PubMed Health⌕ Search

Biomedical subjects

D J Palmer

Publications and source records attributed to D J Palmer.

At least 19 recordsLinked to original sources
PubMed2006

Helper-dependent adenoviral gene therapy mediates long-term correction of the clotting defect in the canine hemophilia A model.

BACKGROUND: Adenoviral vector-mediated gene therapy might have potential for long-term correction of the monogenic disease hemophilia A. OBJECTIVE: In this study, we tested the efficacy of administering a helper-dependent adenoviral vector (HDV) designed for maximal liver-restricted canine factor VIII (cFVIII) expression on three out-bred hemophilia A dogs. METHODS: Three FVIII-deficient animals from the University of North Carolina colony were injected with 1 x 10(12) (Dog A), and 3 x 10(12) (Dog B and C) vp kg(-1) helper-dependent adenoviral vector, and we performed systematic analysis of toxicity, persistence of therapeutic gene expression, and molecular analysis of gene transfer. RESULTS: We observed acute dose-dependent elevation in liver enzymes and thrombocytopenia after injection, although both were transient and resolved within 2 weeks. The whole blood clotting time (WBCT), plasma FVIII concentration, FVIII activity, and activated partial thromboplastin time in all animals improved significantly after treatment, and two animals receiving a higher dose reached near normal WBCT with low-level FVIII activity until terminal sacrifice at 3 months, and 2 years. Importantly, the treated dogs suffered no bleeding events after injection. Moreover, we observed persistent vector-specific DNA and RNA in liver tissue collected from one high-dose animal at days 18 and 79, and could not detect the formation of inhibitory antibodies. CONCLUSION: Although vector-associated toxicity remains an obstacle, a single injection of HDV led to long-term transgene expression and vector persistence in two FVIII-deficient animals with conversion of their severe phenotype to a moderate one.

Adenoviridae↗
PubMed2005

Gene delivery to human sweat glands: a model for cystic fibrosis gene therapy.

Gene therapy vectors are mostly studied in cultured cells, rodents, and sometimes in non-human primates, but it is useful to test them in human tissue prior to clinical trials. In this study, we investigated the possibility of using human sweat glands as a model for testing cystic fibrosis (CF) gene therapy vectors. Human sweat glands are relatively easy to obtain from skin biopsy, and can be tested for CFTR function. Using patients' sweat glands could provide a safe model to study the efficacy of CF gene therapy. As the first step to explore using sweat glands as a model for CF gene therapy, we examined various ex vivo gene delivery methods for a helper-dependent adenovirus (HD-Ad) vector. Gene delivery to sweat glands in skin organ culture was studied by topical application, intradermal injection or submerged culture. We found that transduction efficiency can be enhanced by pretreating isolated sweat glands with dispase, which suggests that the basement membrane is a critical barrier to gene delivery by adenoviral vectors. Using this approach, we showed that Cftr could be efficiently delivered to and expressed by the epithelial cells of sweat glands with our helper-dependent adenoviral vector containing cytokeratin 18 regulatory elements. Based on this study we propose that sweat glands might be used as an alternative model to study CF gene therapy in humans.

Adenoviridae↗
PubMed2005

Effect of cooked and raw egg consumption on ovalbumin content of human milk: a randomized, double-blind, cross-over trial.

BACKGROUND: Maternal avoidance of egg intake has been recommended to treat egg allergy in breastfed infants. OBJECTIVE: To determine if the concentration of ovalbumin (OVA) in human milk is directly related to the quantity and form of egg consumed by breastfeeding mothers. METHODS: Randomized, blinded, cross-over, intervention trial. Breastfeeding women (n = 41) attended four clinic days between 11 and 14 weeks of lactation and on each day were randomly allocated to receive a test breakfast, identical except for the egg content (no egg, one raw egg, half a cooked egg or one cooked egg). Breast milk samples were collected at two hourly intervals for 8 h and their OVA concentration measured by ELISA. RESULTS: There was a direct, dose-response between the amount of cooked egg ingested and the peak OVA concentration (no egg 0.05 ng/mL [95% confidence interval (CI), 0.01-0.11], half a cooked egg 2.24 ng/mL [95% CI, 0.57-3.91], one cooked egg 3.16 ng/mL [95% CI, 1.41-4.91], n = 41, P<0.05) as well as the total OVA excretion (no egg 0.18 ng/mL/h [95% CI, 0.04-0.39], half a cooked egg 4.93 ng/mL/h [95% CI, 1.40-8.46], one cooked egg 9.14 ng/mL/h [95% CI, 4.25-14.03], n = 41, P<0.05). The peak concentration and total OVA excretion in response to one raw egg did not differ from ingesting half a cooked egg. There was no detectable OVA in the breast milk of 24% (10/41) women up to 8 h after any egg challenge. CONCLUSION: OVA was detected in the breast milk of lactating women up to 8 h after a controlled intake of egg. A dose-response correlation was indicated. As excretion of OVA in human milk appears to be a normal phenomenon, further studies need to determine the threshold of OVA excretion that leads to symptoms in egg-allergic breastfed infants.

Adult↗
PubMed2000

B cell- and monocyte-activating chemokine (BMAC), a novel non-ELR alpha-chemokine.

A novel alpha-chemokine, designated KS1, was identified from an EST database of a murine immature keratinocyte cDNA library. The EST has 94% similarity to a recently cloned human gene, BRAK, that has no demonstrated function. Northern analysis of mouse and human genes showed detectable mRNA in brain, intestine, muscle and kidney. Tumour panel blots showed that BRAK was down-regulated in cervical adenocarcinoma and uterine leiomyoma, but was up-regulated in breast invasive ductal carcinoma. KS1 bound specifically to B cells and macrophages, as well as two B cell lines, CESS and A20, and a monocyte line, THP-1. KS1 showed no binding to naive or activated T cells. In addition, KS1 stimulated the chemotaxis of CESS and THP-1 cells but not T cells. The s.c. injection of KS1 creates a mixed inflammatory response in Nude and C3H/HeJ mice. The above data indicates that KS1 and its human homologue represents a novel non-ELR alpha-chemokine that may have important roles in trafficking of B cells and monocytes. We propose the name B cell- and monocyte-activating chemokine (BMAC) for this molecule to reflect the described biological functions.

Amino Acid Sequence↗
PubMed1999

Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: evidence for its association with secretory granules in PC12 cells.

The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline-containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.

Adrenal Medulla↗
PubMed1998

Factors associated with delay in giving thrombolytic therapy after arrival at hospital.

OBJECTIVE: To identify factors associated with delay in administration of thrombolytic therapy for acute myocardial infarction. DESIGN: Retrospective case note review of a six-month period in 1995. Data were obtained on age, sex, hospital arrival time, triage priority, assessment process in the emergency department, grade of emergency doctor, patient history, timing of and findings on electrocardiogram (ECG), type of infarct, timing and site of administration of thrombolytic therapy, and type of thrombolysis given. SETTING: Tertiary referral hospital in Newcastle, New South Wales. PARTICIPANTS: Eighty-five patients given thrombolytic therapy for acute myocardial infarction. OUTCOME MEASURE: Time between hospital arrival and initiation of thrombolytic therapy. RESULTS: The median time from hospital arrival to administration of thrombolytic therapy was 80 minutes (interquartile range [IR], 50-133). Only 26% of patients were triaged to Priority 1 or 2 (to be seen by a doctor within 10 minutes). Patients initially assessed by a specialist emergency physician received thrombolytic therapy a median of 38 (IR, 33-50) minutes after hospital arrival, compared with 65 (IR, 50-107) minutes if initially assessed by a medical registrar, and 148 (IR, 89-185) and 160 (IR, 95-163) minutes, respectively, if initially assessed by an intern or a resident medical officer (P < 0.001). Factors associated with increased delay in receiving thrombolytic therapy (after adjustment for possible confounders) were low triage priority, initial assessment by a junior doctor, atypical presenting history of myocardial infarction, and lesser degrees of ST-segment elevation on the presenting ECG (all P < or = 0.01). CONCLUSIONS: Delay in administration of thrombolytic therapy in hospital results from a combination of hospital and patient factors. Changes in emergency department protocol may reduce these delays in some patients.

Aged↗
PubMed1997

The effects of maternal protein deprivation on the fetal rat pancreas: major structural changes and their recuperation.

There is evidence that low birth weight and poor growth in early life cause a long-term predisposition to non-insulin-dependent diabetes. Morphological changes were assessed in fetal rat pancreas subjected to both pre- and post-natal maternal protein deprivation (LP). Further groups were subjected to purely prenatal maternal protein deprivation (preLP) and purely postnatal maternal protein deprivation (postLP), as well as a control group. The results show that the LP and postLP groups had fewer but larger islets than the control group, while the preLP group had more numerous, smaller islets. All three low protein groups had more irregularly shaped islets than the control group. There was a reduction in the amount of beta cells within each islet in all three protein-deprived groups. The LP and postLP groups showed a reduction in the percentage of islet tissue and beta cells per pancreas, but the percentage of islet tissue expressed per unit body weight was similar in all four groups. These results show that in maternal protein deprivation, homeostatic mechanisms ensure a constant amount of pancreatic endocrine tissue per unit of body weight. However, there remain major structural changes in the size, shape, and composition of the islets. These results support the theory that early development profoundly affects the structure of the pancreas and may play a role in the later development of adult diseases, such as non-insulin-dependent diabetes mellitus.

Animal Nutritional Physiological Phenomena↗
PubMed1997

Pathogenesis of Sendai virus infection in the Syrian hamster.

Young adult male Syrian hamsters were inoculated intranasally with Sendai virus, then killed and examined at postinoculation days (PID) 3, 5, 7, 9, 12, 16, and 21. Evaluation included clinical assessment, histologic examination, immunohistochemistry, viral isolation, and antibody response. Inoculated and control hamsters remained asymptomatic throughout the study. There was a focal to segmental rhinitis involving respiratory tract epithelium lining the dorsal and ventral meatus and nasal septum, and segmental lesions involving all regions of the trachea. At PID 5 and 7, there was focal bronchitis and bronchioloalveolitis, respectively. In general, most lesions had resolved by PID 12, although in hamsters examined at PID 21, residual lesions were present in the nasal passages in one of three, and in the trachea in two of three animals. In immunoperoxidase-stained preparations, viral antigen was present in the respiratory tract epithelium of the nasal passages and trachea beginning at PID 3, with extension to scattered bronchi at PID 5. Sendai virus was recovered from the lungs of inoculated animals at PID 5. Antibodies to Sendai virus were first detected at PID 7, and titers remained high throughout the remainder of the 21-day study. This report provides additional evidence that Syrian hamsters are susceptible to Sendai virus infection, and that the lesions and sites of replication in the upper and lower portions of the respiratory tract are similar to those observed in susceptible strains of laboratory mice.

Animals↗
PubMed1997

Transscleral diode laser cyclophotocoagulation on autopsy eyes with abnormally thinned sclera.

BACKGROUND AND OBJECTIVE: To determine, using autopsy eyes, whether diode laser energy adjustments are indicated in patients with thin sclera. MATERIALS AND METHODS: In the laboratory, the superior 180 degrees of sclera at the limbus was dissected to the level of barely visible anterior uvea and the opposite 180 degrees of sclera served as the control in three human cadaver eyes. A contact G-probe was placed at the limbus, and settings of a diode laser were increased in increments from 1.0 to 9.0 J at 4 burns per setting in each location. RESULTS: On gross examination, circular hypopigmented lesions were seen in the ciliary body (CB) beginning at 3.0 J in thin sclera and at 5.0 J in normal sclera. On light microscopic examination of thin scleral sections, CB damage began at 2.9 J and CB/ciliary body epithelium (CBE) damage occurred beginning at 3.5 J. In normal sclera, minimal CB/CBE changes occurred at 6.0 to 7.5 J. No scleral damage was visible in either the experimental or the control groups. CONCLUSION: Cycloablation energy adjustments are indicated on eyes with abnormally thin sclera to achieve similar histologic end points using the diode laser.

Basement Membrane↗
PubMed1996

Localization of ATP-gated ion channels in cerebellum using P2x2R subunit-specific antisera.

The distribution of the P2x2 purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x2R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x2R mRNA. P2x2R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x2R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Adenosine Triphosphate↗
PubMed1995

Effectiveness of apraclonidine and acetazolamide in preventing postoperative intraocular pressure spikes after extracapsular cataract extraction.

We studied the effectiveness of two prophylactic agents in controlling early postoperative intraocular pressure (IOP) increases after cataract surgery. Fifty-four nonglaucomatous patients received either topical 1% apraclonidine, one drop before and after surgery, or sustained-release acetazolamide, 500 mg, or no medication at the completion of planned extracapsular cataract extraction (ECCE). Mean baseline IOPs were similar among patients randomized to the apraclonidine, acetazolamide, and control groups: 15.29 mm Hg, 15.33 mm Hg, and 14.26 mm Hg, respectively. At 3 hours postoperatively, IOPs were significantly lower in the apraclonidine group (11.13 mm Hg, P = .035), nonsignificantly lower in the acetazolamide group (13.3 mm Hg, P = .17), and significantly increased in the control group (21.32 mm Hg, P = .003). One eye in the apraclonidine group and six in the control group had IOPs greater than 30 mm Hg. At 24 hours, the only statistically significant difference was in the control group, whose mean IOPs remained elevated (21.83 mm Hg, P = .0008). One eye in the apraclonidine group, two in the acetazolamide group, and five in the control group had IOPs greater than 30 mm Hg. We found a significant early IOP reduction with apraclonidine given topically preoperatively and at the completion of planned ECCE.

Acetazolamide↗
PubMed1994

Golgi coatomer binds, and forms K(+)-selective channels gated by, inositol polyphosphates.

Coatomer is a distinct type of coat protein complex involved in the formation of specific Golgi intercisternal transport vesicles. Direct binding studies using purified coatomer isolated from bovine liver cytosol show that coatomer specifically binds both inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP4) and inositol hexakisphosphate (IP6) with subnanomolar affinities (0.1 and 0.2 nM, respectively). Diphosphoinositol pentakisphosphate (PP-IP5) is an efficient competitor for both (1,3,4,5)IP4 and IP6 binding to coatomer. Inositol 1,3,4,5,6-pentakisphosphate ((1,3,4,5,6)IP5) is a poor inhibitor of IP6 binding, whereas little or no competition is detected with inositol 1,4,5-trisphosphate ((1,4,5)I-P3). Coatomer displays ion channel activity when reconstituted into planar bilayers which is preferentially permeable to K+. Permeability ratios of the channel are PK+/PCl- approximately 8.0 and PK+/PNa+ approximately 7.1, indicating a cation-selective channel with selectivity of K+ over Na+. In symmetrical 500 mM KCl, the smallest observable unitary channel conductance is 8.3 picosiemens. The coatomer channel activity is normally active with long open times (0.1 to several seconds) and is selectively blocked by 10 microM (1,3,4,5)IP4, 1 microM IP6, and 0.27 microM PP-IP5; even lower concentrations are sufficient to induce channel flicker. The channel activity is not affected by (1,4,5)IP3, or (1,3,4,5,6)IP5. Thus, the channel activity of coatomer is modulated by the inositol polyphosphates which exhibit tight binding to the complex.

Animals↗
PubMed1993

Coated vesicle assembly in the Golgi requires only coatomer and ARF proteins from the cytosol.

Transport vesicles derived from the Golgi apparatus are thought to mediate biosynthetic transport across the Golgi stack. These vesicles are surrounded by a protein coat whose principal constituents are coatomer (a complex of seven distinct subunits or COPs) and ADP-ribosylation factor (ARF, an N-myristylated small GTP-binding protein). The coat proteins of the COP-coated vesicles were originally defined by ultrastructural criteria, however, and it is possible that important but minor coat proteins or cytoplasmic proteins needed for coat assembly may have been overlooked. Here we show that coatomer and ARF are the only cytoplasmic proteins needed for the assembly and budding of COP-coated vesicles. COP-coated buds may therefore form essentially by self-assembly from Golgi cisternae after an initial step in which GTP is used to allow ARF binding.

ADP-Ribosylation Factors↗
PubMed1993

Binding of coatomer to Golgi membranes requires ADP-ribosylation factor.

Coatomer, a complex of seven proteins, appears to be the precursor of the coat structure of non-clathrin-coated Golgi-derived vesicles. Another component of this vesicle coat is the cytosolic protein ADP-ribosylation factor (ARF). Like coatomer, ARF appears to reversibly associate with Golgi membranes. We now report that ARF is required for coatomer binding to Golgi membranes and that myristoylated, but not non-myristoylated, ARF is the required species. We utilize an antibody directed against the beta-subunit of coatomer (beta-COP) to follow coatomer binding. ARF and beta-COP bind stoichiometrically to Golgi membranes. ARF-dependent beta-COP binding requires a membrane-associated protein, is saturable, and is enhanced in the presence of stable GTP analogues like guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). ARF and beta-COP bind sequentially to Golgi membranes, since beta-COP can be bound to reisolated membranes that had been previously incubated with ARF and GTP gamma S. We conclude that membrane-bound ARF confers to Golgi membranes all of the requirements for specific beta-COP binding.

ADP-Ribosylation Factors↗
PubMed1993

Budding from Golgi membranes requires the coatomer complex of non-clathrin coat proteins.

Do the coats on vesicles budded from the Golgi apparatus actually cause the budding, or do they simply coat buds (Fig. 1)? One view (the membrane-mediated budding hypothesis) is that budding is an intrinsic property of Golgi membranes not requiring extrinsic coat proteins. Assembly of coats from dispersed subunits is super-imposed upon the intrinsic budding process and is proposed to convert the tips of tubules into vesicles. The alternative view (the coat-mediated budding hypothesis) is that coat formation provides the essential driving force for budding. The membrane-mediated budding hypothesis was inspired by the microtubule-dependent extension of apparently uncoated, 90-nm-diameter membrane tubules from the Golgi apparatus and other organelles in vivo after treatment with brefeldin A, a drug that inhibits the assembly of coat proteins onto Golgi membranes. This hypothesis predicts that tubules will be extended when coat proteins are unavailable to convert tubule-derived membrane into vesicles. Here we use a cell-free system in which coated vesicles are formed from Golgi cisternae to show that, on the contrary, when budding diminishes as a result of immunodepletion of coat protein pools, tubules are not formed at the expense of vesicles. We conclude that coat proteins are required for budding from Golgi membranes.

ADP-Ribosylation Factors↗
PubMed1993

Two distinct populations of ARF bound to Golgi membranes.

ADP-ribosylation factor (ARF) is a small molecular weight GTP-binding protein (20 kD) and has been implicated in vesicular protein transport. The guanine nucleotide, bound to ARF protein is believed to modulate the activity of ARF but the mechanism of action remains elusive. We have previously reported that ARF binds to Golgi membranes after Brefeldin A-sensitive nucleotide exchange of ARF-bound GDP for GTP gamma S. Here we report that treatment with phosphatidylcholine liposomes effectively removed 40-60% of ARF bound to Golgi membranes with nonhydrolyzable GTP, presumably by competing for binding of activated ARF to lipid bilayers. This revealed the presence of two different pools of ARF on Golgi membranes. Whereas total ARF binding did not appear to be saturable, the liposome-resistant pool is saturable suggesting that this pool of ARF is stabilized by interaction with a Golgi membrane-component. We propose that activation of ARF by a guanine nucleotide-exchange protein results in association of myristoylated ARF GTP with the lipid bilayer of the Golgi apparatus. Once associated with the membrane, activated ARF can diffuse freely to associate stably with a target protein or possibly can be inactivated by a GTPase activating protein (GAP) activity.

ADP-Ribosylation Factors↗
PubMed1992

Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules.

An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.

Adrenal Medulla↗