Enzymatic Amplification Reaction Research Articles

Microdetermination of 2-deoxyglucose and 2-deoxyglucose 6-phosphate to determine glucose utilization rates in single neurons and small CNS regions after injecting nontracer amounts of 2-deoxyglucose.

A nontracer amount (0.25 mmol/kg of body weight) of 2-deoxyglucose (DG) was intravenously injected into rats, which were frozen 2 and 4 min later in liquid nitrogen. Freeze-dried samples of CNS regions and cell bodies of spinal motor neurons were prepared, and the concentrations of glucose, glucose 6-phosphate, DG, and DG 6-phosphate (DG6P) in them were microassayed after 3,000-1,500,000-fold amplification using an enzymatic amplification reaction, NADP cycling. Based on the time course of glucose, DG, and DG6P concentrations in arterial plasma and the anterior horn of the spinal cord, the Sokoloff-type rate equations for DG and DG6P concentrations were mathematically solved, and the resultant DG and DG6P concentration functions were fitted to the data points using the nonlinear least-squares fitting SALS package program. This fitting provided four rate constants for the functions and supported the theoretical basis for our calculations of glucose utilization rate (GUR) when DG was administered in nontracer amounts. The GUR was highest in the spinal motor neurons and lowest in the white matter of the cerebellum. Neuron-rich structures, such as the cerebellar molecular and granular layers and the anterior horn of the spinal cord, had higher GUR values than the white matter of the cerebellum and spinal cord.

Development of a polymerase chain reaction-probe test for identification of Alloiococcus otitis.

A rapid polymerase chain reaction test was developed for specific identification of the human middle ear pathogen Alloiococcus otitis. Primers for the enzymatic amplification reaction were designed from highly specific sequences within the 16S rRNA gene. In addition, a confirmatory test based on hybridization of the polymerase chain reaction products to a specific internal probe was developed.

Rapid identification of Aerococcus viridans using the polymerase chain reaction and an oligonucleotide probe

A polymerase chain reaction/oligonucleotide probe method was developed for the specific identification of the Gram-positive bacterium Aerococcus viridans. Primers for the enzymatic amplification reaction were designed from specific sequences within the 16S rRNA. The method was also highly sensitive and 10 cfu of A. viridans could be detected in 5 h although the reliability of detection was poor in mixed cultures with Escherichia coli.

An Enzymatic Microdetermination Method for Hydroxylase Cofactor

Abstract An enzymatic microdetermination method is described to determine the concentration of biologically active hydroxylase cofactor in crude samples. A small amount of hydroxylase cofactor was oxidized and reduced in a cyclic fashion by phenylalanine 4-monooxygenase (EC 1.14.16.1, PheOHase) and dihydropteridine reductase (EC 1.6.99.7, DPRase) in the presence of excess phenylalanine (Phe) and NADH. The amount of NAD+, accumulated as a result of this cyclic reaction (first cycling), was in proportion to that of the hydroxylase cofactor. The NAD+ was further amplified by an enzymatic amplification reaction, NAD cycling (second cycling) and determined fluorometrically. This double cycling method with more than a 20,000 fold amplification provided extremely high sensitivity, down to 0.02 pmol per assay, and was successfully applied to crude microsamples of pg wet weight.

Exploitation of the Flow Injection Approach for Analytical Procedures Based on Enzymatic Amplification Reactions

Abstract The concept of using the flow injection approach for determining small amounts of substrates by enzymatic amplification procedures is discussed. Substrate recycling is accomplished by incorporating into the FIA manifold a small column reactor with coimmobilized enzymes allowing the enzymatic degradation step and the ensuing detection of a suitable reaction product to be individually optimized. At a sampling frequency of ca. 60 samples per hour, initial investigations with a model system based on coimmobilized lactate

Prelude to summer products

This week's issue spotlights a set of levers for muscle research, thermal cyclers for enzymatic DNA amplification reactions, and an oven for cooking up high-temperature superconductors.

Glutamate decarboxylase activities in single vertebrate neurons.

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.

Choline acetyltransferase activities in single motor neurons from vertebrate spinal cords.

Single cell bodies of spinal motor neurons were isolated from freeze-dried sections of fresh spinal cords from six species of vertebrates. Single human neurons were also isolated from the spinal cords of three autopsy cases without neurological diseases. Choline acetyltransferase activity of these single neurons was determined by measuring acetyl-CoA formation from CoASH and acetylcholine by use of the enzymatic amplification reactions, CoA and NADP cyclings. The enzyme activity was unevenly distributed in the cytosol of spinal motor neurons of all species, but not measurable in rabbit dorsal root ganglion cells. The specific activity on a dry weight basis varied widely among the individual neurons from the species studied. The average activity was highest with rat neurons and lowest with yellowtail neurons. The neurons from cold-blooded animals (bullfrog and yellowtail) had about one-tenth the activity compared with the warm-blooded animals (cat, rabbit, rat, and hen). Human neurons, obtained under different morbid and post-mortem conditions with three autopsy cases, had very low activities corresponding to those of cold-blooded animals. Since the choline acetyltransferase activity lost from mouse brain after 11 h at 38 degrees C was 50%, the activity in human neurons was believed to actually be low in vivo.

Enzymatic determination of choline acetyltransferase by coenzyme A cycling and its application to analysis of single mammalian neurons.

An enzymatic assay for choline acetyltransferase was developed by measuring acetyl-coenzyme A (acetyl-CoA) formed from CoASH and acetylcholine (ACh). This method is extremely sensitive and may be applied to the analysis of microgram to nanogram crude samples. The method is, however, not useful when choline acetyltransferase is present in very low concentrations. The basis of this method is to amplify a small amount of synthesized acetyl-CoA in the assay mixture by using an enzymatic amplification reaction, CoA cycling. This amplification mechanism made it possible to perform microassays (13 nl-2.2 microliter of assay volume) of freeze-dried sections prepared from cerebral cortex, striatum, and hippocampus of mice and single cell bodies isolated from freeze-dried sections of rabbit spinal cords. These samples were weighed and added directly to the reaction mixture. The activities of the above cerebral regions, assayed with 1,500-2,000-fold amplification, corresponded well to the results previously reported by other workers. The average activity of single anterior horn cells, determined with 64,000-420,000-fold amplification, was 40-fold higher than that of rabbit cerebral cortex, and the specific activities on a dry weight basis were widely distributed among individual neurons. No activity was detected in the noncholinergic dorsal root ganglion cells or in cerebellar cortex.

Application of a galactosylceramidase microassay method to early prenatal diagnosis of Krabbe's disease

An enzymatic microassay method was devised to determine galactosylceramidase activities of microsamples prepared from primary cultures of amniotic fluid cells. A piece of freeze-dried cell mass (0.5–1 μg dry weight) prepared from a colony on the bottom plate of a culture flask, was weighed and incubated in 4.02 μl of the assay mixture. A small amount of galactose hydrolyzed from galactosylceramide was amplified about 10000-fold and determined by using an enzymatic amplification reaction, NAD cycling. Microsamples were also prepared from freeze-dried leukocytes and skin fibroblasts. The present method was applied to prenatal diagnosis using amniotic cells as a sample in a case of high-risk pregnancy for Krabbe's disease. The result was confirmed by analysis of fibroblasts and organs from the aborted fetus.

Enzymatic determination of galactosylceramide galactosidase in tissues by NAD cycling

An enzymatic determination method for galactosylceramide galactosidase (EC 3.2.1.46) was devised by using an enzymatic amplification reaction, NAD cycling. Galactose released by crude enzyme samples (tissue homogenates and cell suspensions) from galactosylceramide quantitatively reduced NAD to NADH by the galactose dehydrogenase reaction; then the NADH was amplified 6000-10,000-fold by NAD cycling and determined fluorometrically. A higher sensitivity of assay was obtained compared with the previous radiometric method. The present method was successfully applied to tissues from patients with Krabbe's disease, whose organs are deficient in galactosidase. The galactosidase reaction rate with a crude sample was not proportional to its concentration. However, the double-reciprocal plot of the reaction rate against the sample concentration became linear and provided a unique value of specific activity to each sample.

Ultramicro analysis of enzymes and substrates by enzymatic amplification reactions "Enzymatic cycling"

Abstract