Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.     

Rapid isolation of triosephosphate isomerase utilizing high-performance liquid chromatography

A new method for the isolation of homogeneous triosephosphate isomerase (TPI, EC 5.3.1.1) has been developed. The method utilizes high-performance liquid chromatography on DEAE 5PW and Hydrophase-polyethyleneimine columns, which results in the rapid isolation and essentially quantitative recovery of the enzyme. The procedure is superior to previous methods with respect to specificity, recovery, and time. In addition, this rapid process minimizes the potential for postsynthetic modifications of the protein. Milligram quantities of TPI can be isolated from 100 g of tissue.     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Pulmonary microcirculatory kinetics of neutrophils deficient in leukocyte adhesion-promoting glycoproteins

The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.     

Convective heat transfer measured directly with a heat flux sensor

A heat flux disk has been developed that directly measures the convective heat transfer in W/m2. When the sensor is calibrated on an aluminum cylinder, the calibration constant obtained is greatest in still air. As air movement increases, the calibration constant is reduced with increasing convective heat transfer coefficient, 0.5%.W-1.m2.K. The influence of wind on the calibration value is greatly reduced when the sensor is attached to a surface with lower thermal conductivity. The local convective heat transfer coefficient (hc) of the human body was measured. The leg acts in a manner similar to that of a cylinder, with the highest hc value at the front facing the wind and the lowest approximately 90 degrees from the wind, and in the wake a value is obtained that is close to the average hc value of the leg. When hc is measured at several angles and positions all over the body, the results indicate that the body acts approximately as a cylinder with a hc value related to the wind speed as hc = 8.6.v0.6 W.m-2.K-1, where v is velocity.     

A statistical power analysis of the 'internal reference' technique for comparing growth and growth depensation of tilapia strains

Experiments were conducted to compare the growth and growth compensation of three strains of juvenile Oreochromis niloticus . Ten full sib families (10 replicates) per strain were split and grown under crowded and uncrowded conditions for 3 weeks (the treatment). Both treatments were then grown an additional 12 weeks under less crowded conditions (the compensation). Standard length measurements were made at the end of crowding and the end of compensation. Each replicate included a size-matched control population of a fourth (red) reference strain. ANCOVA with the reference strain used as a cofactor revealed significant strain effects on specific growth throughout the experiment. The reference strain removed most of the random among-replicate error variance as shown by an increase of r 2 from 0.06 to 0.91 when it was included in the statistical models. If the reference fish had not been used, approximately 450 replicate families would have been needed to achieve the sensitivity of the present experiment (a difference of 7% among strains significant at P =0.05). We conclude that the CLSU strain grows significantly more slowly than the Israel and NIFI strains under the experimental conditions, that the crowding effect was essentially eliminated after 12 weeks of compensation, and that the reference strain greatly improved the resolution of the strain-testing experiment.     

Metabolism of [U-13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling

Abstract: Metabolism of [U-¹³C₅]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify ¹³C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-¹³C₅]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-¹³C₅]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-¹³C₂]glutamate was observed in astrocytes incubated with [U-¹³C₅]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using ¹³CO₂ produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.     

Identification and Determination of 3,4-Dihydroxyphenylacetaldehyde, the Dopamine Metabolite, in In Vivo Dialysate from Rat Striatum

Abstract: 3,4-Dihydroxyphenylacetic acid (DOPAC) is commonly considered to be the main dopamine (DA) metabolite produced by monoamine oxidase (MAO); however, the initial product of DA oxidation is 3,4-dihydroxyphenylacetaldehyde (DOPALD). Owing to technical difficulties in detecting DOPALD from a biological matrix, no studies have so far been performed to measure brain levels of this aldehyde in vivo. In this work, using transstriatal microdialysis in freely moving rats, we identified DOPALD by HPLC coupled to a coulometric detector. In chromatograms obtained from microdialysis samples, DOPALD appeared as a peak with a retention time coincident with that of the standards obtained via enzymatic and chemical synthesis. On the other hand, DOPALD was undetectable ex vivo from rat striatal homogenates. This discrepancy is probably due to the preferential extraneuronal localization together with the high reactivity of the aldehyde, which is rapidly removed by the dialysis probe, whereas the ex vivo procedure allows its condensation and enzymatic conversion. Measurement of DOPALD levels as a routine procedure might represent a reliable tool to evaluate DA oxidative metabolism directly, in vivo. Moreover, parallel detection of DOPALD and DOPAC levels in brain dialysate may make it possible to distinguish between the activity of MAO and aldehyde dehydrogenase. DOPALD, like many endogenous aldehydes, has been shown to be toxic to the cell in which it is formed. Therefore, in vivo measurement of DOPALD levels could highlight new aspects in the molecular mechanisms underlying both acute neurological insults and neurodegenerative diseases.     

Genetics of intracisternal-A-particle-related envelope-encoding proviral elements in mice.

Intracisternal-A-particle-related envelope-encoding (IAPE) proviral elements in the mouse genome encode and express an envelope-like protein that may allow transmission of IAPEs as infectious agents. To test IAPE mobility and potential transmission in mice, we have analyzed the distribution of IAPE elements in the genomes of Mus spretus and Mus musculus inbred strains and wild-caught animals. Potential full-length (IAPE-A) proviral elements are present as repetitive copies in DNA from male but not female animals of M. musculus inbred strains and Mus musculus castaneus. Analysis of IAPE-cellular junction fragments indicates that fixation of most IAPEs in the germ line occurred in M. musculus and M. spretus after speciation but before M. musculus inbred strains were derived.     

Molecular evidence of a lignin peroxidase H8 homologue inFusarium oxysporum

We describe an evidence for the existence of a ligninase isoenzyme H8 in the deuteromyceteFusarium oxysporum on the genomic as well as on the RNA level.