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J P Chambers

Publications and source records attributed to J P Chambers.

At least 55 records · Page 3Linked to original sources
PubMed1988

Lysosomal enzymes in the rat harderian gland are altered by either bromocriptine treatment or hypophysectomy and hormone replacement therapy.

Four groups of adult male hypophysectomized rats were injected subcutaneously twice daily between 0800-0900 hr and 1600-1700 hr with either saline diluent, 150 micrograms sheep prolactin and/or growth hormone (GH); intact rats received either saline or 150 micrograms bromocriptine twice daily. After 4 days of treatment, lysosomal enzyme assays revealed significant elevations in both acid phosphatase and alpha-mannosidase enzyme activities in the Harderian glands of saline-injected hypophysectomized rats compared to those in intact controls. beta-Glucuronidase levels were depressed and hexosaminidase activity unaffected by hypophysectomy treatment alone compared to intact controls. Lysosomal enzyme activities in hypophysectomized animals treated with prolactin were not different from the hypophysectomized control animals. However, treatment with GH alone or in combination with prolactin had a significant inhibitory effect on beta-glucuronidase, hexosaminidase, and alpha-mannosidase enzyme activities in the Harderian gland of hypophysectomized animals. Bromocriptine treatment in intact rats only elevated acid phosphatase activity. In summary, the patterns of responses did not reveal a role for prolactin in the control of Harderian gland lysosomal enzyme activities by the pituitary. However, some of the influence on this target system may be exerted by growth hormone.

Acid Phosphatase↗
PubMed1988

Underfeeding and exposure to short photoperiod alters rat pineal and Harderian gland lysosomal enzyme activities.

Harderian gland (HG) weight and lysosomal enzyme activity were evaluated after 21-day-old female rats were singly caged in a long (LP; 14:10 LD) or short (SP; 8:16 LD) photoperiod and fed on one of two dietary regimens (fed ad libitum or 50% underfed) for 50 days; an additional fed and an underfed group of animals in LP were injected every afternoon with 100 micrograms melatonin. Absolute HG weights were significantly lower in all underfed groups compared to their respective fed controls or to the LP fed control group. Absolute HG weights of underfed rats in SP were significantly lower than the underfed rats in LP. Relative HG weights (mg/100 g body wt) were significantly higher in the underfed saline or melatonin-treated groups compared to their respective fed controls; however, HG of the underfed SP group were not different from SP-fed controls. No significant differences in HG acid phosphatase, hexosaminidase, and beta-glucuronidase activities were observed in any of the treatment groups maintained in LP. Acid phosphatase, hexosaminidase, and beta-glucuronidase activities were significantly elevated in HG of underfed animals maintained in SP compared to their respective fed controls or to the LP-underfed group. Both the underfed control and the underfed-melatonin treated groups had lower pineal protein values than their respective fed groups; underfed animals in 8:16 LD had similar pineal protein values compared to those of the fed control group in SP. Significant effects of photoperiod and underfeeding with no interaction between these variables were observed on pineal acid phosphatase. The fed group maintained in 8:16 LD had significantly higher acid phosphatase activity than the fed group kept in 14:10 LD. In conclusion, underfeeding resulted in severely reduced body weights and absolute Harderian gland weights. Increased activity in certain lysosomal enzymes occurred in both the pineal and Harderian gland and in some instances this was dependent upon the light cycle and dietary regimen to which the animals were exposed.

Acid Phosphatase↗
PubMed1987

Pineal and retinal lysosomal enzyme rhythms.

Pineal glands and the neurosensory portion of the retinae of adult male rats adapted to a 24-h cycle with lights on from 06.00 to 20.00 h were collected at 9 timepoints during the cycle. Significant rhythms in both pineal and retinal hexosaminidase, beta-glucuronidase, acid phosphatase and beta-glucosidase were observed. For each enzyme, pineals had greater overall activity per unit amount of protein than did retinae. All 4 significantly rhythmic pineal enzymes peaked within 30 min of each other (18.30-19.00 h) whereas the retinal enzymes peaked some 6 h earlier, between 11.30 and 13.45 h. To our knowledge, this is the first report demonstrating 24-h rhythms in lysosomal enzymes in the pineal gland and retina. Since the acrophases (peak times) of these enzymes within each tissue are tightly synchronized yet different between tissues, lysosomes may play unique roles in the physiology of different structures in the photoneuroendocrine system with respect to time in the light-dark cycle.

Acid Phosphatase↗
PubMed1987

Partial characterization of two (Ca+2 + Mg+2)-dependent ATPase activities from bovine brain synaptic membrane homogenates.

Synaptic plasma membranes isolated from bovine brain exhibited a low and high affinity (Ca+2 + Mg+2)-dependent ATPase as evidenced by kinetic constants for ATP. One activity which hydrolyzed ATP maximally at pH 7.4 and 7.8 exhibited an 8-fold higher affinity when compared to the second or lower affinity activity which hydrolyzed ATP maximally at pH 7.0. Both activities exhibited submicromolar kinetic constants for Ca+2 (Km = 0.24 micromolar). Km values for magnesium differed significantly; the lower affinity activity being approximately 6.5 times higher (120 microM) than that observed for the high affinity activity (18 microM). Vmax values obtained under optimal assay conditions (low and high) were 110-135 and 43-55 nmol/min/mg protein, respectively. Both activities were KCN, NaN3 and ruthenium red insensitive. Only slight inhibition was observed in the presence of rotenone and oligomycin. Although both activities were observed to be trifluoperazine sensitive, they differed significantly with regard to other parameters. Na+1 and NH4+1 ions preferentially inhibited the low affinity activity greater than 90%. Cs+1 ions completely inhibited the high affinity activity while reducing the low affinity only 22%. Li+1, Al+3 and Mn+2 significantly inhibited the high affinity activity while reducing the low activity only moderately. Both the low and high activity were inhibited by vanadate with half maximum inhibition occurring at 2 and 5 microM, respectively indicating the plasma membrane origin of these activities. Thermal denaturation studies indicated the high affinity activity to be stable for 2 minutes at 45 degrees C after which 50% of the activity is lost at 2.5 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The effects of trimethyltin on the Ca+2, Mg+2 and Ca+2 + Mg+2-dependent ATPases of human neuroblastoma GM 3320.

Data presented here indicate neuroblastoma GM 3320 tissue homogenates exhibit ouabain insensitive Ca+2-dependent, Mg+2-independent, Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities. Inclusion of trimethyltin in homogenate preparations of these cells appears to discriminate between these various ATPase activities. At low concentrations (25 microM), trimethyltin preferentially stimulated the Ca+2-dependent, Mg+2-independent ATPase activity while inhibiting the Ca+2 + Mg+2-ATPase activity approximately 70%. At 75 microM trimethyltin, the Ca+2 + Mg+2-dependent ATPase activity is inhibited greater than 95% while the Ca+2-dependent, Mg+2-independent activity is essentially unchanged from control activity and the Mg+2-dependent, Ca+2-independent activity is inhibited approximately 50%. At concentrations greater than 75 microM, trimethyltin significantly inhibits the Ca+2-dependent, Mg+2-independent ATPase activity. Thus, at trimethyltin concentrations of 50-75 microM, preferential inhibition of the Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities of neuroblastoma GM 3320 is achieved.

Adenosine Triphosphatases↗
PubMed1986

Decreased hepatic retinyl palmitate hydrolase activity in protein-deficient rats.

28-day-old weanling rats were fed a diet containing 3% casein as the only source of protein for eight weeks to induce protein deficiency. When compared to control animals (fed a diet containing 25% casein), these rats had significantly lowered body (5.2-fold reduction) and liver (2.5-fold reduction) weights. The circulatory level of retinol (nmol per ml plasma) as well as retinol (nmol per g tissue) in the liver of these protein-deficient animals were also reduced significantly, although their liver concentration of retinyl palmitate (nmol per g tissue) was comparable to that of the control group. Assay of liver tissue for retinyl palmitate hydrolase activity revealed a 4-fold reduction (compared to that of control animals) of specific enzyme activity (nmol retinol formed per g protein per h). These findings suggest that severe protein deficiency results in a decreased hydrolysis of retinyl esters in the liver, which may be in part responsible for the reduced level of metabolically 'active' retinoids available for normal physiological functions.

Animals↗
PubMed1986

Effects of glycosylation inhibitors on the frog retina.

Effects of the intraocular injection of three inhibitors of glycosylation (tunicamycin, castanospermine, and swainsonine) on the rhodopsin content and the integrity of disc membranes in frog retina were studied. The administration of 10 or 100 micrograms of tunicamycin resulted in a 78% loss of rhodopsin in the frog retina which also exhibited a significant reduction in the length of photoreceptor outer segments (as examined under light microscope). This suggests that the synthesis and/or insertion of rhodopsin into the disc membrane is inhibited by tunicamycin. In contrast, injections of up to 250 micrograms of castanospermine and swainsonine resulted in neither a decrease in rhodopsin content nor a change in the length of photoreceptor outer segments. Examination of retinal tissue homogenates for alpha Mannosidase and alpha-Glucosidase activities revealed homogenates for alpha Mannosidase and alpha-Glucosidase activities revealed these hydrolase activities to be significantly decreased (70%). We suggest the possibility that specific oligosaccharide processing reactions may not be required for the insertion of rhodopsin and subsequent assembly of disc membranes in frog photoreceptors.

Alkaloids↗
PubMed1986

The effects of purified Mojave toxin on rat synaptic membrane (Ca+2 + Mg+2)-ATPase and the dihydropyridine receptor.

Effects of purified Mojave toxin on rat synaptic membrane (Ca+2 + Mg+2)-ATPase and dihydropyridine receptor were determined. The toxin was observed to stimulate specifically (Ca+2 + Mg+2)-ATPase approximately two-fold with no effect on Mg+2 dependent ATPase activity. Examination of the effects of increasing amounts of purified Mojave toxin on binding of the calcium channel blocker, nitrendipine, indicated that the addition of 10 micrograms (4.5 X 10(-10) moles) of toxin resulted in greater than 90% inhibition of nitrendipine binding. Furthermore, binding studies revealed the toxin to have little affinity for the ligand indicating its interaction with calcium channel components. Since Mojave toxin has associated with it a phospholipase A2 activity, we investigated the effects of 4-bromophenacylbromide, a known inhibitor of phospholipase A2 activity in order to discern the possible effects of the purified toxin on synaptic membranes. At concentrations previously shown to be inhibitory of purified phospholipase A2 from cobra venom, both ATPase activity and nitrendipine binding of synaptic membranes were significantly inhibited. Thus we cannot rule out the possibility that the endogenous phospholipase activity of the purified toxin is responsible for its effects on the rat brain synaptic functions studied here. Binding studies conducted in the presence of verapamil and diltiazem indicated that the toxin interacts with allosteric sites responsible for regulation of the binding of nitrendipine. Although we have not tested the effects of Mojave toxin on other ion channels and/or receptors, results presented here suggest the potential usefulness of this toxin as a molecular probe of the calcium channel complex.

Animals↗
PubMed1986

Effects of castanospermine on purified lysosomal alpha-1,4-glucosidase.

The indolizidine alkaloid castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) inhibits hydrolysis of maltose, glycogen and isomaltose by purified lysosomal alpha-glucosidase yielding Ki values of 0.095, 0.10 and 0.30 mumol/l, respectively. Castanospermine exhibited high affinity for both the maltose and isomaltose sites. In distinct contrast, the alkaloid exhibited little or no affinity for the site catalyzing hydrolysis of glycogen as indicated by a noncompetitive mode of inhibition. Kinetic data presented in this report indicate castanospermine to be a very potent inhibitor of lysosomal alpha-glucosidase.

Alkaloids↗
PubMed1985

The molecular basis for charge heterogeneity in human acid alpha-glucosidase.

The molecular basis for charge heterogeneity in human hepatic alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was determined by analysis of the carbohydrate and polypeptide components of the enzyme. Only small remnants of high mannose chains that contained neither sialic acid nor mannose 6-phosphate were detected in the carbohydrate structure. Four enzymatically active forms of alpha-glucosidase separated by chromatofocusing were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were found to contain different polypeptides. The absence of charged residues in oligosaccharide chains and variability in the polypeptide subunits of the charge forms of hepatic alpha-glucosidase suggest that charge heterogeneity results from differences in the protein structure of the charge forms. The pattern of differences in the polypeptide subunits suggests that the charge forms for hepatic alpha-glucosidase may be the product of proteolysis.

Electrochemistry↗
PubMed1985

Protection of suckling mice from heat-stable enterotoxin of Escherichia coli by infant formulas.

Human milk contains factors that protect suckling infants against enteropathogen-induced diarrhea. One nonspecific protective factor found in human milk is a small molecular weight material that protects suckling mice from death due to the heat-stable enterotoxin of Escherichia coli. This study demonstrated that cow's milk and most formulas derived from cow's milk also contain a factor(s) that can protect against heat-stable enterotoxin of E. coli in suckling mice.

Animals↗
PubMed1984

Glutamyl ribose 5-phosphate storage disease. A hereditary defect in the degradation of poly(ADP-ribosylated) proteins.

A patient with a lysosomal storage disease, progressive neurologic degeneration, and renal failure was found to have accumulated a low molecular weight ninhydrin and phenol-H2SO4 reactive compound. Amino acid analysis and gas chromatography-mass spectrometry identified a glutamic acid moiety. Direct insertion mass spectrometry proved the carbohydrate portion to be a sugar phosphate. NaB3H4 reduction and borate electrophoresis, paper chromatography, and enzymatic digestion indicated the presence of ribose 5-phosphate. Quantitative analysis of the intact compound indicated a 1:1:1 ratio for glutamic acid: ribose:phosphate. Brain was found to contain 0.96 mumol/g, wet weight, and kidney 0.60 mumol/g, wet weight, of glutamyl ribose 5-phosphate. This substance is the linkage region in ADP-ribosylation of histones and other proteins. It is suggested that the primary defect in this patient is a genetic abnormality of ADP-ribose protein hydrolase (Okayama, H., Honda, M., and Hayaishi, O. (1978) Proc. Natl. Acad. Sci. U. S .A. 75, 2254-2257).

Amino Acids↗
PubMed1983

Castanospermine, a tetrahydroxylated alkaloid that inhibits beta-glucosidase and beta-glucocerebrosidase.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) was tested against a variety of commercially available glycosidases and found to be a potent inhibitor of almond emulsin beta-glucosidase, and also to inhibit fungal beta-xylosidase. This alkaloid was inactive on yeast alpha-glucosidase, alpha- or beta-galactosidase, alpha-mannosidase, beta-N-acetylhexosaminidase, beta-glucuronidase, alpha-L-fucosidase. Fifty-percent inhibition of beta-glucosidase required about 10 micrograms/ml of castanospermine. The amount of inhibition was uniform throughout the time course, and the inhibition with regard to substrate concentration (p-nitrophenyl-beta-D-glucopyranoside) appeared to be of the mixed type. Castanospermine was also a potent inhibitor of beta-glucocerebrosidase when assayed with fibroblast extracts using either a fluorimetric or a radioactive assay. Interestingly enough, castanospermine also inhibited the lysosomal alpha-glucosidase, and this inhibition required comparable levels of alkaloid to that required for inhibition of beta-glucocerebrosidase. However, a number of other lysosomal glycosidases were not sensitive to castanospermine (i.e., alpha- or beta-galactosidase, alpha- or beta-mannosidase, alpha- or beta-L-fucosidase, beta-N-acetylhexosaminidase, beta-glucuronidase).

Alkaloids↗
PubMed1983

Protection of suckling mice from the heat-stable enterotoxin of Escherichia coli by human milk.

Human milk contained a factor or factors that protected suckling mice from lethal fluid loss due to Escherichia coli heat-stable toxin. This protective effect was present throughout lactation. The factor was heat stable, acid and alkali stable (pH 2 and 12), and of low molecular weight. Ion-exchange chromatography indicated that it was uncharged at neutral pH. The factor was insensitive to proteinase K digestion and partitioned in the aqueous phase in 2:1 (vol/vol) chloroform-methanol. Acid hydrolysis (2 N HCl at 100 C for 3 hr) destroyed the protective effect. Neither an amino acid mix simulating human milk nor lactose in phosphate-buffered saline was protective. These data indicate that the protective factor in human milk is neither a lipid nor a protein but appears to be a carbohydrate.

Amino Acids↗
PubMed1983

Acid alpha-glucosidase from human heart.

An alpha-glucosidase maximally active at acid pH has been purified from human heart some 2,600-fold and its properties compared to a purified alpha-glucosidase from human liver. Molecular weight was evaluated using three different analytical procedures. The effect of various cations was determined. Thermal lability was evaluated using three different substrates. Affinity and hydrolysis velocity constants for maltose, glycogen and 4-methylumbelliferyl-alpha-D-glucose were determined for both preparations at optimal hydrogen ion concentration. Inhibition studies were carried out using the disaccharide turanose. From this study, we conclude there are no significant differences in molecular weight or kinetic properties between the cardiac and hepatic alpha-glucosidase enzymes.

Cations↗