Dissolution test for ivermectin in oral veterinary paste.

A dissolution test for oral veterinary pastes with ivermectin using the Ph. Eur. paddle apparatus was developed. Sink conditions were achieved with sodium lauryl sulphate in a concentration of 0.5% as dissolution medium. By means of HPLC fast degradation of ivermectin was observed in HCl 0.1 M solution. Rotation speed of the paddle at 75 rpm was appropriate as demonstrated in a study comparing two different products.

The choice of lipids and surfactants for injectable extravenous microspheres.

Suspensions of lipid microspheres sizing from 1 to 30 microm, whose fluidity and lipid/surfactant composition is suitable for parenteral administration were developed. None of the formulations prepared with Precirol (palmitostearate), as the only lipid, was physically stable during storage, because liquid suspensions formed semisolid gels within one week. Stable 10% (w/w) suspensions of lipid microspheres were produced using saturated triglycerides in combination with medium chain unsaturated triglycerides (Miglyol) as lipids and polysorbate 80 (2% w/w) as a surfactant.

Interaction of M1 and M2 isozymes pyruvate kinase from human tissues with phospholipids.

The effect of pH and the presence of FBP on the interaction of skeletal muscle (PK-M1) and kidney or tumor meningioma (PK-M2) pyruvate kinase with the phospholipids liposomes were investigated by ultracentrifugation and steady-state kinetics and were compared with those results obtained using the bovine heart (PK-M1) isoenzyme which we previously studied. Pyruvate kinase specific activity increases upon the interaction with liposomes. The activation is specifically sensitive to presence of phosphatidylserine (PS) in liposomes. Liposomes made of phosphatidylcholine + phosphatidylserine mixture are good adsorptive systems for both human and bovine of M-type isozymes at low ionic strength. Interaction of PK-M1 with PS liposomes results in the change of Vmax and K(m) values for PEP without marked effect on Hill coefficients. Addition of PS liposomes to PK-M2 induces hyperbolic saturation curves for PEP.

Evidence for the involvement of ecto-5'-nucleotidase (CD73) in drug resistance.

Increased ecto-5'-nucleotidase (ecto-5'NT) protein expression in several multidrug-resistant (MDR) cell lines, documented previously by our group, suggests that this enzyme is involved in drug resistance. Here, Northern blot analysis of selected cell lines and their MDR variants positively correlated ecto-5'NT protein with its mRNA expression. An inhibitor of ecto-5'NT enzymatic activity, alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP), was used to determine if functionally active enzyme had a role in drug resistance. AMP-CP (0.3 mM) reversed the resistance of ecto-5'NT-positive MDR cells (MCF7/A6, L1210/A) to doxorubicin, whereas it did not affect the doxorubicin sensitivity of the ecto-5'NT-negative parental cell lines or that of 2 ecto-5'NT-negative MDR cell lines (HL60/VCR and A2780/DX5). Furthermore, AMP-CP increased rhodamine uptake and inhibited rhodamine efflux from ecto-5'NT-positive MDR cells without affecting ecto-5'NT-negative MDR cells. The presence of exogenous adenosine (0.5 microM) circumvented AMP-CP-induced inhibition of rhodamine efflux from EL4/ADM cells. AMP-CP inhibited the growth of the ecto-5'NT-positive L1210/A MDR cells but had no effect on the growth of the parental cell line. Determination of intracellular ATP levels indicated that MDR cells which had increased ecto-5'NT expression also had a lower intracellular ATP level than their parental cells. Our results suggest that, in certain MDR cell lines, ecto-5'NT serves as a required accessory molecule in resistance mediated by ATP-dependent mechanisms and that growth-sustaining nucleosides are provided by this salvage pathway.

Differential in vivo and in vitro effect of gentamicin on glutamate synthesis and glutamate deamination in rabbit kidney-cortex tubules and mitochondria.

The effect of gentamicin on both glutamate synthesis and glutamate deamination was studied in kidney-cortex mitochondria and tubules isolated from both control and gentamicin-treated animals. In kidney-cortex mitochondria which were permeabilized in order to make a free access of substrates and antibiotic to the glutamate dehydrogenase, gentamicin appeared to be a very potent inhibitor of glutamate synthesis, resulting in about 60% decrease of the enzyme activity at 5 mM concentration. Other aminoglycoside antibiotics decreased the enzymatic activity, in the following order: gentamicin > neomycin = tobramycin = kanamycin > biodacyna > amikacin > streptomycin. This, in principle, corresponds to their known nephrotoxic potential observed in vivo. The inhibitory action of antibiotics was abolished by neither ADP nor leucine, allosteric activators of glutamate dehydrogenase. Surprisingly, gentamicin did not decrease the rate of ammonia formation from glutamate when added to both renal tubules and mitochondria isolated from control rabbits. This indicates that the antibiotic exerts its inhibitory effect on glutamate dehydrogenase activity in the direction of glutamate synthesis only. In contrast, the rate of both glutamate deamination and glutamate synthesis was about 40% lower in renal tubules and mitochondria isolated from kidney-cortex of animals which were given antibiotics for 10 days. In view of these results it seems that (i) the depression of ammoniagenesis in gentamicin-treated animals may be due to a decrease of glutamate dehydrogenase content and (ii) under conditions in vitro the aminoglycoside inhibits the enzyme activity in the direction of glutamate synthesis while it does not affect the glutamate deamination.

Inactivation of enolase from carp (Cyprinus carpio) muscle by 2,3-butanedione.

Enolase from white muscle of carp (Cyprinus carpio) is inactivated by 2,3-butanedione in borate buffer. Magnesium ions as well as substrate--2-phosphoglycerate markedly altered the rate and extent of inactivation. The partially inactivated enzyme shows unaltered Km but decreased Vmax after 10 min incubation with butanedione, however after 60 min incubation the Km value increased 2.5 fold.

Effect of proteases on the activity of enolase from muscle of carp (Cyprinus carpio) and pig.

The treatment of enolase from pig and carp (Cyprinus carpio) with proteases resulted in a decrease of enzymatic activity, which depended on the kind of protease used. The most active were trypsin and subtilisin. Substrate and magnesium ions protected enolase against inactivation. The enolase from pig muscle was much more resistant to protease action than this enzyme from carp muscle. Some differences in the structure between the two enolases are suggested.

Chemical modification of histidine, tyrosine, tryptophan and cysteine residues in carp (Cyprinus carpio) muscle enolase.

Enolase from carp (Cyprinus Carpio) muscle was modified by diethylpyrocarbonate, tetranitromethane, N-bromosuccinimide and 5,5'-dithiobis(2-nitrobenzoic acid). The extent and rate of modification and its effect on the enzyme activity were determined. Modification of histidine, tyrosine and tryptophan residues caused complete inactivation of the enzyme; Mg2+ as well as 2-phosphoglycerate markedly altered the rates of modification and inactivation. The above-mentioned amino acid residues seem to be essential for the functioning of muscle enolases. Modification of cysteine residues had no effect on the enolase activity.

Studies on immunological properties of enolase from carp muscles (Cyprinus carpio) after chemical modification of some amino-acid residues.

After modification of histidine residues, enolase from carp muscles lost the ability to precipitate with antibodies against native enzyme. This ability remained after nitration of tyrosine residues, but the immunoelectrophoretic pattern--localisation and height of arcs--differed from that of native enolase. Minor changes of this pattern were found in enolase with modified tryptophan residues.

Purification and properties of enolase from carp (Cyprinus carpio). Comparison with enolases from mammals' muscles and yeast.

The enolase (2-phospho-D-glycerate hydrolyase E.C. 4.2.1.11) from carp muscle was obtained, the specific activity--88 U/mg of protein. Km for 2-phosphoglycerate was 0.313 mM and for phosphoenolpyruvate--0.76 mM. The enzyme is active only in the presence of divalent metal ions, Mg2+ being the best activator. The phosphate and fluoride decreased the activity of enzyme. The molecular weight of the dimeric form of enzyme was found to be 93,000. The enzyme is immunologically different from pig muscle enolase.