Application of a microbial sensor to the quality control of meat freshness.

Samples of fresh meat stored at 5 degrees C were periodically removed from storage and washed with water for periods of up to 2 weeks. The amount of amino acids, polyamines and viable counts (number of bacteria) in the washed water were measured by using an HPLC system and a colony counting method. At the same time, the washed water was charged into a flow injection analysis (FIA) system combined a microbial sensor using yeast (Trichosporon cutaneum), which was developed in this work for monitoring the freshness of meat. A relationship between the sensor signals obtained by the FIA system and the amounts of polyamines and amino acids produced from the meat and the number of bacteria which had multiplied in the meat during the aging process was investigated. The sensor signal was found to correspond to increases in amino acid levels and viable counts in the meat with the storage time in the course of the first stage of aging. This is due to the fact that amino acids produced initially by enzymes in the meat serve as a source of nutrition for septic bacteria during the aging process, and as a result, the level of bacterial cells increases with increasing amounts of amino acids with the passage of days. A good correlation, with a correlation factor of 0.908, was obtained between the sensor signal and viable counts obtained by the colony counting method. The present sensor method was more sensitive than the colony counting method at the early stage of the aging process, where viable counts were in the vicinity of 10(4) g(-1).

Determination of hardness in tapwater and upland soil extracts using a long-term stable divalent cation selective electrode based on a lipophilic acrylate resin as a membrane matrix.

A divalent cation-selective electrode, which utilizes a lipophilic resin as a matrix for the sensing membrane, and which has long-term stability has been developed. The sensing membrane is a lipophilic acrylate resin which is impregnated with a solution of 1-decylalcohol and the calcium salt of bis[4-(1,1,3,3-tetramethylbutyl) phenyl] phosphate at concentrations of 0.08 g ml(-1) each. The electrode exhibited nearly equal selectivity to Ca(2+) and Mg(2+) ions and could be used as a water hardness sensor. The electrode shows a Nernstian response with a slope of 29 mV decade(-1) to both Ca(2+) and Mg(2+) ions in the concentration range from 10(-5) M to 10(-1) M and could be used in the pH range from 3 to 10 for the determination of 10(-3) M Ca(2+) and Mg(2+) solutions. The initial performance of the electrode could be maintained for 1 year, since the lifetime test of the electrode was conducted in tapwater at a continuous flow rate of 4 ml min(-1). The hardnesses of tapwater and upland soil extracts were determined using the developed electrode and the analytical results were in good agreement with those obtained by chelatometric titration using an EDTA solution as the titrant. A coefficient factor of correlation 0.998 was obtained between the electrode method and titrimetry. The long-term stability of the electrode was found to be due to strong affinity of 1-decylalcohol to the lipophilic acrylate resin.

Successful radiofrequency catheter ablation from the supravalvular region of the aortic valve in a patient with outflow tract ventricular tachycardia.

Outflow tract ventricular tachycardia (OT-VT) was successfully ablated from the right coronary cusp of the aortic valve. The 12-lead ECG was totally different from the typical right ventricular OT-VT because the R/S ratio in precordial lead V1 was equal to 1 and tall R waves in precordial leads V2-6 were seen. Radiofrequency energy application from the right coronary cusp of the aortic valve successfully ablated this VT without complications. Radiofrequency catheter ablation from the right coronary cusp of the aortic valve can be done safely and effectively.

Electrocardiographic characteristics of left ventricular outflow tract tachycardia.

Catheter ablation of idiopathic left ventricular outflow tract tachycardia (LVOT-VT) is rare because a safe ablation technique at this site has not been described, and serious complications may occur. This study compared the QRS morphology of LVOT-VT with that of idiopathic right ventricular outflow tract tachycardia. A comparison was made between the electrocardiographic characteristics of LVOT-VT originating from the supravalvular region of a coronary cusp (Supra-Ao group) with those of LVOT-VT originating from the infravalvular endocardial region of a coronary cusp of the aortic valve within the LV (Infra-Ao group). After precise mapping of the right ventricle, left ventricle, pulmonary artery, coronary cusps, and proximal portion of the anterior interventricular vein, there were 17 patients in whom VT was thought to be located at the LVOT by both activation and pace mapping. They were divided between a Supra-Ao group (n = 8), and an Infra-Ao group (n = 9). Analysis of the 12-lead electrocardiogram (ECG) revealed an S wave in lead I in all 17 patients. A precordial R wave transition was also observed at V1 or V2 in 16 patients (94%). In 7 of 8 patients (88%) with Supra-Ao LVOT-VT, no S wave was observed in either V5 or V6. In contrast, an Rs pattern was observed in both V5 and V6, or in V6 only, in 100% of the patients with Infra-Ao LVOT-VT. A LVOT-VT should be suspected when the ECG shows an S wave in lead I and an R/S ratio greater than 1 in lead V1 or V2, versus a coronary cusp location if there is no S wave in either lead V5 or V6.

Continuous monitoring for cyanide in waste water with a galvanic hydrogen cyanide sensor using a purge system.

A continuous monitoring system for cyanide with a galvanic hydrogen cyanide sensor and an aeration pump for purging was developed. Hydrogen cyanide evolved from cyanide solution using a purging pump was measured with the hydrogen cyanide sensor. The system showed good performance in terms of stability and selectivity. A linear calibration curve was obtained in the concentrating range from 0 to 15 mg dm(3) of cyanide ion with a slope of -0.24 microA mg(-1) dm(-3). The lower detection limit was 0.1 mg dm(-3). The 90% response time of the sensor system was within 3.5 min for a 0.5 mg dm(-3) cyanide solution, when the flow rate of the purging air was 1 dm(3) min(-1). The system maintained the initial performance for 6 months in the field test. The developed galvanic sensor system was not subject to interference from sulfide and residual chlorine, compared with a potentiometric sensor system developed previously. The analytical results obtained by the present system were in good agreement with those obtained by the pyridine pyrazolone method. The correlation factor and regression line between both methods were 0.979 and Y=2.30 x 10(-4)+1.12X, respectively. This system was successfully applied for a continuous monitoring of cyanide ion in waste water.

Catheter ablation of ventricular tachycardia in patients with right ventricular dysplasia: Identification of target sites by entrainment mapping techniques.

OBJECTIVE: To identify target sites for radiofrequency ablation of ventricular tachycardia (VT) by entrainment mapping techniques in patients with arrhythmogenic right ventricular dysplasia. METHODS: Entrainment mapping and radiofrequency ablation of eight VTs was performed in seven patients. Radiofrequency ablation was applied at 31 reentry circuits sites that were classified based on findings during entrainment. RESULTS: By entrainment criteria the 31 sites were classified as: exit sites (n = 12), proximal sites (n = 6), and outer loop sites (n = 13). Radiofrequency current application terminated VT at 7 of 31 sites: 2 of 12 exit sites (17%), 4 of 6 proximal sites (67%), and 1 of 13 outer loop sites (8%). CONCLUSION: Radiofrequency ablation terminated VTs most often at sites proximal to the exit as opposed to outer loop sites and exit sites (P = 0.05). The critical isthmus for ablation of VT in right ventricular dysplasia often may be distant to the exit.

Flow injection analysis for residual chlorine using Pb(II) ion-selective electrode detector.

A simple flow injection analysis (FIA) system for residual chlorine in tap water has been developed by using a Pb(II) ion-selective electrode (ISE) detector. The method is based on a specific response of the Pb(II)-ISE to residual chlorine. The FIA system consists of a millivolt meter, a peristaltic pump, a Pb(II)-ISE detector and a recorder. A linear working curve between peak height and concentration of residual chlorine was obtained from 0.1 to 1 mg l(-1) for the developed FIA system. The relative standard deviation for repeated injections of a 0.2 mg l(-1) residual chlorine sample was 2%. The regression line and its correlation factor between the conventional o-tolidine colorimetric method and the present method were Y=0.75X+0.17 and 0.967, respectively, for this determination.

Development of pH measurement system for legal traceability of pH standard solutions.

The best reproducible technology of pH measurement for precise pH buffer solutions regulated by Japanese Industrial Standards (JIS) was studied. A pH meter was devised with a high resolution of +/- 0.0001 pH. An 18-bit analog-to-digital converter is used, one-bit resolution corresponding to 0.0019 mV (ca. 0.000032 pH) against an input electrode potential +/- 500 mV. Digital data were treated smoothly for some types of noise, a reproducibility of +/- 0.0002 pH being obtained with a potentiometer. A flow cell was devised to attain temperature control within +/- 0.03 degrees C and air-tight measurement prevented contamination with carbon dioxide. Also, the flow cell has a structure such that potassium chloride (KCl) inner solution effused from a ceramic junction of the reference electrode designed so as not to touch the glass membrane. A combination pH electrode (a glass electrode and a reference electrode) was assembled to minimize the dead volume of sample solution. This highly sensitive pH measuring system, consisting of a pH meter, a flow cell, a combination pH electrode, a circulating water thermostat and a peristaltic pump, was used for the certification of pH standard solutions in Japanese metrological law. The performance of this system was within +/- 0.0006 pH reproducibility and 20-30 min response time (5 min within +/- 0.0002 pH) at a sample flow rate of 3 ml min (-1).

Improvement of the silver/silver chloride reference electrode and its application to pH measurement.

When a silver/silver chloride (Ag AgCl ) reference electrode was used continuously in a low conductivity solution or reductive solution, it was often observed that stability of the liquid junction potential was lost. This phenomenon was remarkable with a Ag AgCl reference electrode compared to a calomel reference electrode. We found that 340 mg l(-1) of silver was dissolved in 3 M potassium chloride (KCl) internal solution as silver complex ions (AgCl(-(x-1))(x)) for x = 2 or 3. However, only 1.93 mg l(-1) of silver chloride (AgCl) can theoretically be dissolved in water. The complex ion that effused into the sample solution through the liquid junction clogged the liquid junction (e.g. porous ceramic) as AgCl, or as metallic silver (Ag) in reducing solution. Therefore, the constant effusion of KCl internal solution was inhibited, and the liquid junction potential became unstable or fluctuating. A new reference electrode was developed, which can eliminate AgCl(-(x-1))(x) in 3 M KCl internal solution by the use of chelating resins. A combination of this reference electrode with a pH electrode made long-term stable pH measurements possible.

Determination of sound speed in biological tissues based on frequency analysis of pulse response.

The sound speed in biological tissues provides important diagnostic and treatment planning information. Conventional methods of sound-speed determination generally require that transducers make physical contact with specimens in order to measure thickness and travel time in the time domain. The physical contact may cause deformation and affect blood flow and the measurement of travel time in the time domain may be sensitive to waveform distortion due to tissue inhomogeneity and surface roughness. A method for determination of the sound speed is proposed in which the sound travel time in the sample and the difference in total travel time from the transducer to the rigid reflector due to the presence of the sample are estimated in the frequency domain and which does not require physical contact of ultrasonic probes to living or freshly excised tissue specimens. Ultrasonic speed measurements in silicone rubber and acrylic resin specimens verified the method validity. The standard deviation of the measurements over a 10- x 10-mm area is less than 4 m/s. Sound-speed distribution measurements of porcine muscle are in agreement with previously published results.

Pulmonary intralobar sequestration accompanied by aneurysm of an anomalous arterial supply.

A 47-year-old woman presented with hemoptysis and her chest X-ray films showed an opacity suggesting a mass in the left lower lung field. Based on radiographic investigations, the mass was diagnosed as an aneurysm develop in an anomalous vessel and was considered to be a Pryce type I pulmonary intralobar sequestration. Resection of the left lower lobes was performed and the aneurysm was found to be filled with thrombus. It is rare for an aneurysm to form in an aberrant vessel. This complication may have been the result of regional sclerosis affecting the anomalous artery as well as systemic atherosclerosis.

Expression of human factor IX in rat capillary endothelial cells: toward somatic gene therapy for hemophilia B.

In aiming to develop a gene therapy approach for hemophilia B, we expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression (0.84 and 3.6 micrograms per 10(6) cells per day for CECs and NIH 3T3 cells, respectively) as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX of 68 kilodaltons identical to purified plasma factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including gamma-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number of cells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.

Prothrombin Himi; an abnormal prothrombin characterized by a defective thrombin activity.

An abnormal prothrombin has been detected in a 26-year-old female, who had no history of excessive bleeding. Prothrombin activity was approximately 10% when measured using either the classical one-stage assay or the assay with Echis carinatus venom, whereas prothrombin antigen level was normal. In keeping with current nomenclature practices, the abnormal prothrombin was designated "Prothrombin Himi". The electrophoretic behavior and calcium binding properties of Prothrombin Himi did not differ significantly from normal. Prothrombin Himi was isolated by chromatography on Q-Sepharose. Electrophoretic migration of the purified abnormal prothrombin on SDS-PAGE was normal. Upon prothrombin activation by Echis carinatus venom, the clotting activity produced from Prothrombin Himi was only 37% of the normal level after 90 minutes of the activation time, where as the amidolytic activity was almost the same as normal. The cleavage patterns of Prothrombin Himi by factor Xa or Echis carinatus venom investigated by SDS-PAGE, were found to be normal. These results indicate that Prothrombin Himi was characterized by a defective thrombin enzymatic activity.

Intracellular pathways of insulin transport across vascular endothelial cells.

Processing and transport of hormones across vascular endothelial cells may modulate hormone action at subendothelial tissue sites. Insulin was transported across cultured rat capillary and bovine aortic endothelial cells, after a delay of 5-10 min, at a constant rate for 60 min at 37 degrees C. 125I-labeled insulin transport was inhibited by 88 +/- 11% (SE, n = 4) and 75 +/- 18% (SE, n = 4) in the presence of anti-insulin receptor antibody and unlabeled insulin (at 10(-7) M), respectively. Reverse phase high-performance liquid chromatography showed 88% of the 125I-insulin transported over 60 min was indistinguishable from the 125I-insulin added to the cells at 4 degrees C. In aortic endothelial cells preincubated with 2.3 x 10(-9) M of insulin for 24 h, insulin receptor binding was downregulated by 67%, and 125I-insulin transport was decreased by 52 +/- 11%. The proton ionophore monensin (0.05 mM) increased the internalized insulin in bovine aortic endothelial cells by 78%, with a corresponding decrease in 125I-insulin released by 76 +/- 2% (SE, n = 4). 125I-insulin transport across the aortic endothelial cell monolayer was similarly decreased (54 +/- 12%, SE, n = 4) by monensin. In contrast, the lysosomal protease inhibitor leupeptin had no effect. Degradation and transport were similarly dissociated by low temperature. At 15 degrees C, no significant insulin degradation was detected, whereas 125I-insulin release from the cells continued at 30 +/- 3% of the rate at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of insulin receptor internalization in vascular endothelial cells by insulin and phorbol ester.

Phorbol 12-myristate 13-acetate (PMA) was used to examine the role of insulin receptor phosphorylation in the regulation of insulin receptor internalization in vascular endothelial cells. Association of 125I-insulin in rat capillary and bovine aortic endothelial cells preincubated with PMA was increased by 80 and 64% over control, respectively. The increase was due to enhanced 125I-insulin internalization as opposed to an effect on surface-bound hormone. PMA had no significant effect on 125I-insulin degradation or on release of internalized insulin from the cells. Internalization of 125I-labeled insulin receptor was determined by the resistance of labeled receptor to trypsinization. At 10 degrees C, nearly all of the labeled receptor was sensitive to removal by trypsin, indicating that it was exposed on the cell surface. Exposure of labeled cells to insulin (100 nM) at 37 degrees C resulted in the rapid appearance of trypsin-resistant insulin receptor, indicating receptor internalization. Steady state for receptor internalization was attained at 10-15 min. When surfaced-labeled cells were preincubated with PMA at 37 degrees C, the rate of insulin receptor internalization was increased by 3.6 +/- 0.2-fold and 2.1 +/- 0.5-fold at 1 and 5 min of insulin exposure, respectively (ED50 at 16 nM PMA). This effect of PMA was associated with an increase in serine phosphorylation of the insulin receptor. Thus, PMA increased insulin internalization in the endothelial cells by modulating the insulin-induced internalization of the receptor. The additive effects of PMA and insulin on insulin receptor phosphorylation suggest that the phorbol ester and insulin act via independent signaling mechanisms.

Altered insulin distribution and metabolism in type I diabetics assessed by [123I]insulin scanning.

Scintigraphic scanning with [123I]insulin provides a direct and quantitative assessment of insulin uptake and disappearance at specific organ sites. Using this technique, the biodistribution and metabolism of insulin were studied in type 1 diabetic patients and normal subjects. The major organ of [123I]insulin uptake in both diabetic and normal subjects was the liver. After iv injection in normal subjects, the uptake of [123I]insulin by the liver was rapid, with peak activity at 7 min. Activity declined rapidly thereafter, consistent with rapid insulin degradation and clearance. Rapid uptake of [123I]insulin also occurred in the kidneys, although the uptake of insulin by the kidneys was about 80% of that by liver. In type 1 diabetic patients, uptake of [123I]insulin in these organ sites was lower than that in normal subjects; peak insulin uptakes in liver and kidneys were 21% and 40% lower than those in normal subjects, respectively. The kinetics of insulin clearance from the liver was comparable in diabetic and normal subjects, whereas clearance from the kidneys was decreased in diabetics. The plasma clearance of [123I]insulin was decreased in diabetic patients, as was insulin degradation, assessed by trichloroacetic acid precipitability. Thirty minutes after injection, 70.9 +/- 3.8% (+/- SEM) of [123I]insulin in the plasma of diabetics was trichloroacetic acid precipitable vs. only 53.9 +/- 4.0% in normal subjects. A positive correlation was present between the organ uptake of [123I]insulin in the liver or kidneys and insulin degradation (r = 0.74; P less than 0.001). Both decreased insulin uptake in the liver and kidneys and decreased insulin degradation were inversely correlated to the binding capacity of antiinsulin antibodies in plasma of diabetics (r = -0.87 to -0.92; P less than 0.001). In summary, type 1 diabetic patients have altered patterns of insulin biodistribution and metabolism, with decreased organ uptake and slow degradation of [123I]insulin, which correlated with the insulin antibody-binding capacity of their serum. Thus, antiinsulin antibodies, even at subclinical concentrations, may modulate the metabolic effects of insulin in the diabetic by prolonging the biological half-life of the hormone as well as by altering its distribution and uptake at specific organ sites.

Comparative studies on insulin-like growth factor II and insulin processing by vascular endothelial cells.

Processing of circulating polypeptide hormones by vascular endothelial cells may be critical to hormone transport from the vascular lumen to tissue sites of action. The comparative processing of insulin-like growth factor II (IGF II) and insulin by cultured bovine aortic endothelial cells was examined. These cells possess high-affinity receptors for IGF II and insulin, as shown by competitive-binding studies. At 37 degrees C, internalization determined by both resistance to an acid wash and electron microscopy was rapid with 50-70% of bound IGF II and insulin internalized at 60 min. Subsequently, between 70 and 75% of the internalized hormones were released from the cells within 60 min. Although most of both hormones were released intact, degradation of IGF II was greater than that of insulin by two- to threefold, as assessed by G-50 chromatography and trichloroacetic acid precipitability. Leupeptin, a specific lysosomal protease inhibitor, increased cell-associated 125I-labeled IGF II by 53.0 +/- 6.0% and decreased degradation by 55%; however, it was without effect on 125I-labeled insulin. Chloroquine and monensin, which act at the lysosomes and at other sites, increased both cell-associated IGF II and insulin and decreased the degradation of both hormones. The increases in cell-associated 125I-IGF II produced by chloroquine (42.0 +/- 7.4%) and monensin (78.3 +/- 8.5%) were quantitatively similar to the decreases in IGF II degradation caused by the agents; however, the increase in cell-associated insulin was approximately threefold greater than could be accounted for simply by decreased insulin degradation.(ABSTRACT TRUNCATED AT 250 WORDS)