The leading established metal-based drugs: a revisitation of their relevant physico-chemical data.

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.

Temperature-mediated absorption of phenylmercuric nitrate on polyethylene and polypropylene containers in chloramphenicol eye drops.

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.

Phenylmercury degradation by heterogeneous photocatalysis assisted by UV-A light.

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.

Site specific identification of endogenous S-nitrosocysteine proteomes.

Cysteine S-nitrosylation is a post-translational modification regulating protein function and nitric oxide signaling. Herein the selectivity, reproducibility, and sensitivity of a mass spectrometry-based proteomic method for the identification of endogenous S-nitrosylated proteins are outlined. The method enriches for either S-nitrosylated proteins or peptides through covalent binding of the cysteine sulfur with phenylmercury at pH=6.0. Phenylmercury reacts selectively and efficiently with S-nitrosocysteine since no reactivity can be documented for disulfides, sulfinic or sulfonic acids, S-glutathionylated, S-alkylated or S-sulfhydrylated cysteine residues. A specificity of 97±1% for the identification of S-nitrosocysteine peptides in mouse liver tissue is achieved by the inclusion of negative controls. The method enables the detection of 36 S-nitrosocysteine peptides starting with 5pmolS-nitrosocysteine/mg of total tissue protein. Both the percentage of protein molecules modified as well as the occupancy by S-nitrosylation can be determined. Overall, selective, sensitive and reproducible enrichment of S-nitrosylated proteins and peptides is achieved by the use of phenylmercury. The inclusion of appropriate negative controls secures the precise identification of endogenous S-nitrosylated sites and proteins in biological samples. BIOLOGICAL SIGNIFICANCE: The current study describes a selective, sensitive and reproducible method for the acquisition of endogenously S-nitrosylated proteins and peptides. The acquisition of endogenous S-nitrosoproteomes provides robust data that is necessary for investigating the mechanism(s) of S-nitrosylation in vivo, the factors that govern its selectivity, the dependency of the modification on different isoforms of nitric oxide synthases (NOS), as well as the physiological functions of this protein modification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Separating and analyzing sulfur-containing RNAs with organomercury gels.

Polyacrylamide gel electrophoresis is a widely used technique for RNA analysis and purification. The polyacrylamide matrix is highly versatile for chemical derivitization, enabling facile exploitation of thio-mercury chemistry without the need of tedious manipulations and/or expensive coupling reagents, which often give low yields and side products. Here, we describe the use of [(N-acryloylamino)phenyl]mercuric chloride in three-layered polyacrylamide gels to detect, separate, quantify, and analyze sulfur-containing RNAs.

Physical and chemical properties and stability of sodium cefazolin in buffered eye drops determined with HPLC method.

The aim of the studies was to analyze the stability of 1% and 5% eye drops containing sodium cefazolin, prepared in citrate buffer of pH 6.11-6.27, which were stored at the temperature of 4 degrees C and 20 degrees C with light protection. The drops were prepared under aseptic conditions by dissolving sodium cefazolin (Biofazolin, IBA Bioton), dry injection form of the drug, in citrate buffer. The viscosity of the drops was increased using polyvinyl alcohol. The drops were preserved with phenylmercuric borate of 0.001% concentration mixed with beta-phenylethyl alcohol of 0.4% concentration in the drops. The concentration of cefazolin was determined at every three days using HPLC method. Besides, the measurements of pH, osmotic pressure and viscosity were performed as well as the organoleptic analysis of the drops (clarity, color, odor). The concentration of cefazolin in 1% drops after the 30-day-storage at the temperature of 4 degrees C, depending on their composition, decreased in the range of 2.17-6.02%. In 5% drops the decrease in cefazolin concentration was similar, i.e., after 30-day-storage at the temperature of 4 degrees C it was 1.62-6.76%. In 1% and 5% drops stored at the temperature of 20 degrees C the stability of the drops determined as the 10% degradation time of cefazolin was in the range of 9-15 days.

Ultrasound gel causes fine needle aspiration artifact? A clear choice.

OBJECTIVES: Ultrasound-guided fine needle aspiration (FNA) is a commonly employed tool in cytopathologic practice. Artifacts resulting in misinterpretation of specimens have been noted with various ultrasound gel media. Our purpose was to perform a prospective human cadaveric study of this phenomenon to identify a low-cost solution that eliminates the artifact. STUDY DESIGN: Three separate ultrasound-guided FNAs were performed on the thyroid and parotid glands in situ of a fresh human cadaver using three different types of ultrasound gel media. Slides were prepared in standard fashion (Quik-Diff and Papanicolaou stains). Two cytopathologists subsequently analyzed the slides for the presence of any artifact interfering with their ability to visualize and interpret the cellular aspirate material. RESULTS: Two of the three gel media revealed significant artifacts mimicking apoptosis, necrosis or colloid, making it difficult to visualize the cellular components and differentiate the artifact from the thyroid colloid. One gel medium did not show any significant artifact, and there was no discernable difference in its quality with regard to the ultrasound image during FNA procedures. CONCLUSIONS: Ultrasound gels can be associated with a significant artifact in FNA specimens. To eliminate this artifact, which may alter the adequacy, diagnosis or cytologic appearance, we confirm a specific gel type that is useful for ultrasound-guided FNAs.

A simple synthesis of APM ([p-(N-acrylamino)-phenyl]mercuric chloride), a useful tool for the analysis of thiolated biomolecules.

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.

Stability of cefuroxime in 1% and 5% buffered eye drops determined with HPLC method.

The aim of the studies was to develop formulary technologies of 1% and 5% eye drops containing cefuroxime with stability of at least 10-12 days. The stability was defined as the time required to reach the cutoff value of 10% degradation of cefuroxime in the drops, as determined using an HPLC assay. The drops should have such properties as optical clarity, pH in the range of 3.5 to 8.5 and osmotic pressure not lower than 280 mOsm/L. Additionally, drops of enhanced viscosity within the range 7-9 mPaxs were developed. Drops (1% and 5%) were prepared under aseptic conditions by dissolving Biofuroksym (Cefuroxime natricum) IBA Bioton--the form of the drug for dry injections--in citrate buffer of pH 6.05-6.28. Polyvinyl alcohol was used to increase the viscosity of the drops. Phenylmercuric borate at the final concentration of 0.001% was used together with beta-phenylethyl alcohol at the final concentration of 0.4% to preserve the drops. The drops were stored for 30 days in tightly closed glass bottles at the temperature of 4 degrees C and 20 degrees C, protected from light. As the course of the infection may differ in intensity, location and the area of the infection in the eye, the composition of the drops was developed at two concentrations (1% and 5%), and five formulary versions for each concentration were prepared. The concentration of cefuroxime in the drops was determined every three days using HPLC. Such properties as pH, osmotic pressure and viscosity were also examined. Additionally, organoleptic analysis (clarity, color, odor) was performed. Physical and chemical properties of all formulations of 1% and 5% drops containing cefuroxime prepared in citrate buffer of pH 6.05-6.28 met the standards set in the objective of the work. The stability of cefuroxime in buffered drops stored at the temperature of 4 degrees C, determined with HPLC as the time of 10% degradation of cefuroxime, was 15 days for 1% and 5% drops. In the drops, which were buffered and of increased viscosity, the times of 10% cefuroxime degradation were 18 days for 1% drops and 30 days for 5% drops. The preservatives added to the buffered drops did not lower their stability. Osmotic pressure, pH and viscosity of the drops during the period of 30-day-storage at the temperature of 4 degrees C met the requirements acceptable for the eye drops. The stability of 1% and 5% buffered drops containing cefuroxime stored at the temperature of 4 degrees C allows preparing the drops in pharmacies on the basis of doctor's prescription. Depending on the character and the course of the infection the drops can be prepared at the concentration of 1% and 5% following the composition of the selected formulation which would meet the individual needs of the patient's therapy.

Stability of ceftazidime in 1% and 5% buffered eye drops determined with HPLC method.

The aim of the studies was to determine with HPLC method the stability of ceftazidime in buffered 1% and 5% eye drops of proposed formulary composition, which were stored for 30 days at the temperature of 4 degrees C and 20 degrees C and protected from light. The 1% and 5% eye drops were prepared under aseptic conditions by dissolving Biotum (ceftazidimum), dry injection formulation, in citrate buffer of pH 6.10-6.24. The viscosity of the eye drops was increased with polyvinyl alcohol, phenylmercuric borate combined with 2-phenylethanol was used to preserve the eye drops. The eye drops were stored for 30 days in sterile glass bottles at the temperature of 4 degrees C and 20 degrees C and protected from light. The concentration of ceftazidime and pyridine was analyzed simultaneously with HPLC method every three days; pH, osmotic pressure and viscosity were examined as well as the organoleptic analysis of the eye drops (clarity, color, odor). Storage temperature had the biggest impact on ceftazidime stability in the eye drops. The stability of the drops depended also on ceftazidime concentration in the eye drops, the presence of preservatives and polyvinyl alcohol. The time of 10% ceftazidime degradation in buffered 1% and 5% eye drops, stored at the temperature of 4 degrees C, was from 27 to 18 days in 1% eye drops and from 21 to 12 days in 5% eye drops, depending on their composition. In the eye drops which were stored at the temperature of 20 degrees C 10% ceftazidime degradation occurred on the 3rd day of storage in all 1% and 5% formulary versions.

Technology of eye drops containing metronidazole.

The aim of the studies was to determine the stability of metronidazole using UV spectrophotometric method in 0.5% w/w eye drops which were prepared under aseptic conditions and thermally sterilized. 0.9% solution of NaCl, 5% glucose and phosphate buffers of pH 6.97 and 6.81 were used as the solvents of metronidazole in the drops. Thiomersal and phenylmercuric borate were used to preserve the drops. The viscosity of the eye drops was increased using the solution of polyvinyl alcohol. The drops were stored in tightly closed glass infusion bottles, protected from light. For the stability of analysis a long-term assay was used under controlled conditions following the requirements of ICH, i.e., the time of storage was 24 months at the constant temperature of 25 +/- 2 degrees C and constant humidity of 60% +/- 2% RH. The eye drops containing metronidazole were significantly physically and chemically stable: after 24 months of storage the metronidazole concentration in the drops was close to 100% of the initial concentration. The drops were colorless and transparent. Physical and chemical properties such as pH, osmotic pressure and viscosity underwent insignificant changes during the storage. The preservation test showed that the degree of reduction of the pharmacopeal strains of micro-organisms in freshly prepared drops and in those stored for 24 months at the temperature of 25 +/- 20 degrees C was in agreement with the requirements of Ph. Eur. 6.

Asymmetric solid-phase alkylation of ketones immobilized via SAMP hydrazone analogue linkers.

The preparation and application of new solid supports with chiral linkers, analogues of SAMP hydrazine on solid-phase, are described. The supports were used for immobilization of ketones (diethylketone, cyclohexanone, 4- tert-butylcyclohexanone), and diastereoselective alkylation of formed chiral ketone hydrazones. The enantiomeric purities of cleaved alpha-alkylated chiral ketones ranged from 10 to 73%. The use of chiral lithium amides for metalation of hydrazones of t-butylcyclohexanone increased the enantiomeric excess of the alkylated product by 25-47%.

Syntheses, crystal and molecular structures, and properties of some new phenylmercury(II) dithiolate complexes.

A series of new phenylmercury(II) dithio complexes [PhHg(Bun2dtc)] (; Bun2dtc-=di-n-butyldithiocarbamate), [PhHg(morphdtc)] (; morphdtc-=morpholinedithiocarbamate), [PhHg(Bz2dtc)] (; Bz2dtc-=dibenzyldithiocarbamate), [PhHg(methoxethxant)] (; methoxethxant-=2-methoxyethylxanthate) [(PhHg)2NED] (; NED2-=1-nitroethylene-2,2-dithiolate) and [(PhHg)2CDC] (; CDC2-=cyanodithioimidocarbonate) have been prepared and characterized by elemental analysis, UV-Vis, IR, 1H and 13C NMR spectra and mass spectrometry. The crystal structures of , and showed a linear Hg(II) core at the center of the molecules. The weak intra- and intermolecular HgS interactions provide a molecular chain framework. The reaction of PhHgO2CCH3 with Bun2dtcH gave the known dimeric complex Hg(Bun2dtc)2 while the Ni(O2CCH3)2 mediated reaction gave instead of the expected heterobimetallic complex [PhHgNi(Bun2CS2)2]O2CCH3 which has been corroborated by natural charges at each atom obtained at the density functional level (DFT) of theory. Upon excitation at 358 nm exhibited a medium strong photoluminescence emission at 420 nm as a consequence of intraligand pi-->pi* transitions. The electronic absorption bands of were assigned from time dependent density functional theory (TD-DFT) calculations. Geometrical configurations of , and have been optimized using the DFT method. All of the complexes are weakly conducting (sigmart approximately 10(-12) S cm(-1)). However and exhibited semiconductivity with band gaps of 0.39 and 0.94 eV respectively.

Treatment of phenylmercury salts by heterogeneous photocatalysis over TiO(2).

UV/TiO(2) photocatalysis of phenylmercury salts in aqueous solutions has been performed starting from both acetate (C(6)H(5)HgCH(3)CO(2), PMA) and chloride (C(6)H(5)HgCl, PMC) salts, in the presence or the absence of oxygen at acidic pH. Removal of Hg(II) in solution took place with the simultaneous deposit of dark or pale gray solids on the photocatalyst, identified as metallic Hg (when starting from PMA) or mixtures of Hg(0) and Hg(2)Cl(2) (when starting from PMC). Partial mineralization of the organic part of both compounds has also been achieved. Hg(II) removal and mineralization were enhanced in the absence of oxygen. PMA photocatalysis followed a saturation kinetics, going from first order at low concentration to zero order at higher concentrations (>0.5mM). For PMA, reaction was faster at high pH (11) with formation of mixtures of Hg and HgO. Phenol was detected as a product of the reaction in both cases, PMA and PMC, and no formation of dangerous methyl- or ethylmercury species was observed in the first case. A mechanism for the photocatalytic reaction has been proposed. The fact that calomel was found as a deposit when starting from PMC under nitrogen suggests that the mechanism of Hg(II) transformation proceeds through successive one-electron transfer reactions passing by mercurous forms.

Discovery of potent inhibitors of pseudomonal quorum sensing via pharmacophore modeling and in silico screening.

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.

ADAMTS-4 (aggrecanase-1): N-terminal activation mechanisms.

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.

Localization of chromophore absorption signals in TEM with an improved prism-mirror-prism filter.

A corrected prism-mirror-prism electron energy filter with curved entrance and exit faces was designed and incorporated into a Zeiss EM902 transmission electron microscope. The installation of the new filter required little modification to the existing microscope geometry and electronics. The filter had an energy resolution of 1.1 eV over the full image plane (acceptance half angle 10 mradian). The improved energy resolution was applied in studies of the low electron energy loss region that includes molecular orbital excitations or chromophore energy absorptions. Chromophore signal behaviour under electron irradiation was characterized using embedded crystals of hematin and of the dye mercury orange. Images of these crystals confirmed the expected decrease in signal intensity on shifting the selected energy loss from the region of molecular orbital excitations (less than approximately 5 eV) to higher energy losses. Electron irradiation-induced fading of the chromophore signal from hematin and mercury orange yielded similar 1/e dose values of 1.1 x 10(5) e(-) nm(-2) and 1.4 x 10(5) e(-) nm(-2) respectively. In a cellular context, chromophore signals were obtained from energy-filtered images of RIF-1 cell sections containing the photosensitizer chlorin e6 and from sections of BS-C-1 cells with cytoskeletal labelling via FITC-conjugated antibodies. The respective signals were extracted using a dose-dependent method or a shift in selected energy. Chromophore signal distributions were in agreement with fluorescence light microscopic images, but provided detail at higher spatial resolution.

Expression, purification, and characterization of blasticidin S deaminase (BSD) from Aspergillus terreus: the role of catalytic zinc in enzyme structure.

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.

The decomposition of phenylmercuric nitrate in sulphacetamide drops during heat sterilization.

Analytical methods have been developed which enable phenylmercuric nitrate, mercuric ion and diphenylmercury to be measured in sulphacetamide drops. Application of these methods demonstrate that during heat sterilization of both 10 and 30% sulphacetamide drops containing phenylmercuric nitrate, together with disodium edetate and sodium metabisulphite, there is a 20-30% overall loss of phenylmercuric nitrate. This loss occurs mainly due to the phenylmercuric ion establishing an equilibrium with equimolar amounts of mercuric ion and diphenylmercury.

Chemical modification and mutagenesis studies on zinc binding of aminoacyl-tRNA synthetases.

Thermus thermophilus methionyl-tRNA synthetase consists of two identical subunits with a potential Zn(2+)-binding sequence of Cys-X2-Cys-X13-Cys-X2-His (Nureki, O., Muramatsu, T., Suzuki, K., Kohda, D., Matsuzawa, H., Ohta, T. Miyazawa, T., and Yokoyama, S. (1991) J. Biol. Chem. 266, 3268-3277). Upon chemical modification of the 3 Cys residues of T. thermophilus MetRS with sodium p-(hydroxymercuri)phenylsulfonate, one Zn2+ ion was released from one subunit of the molecule, as monitored with 4-(2-pyridylazo)resorcinol. Site-directed mutagenesis of Cys and His residues in the Zn(2+)-binding sequence reduced the aminoacylation activity; the kcat value was markedly decreased, and the Km values for L-methionine and tRNAf(Met) were increased. Similarly, Cys modification released two Zn2+ ions from T. thermophilus and Escherichia coli isoleucyl-tRNA synthetases and E. coli threonyl-tRNA synthetase, which have Zn(2+)-binding motifs, and impaired their activities. By contrast, three other aminoacyl-tRNA synthetases that lack Zn(2+)-binding motif neither released Zn2+ ion nor lost their activities upon Cys modification. These results indicate that the Zn(2+)-binding sequences are important for catalysis and recognition in the aminoacylation reactions of a subgroup of aminoacyl-tRNA synthetases.