Cross-linking of DNA in liver and testes of rats fed 1,3-propanediol

1,3-Propanediol (PAD) was fed to rats for 15 weeks, and its effects on hepatic and testicular DNA were studied. The control rats were fed a casein-based diet that contained 10% tocopherol-stripped corn oil with 30 IU of d,l-α-tocopherol acetate/kg; the experimental rats were fed the same diet with 500 ppm of PAD. Homogenates prepared from the livers of each group of rats converted 1,3-propanediol to malondialdehyde (MDA) with equal efficacy, but homogenates of testes did not catalyze this conversion. After 10–15 weeks of feeding the diets, the hepatic DNA of the rats fed PAD had less template activity, more bound tryptophan and more DNA-protein and interstrand DNA cross-links than that of the control rats. As measured by template activity and bound tryptophan, testicular DNA of the experimental rats was not different from that of the control rats; however, there was slightly more cross-linking in the testicular DNA of experimental rats than in that of control rats. Testes of the experimental rats contained more lipid-soluble fluorophores than did those of the control rats. The results are consistent with the conclusion that PAD was converted to MDA in vivo and that MDA is the reactive species that caused the observed biological damage.     

Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.     

类脂过氧化对竹红菌甲素引起膜蛋白光敏交联的影响

本文研究了丙二醛与红细胞膜温育后所形成的交联。当丙二醛浓度在5×10~-4mol/L以上时,膜蛋白发生交联,并且在460nm处有荧光特征峰出现。而甲素光敏所致膜蛋白的交联,在460nm处没有荧光峰,光敏所产生的内源丙二醛量很少,不足以引起红细胞膜反应形成交联。我们还研究了BHT和Vit E两种抗氧化剂对甲素光敏作用的影响。BHT能抑制类脂过氧化,不能抑制膜蛋白巯基的变化和膜蛋白的交联。而Vit E仅不能抑制膜蛋白的交联。以上结果均说明甲素光敏所致膜类脂过氧化是不参与膜蛋白交联的。     

Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin

The NH2-terminal 12-residue peptide of alpha 2-plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Gly-Leu-Lys-NH2 . AcOH, was found to be a good substrate for plasma transglutaminase (activated blood coagulation factor XIII) and rapidly incorporated into fibrin by the enzyme. A high concentration of the peptide inhibited the enzyme-mediated cross-linking of alpha 2-plasmin inhibitor to fibrin probably by competing with the inhibitor for the same site of fibrin alpha-chain.     

Identification of guanine and adenine adducts in DNA alkylated by dibromodulcitol in vitro and in vivo

DNA reacted with dibromodulcitol in neutral solution yielded 3- and 7-alkyl substituted purines after hydrolysis at neutral pH-value at 37°C. The alkylated products were identified by mass spectrometry and by comparison of their UV absorption spectra and chromatographic properties on thin-layer chromatography (TLC) and various columns with those of the corresponding galactitylpurine derivatives obtained by synthetic route from alkylation of the appropriate nucleic bases or nucleosides. The labelled alkylpurines occurring in DNA of Yoshida tumour cells treated with [³H]dibromodulcitol in vivo were also indentified by co-chromatography of labelled DNA hydrolysate with synthetic 3- and 7-alkyl substituted purines. On the basis of the same chromatographic behaviour 3-(1-deoxy-3,6-anhydrogalactit-1-yl)adenine, 7-(1-deoxygalactit-1-yl)guanine, 7-(1-deoxy-3,6-anhydrogalactit-1-yl)guanine and 1,6-di(guanin-7-yl)-1,6-dideoxygalactitol were identified as main alkylated products in tumor cell DNA after in vivo treatment with dibromodulcitol.     

Immobilization of Haloferax mediterranei aldolase by cross-linking in a proteinic matrix: stability and halophilic characteristics

Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) of Haloferax mediterranei was immobilized by treating the cell extract in the presence of 10% BSA, with the cross-linking reagent, 0.5% glutaraldehyde for 15min, with the retention of 60% of its original activity. The immobilized preparation exhibited a shift in the temperature optimum from 55°C to 65°C. The enzyme showed enhanced stability towards inactivation by radiation and storage (0–5°C) on immobilization. Immobilization also made the enzyme less halophilic, reducing its denaturation on prolonged storage in a non-salt medium, as well as exhibiting optimal activity at a lower KCl concentration (0.5m) as compared to the soluble enzyme (1–2m).     

酶标HBV DNA探针的研制与应用

本实验采用一种非放射性物质——碱性磷酸酶标记乙肝病毒HBV DNA制备分子探针。碱性磷酸酶在苯醌作用下与单链DNA联结,形成DNA和酶的共价复合物,即酶标探针。此探针通过分子杂交与待测DNA结合,与酶的底物作用显色,几小时内可观察结果,其最低检测量约为10pg。用此探针检测乙肝病人血清中的HBV DNA,与~(32)P标记的探针比较,酶标探针可检测出~(32)P标记探针检出率的95.7％。结果表明,所合成的酶标探针具有准确、简便、快速、安全而经济的优点,具有应用前景。     

Ca2+-dependent cross-linking processes in human platelets

When platelet cytoplasmic Ca²⁺ is increased by the ionophore A23187 in the presence of the protease inhibitor leupeptin, there is the coincident appearance of a cross-linked polymer and the partial disappearance of monomeric protein and glycoprotein units. In the absence of leupeptin only 30% of the polymer was formed. The disappearance of monomeric protein bands, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is prevented by histamine, which as a pseudodonor amine is a known inhibitor of transglutaminase-catalyzed cross-linking. [¹⁴C]Histamine, at a tracer concentration, is incorporated into the polymer as well as into myosin, glycoproteins IIB and III, actin and tropomyosin. The lose of monomeric protein bands is mostly due to their conversion into polymers. Control measurements show that leupeptin effectively inhibited platelet Ca²⁺-dependent proteases. The cross-linking processes bringing about the observed increase in polymer formation are thus the result of a Ca²⁺-dependent platelet transglutaminase activity. The latter is located in the platelet cytosol and has been identified as platelet factor XIII on the basis of its specific cross-linking of fibrin. Platelet factor XIII, upon activation, may function physiologically to couple membrane proteins to cytoplasmic structural proteins. Thus, a new concept is proposed for the stabilization of platelet membranes and platelets as they form the hemostatic plug.     

Electrospun cellulose acetate phthalate nanofibrous scaffolds fabricated using novel solvent combinations biocompatible for primary chondrocytes and neurons

Electrospun nanofibres have been shown to exhibit extracellular matrix (ECM)-like characteristics required for tissue engineering in terms of porosity, flexibility, fibre organization and strength. This study focuses on developing novel cellulose acetate phthalate (CAP) scaffolds by electrospinning for establishing 3-D chondrocyte and neuronal cultures. Five solvent combinations were employed in fabricating the fibres, namely, acetone/ethanol (9:1), dimethylformamide/tetrahydrofuran/acetone (3:3:4), tetrahydrofuran/acetone (1:1), tetrahydrofuran/ethanol (1:1) and chloroform/methanol (1:1). The electrospun fibres were characterized by scanning electron microscopy (SEM) analysis and confirmed to be within the nanometre range. Based on the morphology of the fibers from SEM results, two solvent combinations such as acetone/ethanol and dimethylformamide/tetrahydrofuran/acetone were selected for stabilization as CAP exhibits a pH dependent solubility. Fourier-Transform Infrared (FTIR) analysis revealed the hydrolysis of CAP which was overcome by EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] and EDC/NHS (N-hydroxysuccinimide) cross-linking resulting in its stability (pH of 7.2) for three months. MTT [3-(4, 5-dimethylthiazol-2-yl)-1, 5-diphenyltetrazolium bromide] assay performed using L6 myoblast confirmed the biocompatibility of the scaffolds. 3-D primary chondrocyte and neuronal cultures were established on the scaffolds and maintained for a period of 10 days. H&E staining and SEM analysis showed the attachment of the chondrocytes and neurons on CAP scaffolds prepared using dimethylformamide/tetrahydrofuran/acetone and acetone/ethanol respectively.     

Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained ⿼50% of the free enzyme activity at 6.4 mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.