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At least 973 records · Page 54Linked to original sources
PubMed1995

Cholecystokinin effect on human lymphocyte ionized calcium and mitogenesis.

Cholecystokinin (CCK) is a peptide present in large amounts in gut, brain, and neurons innervating lymphatic tissues. Plasma CCK levels increase in enterally alimented patients. Enteral alimentation is also associated with enhanced immune function. The effects of CCK and a CCK antagonist were studied on human peripheral blood mononuclear cells (H-PBMC), lymphocyte intracellular ionized calcium ([Ca2+]i), and lymphocyte mitogenesis. CCK receptors transduce their signal via the release of [Ca2+]i. CCK octapeptide caused a specific increase in [Ca2+]i measured by Fura-2 fluorometry in H-PBMC and human T helper lymphocytes. Neither gastrin-17 nor pentagastrin produced a signal. While the highly specific CCK antagonist MK329 blocked the CCK [Ca2+]i signal, it had no effect on the PHA-mediated signal. At high dosages (10(-7)-10(-8) M), CCK was a comitogen with "complete" lymphocyte mitogens such as anti-CD3 monoclonal antibody (mAb) or low-dose PHA, but not for "partial" mitogens such as phorbol esters. CCK comitogenic effect occurred even in the presence of cyclosporine. CCK radioimmunoassay demonstrated that H-PBMC contained CCK and that anti-CD3 mAb- or PHA-mediated H-PBMC mitogenesis caused release of CCK. MK329 blocked PHA and anti-CD3 mAb mitogenesis and CCK comitogenic effects. We conclude that CCK octapeptide may be a coregulator of lymphocyte Ca2+ activation signals. The immunologically beneficial effect of enteral nutrition may, in part, be mediated by increased levels of CCK.

Benzodiazepinones↗
PubMed1997

The potential of 5' nuclease PCR for detecting a single-base polymorphism in Orthopoxvirus.

A fluorogenic 5' nuclease PCR assay was evaluated for its ability to specifically detect and differentiate DNA of two Orthopoxvirus species. A pair of consensus primers that target a DNA segment of the Orthopoxvirus haemagglutinin gene, and two oligonucleotide probes; each labelled with a different fluorescent reporter dye and the same quencher dye, were used in a single-tube assay. The assay is based on the 5'-->3' nuclease activity of AmpliTaq DNA polymerase that cleaves a fluorescein-labelled hybridized probe. Probe cleavage generates specific fluorescent signals whose intensity can be quantified by fluorometry. After evaluating the effects of various annealing temperatures and probe concentrations and normalizing the emission intensities of the reporter dyes, it was possible to detect and differentiate monkeypox and vaccinia virus DNAs on the basis of a single-base polymorphism. The sensitivity of the 5' nuclease PCR assay is comparable to the sensitivity of ethidium bromide-stained gels, but the assay provides higher specificity and virtually eliminates the need for laborious post-PCR processing.

DNA Primers↗
PubMed2000

Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR.

The < > assay (PE Applied Biosystems) combines PCR with concomitant release of fluorogenic nucleotides for immediate product detection by fluorometry. Yersinia pestis, the etiological agent of bubonic plague, expresses species-specific genes known to be located on two unique plasmids (9.6-kb pPCP and 100.9-kb pMT). Pesticin (pst) is a unique gene located on pPCP which encodes for a bacteriocin. Using fluorogenic probe coupled PCR as few as three copies of pst targets were detected from total Y. pestis genomic DNA. The pst probe used in this report was positive only for pesticinogenic isolates and did not show complementarity with Yersiniae nor with other bacteria targeted in this study suggesting, that the pst probe is very specific for Y. pestis. Under optimal conditions of Mg(2+)concentration and thermal cycle number, addition of extraneous DNA to respective assay mixtures had no effect on detection.

Bacteriocins↗
PubMed2000

Adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective IL-4 to murine synovium.

Treatments for rheumatoid arthritis and other inflammatory arthropathies are often ineffective at preventing joint destruction. Long-term genetic modification of the cells lining the joint space (synoviocytes) in vivo represents a potential method for the treatment of these chronic conditions. However, a vector capable of efficiently transducing synoviocytes in vivo for a persistent period has not been available. The present report describes the genetic modification of synoviocytes in vivo using recombinant adeno-associated virus. High-titer adeno-associated virus encoding the gene for Escherichia coli beta-galactosidase was injected into the knee joints of mice. Synovial tissues were then examined for beta-galactosidase transgene expression by in situ staining and by fluorometry. High-efficiency, persistent transgene expression was observed in the synovium with no evidence of vector-induced inflammation. Expression was observed for at least 7 months and was higher in arthritic than nonarthritic mice. Gene transfer of murine IL-4 to the joints of mice with collagen-induced arthritis led to detectable levels of IL-4 in the joint and protection from articular cartilage destruction. These data suggest that adeno-associated virus may be a useful vector for gene delivery to the synovium for the treatment of inflammatory arthropathies.

Animals↗
PubMed1998

Indocyanine green: physicochemical factors affecting its fluorescence in vivo.

This study reinvestigates the spectral properties of ICG (Indocyanine green) in vivo, the role of quenching, and the possibility of an interaction of ICG with blood components and/or vessel walls. ICG quenching as a function of concentration was studied by spectrophotometry on whole blood samples from golden hamsters. Fluorescence ICG characteristics were evaluated by front-face fluorometry. In vivo, fluorescence measurements were performed on the femoral artery of golden hamsters. In vitro, on whole blood samples, fluorescence intensity is modified by ICG quenching as concentration increases above 80 microgram/ml. The maximum fluorescence peak is not affected and remains centered at 832 nm. The in vivo measurements display a similar fluorescence intensity shape, which is affected only by ICG concentrations. However, the maximum fluorescence emission peak is modified significantly with time. Between 0 and 120 min, four phases can be distinguished in which a wavelength shift from 826 to 835 nm is observed. The wavelength shift with change in fluorescence intensity observed in vivo could be due to a localization of ICG molecules in sites more hydrophobic than serum proteins. It is possible to hypothesize the presence of an endothelium-bound form with a specific fluorescence spectrum. The amphiphilic properties of ICG are consistent with fixation of some ICG molecules on sites other than plasmatic proteins after injection. The process of fixation of ICG molecules on surface components or within the vascular endothelium could be due to a change in the microenvironment of some ICG molecules.

Animals↗
PubMed1997

Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin.

We investigated the utility of the oxidant-reactive probe dichlorofluorescin (DCFH) for measurement of NO synthase (NOS) activity in rat alveolar macrophages (AMs) activated by culture for 18 h with interferon-gamma (IFN-gamma, 25 U/ml). Using both microplate-based fluorometry and flow cytometric analysis, AMs treated with l (but not d)-arginine showed a dose- and time-dependent increase in DCFH oxidation above buffer control (e.g., DCF production (nM): control, d-arginine 100 microM, l-arginine 100 microM respectively; 91+/-9, 76+/-11, 396+/-45, mean +/- SE, n = 6, 120 min). Furthermore, the NOS inhibitor (nitro-l-arginine) showed complete inhibition of the l-arginine-dependent DCF production. Parallel assays showed a strong correlation in DCFH oxidation with nitrite production in the same samples (e.g., DCF production (nM): 143, 222, 409; nitrite (microM): 2.2, 3.6, 7.1; for 5, 10, 50 microM l-arginine, respectively). In contrast, the alternate oxidant-reactive probes hydroethidine (HE) and dihydrorhodamine (DHR) did not report increased oxidant production in activated AMs incubated with l-arginine, despite their ability to easily detect intracellular superoxide anion production in cells treated with menadione (100 microM). We conclude that DCFH is a useful probe for quantitation of NOS-2 activity in activated rat lung macrophages.

Animals↗
PubMed1988

Fiber optic surface fluorometry/reflectometry and 31-p-NMR for monitoring the intracellular energy state in vivo.

Various members of the respiratory chain exhibit different optical properties in the reduced and oxidized forms, thus enabling the non-invasive monitoring of various organs in vitro as well as in vivo. Since the pioneering work of Chance, Cohen, Jobsis and Schoener in 1962, many groups of investigators adopted their approach in monitoring NADH oxidation reduction states in vivo for the brain as well as for other body organs. In 1972, we introduced flexible, optical fibers into the surface fluorometry replacing the usual "rigid" optical system used by other groups. During the last decade, this technique has been developed, improved and applied to many experimental setups in brain research and very recently was combined with 31P NMR spectroscopy for the puppy and the adult dog brain in vivo. In our system, the effects of movement artifacts and changes in blood oxygenation are negligible while the effects of tissue absorption or blood volume changes are considerable and could be minimized by subtraction of the reflectance signal from that of the fluorescence (1:1 ratio) providing the corrected fluorescence signal.

Anaerobiosis↗
PubMed2002

The evaluation of brain CBF and mitochondrial function by a fiber optic tissue spectroscope in neurosurgical patients.

The brain of neurosurgical patients are exposed to various manipulations in the ICU or during surgery. Under such conditions brain O2 balance may become negative and as a result brain vitality and function will deteriorate. In order to evaluate brain vitality in real time it is important to measure more than one parameter. The multiparametric monitoring system used in our previous study to monitor comatose patients (Mayevsky et al., Brain Res. 740: 268-274, 1996) was changed into a "simplified" tissue spectroscope for real time monitoring of brain O2 balance. Mitochondrial function was evaluated by monitoring the NADH redox state by surface fluorometry. Microcirculatory blood flow was assessed by laser Doppler flowmetry. The combined optical probe was located on the surface of the brain during various neurosurgical procedures and the responses were recorded and presented in real time to the surgeon. A total of 32 patients were monitored during various procedures. The results could be summarized as follows: 1. Hypercapnia led to 3 different types of responses. In two patients the 'stealing' like event was recorded. In the other 7 patients the responses to high CO2 was not detectable. In the last group of 6 patients a clear CBF elevation was recorded with variable response of mitochondrial NADH. 2. Our monitoring device was able to evaluate the efficacy of the STA-MCA anastomosis during aneurysm surgery. 3. A significant correlation was recorded between CBF and NADH redox state during changes in blood pressure, papaverine injection, spontaneous drop in blood supply to the brain or during releasing of high ICP levels. We conclude that in order to evaluate the metabolic state of the brain during neurosurgical procedures it is necessary to monitor both CBF and mitochondrial NADH by using the tissue spectroscope.

Brain↗
PubMedDate not supplied

Determination of the pseudofacility by fluorophotometry in the human eye.

PURPOSE: Pseudofacility (decrease in aqueous humor flow in response to increase in the intraocular pressure (IOP)) has been evaluated in animals and in humans by a combination of different techniques that seriously alter the ocular physiology, such as the cannulation of the eyeballs, use of radioactive isotopes, perilimbic suction cups or tonography. Very different results have been obtained. Our objective is to relate the value of the aqueous humor flow in humans (as measured by fluorometry) with IOP. In order to obtain the widest range of IOP possible, we have performed this study in both normal as well as glaucoma patients. METHODS: 48 eyes were studied, corresponding to 31 subjects (mean age 58 +/- 14 years). Of these, 21 corresponded to normal subjects and 27 to glaucoma patients. The aqueous humor flow (F) was determined by fluorophotometry (Protocol of Yablonsky) and the IOP was measured thereafter. RESULTS: The mean value of F was 2.1 +/- 0.5 microliter/min for healthy eyes and 2.2 +/- 0.7 microliter/min for glaucoma-affected eyes. The values were not different (p > 0.1). The mean value of the IOP was 15.3 +/- 3.1 mmHg in the normal population and 22.1 +/- 4.3 mmHg in the glaucoma patients. A linear relationship between F and IOP was found (r = -0.41, p < 0.05, decrease in F = 0.081 microliter min-1 mmHg-1). CONCLUSIONS: The value of the pseudofacility in the humans can be estimated at 0.081 microliter min-1, mmHg-1, which constitutes 27% of the total outflow facility. This figure also makes it possible to estimate the role of the ultrafiltration in blood in the production of the aqueous humor since it is assumed that active secretion is pressure-independent. The results obtained are concordant with a secondary but no unimportant role of the plasmatic ultrafiltration with respect to the active secretion.

Aqueous Humor↗
PubMed1991

Blood-retina barrier permeability and its relation to the progression of diabetic retinopathy in type 1 diabetics. An 8-year follow-up study.

Blood-retina barrier (BRB) permeability and its relation to the progression of diabetic retinopathy was studied over an 8-year period in 50 insulin-dependent diabetic patients. Initially, the patients underwent an ophthalmological examination, including measurement of best corrected visual acuity, fundus photography and vitreous fluorometry for determination of BRB permeability. After 8 years the patients were reexamined and their retinal status and clinical course were evaluated. We found a positive correlation between a high initial permeability value and an unfavorable clinical course using the parameter photocoagulation. A decrease in follow-up visual acuity was also associated with high initial permeability; however, this correlation was not statistically significant. A significant difference in mean blood pressure was found between values measured in laser-treated patients vs a group that did not undergo such therapy. Thus, in patients showing the same initial retinal morphology, high permeability seems to indicate an unfavorable disease course. The extent to which BRB permeability can be a valuable supplement to fluorescein angiography and three-mirror examination in the clinical decision process needs to be further evaluated.

Adolescent↗
PubMed1988

Enhancement of blood-brain barrier permeability to sodium fluorescein by stimulation of mu opioid receptors in mice.

The effects of opioids on the permeability of the blood-brain barrier (BBB) were examined in mice with sodium fluorescein as an indicator of the permeability. The brain was perfused with saline 30 min after injection of sodium fluorescein (40 mg/kg, i.v.) and examined by fluorometry. Morphine hydrochloride (0.3-10 mg/kg, s.c.) markedly increased the brain level of sodium fluorescein dose-dependently without influencing the plasma level, when administered 20 min before sodium fluorescein injection. Intracerebroventricularly (i.c.v.) injected morphine hydrochloride (0.5 and 1.0 microgram) increased the brain sodium fluorescein level. Buprenorphine (0.1 and 0.5 mg/kg, s.c.) was also effective. However, pentazocine, ethylketazocine, U-50488H and SKF-10047 had no significant influence. The i.c.v. administration of [D-Ala2, MePhe4, Gly(ol)5]enkephalin (0.1 microgram) and [D-Ala2, D-Leu5]enkephalin (0.5 microgram) but not of [D-Thr2, Leu5]enkephalin-Thr increased the brain level of sodium fluorescein significantly. A small dose of naloxone (i.p.) significantly inhibited the effects of morphine, buprenorphine, [D-Ala2, MePhe4, Gly(ol)5]enkephalin and [D-Ala2, D-Leu5]enkephalin. ICI-174864 coadministered i.c.v. with [D-Ala2, D-Leu5]enkephalin was ineffective in antagonizing the effect of the latter. These findings suggest that the stimulation of mu opioid receptors results in an increase in BBB permeability to sodium fluorescein.

Animals↗
PubMed1995

Transient transport across an inhomogeneous blood-retina barrier.

A mathematical model for transport across the blood-retina barrier and diffusion into the vitreous body of the human eye is formulated. The eye is modeled as a sphere, the surface of which represents the blood-retina barrier. The equations of the model are solved analytically, using an expansion in spherical harmonics and inversion of the Laplace transform in the time variable. The numerical properties of the solution are investigated and the applicability of the model to the analysis of data from three-dimensional vitreous fluorometry is discussed.

Animals↗
PubMed1977

Intrapulmonary neuro-epithelial bodies in newborn rabbits. Influence of hypoxia, hyperoxia, hypercapnia, nicotine, reserpine, L-DOPA and 5-HTP.

Neonatal rabbit neuro-epithelial bodies (NEB) were investigated under various experimental conditions with light microscopy, microspectrography, morphometry and electron microscopy. (1) Hypoxia causes a decreased amine fluorescence intensity and an increased secretory exocytosis of dense core vesicles (DCV). Otherwise the NEB appear structurally normal. (2) Hypercapnia also produces a decreased fluorescence and an increased exocytosis; ultrastructurally, however, the dense core of DCV fragmentizes. (3) Hyperoxia does not appear to affect significantly either fluorescence or exocytosis. (4) The uptake of biogenic amines such as 5-HTP and L-DOPA was demonstrated by fluorometry and electron microscopy. (5) Reserpine, on the other hand, provokes an amine depletion with a decrease of the NEB fluorescence and an ultrastructural palor of the DCV. (6) Intratracheally administered nicotine is accompanied by a decreased fluorescence and a distinct exocytosis of fragmented DCV. The reaction of NEB to hypoxia and hypercapnia suggests that these corpuscles could be intrapulmonary chemoreceptors (in addition to the classically known central and peripheral chemoreceptors), inducing a reflex reaction through the liberation of DCV at the corpuscular sensible nerve endings and via the CNS. In addition, they may subserve a local intrapulmonary effect by modulating directly the hypoxic and hypercapnic pulmonary vasoconstriction and thus the V/Q ratio.

Animals↗
PubMed1992

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles.

Ca2+ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca2+, 2) calmodulin, and 3) Ca2+/calmodulin-dependent phosphorylation. Bell-shaped Ca(2+)-dependence of Ca2+ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca2+ release channel could be regulated by [Ca2+o]. The time- and concentration-dependent inhibition of Ca2+ release from skeletal SR by calmodulin was also studied using passively-Ca2+ loaded SR vesicles. Up to 50% of Ca2+ release was inhibited by calmodulin (0.01-0.5 microM); this inhibition required 5-15 min preincubation time. The hypothesis that Ca2+/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca2+ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca2+-loaded SR vesicles. Approximately 80% of the initial rates of Ca(2+)-induced Ca2+ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg-ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type II) were identified. The translated amino acid sequences show that Type II isoform is SR-form.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Distribution and metabolism of rubidazone and daunorubicin in mice.

A comparative investigation of the distribution and metabolism of rubidazone and daunorubicin was conducted in NMRI mice, after a single IV dose of either rubidazone (8.4 mg/kg) or daunorubicin (7 mg/kg). The anthracyclines were analyzed by high-pressure liquid chromatography and fluorometry. Daunorubicin was the main compound found after daunorubicin administration, except in kidney and urine, where daunorubicinol was the main metabolite with trace amounts of aglycone. In contrast, rubidazone undergoes extensive metabolism into daunorubicin, daunorubicinol, and aglycone. The total drug level is significantly lower after rubidazone administration in kidney, heart, gastrointestinal tract, spleen, and urine, and the biliary excretion of rubidazone is more important than that of daunorubicin. When compared with daunorubicin, significantly less daunorubicinol is produced after rubidazone administration.

Animals↗
PubMed1989

Radiolabelling of monoclonal antibodies: optimization of conjugation of DTPA to F(ab')2-fragments and a novel measurement of the degree of conjugation using Eu(III)-labelling.

F(ab')2 fragments (at concentrations of 5-30 mg/ml) derived from monoclonal antibodies raised against human prostate specific acid phosphatase were derivatized with a bicyclic anhydride of diethylenetriaminepentaacetic acid (cDTPAA) in the molar ratios of cDTPAA/F(ab')2 of 1:1, 5:1, 10:1 or 50:1. The most optimal product, aimed at radioimaging of prostatic cancer was obtained when the antibody fragment concentration was at least 10 mg/ml and the molar ratio of cDTPAA to F(ab')2 was 5:1. cDTPAA was added dissolved in dimethylsulfoxide (DMSO). Under these conditions, 1.8-2.2 DTPA molecules/F(ab')2 molecule were bound, giving a coupling efficiency of 37%-44%, and the labelling efficiency with 111In (3 mCi/1 mg protein) was 95% +/- 3% (n = 7). The antibody fragment completely retained its immunoreactivity measured by radioimmunoassay and showed no aggregation when studied using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). For evaluation of the degree of conjugation of DTPA to the antibody fragment, a novel technique was developed relying on the use of EuCl3, and the measurement of europium fluorescence employing time resolved fluorometry. Results by EuCl3 labelling were identical to those obtained by the conventional 111InCl3 labelling method.

Acid Phosphatase↗
PubMed1979

Fluorescence assay of tissue distribution of 4-demethoxydaunorubicin and 4-demethoxydoxorubicin in mice bearing solid tumors.

The tissue distribution of 4-demethoxydaunorubicin and 4-demethoxydoxorubicin was studied in comparison with that of their patient compounds, daunorubicin and doxorubicin, in mice bearing transplanted tumors. The doses administered were equal or equitoxic to those of their parent compounds. The levels of total fluorescence due to initial drugs and metabolites were determined on tissue extracts by fluorometry. After administration of equal doses of daunorubicin and 4-demethoxydaunorubicin, the calculated Cxt values of 4-demethoxydaunorubicin equivalents were higher than those for daunorubicin in all the organs tested except the heart. In animals treated with equitoxic doses, lower 4-demethoxydaunorubicin levels were found in all the organs tested. In mice treated with equitoxic doses of doxorubicin and 4-demethoxydoxorubicin, 4-demethoxydoxorubicin reached higher drug concentrations than doxorubicin in spleen and liver, whereas in all the other organs tested lower drug levels were found. The rate of drug disappearance from organs was slower in animals treated with 4-demethoxyderivatives than in those treated with their parent drugs.

Animals↗
PubMed1981

Fluorescence assays and pharmacokinetic studies of 4'-deoxydoxorubicin and doxorubicin in organs of mice bearing solid tumors.

The pharmacokinetic of 4'-deoxydoxorubicin, a new analog of doxorubicin, was compared with that of its parent compound in mice treated with equal and equiactive doses. The levels of total fluorescence due to the initial drugs and to metabolites were determined in tissue extracts by fluorometry. 4'-Deoxydoxorubicin reached the same tissue levels as doxorubicin in all the organs tested except in spleen and lung, where a higher peak was found in the animals treated with the new analog. The rate of elimination of 4'-deoxydoxorubicin from the organs tested was higher than that of doxorubicin.

Animals↗