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PubMed1994

Thiotungstate-copper interactions. I. Studies on the metabolism of [185W] tetrathiotungstate and the systemic interactions of labeled pharmacological doses with copper in rats.

[185W] tetrathiotungstate was employed to study the metabolism of thiocompounds in rats after i.v. injection. At tracer levels (12.5 micrograms W) the most important plasma binding protein eluted in the position of ceruloplasmin but the association did not prevent uptake of thiotungstate by the liver. At higher dose levels (1.5 mg W) there was considerable hydrolysis immediately after injection with rapid excretion of label in urine. The [185W] tetrathiotungstate remaining in plasma was associated with albumin and the amount retained was increased by pretreatment of the rats with copper. The increased binding to albumin did not prevent hepatic uptake and over the short-term pretreatment with copper increased the movement of the isotope into subcellular organelles, probably lysosomes. The excretion in bile was increased and the label was associated with high molecular weight proteins. In liver cytosol the 185W was bound by specific, as yet uncharacterized, proteins. At the higher dose levels there was some movement to higher molecular weight proteins and this was greatly increased by the pretreatment with copper. The studies show that the metabolism of 185W tetrathiotungstate is sufficiently similar to 99Mo or 35S tetrathiomolybdate for work on the systemic interactions of thiocompounds and copper in man and animals.

Animals↗

PubMed1994

Thiotungstate-copper interactions II. The effects of tetrathiotungstate on systemic copper metabolism in normal and copper-treated rats.

The intraperitoneal administration of tetrathiotungstate to rats (6-17.4 mg W/Kg BW) caused profound changes in copper metabolism in both normal rats and in rats pretreated with copper. Plasma copper associated with albumin increased, liver copper, particularly cytosol copper, was depleted, and biliary excretion was increased. There was also a movement of copper to higher molecular weight proteins in both liver cytosol and bile. In contrast to penicillamine, tetrathiotungstate did not increase liver cytosolic apometallothionein levels and reduced the rise provoked by copper. Metallothionein-bound copper was removed. Ceruloplasmin oxidase activity was inhibited and there was evidence for increased movement of copper into subcellular organelles, probably lysosomes. It is concluded that tetrathiotungstate has a genuine "decoppering" effect and could be considered as an alternative to thiomolybdates in the treatment of copper storage diseases.

Animals↗

PubMed1996

Modification of Lowry's method for collagen concentration measurement.

A procedure for the determination of collagen concentration is described. It is simple and sensitive and is based on a modification of Lowry's method. The modification includes an initial heating of collagen samples in alkaline solution and changes in the concentrations of reagents used. It may be used for different collagen types, i.e. I, II, V and XI, from various sources as well as for gelatine. The influence of different buffers, salts, detergents, etc. on the precision of the method was studied. The precision of the measurements was not influenced by pH, the turbidity of the sample and the presence of certain salts, osmolytes and preservatives, routinely used in biochemical, microbiological and immunological experimental protocols. The modification is suitable for routine determinations of collagen concentration in a wide variety of samples.

Animals↗

PubMed1992

Electron microscopical demonstration of horseradish peroxidase by use of tetramethylbenzidine as chromogen and sodium tungstate as stabilizer (TMB-ST method): a tracing method with high sensitivity and well preserved ultrastructural tissue.

Until now methods using tetramethylbenzidine (TMB) for electron microscopy (TMB-EM methods) are all unable to provide a maximum demonstration of transported horseradish peroxidase (HRP) while maintaining good ultrastructural tissue preservation. In order to solve this problem, we have attempted to adapt a newly developed, highly sensitive TMB method using sodium tungstate (ST) as the stabilizer (TMB-ST method) for HRP electron microscopic retrograde and anterograde fiber tracing. The present study shows that the TMB-ST method combined with diaminobenzidine-cobalt (DAB-Co) is more sensitive than existing TMB-EM methods and that ultrastructural details are well preserved with this combined method. The resultant reaction product complex after osmication is stable and is observed as characteristic crystal-like structures which are extremely electron dense and often aggregated into clumps. In contrast, the TMB-ST method without the DAB-Co step frequently produces a moderate electron-dense reaction product. Therefore, we recommend the TMB-ST method combined with DAB-Co for HRP electron microscopy.

3,3'-Diaminobenzidine↗

PubMed1993

The tungstate-stabilized tetramethylbenzidine reaction for light and electron microscopic immunocytochemistry and for revealing biocytin-filled neurons.

A peroxidase reaction product that can be easily distinguished from standard diaminobenzidine (DAB) reaction products is needed for pre-embedding electron microscopic double-antibody labelling studies. Benzidine dihydrochloride (BDHC) and gold-substituted silver peroxidase reactions are unsatisfactory for double labelling because they lack sensitivity and reliability and/or compromise ultrastructure. We show here that light and electron microscopic immunocytochemistry can be done with a modification of the tungstate-stabilized tetramethylbenzidine (TMB) reaction (Weinberg and Van Eyck 1991) which yields a crystalline reaction product. With this method, we have obtained excellent immunolabelling for a variety of antigens, including tyrosine hydroxylase, enkephalin, serotonin, Fos protein and retrogradely transported cholera toxin B subunit (CTB). The TMB-tungstate reaction is useful for ultrastructural double labelling because the crystals contrast well with the amorphous product of diaminobenzidine reactions. The TMB-tungstate reaction is more sensitive and reliable for immunocytochemistry than the benzidine dihydrochloride reaction and gives better ultrastructure than the gold-substituted silver peroxidase reaction. We also show that neurons filled with biocytin by intracellular injection can be visualized with TMB-tungstate for either light (LM) or electron (EM) microscopy.

Animals↗

PubMed1991

Inhibition of proliferation of human immunodeficiency virus type 1 by novel heteropolyoxotungstates in vitro.

Fifteen heteropolyoxotungstates were tested for their effects on the proliferation of human immunodeficiency virus type 1 (HIV-1) using an in vitro system consisting of MT-4 cells and HTLV-IIIb. Eight heteropolyoxotungstates (HPOTs) with the Keggin structure or dimerized deficient Keggin structure proved to be potent inhibitors of HIV-1. In contrast, seven non-Keggin HPOTs including HPA 23 did not have significant effects on HIV-1 proliferation at non-toxic doses. [PTi2W10O40]7- (PM-19) was the most potent inhibitor of HIV-1 among the 15 HPOTs tested. The inhibition of HIV-1 replication by PM-19 presumably results from impaired virus adsorption and/or penetration into target cells. Viral spread of HIV-1 and HIV-2 on cell-to-cell basis was also susceptible to PM-19. In combination, PM-19 and 3'-azido-3'-deoxythymidine were synergistic in inhibiting HIV-1 proliferation.

Adsorption↗

PubMed1991

In vitro antiviral activity of polyoxotungstate (PM-19) and other polyoxometalates against herpes simplex virus.

Polyoxotungstates with Keggin-type structure were found to demonstrate marked antiherpetic activity. K7[TiW10PO40].6H2O (PM-19) caused a decrease in plaque formation by several strains of herpes simplex virus (HSV) type 1, including acyclovir-resistant (thymidine kinase-negative) strains, at concentrations which were not toxic to the host cells. The 50% plaque-inhibiting concentration (EC50) for the different strains was between 20 and 50 micrograms/ml. Single-cycle HSV growth was also inhibited by PM-19. PM-19 inhibited viral DNA synthesis in HSV-infected cells at a concentration of 5 micrograms/ml but did not exhibit a virucidal effect, and pretreatment of the host cells with PM-19 did not provide resistance to herpes infection. Yet, virus adsorption to the cells was markedly affected at PM-19 concentrations higher than 25 micrograms/ml. PM-19 was also effective against human cytomegalovirus, but not against adenoviruses and varicella-zoster virus, although it did delay the development of the cytopathic effect of these viruses.

Animals↗

PubMed1987

Modification of the structural and redox properties of cytochrome c by heteropolytungstate binding.

Complex formation between horse heart ferricytochrome c and large three-dimensional polyanions has been investigated, in order to study the influence of surface electrostatic interactions on the structural and redox properties of cytochrome c. Cytochrome c binds the large heteropolytungstates (NaSb9W21O86)18- and (KAs4W40O140)27- with a 1/1 polyanion/cytochrome c ratio, and the smaller ion (SiW11O39)8- with a 2/1 ratio. Upon complexation, cytochrome c undergoes structural changes that are dependent on the size and charge of the polyanion, and on the pH and ionic strength of the medium. Three different forms of complexed cytochrome c have been characterized by optical and EPR spectroscopies, in the pH range 6.5-8: an N form, close to the native structure, an A form, analogous to cytochrome c in acidic medium, and a novel B form in which the heme pocket is open but the iron remains low-spin. The redox potential of cytochrome c is lowered to 250-220 mV (vs. NHE) in the N form, and to 80 mV in the B form.

Animals↗

PubMed1988

The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies.

The inducible trimethylamine-N-oxide reductase which migrates on non-denaturing polyacrylamide gels with an RF of 0.22, has been purified from the soluble fraction of wild-type E. coli K12. The molecular weight of the purified enzyme estimated by molecular-sieve chromatography is about 230,000. It is composed of two subunits of molecular weight 110,000. Antiserum specific for the enzyme has been produced. Gel filtration on Sephadex G-200 of the soluble fraction gave two peaks of trimethylamine-N-oxide reductase, one with an Mr of 230,000 and an RF of 0.22, and another with an Mr of 120,000 and an RF of 0.36. Since the anti-trimethylamine-N-oxide reductase serum recognises the two forms and shows a single subunit with an Mr of 110,000, we conclude that in E. coli there is a single inducible trimethylamine-N-oxide reductase which can exist as a dimer or a monomer. Other immunological studies with anti-trimethylamine-N-oxide reductase serum on crude extracts prepared from cells grown in the absence of inducer showed that the constitutive trimethylamine-N-oxide reductase was not recognised by the antiserum. The same analyses carried out on a tor mutant (defective in the structural gene of the inducible enzyme) confirmed without ambiguity that the constitutive enzyme is immunologically distinct from the inducible enzyme. In the same way, using the anti-trimethylamine-N-oxide reductase serum, rocket immunoelectrophoresis analyses were able to show that the inducible apoenzyme is not regulated by the fnr gene product and that molybdate does not seem necessary for the synthesis or stabilisation of this enzyme.

Aerobiosis↗

PubMed1991

Synthesis and kinetic study of transition state analogs for ribonuclease T1.

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.

Binding Sites↗

PubMed1992

Polytungstate binding to metmyoglobin: an access to various structural forms of the protein.

Complex formation between metMb and three heteropolytungstates, offering various sizes and charges, has been studied in the pH range 6-8. 1:1 complexes are formed with (KAs4W40O140)27- and (NaSb9W21O86)18-, with an association constant of 10(6) and 4 x 10(5) M-1 respectively, at pH 7.3 and 10 mM ionic strength. Resulting structural changes of the metMb moiety have been investigated by absorption, CD and EPR spectroscopies. Besides an acid-denatured-like form, obtained at a pH as high as 6.5 with the largest polyanion, the formation of an hemichrome is generally observed. It can be reduced to the hemochrome, whereas the complexation of deoxyMb by the polytungstates leaves the deoxy structure unaltered.

Antimony↗

PubMed1985

Vanadate, tungstate and molybdate activate rod outer segment phosphodiesterase in the dark.

Anionic activation of rod outer segment phosphodiesterase by vanadate, molybdate and tungstate is demonstrated. Comparisons are made to adenylate cyclase, which is known to be activated by vanadate and molybdate but not by tungstate. In view of the differences in anionic activation between these two important enzymatic regulators of intracellular cyclic nucleotide metabolism, it is possible that tungstate can be used as a selective probe for the effects of phosphodiesterase activity in photoreceptors and other cells. The known electrophysiological stimulation of Limulus photoreceptors by these anions is also interpreted in light of our results. If anionic production of quantum bumps in Limulus photoreceptors is mediated by changes in cyclic nucleotides, then the electrophysiological response of Limulus photoreceptors to tungstate may indicate a role for phosphodiesterase rather than adenylate cyclase in mediating light-induced cyclic nucleotide alterations in this cell.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1995

Contamination of titanium castings by aluminium oxide blasting.

OBJECTIVES: It is desirable that the surfaces of surgical implants be uncontaminated by foreign materials to avoid untoward tissue reactions, and grit blasting is widely assumed to leave clean metal surfaces. SEM examination and X-ray microanalysis of a recovered 'pure' titanium implant casting that was associated with tissue breakdown revealed embedded particles of alumina. The casting had been cleaned of investment by blasting with alumina grit. METHODS: A variety of treatments of cast titanium plates was used: (a) to establish that the observed aluminium was due to the blasting grit, and (b) to determine whether removal of investment could be achieved effectively by other means. SEM examination and X-ray microanalysis were used. RESULTS: The detected aluminium was associated with embedded fragments identified as coming from the blasting grit. Acid-pickling and mechanical (rotary instrument) trimming produced minimally contaminated surfaces. CONCLUSIONS: Whilst unproven, the presence of the alumina is viewed with great concern as a possible causative agent in the observed tissue breakdown and procedures avoiding alumina blasting are recommended as a precautionary measure.

Aluminum Oxide↗

PubMed1994

Involvement of electrostatic interactions in cytochrome c complex formations.

Structural studies on various electron transfer complexes involving the tetrahemic cytochrome c3 provided evidence that one of the hemes (heme 4) is the interacting site on the molecule. The reactivity of this particular heme is allocated to the positive charges found around the heme group which are strongly involved in the electrostatic interaction processes. Electrostatic and hydrophobic effects in complex formation are considered on the basis of two electron transfer complex examples: the soluble cytochrome c-cytochrome c peroxidase and the membrane bound photosynthetic reaction center.

Amino Acid Sequence↗

PubMed1982

pH-dependent effects of sodium tungstate on the steroid-binding properties of the hen oviduct progesterone receptor.

Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37 degrees C for 20 min, its ability to bind [3H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2-3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (greater than 5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [3H]progesterone-receptor complexes; 70-80% of which remained after a 20 min incubation at 37 degrees C in the presence of 2-3 mM tungstate at pH 7. At pH 8, presence of tungstate (1-10 mM) during the 37 degrees C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4 degrees C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.

Animals↗

PubMed1985

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12.

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants.

Bacterial Proteins↗

PubMed1995

Ultrastructure of calcitonin gene-related peptide-immunoreactive, unmyelinated afferents to the cat carotid body: a case of volume transmission.

To relate the ultrastructure of unmyelinated afferents to the cat carotid body with the known electrophysiological properties of cat chemosensory C-fibers, we took advantage of the fact that the calcitonin gene-related peptide is exclusively present in a population of sparsely branched afferents to the carotid body. They have a morphology identical to the afferents originating from carotid sinus nerve unmyelinated axons. Immunoreactive axons were stained using pre-embedding protocols and horseradish peroxidase-labeled secondary antibody. Labeling was present only in unmyelinated axons and boutons distributed in the interstitial and parenchymal tissue. The varicosities had an average diameter of 0.7 micron, and contained both small, clear vesicles and larger dense-core vesicles. No labeled axons were ever seen to contact glomus cells, but could be observed as close as 0.2 micron to a glomus cell, always with an interposed glial process. With a very sensitive protocol, that used tungstate-stabilized tetramethylbenzidine as the chromogen, amorphous deposits of reaction product were often detected in the extracellular space around a labeled bouton. We interpret these findings as indicating that the reciprocal chemical transmission between the oxygen-sensitive glomus cells and the unmyelinated afferents takes place through non-synaptic transmission, via the rather large extracellular space of the carotid body. In addition, the larger distances between glomus cells and unmyelinated afferents could explain the lowered sensitivity and sluggishness of chemosensory C-fibers, compared to the A-fibers.

Afferent Pathways↗

PubMed1994

Vanadate, molybdate and tungstate for orthomolecular medicine.

Recent studies indicate that oxyanions, such as vanadate (V) or vanadyl (IV), cause insulin-like effects on rats by stimulating the insulin receptor tyrosine kinase. Tungstate (VI) and molybdate (VI) show the same effects on rat adipocytes and hepatocytes. Results of uncontrolled trials on volunteers accumulated in Japan also suggest that tungstate effectively regulates diabetes mellitus without detectable side effects. Since these oxyanions naturally exist in organisms, oxyanion therapy, the oral administration of vanadate, vanadyl, molybdate, or tungstate, can be considered to be orthomolecular medicine. Therefore, these oxyanions may provide a viable alternative to chemotherapy. Many diseases in addition to diabetes mellitus might also be treated since the implication of these results is that tyrosine kinases are involved in a variety of diseases.

Adipocytes↗