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C L Wheatley

Publications and source records attributed to C L Wheatley.

10 recordsLinked to original sources
PubMed2001

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1.

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

Alleles↗
PubMed1999

Broad screening test for sphingolipid-storage diseases.

BACKGROUND: Lipid-storage diseases are collectively important because they cause substantial morbidity and mortality, and because they may present as dementia, major psychiatric illness, developmental delay, or cerebral palsy. At present, no single assay can be used as an initial general screen for lipid-storage diseases. METHODS: We used a fluorescent analogue of lactosylceramide, called N-[5-(5,7-dimethylborondipyrromethenedifluoride)-1-pentanoyl]D- lactosylsphingosine (BODIPY-LacCer), the emission of which changes from green to red wavelengths with increasing concentrations in membranes, to examine the intracellular distribution of the lipid within living cells. FINDINGS: During a brief pulse-chase experiment, the fluorescent lipid accumulated in the lysosomes of fibroblasts from patients with Fabry's disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs and Sandhoff forms), metachromatic leucodystrophy, mucolipidosis type IV, Niemann-Pick disease (types A, B, and C), and sphingolipid-activator-protein-precursor (prosaposin) deficiency. In control cells, the lipid was mainly confined to the Golgi complex. In a masked study, replicate samples of 25 of 26 unique cell lines representing ten different lipid-storage diseases, and 18 of 20 unique cell lines representing controls were correctly identified; the sensitivity was 96.2% (95% CI 80.4-99.9) and the specificity 90.0% (68.3-98.8). INTERPRETATION: This method may be useful as an initial general screen for lipid-storage diseases, and, with modification, could be used for large-scale automated screening of drugs to abrogate lysosomal storage in various lipidoses. The unexpected accumulation of BODIPY-LacCer in several biochemically distinct diseases raises important questions about common mechanisms of cellular dysfunction in these disorders.

Antigens, CD↗
PubMed1995

Discrete pathways for arachidonic acid release from tannin versus beta-glucan-stimulated rabbit alveolar macrophages.

Previously, we observed both tannin and beta-glucan to be agonists for arachidonic acid (AA) release from rabbit alveolar macrophages. Although tannin inhibited reincorporation of exogenous AA, beta-glucan had no apparent effect, suggesting separate signal transduction pathways leading to elevated AA levels. In this study alveolar macrophages were pretreated with the tyrosine phosphatase inhibitor sodium orthovanadate then stimulated with either condensed tannin or beta-glucan. Vanadate exerted opposing effects on AA release. Furthermore, vanadate reversed the ability of tannin to inhibit reacylation. Additional studies using the phospholipase A probe bis-BODIPY-C11-PC indicated that although the known phospholipase A2 activators, calcium ionophore A23187, insoluble immune complexes, and beta-glucan, generated an increase in fluorescence consistent with phospholipase A activation, tannin had no effect. These findings suggest the increase in free AA resulting from stimulation of macrophages by either tannin or beta-glucan is produced via two different mechanisms.

Animals↗
PubMed1990

Activation of platelets by eosinophil granule proteins.

Two of the four principal cationic proteins of the eosinophil granule, major basic protein (MBP) and eosinophil peroxidase (EPO), were shown to be platelet agonists. Both MBP and EPO evoked a dose-dependent nonlytic secretion of platelet 5-hydroxytryptamine in unstirred platelet suspensions even in the presence of 10 microM indomethacin. MBP also evoked secretion of platelet alpha granule and lysosome components. Secretion by MBP and EPO was inhibited by 1 microM PGE1, but the nature of the inhibition differed from that observed with thrombin. Thus, MBP and EPO can be classified as strong platelet agonists with a distinct mechanism of activation.

Alprostadil↗
PubMed1989

Anticytoplasmic autoantibodies in the diagnosis and follow-up of Wegener's granulomatosis.

Sixty-five patients with biopsy-proven Wegener's granulomatosis (WG), 54 with systemic vasculitis, 22 with relapsing polychondritis, 20 with sarcoidosis, 20 with malignant pulmonary lesions, and 15 with other conditions underwent determination of anticytoplasmic autoantibodies (ACPA) by the indirect immunofluorescence technique on neutrophil cytospin preparations to assess the specificity of ACPA for WG, their sensitivity in relationship to the extent and activity of the disease, and their value for follow-up of WG. Of these 65 patients with WG, 38 were ACPA positive. Two patients in the vasculitis group, best categorized as having microscopic polyarteritis, were ACPA positive. We obtained 125 serum samples from the 65 patients with WG and assigned them to one of two categories (limited or generalized), based on the extent of disease. Each of these categories was then subdivided into "active" or "in remission." Median ACPA titers were significantly different between active disease and remission in each category, as well as between active limited and active generalized disease. All patients whose disease changed from active to in remission had reductions in ACPA titer levels; those who experienced flares had titer increases. Patients with intercurrent illnesses or complications of treatment, mimicking WG flares, did not have titer increases. We conclude that ACPA determined by the indirect immunofluorescence technique is highly specific for WG. The sensitivity is dependent on the extent and activity of WG, and serial titer determinations are valuable in monitoring disease activity.

Adolescent↗
PubMed1988

Modification of radiation-induced damage to bone marrow stem cells by dose rate, dose fractionation, and prior exposure to cytoxan as judged by the survival of CFUs: application to bone marrow transplantation (BMT).

The relative importance of the effects of dose rate, dose fractionation, and prior exposure to Cytoxan on the recovery of cells in the bone marrow, following conditioning for BMT, remains controversial. Traditionally, bone marrow stem cells and leukemic cells have been considered as having a limited ability to repair radiation-induced damage following total body irradiation (TBI) compared to cells of the lung (the dose-limiting tissue for TBI). We examined the survival response of the bone marrow stem cells of mice (CFUs) at three TBI dose rates (0.47, 0.25 and 0.08 Gy/min). The radiation response of CFUs (compilation Do = 0.75 Gy) was independent of dose rate. One TBI dose fractionation was chosen: two fractions per day, separated by 6 hours, for 3 days. The radiation survival curve of CFUs showed a compilation Do of 1.09 Gy, compared to 0.75 Gy for the one-fraction case. The recovery of CFUs following 2 days of Cytoxan demonstrated an "overshoot," whereas recovery of CFUs was incomplete, even by day 23, following the initiation of the complete conditioning regimen of Cytoxan plus TBI. These data demonstrate no significant effect of dose rate, at least in the range 0.08 to 0.48 Gy/min, on the survival of CFUs following either single or six fractionated TBI doses. However, the statistically significant difference in the Do of CFUs in going from one to six fractions has direct application to bone marrow transplantation techniques. Moreover, Cytoxan, at least at 200 mg/kg for 2 days, prior to TBI, appears to have only a marginal modifying effect on the eventual recovery of CFUs.

Animals↗
PubMed1988

Protein phosphorylation during tannin-mediated activation of human platelets.

Experiments to explore human platelet protein phosphorylation changes and 5-hydroxytryptamine (5-HT) secretion after challenge with cotton bract tannin were performed. Quantitative changes in sodium phosphate phosphorus 32 incorporation in two platelet proteins of 19 kilodaltons (kd) and 47 kd were assessed by measuring protein band densities on autoradiographs of dried polyacrylamide gels. Secretion of 5-HT was assessed by 14C-5-HT release. Results show that tannin causes increases in phosphorylation of discrete platelet proteins that begin in less than 2 seconds. These increases are maximal in 1 minute for the 47 kd protein and in 3 minutes for the 19 kd protein. Fifty percent of maximum response required less than 2 seconds for both of these proteins, and 50% of maximum 5-HT secretion required 48 seconds. Dose-response studies comparing 0 to 50 micrograms/ml tannin with 0 to 1 U/ml human alpha-thrombin showed that tannin caused 5-HT secretion and protein phosphorylation changes that were very similar to those induced by human alpha-thrombin. Fifty micrograms per milliliter of tannin caused increases in 19 kd protein phosphorylation and 47 kd protein phosphorylation to 312% +/- 34% (SEM) and 204% +/- 13% of control, respectively (n = 14). One unit per milliliter of thrombin induced changes of 350% +/- 40% and 221% +/- 17% of control in the 19 kd and 47 kd proteins, respectively. Release of 5-HT by tannin and thrombin was 61% +/- 3% and 69% +/- 3% of total cellular 5-HT, respectively. Indomethacin had little inhibitory effect on activation by these two different agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1983

An in vivo study of the radioprotective effect of diethyldithiocarbamate (DDC).

We have recently shown that diethyldithiocarbamate (DDC) protects plateau-phase cultures of mammalian cells from radiation. Experiments in vivo have extended our knowledge of the radioprotective properties of DDC and show that the LD50/30 in mice is increased from 780 rad to approximately 1400 rad when non-toxic concentrations of DDC are present prior to the irradiation. When DDC is present during irradiation, the pattern of death of the mice is similar to that seen in the absence of the drug and is quite different from that seen in animals dying of a gastrointestinal syndrome (LD50/7 congruent to 1700 rad). To confirm that the LD50/30 data represent bone marrow protection by DDC, we performed bone marrow CFUS assays with and without the presence of DDC, at the time of in vivo irradiation. The DO of the CFUS is increased from 80 rad in the control animals to 120 rad in the animals that have been pretreated with DDC. In experiments using 35S-labelled DDC, the tissue distribution of the drug in tumor-bearing mice indicates a preferential uptake of DDC in kidney, lung and bone marrow compared to tumor tissue. Based on data from both in vitro and in vivo studies, we believe that DDC shows promise as a radioprotective agent and should be considered for clinical trials.

Animals↗