The perils of Primaxin

Primaxin is one of the most effective antibiotics introduced into medical practice in the past few decades. It is a single-pill combination of two agents, an unusual β‎-lactam antibiotic and an inhibitor of an obscure kidney enzyme that abolished the efficacy of the β‎-lactam in that organ. The initial discovery was a β‎-lactam antibiotic (think penicillin, amoxicillin) named thienamycin from a fermentation broth. Thienamycin had marvelous antibiotic properties, potent against many microorganisms resistant to earlier β‎ lactams, but was notoriously unstable. Once the unusual structure of thienamycin was unraveled, structural modifications were started. A critical chemical modification resulted in a new agent, imipenem, that retained the antibiotic properties of thienamycin but was stable. A second drug discovery effort resulted in an inhibitor of the kidney enzyme, cilistatin. A combination of the two created Primaxin.

In vivo and in vitro Study of Probiotic Effect of Lactobacillus acidophilus on Pathogenecity of Proteus mirabilis isolated from Urinary Tract Infection (UTI)

The aim of the study was to treat the pathogenesity and adhesion property of Proteus mirabilis isolate, obtained from urinary tract infection (UTI) patients. After the identification of Proteus mirabilis it was found that the pathogenic bacteria possessed the adhesion ability both in vivo and in vitro bioassays, with lactobacillus acidophilus filtrate as Probiotic. Proteus mirabilis was subjected to different concentrations of lactobacillus acidophilus filtrate to investigate its adhesion property and pathogenesity. Three concentrations (25, 50,100) % of Lactobacillus acidophilus concentrated filtrate were used in vitro bioassays against pathogenic bacteria. Results showed that the third fold 25% was the most effective concentration in reducing the adhesion intensity of the bacteria. This concentration was selected to be used in in vivo for detection of infectivity to the animal tissues (Kidney and liver) and also the effect of Proteus mirabilis and Probiotic on the level of hormones in liver; Serum glutamate pyruvate transaminase, Serum glutamate oxoloacelate transaminase and Alkaline phosphtases (GPT, GOT , ALP) and kidney enzyme (Urea) in serum of mice was investigated. It was found that the levels of liver hormones and kidney enzyme increased at the time of infection, and these levels returned to normal or decrease after treating the animals with 25% of concentrated filtrate of Probiotic.

Localization of S-adenosylhomocysteine Hydrolase in the Rat Kidney

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 ± 0.02 mU/mg in the kidney, 0.32 ± 0.03 mU/mg in the glomeruli, and 0.50 ± 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation.

Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from ∼2 to 15 μmol Pi ⋅ mg−1 ⋅ min−1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the α-subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.

Cloning and overexpression of rat kidney biliverdin IXα reductase as a fusion protein with glutathione S-transferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity

Native biliverdin IXα reductase (BVR-A) is a monomer of molecular mass 34 kDa. We have developed an expression vector that allows the isolation of 40 mg of a glutathione S-transferase (GST)-BVR-A fusion protein from 1 litre of culture. The fusion protein (60 kDa) behaves as a dimer on gel filtration (120 kDa), so that we have artificially created a BVR-A dimer. The recombinant rat kidney enzyme exhibits pre-steady-state ‘burst’ kinetics that show a pH dependence similar to that already described for ox kidney BVR-A. Similar behaviour was obtained in the presence and absence of the GST domain both for the burst kinetics and during initial-rate studies in the presence and absence of albumin. The stereospecificity of the BVR-A-catalysed oxidation of [4-3H]NADH, labelled at the A and B faces, was shown to occur exclusively via the B face.

Evidence for isoforms of 11β-hydroxysteroid dehydrogenase in the liver and kidney of the guinea pig

In the human and in rodents like the rat and mouse, the liver enzyme 11β-hydroxysteroid dehydrogenase type I (11β-HSD-I) is a functional oxidoreductase preferring NADP+/NADPH as cosubstrate, while the renal isoenzyme (11β-HSD-II) prefers NAD+ as cosubstrate, and seems to be a pure oxidase and protects the tubular mineralocorticoid (MC) receptor from occupancy by cortisol and corticosterone. We studied the enzyme kinetics of 11β-HSDs in kidney and liver microsomes of the guinea pig, a species whose zoological classification is still a matter of debate. With a fixed concentration of 10−6 mol/l cortisol, liver and kidney microsomes preferred NAD+ to NADP+ (10−3 mol/l) for the conversion to cortisone. Kidney microsomes converted cortisol to cortisone with Km values of 0·64 μmol/l and 9·8 μmol/l with NAD+ and NADP+ as cosubstrates respectively. The reduction of cortisone to cortisol was slow with kidney microsomes, but could be markedly enhanced by adding an NADH/NADPH regenerating system: with NADPH as preferred cosubstrate, the approximate Km was 7·2 μmol/l. This indicated the existence of both isoenzymes in the guinea pig kidney. Liver microsomes oxidized cortisol to cortisone with similar Km and Vmax values for NAD+ to NADP+ as cosubstrates (Km of 4·3 μmol/l and 5·0 μmol/l respectively). The NAD+ preference for the oxidation of 10−6 mol/l cortisol described above may be due to a second, NAD+-preferring 11β-HSD with a Km of 1·4 μmol/l. In contrast to the kidney, liver microsomes actively converted cortisone to cortisol with a preference for NADPH (Km: 1·2 μmol/l; Vmax: 467 nmol/min per mg protein). Thus, the main liver enzyme is similar to the oxidoreductase of other species (11β-HSD-I) and is also present in the kidney, while the main kidney enzyme is clearly NAD+-preferring. This kidney enzyme (analogous to 11β-HSD-II of other species) seems to be suitable for the protection of the MC receptor from the high free plasma cortisol levels of the guinea pig. Journal of Endocrinology (1997) 153, 291–298

Determination of Na(+)-K(+)-ATPase alpha- and beta-isoforms and kinetic properties in mammalian liver

While Western blot analysis clearly revealed the presence of the alpha- and beta-subunits of Na(+)-K(+)-ATPase in a variety of rat tissues, beta was not readily detectable in liver. This observation was consistent with a previous report indicating that Na(+)-K(+)-ATPase immunoprecipitated from rat liver gives no clear evidence for the presence of a beta-subunit (Hubert et al. Biochemistry 25: 4156-4163, 1986). However, Western blot analysis of density gradient-purified lamb and rat liver microsomes showed the presence of a protein with an approximate molecular mass of 42 kDa that was immunoreactive with beta-specific polyclonal antibodies as well as beta-directed monoclonal antibodies. Deglycosylation of this protein by N-glycosidase F generated a core protein (beta c, M(r) approximately 32,000) that had the identical electrophoretic mobility as the beta c protein of the purified kidney enzyme. Isoform-specific monoclonal and synthetic peptide-directed polyclonal antibodies were used to demonstrate the presence of only the alpha 1- and beta 1-proteins in the liver and the presence of beta 2 in rat brain. Functional studies then showed that although both rat and lamb liver enzymes had sensitivities to cardiac glycoside inhibition similar to that of their corresponding kidney enzyme, the lamb liver enzyme had higher affinities for Na+, K+, and ATP than the kidney enzyme.