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Characterization of the DNA polymerases induced by a group of herpes simplex virus type I variants selected for growth in the presence of phosphonoformic acid.

Five independently derived variants of a herpes simplex virus type I (HSV-1) strain were plaque purified from a virus population passaged in 1 mM phosphonoformic acid (PFA). The DNA polymerase induced by the parent and PFA-resistant viruses were purified and characterized. No differences were observed among the enzymes with respect to their chromatographic properties, specific activities, or polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The variant enzymes exhibited levels of PFA resistance which ranged from 15- to 25-fold. Resistance to PFA was always associated with a similar degree of resistance to its congener phosphonoacetic acid, but cross-resistance to beta-phenylphosphonoacetic acid was only seen with two of the five variant enzymes. PFA and pyrophosphate were mutually competitive in PPi exchange reactions, but in DNA synthetic reactions the levels of resistance to PFA and PPi were not equal. The apparent affinities of the enzymes for Mg2+ did parallel their affinities for PFA. Km values of dNTPs were about 2-fold higher than the parent virus enzyme for all of the variant enzymes except one which was 4-fold higher. The processivity of polymerization was apparently unaffected by the enzyme changes related to PFA resistance although one variant enzyme had a lower value. Resistance among the variant enzymes to the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine was directly related to the level of resistance to PFA. The data presented here indicated that (i) PFA resistance may result from several types of active site alterations, since the PFA-resistant enzymes were of three kinetically distinct types. Also, additional enzyme alterations, probably unrelated to PFA resistance, were detected in one enzyme. (ii) PFA and PPi possess some different binding determinants within the active center of herpes simplex virus type I DNA polymerase. (iii) PFA and the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine may have a common ultimate inhibitory mechanism.

Antiviral Agents↗

Pharmacological studies with almagate, a potent new antacid compound.

Pharmacological screening tests have been done in order to provide an initial assessment of the new antacid compound almagate (aluminium-magnesium hydroxycarbonate hydrate, Al2Mg6(OH)14(CO3)2 X 4 H2O, Almax). In rats with pyloric ligatures, almagate (125-500 mg/kg) was significantly more potent than aluminium hydroxide in raising the pH and reducing the total acidity of the gastric juice produced, without affecting the volume secreted. Pepsin activity in the gastric juice was also significantly inhibited by almagate even after adjustment to the optimal enzyme pH 2, a phenomenon not demonstrable with aluminium hydroxide. Almagate in oral doses up to 3 g/kg was without effects on the central, autonomic and somatic nervous systems in mice, nor at 500 mg/kg did it influence the cardiovascular system or blood pressure responses to agonist drugs in anaesthetised cats. The results confirm almagate to be a potent antacid drug devoid of systemic pharmacological or toxicological effects.

Aluminum Hydroxide↗

Influence of hydrocortisone on the binding of nitrosoureas to nuclear chromatin subfractions.

The effects of steroid-induced modifications of chromatin structure on the extent and sites of chloroethylnitrosourea binding to chromatin were studied using log-phase HeLa cells. The cells were exposed to 0.1 to 2.0 microM hydrocortisone for 22 hr; this resulted in depressed DNA synthesis while transcriptional activity was stimulated. Hydrocortisone had no effect upon cellular or nuclear uptake of the two nitrosoureas under study, 0.6 mM chlorozotocin or 1-(2-chloroethyl-3-cyclohexyl-1-nitrosourea). Both drugs were found to alkylate transcriptional chromatin preferentially, as demonstrated by DNase II and DNase I digestion. This alkylation was stimulated 2-fold by the same micromolar concentrations of hydrocortisone, 0.1 to 2.0 microM, which stimulated transcription. The extent of nuclear RNA alkylation, determined using RNase T2 as a probe, was found to contribute less than 20% of total chromatin alkylation and was unaffected by steroid pretreatment. Instead, the increased alkylation within these chromatin subfractions was attributed to a steroid-induced alteration of chromatin structure. Electron microscopic examination of HeLa nuclear morphology revealed a hydrocortisone-induced disaggregation of nuclear membrane-associated heterochromatin resulting in a more heterogeneous, less condensed distribution of chromatin. Such data are consistent with a relaxation of the supercoiled chromatin structure, resulting in increased transcription and increased accessibility of potential target sites for nitrosourea alkylation.

Alkylation↗

Mg deficiency induces mineral content changes and oxidative stress in rats.

We have studied the effect of Mg deficiency on both tissue mineral content and liver oxidative status. Male Wistar rats kept for eight weeks on a Mg-deficient diet (Mg 152ppm) quickly developed a severe plasma Mg deficiency (73% decrease). The content of Cu and Fe significantly increased in different tissues following the treatment. Liver glutathione, CuZn-superoxide dismutase, and vitamin E were significantly reduced (by about 16%, 18% and 30% respectively). Lipid peroxidation, induced in vitro by NADPH/ADP-Fe3+ and measured as MDA formation, increased by about 100% after 20 min incubation in liver microsomes isolated from Mg-deprived rats. The alterations found in the content of transition metals and in the level of both cytosolic and membrane antioxidants, as well as the higher sensitivity of liver microsomes to lipid peroxidation in vitro, are consistent with an oxidative stress occurring in vivo in the tissues of Mg-deficient animals.

Adenosine Diphosphate↗

Enzymatic inhibition of diphosphonate: a proposed mechanism of tissue uptake.

Enzymes have been proposed as tissue receptors that bine 99mTc-stannous diphosphonate and its analogs. Incubation of diphosphonate with several enzymes demonstrated inhibition of acid and alkaline phosphatase activity but showed no effect on glutamic oxalacetic transaminase and lactate dehydrogenase activity. Complete reversal of the diphosphonate-induced inhibition of alkaline phosphatase activity occurred when calcium ion was added to the reaction. The specificity of calcium to induce reversal was dispelled when magnesium ion gave identical results. Diphosphonate-induced inhibition of acid phosphatase, however, was not reversed by calcium or magnesium.

Acid Phosphatase↗

Serum-free culture of Pasteurella haemolytica optimized for leukotoxin production.

OBJECTIVE: To screen supernatants of Pasteurella haemolytica cultures grown in 4 serum-free culture media for maximal leukotoxin (LKT) production and minimal protein concentration as an optimal source of LKT for purification. SAMPLE POPULATION: One strain of P haemolytica biotype A serotype 1 originally isolated from the pneumonic lung of a calf. PROCEDURE: Pasteurella haemolytica was grown in brain-heart infusion (BHI) broth, yeast-tryptone broth, RPMI-1640 medium, and McCoy's modified 5A medium. Culture biomass and protein concentration, LKT activity, and LKT concentration in culture supernatants were measured. Effects of media pH and supplementation with metal cations and glucose on growth rate of P haemolytica and culture supernatant parameters were evaluated. RESULTS: Pasteurella haemolytica cultivated in BHI broth or RPMI-1640 medium containing 0.1 M phosphate (pH 6.8) produced the highest concentrations of LKT. Supplementation of RPMI-1640 medium with 0.36 mM FeCl3 or 1.0 mM MgSO4 further increased specific activity of LKT in culture supernatant, but addition of 1% glucose did not enhance LKT production. Leukotoxin production in MgSO4-supplemented RPMI-1640 medium was comparable to that in serum protein-supplemented medium. CONCLUSIONS: Although BHI broth was superior to RPMI-1640 medium for P haemolytica growth and LKT production, the higher protein concentration and lower LKT specific activity made BHI broth a less desirable medium, compared with RPMI-1640 medium. Growth rate and LKT production with minimal protein content was optimal in pH 6.8 phosphate-buffered MgSO4-supplemented RPMI-1640 medium. This medium can serve as a source of culture supernatant for purification of LKT.

Animals↗

[The induction and the prevention of Vi antigen of S.typhi loss by three metalic ions of salts].

Three metalic salts Ferrous, Mgnesium and Calcium were used to induce and to prevent Vi antigen of S. typhi loss. All of the 96 Vi-II phage typing standard strains belonged to V type (a recovery rate of 100%). Out of 1320 local strains, 1292 were type V (a recovery rate of 97.7%) and 28 remained type W with a Vi loss rate of 2.1%. The results showed that induction and prevention of Vi antigen of S. typhi loss by the three metalic salts was higher than that of other reports which showed a loss rate of 5% Vi antigen of S. typhi.

Animals↗

Hypermagnesemia. A potential complication during treatment of theophylline intoxication with oral activated charcoal and magnesium-containing cathartics.

Toxic reaction to theophylline compounds is common. Oral activated charcoal (OAC) is a widely accepted mode of therapy for management of moderate to severe cases of theophylline toxicity. Magnesium-containing cathartics are generally recommended in conjunction with OAC in the treatment of drug or toxin ingestions. We report two cases of hypermagnesemia complicating the treatment of theophylline toxicity with OAC and magnesium citrate. In both patients, the hypermagnesemia contributed significantly to morbidity or mortality. In light of these cases and after review of the literature, we suggest that sorbitol be considered the cathartic agent of choice in the treatment of theophylline toxicity with OAC.

Aged↗

Different Mg to Fe ratios in the mixed metal MgFe hydroxy-carbonate compounds and the effect on phosphate binding compared with established phosphate binders.

Due to the side effects of the current oral phosphate binders, there is a need for effective alternatives. A number of mixed metal hydroxy-carbonate compounds (MMHCs) based on Mg and Fe have recently been established as effective phosphate binders. We have now carried out further studies on the MMHCs with different ratios of Mg(2+):Fe(3+) in different forms to assess for phosphate binding efficacy and ionic release in aqueous solution and food slurries. The compounds that provide the most promise are those with Mg(2+):Fe(3+) ratios of 2:1 and 4:1 in the unaged/dry form. Their phosphate binding efficacy was compared with a wide range of established phosphate binders, such as aluminum hydroxide [Al(OH)(3)], calcium carbonate (CaCO(3)), calcium acetate (CaAc(2)), magnesium hydroxide [Mg(OH)(2)], and lanthanum carbonate [La(2)(CO(3))(3)] in various food slurries. The results showed that the MgFe compounds were much more effective (on a weight for weight basis) than the established binders, and their properties were relatively pH independent. Calcium compounds (CaCO(3) and CaAc(2)) were ineffective under the experimental conditions. Mg(OH)(2) was effective at low pH but not at pHs greater than 5.0, and also released two- to threefold more magnesium than the MgFe compounds. Al(OH)(3) showed some degree of efficacy, but the binding capacity was, at best, less than 50% of the MMHCs. La(2)(CO(3))(3) required at least a 10-fold increase in weight to give comparable binding to the MMHCs. In conclusion, MgFe hydroxy-carbonate compounds are effective phosphate binders and may provide a better alternative to both existing and emerging binders for combating hyperphosphataemia.

Binding Sites↗

The development and in-vitro evaluation of novel mixed metal hydroxy-carbonate compounds as phosphate binders.

The currently available phosphate binders are relatively inefficient and suffer from clinical side-effects of increased absorption of calcium and aluminium and the diarrhoea-inducing effects of magnesium. A new class of compounds based on mixed metal hydroxides has been developed and evaluated for their potential as phosphate binders. The mixed metal hydroxides were prepared using a standard procedure for hydrotalcite (Al2Mg6(OH)16.CO3.4H2O) by substituting Fe3+ for Al3+, with Mg2+ or Ca2+ as the divalent metal ion. Phosphate precipitation (binding) was examined at different pH values in aqueous solution and in various food mixtures in comparison with hydrotalcite, Al(OH)3, CaCO3 and Mg(OH)2 on the same weight-to-weight basis. A series of compounds with differing ratios of metal ions (Fe:Mg/Ca 1:2 or 1:3) gave analytically similar ratios to those predicted from the initial amounts added. CTFeCa bound > 90% phosphate in aqueous solution compared with 65% binding with CTFeMg, 85% binding with Mg(OH)2, and less than 30% binding for CaCO3 and Al(OH)3. The mixed metal compounds also bound up to 80% phosphate in various food matrices, which was relatively independent of changes in pH, compared with Mg(OH)2, where binding decreased from 85% at pH 3.0 to 25% at pH 8.0. Al(OH)3 and CaCO3 were relatively ineffective phosphate binders under all the conditions tested. The mixed metal hydroxides compounds show considerable promise as phosphate binders over those currently available and warrant further patient-based in-vivo testing.

Aluminum↗