Biomedical subjects
P Gardner
Publications and source records attributed to P Gardner.
Cloning and analysis of two new isoforms of multifunctional Ca2+/calmodulin-dependent protein kinase. Expression in multiple human tissues.
Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is a mediator of calcium signals in diverse signaling pathways. In human lymphocytes and epithelial tissues, CaM kinase activates a chloride channel via a Ca(2+)-dependent pathway which is preserved in cystic fibrosis. To characterize the CaM kinase present in these tissues we have cloned an isoform of this kinase from human T lymphocytes. We show the cDNA structure of two variants of this human CaM kinase, gamma B and gamma C, which are predicted to translate to 518 and 495 amino acids, respectively. Amino acid differences between these isoforms and the rat brain gamma isoform (which we refer to as gamma A) are localized to the variable domain. We used RNase protection of this variable region to reveal the level of expression of gamma B and gamma C CaM kinase mRNAs in nine human tissues and cell lines. When transfected into Jurkat T cells, the gamma B cDNA encoded a functional kinase which cosedimented on sucrose gradients with endogenous T cell CaM kinase activity and formed a large multimeric enzyme. The recombinant gamma B isoform displayed two phases of autophosphorylation characteristic of CaM kinases, including the phase which converts it to a partially Ca(2+)-independent species. Site-directed mutagenesis of the predicted autoinhibitory domain yielded a mutant which was approximately 37% active in the absence of Ca2+/calmodulin, confirming the region as critical for autoregulation, and suggesting this mutant as a tool for studying the role of CaM kinase in nonneuronal tissues.
Flash photolysis of caged inositol 1,4,5-trisphosphate activates plasma membrane calcium current in human T cells.
Sustained elevation of intracellular Ca2+ by cell surface receptors is often dependent on influx of Ca2+ across the plasma membrane through routes not involving voltage-gated Ca2+ channels. We demonstrate that intracellular release of inositol 1,4,5-trisphosphate (InsP3), either from stimulation of transfected human muscarinic receptors or from photolytic release of caged InsP3, activates whole cell Ca2+ current in the Jurkat T cell line. Whole cell voltage clamp recordings indicate that the current is carried by a Ca(2+)-selective channel that resembles T-type voltage-gated Ca2+ channels in relative conductance of different cation species. Elevation of internal Ca2+ inactivates the channel, whereas internal perfusion with inositol 1,3,4,5-tetrakisphosphate (InsP4) does not affect it. Photolytic release of caged 1-(alpha-glycerophosphoryl)-inositol 4,5-bisphosphate, an analog of InsP3 which activates InsP3 receptors but is not readily metabolized to InsP4, also activates the current. We conclude that generation of InsP3 is sufficient to activate Ca(2+)-selective channels in the plasma membrane of T cells. InsP3 may have its effect indirectly through depletion of Ca2+ stores, or directly with a plasma membrane-associated InsP3 receptor.
Hepatitis B immunization strategies: expanding the target.
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Recombinant human tumor necrosis factor alpha induces calcium oscillation and calcium-activated chloride current in human neutrophils. The role of calcium/calmodulin-dependent protein kinase.
The role of calcium in the action of tumor necrosis factor (TNF) on human neutrophils is not clear. With fluorescent cytometry, using the visible wavelength calcium probe, fluo-3, and patch clamping, we investigated whether TNF induces cytosolic free Ca2+ changes and Ca(2+)-activated Cl- current, respectively. Bath application of 1000 units/ml recombinant human TNF alpha (rhTNF alpha) induced a rise in cytosolic free Ca2+ in 75% of fluo-3-loaded cells, 25% of which displayed irregular patterns of oscillation. Addition of rhTNF alpha activated Cl- current in 80% of tested cells; the activated current was blocked by 10 microM 5-nitro-2-3-phenylpropylamino)benzoic acid, a Cl- channel blocker. The current was similarly activated by 1 microM ionomycin, a Ca2+ ionophore. To study the mechanism by which rhTNF alpha induced Ca(2+)-activated Cl- current, we examined the involvement of calcium/calmodulin-dependent protein kinase (CaM kinase). With intracellular application of the Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (5 mM), the calmodulin antagonist (2 microM), CaM kinase II-(290-309), or the inhibitory peptide (10 microM), CaM kinase II-(273-302), the current was no longer activated by rhTNF alpha. The intracellular application of the control peptide (10 microM), CaM kinase II-(284-302), or the protein kinase C (PKC) inhibitory, PKC-(19-36), or control, [Glu27]PKC-(19-36), peptide (5 microM) did not block the rhTNF alpha-induced Cl- current. These results show that Ca2+ changes are associated with the effects of rhTNF alpha and that CaM kinase plays a role in the mechanism underlying rhTNF alpha-induced activation of Ca(2+)-activated Cl- current in human neutrophils.
Cloning and analysis of the gene encoding hummingbird proinsulin.
Because hummingbirds exhibit the highest mass-specific metabolic rates seen among vertebrates and rely on sugars as their main energy source, we have investigated the structure of hummingbird insulin (Selaphorus rufus) to determine whether it possesses structural adaptations that increase its receptor binding affinity (potency). We report here the nucleotide sequence of hummingbird proinsulin determined from hummingbird genomic DNA. The predicted amino acid sequence of the A-chain of hummingbird insulin is identical to that of chickens and the B-chain differs by only one amino acid at a noncritical position, B2 (Val in hummingbird and Ala in chicken). These findings suggest that alterations in secretory and metabolic dynamics of insulin are of greater importance than changes in binding affinity in the adaptation to states of high carbohydrate flux in these very energetic organisms.
A comparison of visual and automated methods of analyzing fetal heart rate tests.
OBJECTIVES: Our objective in this study was to compare evaluation and clinical implications of visual versus computerized analysis of nonstress tests. METHODS: Nonstress tests of 575 high-risk patients were analyzed visually and by a computer using the Oxford Sonicaid System 8000. Standard reactivity criteria were used for visual assessment; the System 8000 used an algorithm with the Dawes-Redman criteria. RESULTS: Ninety-six percent of nonstress tests that met Dawes-Redman criteria were reactive by visual analysis; 93% of reactive nonstress tests met Dawes-Redman criteria. Only 30% of tests that failed Dawes-Redman criteria were nonreactive, whereas 44% of nonreactive tests failed to meet Dawes-Redman criteria. Sensitivities, specificities, and positive and negative predictive values were similar for both approaches. Additional tests or interventions would have occurred in 9% of the cases analyzed by System 8000 and in 49% of the cases analyzed visually. CONCLUSIONS: Although these approaches rate nonstress tests differently, their diagnostic performances are similar. Automated fetal heart rate testing may become an acceptable alternative to conventional visual analysis.
Chemical shift photoelectron diffraction from molecular adsorbates.
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Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3.
Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction. This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al. [Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, 93-96]. PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction. This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins. These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.
Abnormal chloride conductance in multidrug resistant HL60/AR cells.
Chloride channel currents were measured in drug sensitive parental HL60 and multidrug resistant (MDR) subline HL60/AR cells, using a whole cell patch-clamp technique. In addition, the in vitro effects of 4,4' diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, on intracellular accumulation and sensitivity to daunorubicin and intracellular pH (pH(i)) in HL60 cells were examined. Baseline DIDS blockable Cl- currents were consistently lower in HL60/AR cells (0.9 pA/pF) as compared to HL60 cells (7.0 pA/pF). Similarly cAMP-activated Cl- currents were minimal in HL60/AR cells (0.2 pA/pF) as compared to HL60 cells (8 pA/pF). In vitro treatment of drug sensitive HL60 cells with DIDS resulted in concentration-dependent decreased accumulation and increased resistance to daunorubicin and decreased pH(i). These data show that altered Cl- permeability is associated with MDR and suggest that Cl- channels may play a role in MDR.
Structural determination of a molecular adsorbate by photoelectron diffraction: Ammonia on Ni{111}
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Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.
Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.
Adsorbate structures from photoelectron diffraction: Holographic reconstruction or real-space triangulation?
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Human lymphocytes transcribe the cystic fibrosis transmembrane conductance regulator gene and exhibit CF-defective cAMP-regulated chloride current.
Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians, primarily affecting epithelial tissues of the lung and gut. Mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR), are responsible for this disease. Whether a physiological defect exists in the immune system of CF patients has remained controversial. A chloride ion transport defect has been described in human CF-derived lymphocytes; however, it has not been possible to detect CFTR mRNA in lymphocytes. We report here that normal human B-lymphoblasts display whole cell Cl- conductances induced by calcium-mediated pathways, volume regulation, and cAMP which are equivalent to currents described in epithelial cells. B-lymphoblasts from CF-affected humans demonstrated defective Cl- conductance regulation by cAMP but preserved regulation by calcium-mediated and volume regulation mechanisms. CFTR involvement in cAMP regulation of Cl- conductance in lymphocytes is further supported by our demonstration of the presence of appropriately spliced CFTR mRNA segments in human B and T lymphocytes as detected by an optimized reverse-transcription and polymerase chain reaction approach. The identity of the amplified products was confirmed by hybridization to CFTR-specific probes and DNA sequencing. Furthermore, the 3'-end of the gene was found in a T cell cDNA library. We conclude that CFTR mRNA is expressed in lymphocytes, consistent with the cAMP regulation of chloride transport present in normal lymphocytes but defective in CF-derived lymphocytes.
Functional and immunological responses of Jurkat lymphocytes transfected with the substance P receptor.
1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+ ([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific 125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and 125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with a Kd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10(-11)-10(-6) M SP and was maximal at 10(-7) M SP, with an EC50 of 0.3-0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl- current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will not permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.
Optimum sampling time for detection of dental bacteraemia in children.
A total of 458 blood cultures were taken from 229 children aged 2 to 16 yr. Each 6 ml sample was taken starting at one of the following times after the extraction of a single tooth: 10 s, 30 s, 60 s, 90 s, 120 s, 180 s, 600 s and continuously over 2.5 min. The samples were cultured in the BACTEC radiometric culture system and when positive the bacteria isolated were speciated. At 30 s 56% of the samples were positive, the highest proportion, in contrast to only 38% of positive samples at 90 s and 28% at 600 s. Over 50% of the organisms found were Viridans streptococci.
Site-specific incorporation of [125I]iododeoxyuridine into DNA.
A procedure for the incorporation of [125I]IdU into specific sites in DNA is described. The approach depends upon attachment of radioiododeoxyuridine to a controlled pore glass support which is then used for automated synthesis of an oligomer. The resulting oligomer, containing a terminal 3'[125I]iododeoxyuridine, is used as a primer during DNA synthesis catalyzed by the Taq polymerase employing thermal cycling. The product formed includes the radioiodonucleotide at a single internal site determined by the length of the oligomer.
Refining the biophysical profile with a risk-related evaluation of test performance.
OBJECTIVE: Our objective was to determine the ability of biophysical profile variables to predict bad perinatal outcome in high-risk third-trimester pregnancy. STUDY DESIGN: The outcomes of 1146 fetuses were correlated with abnormal single or multiple variables occurring in biophysical profile done within 72 hours of delivery. Theoretic risks of poor outcomes for different combinations of abnormal variables, calculated with Shortlife's formula, were compared with the actual risks observed (chi 2 tests). RESULTS: Two hundred forty-six fetuses had at least one abnormal biophysical profile variable with the risk of bad outcome, for a single abnormal variable, ranging from 8% (body movements) to 100% (tone) and increasing from 14% (any variable abnormal) to 63% (all variables abnormal). In most (57%) observed combinations of biophysical profile variables, significant differences between theoretic and actual risks of bad outcomes were found. By stepwise logistic regression the best predictive model contained all variables except fetal movement. CONCLUSIONS: Dynamic biophysical profile variables appear to be interdependent. Not all combinations of abnormal variables occur and specific combinations improve prediction of poor outcome. Risk-related scales for biophysical profile outcomes might prove superior to more conventional scoring systems.