Changes in poly(adenosine diphosphate-ribose) and poly(adenosine diphosphate-ribose) polymerase in synchronous HeLa cells.

An antibody has been prepared which is highly specific for poly(adenosine diphosphate-ribose). Neither poly(A), DNA, nor a variety of adenine-containing nucleosides or nucleotides were effective in competing with poly(ADP-ribose) for binding to the antibody. Of all compounds tested, only adenosine diphosphate-ribose competed for binding to the antibody. Unlabeled poly(adenosine diphosphate-ribose) was about 10 000 times more effective in competing with labeled polymer for antibody binding than was adenosine diphosphate-ribose. Using the antibody, the amount of poly(adenosine diphosphate-ribose) was found to increase from early S phase to a peak at mid S with a second, even larger increase seen at the S-G2 transition point in synchronously dividing HeLa cells. Pulse labeling of the polymer with [2-3H]adenosine was also maximal at the same time points. Changes in the levels of poly(adenosine diphosphate-ribose) polymerase activity measured in isolated nuclei coincided with the changes in amounts of polymer present in intact cells during progression from S phase into G2.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

Coordinated activities of human dicer domains in regulatory RNA processing

The conserved ribonuclease Dicer generates microRNAs and short-interfering RNAs that guide gene silencing in eukaryotes. The specific contributions of human Dicer's structural domains to RNA product length and substrate preference are incompletely understood, due in part to the difficulties of Dicer purification. Here, we show that active forms of human Dicer can be assembled from recombinant polypeptides expressed in bacteria. Using this system, we find that three distinct modes of RNA recognition give rise to Dicer's fidelity and product length specificity. The first involves anchoring one end of a double-stranded RNA helix within the PAZ domain, which can assemble in trans with Dicer's catalytic domains to reconstitute an accurate but non-substrate-selective dicing activity. The second entails nonspecific RNA binding by the double-stranded RNA binding domain, an interaction that is essential for substrate recruitment in the absence of the PAZ domain. The third mode of recognition involves hairpin RNA loop recognition by the helicase domain, which ensures efficient processing of specific substrates. These results reveal distinct interactions of each Dicer domain with different RNA structural features and provide a facile system for investigating the molecular mechanisms of human microRNA biogenesis.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.     

Distribution of Drosophila melanogaster transposable element sequences in species of the obscura group

Fifteen species belonging to the obscura group of the genus Drosophila were screened for sequences homologous to Drosophila melanogaster transposable elements (TEs) as an initial step in the examination of the possible occurrence of TEs at chromosomal inversion breakpoints. Blots of genomic DNAs from species of the obscura group were hybridized at three different stringencies with 14 probes representing the major families of TEs described in D. melanogaster. The probe DNAs included copia, gypsy, 412, 297, mdg1, mdg3, 3S18, F, G, I, jockey, P, hobo, and FB3. D. melanogaster TEs were not well represented in the species of the obscura group analyzed. The TEs that were observed generally exhibited heterogeneous distributions, with the exception of F, gypsy and 412 which were ubiquitous, and 297, G, Sancho 2, hobo and FB which were not detected.by A. Bird     

Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines.

A mammary-derived growth factor, MDGF1, which stimulates collagen synthesis and proliferation in mammary epithelial cells was previously detected and purified from human milk and primary human breast tumors. MDGF1 binds to putative cell-surface receptors of 120-140 kDa and stimulates proliferation of normal and malignant human mammary epithelial cells. Partial protein sequence (N-terminal 18 amino acid sequence) shows that MDGF1 has no homology to any other known growth-promoting peptides. Polyclonal antiserum raised against this synthetic peptide recognizes native milk-derived MDGF1. We hypothesize that MDGF1 might be an autocrine or paracrine factor produced by and acting on normal and malignant human breast epithelial cells possessing MDGF1 receptors. As a first step in testing this possibility, we examined whether human breast epithelial cells in culture produce the growth factor. A protein with the size of MDGF1 was immunologically detected in the concentrated conditioned medium prepared from human breast cancer cell line MDA-MB 231, the mammary-derived but nontumorigenic HBL-100 line, and the normal reduction mammoplasty-derived, nonimmortalized 184 cell strain. A competitive radioreceptor assay (RRA) was used to estimate the level of MDGF1 in the conditioned medium. MDGF1 was present in the nanogram range per 1 million cells. A 62-kDa protein was detected in the above cell lysates by Western immunoblotting or by immunoprecipitation of metabolically labeled cell-conditioned media. The polyclonal antisera directed against the 18 amino acid peptide sequence from milk-derived MDGF1 could adsorb MDGF1 biological activity from conditioned medium. In vitro translation of cell mRNA yielded a protein of 55 kDa which was immunoprecipitated by anti-MDGF1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drosophila mediopunctata P elements: a new example of horizontal transfer.

Sequences homologous to the P element of Drosophila melanogaster were previously identified in Drosophila mediopunctata, a member of the tripunctata group, subgenus Drosophila. We report here that the P element is present in about three to five copies in the D. mediopunctata genome. While one of the insertion sites appears to be fixed, others may be polymorphic, indicating relatively recent P element activity. Phylogenetic analysis revealed that the D. mediopunctata element belongs to the canonical subfamily of P elements and that divergence of the D. mediopunctata element from other members of this subfamily ranges from 2% to 5% at the nucleotide level. This is the first report of a canonical P element outside the subgenus Sophophora. Based primarily on the striking incongruence between P element and host species phylogenies, the presence of a canonical P element in D. mediopunctata is most likely explained by horizontal transfer between species.     

Mass spectrometric differentiation of leucine and isoleucine in proteins derived from bacteria or cell culture

The differentiation of leucine and isoleucine is a well known difficulty in mass spectrometric peptide sequencing. A technique has been developed which allows these two amino acids to be distinguished by growing a bacterial or cell culture in a medium containing γ,δ-dl-dideuteroleucine. The isotopically labelled residue is incorporated into the cell's proteins, and the resulting mass spectra of leucine containing peptides exhibit sequence ions 2 amu higher than the corresponding isoleucine peptides.