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PubMed1984

[Restriction analysis of the plasmid and chromosomal DNA of the strain Streptomyces fradiae--a producer of neomycin].

Plasmid DNA with a molecular weight of 53-55 Md was isolated from Streptomyces fradiae strain 676 producing neomycin on centrifugation of the DNA preparation at the density gradient of cesium chloride-ethidium bromide. The extrachromosomal DNA had 6 recognition sites for Bgl II, more than 13 recognition sites for Bam HI, more than 14 recognition sites for Kpn I, more than 18 recognition sites for Pst I, more than 20 recognition sites for Sac I, more than 21 recognition sites for Pvu II and no recognition sites for Eco RI, Eco RV, Hind III and Sla I.

Chemical Phenomena↗
PubMedDate not supplied

Microbiological determination of neomycin in feeds and formulated products.

An AOAC modified method is described for the microbiological assay of neomycin, which has been adapted to include complete feeds, supplements, premixes, liquids, oil suspensions, boluses, and antibiotic-impregnated paper. The method features a more sensitive standard response line with a monolayer plating system. The use of a buffered plating medium in place of the water-prepared medium results in a curve with less degree of slope, which allows for more accurate interpretation of the standard response. The feed extract diluent used for standard response line dilution, which is prepared from exposure of the feed extract fluid to pH changes, heat, and sodium hypochlorite, has been eliminated. The constant salt concentration diluent used for the preparation of standards is the same as the salt concentration of the sample extract solution to be tested. Results for 50 commercial complete feeds and 50 commercial premixes received over the last 5 years produced an overall mean recovery of 101% with a mean percent recovery range of 80-112%. A statistical analysis of these 100 commercial, complete feeds and premixes, ranging in concentration from 47 g/ton to 70 g/lb, indicates the assay has little, if any, concentration-related bias. Precision and accuracy of the method was supported by laboratory studies of 20 assays that produced a mean recovery of 101% and standard deviation of 3.

Animal Feed↗
PubMed1983

Pharmacokinetic study of neomycin in calves following intravenous and intramuscular administration.

Neomycin sulfate was administered to calves by the intravenous and intramuscular routes. Serum drug levels were determined and the intravenous pharmacokinetic parameters derived using the Gauss-Newton nonlinear fitting algorithm and the two compartment open model. The kinetic parameters determined were as follows: zero time intercept, serum drug level 68.045 +/- 15.894 micrograms/mL, alpha slope intercept 37.666 +/- 13.874 micrograms/mL and beta slope intercept 30.379 +/- 12.638 micrograms/mL; equilibration rate (pool I and II) 0.081 +/- 9.064 min-1; elimination rate 0.004 +/- 0.001 min-1; half-time alpha 14.774 +/- 11.236 min, half-time beta 166.596 +/- 47.576 min; first order elimination constant 0.009 +/- 0.002 min+; transfer rate constants, central to peripheral, 0.032 +/- 0.026 min+ and peripheral to central 0.045 +/- 0.037 min-1; volume of central compartment 0.186 +/- 0.047 L/kg; volume of distribution 0.388 +/- 0.130 L/kg; body clearance 0.002 +/- 0.001 L/kg/min.

Animals↗
PubMed1983

The effect of lincomycin-neomycin treatment on experimental anaerobic bacterial bovine mastitis.

Three healthy lactating quarters of a Friesland cow were each experimentally infected with a pure culture of a strain of either Bacteroides fragilis, Eubacterium lentum or a Peptostreptococcus sp. respectively. The onset and progression to clinical mastitis was monitored 12 hourly by examination for clinical signs of inflammation, bacterial culture, somatic cell counts and with a strip cup. All infected quarters developed clinical mastitis within 24 hours. The 2 quarters infected with B. fragilis and E. lentum respectively were treated 4 times consecutively at 12 hour intervals, commencing at 24 h by intramammary instillation of 10 ml of a mixture containing 200 mg lincomycin hydrochloride, 200 mg neomycin sulphate and 5 mg methylprednisolone (Lincocin Forte, Upjohn). Both quarters became clinically normal and no bacteria could be detected in the secretions 12 hours after the first treatment. At 36 hours the strip cup became negative, and the somatic cell count dropped to less than 500 X 10(3) at 72 hours after the initial treatment. The quarter infected with a Peptostreptococcus sp. was unable to overcome the infection by natural means when intramammary treatment was delayed for the first 36 hours after the onset of clinical mastitis. Subsequent treatment of this quarter gave results similar to those treated earlier.

Animals↗
PubMed1983

[Use of neomycin-bacitracin irrigating solution with intraoperative autotransfusions during orthopedic operations].

Although there is doubt about the importance of rinsing the operative field with a solution containing locally acting antibiotics, it is frequently done. In this paper we tried to answer the question, wether intraoperative autotransfusion (IAT) with the Haemonetics Cell Saver is contraindicated during rinsing the operative area with locally acting antibiotics or vice versa. The measured serum concentrations for neomycin and bacitracin (Nebacetin) were extremely low. Therefore IAT is recommended with a system separating and washing the autologous red blood cells even under circumstances of rinsing the operative field with a solution containing locally acting antibiotics.

Adult↗
PubMed1978

[Detection of plasmids in actinomycetes that produce oxytetracycline and neomycin].

Crude preparations of DNA isolated from Act. rimosus 907, an oxytetracycline-producing organism and Act. fradiae 676, a neomycin-producing organism were analyzed with electrophoresis in agarose gel followed by electron microscopic analysis of DNA isolated from the gel. It was found that Act. rimosus and Act. fradiae contained ring molecules of DNA with the molecular masses of 37 . 10(6) and 59 . 10(6) dalton respectively.

DNA, Bacterial↗
PubMed1975

[State of certain indices of natural, nonspecific immunity in workers engaged in the manufacture of neomycin and monomycin].

Women occupied at various stages of neomycin and monomycin production were examined immunologically. The examination revealed insignificant changes in the phagocytic activity of the blood neutrophils, however no relation between the changes level and the level of contact with the antibiotics was observed. Changes in composition of the microflora in the deep skin layers in direction of an increase in the number of pathogenic microbes and a decrease in the bactericidal function of the skin were found in the women occupied at the final stages of the technological process, i.e. drying, packing and control.

Drug Industry↗
PubMed1993

Synergistic effects of monensin in combination with permethrin or neomycin on neuronal activity.

Drug combinations have the potential to produce novel and unpredictable responses on nervous tissue. This study was designed to test the hypothesis that the effects of combinations of monensin (an ionophore antibiotic) and either neomycin (an aminoglycoside antibiotic) or permethrin (synthetic pyrethroid) are synergistic. Effects of the drug combinations upon the electrical properties and membrane activities of an in vitro sensory neuron preparation were found to be greater than expected from addition of the effects of the same drugs acting individually, indicating synergism and thus supporting the hypothesis. It was concluded that drugs acting at different neuronal membrane sites and applied in combination produce unpredictable responses. Such drug combinations behave as if they were novel drugs.

Animals↗
PubMed1992

Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes.

A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the protein-coding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin. Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants. The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing. The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability. The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.

Base Sequence↗
PubMed1994

Membrane-bound neomycin phosphotransferase confers drug-resistance in mammalian cells: a marker for high-efficiency targeting of genes encoding secreted and cell-surface proteins.

An efficient method for inactivating genes is the use of silent selectable markers that are expressed only after homologous recombination into the active target gene. However, use of this approach for genes encoding secreted or membrane-anchored proteins may produce hybrid proteins comprising the N-terminal signal sequence from the target gene linked to the protein conferring drug resistance. Such chimeric enzymes will be secreted, precluding selection for drug resistance. To overcome this problem, we tested the possibility of anchoring in the membrane the cytoplasmic neomycin phosphotransferase (NPT). We constructed a fusion gene with a transmembrane domain connecting the N-terminal signal sequence of a membrane-targeted protein and the neo gene. Expression of this gene yielded G418-resistant colonies of C2C12 cells which contained assayable NPT activity. Comparison of enzyme activity in cell extract fractions verified that the active fusion protein was insoluble, presumably through localization to a membrane compartment. Transmembrane neo cassettes should serve as integration-activated markers capable of targeting genes encoding secreted or cell surface proteins.

Amino Acid Sequence↗
PubMed1985

Quantitative in situ gel electrophoretic assay for neomycin phosphotransferase activity in mammalian cell lysates.

A method for the assay of neomycin phosphotransferase activity in eucaryotic cell lysates is described. Total cytoplasmic proteins are fractionated in nondenaturing polyacrylamide gels and then allowed to react in situ with [gamma-32P]ATP and kanamycin. The reaction products are detected by blotting to phosphocellulose paper and autoradiography. The assay is linear with protein concentration and sensitive enough to detect expression in transient assays.

Adenosine Triphosphate↗
PubMed1987

Dot assay for neomycin phosphotransferase activity in crude cell extracts.

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.

Collodion↗
PubMed1990

A quantitative assay for neomycin phosphotransferase activity in plants.

A sensitive, simple, and quantitative assay for determining neomycin phosphotransferase (NPT) activity in plant cell extracts is described. The procedure retains the simplicity of previously published methods, yet offers up to a 140-fold increase in sensitivity. This increase is due to (1) the addition of bovine serum albumin (BSA) to the assay mixture, (2) desalting of crude maize extracts to remove a low-molecular-weight inhibitor of the enzyme, and (3) use of a different extraction buffer and an improved extraction procedure to liberate more enzyme from the cells. This method has been used successfully to detect and quantitate both stable and transient expression of NPT in transgenic tobacco and maize tissue.

Animals↗
PubMed1990

High-sensitivity protein detection by a new "contact-copy" method using a protein A-neomycin phosphotransferase II fusion protein.

A new system for high-sensitivity protein detection by an immunoenzymatic "contact-copy" procedure is described. It is based on two components: (i) a microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (protein A-NPT II) in which the protein A moiety acts as a second immunological reagent while NPT II catalyzes the detection reaction and (ii) a novel kanamycin-loaded substrate matrix (kanamycin-cyanuric chloride-activated and sulfanilic acid-derivatized paper) brought into direct contact with a protein-carrying matrix after blot or dot application and initial immunoreaction--the NPT II enzyme reaction with [gamma-32P]ATP as cosubstrate leads to phosphorylation of the substrate kanamycin on the substrate matrix, which is used for further analysis. The contact-copy method has at least the same detection sensitivity as procedures employing 125I-protein A, but allows extremely short exposure times and avoids probe prelabeling. Twenty-five picograms of specific protein blotted from sodium dodecyl sulfate-polyacrylamide gels onto nitrocellulose is detected after 15 min of autoradiography. The limit of detection in dot tests was found to be 10 pg per dot (3 mm2). The method is suitable for quantitative determination of antigens in the range down to 100 pg. Several contact copies of the same original protein-carrying matrix can be produced and used for detection or quantitative analysis without destroying the original matrix.

Collodion↗
PubMed1991

Assay of neomycin phosphotransferase activity in cell extracts.

A simple method for the measurement of neomycin phosphotransferase (NPT) activity in crude extracts of eukaryotic cells is described. This method is based on the elimination of interfering phosphorylated proteins by using phenol-chloroform extraction. This solution phase assay allows the detection of greater than or equal to 0.01 ng of NPT in the crude cell extract. Rapid screening of a large number of cell cultures generated in gene-transfer experiments, using NPT as a selective marker, is made possible by this simple technique. Further, the promoter strength of vector constructs used in gene therapy may also be estimated by this procedure.

Cell Extracts↗
PubMed1994

Inhibition of phospholipase C with neomycin improves metabolic and neurologic outcome following traumatic brain injury.

Activation of phospholipase C has been implicated as a factor in the development of irreversible tissue damage following injury to the central nervous system. We have used phosphorus magnetic resonance spectroscopy and a battery of postinjury motor function tests to characterize the role that phospholipase C activity may play in determining biochemical and neurologic outcome following traumatic brain injury in rats. Moderate (2.7 atmospheres) fluid percussion induced lateral brain injury caused a decline in free magnesium concentration, phosphorylation potential, and increased mitochondrial rate of oxidative phosphorylation. Neurologic motor score at 24 h and 1 week posttrauma in these animals was consistent with moderate injury. In contrast, treatment with the phospholipase C inhibitor neomycin B (15 mg/kg i.v.) immediately prior to injury significantly improved free magnesium status, bioenergetic state and neurological outcome (P < 0.01) after injury. We propose that phospholipase C activated second messenger pathways affecting magnesium homeostasis are involved in determining outcome after brain injury.

Animals↗