Characterization of NAD:arginine ADP-ribosyltransferases

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.     

Optimization of alpha-acylaminoketone ecdysone agonists for control of gene expression

Fifteen new alpha-acylaminoketones were prepared by four different routes in an initial effort to optimize the potency of these compounds as ecdysone agonists. The compounds were assayed in mammalian cells expressing the ecdysone receptors from Bombyx mori (BmEcR) and Choristoneura fumiferana (CfEcR) for their ability to cause expression of a reporter gene downstream of an ecdysone response element. A new alpha-acylaminoketone was identified which had activity equal to that of the standard dibenzoylhydrazine ecdysone agonist GS()-E in the assay based on CfEcR.     

Metastasis of bronchogenic carcinoma to the thumb

Bone metastasis in the hand is rare. The etiology is quite different from that of metastasis to other bones; bronchogenic carcinoma is by far the most frequent case. Distal phalanges are mainly involved with irregular osteolysis and cortical destruction. Differential diagnosis of phalangeal metastasis includes osteomyelitis, rheumatoid arthritis and gout. The prognosis is always that of metastatic bronchial cancer with an average survival of three months. Treatment may involve distal digital amputation or antalgic radiotherapy. A case of bronchogenic carcinoma with metastasis to the thumb is presented. The metastasis was located in the distal phalanx of the left thumb. The primary tumor was located in the lung. Treatment consisted of amputation. The overall survival was five months.     

Two New Mycobacterium Strains and Their Role in Toluene Degradation in a Contaminated Stream

Two toluene-degrading strains, T103 and T104, were isolated from rock surface biomass in a freshwater stream contaminated with toluene. The strains exhibit different capacities for degradation of toluene and other aromatic compounds and have characteristics of the genus Mycobacterium. Both are aerobic, rod-shaped, gram-positive, nonmotile, and acid-alcohol fast and produce yellow pigments. They have mainly straight-chain saturated and monounsaturated fatty acids with 10 to 20 carbon atoms and large amounts of tuberculostearic acid that are typical of mycobacteria. Fatty acid analyses indicate that T103 and T104 are different mycobacterial strains that are related at the subspecies level. Their identical 16S rDNA sequences are most similar to Mycobacterium aurum and Mycobacterium komossense, and they constitute a new species of fast-growing mycobacteria. Ecological studies reveal that toluene contamination has enriched for toluene-degrading bacteria in the epilithic microbial community. Strains T103 and T104 play only a small role in toluene degradation in the stream, although they are present in the habitat and can degrade toluene. Other microorganisms are consequently implicated in the biodegradation.     

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.      

Independent phylogenetic origins of methanotrophic and chemoautotrophic bacterial endosymbioses in marine bivalves.

The discovery of bacterium-bivalve symbioses capable of utilizing methane as a carbon and energy source indicates that the endosymbionts of hydrothermal vent and cold seep bivalves are not restricted to sulfur-oxidizing chemoautotrophic bacteria but also include methanotrophic bacteria. The phylogenetic origin of methanotrophic endosymbionts and their relationship to known symbiotic and free-living bacteria, however, have remained unexplored. In situ localization and phylogenetic analysis of a symbiont 16S rRNA gene cloned from the gills of a recently described deep-sea mussel species demonstrate that this symbiont represents a new taxon which is closely related to free-living, cultivable Type I methanotrophic bacteria. This symbiont is distinct from known chemoautotrophic symbionts. Thus, despite compelling similarities between the symbioses, chemoautotrophic and methanotrophic symbionts of marine bivalves have independent phylogenetic origins.     

Prospects for estimating nucleotide divergence with RAPDs

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.      

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf. © 1993 Wiley-Liss, Inc.