Microdose Lithium Treatment Reduced Inflammatory Factors and Neurodegeneration in Organotypic Hippocampal Culture of Old SAMP-8 Mice.

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.

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.

Early biochemical effects of an organic mercury fungicide on infants: "dose makes the poison".

Phenylmercury absorbed through the skin from contaminated diapers affected urinary excretion in infants in Buenos Aires. The effects were reversible and quantitatively related to the concentration of urinary mercury. Excretion of gamma-glutamyl transpeptidase, an enzyme in the brush borders of renal tubular cells, increased in a dose-dependent manner when mercury excretion exceeded a "threshold" value. Urine volume also increased but at a higher threshold with respect to mercury. The results support the threshold concept of the systemic toxicity of metals. gamma-Glutamyl transpeptidase is a useful and sensitive marker for preclinical effects of toxic metals.

Permeability of rat and rabbit erythrocyte membranes for a series of amides.

Permeability coefficients of rat and rabbit erythrocyte membranes for a series of amides, as well as for erythrocytes treated with p-chloromercuribenzenesulfonic acid monosodium salt (pCMBS) have been determined at 25 and 37 degrees C. Directly proportional dependence of the pCMBS treated erythrocyte permeability for investigated substances and their partition coefficients between the hydrophobic phase and water as well as the values of activation energy of this process indicate that penetration of small hydrophilic molecules is realized by passive diffusion through the lipid bilayer. The results obtained indicate that penetration of small hydrophilic molecules of formamide through lipids is determined by the existence of a free space between hydrocarbon chains that arises from kink formation. The differences in permeability between rat and rabbit erythrocyte membranes could arise in particular as a result of the differences in lipid composition.

Phytodetoxification of hazardous organomercurials by genetically engineered plants.

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.

Influence of additives and storage temperature on physicochemical and microbiological properties of eye drops containing cefazolin.

The purpose of the studies was to choose additives for eye drops containing cefazolin and the assessment of the influence of used additives and the storage temperature on the physicochemical properties and the stability of the eye drops. The drops were 1% sterile solutions of cefazolin in citrate buffer of pH 6.15-6.20. The drops were preserved with 0.002% thiomersal or 0.001% phenylmercuric borate mixed with 0.4% beta-phenylethyl alcohol. Viscosity of the eye drops was increased using polyvinyl alcohol (PVAL). The pharmaceutical compatibility test of selected additives with cefazolin showed the pharmaceutical interaction of 1% solution of cefazolin with higher than 0.003% concentration of thiomersal, 0.005% benzalkonium chloride and 0.01% chlorhexidine diacetate. The drops, protected from light, were stored at the temperature of 4 degrees C and 20 degrees C. As the criteria of the qualitative assessment of freshly prepared drops and during their storage, the following properties were considered: organoleptic analysis, sterility, pH, osmotic pressure, density, viscosity, antimicrobial activity of cefazolin and preservation efficiency of thiomersal and phenylmercuric borate in the eye drops. The studies showed that the storage temperature did not influence the physicochemical properties of the drops or the antimicrobial activity of cefazolin in the drops, which was not influenced by the used additives either. After 30 days of storage at both temperatures, cefazolin in the eye drops retained 100% of its initial activity. Phenylmercuric borate, whose antimicrobial activity in the eye drops was compatible with the preservation assay cited in the Polish Pharmacopoeia (PPh V), can be used to preserve the drops containing cefazolin.

Creatine kinase is modified by 2-chloromercuri-4-nitrophenol at the active site thiols with complete inactivation.

Creatine kinase modified by mercurials has been reported to be fully reactive as the native enzyme. This was ascribed to the modification of a second class of thiol groups instead of the reactive thiols at the active site (Laue, M.C. and Quiocho, F.A. (1977) Biochemistry 16, 3838-3845). It has now been shown by spectrophotometric titration and fluorescence studies that 2-chloromercuri-4-nitrophenol (MNP) reacts preferentially with the active-site thiol. Moreover, if the activity of the modified enzyme is determined in the absence of added bovine serum albumin or other enzymes, as usually employed in coupled activity assay systems for creatine kinase, the modified enzyme is completely inactive. Addition of an excess of bovine serum albumin or rabbit muscle glyceraldehyde-3-phosphate dehydrogenase restores the activity of the enzyme to over 80% of its original level. It appears that the active thiol groups at the active site of creatine kinase are after all modified by MNP with complete inactivation.

Solubilization and denaturation of monomeric actin from erythrocyte membranes by p-mercuribenzenesulfonate.

Solutions of p-mercuribenzenesulfonate extract the peripheral proteins from the red cell membrane in a water-soluble form. Low concentrations of the reagent selectively solubilize actin, while at higher concentrations, spectrin, ankyrin and bands 4.2 and 4.1 are extracted. After brief exposure to the reagent, followed by displacement of the mercurial with dithiothreitol or 2-mercaptoethanol, the soluble actin is capable of inhibiting DNAse I activity. With prolonged exposure or with higher concentrations of the reagent, the ability to inhibit DNAse is gradually lost. The kinetics of both the release of actin capable of DNAse inhibition and the subsequent loss of that capability are pseudo-first-order with respect to time, but show second-order dependence on the concentration of mercurial. These data suggest that dissociation of the actin from protofilaments in the cytoskeleton requires exposure of more than one sulfhydryl group to the reagent. Subsequent inactivation also appears to be dependent on the reaction of further multiple sulfhydryl groups, possibly in buried regions of the actin molecule.

Specific interaction of the water transport inhibitor, pCMBS, with band 3 in red blood cell membranes.

The human red cell anion transport protein, band 3, contains six pCMBS (p-chloromercuribenzene sulfonate) reactive SH groups, five of which react with N-ethylmaleimide. We have carried out equilibrium binding experiments using N-ethylmaleimide-treated red cell ghosts and found that the sulfhydryl reactive water transport inhibitor, pCMBS, inhibits the binding to band 3 of the specific anion exchange inhibitor DBDS (4,4'-dibenzoamido-2,2'-disulfonic stilbene) in a non-competitive manner. Stopped-flow kinetic studies, in which DBDS is mixed with ghosts in the presence of pCMBS, show that pCMBS slows the DBDS induced conformational change in band 3. A non-competitive reaction scheme has been developed which incorporates the quantitative results of equilibrium and kinetic studies. The pCMBS effect on DBDS binding and kinetics is reversed with 5 mM cysteine suggesting a sulfhydryl bond is involved in pCMBS binding to band 3. These data suggest that pCMBS has a specific binding site on band 3, consistent with the hypothesis that band 3 mediates red cell water transport.

Distinct properties of uridine transport systems in growing, quiescent and serum-stimulated hamster embryo cells.

The kinetics of uridine uptake in growing, quiescent and serum-activated hamster embryo cells are investigated. The maximum velocity of uridine uptake in growing hamster embryo cells, is lower than in the methylcholanthrene transformed hamster cell line (MCT). This kinetic constant is further reduced in quiescent cells. The Km values in growing and in quiescent hamster embryo cells, as well as in MCT cells are of the same magnitude. Distinct alterations in the pattern of inhibition by nitrobenzyl 6-mercaptoinosin (NBMI) are detected as growing hamster embryo cells become quiescent. In quiescent cells the maximum level of inhibition is lower and the apparent Ki value for the inhibition is much higher. These changes are due to the lower apparent K'm values of NBMI-bound carriers and to the slower rate of formation of the carrier-inhibitor complex. The changes in the kinetic properties of the carriers are partly reversed by serum-activation. The number of inhibitor binding sites (i.e. nucleoside carriers) does not increase by serum-stimulation of quiescent cells (0.36 and 0.34-10(5) sites/cell in quiescent and serum-stimulated cells, respectively). It is implied that the reduction in uridine transport in quiescent cells is probably due to changes in the turnover of the carriers. These changes may be connected with the observed alterations in the properties of carriers or their immediate environment in quiescent cells.

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate.

The disruption of erythrocyte membrane cytoskeletons brought about by treatment with p-mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of 203Hg-labelled PMBS. After pretreatment with N-ethylmaleimide to block readily reactive sulphydryl groups, incubation with [203Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal pKa value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.

Sulfhydryl groups of rabbit liver arylsulfatase A.

Rabbit liver arylsulfatase A (aryl-sulfate sulfhydrolase, EC 3.1.6.1) monomers of 130 kDa contain two free sulfhydryl groups as determined by spectrophotometric titration using 5,5'-dithiobis(2-nitrobenzoate) and by labeling with the fluorescent probe 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Fluorescence quenching data indicate that the reactive sulfhydryl is present in proximity to one or more tryptophan residues. Chemical modification of the sulfhydryl groups does not alter the distinctive pH-dependent aggregation property of the arylsulfatase A. The free sulfhydryls of the enzyme react with numerous sulfhydryl reagents. Based on the reactions of iodoacetic acid, methyl methanethiosulfonate, 5,5'-dithiobis(2-nitrobenzoate) and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid with the sulfhydryl groups of arylsulfatase A, it is concluded that free sulfhydryls are not essential for the enzyme activity. In contrast, the observed inactivation of the enzyme by p-hydroxymercuribenzoate or p-hydroxymercuriphenylsulfonate is probably due to a modification of a histidine residue, consistent with previous reports that histidine is near the active site of arylsulfatase A. p-Hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate are able to react both with cysteine and with histidine residues of the protein molecule.

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).

Active-site residues of procarboxypeptidase Y are accessible to chemical modification.

The accessibility of the active-site cleft of procarboxypeptidase Y from Saccharomyces cerevisiae has been studied by chemical modifications of two specific amino-acid residues. Previous studies have shown that these residues, Cys-341 and Met-398 in the mature enzyme, are located in the S1 and S'1 substrate binding sites, respectively, of carboxypeptidase Y. We have found that these residues also in proCPY are accessible to modification with fairly bulky reagents and in the case of Met-398 the rate of modification is even faster than in carboxypeptidase Y. While the catalytic serine in the mature enzyme reacts with diisopropylfluorophosphate, this is not the case for procarboxypeptidase Y.

Effects of glucose, chloromercuribenzene-p-sulphonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid on phosphate efflux from pancreatic islets.

Collagenase-isolated pancreatic islets of non-inbred ob/ob mice, containing more than 90% beta-cells, were labelled with radioactive orthophosphate (32P or 33P) and then subjected to non-recirculating perifusion. The basal D-glucose concentration in the perifusion medium was 2.8 mM. When the concentration was suddenly raised to 5.6, 8.3 or 16.7 mM, D-glucose promptly elicited a transient and dose-dependent release of radiophosphate. In the presence of 2.8 mM D-glucose, 0.1 mM of the poorly permeating sulphydryl blocker, chloromercuribenzene-p-sulphonic acid, also evoked a phosphate flush resembling the one induced by D-glucose. The basal radiophosphate release was partially inhibited by 1 mM 4-acetamido-4-'-isothiocyanostilbene-2,2'-disulphonic acid. However, the phosphate flush induced by 16.7 mM D-glucose was not noticeably inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. It is concluded that the phosphate flush emanates from beta-cells and that membrane sulphydryl groups may participate in its regulation. Although at least the basal phosphate release may in part represent transmembrane transport through 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid-sensitive anion channels, other mechanisms are also likely to participate in the glucose-induced phosphate flush.

Titratable effects of p-chloromercuriphenyl sulfonate, a thiol-attacking reagent, on glucocorticoid receptor binding.

Cytosol prepared from cultured AtT-20 mouse pituitary cells or mouse liver was treated with concentrations of p-chloromercuriphenyl sulfonate (PCMPS) which reduced but did not abolish receptor-binding activity. Scatchard analysis of triamcinolone acetonide binding to the treated cytosol showed that the PCMPS effect was caused by a reduction of binding affinity with little effect on the apparent binding site concentration. The effect on affinity was dose-dependent. Binding specificity appeared unaffected since the relative abilities of triamcinolone acetonide, dexamethasone, cortisol, progesterone, and corticosterone to compete with labeled triamcinolone were similar at various PCMPS concentrations which caused a progressive reduction of detectable cytosol binding. The PCMPS effect was reversible since cytosol treated with up to 200 microM PCMPS followed by dithiothreitol 15 min later showed nearly complete recovery of binding sites (62-100%). The possibility that several sulfhydryl groups were involved in this phenomenon was further explored in experiments using AtT-20 cytosol labeled with [3H]dexamethasone-mesylate, a glucocorticoid affinity label which binds covalently to sulfhydryl groups. Chromatography of dexamethasone-mesylate labeled receptor on a sulfhydryl affinity column resulted in binding, indicating that the receptor had at least two sulfhydryl groups, one bound to the mesylate moiety of the steroid and the other capable of binding to the affinity column.

Transport of methotrexate in L1210 cells. Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide.

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 microM CMPS or 175 microM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4 degrees C) after exposure to these inhibitors (at 37 degrees C). Methotrexate efflux was also inhibition by CMPS (50% at 4 microM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 microM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.

Effect of thiourea on PCMBS inhibition of osmotic water transport in human red cells.

The organomercurial reagent p-chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane. We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether. For a 2 mM PCMBS concentration Ki = 3 +/- 1 mM. When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time. Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when [thiourea] = 50 mM. Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.

Stimulation of Na+ transport across the toad urinary bladder by p-chloromercuribenzene sulfonate.

The sulfhydryl reagent p-chloromercuribenzene sulfonate increased the ISC across substrate-replete toad urinary bladder when applied to the mucosal (apical) surface. This increase was accounted for by an increased mucosal to serosal net flux of Na+. In the absence of substrate, the rise in ISC was accompanied by an irreversible increase in tissue conductance which was not apparent in the replete preparation. These findings suggest that p-chloromercuribenzene sulfonate may be useful in marking mucosal functions associated with the Na+ transport apparatus.

The action of organic mercurials on the erythrocyte membrane.

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although N-ethylmaleimide and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.