Degradation of arylarsenic compounds by microorganisms

Microorganisms were not directly accumulated when soil contaminated to about 0.5 mM with diphenylarsinic acid (DPAA) was used as the sole source of carbon. However, using toluene as the carbon source yielded several isolates, which were then used in cultivation with DPAA as the sole source of carbon. By these methods, Kytococcus sedentarius strain NK0508, which can grow in up to 0.038 mM DPAA, was isolated. The toxicity of DPAA retarded the growth of K. sedentarius and the direct accumulation of DPAA-utilizing microorganisms from environmental samples. This strain can utilize about 80% of DPAA and phenylarsonic acid as the sole source of carbon for 3 days. Degradation products of DPAA were determined to be cis, cis, muconate and arsenic acid. When K. sedentarius was cultivated with methylphenylarsinic acid and diphenylmethylarsine, about 90% and 10% degradation of the two compounds, respectively, were observed. Diphenylmethylarsine oxide, possibly synthesized by methylation of DPAA, was detected as one of the transformation products. These results suggest that degradation is initiated by splitting of the phenyl groups from the arylarsenic compounds with subsequent hydroxylation of the phenyl groups and ring opening to yield cis, cis, muconate.     

Biodegradation of diphenylarsinic acid to arsenic acid by novel soil bacteria isolated from contaminated soil

Microorganisms capable of degrading diphenylarsinic acid (DPAA) were enriched from contaminated soil using the soil-charcoal perfusion method. Two novel bacterial strains, L2406 and L2413, that can degrade DPAA in a mineral salt medium supplemented with DPAA as the sole carbon source were isolated. Based on comparative morphology, physiology, and comparison of the 16S rRNA gene sequences, both were presumed to be species closely related to Ensifer adhaerens. As the metabolites, phenylarsonic acid (PAA) was determined by liquid chromatography-mass spectrometry analysis as well as three unknown peaks all of whose molecular weights were estimated to be 278. The increase of m/z = 16 from DPAA in the unknowns suggests monohydroxylation of DPAA at the 2-, 3- and 4-positions. The ability of strains L2406 and L2413 to degrade DPAA was suppressed in iron insufficient conditions, e.g. less than 7.2 μM iron in the culture medium. These facts strongly suggest the following hypothesis: Monooxygenase works at the initial degradation step of DPAA degradation by the isolates; and direct hydrolysis from DPAA to PAA is not likely to occur. In addition, release of arsenic acid from PAA by strain L2406 was confirmed by liquid chromatography-inductively coupled plasma mass spectrometry. From these results, strain L2406 was considered to be capable of degrading DPAA to arsenic acid via PAA when DPAA was supplied as the sole carbon source.     

MOBILIZATION OF B AND T LYMPHOCYTES AND HAEMOPOIETIC STEM CELLS BY POLYMETHACRYLIC ACID AND DEXTRAN SULPHATE

The leucocytosis which can be evoked by the polyanions dextran sulphate (DS), polymethacrylic acid (PMAA) and the copolymer of PMAA and styrene (PMAA—STYR) was studied in mice. After intravenous administration of these polyanions peak numbers of leucocytes were found in the peripheral blood 3 hr after injection. All three types of polyanions increased the number of lymphocytes, granulocytes and monocytes. Dose—response studies revealed that the nature of the polyanion determined the degree of leucocyte mobilization. The most potent mobilizer was found to be DS. This polyanion could evoke a six-fold increase of the number of peripheral blood leucocytes. By means of the membrane fluorescence technique it could be demonstrated that optimal doses of DS, PMAA and PMAA—STYR mobilized both B and T lymphocytes. The ratio between the number of B and T cells mobilized was greater for DS than for the other two polyanions. Intravenous injection of DS, PMAA and PMAA—STYR also increased the number of circulating haemopoietic stem cells (CFU-S). The most potent stem cell mobilizer appeared to be PMAA—STYR. This polyanion evoked a twenty-five-fold increase in the number of CFU-S.     

Interaction of myoglobin with poly(methacrylic acid) at different pH in their layer-by-layer assembly films: an electrochemical study

Myoglobin (Mb), with net positive surface charges at pH 5.0, was successfully assembled into layer-by-layer films on various solid surfaces with poly(methacrylic acid) (PMAA) at different pH, designated as {PMAA(pH 5.0)/Mb}n, {PMAA(pH 6.5)/Mb}n, and {PMAA(pH 8.0)/Mb}n, respectively. As a weak polycarboxylic acid with pKa=6 - 7, PMAA carried different negative charges at different pH due to different ionization degree of its carboxylic acid groups. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor and confirm the assembly of {PMAA/Mb}n films. All the results showed that the adsorption amount of Mb in each bilayer had an "unexpected" sequence of {PMAA(pH 5.0)/Mb}n>{PMAA(pH 6.5)/Mb}n>{PMAA(pH 8.0)/Mb}n, which could be explained by the formation of soluble complex of PMAA-Mb at pH 8.0 and the cooperative effect of hydrogen bonding and induced electrostatic interaction between Mb and PMAA at pH 5.0. The influence of ionic strength in exposure solution and in Mb adsorbate solution was investigated, and the results supported the above explanations. The {PMAA/Mb}n films provided a suitable microenvironment for Mb to retain its near-native structure and transfer electron with underlying electrodes. The reversible CV peak pair for Mb Fe(III)/Fe(II) redox couple could be used to catalyze reduction of hydrogen peroxide electrochemically, showing the potential applicability of the films as the new type of biosensors or bioreactors based on the direct electrochemistry of Mb. The electrochemical and electrocatalytic behaviors of protein layer-by-layer films with weak polyelectrolytes could thus be controlled by adjusting the solution pH of weak polyelectrolytes.     

Antiviral Activity of Polyacrylic and Polymethacrylic Acids: II. Mode of Action in Vivo.

A marked virus-inhibiting potency is obtained in the serum after intraperitoneal injection of polyacrylic acid (PAA) and polymethacrylic acid (PMAA) in mice. Much higher antiviral levels were reached than for other related polymers including dextran sulfate, heparin, polyvinyl sulfate, pyran copolymer, polystyrene sulfonate, and macrodex. The broad antiviral action of PAA and PMAA was attributed both to a direct interference with the virus-cell interaction and the viral ribonucleic acid metabolism and to the formation of an interferon-like factor. Both polyanions differed in interferon-inducing ability: highest serum interferon titer was obtained 18 hr after the intraperitoneal injection of PAA. The mechanism of interferon production by PAA and PMAA is discussed. As described previously for Sindbis virus and endotoxin, the animals also became hyporeactive after injection of PAA.     

Antiviral Activity of Polyacrylic and Polymethacrylic Acids: I. Mode of Action in Vitro.

Polyacrylic acid (PAA) and polymethacrylic acid (PMAA) were investigated for their antiviral properties in tissue culture. Compared to other related polyanions, as dextran sulfate, polystyrene sulfonate, polyvinyl sulfate, and polyphloroglucinol phosphate, PAA and PMAA were found to be significantly more antivirally active and less cytotoxic. PMAA added 24 hr prior to virus inoculation inhibited viral growth most efficiently but it was still effective when added 3 hr after infection. Neither a direct irreversible action on the virus nor inhibition of virus penetration into the cell could explain the antiviral activity of PMAA. PMAA inhibited the adsorption of the virus to the host cell and suppressed the one-cycle viral synthesis in tissue cultures inoculated with infectious RNA.     

Cyclic diarylheptanoids from Myrica nana inhibiting nitric oxide release

Investigation of the roots of Myrica nana afforded five new cyclic diarylheptanoids, myricananins A-E (1-5), two new artifacts of myricananins A and B (6-7), and four known compounds, 12-hydroxymyricanone (8), alnusonol (9), myricatomentogenin (10), and actinidione (11). The structures of these new compounds were established by detailed spectroscopic methods. The stereochemistry of compounds 1 and 2 were determined by single-crystal X-ray diffraction. In exception of compounds 2, 6 and 10, all the other compounds were examined for their inhibitory effects on nitric oxide production in lipopolysaccharides-activated macrophages. Compounds 1, 3, 7, 8 and 9 inhibited the release of nitric oxide with IC(50) values of 45.32, 63.51, 52.81, 30.19 and 46.18muM, respectively. Furthermore, compound 1 was found to inhibit the expression of inducible nitric oxide synthase.     

Enzymatic and conformational stability of polymeric complexes of alpha-amylase

The enzymatic and conformational stability of Bacillus subtilis alpha-amylase and its polymeric complexes in acid media and subsequent renaturation in weakly alkaline media were investigated. The following parameters of alpha-amylase secondary structure were determined from circular dichroism spectra: helical units -25%, beta-structures -9%; beta-turns -13%; disordered conformations -53%. After complexation with polymethacrylic acid (PMAA) the alpha-amylase secondary structure did not change, and the tertiary structure underwent only small local changes. Complexation of alpha-amylase with linear and cross-linked PMAA led to an increase in both enzymatic and conformational stabilities in acid media. Purification of alpha-amylase using a biosorbent resulted in higher acid resistance of the free enzyme and of that in the complex with PMAA. Moreover, the degree of reversibility of the acid inactivation also increased.     

Mobilization of B and T lymphocytes and haemopoietic stem cells by polymethacrylic acid and dextran sulphate.

The leucocytosis which can be evoked by the polyanions dextran sulphate (DS), polymethacrylic acid (PMAA) and the copolymer of PMAA and styrene (PMAA--STYR) was studied in mice. After intravenous administration of these polyanions peak numbers of leucocytes were found in the peripheral blood 3 hr after injection. All three types of polyanions increased the number of lymphocytes, granulocytes and monocytes. Dose--response studies revealed that the nature of the polyanion determined the degree of leucocyte mobilization. The most potent mobilizer was found to be DS. This polyanion could evoke a six-fold increase of the number of peripheral blood leucocytes. By means of the membrane fluorescence technique it could be demonstrated that optimal doses of DS, PMAA and PMAA--STYR mobilized both B and T lymphocytes. The ratio between the number of B and T cells mobilized was greater for DS than for the other two polyanions. Intravenous injection of DS, PMAA and PMAA--STYR also increased the number of circulating haemopoietic stem cells (CFU-S). The most potent stem cell mobilizer appeared to be PMAA--STYR. This polyanion evoked a twenty-five-fold increase in the number of CFU-S.     

Bronchoalveolar lavage examined by solid phase microextraction, gas chromatography--mass spectrometry and selected ion flow tube mass spectrometry

Samples (210 in total) of broncholaveolar lavages (BALs), obtained from patients hospitalized with pneumonia in various departments of two hospitals, were analysed using the method of solid phase microextraction-gas chromatography (SPME-GC) with FID detection. Up to 20% (9% unequivocally, 11% probably) of these samples was found to contain volatile fatty acids (VFAs) in the series from acetic acid to heptanoic acid. Importantly, the presence of these acids indicates the presence of fermenting anaerobic bacteria, which were not detected by the conventional microbiological examination. Other compounds, namely the heptanol and cyclohexanone, were also detected by this method in some samples. Cyclohexanone occurred almost exclusively in samples from patients receiving intensive care with mechanical ventilation, and is suspected to originate from plastic parts of ventilators. Selected representative samples were also analysed using further methods, namely gas chromatography-mass spectrometry (GC-MS) of native and silylated samples, and selected ion flow tube mass spectrometry (SIFT-MS). These methods confirmed the identities of above mentioned compounds, and detected numerous other compounds tentatively identified as various alcohols, aldehydes, ketones, esters and hydrogen cyanide, HCN. Most of these compounds occurred in small amounts and their origin and diagnostic significance remains uncertain, except, that is, for the HCN, which indicates the presence of Pseudomonas aeruginosa.     

Protonation of two adjacent tyrosine residues influences the reduction of cytochrome c by diphenylacetaldehyde: a possible mechanism to select the reducer agent of heme iron

We have shown that diphenlacetaldehyde (DPAA) is able to promote mitochondrial DeltaPsi disruption accompanied by damage in mitochondrial DNA, lipids, and proteins [Almeida, A. M.; Bechara, E. J. H.; Vercesi, A. E.; Nantes, I. L. Free Radic. Biol. Med. 27:744-747; 1999]. In this work, DPAA was used as a model of carbonyl reagent for cytochrome c. The results suggest that DPAA is a redox cytochrome c modifier. Conversion of Fe(III) to Fe(II) cytochrome c promoted by DPAA is pH dependent. The second-order rate determined for heme iron reduction (k2) is 698 M(-1) s(-1) and this process occurs with an activation energy of 8.5 +/- 0.8 kcal/mol. Analysis of the pH profile suggests the presence of two ionizable cytochrome c groups (pKa1 = 8.9 and pKa2 = 11.4) related to the electron transfer from DPAA to heme iron. The heats of ionization of the two prototropic groups, pKa1 (DeltaH(ion) = 6.5 kcal/mol, DeltaS(ion) = -29.0 cal/mol.K), and pKa2 (DeltaH(ion) = 5.0 kcal/mol, DeltaS(ion) = -24.0 cal/mol.K), suggest involvement of two tyrosine residues, probably Y67 and Y74, related to DPAA-promoted heme iron reduction. The cytochrome c chemical modification by iodination of tyrosine groups significantly decreased the reduction rate promoted by DPAA, and shifted the pH(opt) value from 10.0 to 9.25. The cytochrome c-promoted DPAA electron abstraction quickly produces the expected enol-derived radical, as indicated by 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) spin trapping EPR measurements. This radical reacts with molecular oxygen, producing a peroxyl intermediate radical that, via a putative dioxetane intermediate, promotes formation of benzophenone as the main final product of this reaction, detected by high-performance liquid chromatography coupled with tandem mass spectrometry.     

Phytoremediation of diphenylarsinic-acid-contaminated soil by Pteris vittata associated with Phyllobacterium myrsinacearum RC6b

A pot experiment was conducted to explore the phytoremediation of a diphenylarsinic acid (DPAA)-spiked soil using Pteris vittata associated with exogenous Phyllobacterium myrsinacearum RC6b. Removal of DPAA from the soil, soil enzyme activities, and the functional diversity of the soil microbial community were evaluated. DPAA concentrations in soil treated with the fern or the bacterium were 35–47% lower than that in the control and were lowest in soil treated with P. vittata and P. myrsinacearum together. The presence of the bacterium added in the soil significantly increased the plant growth and DPAA accumulation. In addition, the activities of dehydrogenase and fluorescein diacetate hydrolysis and the average well-color development values increased by 41–91%, 37–78%, and 35–73%, respectively, in the treatments with P. vittata and/or P. myrsinacearum compared with the control, with the highest increase in the presence of P. vittata and P. myrsinacearum together. Both fern and bacterium alone greatly enhanced the removal of DPAA and the recovery of soil ecological function and these effects were further enhanced by P. vittata and P. myrsinacearum together. Our findings provide a new strategy for remediation of DPAA-contaminated soil by using a hyperaccumulator/microbial inoculant alternative to traditional physicochemical method or biological degradation.     

Blood Plasma TGF- β1 Concentration in Sporadic Dilatative Pathology of Ascending Aorta: More Questions than Answers

Transforming growth factor β1 (TGF- β1) is a cytokine that participates in a broad range of cellular regulatory processes and is associated with various diseases including aortic aneurysm. Increased TGF- β1 levels are associated with Marfan syndrome (MFS) caused by FBN1 mutations and subsequent defects in signaling system. We studied TGF- β1 levels in 62 patients with sporadic, non syndromic, dilatative pathology of ascending aorta (DPAA) and in reference group subjects (n = 212). An initial screening of 212 reference individuals identified TGF- β1 gender discrepancies and age-dependent cytokine increase in women. Patients with DPAA had increased levels of TGF- β1 in comparison to reference group subjects (median 7.7 ng/ml, range 2.1–25.3, and median 6.2 ng/ml, range 1.0–33.1, respectively). There is a significant association between TGF-β1 concentration and DPAA (OR 1.084, CI 1.027–1.144, p = 0.004) but the mechanisms of cause and effect have not been established yet. Slightly increased TGF-β1 concentrations in patients with sporadic DPAA in comparison to the reference subjects show a potential use of TGF-β1 as a biomarker for the disease. However, cytokine dependence on age, gender, and other unknown factors among individuals with no cardiovascular complains reduces its specificity for DPAA. We would also like to raise awareness regarding the choice of methods when measuring TGF-β1 levels with an emphasis on preanalytical phase and the choice of sample.     

CHANGES OF LYMPHOCYTE KINETICS IN THE NORMAL RAT, INDUCED BY THE LYMPHOCYTE MOBILIZING AGENT POLYMETHACRYLIC ACID

The changes in lymphocyte kinetics induced by the lymphocyte mobilizing agent polymethacrylic acid (PMAA) were studied in the normal rat. Quantitative data are presented concerning the degree of lymphocyte mobilization in the spleen and in various lymph nodes at different times after PMAA administration. Data were also obtained regarding the exact site of lymphocyte mobilization in the spleen. Evidence is given that PMAA mobilizes both T and B lymphocytes.
Furthermore, results are presented on the different routes along which mobilized lymphocytes reach the blood. It is concluded that lymphocytes mobilized from the various lymph nodes are transported to the peripheral blood mainly by way of the efferent lymphatics ('indirect' route) while lymphocytes mobilized from the spleen will enter the blood chiefly via the so-called 'direct' route.
The relevance of these data to lymphocyte kinetics is discussed in relation to the planning of effective irradiation schedules for extra-corporeal irradiation of the blood during induced lymphocyte mobilization.     

Chromatographic analysis of lipoic acid and related compounds

The analysis of lipoic acid and related compounds, such as its reduced form dihydrolipoic acid, its amide form lipoamide and other analogues, in biological and food samples is important in biochemistry, nutritional and clinical chemistry. This review summarizes the chromatographic methods for the determination of lipoic acid and related compounds, and their applications to various samples such as bacteria, tissues, drugs and food. Gas chromatographic methods with flame ionization detection and flame photometric detection are commonly used for the quantification of lipoic acid present as its protein-bound form, after acid or base hydrolysis of these samples. High-performance liquid chromatographic methods with ultraviolet, fluorescence and electrochemical detection are mainly used for the determination of free lipoic acid and related compounds, such as dihydrolipoic acid, lipoamide and other analogues. Moreover, gas chromatography–mass spectrometry and capillary electrophoresis methods are also developed.     

Leukocyte mobilization in mice by polyanions. Decline of leukocyte mobilizing capacity after transplantation of lymphoma

The leukocyte mobilizing polyanions dextran sulphate (DS) and polymethacrylic acid (PMAA) were administered to AKR and (C57BL x CBA) F1 mice at various times after transplantation of syngeneic lymphoma cells. In nonleukaemic mice DS and PMAA increased the number of circulating leukocytes 3--4-fold. The extent of leukocyte mobilization in leukaemic mice depended on the interval between transplantation of the lymphoma cells and injection of the polyanion. During the development of leukaemia in AKR as well as in (C57BL x CBA) F1 mice the capacity to react upon injection of polyanions with leukocyte mobilization gradually decreased. For DS, this decrease started before the number of leukocytes increased in the peripheral blood. On the other hand, the capacity for PMAA-induced leukocyte mobilization was fully preserved for several more days. In heavily leukaemic mice neither DS nor PMAA could further increase the number of peripheral blood leukocytes. In such mice the distribution pattern of leukaemic blast cells, small lymphocytes, granulocytes and monocytes was also hardly or not affected by injection of the polyanion.     

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.     

Preparation and recognition performance of creatinine-imprinted material prepared with novel surface-imprinting technique

By adopting the novel surface molecular imprinting technique put forward by us not long ago, a creatinine molecule-imprinted material with high performance was prepared. The functional macromolecule polymethacrylic acid (PMAA) was first grafted on the surfaces of micron-sized silica gel particles in the manner of “grafting from” using 3-methacryloxypropyltrimethoxysilane (MPS) as intermedia, resulting in the grafted particles PMAA/SiO₂. Subsequently, the molecular imprinting was carried out towards the grafted macromolecule PMAA using creatinine as template and with ethylene glycol diglycidyl ether (EGGE) as crosslinker by right of the intermolecular hydrogen bonding and electrostatic interaction between the grafted PMAA and creatinine molecules. Finally, the creatinine-imprinted material MIP-PMAA/SiO₂ was obtained. The binding character of MIP-PMAA/SiO₂ for creatinine was investigated in depth with both batch and column methods and using N-hydroxysuccinimide and creatine as two contrast substances, whose chemical structures are similar to creatinine to a certain degree. The experimental results show that the surface-imprinted material MIP-PMAA/SiO₂ has excellent binding affinity and high recognition selectivity for creatinine. Before imprinting, PMAA/SiO₂ particles nearly has not recognition selectivity for creatinine, and the selectivity coefficients of PMAA/SiO₂ for creatinine relative to N-hydroxysuccinimide and creatine are only 1.23 and 1.30, respectively. However, after imprinting, the selectivity coefficients of MIP-PMAA/SiO₂ for creatinine in respect to N-hydroxysuccinimide and creatine are remarkably enhanced to 11.64 and 12.87, respectively, displaying the excellent recognition selectivity and binding affinity towards creatinine molecules.