Bovine serum albumin-bovine hemoglobin conjugate as a candidate blood substitute

A bovine serum albumin-bovine hemoglobin conjugate was prepared using 3-maleimidobenzoic acid N-hydroxysuccinimide ester as cross-linker. The conjugate was purified using DEAE Sepharose. It had an M _r of 127 kDa. Its P₅₀ (half-saturated O₂ pressure) value and Hill coefficient were 27 mm Hg and 2, respectively.     

The synthesis of a cobalt(II) tetracarboxyphthalocyanine- deoxyribooligonucleotide conjugate as a reagent for the directed DNA modification

The cobalt(II) tetracarboxyphthalocyanine-deoxyribonucleotide pd(TCTTCCCA) conjugate was synthesized. The phthalocyanine N-succinimide ester prepared from phthalocyanine using DCC was mixed in DMF with an aqueous solution of the oligonucleotide bearing a 1,3-diaminopropane linker at the 5'-phosphate. The resulting conjugate was tested in the intraduplex reaction with target 14-mer and 22-mer oligonucleotides containing conjugate-complementary sequences. In the presence of O2 and a thiol (2-mercaptoethanol or DTT) as a coupled reducer or H2O2, sequence-specific DNA modification was observed that caused the cleavage of the target upon treatment with piperidine.     

Synthesis of 3′-O-Alkyl Homologues and a Biotin Probe of Isorhamnetin and Evaluation of Cytotoxic Efficacy on Cancer Cells

Isorhamnetin is a natural flavonoid which shows a variety of biological activities such as antioxidant, anti-inflammatory and antitumor. In order to identify the cellular binding protein of isorhamnetin as potential anti-cancer target, we first synthesized 3′-O-substituted quercetin as isorhamnetin homologues and evaluated the growth inhibitory activity of these derivatives on breast, colon and prostate cancer cell lines. The preliminary results showed that the 3′-O modification did not affect the cytotoxic activity of the scaffold. Analysis of the co-crystal structure and the docking pose of isorhamnetin with reported binding protein of isorhamnetin or quercetin indicated the 3′-O-substitution groups located outside of the binding pocket, which is in accordance with activity of 3′-O derivatives. Then a biotin conjugate of isorhamnetin with a tetraethylene glycol (PEG)₄ linker at the 3′ position was synthesized and the resulting probe retained the anti-proliferative activity on cancer cell lines, while the cellular fluorescence analysis showed the distribution of probe inside the cells which indicated the probe had limited cell permeability. Finally, pull down assay both in situ inside cells and in the cell lysates indicated the isorhamnetin biotin probe was capable of protein labeling in cell lysates. These findings provide the isorhamnetin 3′-O-biotin probe as a tool to reveal the target proteins of isorhamnetin.     

Chemically Modified Short Interfering Hybrids (siHYBRIDS): Nanoimmunoliposome Delivery In Vitro and In Vivo for RNAi of HER-2

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC₅₀ value of 37 nM. The inhibitory potency of this siHybrid was increased (IC₅₀ value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 ′-deoxyinosine (dI) and 2 ′-O-methylribonucleotide (2 ′-OMe) residues: 5′-d(TITIT)-2′OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.     

Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle

Steric blocking peptide nucleic acid (PNA) oligonucleotides have been used increasingly for redirecting RNA splicing particularly in therapeutic applications such as Duchenne muscular dystrophy (DMD). Covalent attachment of a cell-penetrating peptide helps to improve cell delivery of PNA. We have used a HeLa pLuc705 cell splicing redirection assay to develop a series of PNA internalization peptides (Pip) conjugated to an 18-mer PNA705 model oligonucleotide with higher activity compared to a PNA705 conjugate with a leading cell-penetrating peptide being developed for therapeutic use, (R-Ahx-R)₄. We show that Pip–PNA705 conjugates are internalized in HeLa cells by an energy-dependent mechanism and that the predominant pathway of cell uptake of biologically active conjugate seems to be via clathrin-dependent endocytosis. In a mouse model of DMD, serum-stabilized Pip2a or Pip2b peptides conjugated to a 20-mer PNA (PNADMD) targeting the exon 23 mutation in the dystrophin gene showed strong exon-skipping activity in differentiated mdx mouse myotubes in culture in the absence of an added transfection agent at concentrations where naked PNADMD was inactive. Injection of Pip2a-PNADMD or Pip2b-PNADMD into the tibealis anterior muscles of mdx mice resulted in ~3-fold higher numbers of dystrophin-positive fibres compared to naked PNADMD or (R-Ahx-R)₄-PNADMD.     

The characterization of <Emphasis Type="Italic">Thermotoga maritima</Emphasis> ferritin reveals an unusual subunit dissociation behavior and efficient DNA protection from iron-mediated oxidative stress

Ferritin from the hyperthermophilic anaerobe Thermotoga maritima, a bacterium of ancient phylogenetic origin, is structurally similar to known bacterial and eukaryotic ferritins: 24 identical subunits assemble into a shell having octahedral symmetry and a Mr of about 460 kDa. T. maritima ferritin (TmFtn), purified to homogeneity as a recombinant protein, contains approximately 2–3 iron atoms and can incorporate efficiently up to 3,500 atoms in the form of a ferric oxy-hydroxide mineral at 80°C, the optimal growth temperature of the bacterium. The 24-mer unexpectedly dissociates reversibly into dimers at low ionic strengths. In turn, dimers re-associate into the native 24-mer assembly at high protein concentrations and upon incorporation of iron micelles containing at least 500 Fe(III). TmFtn uses O₂ as efficient iron oxidant. The reaction stoichiometry is 3–4 O₂:Fe(II) as in all bacterial ferritins. Accordingly no H₂O₂ is released into solution, a feature reflected in the in vitro ability of TmFtn to reduce significantly iron-mediated oxidative damage to DNA at 80°C. A similar TmFtn-mediated ROS detoxifying role likely occurs in the bacterium which lacks the SOD/catalase defense systems of the aerobic world.     

Mapping oxidative DNA damage at nucleotide level

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the in vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50 mM H₂O₂, or purified genomic DNA was treated with 5 mM H₂O₂, 100 μM Ascorbate, and 50 μM, 100 μM, or 100 μM of Cu(II), Fe(III), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H₂O₂-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H₂O₂.     

Biofilm control in water by a UV-based advanced oxidation process

An ultraviolet (UV)-based advanced oxidation process (AOP), with hydrogen peroxide and medium-pressure (MP) UV light (H₂O₂/UV), was used as a pretreatment strategy for biofilm control in water. Suspended Pseudomonas aeruginosa cells were exposed to UV-based AOP treatment, and the adherent biofilm formed by the surviving cells was monitored. Control experiments using H₂O₂ or MP UV irradiation alone could inhibit biofilm formation for only short periods of time (<24 h) post-treatment. In a H₂O₂/filtered-UV (>295 nm) system, an additive effect on biofilm control was shown vs filtered-UV irradiation alone, probably due to activity of the added hydroxyl radical (OH?). In a H₂O₂/full-UV (ie full UV spectrum, not filtered) system, this result was not obtained, possibly due to the germicidal UV photons overwhelming the AOP system. Generally, however, H₂O₂/UV prevented biofilm formation for longer periods (days) only when maintained with residual H₂O₂. The ratio of surviving bacterial concentration post-treatment to residual H₂O₂ concentration played an important role in biofilm prevention and bacterial regrowth. H₂O₂ treatments alone resulted in poorer biofilm control compared to UV-based AOP treatments maintained with similar levels of residual H₂O₂, indicating a possible advantage of AOP.     

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (¹O₂) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to ¹O₂. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with ¹O₂ or superoxide (O^–₂). One sRNA was specifically induced by ¹O₂ and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by ¹O₂ and O^–₂ were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH_I and RpoH_II. The most abundant sRNA was processed in the presence of ¹O₂ but not by O^–₂. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of ¹O₂‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by ¹O₂ exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.     

Access to fluorescent probes via allyl glycosides: the synthesis of aBrucella trisaccharide epitope linked to a coumarin

Oligosaccharide allyl glycosides are demonstrated to provide a route to fluorescent probes and simple inhibitors. Ethyl 2-O-acetyl-4-azido-3-O-benzoyl-4,6-dideoxy-1-thio--d-mannopyranoside (6) was used as glycosyl donor in the preparation of the trisaccharide [-d-Rhap4NFo(1 2)-]₂--d-Rhap4NFo-O-allyl (16). Thioglycoside6 was activated withN-iodosuccinimide and triflic acid or by bromine in the glycosylations and the inhibitor16 was obtained after deprotection by transesterification, reduction of the azido groups with hydrogen sulfide, andN-formylation with ethyl formate. Ozonolysis of the allyl glycoside in16 and reductive amination with 7-amino-4-methylcoumarin then gave the target fluorescent trisaccharide conjugate.Issued as NRCC 31913.     

Computer simulation in template-directed oligonucleotide synthesis

Summary A computer simulation (KINSIM) modeling up to 33 competing reactions was used in order to investigate the product distribution in a template-directed oligonucleotide synthesis as a function of time and concentration of the reactants. The study is focused on the poly(C)-directed elongation reaction of an oligoguanylate (a 7-mer is chosen) with guanosine 5-monophosphate-2-methylimidazolide (2-MeImpG), the activated monomer. It is known that theelongation of oligoguanylates to form oligomeric products such as 8-mer, 9-mer, 10-mer, etc., is in competition with (1) thedimerization and further oligomerization reaction of 2-MeImpG that leads to the formation of dimers and short oligomers, and (2) thehydrolysis of 2-MeImpG that forms inactive guanosine 5-monophosphate, 5-GMP. Experimentally determined rate constants for the above three processes at 37°C and pH 7.95 were used in the simulation; the initial concentrations of 2-MeImpG, [M]_o, and of the oligoguanylate primer, [7-mer]_o, were varied, and KINSIM calculated the distribution of products as a function of time until equilibration was reached, i.e., when all the activated monomer has been consumed. In order to sort out how strongly the elongation reaction may be affected by the competing hydrolysis and dimerization, we also simulated the idealized situation in which these competing reactions do not occur. Simulation of the idealized system suggests that (1) the fraction of [7-mer]_o that has reacted as well as the product distribution after equilibration do not depend on the absolute concentrations of the reactants, but only on their ratio, [M]_o/[7-mer]_o; (2) the rate of elongation is proportional to [7-mer]_o and not to [M]_o; and (3) as the [M]_o/[7-mer]_o ratio increases longer oligomers are formed. The results of the computer simulation with the experimental system, i.e., elongation in the presence of both hydrolysis and dimerization, are similar to the ones obtained with the idealized system as long as dimerization and hydrolysis are not responsible for consuming a substantial fraction of 2-MeImpG.     

Measuring maximum oxygen uptake with an incremental swimming test and by chasing rainbow trout to exhaustion inside a respirometry chamber yields the same results

This study hypothesized that oxygen uptake (ṀO₂) measured with a novel protocol of chasing rainbow trout Oncorhynchus mykiss to exhaustion inside a static respirometer while simultaneously monitoring ṀO₂ (ṀO_2chase) would generate the same and repeatable peak value as when peak active ṀO₂ (ṀO_2active) is measured in a critical swimming speed protocol. To reliably determine peak ṀO_2chase, and compare to the peak during recovery of ṀO₂ after a conventional chase protocol outside the respirometer (ṀO_2rec), this study applied an iterative algorithm and a minimum sampling window duration (i.e., 1 min based on an analysis of the variance in background and exercise ṀO₂) to account for ṀO₂ dynamics. In support of this hypothesis, peak ṀO_2active (707 ± 33 mg O₂ h⁻¹ kg⁻¹) and peak ṀO_2chase (663 ± 43 mg O₂ h⁻¹ kg⁻¹) were similar (P = 0.49) and repeatable (Pearson's and Spearman's correlation test; r ≥ 0.77; P < 0.05) when measured in the same fish. Therefore, estimates of ṀO_2max can be independent of whether a fish is exhaustively chased inside a respirometer or swum to fatigue in a swim tunnel, provided ṀO₂ is analysed with an iterative algorithm and a minimum but reliable sampling window. The importance of using this analytical approach was illustrated by peak ṀO_2chase being 23% higher (P < 0.05) when compared with a conventional sequential interval regression analysis, whereas using the conventional chase protocol (1-min window) outside the respirometer increased this difference to 31% (P < 0.01). Moreover, because peak ṀO_2chase was 18% higher (P < 0.05) than peak ṀO_2rec, chasing a fish inside a static respirometer may be a better protocol for obtaining maximum ṀO₂.     

Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN₈+pN₄+pN′₈ Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN₈ is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′₈ Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10⁻¹⁴ mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.     

Synthesis and characterization of a pH-sensitive conjugate of isoniazid with Fe3O4@SiO2 magnetic nanoparticles

The Letter describes the preparation and characterization of a conjugate of isoniazid (INH) with magnetic nanoparticles Fe₃O₄@SiO₂ 115 ± 60 nm in size. The INH molecules were attached to the surface of nanoparticles by a covalent pH-sensitive amidine bond. The conjugate was characterized by X-ray diffraction, SEM, dynamic light scattering, IR spectroscopy and microanalysis. The conjugate released isoniazid under in vitro conditions (pH = 4; 37 °C; t_1/2 ≈ 115 s). In addition, the cytotoxicity of the Fe₃O₄@SiO₂–INH conjugate was evaluated in SK-BR-3 cells using the xCELLigence system.     

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H₂O₂/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H₂O₂ and o-phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.     

Superactive Oligonucleotide Derivatives

Abstract

The conjugate of antitumor antibiotic bleomycin A5 with the tetranucleotide catalytically cleaves 20-mer ssDNA target in the presence of flanking octanucleotides. Each molecule of the conjugate cleaves on the average three molecules of the target.     

LNA and α-L-LNA: Towards Therapeutic Applications

Abstract

LNA and α-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we report dose-dependent inhibition of HIV-1 Tat-dependent trans activation by a 12-mer chimeric α-L-LNA/DNA oligomer. This oligomer exhibits a dose-dependency similar to that of the corresponding 12-mer chimeric LNA/2′-O-Me-RNA oligomer. In addition, we show that incorporation of α-L-LNA or LNA monomers into each of the two binding arms of a “10–23” DNAzyme markedly increases cleavage of the target RNA.