Co-localization of beta1,6-branched oligosaccharides and coarse melanin in macrophage-melanoma fusion hybrids and human melanoma cells in vitro

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of beta1,6-N-acetylglucosaminyltransferase V and beta1,6-branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced 'coarse melanin'- autophagic vesicles with multiple melanosomes. As beta1,6-branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin- and immunohistochemistry, we analyzed cell lines producing coarse melanin for beta1,6-branched oligosaccharides: gp100/pmel-17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94-H48, a highly metastatic, macrophage-melanoma experimental fusion hybrid; (ii) 6(neo) mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel-23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel-23 cells, and co-localized with stains for beta1,6-branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co-expression of beta1,6-branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.     

Skin waste, vertex angle, and scar length in excisional biopsies: comparing five excision patterns--fusiform ellipse, fusiform circle, rhomboid, mosque, and S-shaped

The common excision skin pattern is either a fusiform ellipse or another pattern with dissimilar length and width. The purpose of this study was to define the most advantageous skin pattern regarding skin waste, vertex angle, and scar length. Five skin excision patterns used traditionally for closure of round lesions were analyzed: fusiform ellipse, fusiform circle, rhomboid, mosque, and S-shaped. In the analysis, the pattern characteristics were formulated by geometric principles, from which the results were compared. The smallest skin waste was found in rhomboid and mosque patterns, whereas the largest skin waste was found in the fusiform circle and ellipse. The vertex angle was found to decrease monotonously with the excision length-to-width ratio for all patterns except the mosque shape, which is zero per definition. The paradigm stating that a vertex angle of 30 degrees or less is maintained for length-to-width ratios below 4 in the surgical ellipse was found incorrect. It holds only for rhomboid and S-shaped excisions. The scar length was found almost independent of the pattern, with a variance of 3 percent. The authors conclude that the most advantageous surgical skin patterns are the rhomboid and mosque excisions.     

Thyroid receptor ligands. Part 2: Thyromimetics with improved selectivity for the thyroid hormone receptor beta

A set of thyromimetics having improved selectivity for TR-beta1 were prepared by replacing the 3'-isopropyl group of 2 and 3 with substituents having increased steric bulk. From this limited SAR study, the most potent and selective compounds identified were derived from 2 and contained a 3'-phenyl moiety bearing small hydrophobic groups meta to the biphenyl link. X-ray crystal data of 15c complexed with TR-beta1 LBD shows methionine 442 to be displaced by the bulky R3' phenyl ethyl amide side chain. Movement of this amino acid side chain provides an expanded pocket for the bulky side chain while the ligand-receptor complex retains full agonist activity.     

The EMBL Nucleotide Sequence Database: major new developments

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) incorporates, organizes and distributes nucleotide sequences from all available public sources. The database is located and maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK. In an international collaboration with DDBJ (Japan) and GenBank (USA), data are exchanged amongst the collaborating databases on a daily basis to achieve optimal synchronization. Webin is the preferred web-based submission system for individual submitters, while automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via FTP, Email and World Wide Web interfaces. EBI's Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases plus many other specialized molecular biology databases. For sequence similarity searching, a variety of tools (e.g. Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. All resources can be accessed via the EBI home page at http://www.ebi.ac.uk.     

Melatonin and activity rhythm responses to light pulses in mice with the Clock mutation

Melatonin and wheel-running rhythmicity and the effects of acute and chronic light pulses on these rhythms were studied in Clock(Delta19) mutant mice selectively bred to synthesize melatonin. Homozygous melatonin-proficient Clock(Delta19) mutant mice (Clock(Delta19/Delta19)-MEL) produced melatonin rhythmically, with peak production 2 h later than the wild-type controls (i.e., just before lights on). By contrast, the time of onset of wheel-running activity occurred within a 20-min period around lights off, irrespective of the genotype. Melatonin production in the mutants spontaneously decreased within 1 h of the expected time of lights on. On placement of the mice in continuous darkness, the melatonin rhythm persisted, and the peak occurred 2 h later in each cycle over the first two cycles, consistent with the endogenous period of the mutant. This contrasted with the onset of wheel-running activity, which did not shift for several days in constant darkness. A light pulse around the time of expected lights on followed by constant darkness reduced the expected 2-h delay of the melatonin peak of the mutants to approximately 1 h and advanced the time of the melatonin peak in the wild-type mice. When the Clock(Delta19/Delta19)-MEL mice were maintained in a skeleton photoperiod of daily 15-min light pulses, a higher proportion entrained to the schedule (57%) than melatonin-deficient mutants (9%). These results provide compelling evidence that mice with the Clock(Delta19) mutation express essentially normal rhythmicity, albeit with an underlying endogenous period of 26-27 h, and they can be entrained by brief exposure to light. They also raise important questions about the role of Clock in rhythmicity and the usefulness of monitoring behavioral rhythms compared with hormonal rhythms.     

Plastic reproductive strategies in a clonal marine invertebrate

Plastic reproductive allocation may allow individuals to maximize their fitness when conditions vary. Mate availability is one condition that may determine the fitness of an individual's allocation strategy. Using a variety of methods, I detected evidence of plastic allocation to asexual (clonal) reproduction in response to mate availability in the brittle star Ophiactis savignyi. There were more mature individuals in populations in which both sexes were present, and clones from these populations had fewer clone-mates than clones from single-sex populations. Animals placed with mates in a field experiment divided less frequently than animals without a mate. These findings demonstrate that animals reduce their allocation to asexual reproduction when mates are present and when a loss of fecundity associated with cloning would decrease offspring production. This plasticity is probably adaptive because it maximizes sexual-reproductive potential when fertilization is more likely, but maximizes survival of the clone when mates are absent and gametes are unlikely to be converted to offspring.     

Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabolites

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i). minimal interference with the exponential cell growth, (ii). minimal reduction in cellular host RNA levels, and (iii). reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.     

Ether phospholipids and glycosylinositolphospholipids are not required for amastigote virulence or for inhibition of macrophage activation by Leishmania major

Ether phospholipids are major components of the membranes of humans and Leishmania. In protozoan parasites they occur separately or as part of the glycosylphosphatidylinositol (GPI) anchor of molecules implicated in virulence, such as lipophosphoglycan (LPG), smaller glycosylinositolphospholipids (GIPLs), and GPI-anchored proteins. We generated null mutants of the Leishmania major alkyldihydroxyacetonephosphate synthase (ADS), the first committed step of ether lipid synthesis. Enzymatic analysis and comprehensive mass spectrometric analysis showed that ads1- knock-outs lacked all ether phospholipids, including plasmalogens, LPG, and GIPLs. Leishmania ads1- thus represents the first ether lipid-synthesizing eukaryote for which a completely null mutant could be obtained. Remarkably ads1- grew well and maintained lipid rafts (detergent-resistant membranes). In virulence tests it closely resembled LPG-deficient L. major, including sensitivity to complement and an inability to survive the initial phase of macrophage infection. Likewise it retained the ability to inhibit host cell signaling and to form infectious amastigotes from the few parasites surviving the establishment defect. These findings counter current proposals that GIPLs are required for amastigote survival in the mammalian host or that parasite lyso-alkyl or alkylacyl-GPI anchors are solely responsible for inhibition of macrophage activation.