusing of peripheral ultraviolet and visible radiation albedo

Concern about short- and long-term ultraviolet radiation (particularly UVB) damage to the eye has led to increased research in this area. Numerous studies have confirmed the pathogenic enhancing roles of reflected ultraviolet (UV) and visible radiation in our environment. There is concern that conventional sunglasses do not protect the eye adequately from reflected rays (albedo), especially on the lateral aspect, from behind and from below. Using eye models and computer ray tracing methods, the pathways of oblique rays incident at the temporal peripheral cornea have been plotted by Maloof, Ho and Coroneo.¹ These rays are refracted and focused and theoretically can result in up to 20 times the concentration of incident irradiance at the nasal anterior chamber angle and nasal equatorial cortex of the crystalline lens. The purpose of this study was to determine the limits of angular subtense of the incident peripheral light which is refracted in this manner in human subjects and to investigate the relation between corneal shape and certain ocular parameters to the limits. A statistically significant positive correlation was found between temporal entrance angle and anterior chamber depth (r = 0.70, P< 0.0006). The entrance angle ranged from 15 degrees to 30 degrees and was located 10 degrees to 45 degrees posterior to the coronal plane. Our results support Maloof and colleagues' predictions for the implication of focused peripheral UV and high intensity visible radiation in the pathogenesis of pterygium and cortical cataract and emphasise the need for lateral eye protection in conditions of high ultraviolet albedo.     

Generation of antigen-specific CD4+ T cell lines from naive precursors

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4⁺ T cells from previously unprimed individuals. Freshly isolated CD4⁺ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp 160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (MΦ). In short-term (< 8 days) cultures, CD4⁺ T cells or their CD4⁺, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4⁺, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or MΦ. KLH- and SWM-specific CD4⁺ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and MΦ in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4⁺ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.     

Molecular breakpoint analysis and relevance of variable mosaicism in a woman with short stature,primary amenorrhea,unilateral gonadoblastoma,and a 46,X,del(Y)(q11)/45,X karyotype

Congenital hydrocephalus associated with aqueductal stenosis and/or agenesis of the corpus callosum has been described in newborn males with mutations in L1CAM, a gene that encodes a neural cell adhesion molecule. These males usually have severe mental retardation and may have spastic paraplegia and adducted thumbs. In contrast, Hirschsprung disease, or absence of ganglion cells in the distal gut, has rarely been described in such individuals. We report a male infant who had severe hydrocephalus identified in the prenatal period with evidence of aqueductal stenosis and adducted thumbs at birth. He developed chronic constipation, and rectal biopsy confirmed the diagnosis of Hirschsprung disease. Molecular testing of the L1CAM gene revealed a G2254A mutation, resulting in a V752M amino acid substitution. A common polymorphism in RET, but no mutation, was identified. Our patient represents the third example of coincident hydrocephalus and Hirschsprung disease in an individual with an identified L1CAM mutation. We hypothesize that L1CAM‐mediated cell adhesion may be important for the ability of ganglion cell precursors to populate the gut, and that L1CAM may modify the effects of a Hirschsprung disease–associated gene to cause intestinal aganglionosis. © 2002 Wiley‐Liss, Inc.     

Synthesis of alpha-methyl-benzamido-alpha'-substituted styryl cyclohexanone thiosemicarbazones as potential antifertility agents

Cyclohexanone was condensed with N-hydroxymethyl benzamide in conc. sulphuric acid to give alpha-methyl-benzamido-cyclohexanone (I). The reaction of (I) with thiosemicarbazide in ethanol resulted in alpha-methyl-benzamido-cyclohexanone thiosemicarbazone (II). Condensation of (II) with various aromatic aldehydes in the presence of ethanol afforded alpha-methyl-benzamido-alpha'-substituted-styryl-cyclohexanone thiosemicarbazones (III) in yields ranging from 40 to 50 percent. The compounds exhibited pronounced antiimplantation activity in female albino rats.