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     

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.     

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%。     

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.     

Activation of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine with poly(salicylic acid 5-aminodisulfide)

5-Aminosalicylic acid (5-ASA) inhibited by a mixed mechanism the peroxidase catalyzed oxidation of tetramethylbenzidine (TMB) in 0.015 M phosphate-citrate buffer (pH 6.4) supplemented with 5% DMSO and 5% DMF. Poly(salicylic acid 5-aminodisulfide) (poly(SAADS)) in 0.01 M phosphate buffer (pH 6.2-7.4) supplemented with 5% DMSO and 5% DMF effectively activated the peroxidase-catalyzed oxidation of TMB. The activation was quantitatively characterized by coefficients (M^–1) determined at different pH values: increased linearly with increase in pH up to the maximal value of 2.44·10⁵ M^–1 at pH 7.0. The activating effect of poly(SAADS) on the peroxidase-catalyzed oxidation of TMB is explained by the activator properties of polyelectrolyte, with its anionic form interacting with peroxidase sites responsible for the acid-base catalysis.     

Double-stranded RNA-stimulated enzyme activities isolated from human placental extracts

A (2′–5′)A_n synthetase activity was isolated from human placental extracts by affinity chromatography on poly(rI)·poly(rC)-agarose. The oligonucleotide (2′–5′)A_n was identified by (1) chromatography on PEI-cellulose and DEAE-cellulose, (2) inhibition of polypeptide synthesis in lysed rabbit reticulocytes (3) competition of the binding of pppA(pA)3,3′-[³²P]pCp to rabbit reticulocyte lysates, and (4) alkaline phosphatase digestion. The synthetase activity in most placental preparations is activated by natural or synthetic dsRNA. However, in a few placental synthetase preparations, dsRNA is only marginally stimulatory and only becomes effective by prior treatment of the enzyme preparations with the calcium-dependent micrococal nuclease. This suggeststhat there is an endogenous placental dsRNA contaminant in the enzyme preparations. In some synthetase preparations, a second dsRNA-stimulated product, tentatively identified as the nucleotide 5′-IMP, is also observed. Because the specific AMP deaminase inhibitor coformycin (10 μM) blocks the formation of IMP from ATP and causes a quantitative accumulation of AMP, and because the formation of IMp becomes independent of dsRNA when ADP or AMP is used in plase of ATP, the presence of a dsRNA-stimulated ATP phosphohydrolase (ATPase) activity in human placenta is suggested.     

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Systems Metabolic Engineering Strategies for Non‐Natural Microbial Polyester Production

Plastics, used everyday, are mostly synthetic polymers derived from fossil resources, and their accumulation is becoming a serious concern worldwide. Polyhydroxyalkanoates (PHAs) are naturally produced polyesters synthesized and intracellularly accumulated by many different microorganisms. PHAs are good alternatives to petroleum‐based plastics because they possess a wide range of material properties depending on monomer types and molecular weights. In addition, PHAs are biodegradable and can be produced from renewable biomass. Thus, producing PHAs through the development of high‐performance engineered microorganisms and efficient bioprocesses gained much interest. In addition, non‐natural polyesters comprising 2‐hydroxycarboxylic acids as monomers have been produced by fermentation of metabolically engineered bacteria. For example, poly(lactic acid) and poly(lactic acid‐co‐glycolic acid), which have been chemically synthesized using the corresponding monomers either fermentatively or chemically produced, can be produced by metabolically engineered bacteria by one‐step fermentation. Recently, PHAs containing aromatic monomers could be produced by fermentation of metabolically engineered bacteria. Here, metabolic engineering strategies applied in developing microbial strains capable of producing non‐natural polyesters in a stepwise manner are reviewed. It is hoped that the detailed strategies described will be helpful for designing metabolic engineering strategies for developing diverse microbial strains capable of producing various polymers that can replace petroleum‐derived polymers.     

Immunological studies on the interaction of polyadenylic acid and human liver ribonuclease

The effect of polyadenylic acid, a potent inhibitor of mammalian and bacterial RNAses, on the binding of human liver RNAse to its antibody was studied. To do this, a human liver RNAse antibody was immobilized on Sepharose 4B. Examination of the ability of the enzyme to bind to the immobilized anti-RNAse in the presence or absence of polyadenylic acid indicated that enzyme-antibody binding was more sensitive to the presence of polyadenylic acid than was enzyme activity. Furthermore, the effect of polyadenylic acid on enzyme-antibody binding was specific since neither polycytidylic acid nor polyuridylic acid had much effect on the antigenicity of the enzyme. The metal cation, Mg2+, and the polyamine, spermidine, but not putrescine, readily reversed the effects of polyadenylic acid on enzyme-antibody binding.     

Poly(glutamic Acid) for Biomedical Applications

ABSTRACT:?

Paclitaxel is a widely used anti-cancer agent. Conjugates of paclitaxel with poly(glutamic acid) have shown great promise in preclinical trials, and clinical trials are now underway. Preclinical data suggest that more paclitaxel is preferentially delivered to tumor sites vs. nonconjugated paclitaxel. When poly(glutamic acid) is conjugated to other families of cancer drugs, similar improvements in effectiveness and reduced toxicity are observed. Optimization of poly(glutamic acid) for use in drug delivery applications is a key step in making this technology viable.     

The effect of thiols on the dehalogenation of 5-iodo and 5-bromouracil

The 5-iodo- and 5-bromo- analogs of uracil are dehalogenated in the presence of both cysteine and 2-mercaptoethanol to yield uracil. Presumably, the reaction involves the initial addition of the thiol group across the 5,6 double bond of the halopyrimidine to yield the corresponding 5-halo, 5,6-dihydrouracil-6-thioether which then dehalogenates to yield uracil. Under comparable conditions, cysteine causes more rapid dehalogenation of both halouracils than does 2-mercaptoethanol.Thiol containing compounds catalyze hydrogen-deuterium exchange at carbon five of uracil (1–3) and have been implicated as having a catalytic effect in the deamination of cytosine (4,5). Presumably, these reactions involve the reversible nucleophilic addition of the thiol group across the 5,6 double bond of the pyrimidine to yield the corresponding 5,6 dihydropyrimidine with a substituted thioether group on carbon six. This pathway is supported by comparable reactions involving the addition of bisulfite to the pyrimidine ring system (6–10). Different from the bisulfite addition compounds, the thioether containing dihydropyrimidine adducts have not been isolated and characterized; however, 5′-deoxy-5′,6-epithio-5,6-dihydro-2′,3′-0-isopropylideneuridine resulting from the intramolecular attack of the 5′ thiol group on carbon six of the uracil ring system of 5′-deoxy-5′-thio-2′,3′-0-isopropylideneuridine has been isolated and characterized (11).In a recent communication, we reported that bisulfite buffer systems catalyze the dehalogenation of 5-iodo-, 5-bromo-, and 5-chlorouracil (12). The object of this work is to demonstrate that cysteine and 2-mercaptoethanol, sulfur nucleophiles with more physiological importance than bisulfite, also cause halopyrimidine dehalogenation under nearly physiological conditions of temperature and pH.     

A homopolymer of the potent antitumor drug 2′-deoxy-5-fluorouridylate

Poly(5-fluoro-2′-deoxyuridylic acid) was synthesized and its properties were compared with those of poly(dT) and poly(dU). It readily complexed with poly(dA). The 1:1 complex melted at about 20°C lower than poly(dA) · poly(dT). A triple-stranded helix, poly(dA)·2 poly(dF⁵U) was formed only in high salt (2.0 M NaCl).     

Structures of cell wall teichoic acids of <Emphasis Type="Italic">Brevibacterium iodinum</Emphasis> VKM Ac-2106<Superscript>T</Superscript>

Structures of two cell wall teichoic acids of Brevibacterium iodinum VKM Ac-2106^T were studied. The structure of mannitol teichoic acid described earlier was mainly confirmed. This polymer is 1,6-poly(mannitol phosphate) bearing -D-glucopyranosyl residues at the C-2 of mannitol and pyruvic acid residues at the C-4 and C-5. The absolute configurations of D-mannitol and S-pyruvic acid were found. The following distinctions from the earlier described structure were found: unsubstituted 1,6-poly(mannitol phosphate) residues and residues substituted only by -D-glucopyranosyl at the C-2 of mannitol but unsubstituted by pyruvic acid are present in the chain. The structure of glycerol teichoic acid present in the cell wall as a minor component (7%) is also described. This acid is identified as 1,3-poly(glycerol phosphate) substituted at the C-2 of glycerol by 2-acetamido-2-deoxy--D-galactopyranosyl residues bearing R-pyruvic acid residues at the C-4 and C-6 of galactose. This polymer is for the first time described in the cell wall of Gram-positive bacteria.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1659–1666.Original Russian Text Copyright © 2004 by Potekhina, Evtushenko, Senchenkova, Shashkov, Naumova.     

极性生长的细胞中胞嘧啶甲基化的动态变化及其对GABA信号的响应

DNA胞嘧啶(C)的甲基化(5m C)在植物发育过程中具有重要的调节作用,多种环境因子如逆境胁迫、植物内/外源性因子等均会触发DNA甲基化的变化。为探讨γ-氨基丁酸(GABA)对植物发育的可能调节机制,本研究以极性生长的烟草花粉管和拟南芥根为材料,分析5m C的含量及其对GABA信号的响应。结果表明,1.0 mmol/L GABA能显著促进烟草花粉管和拟南芥根的极性生长;同时,GABA处理使烟草花粉管和拟南芥根的基因组中5m C含量显著降低、5-羟基胞嘧啶(5hm C)含量显著增加。5hm C是5m C去甲基化途径中的一个重要中间产物,本研究证实了GABA可以作为一种重要的外源信号调节DNA甲基化的动态变化。     

Nano carriers that enable co-delivery of chemotherapy and RNAi agents for treatment of drug-resistant cancers

Tumor cells exhibit drug resistant phenotypes that decrease the efficacy of chemotherapeutic treatments. The drug resistance has a genetic basis that is caused by an abnormal gene expression. There are several types of drug resistance: efflux pumps reducing the cellular concentration of the drug, alterations in membrane lipids that reduce cellular uptake, increased or altered drug targets, metabolic alteration of the drug, inhibition of apoptosis, repair of the damaged DNA, and alteration of the cell cycle checkpoints ( and ). siRNA is used to silence the drug resistant phenotype and prevent this drug resistance response. Of the listed types of drug resistance, pump-type resistance (e.g., high expression of ATP-binding cassette transporter proteins such as P-glycoproteins (Pgp; also known as multi-drug resistance protein 1 or MDR1, encoded by the ATP-Binding Cassette Sub-Family B Member 1 (ABCB1) gene)) and apoptosis inhibition (e.g., expression of anti-apoptotic proteins such as Bcl-2) are the most frequently targeted for gene silencing. The co-delivery of siRNA and chemotherapeutic drugs has a synergistic effect, but many of the current projects do not control the drug release from the nanocarrier. This means that the drug payload is released before the drug resistance proteins have degraded and the drug resistance phenotype has been silenced. Current research focuses on cross-linking the carrier's polymers to prevent premature drug release, but these carriers still rely on environmental cues to release the drug payload, and the drug may be released too early. In this review, we studied the release kinetics of siRNA and chemotherapeutic drugs from a broad range of carriers. We also give examples of carriers used to co-deliver siRNA and drugs to drug-resistant tumor cells, and we examine how modifications to the carrier affect the delivery. Lastly, we give our recommendations for the future directions of the co-delivery of siRNA and chemotherapeutic drug treatments.     

Biosynthesis of poly(3-hydroxyalkanoate) from amino acids

It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions. The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.