RESUMEN

A new reagent strip for the determination of leukocytes in urine (LEUKOSTIX; Ames) is described. The test is based on the esterase activity in leukocytes as a marker. Upon contact between the reagent matrix and a urine containing leukocytes, an amino acid ester is hydrolyzed by the esterase to its corresponding alcohol. The free alcohol then couples with a diazonium salt to produce a purple azo dye. The relative concentration of leukocytes in the urine is obtained by visually comparing the strip reaction with a color chart. Performance of the strip was evaluated in a clinical study involving eight different sites and 867 urine specimens. The comparison method was sediment microscopy; specimens containing five cells or more per high-power field were considered to be positive. Sensitivity was 76.3%, specificity 80.8%. Performance was comparable with that of the CHEMSTRIP LN (Boehringer-Mannheim Diagnostics, Inc.) leukocyte test, which we evaluated concurrently.

Asunto(s)

RESUMEN

The control system described here is used to monitor the performance of urinary reagent-strip tests for leukocytes. The presence of leukocyte esterase in the urine is used as a marker for leukocytes in urine. The control system is based on sonicated leukocytes, isolated from whole blood. The esterase activity of this sonicate is determined by spectrophotometry with the N-tosyl-L-alanine ester of 5-phenyl-3-hydroxypyrrole as substrate. The assay result is used to determine the amount of sonicate needed to prepare buffered esterase-containing solutions. Such control solutions mimic leukocytic urines and are stable for 6 h at room temperature. The variability of the control system was tested by preparing it five times in a day on five separate days. The overall CV for Leukostix Reagent Strips (Ames Division, Miles Laboratories, Inc.) when tested with these solutions and analyzed with a reflectance spectrophotometer was 8%; for visual readings it was 10%. The overall CV for Chemstrip LN Reagent Strips (Biodynamics, Indianapolis, IN) was 10%.

Asunto(s)

RESUMEN

Escherichia coli K-12 strains have deletions for the normal lambda integration site were lysogenized with bacteriophage lambda at a site within the L-fucose utilization system (fuc). The frequency of lambda integration at this site is approximately 2 X 10(-8) to 5 X 10(-7). Studies of the lytic properties of these strains indicated very infrequent cell lysis with a relatively low phage burst size. Transductional ability of the phage lysates was found to be normal, comparable to that found in conventional low-frequency transducing lysates. Two major classes of transducing phage were found. One carried the markers argA and fucA (a fucose utilization gene of unknown function previously referred to as fuc-1) and the gene for D-arabinose utilization (dar+). The other carried only fucC, the gene specifying L-fuculose-1-phosphate aldolase. A minor class of phage was found that carried fucA, but not argA or dar+. Upon consideration of the transductional nature of these phage classes, we are proposing that the gene order for the L-fucose utilization system is dar, fucA, (lambda), fucC.

Asunto(s)

RESUMEN

Studies involving lambda phage transduction of the D-arabinose utilization gene (dar+) in Escherichia coli K-12 indicated the product of this gene to be a transdominant activator. An apparent anomaly regarding this hypothesis exists in that a diploid recessive lysogen (lambda dar-/dar-) can spontaneously become capable of growth on D-arabinose.

Asunto(s)

RESUMEN

The Prostatic-Group-Label Immunoassay (PGLIA) technique has been incorporated into a reagent-strip format. We report use of flavin N6-(N'-2,4-dinitrophenyl-6-aminohexyl)adenine dinucleotide (DNP-FAD) as the prosthetic group derivative and 6-N-(2,4-dinitrophenyl)aminohexanoic acid (DNP-caproate) as the competing ligand. DNP-FAD not bound by antibody combines with glucose oxidase apoenzyme, which then reacts with glucose and oxygen, and gives color through a peroxidase-linked system. The rate of color generation is thus a function of the DNP-caproate concentration. PGLIA reagent strips are prepared by sequential impregnations of filter paper with an acetone solution of indicator (3,3',5,5'-tetramethylbenzidine); an aqueous solution containing glucose oxidase apoenzyme, the rest of the color generation system, stabilizers, and antibody to DNP; and a solution of DNP-FAD in n-propanol. This preparation permits effective antibody binding, and prevents premature interaction of immunoassay components. A quantitative color response to concentrations of DNP-caproate in the range of 1 to 8 mumol/L was demonstrated with these reagent strips. Prototype PGLIA reagent strips for theophylline and phenytoin have been successfully developed by substituting the appropriate FAD derivative and antibody for the corresponding reagents in the DNP model system.

Asunto(s)

Asunto(s)

RESUMEN

When treated with chloramphenicol, Escherichia coli 15T minus produces two new species (IV and V) of transfer ribonucleic acid specific for phenylalanine in addition to the major normal species (II) and two minor normal species (I and III), which are seen as distinct components upon fractionation by chromatography on columns of benzoylated diethylaminoethyl-cellulose. Species IV is produced when cells are grown in iron-deficient medium and is, therefore, probably deficient in the 2-methylthio modification of N-6-(delta-2-isopentenyl) adenosine. A new minor species (Va) also appears under those conditions. All of the new components elute earlier than the major normal species. Addition of chloramphenicol to iron-deficient cells leads to the production of species V, and that production is blocked by rifampin, as is the production of species IV. Thus, species IV and V appear to be transcriptional products. Although E. coli 15T minus appears to be rel plus, starvation for methionine or cysteine leads to the accumulation of species IV (without addition of chloramphenicol); rifampin blocks the accumulation. Species V is still produced on addition of chloramphenicol to starved cultures. Starvation for arginine or tryptophan does not alter the chromatographic profile from the normal case. Treatment with permanganate indicates that species II and IV contain isopentenyladenosine but that species V does not. Species V appears to be deficient in both isopentenyl and methylthio modifications of adenosine and perhaps at least one other modification, because removing the isopentenyl moiety from adenosine does not convert species IV into species V, but converts it into species Va. A precursor relationship among species V, VI, and II is suggested by following the chromatographic profile of phenylalanine transfer ribonucleic acid during recovery of E. coli from treatment with chloramphenicol; the various species increase and decrease in a sequential manner.

Asunto(s)

RESUMEN

The levels of macromolecules in Escherichia coli 15T(-) growing in broth, glucose, succinate, and acetate media were determined to compare relationships among deoxyribonucleic acid (DNA), ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA), and protein in cells at different growth rates. DNA and protein increased in relative amounts with decreasing growth rate; relative amounts of rRNA and tRNA decreased, tRNA making up a slightly larger proportion of RNA. For several amino acid-specific tRNAs studied, acceptor capacities per unit of DNA increased with increasing growth rate. The syntheses of tRNA and rRNA are regulated by similar, yet different, mechanisms. Chromatographic examination on columns of benzoylated diethylaminoethyl-cellulose of isoaccepting tRNAs for arginine, leucine, lysine, methionine, phenylalanine, serine, and valine did not reveal differences in the isoaccepting profiles for rapidly (broth culture) and slowly growing (acetate culture) cells. Therefore, isoacceptors for individual amino acids appear to be regulated as a group. Lower efficiencies of ribosomal function in protein synthesis can be explained, in part, by a low ratio of tRNA to the number of ribosomes available and by a decreasing concentration of tRNA with decreasing growth rate. Data on the tRNAs specific for seven amino acids indicate that the decreasing concentration of tRNA is a general event rather than a severe limitation of any one tRNA or isoaccepting tRNA.

Asunto(s)