Template specificities of Aclacinomycin B on the inhibition of DNA-dependent RNA synthesis in vitro

Summary The effect of Aclacinomycin B (ACM-B), an anthracycline antitumor antibiotic, on the DNA-dependent RNA synthesis using single- and double-stranded DNAs of known base content and sequence is studied. The data show that ACM-B effectively inhibits the double-stranded DNA-directed RNA synthesis with a preference of poly[d(A-T)] > poly[d(G-C)] > poly[d(I-C)]. In contrast, it has no inhibitory effect on the template function of single-stranded DNA (e.g. poly dA, poly dT, and poly dC). These results suggest that the mechanism of ACM-13 inhibition, like other anthracycline antibiotics, is by intercalation. In addition to the base specificity, there are also dramatic differences in inhibition depending on the base sequence in the DNA template. Thus, ACM-13 preferentially inhibits the alternating double-stranded copolymers over the double-stranded homopolymers; e.g. poly [d(A-T)] is inhibited to a greater extent than poly dA · poly dT and poly [d(G-C)] is inhibited more than poly dG · poly dC. Since the inhibition by ACM-13 can be totally abolished when assayed in excess amount of DNA, this result suggests that ACM-B inhibition of RNA synthesis is solely on the DNA template (which is in support of the intercalation model), and has ruled out the possibility that ACM-B may also exert an inhibitory effect on the activity of RNA polymerase per se.     

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A⁺) mRNA detection by hybridization to [³H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000–120000 range. The Mr 120000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.Abbreviations ADP-ribose adenosine (5) diphospho(5)--D ribose - poly(ADP-ribose) polymer of ADP-ribose - mRNP messenger ribonucleoprotein particles - PMSF phenylmethylsulfonyl fluoride - LDS lithium dodecyl sulfate - TCA trichloroacetic acid     

On the interaction of the synthetic 53–58 fragment of lac repressor with DNA

The hexapeptide fragment 53–58 of the lac repressor protein (Ala-Gln-Gln-Leu-Ala-Gly) was synthesized. Circular dichroic spectrum of the peptide has negative bands at 195 and 225 nm (weak). The hexapeptide, which has no basic residues, stabilized the AT rich Clostridium perfringens DNA, but had no effect, significantly, on the melting profile of either poly dA · poly dT or poly d(A-T)·poly d(A-T). The melting temperature of Micrococcus lysodeikticus DNA with a high GC content, was unaffected by the peptide.     

一个独特的RNA酶A抗性复合物在腺病毒L1 poly(A)位点的上游形成

在腺病毒交替的ｐｏｌｙ（Ａ）位点使用过程中,靠近主要晚期启动子的Ｌ１ ｐｏｌｙ（Ａ）位点起着主导的作用。前期的实验已经发现,在Ｌ１ ｐｏｌｙ（Ａ）位点的上游存在一个ＲＮＡ的抑制元件叶ＵＲＥ,缺失ＵＲＥ可以使模拟小基因的ｐｏｌｙ（Ａ）进入病毒晚期的感染方式。现将Ｌ１ ｐｏｌｙ（Ａ）位点单独游离出来,用体外的紫外交联的方法对其进行研究,结果发现在没有紫外光照射的情况下,仍有一组小于３０ｋＤ独特的ＲＮＡ     

Effects of 3′deoxyadenosine and actinomycin D on RNA synthesis in toad bladder: Analysis of response to aldosterone

Summary Previous studies have shown that aldosterone increases transepithelial active Na⁺ transport after a latent period of about 60 min and incorporation of³H-uridine into polyadenylated RNA (poly(A)(+)RNA) (putatively poly(A)(+)mRNA) as early as 30 min after aldosterone addition. To assess the physiological importance of this pathway, the effects of 3deoxyadenosine and actinomycin D were compared in studies on the urinary bladder of the toadBufo marinus. 3deoxyadenosine (30 g/ml) only partially, though significantly, inhibited the aldosterone-dependent increase in Na⁺ transport measured as short-circuit current (scc). The incorporation of³H-uridine into poly(A) (+)RNA was inhibited by 70 to 80%. In contrast, Actinomycin D (2 g/ml) totally inhibited the aldosterone-dependent increase in scc, and the incorporation of³H-uridine into poly(A)(+)RNA by 68 to 75%. 3deoxyadenosine or actinomycin D alone had no significant effects on baseline scc, while inhibiting poly(A)(+)RNA to the same extent. The differential effects of deoxyadenosine and actinomycin on aldosterone-dependent Na⁺ transport may be related to their different sites of action on RNA synthesis: both drugs inhibited, to a similar extent, cytoplasmic poly(A)(+)mRNA; however, 3deoxyadenosine, in contrast to Actinomycin D, failed to inhibit poly(A)(-)RNA, sedimenting between 4S and 18S (putatively poly(A)(-)mRNA). We conclude that the mineralocorticoid action of aldosterone during the first three hours depends on the synthesis of both poly(A)(+)mRNA and poly(A)(-)mRNA.     

The interaction of chromium with nucleic acids

Native and denatured calf thymus DNA, and homopolyribonucleotides were compared with respect to chromium and protein binding after an in vitro incubation with rat liver microsomes, NADPH, and chromium(VI) or chromium(III). A significant amount of chromium bound to DNA when chromium(VI) was incubated with the native or the denatured form of DNA in the presence of microsomes and NADPH. For both native and denatured DNA the amount of protein bound to DNA increased with the amount of chromium bound to DNA. Denatured DNA had much higher amounts of chromium and protein bound than native DNA. There was no interaction between chromium(VI) and either form of DNA in the absence of the complete microsomal reducing system. The binding of chrornium(III) to native or denatured DNA was small and relatively unaffected by the presence of microsomes and NADPH. The binding of chromium and protein to polyriboadenylic acid (poly(A)), polyribocytidylic acid (poly(C), polyri-boguanylic acid (poly(G)) and polyribouridylic acid (poly(U)) was determined after incubation with chromium(VI) in the presence of microsomes and NADPH. The magnitude of chromium and protein binding to the ribo-polymers was found to be poly(G) ? poly(A) ? poly(C) ? poly(U). These results suggest that the metabolism of chromium(VI) is necessary in order for chromium to interact significantly with nucleic acids. The metabolically-produced chromium preferentially binds to the base guanine and results in DNA-protein cross-links. These findings are discussed with respect to the proposed scheme for the carcinogenicity of chromium(VI). Keywords: DNA-protein cross-links — Chromium-guanine interaction-Microsomal reduction of chromate     

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB–DEG, PHB–TEG, PHB–PEG, and PHB–HV–PEG copolymers using short-chain PEGs (with n?≤?9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB–HV–PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.     

三乙酸甘油酯对PLA/PBAT共混体系性能影响

利用转矩流变仪将聚乳酸(PLA)、聚己二酸对苯二甲酸丁二酯(PBAT)和三乙酸甘油酯(GTA)熔融共混,利用差示扫描量热仪(DSC)、动态热机械分析仪(DMA)、万能材料试验机、冲击试验机、扫描电子显微镜(SEM)对共混物的热力学性能、力学性能以及微观形态结构进行测试和表征。实验发现,加入GTA后共混物的两相玻璃化转变温度呈相互靠近趋势,冷结晶温度和熔融温度都降低。当GTA加入量为3份时,共混物中分散相粒径减小,PLA/PBAT/GTA(80/20/3)组分的断裂伸长率得到明显提升,增加了2.6倍,由未加入GTA时的17.7%增长到64.1%。     

Dynamic compressive loading influences degradation behavior of PEG-PLA hydrogels

Biodegradable hydrogels are attractive 3D environments for cell and tissue growth. In cartilage tissue engineering, mechanical stimulation has been shown to be an important regulator in promoting cartilage development. However, the impact of mechanical loading on the gel degradation kinetics has not been studied. In this study, we examined hydrolytically labile gels synthesized from poly(lactic acid)-b-poly(ethylene glycol)-b-poly-(lactic acid) dimethacrylate macromers, which have been used for cartilage tissue engineering. The gels were subject to physiological loading conditions in order to examine the effects of loading on hydrogel degradation. Initially, hydrogels were formed with two different cross-linking densities and subject to a dynamic compressive strain of 15% at 0.3, 1, or 3 Hz. Degradation behavior was assessed by mass loss, equilibrium swelling and compressive modulus as a function of degradation time. From equilibrium swelling, the pseudo-first-order reaction rate constants were determined as an indication of degradation kinetics. The application of dynamic loading significantly enhanced the degradation time for the low cross-linked gels (P < 0.01) while frequency showed no statistical differences in degradation rates or bulk erosion profiles. In the higher cross-linked gels, a 3 Hz dynamic strain significantly increased the degradation kinetics resulting in an overall faster degradation time by 6 days compared to gels subject to the 0.3 and 1 Hz loads (P < 0.0001). The bioreactor set-up also influenced overall degradation behavior where the use of impermeable versus permeable platens resulted in significantly lower degradation rate constants for both cross-linked gels (P < 0.001). The compressive modulus exponentially decreased with degradation time under dynamic loading. Together, our findings indicate that both loading regime and the bioreactor setup influence degradation and should be considered when designing and tuning a biodegradable hydrogel where mechanical stimulation is employed.     

Biodegradation of poly(l-lactide)

The biodegradation of poly(L-lactide) (PLA) is reviewed. The important role of actinomycetes in PLA degradation is emphasized. These PLA-degrading actinomycetes belong phylogenetically to the Pseudonocardiaceae family and related genera, including Amycolatopsis, Lentzea, Streptoalloteichus, Kibdelosporangium and Saccharothrix. A PLA-degrading enzyme purified from an isolated Amycolatopsis strain-41 has substrate specificity on PLA higher than proteinase K. The application of these strains and their enzymes can be effectively used for biological treatment of plastic wastes containing PLA.     

An S-like ribonuclease gene is used to generate a trap-leaf enzyme in the carnivorous plant Drosera adelae

Carnivorous plants usually grow in nutrient-deficient habitats, and thus they partly depend on insects for nitrogen and phosphate needed for amino acid and nucleotide synthesis. We report that a sticky digestive liquid from a sundew, Drosera adelae, contains an abundant amount of an S-like ribonuclease (RNase) that shows high amino acid-sequence similarity to S-like RNases induced by phosphate starvation or wounding in normal plants. By giving leaves an RNase "coat", D. adelae seems to achieve two requirements simultaneously to adapt itself to its specific surroundings: it obtains phosphates from insects, and defends itself against pathogen attack.     

Secondary association of exogenous polynucleotides with cells and nuclei in uptake experiments

Manipulation of [³H]polynucleotide-treated cells to remove them from the substrate or to isolate nuclei has been shown to result in secondary association of the exogenous polynucleotide with the cells or nuclei. Experiments have shown untreated (control) cultures, when processed with supernatants from [³H]polynucleotide-treated cell monolayers, exhibited a significant amount of radioactive label associated with the nuclei from control cells. In spite of thorough washing of polynucleotide-treated cell monolayers prior to the manipulation, the association was extensive. It is likely to overshadow the association resulting solely from the exposure of monolayer cells to the polynucleotide.     

The uptake of liposome-entraped 125I-labelled poly(vinylpyrrolidone) by rat jejunum in vitro

The uptake of free and liposome-entrapped ¹²⁵I-labelled poly(vinylpyrrolidone) was measured in an intestinal sac preparation from adult rats. An an equal concentration of ¹²⁵I-labelled poly(vinylpyrrolidone), the rate of uptake of the liposome-entrapped material was four times that of the free macromolecule.     

Cis-aconityl spacer between daunomycin and macromolecular carriers: A model of pH-sensitive linkage releasing drug from a lysosomotropic conjugate

N-cis-Aconityl and N-maleyl derivatives of daunomycin prepared from the respective anhydrides were conjugated to Affi-Gel 701 (aminoethyl polyacrylamide beads) and to poly(D-lysine). The cis-aconityl linkage between the drug and Affi-Gel 701 is pH-sensitive with a hydrolysis half-life of less than 3 h at pH 4 and more than 96 h at pH 6 or higher. Thin-layer chromatography and cytotoxic tests in cultured cells indicate that the product of hydrolysis is unaltered daunomycin. These Affi-Gel conjugates present for 3 days in the culture medium of WEHI-5 cells at neutral pH have little or no growth inhibitory effect. N-cis-aconityl daunomycin-poly(D-lysine) conjugates, however, added to WEHI-5 cells under comparable conditions cause a 90% inhibition of cell growth. In contrast, comparable addition of N-maleyl daunomycin-poly(D-lysine) conjugates is not inhibitory. We conclude that unlike the Affi-Gel conjugate, N-cis-aconityl daunomycin-poly(D-lysine) enters cells and reaches the lysosomal compartment, and that the cis-aconityl spacer releases daunomycin from poly(D-lysine) in the acidic milieu of lysosomes due to the participation of a free cis-carboxylic group. This releasing mechanism should be applicable to other drug-macromolecular conjugates.