Oxygen affinity and electrolyte distribution of human blood: changes induced by propranolol.

Propranolol causes a massive leakage of potassium ions from red cells, which results in an alteration of the Gibbs-Donnan equilibrium across the red cell membrane. According to such a mechanism, the presence of propranolol significantly increases the hydrogen ion activity of the interior of the red cell, causing a decreased oxygen affinity of hemoglobin according to the classical Bohr effect. No release of 2,3-diphosphoglycerate which may be bound to the membrane is thus necessary to explain the effect of propranolol on the oxygen dissociation curve of blood.

Technical note: Improved method for rapid DNA extraction of mastitis pathogens directly from milk.

Efficient control against bovine mastitis requires sensitive, rapid, and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen. Therefore, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment.

Electrode measurement of oxygen tension with 1-ms time resolution.

A continuous flow device utilizing a Clark oxygen electrode was constructed; this device had a dead time and resolution of 1 ms. Mixing was tested by observing the neurtralization of acid with base, and at the maximal flow rate, the mixing was 94% complete within 1 ms and better than 98% complete within 2 ms after initial mixing. Observation o of the oxygenation of hemoglobin gave data which agreed with previous data obtained by a stopped-flow optical experiment. The respiration of phosphorylating submitochondrial particles was measured utilizing this device. The burst of respiration in submitochondrial particles was triphasic, with a very rapid burst lasting some 60 ms, followed by a longer burst of respiration lasting more than 4 s.

Solid phase purification and SSCP analysis of amplified genomic DNA by capillary electrophoresis.

Detection and identification of point mutations in genomic DNA has proven increasingly important in biomedical research. A variety of methods for the analysis of single base substitutions have been proposed among which Single Strand Conformational Polymorphism (SSCP) quickly gained success due to its simplicity. In this work we present an analytical on-line tool which combines the ease of solid phase purification of amplified genomic DNA, the simplicity of SSCP and the significant potential advantages offered by capillary electrophoresis (CE).

Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer.

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.

Fluorescence-based automated DNA sequencing by limited primer labeling.

The applicability of automated DNA sequencing systems to sequencing strategies that require a large number of primers is limited by the necessity for expensive fluorescence-labeled oligonucleotides. Here we present a simple procedure that allows the use of unlabeled oligonucleotides to perform fluorescence-based DNA sequencing. This method is based on a limited primer extension that incorporates three deoxynucleotides, one of which carries a fluorescent moiety. The elongated fluorescent primer is then used in a standard T7 sequencing reaction. This labeling procedure is both economical and straightforward and offers a valid alternative to current fluorescence-labeling protocols. Results of this method with different DNA templates demonstrate the reliability of the protocol.

Anion-exchange HPLC analysis of biotinylated oligonucleotides.

Biotinylated oligonucleotides combined with streptavidin-coated magnetic beads are commonly used in current molecular biology. Their quality and the level of incorporated biotin are essential for yielding good results in either solid-phase DNA sequencing or solid-phase purification procedures. This paper presents a very simple analytical test using anion-exchange HPLC and avidin to ascertain the quality of biotinylated oligonucleotides and to predetermine their ability to bind to avidin, which is a prerequisite for functionality in some solid-phase methods.

Mixed-mode fluorescent DNA sequencing.

Fluorescence-based, automated DNA sequences represent one of the major advances in recent molecular biology. Two main technologies have been developed in this field: the single-label/four-lane system and the four-label/one-lane system. The following present the use of single-label-sequencing chemistry, which resembles traditional radioactive DNA sequencing, using the four-label system ABI 373A that expands its flexibility and obtains data that are immediately interpretable without software manipulation. This method has been named mixed-mode fluorescent DNA sequencing. Here we show one of its possible applications in molecular genetic analysis.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 1).

The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 2).

The aim of this study was to evaluate the reproducibility, the accuracy and the reliability of a continuous subcutaneous glucose measuring system. The GlucoDay system (A. Menarini I.F.R. S.r.l.-Florence, Italy) is a portable instrument provided with a micro-pump and a biosensor, coupled to a microdialysis system (see part 1). This instrument has demonstrated high reliability coupled with a low degree of invasivity. The profiles of glucose monitoring allow to achieve an excellent knowledge of the real variation of glucose in diabetic patients. The reproducibility study showed a bias lower than 10% between instruments. The accuracy study showed a difference from the reference method lower than 15%.

Rapid potentiometric determination of cholinesterases in plasma and red cells: application to eptastigmine monitoring.

Eptastigmine (MF 201) is a new physostigmine derivative with potent inhibitory activity on cholinesterases. Here we present a new potentiometric cholinesterase activity assay suitable for MF 201 monitoring. The analysis is performed on a differential pH system and has the following characteristics: (a) within-run precision: C.V. 2.0% (plasma cholinesterase), 1.8% (red cell cholinesterase); (b) between-run precision: C.V. 4.0% (plasma cholinesterase); (c) linearity: 1-10 kU/l (plasma cholinesterase), 6-70 U/g Hb (red cell cholinesterase); (d) comparison with a reference method (x, HITACHI 737 Boerhinger Mannheim, Italy): y = 0.785x - 0.07; n = 37; r = 0.998. The assay has been applied to the determination of plasma and red cell cholinesterase activity in volunteers over 60 years of age treated with a single oral dose of 30 mg eptastigmine. We found that red cell cholinesterase is selectively inhibited after MF 201 administration with the following kinetics (time, % of inhibition, mean +/- S.E., n = 6): 0 h, 0; 1 h, 17 +/- 4.6; 2 h, 24 +/- 4; 4 h, 23 +/- 4.4; 12 h, 14 +/- 3. Eptastigmine plasma levels were also determined by a HPLC method: maximum concentration was found one hour after drug administration.

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30 degrees C and correlate well with the manual spectrophotometric method of Ellman for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient.

A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.

Urea determination in dialysis, based on a differential pH technique.

The application of a new technique, based on differential measurements of pH, to determine urea concentration in patients of a dialysis center, is reported. Urea in plasma, whole blood or dialysis fluids is measured by an enzymatic reaction, with urease; the procedure, requiring 10 microL of sample, is simple, fast and correlates well with a reference spectrophotometric method, in the 0-300 mg/dL concentration range, according to the equation y = 1.0291 X -0.0777; r = 0.9991; n = 73.

Introduction of magnetic beads in diagnosis: a simple and rapid method to detect mutations of beta globin gene, directly amplified from blood.

In this communication we present the results obtained by the use of magnetic beads in diagnosis, for the identification of genetic variants at the molecular level by sequencing, in comparison with the more laborious method of the production of ssDNA with asymmetric PCR. We compared the two techniques studying variants of beta globin gene: Hb Abruzzo [beta 143 (H21) His -> Arg] and Hb D Los Angeles [beta 121 (GH4) Glu -> Gln].

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese.

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.