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Biomedical subjects

J P Chambers

Publications and source records attributed to J P Chambers.

At least 37 records · Page 2Linked to original sources
PubMed1994

Altered sarcolemmal calcium channel density and Ca(2+)-pump ATPase activity in tachycardia heart failure.

Whether sarcolemmal (SL) calcium handling is altered in endstage heart failure produced by chronic rapid pacing is not known. To investigate this we paced 7 rabbits at a rate of 400 beats/min for 35 +/- 11 days. 6 animals served as non-paced controls. Purified left ventricular SL membranes were then prepared and tested for [3H]-nitrendipine binding and (Ca(2+) + Mg2+)-dependent ATPase (Ca(2+)-pump) activity. Results show a 50% reduction in calcium channel antagonist binding sites with Bmax values reduced from 450 +/- 40 to 230 +/- 8 fmoles/mg protein in response to chronic rapid pacing (P < 0.01). This change was accompanied by a modest decrease in Kd from 0.29 +/- 0.09 to 0.22 +/- 0.03 nM (not significant). Vmax values for the SL Ca(2+)-pump ATPase were decreased from 387 to 164 nmoles/mg protein/min (P < 0.01) with KCa2+ values reduced from 0.91 to 0.28 microM Ca2+ (P < 0.05) in response to tachycardia induced failure as compared to controls. ATPase activity in both groups was very sensitive to 25 microM calmidazolium and 5 microM vanadate. Results from this study indicate that both a reduction in SL calcium channel density and decrease in SL Ca(2+)-pump ATPase activity are evident in tachycardia heart failure. We conclude that sarcolemmal calcium handling is altered in heart failure induced by chronic rapid pacing and that such changes may contribute to systolic dysfunction associated with this model to heart failure.

Animals↗
PubMed1994

Detection of botulinum toxin using an evanescent wave immunosensor.

Using fluorescein isothiocyanate (FITC)-streptavidin, quartz fibre-immobilized antibody (FiAb) and the evanescent wave component of a light beam, detection of Botulinum Toxin-B (BoTX) is described. Exposure of 3-aminopropyltriethoxysilane/glutaraldehyde (APTS/GA) treated quartz fibres to increasing amounts of anti-BoTX Ab indicated toxin binding to increase in a linear fashion up to approximately 125 ng added Ab. Quantitation of bound BoTX and FiAb by Dot-Blot analysis using avidin-Horseradish peroxidase (HRP) conjugation indicated the presence of 0.27 and 0.67 pmoles, respectively. Inclusion of nonbiotinylated BoTX in sampling mixtures reduced fluorescence in a dose-dependent manner over a narrow concentration range (0-300 ng). Exposure of FiAb to a variety of venoms resulted in no reduction of BoTX binding suggesting detection of BoTX via immobilized anti-BoTX Ab to be very specific.

Biosensing Techniques↗
PubMed1994

Pineal acid phosphatase activity in Syrian hamsters: sex differences and effects of castration and androgen replacement therapy.

Pineal acid phosphatase (ACP) activity was examined in seven experiments involving young intact or castrated male and female Syrian hamsters. Nine-week old female hamsters had a 3-fold (p < 0.001) higher ACP activity in their pineal glands than did males. After three weeks of castration, a significant increase (p < 0.001) in ACP activity occurred in castrated male pineal glands. In males, pellets with 5 alpha-dihydrotestosterone (p < 0.01) but not testosterone or progesterone suppressed pineal ACP activity. In females, no changes in pineal ACP activity were noted due to the estrous cycle. Pineal ACP activity was not affected by testosterone, dihydrotestosterone or androstenedione pellets in intact females or by testosterone pellets in ovariectomized animals.

Acid Phosphatase↗
PubMed1994

Melatonin prevents the suppression of cardiac Ca(2+)-stimulated ATPase activity induced by alloxan.

The effects of melatonin treatment on cardiac sarcolemmal membrane function were investigated in alloxan-injected rats. Ca(2+)-stimulated adenosine-triphosphatase (ATPase, Ca2+ pump) and Mg(2+)-ATPase activities were depressed significantly in sarcolemmal preparations from alloxan-injected rats compared with levels in control rats. These deficits were observed 2 days after alloxan injection, and they were accompanied by an increase in the density of voltage-sensitive calcium channels, as measured by the [3H]nitrendipine-binding assay. In a dose-dependent manner, treatment of rats with melatonin before alloxan injection significantly overcame the suppression of Ca(2+)-stimulated ATPase in cardiac sarcolemma. Melatonin (1, 5, and 10 mg/kg) overcame Ca(2+)-stimulated ATPase suppression by 13, 35, and 70%, respectively. In addition, melatonin at a dose of 10 mg/kg also prevented the suppression of the Mg(2+)-ATPase by 31%. The number of [3H]nitrendipine-binding sites was not influenced by melatonin. The patent Na(+)-K(+)-ATPase and ouabain-sensitive Na(+)-K(+)-ATPase activities were not different between the control and experimental groups. The results indicate that Ca2+ pump activity is suppressed by acute alloxan treatment, whereas the density of voltage-sensitive calcium channels is increased. These changes may be a consequence of alloxan toxicity to the cardiac sarcolemma. Melatonin, likely because of its antioxidant capacity, exerts a protective effect on heart sarcolemmal membrane function in alloxan-injected rats.

Adenosine Triphosphatases↗
PubMed1993

Analgesic effects of detomidine in thoroughbred horses with chronic tendon injury.

This study was undertaken to assess the analgesia provided by detomidine (20 micrograms kg-1 intravenously) in thoroughbred horses. Pain thresholds to a mechanical noxious stimulus were measured before and after a period of mild chronic pain in one foreleg. Detomidine was a good analgesic in control animals; their pain thresholds were significantly elevated for about 60 minutes. After injury, the injured leg had a significantly lower pain threshold and the intensity and duration of analgesia provided by detomidine were significantly reduced. The analgesia in the opposite (sound) leg was also reduced, indicating that there were both central and peripheral aspects to this increased sensitivity to painful stimuli. Detomidine deserves to be considered as a potent analgesic in the horse rather than a sedative with analgesic side effects.

Analgesics↗
PubMed1993

Detection of sub-nanogram quantities of Mojave toxin via enzyme immunoassay with light addressable potentiometric detector.

Sensitive detection of Mojave toxin (MoTX), a potent neurotoxin isolated from the venom of Crotalus scutulatus scutulatus, was developed using an enzyme immunoassay (EIA) and light addressable potentiometric detection. This EIA utilizes both biotin- and fluorescein-labeled anti-MoTX antibodies to immobilize and detect sub-nanogram to nanogram quantities of toxin. Labeled mono- and polyclonal anti-MoTX antibodies were used alone and/or in combination to determine maximum assay sensitivity. Assays performed using a combination of biotinylated poly- and fluoresceinated monoclonal antibodies produced an assay with a lower detection limit near 2.5 ng. Assays performed using labeled polyclonals alone, or in combination with biotinylated mono- and fluoresceinated polyclonal antibodies, indicated increased sensitivity with a detection limit near 300 pg. In conclusion, we describe an enzyme immunoassay using different labeled antibody schemes which detects sub-nanogram quantities of MoTX via light addressable potentiometric detection.

Antibodies↗
PubMed1993

In vivo and in vitro effects of the pineal gland and melatonin on [Ca(2+) + Mg2+]-dependent ATPase in cardiac sarcolemma.

The possible diurnal variation in cardiac [Ca(2+) + Mg2+]-dependent ATPase (Ca2+ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemmal membrane [Ca(2+) + Mg2+]-dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time-dependent changes in [Ca(2+) + Mg2+]-dependent ATPase activity did not occur in either pinealectomized or light-exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca(2+) + Mg2+]-dependent ATPase activity, In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca2+ pump. The half-maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca(2+) + Mg2+]-dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.

Animals↗
PubMed1992

Hydrolysis of 11-cis- and all-trans-retinyl palmitate by retinal pigment epithelium microsomes.

A partial characterization of the enzymatic hydrolysis of 11-cis- and all-trans-retinyl palmitate by bovine retinal pigment epithelium microsomes was carried out using a micro-radiometric method to quantitate liberated palmitic acid. Retinyl ester hydrolase (REH) activity was examined in the absence of detergent. Hydrolysis of 11-cis- and all-trans-retinyl palmitate was protein- and time-dependent. Optimal enzyme activity occurred at slightly alkaline pH (8-9). Apparent kinetic constants (Vmax and Km) for the 11-cis-REH were 2.1 nmol/min/mg protein and 66 microM, respectively. All-trans-REH demonstrated a lower maximum velocity of 0.3 nmol/min/mg protein and a slightly higher substrate affinity of 27 microM. Further characterization of 11-cis-retinyl palmitate hydrolysis involved monitoring formation of reaction products, 11-cis retinol and palmitic acid, which were found to be released in essentially a 1:1 stoichiometry. Addition of all-trans retinyl bromoacetate, a known inhibitor of lecithin:retinol acyltransferase reduced both 11-cis and all-trans-REH activities but to significantly different degrees (50 and 76%, respectively). Although the microsomal preparation exhibited LRAT activity, acyl transfer was not readily reversible as labeled palmitic acid was not transferred to added acyl acceptor compounds. These findings suggest that hydrolysis of 11-cis-retinyl palmitate by bovine retinal pigment epithelium microsomes may occur at a catalytic site distinct from that for the all-trans isomer and that this hydrolysis is not representative of a reverse transesterification reaction.

Animals↗
PubMed1992

Testosterone sensitive dihydropyridine binding in the Harderian gland of the male hamster.

It is well known that in different tissues, dihydropyridines bind at nanomolar concentrations to a receptor and block voltage-operated Ca2+ channels. In studies reported here, Harderian gland tissue homogenates from intact male hamsters exhibited significant dihydropyridine binding (Bmax = 1700 fmoles/mg protein) of high affinity (Kd = 1.1 nM). Tissue homogenates from female animals exhibited a similar Kd value (1.35 nM) but receptor density per mg protein was significantly reduced (Bmax = 270 fmoles). Dihydropyridine binding of Harderian gland tissue homogenates from castrated males was reduced greater than 80% (Bmax = 225 fmoles/mg protein). Treatment of castrated males with subcutaneous testosterone pellets resulted in significant restoration of dihydropyridine binding activity (approximately 80%, Bmax = 1630 fmoles/mg protein) with a comparable binding constant (Kd = 1.50 nM) as observed for noncastrated, control animals. Addition of testosterone (ex vivo) to homogenates from castrated hamsters did not restore dihydropyridine binding to control levels. These data indicate: (a) the Harderian gland from male hamsters exhibits significant dihydropyridine binding; (b) ligand binding is abolished following castration; and (c) significant restoration of dihydropyridine binding occurs following in vivo testosterone treatment. The dependence of dihydropyridine binding restoration upon in vivo steroid hormone administration suggests probable involvement of the steroid at the transcriptional level although non-genomic mechanisms such as the binding of testosterone to a receptor resident in the plasma membrane and subsequent activation of Ca2+ channels can not be ruled out.

Animals↗
PubMed1991

Harderian gland porphyrin, lysosomal and type II 5'-deiodinase rhythms in Sprague-Dawley and Fischer-344 rats kept under chronic long- or short-photoperiod conditions.

1. Harderian gland porphyrin concentrations were 1.5-fold higher in Fischer-344 male rats than in Sprague-Dawley male rats and there were no differences due to exposure to LD 14:10 (LP) or LD 10:14 (SP) lighting conditions for 8 weeks in either strain. 2. 24-hr periodic regression analyses of porphyrin concentration detected a significant rhythm in both lighting conditions in both strains, with no differences in acrophase due to lighting conditions. 3. In both strains, porphyrin levels were highest in the late phase-early dark period and fell during the early part of the dark period. 4. Acid phosphatase activity did not vary with time (circadian rhythm), strain or photoperiodic lighting condition. 5. Circadian rhythms in beta-glucuronidase, alpha-mannosidase and hexosaminidase activity were present in some instances, but, probably due to the low amplitude to these rhythms, a consistent effect of strain or housing condition was not found. When 24-hr rhythms were observed in either strain, the acrophase occurred during the afternoon-early evening daylight period. 6. A significant effect of strain on mean values of type II 5'-deiodinase activity was noted in Fischer-344 rats. 7. Significant rhythms in type II 5'-deiodinase activity were found in both strains exposed to LD 10:14.

Analysis of Variance↗
PubMed1991

Effects of three reputed carboxylesterase inhibitors upon rat serum esterase activity.

Rats have very high endogenous levels of serum carboxylesterase (CAE) compared to primates. This difference accounts for the lower sensitivity of rats to toxic organophosphates, which interact with CAE instead of the more critical acetylcholinesterase. Pretreatment of rats with CAE inhibitors potentiates the effects of organophosphates. In this study, the effects of three putative CAE inhibitors, 2-(o-Cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP), bis-p-nitrophenyl-phosphate (BNPP), and tetraisopropyl pyrophosphoramide (Iso-OMPA), on the hydrolysis of several commercially available substrates were determined. Respective kinetic constants Km and Vmax were derived and effects of inhibitors compared using saturating amounts of substrate. Data presented here indicate significant differences in substrate affinity (Km), reactivity (Vmax), as well as effects of inhibitors. CBDP inhibits hydrolysis of specific naphthyl and paranitrophenyl esters at relatively low concentrations (1-10 microM). In contrast, significantly higher concentrations (mM) of BNPP and Iso-OMPA were required for inhibition of serum esterase activity. Of the inhibitors tested, Iso-OMPA in general exhibited the smallest inhibitory effect on ester hydrolysis. Although inhibition of hydrolysis of specific paranitrophenyl and naphthyl esters occurred in the presence of similar amounts of CBDP, the degree of inhibition differed significantly (50-75% vs. greater than 90%, respectively). These data suggest that there exists in rat serum, a pool of naphthyl ester esterase activity that is very sensitive ex vivo (greater than 90% inhibition) to CBDP and may be very useful in validating a rodent model for soman toxicity.

Animals↗
PubMed1990

Partial characterization of a high affinity [Ca2+ + Mg2+]-dependent adenosinetriphosphatase from bovine retina.

Examination of retinal tissue homogenates indicated the presence of a [Ca2+ + Mg2+]-dependent adenosinetriphosphatase activity that exhibited high affinity for Ca2+ (K0.5 = 0.17 microM) and moderately high affinity for Mg2+ and ATP (K0.5 = 12.5 microM and Km = 22.8 microM, respectively). Maximum ATP hydrolysis occurred at pH 7.4. Under conditions of optimal substrate, cation and hydrogen ion concentrations, specific activity ranged from 15 to 18 nmol phosphate released min-1 mg-1 protein. Although the retinal [Ca2+ + Mg2+] adenosinetriphosphatase hydrolyzes both ATP and dATP, other nucleotides (CTP, GTP, ITP and UTP) were not hydrolyzed to any great extent. The monovalent cations, Li+, K+ and Na+, had no effect upon hydrolysis of ATP; whereas Cs+ and NH4+ ions were moderately (approximately 30%) inhibitory. All divalent cations tested were stimulatory. With the exception of rotenone which inhibited ATP hydrolysis approximately 25%; retinal adenosinetriphosphatase activity was insensitive to mitochondrial inhibitors (NaN3, KCN, ruthenium red and oligomycin). Adenosinetriphosphatase activity was observed to be very sensitive to low concentrations (I50 approximately 2 microM) of vanadate; whereas, lanthanum administration resulted in no inhibition. Removal of calmodulin (80%) resulted in reducing adenosinetriphosphatase activity 60% but addition of exogenous calmodulin back to calmodulin deficient membranes did not restore activity to starting levels. Calmodulin antagonists trifluoperazine and calmidazolium reduced significantly Ca2+ stimulated, Mg2+ dependent ATP hydrolysis. We conclude that the [Ca2+ + Mg2+]-dependent adenosinetriphosphatase of bovine retina is a non-mitochondrial protein exhibiting very high affinity for Ca2+ and appears to require calmodulin for maximum activity. Because of its high affinity for Ca2+, this protein may play an important role in reducing intracellular Ca2+ to nanomolar levels.

Adenosine Triphosphate↗
PubMed1990

Pineal lysosomal enzyme circadian rhythms in male hamsters exposed to natural decreasing photoperiod and temperature conditions.

Circadian rhythms in acid phosphatase (ACP), hexosaminidase (HEX) and beta-glucuronidase (RON) activity were studied in the pineal glands of adult male Syrian hamsters exposed to control (20 +/- 2 degrees C and 14:10 LD) conditions or to naturally decreasing autumn photoperiod and temperature conditions (outside) for 8 weeks. Testes and testosterone levels (p less than 0.001 in both instances) were severely depressed in animals exposed to natural environmental conditions illustrating that the treatment period was of sufficient length to produce pineal-mediated gonadal atrophy. Significant rhythms were found in all enzymes in the pineal glands of control and outside animals with the exception of HEX activity in the control animals. Significant acrophase differences in outside vs. control animals were noted in ACP (7.9 hr earlier, p less than 0.001) and RON (9.8 hr later, p less than 0.001). A significant drop in RON and HEX activity (p less than 0.01 in both instances) was noted in association with the acute lights exposure in the morning to which control animals were exposed. The around-the-clock mean value of each enzyme was significantly lower in outside vs. control hamsters. These results demonstrate that environmental changes which provoke the pineal-mediated depression in gonadal activity also alter the activity of and shift the circadian rhythmicity of lysosomal enzymes in the pineal itself.

Acid Phosphatase↗
PubMed1989

Pineal lysosomal enzymes in the Syrian hamster: circadian rhythm and effects of castration or short photoperiod treatment.

A circadian rhythm in acid phosphatase and hexosaminidase was found in adult male hamsters exposed to a long photoperiod (14:10 h light/dark [LD]; lights on 06.00 h) and killed at 08.00, 14.00, 20.00, 02.00, 04.00, 05.50 and 0.615 h. Hexosaminidase and beta-glucuronidase activity at 02.00, 04.00 and 05.50 h (values pooled for these times before lights on) were significantly elevated compared to enzyme activity at 06.15 and 08.00 h (pooled values after lights on), suggesting a fall in activity associated with lights on. Hypogonadism was induced in female Syrian hamsters by exposure to a short photoperiod (10:14 h LD) until a majority of them were vaginally acyclic. Pineal lysosomal enzyme activities (acid phosphatase, beta-glucuronidase, hexosaminidase, alpha-arabinosidase and beta-galactosidase) were significantly elevated in short photoperiod-exposed animals compared to animals in 14:10 LD, when measured near the middle of the light phase. In the third experiment, castrated animals were used to determine if lowered androgen levels might also affect pineal lysosomal enzyme activity. The results indicated that light phase beta-glucuronidase, hexosaminidase and beta-glucosidase activities were lower in castrated males compared to their intact controls. In summary, these results demonstrate that (1) lysosomal enzyme activity is present in the Syrian hamster pineal, (2) changes can be observed which suggest involvement of this activity in pineal function and, (3) a circadian rhythm in enzyme activity is present with peak activity occurring during the night. In the short photoperiod and castration experiments, the changes in lysosomal enzyme activity could reflect either a hormonal manipulation or a change in circadian regulation of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1988

The fluorescence quantum yield of vitamin A2.

The fluorescence quantum yield of all-trans 3,4-didehydroretinol (vitamin A2) was measured in hexane at room temperature, using quinine sulfate as a standard. Unlike all-trans retinol (vitamin A1) which possessed a relative quantum yield of 0.0298, 3,4-didehydroretinol was 37 times lower in fluorescence (i.e. 0.0008). In addition, a significant bathochromic shift (both excitation and emission maxima) and a general broadening of the fluorescence spectra were noted for 3,4-didehydroretinol. This information is important not only for the understanding of the basic structure of vitamin A but also the photochemistry of vision.

Chromatography, High Pressure Liquid↗
PubMed1988

Kinetic properties of the [Ca2+ + Mg2+]-ATPase in Alzheimer and normal fibroblasts at low free calcium.

Kinetic properties of the [Ca2+ + Mg2+]-ATPase from one Alzheimer (AG0364B, 53-year-old donor) and one control fibroblast cell line (AG6009, 59-year-old donor) were examined. A saturation plot revealed the control fibroblast ATPase activity to saturate at approximately 500 nM free Ca2+; whereas, the Alzheimer activity saturated at approximately 1000 nM. Eadie Hofstee graphical analysis indicated almost identical Vmax values, 19.5 and 21.0 nmoles phosphate released/min/mg protein for the Alzheimer and control fibroblasts, respectively. However, an approximately two-fold higher Km value of 350 nM was observed for the Alzheimer fibroblast homogenate in contrast to 220 nM for the control. In a previous study, a kinetic difference in Alzheimer fibroblast [Ca2+ + Mg2+]-ATPase activity at high free Ca2+ concentration was observed. The difference in affinity reported in this study at low free Ca2+ concentrations supports further the hypothesis that abnormalities in Ca2+ homeostasis might be involved in the etiology of Alzheimer's disease.

Alzheimer Disease↗