Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba.

Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli (E. coli). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei. Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA's enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA.

Synthesis, characterization and kinetic study of silver and gold nanoparticles produced by the archaeon Haloferax volcanii.

AIMS: To evaluate the ability of the haloarchaeon Haloferax volcanii to produce Ag and Au nanoparticles (NPs) and to characterize the obtained material in order to find relevant properties for future potential applications. METHODS AND RESULTS: Nanoparticles were produced by incubating H. volcanii cells with the corresponding metal salt. In the presence of precursor salts, cultures evidenced a colour change associated to the formation of metallic nanostructures with plasmonic bands located in the visible range of the spectrum. X-ray fluorescence analysis confirmed the presence of Ag and Au in the NPs which were spherical, with average sizes of 25 nmol l-1 (Ag) and 10 nmol l-1 (Au), as determined by electronic microscopy. Fourier transformed infrared spectroscopy indicated that both types of NPs showed a stable protein capping. Ag NPs evidenced antibacterial activity and Au NPs improved the specificity of polymerase chain reaction reactions. Au and Ag NPs were able to reduce 4-nitrophenol when incubated with NaBH4 . CONCLUSIONS: Haloferax volcanii is able to synthesize metallic NPs with interesting properties for technological applications. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data demonstrate the ability of H. volcanii to synthesize metal NPs and constitutes a solid starting point to deepen the study and explore novel applications.

Asialoglycoprotein receptor targeted micelles containing carborane clusters for effective boron neutron capture therapy of hepatocellular carcinoma.

The asialoglycoprotein receptor (ASGP-R) is viewed as an ideal target for hepatocyte-specific delivery. And the galactose residue is a promising ASGP-R ligand because of its high receptor affinity. Herein, a novel polymer based on PEGylated galactose was developed to achieve boron neutron capture therapy (BNCT) for active targeting hepatocellular carcinoma (HCC) by loading carborane clusters. Notably, the polymer could self-assemble into micelles with an average diameter of 135 nm under physiological conditions. The micelle had the high selectivity and low cytotoxicity to HepG2 cells (IC50 >1000 µM). Kinetically, the micelle had the higher uptake in HepG2 cells than the positive control group sodium borocaptate (BSH) in vitro. After the HepG2 cells were treated with the micelle, the cytoskeleton was changed and the migration ability was weakened during BNCT. Apoptosis was remarkably induced by breaking of DNA double strands of cancer cells. In addition, the concentration of 10B in the tumor was 4.5 times higher than that of the BSH group at 4 h after the micelle administration in the tumor-bearing mice. The tumor/blood ratio of 10B concentration reached over 25 at 24 h after micelle injection. In the normal mice, the micelles were mainly distributed among the liver and kidney tissues and could be effectively eliminated from the body within 24 h. No systemic toxicity was observed after administration. Thus, the carborane-containing PEGylated galactose micelles with ASGP-R targeting can be used as a promising therapeutic vector for effective boron neutron capture therapy of hepatocellular carcinoma.

Synthesis of cerium and nickel doped titanium nanofibers for hydrolysis of sodium borohydride.

A recyclable titanium nanofibers, doped with cerium and nickel doped was successfully synthesized by using sol-gel and electrospinning method for hydrogen generation from alkali free hydrolysis of NaBH4. The resultant nanocomposite was characterized to find out the structural and physical-chemical properties by a series of analytical techniques such as FT-IR (Fourier transform infrared spectroscopy), XRD (X-ray diffraction), SEM (scanning electron microscope), EDX (energy-dispersive X-ray spectroscopy),N2 adsorption-desorption and BET (Brunauer-Emmett-Teller), etc. The results revealed that cerium and nickel nanoparticles were homogeneously distributed on the surface of the TiO2 nanofibers due to having similar oxidation state and atomic radium of TiO2nanofibers with CeO2 and NiO for the effective immobilization of metal ions. The NiO doped catalyst showed superior catalytic performance towards the hydrolysis reaction of NaBH4 at room temperature. These catalysts have ability to produce 305 mL of H2 within the time of 160 min at room temperature. Additionally, reusability test revealed that the catalyst is active even after five runs of hydrolytic reaction, implying the as-prepared NiO doped TiO2 nanofibers could be considered as a potential candidate catalyst for portable hydrogen fuel system such as PEMFC (proton exchange membrane fuel cells).

Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy.

Significant autofluorescence (AF) of renal tissue is one of the major causes restricting the use of immunofluorescent staining. This study aimed at controlling renal tissue AF and testing an effective method for optimizing specific signals. In the present study, we observed emergence of strong AF in all renal cells under different fluorescent channels. Significant concentration-dependent reduction in AF of kidney tissue was observed with the use of sodium borohydride (NaBH4) and Sudan black B (SBB) alone (p < 0.05). Under maximum effective concentration, semi-quantitative analysis revealed that inhibitory effect of SBB on AF was superior to that of NaBH4 (P < 0.01). When the two chemicals were combined, we observed that background can be reduced, and specific staining can be optimized at optimum concentration. Intensity of renal tissue was examined by confocal λ scanning, which showed that peaks were located at the range of approximately 480 - 590 nm and similar to those of flavin and lipofuscin. These results indicated that combined use of NaBH4 and SBB, when targeted at different sources of AF in renal tissue, is the most effective means of reducing background and preserving specificity of fluorescent labels. In addition, this method does not interfere with various steps of immunofluorescence experiments.

Thiol/disulfide homeostasis in patients with idiopathic recurrent pregnancy loss assessed by a novel assay: Report of a preliminary study.

AIM: To evaluate the relationship between idiopathic recurrent pregnancy loss (RPL) and oxidative stress (OS) by means of thiol/disulfide homeostasis via a novel technique. METHODS: Thirty-nine pregnant women diagnosed with idiopathic RPL were compared with 50 healthy pregnant women without a history of abortion. Idiopathic RPL was defined as experiencing two or more consecutive miscarriages prior to 20 weeks of gestation with the presence of normal karyotypes of couple and/or abortus materials, negative maternal screening for anticardiolipin, anti ß 2 glycoprotein antibodies and lupus anticoagulant, normal thyroid stimulating hormone, prolactin and hemoglobin A1C levels and normal pelvic sonography and/or hysterosalpingography. A new and fully automated method was used to measure plasma native thiol, total thiol and disulfide levels, based on the reduction of dynamic disulfide bonds to functional thiol groups by sodium borohydrate. RESULTS: Women with idiopathic RPL had significantly lower plasma levels of native thiol (341.89 ± 50.0 µmol/L vs. 390.84 ± 38.5 µmol/L, P < 0.001) and total thiol (386.18 ± 51.7 µmol/L vs. 435.78 ± 42.3 µmol/L, P < 0.001). Disulfide/thiol and disulfide/total thiol ratios were significantly higher in the study group. The native thiol/total thiol ratio was significantly lower in patients with idiopathic RPL. No difference was measured in disulfide, albumin and total protein plasma levels. CONCLUSIONS: The main outcome of our study indicates a relation between idiopathic RPL and OS. More importantly, the new method used in our study proposes a promising, practical and daily applicable test for evaluating patients with idiopathic RPL.

Role for Tetrahydrobiopterin in the Fetoplacental Endothelial Dysfunction in Maternal Supraphysiological Hypercholesterolemia.

Maternal physiological hypercholesterolemia occurs during pregnancy, ensuring normal fetal development. In some cases, the maternal plasma cholesterol level increases to above this physiological range, leading to maternal supraphysiological hypercholesterolemia (MSPH). This condition results in endothelial dysfunction and atherosclerosis in the fetal and placental vasculature. The fetal and placental endothelial dysfunction is related to alterations in the L-arginine/nitric oxide (NO) pathway and the arginase/urea pathway and results in reduced NO production. The level of tetrahydrobiopterin (BH4), a cofactor for endothelial NO synthase (eNOS), is reduced in nonpregnant women who have hypercholesterolemia, which favors the generation of the superoxide anion rather than NO (from eNOS), causing endothelial dysfunction. However, it is unknown whether MSPH is associated with changes in the level or metabolism of BH4; as a result, eNOS function is not well understood. This review summarizes the available information on the potential link between MSPH and BH4 in causing human fetoplacental vascular endothelial dysfunction, which may be crucial for understanding the deleterious effects of MSPH on fetal growth and development.

RNA structure determination using chemical methods.

Information about the secondary structure of RNA is often useful when assessing the potential for certain RNAs to interact with proteins or when determining whether RNAs that are dissimilar in sequence can form the same structure. In this protocol we discuss chemical methods for RNA structure determination. These methods rely on the fact that certain reagents interact with RNA bases when they are single stranded, but do not react when the bases are involved in Watson-Crick base pairs. For example, dimethylsulfate (DMS) methylates the N1 position of adenosine, the N7 position of guanine, and the N3 position of cytosine only when these bases are in single-strand regions. Modifications of adenosine and cytosine create blocks to reverse transcriptase; accordingly, these modifications are detected as stops to primer extension. Modification of guanine does not create reverse transcriptase stops, but these modifications can be detected by cleavage of the modified RNA after borohydride reduction and aniline cleavage. Because DMS and other chemical reagents modify only single-stranded RNA, double-stranded regions are inferred by the lack of modification.

Time-dependent slowly-reversible inhibition of monoamine oxidase A by N-substituted 1,2,3,6-tetrahydropyridines.

A novel class of N-substituted tetrahydropyridine derivatives was found to have multiple kinetic mechanisms of monoamine oxidase A inhibition. Eleven structurally similar tetrahydropyridine derivatives were synthesized and evaluated as inhibitors of MAO-A and MAO-B. The most potent MAO-A inhibitor in the series, 2,4-dichlorophenoxypropyl analog 12, displayed time-dependent mixed noncompetitive inhibition. The inhibition was reversed by dialysis, indicating reversible enzyme inhibition. Evidence that the slow-binding inhibition of MAO-A with 12 involves a covalent bond was gained from stabilizing a covalent reversible intermediate product by reduction with sodium borohydride. The reduced enzyme complex was not reversible by dialysis. The results are consistent with slowly reversible, mechanism-based inhibition. Two tetrahydropyridine analogs that selectively inhibited MAO-A were characterized by kinetic mechanisms differing from the kinetic mechanism of 12. As reversible inhibitors of MAO-A, tetrahydropyridine analogs are at low risk of having an adverse effect of tyramine-induced hypertension.

An in planta technique for cis-/trans-stereochemical analysis of jasmonoyl isoleucine.

A novel technique for determining the cis-/trans-stereochemistry of jasmonoyl-isoleucine by coupling its alcoholic derivatives by sodium borohydride with high performance liquid chromatography-tandem mass spectrometry is described. Resolving cis- and trans-stereochemistry of the jasmonates in Achyranthes plants exposed to airborne (exogenous) trans-d(2)MeJA was demonstrated as an example. This novel application firmly establishes for the first time that trans-d(2)MeJA is converted exclusively into trans-JA-Ile in Achyranthes leaves, whereas the subsequent de novo biosynthesized JA-Ile possesses cis-stereochemistry. The method is simple, reproducible and could be employed for in vivo cis-/trans-stereochemistry analysis of jasmonates in plants.

Metabolism of borono-phenylalanine-fructose complex (BPA-fr) and borocaptate sodium (BSH) in cancer patients--results from EORTC trial 11001.

Within the clinical trial EORTC 11001, patients were infused with (10)B-enriched borono-phenylalanine-fructose complex (BPA-fr), or borocaptate sodium (BSH) solutions, which are used as boron carriers for boron neutron capture therapy. Urine samples were periodically collected and analyzed by (10)B NMR spectroscopy. The results revealed time-dependent metabolic changes of the administered compounds. BPA-fr dissociated to the constituents BPA and fructose, and the borate group was partly cleaved from BPA. BSH was partly aggregated to a dimer form, BSSB. These observations were previously reported for cultured cells and animal models, and are confirmed here in human cancer patients.

A bystander effect observed in boron neutron capture therapy: a study of the induction of mutations in the HPRT locus.

PURPOSE: To investigate bystander mutagenic effects induced by alpha-particles during boron neutron capture therapy, we mixed cells that were electroporated with borocaptate sodium (BSH), which led to the accumulation of (10)B inside the cells, and cells that did not contain the boron compound. The BSH-containing cells were irradiated with alpha-particles produced by the 10B(n,alpha)7Li reaction, whereas cells without boron were affected only by the 1H(n,gamma)2H and 14N(n,rho)14C reactions. METHODS AND MATERIALS: The lethality and mutagenicity measured by the frequency of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase locus were examined in Chinese hamster ovary cells irradiated with neutrons (Kyoto University Research Reactor: 5 MW). Neutron irradiation of 1:1 mixtures of cells with and without BSH resulted in a survival fraction of 0.1, and the cells that did not contain BSH made up 99.4% of the resulting cell population. The molecular structures of the mutations were determined using multiplex polymerase chain reactions. RESULTS: Because of the bystander effect, the frequency of mutations increased in the cells located nearby the BSH-containing cells compared with control cells. Molecular structural analysis indicated that most of the mutations induced by the bystander effect were point mutations and that the frequencies of total and partial deletions induced by the bystander effect were less than those induced by the original neutron irradiation. CONCLUSION: These results suggested that in boron neutron capture therapy, the mutations caused by the bystander effect and those caused by the original neutron irradiation are induced by different mechanisms.

Mechanism of interaction between human 8-oxoguanine-DNA glycosylase and AP endonuclease.

Human 8-oxoguanine-DNA glycosylase (OGG1) is the main human base excision protein that removes a mutagenic lesion 8-oxoguanine (8-oxoG) from DNA. Since OGG1 has DNA glycosylase and weak abasic site (AP) lyase activities and is characterized by slow product release, turnover of the enzyme acting alone is low. Recently it was shown that human AP endonuclease (APE1) enhances the activity of OGG1. This enhancement was proposed to be passive, resulting from APE1 binding to or cleavage of AP sites after OGG1 dissociation. Here we present evidence that APE1 could actively displace OGG1 from its product, directly increasing the turnover of OGG1. We have observed that APE1 forms an electrophoretically detectable complex with OGG1 cross-linked to DNA by sodium borohydride. Using oligonucleotide substrates with a single 8-oxoG residue located in their 5'-terminal, central or 3'-terminal part, we have demonstrated that OGG1 activity does not increase only for the first of these three substrates, indicating that APE1 interacts with the DNA stretch 5' to the bound OGG1 molecule. In kinetic experiments, APE1 enhanced the product release constant but not the rate constant of base excision by OGG1. Moreover, OGG1 bound to a tetrahydrofuran analog of an abasic site stimulated the activity of APE1 on this substrate. Using a concatemeric DNA substrate, we have shown that APE1 likely displaces OGG1 in a processive mode, with OGG1 remaining on DNA but sliding away in search for a new lesion. Altogether, our data support a model in which APE1 specifically recognizes an OGG1/DNA complex, distorts a stretch of DNA 5' to the OGG1 molecule, and actively displaces the glycosylase from the lesion.

Optimization of penicillin G acylase multipoint immobilization on to glutaraldehyde-chitosan beads.

The objective of this work was to study the immobilization of penicillin G acylase from Escherichia coli on to chitosan-glutaraldehyde beads by multipoint covalent binding. This process was optimized using a 2(3) experimental design. The parameters selected for the present study were the concentrations of glutaraldehyde, phenylacetic acid and sodium borohydride. Three responses were chosen, namely immobilization yield and stabilization factors of enzyme derivatives at high temperature and at alkaline pH. All the runs at the maximum (+1) and minimum (-1) levels were performed at random. Three experiments were performed at the centre point, coded as zero, for experimental-error estimation. With respect to immobilization yield, the main effectors were the concentrations of glutaraldehyde and phenylacetic acid. For stabilization factors at 50 degrees C and at alkaline pH, the main effectors were the concentrations of glutaraldehyde and sodium borohydride and the interaction between them.

Enhancement of sodium borocaptate (BSH) uptake by tumor cells induced by glutathione depletion and its radiobiological effect.

Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. However, the mechanism of uptake by the tumor remains unclear. We investigated the sulfhydryl moiety of this compound. Down regulation of glutathione (GSH) by buthionine sulfoximine in cultured cells resulted in increase of BSH uptake (7.9-36.5%) compared to the control group and consequently the cytocidal effect of neutron irradiation also increased. On the other hand, the radiation caused damage by gamma-ray irradiation was suppressed when BSH uptake increased. These findings suggested that modulation of GSH enhanced the effect of B (n, alpha) reaction and the protective effect of secondary gamma-ray in BNCT.

Linoleic acid-induced endothelial activation: role of calcium and peroxynitrite signaling.

Hypertriglyceridemia, an important risk factor of atherosclerosis, is associated with increased circulating free fatty acids. Research to date indicates that linoleic acid (LA), the major fatty acid in the American diet, may be atherogenic by activating vascular endothelial cells. However, the exact signaling mechanisms involved in LA-mediated proinflammatory events in endothelial cells still remain unclear. We previously reported increased superoxide formation after LA exposure in endothelial cells. The objective of the present investigation is to determine the role of calcium and peroxynitrite in mediating the proinflammatory effect of LA in vascular endothelial cells. LA exposure increased intracellular calcium, nitric oxide, and tetrahydrodiopterin levels as well as the expression of E-selectin. Inhibiting calcium signaling using 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and heparin decreased the expression of E-selectin. Also, LA-mediated nuclear factor kappa B activation and E-selectin gene expression were suppressed by Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (a superoxide scavenger), N(G)-monomethyl-l-arginine (an endothelial nitric oxide synthase inhibitor), and 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) chloride (a peroxynitrite scavenger). LA exposure resulted in increased nitrotyrosine levels, as observed by Western blotting and immunofluorescence. Our data suggest that the proinflammatory effects of LA can be mediated through calcium and peroxynitrite signaling.

Detoxification of 4-hydroxynonenal (HNE) in keratinocytes: characterization of conjugated metabolites by liquid chromatography/electrospray ionization tandem mass spectrometry.

Keratinocytes are potential targets of lipid peroxidation products (alpha,beta-unsaturated aldehydes) generated in the skin following UV exposure, among which the most abundant and toxic product is 4-hydroxy-trans-2,3-nonenal (HNE). The aim of this study was to investigate the ability of keratinocytes (NCTC2544 cell lines) to detoxify HNE, through characterization of metabolites, until now never demonstrated, using a combined analytical approach (liquid chromatography (LC) and liquid chromatography/mass spectrometry (LC/MS)). Incubation of cells with HNE (up to 200 micro M) was performed in order to evaluate the ability of the cells to detoxify this toxic aldehyde, and indicated that the cell viability was maintained under these conditions. LC analysis of the extracellular media from keratinocytes incubated with 100 micro M HNE shows a time-dependent decrease of HNE, disappearance from the medium within 2 h and concomitant formation of two unconjugated (phase I) metabolites, 4-hydroxy-2-nonenoic acid (HNA) and 1,4-dihydroxy-2-nonene (DHN), which were both identified and quantified by LC and accounted for 48.8 +/- 4.6% of the HNE dose. Four additional metabolites were identified in the extracellular medium by reversed-phase LC coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with positive and negative ion detection as Michael adducts (phase II metabolites), arising by the addition of the nucleophilic sulfur of glutathione (GSH) to the electrophilic C-3 of HNE, followed by oxidation-reduction enzymatic processes. The GSH-HNE conjugates were (a) S-(4-hydroxynonanal-3-yl)glutathione, (b) S-(1,4-dihydroxy-nonane-3-yl)glutathione, (c) S-(4-oxononanal-3-yl)glutathione and (d) S-(4-oxo-nonan-1-ol-3-yl)glutathione, and accounted for 52.3 +/- 5.8% of the HNE dose (35 nmol mg(-1) protein), as estimated indirectly by measuring the extent of cellular GSH consumption (18.7 +/- 1.8 nmol mg(-1) protein). The time course of HNE biotransformation was then determined by monitoring the formation of metabolites inside and outside the cell at different times after HNE addition (5-120 min). A time-dependent and almost linear formation inside the cell was observed for all the metabolites (plateau after 15 min of incubation), followed by a rapid decay and a concomitant increase in the extracellular medium (plateau of formation after 60 min). This confirms that HNE diffuses into the cell where is totally metabolized through phase I and phase II reactions to unreactive products, which are then exported outside the cell. This is the first demonstration that skin epidermal cells are able to detoxify the cytotoxic products of lipid peroxidation.

Rapid screening by MALDI-TOF mass spectrometry to probe binding specificity at enzyme active sites.

The binding affinity of aspartate decarboxylase has been probed using MALDI-TOF spectrometry; adducts formed covalently in the active site were detected by MALDI-TOF mass spectrometry after incubation of the enzyme with a range of potential ligands in the presence of NaCNBH3; this has highighted key structural features which will aid design of potential inhibitors.

Structural analysis of the oligosaccharide alditols released from the jelly coat of Rana dalmatina eggs by reductive beta-elimination.

A combination of ion-exchange chromatography and high performance liquid chromatography (HPLC) has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of oviducal mucins obtained from the jelly coat of Rana dalmatina. The primary structures of 26 O-glycans were determined by one-dimensional and two-dimensional 1H and 1H13C NMR spectroscopy. As observed for 20 other amphibian species, these carbohydrate chains are highly species-specific. The main typical feature of the species R. dalmatina consists in the presence of the backbone Gal(beta1-3)[Gal(beta1-4)]Gal(beta1-3)GalNAc-ol, previously observed among Ranidae, such as R. temporaria and R. ridibunda. Nevertheless, the nature of carbohydrates present at the periphery of the glycans perfectly differentiates the three species.