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At least 1,243 records · Page 69Linked to original sources
PubMed1990

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from non interdigitated gel phases of symmetric diacylphosphatidylcholines. These results, combined with those obtained with dehydroergosterol (Kao, Y. L., P. L.-G. Chong, and C. Huang. 1990. Biochemistry. 29:1315-1322), suggest that considerable rotational mobility of small molecules can be sustained in an intramolecularly highly ordered interdigitated lipid matrix, implying that the membrane maintains a fluid environment around membrane perturbants even when the lipid matrix is extensively interdigitated.

Biophysical Phenomena↗
PubMed1990

Mapping of fluorescence anisotropy in living cells by ratio imaging. Application to cytoplasmic viscosity.

Steady-state and time-resolved fluorescence properties of probes incorporated into living cells give information about the microenvironment near the probe. We have extended studies of spatially averaged fluorescence anisotropy (r) by using an epifluorescence microscope, equipped with excitation and emission polarizers and an image analysis system, to map r of nonoriented fluorophores incorporated into cultured cells. With this imaging system, r for reflected light or glycogen scattering solutions was greater than 0.98. Measurement of r over the range 0.01-0.35 for fluorophores in bulk solution and in thin capillary tubes placed side-by-side gave values equivalent to r measured by cuvette fluorometry. Cytoplasmic viscosity (eta) in Madin-Darby canine kidney (MDCK) cells and Swiss 3T3 fibroblasts was examined from anisotropy images and time-resolved fluorescence decay of the cytoplasmic probes 2,7-bis-carboxyethyl-5 (and 6)-carboxy-fluorescein (BCECF) and indo-1. Nanosecond lifetimes and anisotropy decay were measured using a pulsed light source and gated detector interfaced to the epifluorescence microscope. Anisotropy images of BCECF in MDCK cells revealed two distinct regions of r: one from the cytoplasm (r = 0.144 +/- 0.008) and a second appearing at late times from the interstitial region (r = 0.08 +/- 0.03), representing BCECF trapped beneath the tight junctions. Anisotropy values, taken together with intracellular life-times and the calibration between r and eta/tau f for water/glycerol mixtures, gave eta values of 10-13 cP at 23 degrees C. These values assume little fluorophore binding to intracellular components and are therefore upper limits to cytoplasmic viscosity. These data establish a new methodology to map anisotropy in intact cells to examine the role of fluidity in cellular physiology.

Animals↗
PubMed1990

Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.

Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.

Animals↗
PubMed1991

Measurement of mass transport and reaction parameters in bulk solution using photobleaching. Reaction limited binding regime.

Fluorescence recovery after photobleaching (FRAP) has been used previously to investigate the kinetics of binding to biological surfaces. The present study adapts and further develops this technique for the quantification of mass transport and reaction parameters in bulk media. The technique's ability to obtain the bulk diffusion coefficient, concentration of binding sites, and equilibrium binding constant for ligand/receptor interactions in the reaction limited binding regime is assessed using the B72.3/TAG-72 monoclonal antibody/tumor associated antigen interaction as a model in vitro system. Measurements were independently verified using fluorometry. The bulk diffusion coefficient, concentration of binding sites and equilibrium binding constant for the system investigated were 6.1 +/- 1.1 x 10(-7) cm2/s, 4.4 +/- 0.6 x 10(-7) M, and 2.5 +/- 1.6 x 10(7) M-1, respectively. Model robustness and the applicability of the technique for in vivo quantification of mass transport and reaction parameters are addressed. With a suitable animal model, it is believed that this technique is capable of quantifying mass transport and reaction parameters in vivo.

Animals↗
PubMed1991

A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in pure solvents and in phospholipid vesicles has been measured using frequency domain fluorometry. Data analysis uses a model with two energetically close excited states. The model explains the high quantum yield and the double exponential decay of DPH observed in some pure solvents and in phospholipid vesicles. This model assumes that after excitation to a first excited state, there is a rapid interconversion to a lower excited state and that most of the emission occurs from this state. The interconversion rates between the two excited states determine the average lifetime. For DPH in solvents, we find that the interconversion rates are solvent and temperature dependent. For DPH in phospholipid vesicles, we find that the back reaction rate from excited state 2 to excited state 1 (R12) is what determines the fluorescence properties. The phospholipid phase transition affects only this back reaction rate. The model was analyzed globally for a range of solvents, temperatures and vesicle composition. Of the six parameters of the model, only two, the interconversion rates between the two excited states, varied in all different samples examined. For DPH in phospholipid vesicles, there is an additional feature of the model, which is related to the apparent distribution of the rate R12. Significantly better fits were obtained using a continuous lorentzian distribution of interconversion rates. The resulting lifetime distribution was asymmetric and showed a definite narrowing above the phase transition.

Diphenylhexatriene↗
PubMed1993

Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts.

The goal of these studies was to examine the effects of several factors that may artifactually influence quantitation of cytosolic [Ca2+], [Ca2+]c, while using the fluorescent calcium indicator Indo-1. The following factors were investigated: 1) a possible fluorescence contribution from unhydrolized Indo-1/AM (by Mn2+ quenching), 2) Ca2+ buffering by Indo-1 (by varying [Indo-1]), 3) endothelial and mitochondrial Indo-1 loading (by bradykinin stimulation and calculations), and 4) effects of changing tissue fluorescence (predominantly NAD(P)H) on calculated [Ca2+]c during hypoxia (by a new method which allowed simultaneous determination of [Ca2+]c and changes in [NAD(P)H]). No significant contribution of Indo-1/AM was found. With increasing [Indo-1], calculated systolic [Ca2+]c fell significantly. Indo-1 incorporation (< 18%) into endothelial cells, caused a slight underestimation of systolic [Ca2+]c, while mitochondrial Indo-1 loading may cause overestimation of [Ca2+]c. With increased tissue fluorescence, during hypoxia, systolic [Ca2+]c may be underestimated by approximately 27% (for Indo-1 loading factors three to five times original tissue fluorescence). These studies suggest conditions in which experimental artifacts could be minimized to allow reliable quantitation of [Ca2+]c in intact perfused hearts using Indo-1 fluorometry. The major problem of obtaining reliable results depended on the ability to correct for changing NAD(P)H fluorescence while keeping [Indo-1] low.

Animals↗
PubMed1994

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes.

Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered.

Binding Sites↗
PubMed1994

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.

Animals↗
PubMed1995

Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length.

Stopped-flow fluorometry has been employed to study the effects of melittin, the major protein component of bee venom, on dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) on the millisecond time scale, before melittin-induced vesicle fusion takes place. Use is made of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented fluorescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluorescence anisotropy reports on the "fluidity" of the bilayer. For DMPC bilayers, melittin is found to decrease their fluidity only at their melting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measurements, as deduced from the fact that its rate is comparable to that obtained by following the change in the fluorescence of the single tryptophan residue of melittin upon inserting itself into the bilayer. The perturbation is felt in the bilayer over a distance of at least 50 A, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The length of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, melittin does not alter the fluidity of DPPC SUVs and has a considerably greater affinity for them. These results are discussed in terms of the concept of elastic distortion of the lipids, which results from a mismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (approximately 10%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

DNA minor groove binding of cross-linked lexitropsins: experimental conditions required to observe the covalently linked WPPW (groove wall-peptide-peptide-groove wall) motif.

A theoretical analysis of binding interactions between covalently cross-linked lexitropsins and DNA is undertaken, in which a novel cyclic symmetric 2:2 dimeric lexitropsin-DNA-binding model is proposed. Applicability of commonly used techniques including NMR, quantitative footprinting, CD, and ethidium fluorometry to differentiate the covalently linked WPPW (groove Wall-Peptide-Peptide-groove Wall) from a 2:2 cross-linked lexitropsin-DNA duplex structure is examined.

Antineoplastic Agents↗
PubMed1997

Conformational changes in actin induced by its interaction with gelsolin.

Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer.

Actins↗
PubMed2000

Brain multiparametric responses to carbon monoxide exposure in the aging rat.

The multiparametric monitoring system was applied to study the effects of 2000 ppm carbon monoxide (CO) on brain functions in vivo in the aging rat. The vasodilatory (non hypoxic) effects of CO on CBF in normal adult rats, which were shown in concentrations of 1000-2000 ppm involved the effect of nitric oxide (NO). Energy metabolism was evaluated by optical monitoring of CBF and mitochondrial function by fluorometry of NADH. Ionic homeostasis was evaluated by monitoring the extracellular level of K(+) and H(+) and the DC steady potential. Seven aging rats (24 months) were exposed to 2000 ppm for 60 min and 120 min of recovery, while five control rats were exposed to air under the same conditions. A comparison between the CO group and the control group showed that the changes in CBF, NADH and light reflectance were not statistically significant while extracellular K(+) was elevated and tissue pH became more acidic. Thus, the typical CO induced increase in CBF, was not recorded in the aging rats. We concluded that the brain vasodilatory response to CO was not active in the aging rat, while the ionic homeostasis responses were similar to those found in the adult rat.

Aging↗
PubMed2001

Adrenalectomy causes loss of zinc ions in zinc-enriched (ZEN) terminals and decreases seizure-induced neuronal death.

Chelatable zinc ions from synaptic vesicles have been suggested to be involved in neuronal death caused by stroke, epilepsy and head trauma. Elevated glucocorticoid concentration exacerbates such neuron loss, while low levels protect. We have tested the notion that the neuroprotective effect of prior glucocorticoid reduction is mediated by a reduction of zinc ions contained in zinc-enriched (ZEN) synaptic vesicles. The level of vesicular zinc ions was evaluated by toluene sulfonamide quinoline (TSQ) fluorometry and zinc autometallography (ZnS(AMG)) 10 and 30 days, respectively, after adrenalectomy. The hippocampus showed significant vesicular zinc ion depletion following adrenalectomy. After the kainate injection, adrenalectomized rats showed proconvulsive seizure behavior, i.e. shortened latency to seizure onset time and increased seizure score. Additionally they showed decreased hippocampal CA3 neuronal death as compared to control animals. The present data suggest that zinc ions released from damaged ZEN terminals are involved in seizure-induced neuronal death.

Adrenalectomy↗
PubMed1998

Suppression of glomus cell K+ conductance by 4-aminopyridine is not related to [Ca2+]i, dopamine release and chemosensory discharge from carotid body.

The hypothesis that suppression of O2-sensitive K+ current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine (4-AP), a known suppressant of K+ current, on intracellular [Ca2+]i, dopamine secretion and chemosensory discharge in cat carotid body (CB). In vitro experiments were performed with superfused-perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP (0.2, 1.0 and 2.0 mM in CO2-HCO3- buffer) and recording [Ca2+]i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in [Ca2+]i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered [Ca2+]i by 4-AP are not consistent with the implied meaning of the suppressant effect on K+ current of glomus cells.

4-Aminopyridine↗
PubMed1998

Vasopressin-induced calcium signaling in cultured cortical neurons.

Earlier autoradiographic studies from our laboratory detected vasopressin recognition sites in the mammalian cerebral cortex [R.E. Brinton, K.W. Gee, J.K. Wamsley, T.P. Davis, H.I. Yamamura, Regional distribution of putative vasopressin receptors in rat brain and pituitary by quantitative autoradiography, Proc. Natl. Acad. Sci. U. S.A., 81 (1984) 7248-7252; C. Chen, R.D. Brinton, T.J. Shors, R.F. Thompson, Vasopressin induction of long-lasting potentiation of synaptic transmission in the dentate gyrus, Hippocampus, 3 (1993) 193-204]. More recently, we have detected mRNA for the V1a vasopressin receptors (V1aRs) in cultured cortical neurons [R.S. Yamazaki, Q. Chen, S.S. Schreiber, R.D. Brinton, V1a Vasopressin receptor mRNA expression in cultured neurons, astroglia, and oligodendroglia of rat cerebral cortex, Mol. Brain Res., 45 (1996) 138-140]. To determine whether these recognition sites are functional receptors, we have pursued the signal transduction mechanism associated with the V1a vasopressin receptor in enriched cultures of cortical neurons. Results of these studies demonstrate that exposure of cortical neurons to the selective V1 vasopressin receptor agonist, [Phe2,Orn8]-vasotocin, (V1 agonist) induced a significant accumulation of [3H]inositol-1-phosphate ([3H]IP1). V1 agonist-induced accumulation of [3H]IP1 was concentration dependent and exhibited a linear dose response curve. Time course analysis of V1 agonist-induced accumulation of [3H]IP1 revealed a significant increase by 20 min which then decreased gradually over the remaining 60 min observation period. V1 agonist-induced accumulation of [3H]IP1 was blocked by a selective V1a vasopressin receptor antagonist, (Phenylac1, D-Tyr(Me)2, Arg6,8, Lys-NH29)-vasopressin. Results of calcium fluorometry studies indicated that V1 agonist exposure induced a marked and sustained rise in intracellular calcium which was abolished in the absence of extracellular calcium. The loss of the rise in intracellular calcium was not due to a failure to induce PIP2 hydrolysis since activation of the phosphatidylinositol pathway occurred in the absence of extracellular calcium. V1 agonist activation of calcium influx was then investigated. V1 agonist-induced 45Ca2+ uptake was concentration dependent with a biphasic time course at 250 nM. Preincubation with the L-type calcium channel blocker, nifedipine, blocked V1 agonist-induced calcium influx suggesting V1 agonist-induced L-type calcium channel activation in cortical neurons. Furthermore, V1 agonist-induced calcium influx was blocked by both bisindolyleimide I (PKC inhibitor) and U-73122 (PLC inhibitor) suggesting a modulation of V1 agonist-induced L-type calcium channel activation by downstream components of the phosphatidylinositol signaling pathway such as protein kinase C. These results indicate that in cultured cortical neurons, V1a vasopressin receptor activation leads to induction of the phosphatidylinositol signaling pathway, influx of extracellular calcium via L-type calcium channel activation, and a rise in intracellular calcium which is dependent on V1a receptor activated influx of extracellular calcium. These data are the first to demonstrate an effector mechanism for the V1 vasopressin receptor in the cerebral cortex and provide a potential biochemical mechanism that may underlie vasopressin enhancement of memory function.

Animals↗
PubMed1982

Preparation and properties of fluorescent glycosaminoglycuronans labeled with 5-aminofluorescein.

The uronic acid residues of all known glycosaminoglycuronans reacted with 5-aminofluorescein to yield fluorescent glycosaminoglycuronan derivatives, which showed fluorescence characteristics identical to those of fluorescein or 5-acetamidofluorescein. The fluorescent products could be purified by chromatography on Octyl-Sepharose; three preparations of labeled chondroitin 6-sulfate having different degrees of substitution, and a labeled heparin were obtained. Fluorescent hyaluronic acid containing labeled and unlabeled molecules was digested with testicular hyaluronidase to give fluorescent oligosaccharides. Fluorescent chondroitin 6-sulfate was treated with chondroitinase AC to give a nonfluorescent disaccharide and minor proportion of fluorescent octasaccharide. Fluorescent heparin retained its anticoagulant activity, which was similar to that of the starting heparin; its half-life in circulating rabbit blood was 36 min (by fluorometry) and 45 min (by clotting-time assay).

Animals↗
PubMed1997

Down-regulation of endothelin B receptors in autogenous saphenous veins grafted into the arterial circulation.

OBJECTIVES: It has been postulated that endothelin (ET) might be involved in the development of atherosclerotic vascular lesions. The present study was done to characterize changes in the contractility and ET receptor subtypes in the autogenous saphenous vein graft (VG). METHODS: The rabbit saphenous vein (SV) was grafted into the ipsilateral femoral artery (FA), and at 4 weeks after the operation, VG was harvested. In the medial layer samples of SV, VG and FA, the cytosolic Ca2+ concentration ([Ca2+]i) and force were monitored using front-surface fluorometry of fura-PE3, and mRNA expression of ET receptors was evaluated using the reverse transcription polymerase chain reaction. RESULTS: ET-1 (10(-7) M) developed force in SV, VG and FA, to the same extent. Sarafotoxin (S6c; 10(-7) M) developed force in the SV to the same extent as ET-1. However, S6c did not develop force in FA, and slight force developed in VG. Contractions induced by ET-1 were associated with increases in [Ca2+]i. FA expressed ETA receptor mRNA predominantly, and SV expressed both ETA and ETB receptors mRNAs. In VG, the expression of ETB receptor mRNA was markedly reduced, but expression of ETA receptor mRNA remained unchanged. CONCLUSIONS: Functioning ETB receptors and their mRNA are down-regulated when veins are grafted into the arterial circulation. All these changes in gene expression and function are part of adaptive responses known as 'arterialization'.

Animals↗
PubMed2002

Neonatal biochemical screening for disease.

BACKGROUND: The practice of screening the neonatal population for certain diseases by biochemical testing of a dried blood spot is an established public health initiative in many countries. The diseases for which screening is done vary from region to region, based on ethnic, financial and political considerations. Criteria have been established to identify diseases suitable for neonatal screening. DISEASES SCREENED: In Western countries, screening for phenylketonuria (PKU) and congenital hypothyroidism (CH) has been introduced throughout. Subsequently, cost-benefit analysis has confirmed the existence of a financial benefit. Other diseases screened for in some regions include galactosemia, aminoacidemias and organic acidemias, cystic fibrosis, congenital adrenal hyperplasia, biotinidase deficiency, hemoglobinopathies, glucose-6-phosphate dehydrogenase deficiency, and Duchenne muscular dystrophy, although in no case has a clear financial benefit been established. CONCLUSIONS: Since the introduction of neonatal screening over 40 years ago, new methods have resulted in an increase in the number of diseases amenable to screening, better automation and greater specificity. Methods currently used include growth of an inhibited bacterial auxotroph (the original phenylalanine (phe) method of Dr. Robert Guthrie), spectrophotometry, fluorometry, immunoassay, and tandem mass spectrometry with electrospray ionization.

Adrenal Hyperplasia, Congenital↗