The copper binding domain of SPARC mediates cell survival in vitro via interaction with integrin beta1 and activation of integrin-linked kinase

Secreted protein acidic and rich in cysteine (SPARC) is important for the normal growth and maintenance of the murine lens. SPARC-null animals develop cataracts associated with a derangement of the lens capsule basement membrane and alterations in lens fiber morphology. Cellular stress and disregulation of apoptotic pathways within lens epithelial cells (LEC) are linked to cataract formation. To identify molecular targets of SPARC that are linked to this disorder, we stressed wild-type (WT) and SPARC-null LEC by serum deprivation or exposure to tunicamycin. SPARC enhanced signaling by integrin-linked kinase (ILK), a serine/threonine kinase known to enhance cell survival in vitro. In response to stress, an ILK-dependent decrease in apoptosis was observed in WT relative to SPARCg-null LEC. Co-immunoprecipitation and cross-linking of cell lysates revealed enhanced levels of a SPARC-integrin beta1 complex during stress. Competition with monoclonal antibodies and peptides indicated that the copper binding domain of SPARC is required for SPARC-mediated response to stress. Inhibiting the binding and/or activity of ILK, integrin beta1, or SPARC resulted in increased apoptosis of stressed LEC. We conclude that SPARC protects cells from stress-induced apoptosis in vitro via an interaction with integrin beta1 heterodimers that enhances ILK activation and pro-survival activity.     

Runoff and drainage water quality from geotextile and gravel pads used in livestock feeding and loafing areas

Geotextile and gravel pads offer a low-cost alternative to concrete for providing all-weather surfaces for cattle and vehicle traffic, and are used in many livestock facilities to minimize mud, runoff and erosion of heavy traffic areas. The objective of this study was to compare different combinations of geotextile and gravel used in heavy livestock traffic areas that minimize the potential for water pollution. Three different pad combinations were constructed in 2.4 x 6-m plots as follows: (i) woven geotextile+100mm of gravel+50mm Dense Grade Aggregate (DGA); (ii) woven geotextile + geoweb+100 mm DGA; and (iii) non-woven geotextile+152 mm of gravel+50mm DGA; (iv) mud lots as control. The third combination was equivalent to one of the base treatments specified by the Kentucky Natural Resource and Conservation Service (NRCS). All treatment combinations were duplicated. Lysimeter pans were installed in four out of eight plots for the collection of leachate or drainage water. Runoff was collected at the lower end of the plots. About 14 kg of beef cattle manure were added evenly to the plots. Rainfall at 50mm/h was applied using rainfall simulators. In the first five of ten experiments, manure was removed from the surface of the pads after each experiment. In the remaining five experiments manure accumulated on the surface of the pads. The effect of pad treatment was significant on the electrical conductivity (EC), total solids (TS), chemical oxygen demand (COD), nitrite (NO2-N), total nitrogen (TN) and total phosphorus (TP) values in surface runoff at the 5% level. Manure removal did not have any significant effect on the nutrient content of runoff or leachate samples except for ammonia (NH4-N) values. Although a mass balance indicated relatively small amounts of organic matter and nutrients were lost by runoff and leaching, the actual contamination level of both runoff and leachate samples were high; TP levels as high as 12 mg/l (5.4 mg/m2) in runoff and nitrate (NO3-N) values as high as 10.8 mg/l (1.6 mg/m2) in leachate were observed.     

High affinity binding of thrombin to platelets. Inhibition by tetranitromethane and heparin

[¹²⁵I] iodo-α-thrombin has been modified at the macromolecular substrate binding site in order to study the importance of this region in the platelet-thrombin interaction. Modification was effected by the nitration of tyrosine residues with tetranitromethane. This chemical modification abolished the ability of the enzyme to bind with a high affinity to the platelet surface but did not significantly alter low affinity binding. The presence of heparin was also found to inhibit high affinity binding. These results indicate that the high affinity binding site interacts with the fibrinogen binding region of the thrombin molecule and suggests that there are two distinct classes of binding sites for thrombin on the platelet membrane.     

Elemental mapping of fluorine using electron spectroscopic imaging for the intracellular localisation of an anthracycline drug (ME2303) in human ovarian cancer cells.

A major limitation to the clinical usefulness of cancer chemotherapy is the onset of acquired drug resistance, in which the effectiveness of a drug such as Doxorubicin (DOX), used in a wide variety of neoplasms, diminishes following repeated administration. Resistance is associated with drug exclusion from tumour cell nuclei. New analogues of DOX have been synthesised to reduce patient cardiotoxicity and to increase anti-tumour activity. More recently, a 2-fluoroglycoside of DOX (ME2303) has been shown to be more resistant to glycolysis and has marked anti-proliferative effects on DOX-resistant tumours. The aim of the current study was therefore to determine the intracellular localisation of ME2303 in drug sensitive and resistant human ovarian cancer cell aggregates by mapping fluorine, as a means of understanding the complex mechanisms of drug resistance. Cell aggregates of the human ovarian cell line A2780 and its DOX-resistant subline 2780AD were cultured for 1hr in 5μM of the drug ME2303, then chemically fixed and embedded in Lowicryl K4M using alcohol dehydration at ?20°C. Ultrathin sections (40-50nm) were examined on a Zeiss TEM 902 energy filtering electron microscope using 80Kv at a magnification of 12,000x. Fluorine maps were generated using the two window method by collecting two integrated images above and two integrated images below the K ionisation edge for fluorine (685eV) with a 15eV window. Image sequences were collected within 20sec to minimise the effects of mass loss from the specimen via a Dage SIT 66 video camera connected to a DT 2861 video interface board (512x512 pixels with 256 grey levels) within a 486 PC. In the A2780 cell line, fluorine was found to be distributed diffusely within the cytoplasm and at discrete sites within the nucleus. In contrast, fluorine in the 2780AD subline (co-cultured with 1.4μM DOX to maintain resistance) was found to be largely associated with the peri-nuclear Golgi region and with mitochondria, but was also found within cell nuclei, along the inner nuclear envelope and in the nucleolus. The intra-nuclear localisation of fluorine suggests that even in the presence of DOX, ME2303 can mediate anti-proliferative activity in DOX resistant human ovarian cancer cells by effective nuclear translocation.     

The effect of monovalent cations on the catalytic activity of thrombin

The effect of monovalent cations on the catalytic action of thrombin has been examined utilizing a variety of substrates. Sodium chloride noncompetitively inhibited the action of thrombin on α-tosyl-l-arginine methyl ester and α-N-benzoyl-l-arginine-p-nitroanilide. No inhibition was noted when α-N-benzoyl-l-arginine ethyl ester was the substrate. The extent of inhibition was considerably less with either potassium chloride or lithium chloride. The rate of inactivation of thrombin by 1-chloro-3-tosylamido-7-amino-l-2-heptanone was reduced in the presence of sodium ions. The results are interpreted to show a specific effect of sodium ions on the ability of the active-site histidine residue to participate in thrombic catalysis.     

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.     

Biosynthesis and incorporation into protein of norleucine by Escherichia coli

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.