Gait dysfunction in Huntington's disease: parkinsonism and a disorder of timing. Implications for movement rehabilitation.
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Aged↗
Biomedical subjects
M E Morris
Publications and source records attributed to M E Morris.
A randomized, double-blind trial of filgrastim (granulocyte colony-stimulating factor) versus placebo following allogeneic blood stem cell transplantation.
Blood stem cell transplantation (BSCT) results in rapid hematopoietic recovery in both the allogeneic and autologous transplant settings. Because of the large numbers of progenitor cells in mobilized blood, the administration of growth factors after transplantation may not provide further acceleration of hematopoietic recovery. A randomized, double-blind, placebo-controlled study was performed to determine the effects of filgrastim (granulocyte colony-stimulating factor; G-CSF) administration on hematopoietic recovery after allogeneic BSCT. Fifty-four patients with hematologic malignancies undergoing a related, HLA-matched allogeneic BSCT were randomly assigned to receive daily filgrastim at 10 microg/kg or placebo starting on the day of transplantation. A minimum of 3 x 10(6) CD34(+) cells/kg in the allograft was required for transplantation. All patients received a standard preparative regimen and a standard regimen for the prevention of graft-versus-host disease (GVHD). The median time to achieve an absolute neutrophil count greater than 0.5 x 10(9)/L was 11 days (range, 9-20 days) for patients who received filgrastim compared with 15 days (range, 10-22 days) for patients who received placebo (P =.0082). The median time to achieve a platelet count greater than 20 x 10(9)/L was 13 days (range, 8-35 days) for patients who received filgrastim compared with 15.5 days (range, 8-42 days) for patients who received placebo (P =.79). There were no significant differences for red blood cell transfusion independence, the incidence of acute GVHD, or 100-day mortality between the groups. The administration of filgrastim appears to be a safe and effective supportive-care measure following allogeneic BSCT.
Adolescent↗
Clinical tests of standing balance: performance of persons with multiple sclerosis.
OBJECTIVE: To investigate differences in performance between people with multiple sclerosis (MS) and control subjects on clinical tests of balance, and to assess performance consistency on balance tests in people with MS from morning to afternoon. STUDY DESIGN: Two factor repeated measures design with a two group sample of convenience. SETTING: Kingston Centre and the Camberwell Centre of the MS Society of Victoria, Australia. SUBJECTS: Fourteen people with MS and 14 control subjects matched for age, height, and sex. MAIN OUTCOME MEASURES: Subjects were measured on their ability to maintain standing balance in steady stance, (feet apart, feet together, stride stance, tandem stance, and single leg stance), during self-generated perturbations (functional reach, arm raise, and step tests) and in response to an external perturbation. Participants with MS were also asked to rate their fatigue level in the morning and afternoon. RESULTS: There were no differences between MS and control groups on the ability to maintain standing balance with feet apart, feet together, or in stride stance. Participants with MS performed more poorly than control subjects in tandem stance and single leg stance and in the functional reach test, arm raise test, step test, and in response to an external perturbation. There was little change in balance from morning to afternoon in participants with MS (ICCs (2,1) .70 to .94), despite an increase in self-rated fatigue (t(14) = -3.14, p = .008). CONCLUSION: The ability to maintain balance in standing is a marked problem in people with MS despite the consistency of their performance from morning to afternoon.
Adolescent↗
A rapid spectrofluorimetric technique for determining drug-serum protein binding suitable for high-throughput screening.
PURPOSE: To develop and validate a rapid method for determining the dissociation constants with which pharmaceutical candidates and drugs bind to serum albumin and to alpha1-acid glycoprotein with the goal of deducing the extent of binding. METHODS: The quenching of the intrinsic tryptophan fluorescence of serum albumin and alpha1-acid glycoprotein was monitored by spectrofluorimetry and the data were used to calculate the apparent dissociation constant. Sodium warfarin was used to probe the warfarin-binding site of serum albumin and diazepam was used to probe the benzodiazepine binding site. Additionally, the binding of sodium salicylate, phenylbutazone, sulfinpyrazone, iophenoxic acid, theophylline, chloramphenicol, acetaminophen, lithium chloride and ampicillin were also investigated. Chlorpromazine hydrochloride and imipramine hydrochloride were used as probes for alpha1-acid glycoprotein. The assays were also extended to the multiwell format. The quenching curves were fitted to the quadratic binding equation to determine the dissociation constants. RESULTS: Intrinsic fluorescence measurements are an excellent predictor of the drug binding to human serum albumin and to alpha1-acid glycoprotein. These measurements detect binding to the warfarin and benzodiazepine binding sites of human serum albumin. The dissociation constants estimated using the method compare favorably to the dissociation constants previously reported by Epps et al. using extrinsic fluorescence methodology, and the results correlate well with equilibrium dialysis using drug displacement endpoints. CONCLUSIONS: These measurements can be carried out with small samples and do not require separation of the bound and unbound species. Additionally, the proposed methods eliminate membrane separations, are not compound specific and do not require analytical chromatography or mass spectrometry for quantitation. Spectrofluorimetry may prove to be a useful method for rapidly determining the protein binding of combinatorial libraries.
Anticoagulants↗
Hormonal regulation of sodium/sulfate co-transport in renal epithelial cells.
Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17beta-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.
Animals↗
Molecular mechanisms of renal sulfate regulation.
Inorganic sulfate is an important physiological anion that is a required cofactor for sulfate conjugation reactions of both endogenous and exogenous compounds. It is necessary for the detoxification of xenobiotics and endogenous compounds (catecholamines, steroids, bile acids), for the synthesis of structural components of membranes and tissues (sulfated glycosaminoglycans), and for the biologic activity of endogenous compounds (heparin and cholecystokinin). Inorganic sulfate homeostasis is largely maintained by reabsorption in the renal proximal tubule. Sodium-dependent sulfate cotransport in the brush border membrane is of primary importance in the regulation of plasma inorganic sulfate concentrations. Altered renal reabsorption of sulfate has been observed under different physiological (age, pregnancy, low dietary intake), pathological (hypothyroidism, trace metal excess), and pharmacological conditions (treatment with nonsteroidal antiinflammatory agents). The recent identification of the sulfate transporter genes has allowed the investigation of the molecular mechanisms of altered sulfate transport. Although the regulation of sulfate homeostasis is not fully understood, recent investigations have explored the cellular mechanisms of some of these alterations. In this review, the physiological importance of inorganic sulfate, the availability of this anion, and the regulation of sulfate homeostasis are discussed.
Aging↗
Filgrastim as an alternative to donor leukocyte infusion for relapse after allogeneic stem-cell transplantation.
PURPOSE: Donor leukocyte infusion (DLI) effectively treats relapse after allogeneic stem-cell transplantation (alloSCT), but the response may require several months and may be associated with significant toxicity. Filgrastim has also been observed to effectively treat leukemic relapse after alloSCT. A retrospective analysis was performed to determine the effectiveness of filgrastim in treating relapses after alloSCT. PATIENTS AND METHODS: Fourteen patients with hematologic malignancies were treated with filgrastim at relapse after alloSCT. Filgrastim was given at 5 mcg/kg/d subcutaneously for 21 consecutive days. Response was evaluated at 7 days after completion of filgrastim. Immunosuppressants, if present, were rapidly tapered to complete discontinuation at the time of relapse. RESULTS: Three patients were not assessable for response because additional therapy was necessary before completion of filgrastim. Six patients (43%) achieved a complete response on an intent-to-treat basis. When response was evaluated based on relapse type, three of four cytogenetic relapses, two of three morphologic relapses, and one of four hematologic relapses achieved a complete remission. Two responses were observed in patients who were completely off of any immunosuppression at the time of relapse. Six patients developed chronic graft-versus-host disease. The event-free and overall survival rates for all 14 patients are 43% and 73%, respectively. CONCLUSION: The use of filgrastim with rapid discontinuation of immunosuppression results in response rates that are similar to results using DLI. Filgrastim could be considered as an alternative or an adjunct to DLI for relapses after alloSCT.
Adult↗
Comparison of changes evoked by GABA (gamma-aminobutyric acid) and anoxia in [K+]o, [Cl-]o, and [Na+]o in stratum pyramidale and stratum radiatum of the guinea pig hippocampus.
Ion-selective microelectrode recordings were made to assess a possible contribution of extracellular gamma-aminobutyric acid (GABA) accumulation to early responses evoked in the brain by anoxia and ischemia. Changes evoked by GABA or N2 in [K+]o, [Cl-]o, [Na+]o, and [TMA+]o were recorded in the cell body and dendritic regions of the stratum pyramidale (SP) and stratum radiatum (SR), respectively, of pyramidal neurons in CA1 of guinea pig hippocampal slices. Bath application of GABA (1-10 mM) for approximately 5 min evoked changes in [K+]o and [Cl-]o with respective EC50 levels of 3.8 and 4.1 mM in SP, and 4.7 and 5.6 mM in SR. In SP 5 mM GABA reversibly increased [K+]o and [Cl-]o and decreased [Na+]o; replacement of 95% O2 -5% CO2 by 95% N2 -5% CO2 for a similar period of time evoked changes which were for each ion in the same direction as those with GABA. In SR both GABA and N2 caused increases in [K+]o and decreases in [Cl-]o and [Na+]. The reduction of extracellular space, estimated from levels of [TMA+]o during exposures to GABA and N2, was 5-6% and insufficient to cause the observed changes in ion concentration. Ion changes induced by GABA and N2 were reversibly attenuated by the GABA(A) receptor antagonist bicuculline methiodide (BMI, 100 microM). GABA-evoked changes in [K+]o in SP and SR and [Cl-]o in SP were depressed by > or =90%, and of [Cl-]o in SR by 50%; N2-evoked changes in [K+]o in SP and SR were decreased by 70% and those of [Cl-]o by 50%. BMI blocked delta [Na+]o with both GABA and N2 by 20-30%. It is concluded that during early anoxia: (i) accumulation of GABA and activation of GABA(A) receptors may contribute to the ion changes and play a significant role, and (ii) responses in the dendritic (SR) regions are greater than and (or) differ from those in the somal (SP) layers. A large component of the [K+]o increase may involve a GABA-evoked Ca2+-activated gk, secondary to [Ca2+]i increase. A major part of [Cl-]o changes may arise from GABA-induced g(Cl) and glial efflux, with strong stimulation of active outward transport and anion exchange at SP, and inward Na+/K+/2Cl- co-transport at SR. Na+ influx is attributable mainly to Na+-dependent transmitter uptake, with only a small amount related to GABA(A) receptor activation. Although the release and (or) accumulation of GABA during anoxia might be viewed as potentially protectant, the ultimate role may more likely be an important contribution to toxicity and delayed neuronal death.
Animals↗
Movement disorders in people with Parkinson disease: a model for physical therapy.
People who are diagnosed with idiopathic Parkinson disease (PD) experience movement disorders that, if not managed, can lead to considerable disability. The premise of this perspective is that physical therapy for people with PD relies on clinicians having: (1) up-to-date knowledge of the pathogenesis of movement disorders, (2) the ability to recognize common movement disorders in people with PD, (3) the ability to implement a basic management plan according to a person's stage of disability, and (4) problem-solving skills that enable treatment plans to be tailored to individual needs. This article will present a model of physical therapy management for people with idiopathic PD based on contemporary knowledge of the pathogenesis of movement disorders in basal ganglia disease as well as a review of the evidence for physical therapy interventions. The model advocates a task-specific approach to training, with emphasis on treating people with PD-related movement disorders such as hypokinesia and postural instability within the context of functional tasks of everyday living such as walking, turning over in bed, and manipulating objects. The effects of medication, cognitive impairment, the environment, and coexisting medical conditions are also taken into consideration. An argument is put forward that clinicians need to identify core elements of physical therapy training that apply to all people with PD as well as elements specific to the needs of each individual. A case history is used to illustrate how physical therapy treatment is regularly reviewed and adjusted according to the changing constellation of movement disorders that present as the disease progresses.
Accidental Falls↗
Glucocorticoid-induced alterations of renal sulfate transport.
Glucocorticoid administration decreases renal sodium/phosphate cotransport in the proximal tubule due to a down-regulation of the sodium/phosphate cotransporter but has no effect on the sodium-dependent transport of glucose or proline. The objectives of the present investigation were to determine the effects of the glucocorticoid methylprednisolone (MPL) on 1) inorganic sulfate renal clearance in rats in vivo, 2) sodium/sulfate cotransport in kidney cortex membrane vesicles, and 3) the cellular mechanism of the MPL-induced alterations in sulfate renal transport. Male adrenalectomized Wistar rats received an i.v. dose of 50 mg/kg MPL or the vehicle. Urine samples were collected for 12 h after the administration of MPL, and blood samples were collected at the midpoint of the urine collection. Other animals were sacrificed at 4, 6, and 12 h after MPL administration, and the kidney cortex was removed for RNA or membrane preparations. Kidney cortex sodium/sulfate cotransporter (NaSi-1) mRNA levels were determined by reverse transcription-polymerase chain reaction and NaSi-1 protein levels were determined by enzyme-linked immunosorbent assay. The urinary excretion rate and renal clearance of sulfate were significantly increased in MPL-treated animals (144.0 +/- 27.0 versus 65.3 +/- 21.3 micromol/12 h/kg and 0.208 +/- 0.038 versus 0. 078 +/- 0.025 ml/min/kg, mean +/- S.E., n = 9-12 in treated versus control). The V(max) value for sodium-dependent sulfate transport in brush border membrane vesicles (representing reabsorption in the proximal tubules) was significantly decreased in MPL-treated animals compared with controls (0.68 +/- 0.07 versus 0.88 +/- 0.05 nmol/mg of protein/10 s, mean +/- S.E.). There was no change in the K(m) value for sodium/sulfate cotransport in brush-border membrane and no change in sulfate/anion exchange in basolateral membrane vesicles. Membrane fluidity in brush border membrane and basolateral membrane vesicles, determined by the fluorescence polarization of 1, 6-diphenyl-1,3,5-hexatriene was unaltered by MPL treatment. NaSi-1 mRNA levels were significantly decreased at 4 and 6 h, but not 12 h, after MPL administration, whereas NaSi-1 protein expression was significantly decreased at 4, 6, and 12 h. Therefore, MPL treatment increases the renal clearance of inorganic sulfate, at least in part, due to down-regulation of NaSi-1 mRNA and protein expression in the kidney.
Animals↗
Gait consistency over a 7-day interval in people with Parkinson's disease.
OBJECTIVE: To evaluate the consistency of temporal and spatial parameters of the walking pattern in subjects with idiopathic Parkinson's disease (PD) over a 7-day interval during the "on" phase of the levodopa medication cycle. SETTING: Walking patterns were measured on a 12-meter walkway at the Kingston Gait Laboratory, Cheltenham, using a computerized stride analyzer. SUBJECTS: Sixteen subjects (7 women, 9 men) with PD recruited from the Movement Disorders Clinic at Kingston Centre. MAIN OUTCOME MEASURES: Speed of walking, stride length, cadence, and the percentage of the walking cycle spent in the double limb support phase of gait were measured, together with the level of disability as indexed by the modified Webster scale. RESULTS AND CONCLUSIONS: Product-moment correlation coefficients and intraclass correlation coefficients (ICC 2,1) for repeat measures over a 7-day interval were high for speed (r = .90; ICC = .93), cadence (r = .90; ICC = .86), and stride length (r = 1.00; ICC = .97) and moderate for double limb support duration after removal of outliers (r = .75; ICC = .73); 95% confidence intervals for the change scores were within clinically acceptable limits for all variables. The mean modified Webster score was 11.4 on the first day and 10.1 7 days later. The gait pattern and level of disability in subjects with PD without severe motor fluctuations remained stable over a 1-week period when optimal medication prevailed.
Aged↗
Modulation of sulfate renal transport by alterations in cell membrane fluidity.
Changes in membrane fluidity have been shown to alter the sodium-dependent renal transport of glucose and phosphate; however, this has not been examined for sodium/sulfate cotransport in the renal proximal tubule. Sodium/sulfate cotransport regulates the homeostasis of sulfate in mammals. The objective of this study was to investigate the influence of alterations of membrane fluidity on sodium-coupled sulfate transport in the Madin-Darby canine kidney cells, which have been stably transfected with sodium/sulfate cotransporter (NaSi-1) cDNA (MDCK-Si). Preincubation of cells with 0. 2 mM cholesterol significantly decreased the V(max) for sodium/sulfate cotransport (13.69 +/- 1.11 vs 10.15 +/- 1.17 nmol/mg protein/5 min, mean +/- SD, n = 4, p < 0.01) with no significant alteration in K(m). The addition of benzyl alcohol (20 mM) to cells increased the V(max) of sulfate uptake by 20% (11.97 +/- 0.91 vs 14. 35 +/- 0.56 nmol/mg protein/5 min, mean +/- SD, n = 3, p < 0.05) with no significant change in K(m). Membrane fluidity, as measured by the fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH), was significantly increased in MDCK-Si cells treated with 20 mM benzyl alcohol and decreased in the cells preincubated with 0.2 mM cholesterol, compared with control cells. Our results suggest that alterations in membrane fluidity that may occur as a result of disease states, aging, and pregnancy may play an important role in the modulation of renal sodium/sulfate cotransport.
Animals↗
Effect of pregnancy, postnatal growth, and gender on renal sulfate transport.
Serum sulfate concentrations are increased in infants, young children, and pregnant women, compared with adult values. The objective of this investigation was to examine the influences of age, gender, and pregnancy on renal sulfate transport using guinea pigs as an animal model. Membrane vesicles were isolated from the kidney cortex of male animals at four different ages, from male and female adult animals, and from pregnant and nonpregnant female animals. There were no significant differences in marker enzymes for the brush-border membrane (BBM) or basolateral membrane (BLM) among all groups examined. Uptake was determined by a rapid filtration method and membrane fluidity by measuring the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The Vmax values for Na+ /sulfate co-transport in BBM were significantly increased with decreasing age, whereas the Km for this process was unchanged. The Vmax and Km for Na + /sulfate co-transport in BBM of pregnant animals were significantly higher than the values in the nonpregnant group. Bicarbonate-driven anion exchange of sulfate in BLM was not different among the different age groups. The Vmax for the bicarbonate/sulfate exchange process in BLM was not different between pregnant and nonpregnant groups; however, the Km for this process in BLM of pregnant animals was significantly greater than the value in nonpregnant animals. There were no gender-related differences in sulfate transport in BBM or BLM isolated from adult male and female animals. Renal BBM fluidity was increased with decreasing age and in pregnant animals, suggesting that altered membrane fluidity may represent one possible mechanism to explain the increased sodium/sulfate uptake in young and pregnant animals. The higher Vmax for Na+/sulfate co-transport in young and pregnant animals suggests that there is an increased density of co-transporter protein or an increase in the rate of movement of the carrier protein (i.e., turnover) once loaded with sodium and sulfate. This increased conservation of inorganic sulfate in young and pregnant guinea pigs may be related to the increased demand for sulfated substrates, such as sulfated glycosaminoglycans, during growth and development.
Age Factors↗
Effect of experimentally induced hypothyroidism on sulfate renal transport in rats.
Decreased serum sulfate concentrations are observed in hypothyroid patients. However, the mechanism involved in thyroid hormone-induced alterations of renal sulfate homeostasis is unknown. The objectives of this investigation were to determine the effect of 6-propyl-2-thiouracil (PTU)-induced hypothyroidism in rats on 1) the in vivo serum concentrations, renal clearance, and renal reabsorption of sulfate, 2) the in vitro renal transport in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, and 3) the cellular mechanism of the hypothyroid-induced alteration in sulfate renal transport. Serum sulfate concentrations, renal fractional reabsorption of sulfate, and creatinine clearance were decreased significantly in the hypothyroid group. The Vmax values for sodium-sulfate cotransport in BBM were significantly decreased in the kidney cortex from the hypothyroid animals (0.90 +/- 0.31 vs. 0.49 +/- 0.08 nmol. mg-1. 10 s-1, n = 5-6, P < 0.05) without changes in Km. There were no significant differences in Vmax and Km for sulfate/anion exchange transport in BLM. Sodium-dependent sulfate transporter (NaSi-1) mRNA and protein levels were significantly lower in the kidney cortex from hypothyroid rats. Hypothyroidism did not alter the membrane motional order (fluidity) in BBM and BLM, which indicates that the changes in the membrane fluidity do not represent the mechanism for the altered renal transport. These results demonstrate that PTU-induced hypothyroidism decreases sodium-sulfate cotransport by downregulation of the NaSi-1 gene.
Animals↗
Age- and growth hormone-induced alterations in renal sulfate transport.
The effects of growth hormone (GH) treatment on renal sodium sulfate cotransport (NaSi-1) were studied in adult (9-10 months) and old (22-23 months) male Fischer 344 rats. All animals received twice-daily s.c. injections of recombinant human GH (hGH; 4 mg/kg) for up to 6 days. Animals were sacrificed by exsanguination on days 0, 1, 2, 3, 4, 5, and 6. Kidneys were removed, and kidney cortex was trimmed immediately and used for RNA and membrane preparations. Plasma hGH concentrations were significantly lower in old rats during the hGH treatment (P <.05). Insulin-like growth factor-I (IGF-I) levels were significantly increased and remained stable after day 2 of hGH treatment in both age groups (P <.05). There was no significant difference in plasma IGF-I levels between age groups. Plasma IGF-I binding protein 3 (IGFBP-3) concentrations were significantly higher in 9- to 10-month-old rats compared with that in 22- to 23-month-old animals (P <.001). There were no significant differences in plasma IGFBP-3 concentrations between days of hGH treatment. The NaSi-1 mRNA levels were significantly lower in 22- to 23-month-old rats compared with that in 9- to 10-month-old animals (P <.001). The NaSi-1 mRNA levels were significantly increased on days 2 and 3 of hGH treatment (P <.05) and then gradually decreased to the control value. The NaSi-1 protein levels in old animals (22-23 months) were also significantly lower than that of 9- to 10-month-old animals and were significantly increased from day 2 of hGH treatment, reaching a maximum level on day 3 or 4 and then returning to the baseline level in both age groups. From these results, it was concluded that 1) NaSi-1 mRNA and protein levels are lower in old animals and increase in both adult and aged rats after hGH treatment, 2) plasma IGF-I levels are similar in adult and aged rats and increase after hGH treatment, and 3) plasma IGFBP-3 levels are lower in old rats and remain unchanged after hGH treatment.
Aging↗
A screen for genes induced in the suprachiasmatic nucleus by light.
The mechanism by which mammalian circadian clocks are entrained to light-dark cycles is unknown. The clock that drives behavioral rhythms is located in the suprachiasmatic nucleus (SCN) of the brain, and entrainment is thought to require induction of genes in the SCN by light. A complementary DNA subtraction method based on genomic representational difference analysis was developed to identify such genes without making assumptions about their nature. Four clones corresponded to genes induced specifically in the SCN by light, all of which showed gating of induction by the circadian clock. Among these genes are c-fos and nur77, two of the five early-response genes known to be induced in the SCN by light, and egr-3, a zinc finger transcription factor not previously identified in the SCN. In contrast to known examples, egr-3 induction by light is restricted to the ventral SCN, a structure implicated in entrainment.
Animals↗
Detection and quantitation of a sodium-dependent sulfate cotransporter (NaSi-1) by sandwich-type enzyme-linked immunosorbent assay.
The sodium-dependent sulfate transporter (NaSi-1) DNA has been recently identified from rat kidney cortex. The objective of this study was to develop a quantitative assay for the NaSi-1 transporter protein. The NaSi-1 antigen was prepared by fusion protein techniques following analysis of the primary sequence for antigenicity. Polyclonal and monoclonal antibodies against the NaSi-1 antigen were raised in rabbits and mice, respectively. The specificity of the raised antibodies was examined by Western analysis using brush-border membrane (BBM) and basolateral membrane (BLM) purified from rat kidney cortex. Both NaSi-1 polyclonal and monoclonal antibodies detected a 69-kDa protein in the BBM. Using the purified monoclonal antibody as the capture antibody and the polyclonal antibody as the detecting antibody, a simple and sensitive sandwich-type enzyme-linked immunosorbent assay was developed to quantitate NaSi-1 transporter protein levels in tissue. The specificity of the assay was examined using BBM, BLM and NaSi-1-transfected Madin-Darby canine kidney cells. The assay was capable of detecting NaSi-1 at levels as low as 6.58 fmol. The concentration of NaSi-1 transporter protein in crude membrane isolated from rat kidney cortex was 0.094+/-0.014 fmol/ microg protein (mean+/-SD of three preparations).
Animals↗
Functional expression of P-glycoprotein in the hepatic canalicular membrane of developing rats.
P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, gamma) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.
ATP Binding Cassette Transporter, Subfamily B, Mem↗