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Psychological stress has been previously reported to worsen symptoms of inflammatory bowel disease (IBD). Similarly, intestinal tertiary lymphoid organs (TLOs) are associated with more severe inflammation. While there is active debate about the role of TLOs and stress in IBD pathogenesis, there are no studies investigating TLO formation in the context of psychological stress. Our mouse model of Crohn’s disease–like ileitis, the SAMP1/YitFc (SAMP) mouse, was subjected to 56 consecutive days of restraint stress (RS). Stressed mice had significantly increased colonic TLO formation. However, stress did not significantly increase small or large intestinal inflammation in the SAMP mice. Additionally, 16S analysis of the stressed SAMP microbiome revealed no genus-level changes. Fecal microbiome transplantation into germ-free SAMP mice using stool from unstressed and stressed mice replicated the behavioral phenotype seen in donor mice. However, there was no difference in TLO formation between recipient mice. Stress increased the TLO formation cytokines interleukin-23 (IL-23) and IL-22 followed by up-regulation of antimicrobial peptides. SAMP × IL-23r −/− (knockout [KO]) mice subjected to chronic RS did not have increased TLO formation. Furthermore, IL-23, but not IL-22, production was increased in KO mice, and administration of recombinant IL-22 rescued TLO formation. Following secondary colonic insult with dextran sodium sulfate, stressed mice had reduced colitis on both histology and colonoscopy. Our findings demonstrate that psychological stress induces colonic TLOs through intrinsic alterations in IL-23 signaling, not through extrinsic influence from the microbiome. Furthermore, chronic stress is protective against secondary insult from colitis, suggesting that TLOs may function to improve the mucosal barrier.

It was already shown that microdoses of lithium carbonate (Li2CO3) promoted memory stabilization in humans and mice. Prolonged treatment also reduced neuronal loss and increased the density of the neurotrophin BDNF in transgenic mice for Alzheimer’s disease. The aim of this study was to evaluate whether lithium ions affect inflammatory profiles and neuronal integrity in an animal model of accelerated senescence (SAMP-8). Organotypic hippocampal cultures obtained from 11 to 12-month-old SAMP-8 mice were treated with 2 µM, 20 µM and 200 µM Li2CO3. 2 µM Li2CO3 promoted a significant reduction in propidium iodide uptake in the CA2 area of hippocampus, whereas 20 µM promoted neuroprotection in the CA3 and GrDG areas. 200 µM caused an increase in cellular death, showing toxicity. Measured with quantitative PCR, IL-1α, IL-6 and MIP-1B/CCL-4 gene expression was significantly reduced with 20 µM Li2CO3, whereas IL-10 gene expression was significantly increased with the same concentration. In addition, 2 µM and 20 µM Li2CO3 were also effective in reducing the activation of NFkB and inflammatory cytokines densities, as evaluated by ELISA. It is concluded that very low doses of Li2CO3 can play an important role in neuroprotection as it can reduce neuronal loss and neuroinflammation in older individuals.

Infections with multidrug-resistant (MDR) pathogens are increasingly concerning for public health. Synthesized antimicrobial peptide A4 (SAMP-A4), a peptide computationally designed by our research team, is a potential drug candidate. However, the antimicrobial peptide SAMP-A4 is easily degraded in serum. To obtain SAMP-A4 analogues with high biostability, chemical modifications at its N-terminus, including fatty acid conjugation, glycosylation and PEGylation, were carried out. The results showed that the introduction of hydrophobic fatty acids at the N-terminus of SAMP-A4 is better than hydrophilic glycosylation and PEGylation. With increasing fatty acid chain length, the stability of SAMP-A4 analogues in serum and trypsin solutions is significantly improved, and the activities against MDR bacteria and Candida are significantly enhanced. There is no obvious change in haemolysis even when hexanoic acid is coupled with SAMP-A4, so the resulting analogue SAMP-A4-C6, SAMP-A4 conjugated with hexanoic acid, is the most likely of the analogues to become a drug.

The study of metal-based drugs represents an important branch of modern bioinorganic chemistry. The growing importance of this field is linked to the large success in medicine of a few metal based drugs, either in clinical use or still experimental. Moreover, these metal-based drugs are frequently used as reference compounds to assess comparatively the behavior of newly synthesized metallodrugs. For the convenience of researchers working in this area we report here a compilation of the relevant analytical and spectroscopic data of ten representative metallodrugs based on Platinum, Ruthenium, Gold and Mercury. The selected compounds, namely Cisplatin, Oxaliplatin, Carboplatin, Auranofin, Sodium Aurothiomalate, NAMI-A, KP1019, Thimerosal, Merbromin and Phenylmercury Acetate, were chosen owing to their importance in the field. We believe that this compilation may turn very helpful to researchers as these data are difficult to find and generally scattered over a large number of (old) publications.

Zirconium-based metal−organic frameworks, namely Zr-based MOF, was employed as adsorbent material in the miniaturized solid phase extraction of organic mercury compounds in food prior to capillary electrophoresis-diode array detector analysis. The synthesized adsorbent was characterized by different spectroscopic techniques. Parameters influencing the extraction and complexation of methylmercury chloride, ethylmercury chloride and phenylmercury chloride such as type of eluent solvent, type and amount of adsorbent were investigated. In addition, linear ranges contained 2.00–300.00 ng mL−1 for MeHg+, 5.00–500.00 ng mL−1 for EtHg+ and PhHg+, and the established method presented good linearity (R2 ≥ 0.998). Under the optimized experimental conditions, the ranges of detection limit and quantitation limit were 0.022–0.067 ng mL−1 and 0.073–0.220 ng mL−1, respectively. The relative standard deviations of intra- and inter-day analysis were less than 3.2 and 3.1%, respectively. Trueness of the present method was successfully accomplished by means of the recovery assays (81.4–98.5%) in the blank samples with two concentration levels. The repeatability %RSD of the method was lower than 2.7%. Overall, the developed approach proved to have the latent capability to be utilized in routine analysis of organic mercury compounds in fish and Dendrobium officinale.

Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg2+) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.

A sensitive determination method for mercury speciation analysis was developed. Four mercury species, mercury ion, methylmercury, ethylmercury, and phenylmercury, were complexed with emetine-dithiocarbamate (emetine-CS(2)), and then injected onto a HPLC instrument coupled with a tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection system. The emetine-CS(2) complexing agent was effectively used to measure the concentration in addition to serving as a separation and detection reagent. The calibration curves for these mercury complexes were linear in the range of 0.050 - 10 μg L(-1) (as Hg). The limit of detection for (emetine-CS(2))(2)Hg, emetine-CS(2)-methylmercury, emetine-CS(2)-ethylmercury, and emetine-CS(2)-phenylmercury were 30, 17, 21, and 22 ng L(-1), respectively. The sensitivity of this method enables the determination of mercury species in water samples at sub-ppb levels. Furthermore, the method was applied to biological samples in combination with acid leaching and liquid-liquid extraction using emetine-CS(2) as an extraction reagent. The determination results were in good agreement with the values of the certified reference materials.

Background: The finding of a unilaterally dilatated pupil in a patient who is otherwise alert and unimpaired can pose an interesting diagnostic problem. Algorithms have outlined the approach to evaluating anisocoria, but do not stress the importance of toxic exposures. Case Report: A patient with a history of depression and chronic headaches was referred to the emergency department from an ophthalmologist's clinic with the findings of asymmetrically dilatated pupils and blurred vision. Detailed history revealed that for several weeks before the onset of symptoms, the patient applied to her eyelids hemorrhoidal ointment that contained an active ingredient known to produce mydriasis. Conclusion: In the absence of neurologic deficits, clinical history should search for a toxic or pharmacologic etiology of abnormal mydriasis. A detailed physical examination and bedside diagnostic maneuvers can help distinguish benign causes of an abnormally dilatated pupil from more serious causes relating to central nervous system lesions, potentially sparing the patient from unnecessary neuroimaging.

The Less Invasive Stabilization System (LISS; Synthes, West Chester, PA, USA) is a popular implant for managing proximal tibia fractures and distal femur fractures. However, concern has been raised with difficulty in implant removal. The locked titanium screws have a propensity to become fixed to the plate. This complication and techniques for removing the jammed screws are well described. Most of the effective techniques for removing jammed or cold-welded screws involve using a metal saw to cut the plate or carbide burr to obliterate the screwhead. Both techniques generate a significant amount of metal debris. Given that there is known soft-tissue reaction to these particles, it is sensible to remove as much of the debris as possible. However, removal of the metallic debris can be quite difficult and time consuming. The purpose of this article is to describe a novel technique to manage metal debris generated during jammed locking screw removal. Although we describe this technique for titanium locking screw removal, it is likely useful anytime implant removal generates significant debris.

Kidney injury molecule-1 (KIM-1) is markedly upregulated in renal proximal tubule cells by stimuli that promote dedifferentiation, including ischemic or toxic injury, as well as in cases of tubulointerstitial disease, polycystic kidney disease, and renal cell carcinoma. Structurally, KIM-1 possesses a single transmembrane domain and undergoes membrane-proximal cleavage, which leads to the release of soluble KIM-1 ectodomain into the urine. Urinary KIM-1 ectodomain is a promising sensitive and specific biomarker for acute kidney injury in humans, and therefore it is important to determine what regulates KIM-1 shedding. We found that constitutive cleavage of KIM-1 is mediated by ERK activation, and that cleavage is accelerated by p38 MAP kinase activation. After cleavage, a 14-kD membrane-bound fragment of KIM-1, which contains two highly conserved tyrosine residues, was tyrosine-phosphorylated. Mutagenesis studies demonstrated that the juxtamembrane secondary structure, not the primary amino acid sequence, was critical to the cleavage of KIM-1.

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 microg g(-1) creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H2SO4, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5-50 microg 1(-1). The average SPMA in pH 2 samples is 45-60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 microg g(-1) creatinine.

HipHop-Refine was employed to derive a binding hypothesis for pseudomonal quorum sensing (QS) antagonists. The model was employed as 3D search query to screen the National Cancer Institute (NCI) database. One of the hits illustrated nanomolar QS inhibitory activity. The fact that this compound contained tetravalent lead (Pb) prompted us to evaluate the activities of phenyl mercuric nitrate and thimerosal, both fit the binding pharmacophore. The two mercurials illustrated nanomolar to low micromolar IC50 inhibitory values against pseudomonal QS. The three compounds represent a new class of QS inhibitors.

A new concept is presented for green analytical applications based on coupling on-line high-intensity focused ultrasound (HIFU) with a sequential injection/flow injection analysis (SIA/FIA) system. The potential of the SIA/HIFU/FIA scheme is demonstrated by taking mercury as a model analyte. Using inorganic mercury, methylmercury, phenylmercury, and diphenylmercury, the usefulness of the proposed methodology for the determination of inorganic and total mercury in waters and urine was demonstrated. The procedure requires low sample volumes and reagents and can be further applied to all chemical reactions involving HIFU. The inherent advantages of SIA, FIA, and HIFU applications in terms of high throughput, automation, low reagent consumption, and green chemistry are accomplished together for the first time in the present work.

ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that may require activation through removal of its prodomain before it can exert catalytic activity. We examined various proteinases as well as auto-activation under a wide range of conditions for removal of the prodomain and induction of enzymatic activity. The proprotein convertases, furin, PACE4, and PC5/6 efficiently removed the prodomain through cleavage at Arg(212)/Phe(213), generating an active enzyme. Of a broad range of proteases evaluated, only MMP-9 and trypsin were capable of removing the prodomain. In the presence of mercuric compounds, removal of the prodomain through autocatalysis was not observed, nor was it observed at temperatures from 22 to 65 degrees C, at ionic strengths from 0.1 to 1M, or at acidic/neutral pH. At basic pH 8-10, removal of the prodomain by autocatalysis occurred, generating an active enzyme. In conclusion, the pro-form of ADAMTS-4 is not catalytically active and only a limited number of mechanisms mediate its N-terminal activation.

Capillary electrophoresis (CE) was directly interfaced to flame-heated furnace atomic absorption spectrometry (FHF-AAS) via a laboratory-made thermospray interface for nanoliter trace element speciation. The CE-FHF-AAS interface integrated the superiorities of stable CE separation, complete sample introduction, and continuous vaporization for AAS detection without the need of extra external heat sources and any post-column derivation steps. To demonstrate the usefulness of the developed hybrid technique for speciation analysis, three environmentally significant and toxic forms of methylmercury (MeHg), phenylmercury (PhHg), and inorganic mercury (Hg(II)) were taken as model analytes. Baseline separation of the three mercury species was achieved by CE in a 60 cm long x 75 microm inner diameter fused-silica capillary at 20 kV and using a mixture of 100 mM boric acid and 10% v/v methanol (pH 8.30) as running electrolyte. The precision (relative standard deviation, RSD, n = 7) of migration time, peak area and peak height for the mercury species at 500 microg x L(-1) (as Hg) level were in the range of 0.9-1.2%, 1.5-1.9%, and 1.4-2.0%, respectively. The detection limit (S/N = 3) of three mercury species was 3.0 +/- 0.15 pg (as Hg), corresponding to 50.8 +/- 2.4 microg x L(-1) (as Hg) for 60 nL sample injection, which was almost independent on specific mercury species. The developed hybrid technique was successfully applied to the speciation analysis of mercury in a certified reference material (DORM-2, dogfish muscle).

Purpose The non–protein sulfydryls (NPSH) glutathione and cysteine are able to effect chemical repair of radiation-induced DNA damage, particularly under hypoxic conditions, and are implicated in radioresistance. The levels of NPSH in the nucleus are predicted to be more important than those in cytoplasm. We, therefore, investigated the relation between nuclear NPSH and the clinical outcome of radical radiotherapy (RT). Methods and materials A fluorescence image analysis technique to measure nuclear NPSH was developed, based on the SH-reactive probe 1(4-chloromercuryphenyl-azo-2-naphthol) and the DNA dye 4,6-diamidino-2-phenylindole. This was used to measure nuclear and tissue NPSH levels in biopsies obtained from 58 patients with locally advanced uterine cervical carcinoma, treated with radical RT. Results An approximately twofold range in the nuclear and tissue NPSH levels between individual tumors was found, although the intratumoral heterogeneity was much smaller. Nuclear and tissue NPSH values correlated closely in all cases. No statistically significant associations were noted between NPSH levels and tumor size, stage, or tumor hypoxia as determined at the time of biopsy using an Eppendorf p o 2 probe. The response to RT and patient survival did not correlate with tumor NPSH. Conclusion These results did not support the existence of an independently regulated nuclear pool of NPSH and showed that tissue and nuclear NPSH are not predictive of the outcome of patients with locally advanced cervical carcinomas treated with RT.

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its smooth muscle plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction of bovine pulmonary artery smooth muscle to homogeneity using a combination of purification steps. Heparin sepharose purified preparation of 72 kDa progelatinase is composed of two distinct population of zymogens: a 72 kDa progelatinase tightly complexed with TIMP-2 ∥an ambient tissue inhibitor of metalloprotease in the smooth muscle plasma membrane), and a native 72 kDa progelatinase free of any detectable TIMP-2. The homogeneity of the native 72 kDa progelatinase form is demonstrated by SDS-PAGE under nonreducing condition, nondenaturing native gel electrophoresis. The purified TIMP-2 free proenzyme electrophoresed as a single band of 72 kDa which could be activated by APMA with the formation of 62 and 45 kDa active species. The proenzyme is activated poorly by trypsin but not by plasmin. The purified 72 kDa progelatinase is stable at aqueous solution and does not spontaneously autoactivate. The purified 72 kDa gelatinase exhibited properties that are typical of MMP-2 obtained from other sources. These are: (i) its activity is dependent on the divalent cation, Ca2+, and is inhibited by EDTA, EGTA and 1:10-phenanthroline; (ii) it was inhibited by α2 macroglobulin but not by the inhibitors of serine, cysteine, thiol, aspartic proteinases and calpains; (iii) it was found to be inhibited by TIMP-2, the specific inhibitor of MMP-2; (iv) like MMP-2, obtained from other sources, its major substrates were found to be collagens (type IV and V) and gelatins (type I, IV and V). Additionally, the purified MMP-2 degrades Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gn-D-Arg-OH (dinitrophenyl labelled peptide), a well known synthetic substrate for the MMP-2.

A new carbon paste electrode (CPE) for the determination of iodide ion based on a cetyltrimethylammonium iodide (CTMAI) ion pair as an electroactive material is described. The electrode shows a linear response for iodide ion over the concentration range of 4 x 10(-5) M to 1 x 10(-1) M with a lower detection limit of 4 x 10(-5) M at 25 degrees C. The electrode has a Nemstian slope of -55.0 +/- 0.4 mV/decade and a fast potential response of 45 s, which is almost constant over a pH range of 5.0 - 9.0. Selectivity coefficient data of the CTMAI-CPE for some common ions show negligible interference, and the electrode has high selectivity towards the iodide ion. An average recovery of 101.83% with a relative standard deviation of 1.53% has been achieved for the determination of iodide in Flaxedil (gallamine triethiodide) ampoules, a muscle relaxant drug. The electrode has been examined for the determination of iodide in saline water; the results were found to compare favorably with those obtained using Metrohm iodide ISE. The electrode has been utilized as an end-point indicator electrode for the determination of Hg(II) and phenylmercury(I) in their aqueous solutions using potentiometric titration with a potassium iodide standard solution.

Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA. In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000). To optimize reaction kinetics for organic mercury compounds, themerB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall. Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme. MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells. These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification. In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments.

This study describes two novel synthetic procedures to prepare APM, a useful tool for the analysis and the purification of thiolated biomolecules. The methods developed are technically simple and robust and allowed the first full characterization of pure APM. Moreover, the efficacy of APM, as a biochemical tool, was demonstrated by analysis of tRNA thiolation by APM-PAGE.

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell (MEC) function and is remodeled by matrix metalloproteinases (MMPs). To investigate the significance and regulation of MMP activity in normal MEC, we utilized a primary culture model in which rat MEC were grown three dimensionally within a reconstituted basement membrane (RBM) in defined serum-free medium. Zymograms of culture medium demonstrated that five major gelatinases of 97, 80, 74, 69, and 65 kDa were secreted by MEC and were distinct from gelatinases of RBM origin. Based on molecular weight, p-aminophenylmercuric acid activation, immunoblotting with MMP-specific antibodies, inhibition by EDTA, a peptide containing the prodomain sequence of MMP (TMRKPRCGNPDVAN) and two synthetic MMP inhibitors (BB-94 and CGS 27023A), these were classified as inactive and active forms of MMP-9 and MMP-2. The maximal MMP activities occurred when MEC were in a rapid proliferation and branching phase and declined after they underwent functional differentiation. Known regulators of MEC growth and differentiation were evaluated for their ability to modulate gelatinase activity in primary culture. Secretion of one or both MMPs was inhibited by EGF, TGFalpha, prolactin, and hydrocortisone and stimulated by progesterone. Furthermore, the functional significance of MMPs was demonstrated since three MMP inhibitors blocked branching morphogenesis elicited by the absence of hydrocortisone. Additionally, two synthetic MMP inhibitors not only inhibited epithelial cell growth but also inhibited normal alveolar development of the MEC. Finally, these drugs were found to enhance MMP secretion from MEC, although the activity of the secreted MMPs was inhibited as long as the drug was present.

We established an efficient overproduction-purification system for blasticidin S deaminase (BSD) using the cDNA cloned from Aspergillus terreus. The estimated molecular mass of the purified enzyme indicated BSD was a tetramer. This tetrameric form was very resistant to denaturation by SDS and showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much slower (as a single band of 36 kDa) in its active conformation than its completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS was not performed before electrophoresis. As predicted from the presence of the catalytic zinc-coordinating sequence motif conserved in the cytosine nucleoside/nucleotide deaminase family, BSD also contained one zinc per deaminase subunit. However, the predicted catalytic function appeared not to be the only role of this zinc in the enzyme. First, titration of the zinc-chelating -SH groups with p-hydroxymercuriphenylsulfonate led to dissociation of the BSD tetramer into unstable monomers or dimers. Second, depletion of zinc on reconstitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) resulted in improper folding of the polypeptide. These results suggest that zinc also plays a structural role in maintenance of the protein structure. When we introduced mutations at Glu-56 (the proposed active site) and Cys-91 (a proposed catalytic zinc-binding Cys) in BSD, none of the resulting mutants (E56D, E56Q, C91A, C91S, and C91H) showed any detectable activity, as judged with the spectrophotometric assay. Replacements of Cys-91 resulted in gross perturbation of the enzyme structure although the catalytically essential Glu-56 was not necessarily required for proper folding of the enzyme. These results further support our proposal that the catalytic zinc coordinated by the conserved sequence motif is also structural in BSD.

Methylmercury is a highly toxic, organic derivative found in mercury-polluted wetlands and coastal sediments worldwide. Though commonly present at low concentrations in the substrate, methylmercury can biomagnify to concentrations that poison predatory animals and humans. In the interest of developing an in situ detoxification strategy, a model plant system was transformed with bacterial genes (merA for mercuric reductase and merB for organomercurial lyase) for an organic mercury detoxification pathway. Arabidopsis thaliana plants expressing both genes grow on 50-fold higher methylmercury concentrations than wild-type plants and up to 10-fold higher concentrations than plants that express merB alone. An in vivo assay demonstrated that both transgenes are required for plants to detoxify organic mercury by converting it to volatile and much less toxic elemental mercury.

To investigate the individual role of MerT and MerP encoded by Pseudomonas K-62 pMR26 in the transport of phenylmercury, a series of mutants with a specific point mutation in merT and/or genetic deletion in merP were constructed and transformed into Escherichia coil XL-1-Blue. Transport of phenylmercury across the cytoplasmic membrane of E. coli mediated by MerT and MerP was inhibited by NaCN and by cold temperatures. Deletion of merP reduced, but did not completely abolish the C6H5Hg+-hyperuptake and -hypersensitive phenotypes suggesting that transport of phenylmercury into the cytoplasm of E. coli is still occurring. Mutations of the vicinal cysteine residues (Cys24 and Cys25) in the first transmembrane region of MerT to serine caused complete loss of Hg2+-hyperuptake and -hypersensitivity, whereas the mutations did not affect the C6H5Hg+-hyperuptake and -hypersensitive phenotypes. In addition, no additive effect on the C6H5Hg+-hyperuptake and -hypersensitive phenotypes was found, when mutations of the two cysteines in MerT to serine were further introduced in the merP-deleted mutants. These results clearly demonstrated that the vicinal cysteine residues of MerT are not involved in the transport of C6H5Hg+, but indeed are involved in the transport of Hg2+ as previously reported.

Pseudomonas putida (UWC1), containing a genetically-engineered plasmid (pQM899), that encodes for the production of catechol 2,3-dioxygenase (C230), was used as a potential means of rapidly estimating bactericidal activity of chlorhexidine diacetate (CHA), phenol, cetylpyridinium chloride (CPC) and phenylmercuric nitrate (PMN). Enzyme C230 converts catechol to 2-hydroxymuconic semialdehyde (2-HMS), which is yellow in colour, via a meta cleavage pathway. Ideal conditions for production and measurement spectrophotometrically of 2-HMS were determined. However, the correlation between this method and viable plate counts was not sufficiently accurate to enable 2-HMS production to provide a sufficiently sensitive determination of biocidal activity. An alternative method, synchronous scanning fluorimetry, in which the decrease in catechol concentration was measured under standardized conditions, provided a good dose-response histogram for all the biocides tested. Although, in comparison with plate counts, there was an underestimation of the bactericidal effects of phenol an PMN, the results of this study suggest that this method has potential in determining the bactericidal efficacy of agents such as CHA and CPC.

The physiological function of a new gene, hereby designated merG , located between merA and merB on the broad-spectrum mer operon of Pseudomonas strain K-62 plasmid pMR26 was investigated. The 654-bp merG gene encodes a protein with a canonical leader sequence at its N terminus. The processing of the signal peptide of this protein was dose-dependently inhibited by sodium azide, a potent inhibitor of protein export. These results suggest that the mature MerG protein (ca. 20 kDa) may be located in the periplasm. Deletion of the merG gene from the broad-spectrum mer operon of pMR26 had no effect on the inorganic mercury resistance phenotype, but rendered the bacterium more sensitive to phenylmercury than its isogenic wild-type strain. Escherichia coli cells bearing pMU29, which carries a deletion of the merG gene, took up significantly more phenylmercury than the bacteria with the intact plasmid pMRA17. When the merG gene in a compatible plasmid was transformed into the E. coli strain carrying pMU29, the high uptake of and high sensitivity to phenylmercury were almost completely restored to their original levels. These results demonstrate that the merG gene is involved in phenylmercury resistance, presumably by reducing in-cell permeability to phenylmercury.

Membrane lactate transport was studied in skeletal muscle cells and membrane vesicles from the L6 line in relation to in vitro myogenesis. In myoblasts, lactate was transported by simple diffusion and insensitive to classical inhibitors: a positive correlation between onset of creatine kinase activity and lactate transport in differentiated myotubes was observed and could be considered to be a functional marker of cell differentiation. In myotubes, complete analysis of the velocity curves (direct coordinates, Eadie-Scatchard plots, Hill plots) gave parameters showing that lactate was carried by an allosteric or multisite system. This was confirmed by using sarcolemmal vesicles and specific inhibitors. In whole cells, alpha-cyano-4-hydroxycinnamic acid (CIN) and parachloromercuribenzylsulphonic acid (pCMBS) inhibited the maximal velocity without modifying the global cooperativity of the system. The weak effect of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), which has a low affinity constant (Ki = 22.5 microM), implicated the monocarboxylate system rather than the anionic exchanger as a carrier system in muscle cells. CIN and DIDS exhibited one type of interaction with lactate carriers, and the curvilinear shape of the lactate Hill plot with or without inhibitors suggested that inhibitors were active at the same family of interaction sites and had a common range of affinities. The apparent competitive inhibition of pyruvate (Ki = 3.2 mM) did not modify the transport pathway of lactate in L6 myotubes. In conclusion, kinetic analysis of lactate transport in the presence or absence of inhibitors gave evidence for a multisite lactate carrier activity in myotubes composed of two systems at least, related to two or three isoforms of lactate carriers.

The capacity of flow-cytometric techniques to detect drug-specific biochemical targets and side effects in Leishmania infantum promastigotes was estimated by assessing the effects of three antileishmanial drugs (pentamidine, allopurinol, and amphotericin B) on parasite metabolism. Cell cycle and total protein content were estimated by staining cells with propidium iodide and fluorescein isothiocyanate, nonprotein thiols were stained by mercury orange, and membrane potential was measured by the accumulation of 3,3'-dipenthyloxacarbocyanine iodide inside the cell. Results showed that dynamic studies in parasites treated with subtoxic concentrations of drugs allowed the detection of drug-specific targets: pentamidine primarily affected nonprotein thiol contents and DNA synthesis, allopurinol primarily affected intracellular protein contents, and amphotericin B primarily affected membrane potential. Moreover, the assessment of cellular functions in parasites treated with increasing concentrations of drugs certified the capacity of these techniques to establish dose-response curves and to permit the detection of side effects.

It has been recently demonstrated that the Mg(2+)-dependent 3'-processing activity of purified human immunodeficiency virus type-1 (HIV-1) integrase is stimulated by the addition of exogenous Zn2+ [Lee, S. P.,Han, M. K. (1996) Biochemistry 35, 3837-3844]. This activation was hypothesized to result from integrase self-association. In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol. We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification. In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition. These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates Mg(2+)-dependent 3'-processing. This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+. It should be noted, however, that the content of Zn2+ did not significantly affect the 3'-processing and strand transfer reactions with Mn2+ in vitro.

The aim of this work was to find the effect of temperature and manufacturing source of phenylmercuric nitrate (PMN) on PMN absorption on low-density polyethylene (LDPE) and polypropylene containers in chloramphenicol eye drops. Two factorial experiments were designed to study the effect of temperature on PMN assay in chloramphenicol eye drops stored in LDPE and prepared from two different PMN sources. PMN source had no effect on PMN assay at 2-8 °C, however at stress conditions (30 °C/75%RH) for 3 weeks, the effect of PMN source on PMN assay was found significant (p 0.05) in formulations stored in LDPE bottles. Temperature was the major contributor to decreased PMN assay. In formulations stored in polypropylene containers, PMN source had significant effect on PMN assay at 2-8 °C and 30 °C/75%RH. Overall, new PMN and polypropylene bottles performed better. The eye drops complied with preservative efficacy test both initially and at the end of shelf life. The concentration exponent of PMN is very low and in spite of its high absorption by container/closure, PMN was still able to protect the eye drops at the end of shelf life. It can be inferred that preservative efficacy test is the better indicator of preservative activity.

The characteristics of bacteria take up mercury into cells via membrane potential-dependent sequence-divergent members of the mercuric ion (Mer) superfamily, i.e., a periplasmic mercuric ion scavenging protein (MerP) and one or more inner membrane-spanning proteins (MerC, MerE, MerF, and MerT), which transport mercuric ions into the cytoplasm, have been applied in engineering of bioreactor used for mercurial bioremediation. We engineered bacteria to express MerC, MerE, MerF, or MerT with or without MerP to clarify their individual role and potential in transport of mercurial. By immunoblot analysis using specific polyclonal antibody, the proteins encoded by merC, merE, merF, merT or merP, were certainly expressed and identified in the membrane fraction. Bacteria expressing MerC, MerE, MerF or MerT in the absence of MerP transported significantly more C6H5Hg(I) and Hg(II) across bacterial membrane than their isogenic strain. In vivo expression of MerP in the presence of all the transporters did not cause apparent difference to the C6H5Hg(I) transport, but gives an apparently higher Hg(II) transport than that did by MerE, MerF or MerT but not by MerC. Among the four transporters studied, MerC showed more potential to transport Hg(II) across bacterial membrane than MerE, MerF and MerT. Together these findings, we demonstrated for the first time that in addition to MerE and MerT, MerF and MerC are broad-spectrum mercury transporters that mediate both Hg(II) and phenylmercury transport into cells. Our results suggested that MerC is the most efficient tool for designing mercurial bioremediation systems, because MerC is sufficient for mercurial transport into cells.

The therapeutic use of stem cells to treat diseases and injuries is a promising tool in regenerative medicine. The umbilical cord provides a rich source of stem cells; we have previously reported a population of stem cells isolated from Wharton's jelly. In this report, we aimed to isolate a novel cell population that was different than those found in Wharton's jelly. We isolated stem cells from the subepithelial layer of the umbilical cord; the cells could be expanded for greater than 90 population doubling and had mesenchymal stem cell characteristics, expressing CD9, SSEA4, CD44, CD90, CD166, CD73, and CD146 but were negative for STRO-1. The cells can be directionally differentiated and undergo osteo-, chondro-, adipo-, and cardiogenesis. In addition, we have identified for the first time that mesenchymal stem cells isolated from umbilical cord can produce microvesicles, termed exosomes. This is the first report describing a stem cell population isolated from the subepithelial layer of the umbilical cord. Given the growth capacity, multilineage potential, and most importantly the low levels of HLA-ABC, we propose that this novel cell isolated from the subepithelial layer of umbilical cord is an ideal candidate for allogeneic cell-based therapy.

A simple and green technique named polymer-supported ionic liquid solid phase extraction (PSIL-SPE) was developed for mercury (Hg) species determination. Inorganic Hg (InHg) species was complexed with chloride ions followed by its introduction into a flow injection on-line system to quantitatively retain the anionic chlorocomplex (HgCl42−) in a column packed with CYPHOS® IL 101-impregnated resin. The trapped InHg was then reduced with stannous chloride (SnCl2) and eluted with the same flow of reducing agent followed by cold vapor atomic absorption spectrometry (CV-AAS) detection. Organic mercury species (OrgHg) did not interact with the impregnated resin and were not retained into the column. Total concentration of OrgHg was evaluated by difference between total Hg and InHg concentration. A 95% extraction efficiency was achieved for InHg when the procedure was developed under optimal experimental conditions. The limit of detection obtained for preconcentration of 40 mL of sample was 2.4 ng L−1 InHg. The relative standard deviation (RSD) was 2.7% (at 1 µg L−1 InHg and n=10) calculated from the peak height of absorbance signals (Gaussian-shape and reproducible peaks). This work reports the first polymer-supported IL solid phase extraction approach implemented in a flow injection on-line system for determination of Hg species in mineral, tap and river water samples. Fil: Escudero, Leticia Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Química de San Luis; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales. Laboratorio de Química Analítica para Investigación y Desarrollo; Argentina Fil: Olsina, Roberto Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Química de San Luis; Argentina Fil: Wuilloud, Rodolfo German. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales. Laboratorio de Química Analítica para Investigación y Desarrollo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza; Argentina

Photocatalytic degradation of phenylmercury was studied using TiO2 in aqueous suspension assisted by UV-A irradiation. Reaction conditions, such as pH and amount of TiO2 were set using a factorial design of experiments resulting in a greater influence of pH on phenylmercury degradation. Hg (II) reduction and simultaneous oxidation of aromatic group was observed. Optimum reaction conditions were obtained under nitrogen atmosphere at pH 10 and 0.35 g/L(-1) TiO2. Under these conditions almost 100% reduction of mercury was reached after 30 min UV irradiation. Total mercury reduction was achieved after 40 min reaction under saturated oxygen. Furthermore, phenol and diphenylmercury were identified as intermediate products of oxidation. It was observed that a major fraction of the reduced mercury was removed as metallic vapor by gas stripping, whereas a minor fraction was adsorbed on the catalyst surface, probably as Hg(OH)2. Under optimal conditions obtained by multivariable analysis, total mineralization of organic matter was achieved after about 60-min irradiation.

Brain ischemia reperfusion causes increased formation of reactive oxygen species (ROS). Activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) has been shown to undergo a significant decrease following reperfusion of the ischemic tissue. We have examined the effect of a superoxide radical-generating system (xanthine oxidase/hypoxanthine, XO/HX) on the activity of this enzyme. Incubation of PDH in the presence of XO/HX resulted in its inactivation. The degree of the inactivation was dependent on the amount of XO present, which correlated linearly with the concentration of superoxide radical generated by this system. The activity of lactate dehydrogenase, an enzyme resistant to inactivation by ischemia reperfusion, was not affected by this system. Superoxide dismutase partially prevented and catalase exerted a nearly complete protective effect against the inactivation of PDH. Deferoxamine was partially protective. The sulfhydryl protective reagents, dithiothreitol and glutathione, prevented the inactivation of PDH, even though to varying degrees, which implicates sulfhydryl oxidation. A hydroxyl radical-generating system (hydrogen peroxide irradiated with ultraviolet radiation) effectively inactivated PDH. These results demonstrate that PDH is susceptible to damage and inactivation by ROS and point to the involvement of Fenton chemistry and hydroxyl radicals formed through it in PDH inactivation by XO/HX. A similar mechanism may be responsible for the PDH inactivation during ischemia/reperfusion.

The transport and uptake of TRH was investigated in the maternal-fetal-placental unit of perfused human term placenta. The degradation of TRH in biological fluid was first determined by incubating [125I]TRH with 100 microL 50% maternal or cord sera with or without pretreatment with 200 microM of p-hydroxymercuriphenyl sulfonic acid (p-HMSA), a proline dipeptidase inhibitor. Transplacental transfer of TRH was then studied by adding 10 microCi of [125I]- or [3H]TRH to the maternal circulation of dually perfused isolated lobule of human term placenta with or without 200 microM p-HMSA. Creatinine was used as an internal marker. The rate of degradation of TRH (P0.001) and inhibition by p-HMSA were significantly higher in maternal than cord sera (P0.05). In the maternal circulation, TRH concentration declined rapidly from 100% at time 0 to 33.5 +/- 1.2% at 120 min. The fetal concentration increased from undetectable levels to a maximum of 1.8 +/- 0.3% at 120 min with a low feto-maternal ratio (0.08 +/- 0.02). Perfusion in the presence of p-HMSA, however, did not significantly change fetal concentration, or the maternal and fetal concentration-time integral levels of TRH. Chromatography of maternal, fetal, and placental homogenates showed that TRH was metabolized by the placenta into small molecular weight fragments predominantly released in the maternal circulation. These results suggest that human placenta acts as an enzymatic barrier to the free passage of TRH.

A novel plasma jet desorption atomization (PJDA) source was developed for atomic fluorescence spectrometry (AFS) and coupled on line with thin layer chromatography (TLC) for mercury speciation. An argon dielectric barrier discharge plasma jet, which is generated inside a 300 μm quartz capillary, interacts directly with the sample being analyzed and is found to desorb and atomize surface mercury species rapidly. The effectiveness of this PJDA surface sampling technique was demonstrated by measuring AFS signals of inorganic Hg(2+), methylmercury (MeHg), and phenylmercury (PhHg) deposited directly on TLC plate. The detection limits of the proposed PJDA-AFS method for inorganic Hg(2+), MeHg, and PhHg were 0.51, 0.29, and 0.34 pg, respectively, and repeatability was 4.7%, 2.2%, and 4.3% for 10 pg Hg(2+), MeHg, and PhHg. The proposed PJDA-AFS was also successfully coupled to TLC for mercury speciation. Under optimized conditions, the measurements of mercury dithizonate (Hg-D), methylmercury dithizonate (MeHg-D), and phenylmercury dithizonate (PhHg-D) could be achieved within 3 min with detection limits as low as 8.7 pg. The combination of TLC with PJDA-AFS provides a simple, cost-effective, relatively high-throughput way for mercury speciation.

Oral lichenoid lesions (OLL) or lichen-planus-like lesions are often idiopathic. Our aim was to determine whether OLL can be caused by allergy to mercury in amalgam fillings, and whether resolution of OLL occurs after replacement of amalgam with other dental fillings. Patients with only OLL (except for 1 case with cutaneous lichen planus) referred for patch testing during 1985-1994 to the Contact and Occupational Dermatitis Clinic of the Skin & Cancer Foundation, Darlinghurst, were reviewed. Patch tests were performed with 1% mercury, 1% ammoniated mercury, 0.1% thimerosal, 0.1% mercuric chloride, 0.05% phenylmercuric nitrate and an amalgam disc, using Finn Chambers occluded for 2 days, 19 patients (17 women and 2 men; age range: 28-72 years) had OLL in close contact with amalgam fillings and showed positive patch test reactions to mercury compounds, 16 out of 19 patients had their amalgam fillings replaced. In 13 patients, the OLL healed. 1 patient had marked improvement. 1 patient had no improvement and developed multiple oral squamous cell carcinoma. In conclusion, OLL can be caused by allergy to mercury in amalgam fillings. Replacement of amalgam with other dental fillings usually results in resolution of OLL and is recommended for cases with positive patch test reactions to mercury compounds.

The intracellular distribution of glutathione (GSH) was measured by a quantitative image cytometry method, using the sulphydryl-reactive agent mercury orange. This readily forms fluorescent adducts with GSH and other non-protein sulphydryls (NPSH), but reacts much more slowly with protein sulphydryls. Under optimum staining conditions mean integrated mercury orange fluorescence per cell was closely correlated with a standard biochemical assay for GSH. Use of the DNA dye DAPI as a counterstain allowed measurement of nuclear NPSH. The mean nuclear-cytoplasmic ratio was 0.57 +/- 0.05. Isolation of nuclei under aqueous conditions resulted in the loss of approximately 90% of mercury orange fluorescence, compared with nuclear fluorescence from intact cells, suggesting that background labelling of protein sulphydryls or other macromolecules is low. Depletion of GSH with N-ethylmaleimide or diethylmaleate decreased mercury orange fluorescence in the nucleus and cytoplasm to a similar extent. In contrast, mercury orange fluorescence in the nucleus was much more resistant to DL-buthionine-S,R-sulphoximine (BSO) depletion than that in the cytoplasm. This finding is compatible with a distinct pool of GSH in the nucleus that is comparatively resistant to BSO depletion. Alternatively, the retention of fluorescence in the nucleus following GSH depletion by BSO treatment might be due to accumulation of cysteine. These findings have implications for cancer treatment since the level of NPSH in the nucleus might be a more important determinant of resistance to DNA-damaging agents than that in cytoplasm. The image cytometry method described here is quantitative, allows a measure of tumour cell heterogeneity and can be applied to small biopsy samples obtained by fine-needle aspiration. Thus it appears suitable for prospective clinical studies in cancer patients, and for monitoring the effects of GSH-depleting agents used as adjuncts to cancer chemotherapy or radiotherapy. Images Figure 1

The aim is to present a patient with severe bilateral corneal complications after long-term antiglaucoma treatment with 1% pilocarpine hydrochloride (Pilokarpin, Pliva, Zagreb, Croatia) and its management. A patient with narrow-angle glaucoma treated with 1% topical pilocarpine hydrochloride eye drops for the last twenty years complained of impaired vision, intermittent visual haloes and eye redness. Ophthalmologic examination showed bilateral band keratopathy, peripheral laser iridotomy, medicamentous myosis, brown nuclear cataract, and synchysis scintillans of his right eye. Band keratopathy was thought to have resulted from the presence of the preservative phenylmercuric nitrate in the pilocarpine hydrochloride eye drops. Treatment of the patient consisted of two separate procedures for both eyes, i.e. phaco trabeculectomy and six months later corneal procedure including abrasion of corneal epithelium followed by removal of the superficial stromal calcium deposits by means of a 3.75% ethylenediaminetetraacetic (EDTA) solution. After phaco trabeculectomy, visual acuity was 0.8 on both eyes. Bilateral visual improvement with visual acuity 1.0 was recorded after corneal treatment with EDTA. In conclusion, one must be aware of preservative complications in long-term topical use, such as band keratopathy that can be visually incapacitating. Surgical treatment using EDTA is safe and effective treatment for band keratopathy.