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Gel diffusion regularities with respect to neomycin sulfate substances differing in their composition and the antibiotic separate components were studied on nutrient media of diverse composition. Optimal conditions for standardization of neomycin sulfate antimicrobial activity with using the agar diffusion method and synthetic medium are determined.
Colonic pH was continuously monitored by a pH electrode implanted in the transverse colon of rats over a 24-hour period. Lactulose significantly reduced the colonic pH to 5.37 +/- .39 from a fasting value of 6.77 +/- .75. Pretreatment with neomycin or concomitant use of a nonabsorbable antacid prevented lactulose-induced reduction in the colonic pH. The clinical implication of the study is that combined use of lactulose with neomycin or high doses of non-absorbable antacids in the treatment of chronic portasystemic encephalopathy may be undesirable.
The blocking effects of streptomycin and neomycin upon adrenal neurotransmission were studied in dogs. The pressor responses to splanchnic nerve stimulation (SNS) or 50 micrograms/kg nicotine were both blocked by 100 mg/kg but not by 50 mg/kg of the antibiotics injected systemically through the femoral vein. The effect was reversed by intravenous infusion of calcium chloride. The pressor response to SNS, but not that to nicotine, was blocked by the antibiotics injected into the adrenal gland through the adrenolumbar vein at doses up to 10 mg/kg. This effect was reversed by 50 micrograms/kg calcium chloride. Higher doses of antibiotics blocked the response to both stimuli, with only the response to nicotine being reversed by calcium chloride. These data indicate that streptomycin and neomycin block adrenal neurotransmission by interfering with calcium ions at pre- or pre-plus postsynaptic levels depending on the dose and route of administration.
Observations were made for two consecutive days on the disposition of oxytetracycline, chloramphenicol, neomycin and gentamicin through the digestive tract of pigs given an oral drench. The effect of these antibiotics upon the colibacilli flora and upon an Escherichia coli 127 strain in small filter membrane bags placed into the caecum was also investigated. For chloramphenicol, inactivation by antibiotic resistant colibacilli and, for neomycin, losses of activity by digestive content may explain the lower concentrations obtained in the gut and the correlative lack of curative or preventive action against E coli 127 in small bags. Tetracycline and gentamicin prevented growth of E coli 127 from the second day.
21 patients suffering from microbial eczema were locally treated with antibiotics over a period of 14 days. As antibiotics we chose rifamycin SV and a combination of neomycin and bacitracin. S. aureus--most frequently traceable on microbial eczema--is usually sensitive to rifamycin SV as well as neomycin and bacitracin in combination.
Renal tubular transport of organic anions is immature at birth and can be stimulated in adult rats by repeated administration of xenobiotics. There is some evidence of an increased synthesis of carrier proteins in renal tubular cells following stimulation as well as during postnatal development of renal tubular transport processes. The effect of pretreatment with an inhibitor of protein biosynthesis (neomycin) on stimulated and maturative transport of p-aminohippurate (PAH) was measured. Neomycin has a dose dependent long acting and reversible effect on stimulated PAH transport and on postnatal maturation of PAH excretion, and increased protein biosynthesis is the likely common basic phenomenon of both processes.
Two series of 50 consecutive elective colonic resections with bowel preparation either with Neomycin (group A) or Neomycin combined with Metronidazole (group B) are presented. 17 bacterial inflammatory complications in group A versus 10 in group B were noted. It is concluded that the addition of an antianaerobic antibiotic agent in elective colonic surgery might be of beneficial effect.
Comparative investigation of the neomycin-producing strain and a strain which has spontaneously lost the capacity for the antibiotic synthesis suggested participation of the plasmid with the molecular weight of 59 X 10(6) dalton in determination of the biosynthesis of neomycin and phosphotransferase inactivating the antibiotics. This plasmid factor is not eliminated under the effect of the sublethal concentrations of acridine orange and ethidium bromide.
Columns of immobilized neomycin (reductively coupled to porous glass beads) were used to separate weakly acidic lipids as well as the polyphosphoinositides. All anionic lipids present in chloroform-methanol extracts, which had been washed first with acid and then with neutral salt solutions, were adsorbed. Phosphatidylserine and phosphatidic acid were eluted with chloroform-methanol-formic acid mixtures. Phosphatidylinositol and cardiolipin were eluted sequentially by very low concentrations (10-100 mM) of ammonium formate in chloroform-methanol-water. All three phosphoinositides were isolated from washed chloroform-methanol-KCl extracts of brain. Sulfatides eluted with the phosphatidylinositol. Phosphatidylinositol phosphate and phosphatidylinositol bisphosphate were eluted in turn by higher salt concentrations (0.4-1 M). The immobilized neomycin was used repeatedly but the capacity eventually declined. This loss was reversed by sodium borohydride reduction.
Human adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on chromosome 19q13.3-qter. We report that both transfected and infected AAV-vectors will target a foreign gene preferentially to AAVS1. Of 11 infected cell lines studied, nine (82%) showed integration of the neomycin resistance marker into AAVS1, which confirms the findings of previous investigations. We also show site-specific integration (nine out of 12 cell lines, 75%) following transfection of an AAV vector containing the neomycin gene. The ability of transfected AAV sequences to preferentially target and express selectable genes at a known site in the human genome extends the potential use of AAV as a vector in gene therapy.
This study has been undertaken to verify if neomycin, known to be toxic because it interacts with membrane-bound phosphatidylinositol-4,5-biphosphate, may lead to an extensive perturbation of the membrane organization of human erythrocytes. By comparison, the effects induced by a non toxic aminocyclitol antibacterial agent as spectinomycin were examined. Our data exclude extended alterations of the membrane and hence large structural changes of the whole cell for both the drugs probably as a consequence of the extreme difficulty of penetration because of their selective hydrophilic features. However, only neomycin seems to present a binding on negatively charged regions of erythrocyte membranes, that could be responsible of its remarkable toxicity.
The effect of oral administration of cholestyramine, neomycine, calcium, and bile acids on intestinal 14C-oxalate absorption and urinary oxalate excretion was assessed in patients with normal or increased ('enteric' hyperoxaluria) urinary oxalate excretion. Cholestyramine, neomycine and acute administration of chenodeoxycholic acid (2.75 g/24 h) did not affect significantly 14C-oxalate absorption or urinary oxalate excretion. Oral administration of calcium markedly reduced 14C-oxalate absorption (88.2%) and urinary oxalate excretion (46%). Calcium-induced reduction of intestinal oxalate absorption was more pronounced in patients with hyperabsorption of oxalate than in subjects with normal oxalate absorption or excretion. Patients on treatment with high doses of chenodeoxycholic acid (1.5-2.0 g/day) for dissolution of cholesterol gallstones had increased oxalate absorption and excretion, patients on long-term treatment with lower doses of chenodeoxycholic acid (0.75-1.0 g/day) exhibited normal absorption or urinary excretion of oxalate. The results do suggest that calcium and bile acids do play an important role in the pathogenesis of 'enteric' hyperoxaluria. It is suggested that the beneficial therapeutic effect of cholestyramine in hyperoxaluria due to ileal resection is rather caused by its bile acid binding property than by direct binding of oxalate within the intestinal lumen.
When in low concentration (0.38%) in an ointment base neomycin within the middle ear does not damage the inner ear. Animal experiments show that doses 10 times greater do not damage the hair cells, but when in aqueous solution all hair cells are destroyed. Many years experience has confirmed that neomycin ointment packing for postoperative ears and infected radical mastoid cavities does not produce inner ear damage. These patients have been carefully monitored by audiometry.
The gene for the integral membrane protein glycophorin A (GPA) was cloned in frame to the 5' end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT). Protein expression was achieved in Escherichia coli as well as in mammalian cells. In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks after transfection; the amount of GPA-NPT fusion protein produced was constant from experiment to experiment. Neomycin resistance was directly correlated with GPA expression, thus allowing the direct selection for a stable GPA-expressing cell population without the need of a cloning step. The amount of GPA-NPT produced was further increased by weakening the specific NPT enzymatic activity via site-directed mutagenesis. Detection was simplified by the fact that all different fusion proteins could be detected by the same anti-NPT antibody. This approach may be also applicable to other membrane proteins.
Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high-affinity Mn(II) binding site in the hammerhead ribozyme. This binding site exhibits a dissociation constant Ke of 4.4 microM in buffer solutions containing 1 M NaCl, as shown by titrations monitored by continuous wave (cw) EPR. A combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constant kappa of 0.7 MHz, an asymmetry parameter eta of 0.4, and an isotropic hyperfine coupling constant of Aiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the Mn(II) ligand sphere and can therefore be used to determine the structure of the binding site. A possible location for this binding site may be at the G10.1, A9 site found to be occupied by Mn(II) in crystals (MacKay et al., Nature 1994, 372, 68 and Scott et al., Science 1996, 274, 2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the Mn(II) site in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese(II) center and the Iglo-II basis set for all other atoms yielded values of kappa(14N)=+0.80 MHz, eta=0.324, and Aiso(14N)=+2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that Mn(II) is released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 microM) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry 1995, 34, 11 186).
We describe the construction of a transposable element derived from the Mu phage that upon insertion is able to create a gene fusion between the region of Tn903 coding for neomycin phosphotransferase (NPT I), which confers resistance to aminoglycosides including kanamycin (KmR), neomycin and G418, and the control elements of the gene where the insertion occurs. A chloramphenicol (Cm) transacetylase gene (cat) that confers resistance to Cm is present in the transposon so that transposition events can be monitored even when no active fusions with the nptI coding region occur. The transposase gene is deleted and, therefore, this transposon is perfectly stable upon insertion. The properties of this new transposable element were studied by obtaining gene fusions between the Escherichia coli L-arabinose operon and 'nptI gene. In some of them the KmR phenotype is induced by arabinose. Insertions of this element in cloned fragments of the T-DNA region of Agrobacterium rhizogenes were also isolated. Some of them confer a KmR phenotype upon its E. coli carriers, which indicates that portions of the T-DNA are expressed in these cells.
The analysis of the antibiotics neomycins A, B and C was investigated. The separation of the components was studied using reversed-phase and reversed-phase ion-pair chromatography. The optimum separation was obtained utilizing a Lichrosorb RP-2 column with a mobile phase consisting of 75 mg/l sodium dodecyl sulphate, 0.5M Na2SO4 and 0.015 M sodium acetate buffer at pH 7.0. Using this mobile phase, baseline separation was obtained for all three compounds in approximately 20 min. Detection was via post-column derivatization of the analytes with ortho-phthalaldehyde in the presence of mercaptoethanol to form fluorescent iso-indole products. This system is applied to the analysis of a number of formulated products containing neomycin.
The C5''-OH group in neomycin B was glycosylated with a variety of mono- and di-saccharides to probe the effect of introduction of additional binding elements on antibacterial activity and interaction with the aminoglycosides modifying enzyme APH(3')-IIIa. The designed structures show antibacterial activity superior to that of neomycin B against pathogenic and resistant strains, while in parallel they demonstrate poor substrate activity with APH(3')-IIIa.