The pyridinium cross-links of collagen, pyridinoline (Pyd) and deoxypyridinoline (Dpd), provide structural integrity and rigidity to collagen fibrils in bone. During bone degradation (resorption), the cross-links are released into the circulation and eventually excreted in urine. Pyridinium cross-link measurements in urine have been shown to be sensitive and specific indicators of resorption by both established HPLC and newer enzyme immunoassay (EIA) techniques. We have developed a monoclonal antibody that preferentially binds to the non-peptide-bound free forms of Pyd & Dpd. We have incorporated the antibody conjugated to alkaline phosphatase in a competitive EIA by using Pyd-coated microtiter strip wells. After a 3-h incubation of sample and antibody-enzyme conjugate, color is developed for 1 h with p-nitrophenyl phosphate as the substrate. The intraassay (n = 52) CVs were 3.0-7.6%, and interassay (n = 8) CVs were 6.1-7.4%. Comparisons of the assay (y) with HPLC (x) and a polyclonal antibody-based EIA (x') gave regression equations of y = 0.46x + 4, r = 0.96, and y = 0.56x' + 8, r = 0.96. The EIA detected increased Pyd & Dpd concentrations in urine from postmenopausal women and patients with osteoporosis, hyperthyroidism, hyperparathyroidism, and Paget disease of bone. EIA concentrations also reflected the reduction in Pyd&Dpd excretion resulting from estrogen replacement in surgically menopausal women. Measurement of pyridinium cross-links with this simple EIA appears to provide an accurate index of the rate of resorption and may be useful for metabolic bone disease assessment and monitoring the effects of antiresorptive therapy.

Adult↗

PubMed1995

Endothelin-1-induced in vitro cerebral venoconstriction is mediated by endothelin ETA receptors.

The in vitro effects of endothelin-1 on cerebral veins were studied using cylindrical segments, 5 mm long, from dog pial veins. Isometric responses to endothelin-1 (10(-12)-10(-7) M) and to the endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21), 10(-12) -10(-7) M), were recorded in veins under control conditions and pretreated with the endothelin ET(A) receptor antagonist, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp), 10(-8) -10(-5) M), and the endothelin ETB receptor antagonist, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(me thoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium, 10(-6) and 10(-5) M). The response to endothelin-1 was also recorded in veins pretreated with the nitric oxide synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), or the cyclooxygenase inhibitor, meclofenamate (10(-5) M), and in veins without endothelium or placed in medium without Ca2+ but with EDTA (0.1 mM). In control veins, endothelin-1 produced a concentration-dependent contraction (EC50 = 2.0 x 10(-10) M; maximal contraction = 113 +/- 6 mg) and IRL 1620 induced no effects or a small contraction only with high concentrations (10(-8) - 10(-6) M) (EC50 = 1.5 x 10 (-8) M; maximal contraction = 9 +/- 3 mg). BQ-123 shifted the response to endothelin-1 to the right in a parallel, concentration-dependent way, whereas BQ-788, L-NAME or meclofenamate did not modify the response to endothelin-1. Compared with the control, veins in a medium without Ca2+ had similar EC50 values, but a lower maximal contraction induced by endothelin-1 (57 +/- 10 mg, P < 0.05), and veins without endothelium exhibited similar EC50 values. Thus, endothelin-1 produces marked cerebral venoconstriction that could be mainly mediated by activation of endothelin ETA receptors, may be dependent on extracellular Ca2+, and may be independent of endothelium, nitric oxide and prostanoids.

Animals↗

PubMed1995

Cerebral reactive hyperaemia and arterial pressure in anaesthetized goats.

The effects of arterial pressure on cerebral reactive hyperaemia were studied in anaesthetized goats measuring electromagnetically middle cerebral artery flow and performing arterial occlusions of 5-30 s. Under normotension (mean arterial pressure, MAP = 11 +/- 0.3 kPa), reactive hyperaemia (peak hyperaemic flow to control flow and repayment to debt ratios) increased, and cerebrovascular resistance during peak hyperaemic flow decreased, as ischaemia duration lengthened; the virtual maximal changes were obtained after 20 s ischaemia. During hypertension by aorta constriction (MAP = 18 +/- 0.7 kPa) or by i.v. infusion of noradrenaline (MAP = 19 +/- 0.8 kPa) middle cerebral artery flow did not change significantly and cerebrovascular resistance increased 25 and 46%, respectively (P < 0.05). During both types of hypertension reactive hyperaemia was over 50% higher, and the decrement in cerebrovascular resistance during peak hyperaemic flow was also higher, than under normotension. During hypotension by constriction of the inferior vena cava (MAP = 5 +/- 0.5 kPa) or by i.v. infusion of isoproterenol (MAP = 6 +/- 0.5 kPa), middle cerebral artery flow decreased 35% or did not change, and cerebrovascular resistance decreased 41 and 45%, respectively (P < 0.05). In these conditions, reactive hyperaemia and the decrement in cerebrovascular resistance during peak hyperaemic flow were reduced 80%, and it was similar in both types of hypotension. The absolute levels of cerebrovascular resistance obtained during peak hyperaemia were similar during normotension, hypertension and hypotension. Thus, arterial pressure is a main determinant of postocclusive cerebral reactive hyperaemia, and myogenic mechanisms may be of significance in determining the early stage of cerebral reactive hyperaemia after brief ischaemias. Adrenergic mechanisms might be of minor significance in this type of cerebral reactive hyperaemia.

Anesthesia↗

PubMed1995

Ambulatory Patient Group System (APGS) impact in radiology.

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Ambulatory Care↗

PubMed1995

Monoclonal antibody assay for measuring bone-specific alkaline phosphatase activity in serum.

Alkaline phosphatase (ALP) is present in human serum in the form of several isoenzymes. The two major circulating ALP isoenzymes, bone and liver, are difficult to distinguish because they are the products of a single gene and differ only by posttranslational glycosylation. Quantitative measurement of bone ALP (BAP) activity in serum can provide an index for the rate of bone formation. Furthermore, increased BAP activity in serum is indicative of bone disorders. We describe a method in which serum samples are added to a microtiter plate coated with monoclonal anti-BAP antibody and incubated 3 h at room temperature. After the unbound materials are washed off, the bound BAP activity is measured by adding p-nitrophenyl phosphate substrate. The assay demonstrated no cross-reactivity to intestinal or placental ALP and only 3-8% cross-reactivity to liver ALP. The intraassay (n = 21) CVs were 3.9-5.9%, and interassay (n = 8) CVs were 4.4-7.0%. Comparisons of the assay (y) with an IRMA (x) and a wheat germ agglutinin precipitation method (x') gave regression equations of y = 1.32x-6.4, r = 0.99, and y = 1.41x' + 4.8, r = 0.99. The assay detected increased BAP in sera from patients with osteoporosis, Paget disease, osteomalacia, or primary hyperparathyroidism.

Adult↗

PubMed1995

Occurrence of respiratory syncytial virus subtypes in Mexican infants with acute lower respiratory tract disease.

The occurrence of respiratory syncytial virus (RSV) subtypes in Mexico City was studied by searching for the virus in nasopharyngeal aspirates of 82 infants, less than 12 months of age, hospitalized for acute lower respiratory tract disease with diagnosis of bronchitis, bronchiolitis, pneumonia and laryngotracheobronchitis. The presence of respiratory virus was determined by observation of cytopathic effect in cell monolayers; samples which produced a clear syncytia were considered positive and were treated with anti-respiratory syncytial virus serum. Viral infectivity was neutralized in 21 samples which were regarded as having RSV. These specimens were characterized into subtypes A and B with specific monoclonals by indirect immunofluorescence. Subtype A predominated over subtype B, 19 vs. 2. Patients with pneumonia and laryngotracheobronchitis had subtype A and both subtypes were found in infants with bronchitis and bronchiolitis.

Acute Disease↗

PubMed1994

Inhibitory effect of deferoxamine or macrophage activation on transformation of Paracoccidioides brasiliensis conidia ingested by macrophages: reversal by holotransferrin.

Conidia of P. brasiliensis ingested by murine macrophages at 37 degrees C showed enhanced transformation to yeast cells and further intracellular growth compared with conidia in culture medium alone. Treatment of macrophages with the iron chelator deferoxamine inhibited the intracellular conidium-to-yeast transformation. Cytokine-activated macrophages could also exert this inhibitory effect. Holotransferrin reversed the inhibitory effect of either deferoxamine or activated macrophages on intracellular conidium-to-yeast transformation. These results indicate that iron restriction is one of the mechanisms by which activated macrophages control the intracellular transformation of ingested conidia and growth of yeast cells.

Animals↗

PubMed1993

Immunoassay for urinary pyridinoline: the new marker of bone resorption.

Urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd) are markers of bone resorption that are elevated above normal in subjects with metabolic bone disease. Total Pyd and Dpd, both free and peptide-bound forms, can be measured by HPLC after hydrolysis and cellulose chromatography. Since free Pyd is the major component of total Pyd in urine, we developed an immunoassay using free Pyd as an immunogen. This assay is much easier to perform than HPLC, requires no sample preparation, and correlates well with total Pyd measurement by HPLC (r = 0.97) and with urinary hydroxyproline (r = 0.90). The antiserum reacts most strongly with free Pyd and Dpd and minimally with glycosylated and large peptide-bound forms. The sensitivity of the Pyd immunoassay is less than 25 nM. The intraassay CV is 5-10%; the interassay CV is 10-15%. Analytic recovery studies indicated negligible sample interference. Furthermore, measurement of the Pyd in the same individuals over a 30 day time period exhibited minimal day-to-day variation. Thus, the Pyd immunoassay provides a rapid and easy method for evaluation of Pyd in urine. Pyd immunoassay may serve as a practical method of screening for metabolic bone disease and for monitoring therapeutic treatment.

Adult↗

PubMed1992

Measurement of rho, the ratio of the real to the imaginary part of the p-barp forward elastic-scattering amplitude, at sqrt s =1.8 TeV.

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Journal Article↗

PubMed1992

The canine internal carotid system: regional differences in reactivity to nerve stimulation, noradrenaline and 5-hydroxytryptamine.

Reactivity of the canine internal carotid system to field electrical stimulation, noradrenaline and 5-hydroxytryptamine was determined isometrically using 4-mm cylindrical rings from the cervical and cavernous portions and middle cerebral artery. Electrical stimulation at supramaximal voltage (0.5-4 Hz, 0.2 ms duration) produced a frequency-dependent contraction that was similar in cervical and cavernous portions and lower in the middle cerebral artery. This response was blocked by tetrodotoxin (10(-6) M) or phentolamine (10(-6) M). Noradrenaline (10(-9) - 3 x 10(-4) M) produced concentration-dependent contractions and the sensitivity was: cervical portion = cavernous portion greater than middle cerebral artery, and this effect was blocked by phentolamine (10(-6) M). 5-hydroxytryptamine (10(-9) - 10(-5) M) caused concentration-dependent contractions and the sensitivity was: cervical portion less than cavernous portion less than middle cerebral artery. This response was depressed by lysergic acid diethylamide (10(-6) M). The results indicate that reactivity of the internal carotid system: (a) to alpha adrenergic activation by endogenous and exogenous noradrenaline is lower in the distal segment (middle cerebral artery) than in the proximal segments (cervical and cavernous internal carotid artery) and (b) to tryptaminergic activation increases as it courses toward the brain. Therefore, functional characteristics could be related to embryological origin which the segments of this vascular system are derived from.

Animals↗

PubMed1990

A fluorometric method for determining the degree of biotinylation of proteins.

A method to determine the number of biotin moieties attached to a protein has been developed based on quenching the natural fluorescence of avidin or streptavidin by biotin. The assay consists of titrating the number of biotin combining sites on streptavidin/avidin before and after adding the biotinylated protein. With this method only those biotin moieties capable of binding to streptavidin/avidin are detected. The assay is simple and sensitive, requiring only 1-10 micrograms of biotinylated protein per determination.

Animals↗

PubMed1990

Picogram quantitation of total DNA using DNA-binding proteins in a silicon sensor-based system.

We report a rapid and reproducible method to quantify total DNA at picogram levels. Two high-affinity DNA-binding proteins are used to construct a sandwich assay and a semiconductor sensor is used for quantitation. Single-stranded DNA-binding protein (SSB) from Escherichia coli is conjugated with a linker molecule, biotin, for specific capture of the DNA complex onto a membrane. Monoclonal anti-DNA antibody is conjugated with an enzyme, urease, for signal generation. To detect DNA, a sample is denatured to form single-stranded DNA and then incubated with a reagent containing both DNA-binding protein conjugates and streptavidin. After incubation of the reagent with the DNA sample for 1 h at 37 degrees C to form a complex of streptavidin--biotin--SSB--DNA--anti-DNA--urease, the mixture is filtered through a biotin-coated nitrocellulose membrane which binds the streptavidin component of the complex. The unbound reagent is washed off the membrane, and then the captured DNA complex is detected with a light-addressable potentiometric sensor which measures the pH change catalyzed by the urease in the complex. This assay can detect 2 pg of DNA with a quantitation coefficient of variation of less than 10% in the range 10 to 200 pg.

Animals↗