A protocol for the purification of bovine serum albumin free of deoxyribonuclease activity

Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a linear gradient 0-1.0 M NaCl and subsequently acetylated, proved to be the most effective method for obtaining deoxyribonuclease-free bovine serum albumin.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.     

Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH₂-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58050. Address correspondence and offprint requests to: D. F. J. Purcell.     

The secY protein can act post-translationally to promote bacterial protein export

Conditionally lethal Escherichia coli mutants in secY (prlA) show defective export of proteins to the periplasm and outer membrane. It has been proposed that this gene and other sec genes must act on pro-OmpA at an early stage of protein synthesis in order to allow later translocation to occur. We have described a temperature-sensitive mutation in which the secYts function is impaired at the nonpermissive temperature (Ito, K. (1984) Mol. Gen. Genet. 197, 204-208). A plasmid bearing the wild-type secY gene under the control of the lactose operon (Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631-635) has been introduced into this mutant strain. We now report that the in vivo chase of pulse-labeled full length pro-OmpA to mature OmpA is accelerated by inducing the synthesis of the wild-type secY protein at the end of the period of pulse labeling. We have also assayed the requirements for secY function for in vitro protein translocation. Membranes derived from secY ts cells which were incubated at 42 degrees C were inactive in vitro in the post-translational uptake and processing of pro-OmpA. Thus, the secY protein can act post-translationally, enhancing the translocation of completed pro-OmpA polypeptide chains across the plasma membrane.     

The synergistic lethal interaction of cis-diamminedichloroplatinum and natural nucleosides is related to increased DNA cross-links

Human tumor cells were treated in vitro with combinations of cis- or trans-dichlodiammineplatinum (DDP) and natural nucleosides (thymidine, uridine, cytidine and adenosine). Effects were measured by inhibition of colony-formation (cell survival) and DNA alkaline elution (DNA cross-links). No increments in cell lethality or DNA cross-links were elicited by any combination of trans-DDP and nucleosides. In contrast, every combination of cis-DDP and nucleoside was eminently synergistic with 5- and 10-fold increases in cell lethality over the predicted sum of each agent alone. These increments in cell kill correlated linearly with increases in DNA crosslinks suggesting that the nucleosides interact with cis-DDP to enhance its cytotoxic crosslinking mode of action.     

Cultured human tumour cells may be arrested in all stages of the cycle during stationary phase: demonstration of quiescent cells in G1, S and G2 phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9-18%) were located in G2 phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). To substantiate these findings, cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located 'somewhere' in G1 phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Qs and QG2). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following 'recruiting' or 'synchronizing' clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

The Electrical Conductivity of Bovine Milk in Mastitis Diagnosis

The electrical conductivity (EC) of milk is mainly a function of the electrolyte concentration in the milk and therefore raised in mastitis. The present investigation was aimed at elaborating, if possible, a diagnostic model for screening purposes based on EC determinations and consistent with the diagnostic procedures and interpretations commonly used in laboratory milk diagnosis in the Nordic countries (Klastrup 1975). According to this diagnosis (here called reference diagnosis) cell numbers above 300,000/ml (cell count or the corresponding CMT-score) in foremilk quarter samples during the main part of the lactation period and significantly above the lowest value on within-udder comparison during late lactation are considered indicative of mastitis and bacteriological examinations are made when called for.     

Crystals of virus-like particles in the metacestodes of Taenia solium and T. crassiceps

Crystals of virus-like particles (VLP) are described as occurring in the nuclei of damaged tegumentary cytons from carcasses of Taenia solium metacestodes that had been stripped of their teguments. The VLP are grouped as parallel lines of round particles in an hexagonal packaging of spheroids forming small or large crystals. The individual particles have an external diameter of 36-37 nm and a wall of 5-6 nm thick, which surround a cavity of lower electron density. As identical crystals were also observed in normal tissues of T. solium and of T. crassiceps, it is suggested that both species of cysticerci are normal carriers of a similar species of virus. The possible biological implications of this condition are discussed.