Composition and structure of the GaN{0001-bar}-(1 x 1) surface.
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Journal Article↗
Biomedical subjects
J Ahn
Publications and source records attributed to J Ahn.
Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.
The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.
Animals↗
Identification of the cysteine residues implicated in the formation of alpha 2 and alpha/beta dimers of rat meprin.
Meprin (endopeptidase-24.18; EC 3.4.24.18) is a multisubunit zinc-metallopeptidase found in the brush-border membranes of rodent kidney and human intestine. The alpha and beta subunits of meprin are disulphide-linked to form either soluble alpha 2 homodimers or membrane-associated alpha/beta heterodimers. The aim of the present study was to identify the cysteine residue(s) implicated in the formation of alpha 2 and alpha/beta dimers and to investigate the effects of dimerization on intracellular transport and processing of the alpha subunit. Three cysteine residue candidates for the formation of disulphide bonds in the alpha subunit were selected by hydrophobic cluster analysis. These residues, located at positions 309, 560 and 562, were mutated to serine residues. When the resulting alpha subunit mutants were expressed alone in COS-1 cells, the alpha C560S and alpha C562S mutants were found to be secreted as alpha 2 homodimers whereas the alpha C309S mutant was found as monomers in the culture medium. In double-transfection experiments with the wild-type beta subunit, the alpha C560S and alpha C562S mutants behaved exactly as the wild-type alpha subunit and formed membrane-bound alpha/beta heterodimers. In contrast, the alpha C309S mutant was not retained at the cell surface but rather secreted as monomers in the culture medium, as was found in the simple transfection experiment. These results show that, despite the normal expression level and folding of the protein in a transport-competent from, the alpha C309S mutant is unable to form alpha 2 homodimers or alpha/beta heterodimers. This suggests that Cys309 is the unique residue of the alpha subunit implicated in the alpha 2 and alpha/beta dimerizations. Hydrophobic cluster analysis of the alpha and beta subunit sequences predicts that Cys309 is similar to Cys306 of the beta subunit. We mutated the latter residue to a serine and expressed the beta C306S mutant and the wild-type alpha subunit in the same COS-1 cells. No beta 2 or alpha/beta dimers were observed on immunoblotting, showing that Cys306 of the beta subunit is required for the formation of intermolecular disulphide bonds both in beta 2 homodimers and in alpha/beta heterodimers. Taken together, these results suggest that the alpha/beta heterodimeric form of meprin is held together by a single disulphide bond linking Cys309 in the alpha subunit to Cys306 in the beta subunit.
Amino Acid Sequence↗
Cell-specific sorting of biogenic amine transporters expressed in epithelial cells.
We have utilized polarized epithelial cells stably expressing neurotransmitter transporters to analyze the sorting behavior of these membrane proteins. The transporters for serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are expected to be present in situ in the most distal extremities of axonal membranes, where they terminate the action of their biogenic amine substrates. Both Madin-Darby canine kidney (MDCK) and LLC-PK1 cells were stably transfected with cDNAs encoding either the rat 5-HT transporter (SERT), the human NE transporter (NET), or the rat or human DA transporter (DAT). These cells were grown on permeable filter supports, and the transporters were localized by three independent techniques. Confocal immunofluorescence microscopy indicated that each of the transporters expressed in LLC-PK1 cells was sorted to the basolateral membrane, co-localizing with the Na+/K+-ATPase. In MDCK cells, however, DAT was located primarily on the apical surface, while SERT and NET were found on the basolateral membranes. Cell surface biotinylation using an impermeant biotinylating reagent confirmed the immunocytochemistry results. Thus, SERT and NET in MDCK cells were labeled more efficiently from the basolateral medium than the apical medium, and DAT in MDCK cells was labeled more efficiently from the apical side than the basolateral side. Transport measurements in transfected MDCK cells agreed with the immunocytochemistry and biotinylation results. These results suggest the existence of cell-specific mechanisms that discriminate between neurotransmitter transporters for surface expression and render unlikely any simple hypothesis that sorting mechanisms in neurons and epithelia are identical.
Animals↗
DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity.
DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.
Animals↗
Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons.
At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations.
Amino Acid Sequence↗
Clinical predictors of tuberculosis as a guide for a respiratory isolation policy.
An expanded respiratory isolation policy was implemented in a public hospital that cares for about 200 patients with active tuberculous each year. This led to proper isolation of > or = 95% of patients with tuberculosis on admission but involved an 8-fold overuse of isolation rooms. We developed a model policy to decrease overisolation of nontuberculous patients. Clinical findings in 295 patients admitted to respiratory isolation during a 3-mo period were evaluated for their usefulness in determining which patients had tuberculosis. Multivariate analysis identified five predictive variables: chest radiograph with upper lobe infiltrate (odds ratio, 5.00; CI, 2.38 to 10.51; p = 0.001) or cavity (odds ratio, 3.93; CI, 1.06 to 14.62; p = 0.041), history of having known someone with tuberculosis (odds ratio, 2.42; CI, 1.10 to 5.32, p = 0.027), self-reported positive tuberculin skin test (odds ratio, 5.67; CI, 1.57 to 22.01; p = 0.009), self-reported isoniazid preventive therapy (odds ratio, 0.18; CI, 0.04 to 0.82; p = 0.027). Using these variables to determine which patients required isolation would have decreased the number of isolated nontuberculous patients from 253 to 95, but it would have missed eight of 42 patients with tuberculosis. Further work is needed to identify clinical predictors that would decrease overuse of isolation beds while maintaining satisfactory sensitivity for patients with tuberculosis.
Adult↗
Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1).
Hereditary multiple exostoses is an autosomal dominant disorder that is characterized by short stature and multiple, benign bone tumours. In a majority of families, the genetic defect (EXT1) is linked to the Langer-Giedion syndrome chromosomal region in 8q24.1. From this region we have cloned and characterized a cDNA which spans chromosomal breakpoints previously identified in two multiple exostoses patients. Furthermore, the gene harbours frameshift mutations in affected members of two EXT1 families. The cDNA has a coding region of 2,238 bp with no apparent homology to other known gene sequences and thus its function remains elusive. However, recent studies in sporadic and exostosis-derived chondrosarcomas suggest that the 8q24.1-encoded EXT1 gene may have tumour suppressor function.
Amino Acid Sequence↗
Ensemble competitive learning neural networks with reduced input dimension.
Conventional neural networks utilize all the dimensions of the original input patterns for training and classification. However, a particular attribute of the input patterns does not necessarily contribute to classification and may even cause misclassification in certain cases. A new ensemble competitive learning method using the reduced input dimension is proposed. In contrast to the previous ensemble neural networks which adjust learning parameters, the proposed method takes advantage of the information in each dimension of the input patterns. Since the degree of contribution of each attribute to classification is not known beforehand, the different input data sets with one dimension reduced are presented to multiple neural networks. The classification information from each competitive learning neural network is then combined to make a final decision for classification. In order to improve classification accuracy, the ambiguous output neurons are eliminated which cannot be assigned to any class after training. We use three consensus schemes to judge the classification using ensemble neural networks. The experimental results with remote sensing and speech data indicate the improved performance of the proposed method.
Algorithms↗
Case of the day. Post-renal biopsy complication: perinephric hematoma and arteriovenous fistula.
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Arteriovenous Fistula↗
Effect of glucose and cytokines on the expression of cell adhesion molecules on mesangial cells.
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Cell Adhesion Molecules↗
Helicobacter pylori infection does not increase gastric antrum mucosal cell proliferation.
OBJECTIVES: Gastric carcinoma is the world's second most common cancer. Recent studies suggest an association between Helicobacter pylori and gastric carcinoma. The aim of this study was to address the effects of H. pylori infection on gastric antrum mucosal cell proliferation. METHODS: Forty patients undergoing upper endoscopy for standard indications were included in the study. A rapid urease test was used to determine the presence of H. pylori. Epithelial cell proliferation was determined by immunohistochemical techniques utilizing monoclonal antibody to bromodeoxyuridine. RESULTS: There were no significant differences in the number of labeled cells and in the proliferation fraction (p > 0.1) when patients with H. pylori were compared with those without, and when those over the age of 50 were compared to those under 50. The presence of ulcers similarly had no effect (p > 0.1). CONCLUSION: Helicobacter pylori infection does not increase gastric antrum mucosal cell proliferation.
Age Factors↗
Ion transport in rabbit proximal colon: effects of sodium, amiloride, cAMP, and epinephrine.
Effects on ion transport of extracellular Na+ and Cl- ([Na+]o and [Cl-]o, respectively), HCO3(-)-CO2, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), epinephrine, and transport inhibitors were examined in short-circuited rabbit proximal colonic mucosa. Net Na+ flux was independent of Cl- but partially (60%) dependent on HCO3(-)-CO2. Net Cl- flux was partially (70%) dependent on Na+ and totally dependent on HCO3(-)-CO2. Both fluxes peaked between 25 and 60 mM and decreased at higher concentrations. Apical Na+ influx but not Cl- influx obeyed the same pattern. The inhibition resulted from increases in mucosal but not serosal [Na+]o and not from increases in [Cl-]o. Amiloride and benzamil (0.2-0.3 mM) partially inhibited net Na+ absorption, as did 8-BrcAMP, but these effects were independent of the inhibition seen at high [Na+]o. Net Cl- absorption was inhibited by 8-BrcAMP but not by 0.2 mM amiloride. At high [Na+]o and [Cl-]o, there were a residual ion flux suggesting HCO3- secretion and, in the presence of 8-BrcAMP and amiloride or benzamil, net secretions of Na+ and Cl-, the former larger than the latter. Epinephrine, via alpha 2-receptors, reversed the ion-transport effects of high [Na+]o but did not stimulate a mucosal-to-serosal unidirectional HCO3- flux (as shown in rabbit ileum). 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (0.5 mM) and bumetanide (10 microM) had no effect on ion transport. The results suggest 1) Na+ entry via two Na(+)-H+ exchangers, one inhibited by amiloride and cAMP and the other inhibited by high [Na+]o and stimulated by epinephrine; 2) Cl- entry via Cl(-)-HCO3- exchange; 3) HCO3- secretion at high [Na+]o and [Cl-]o; and 4) cAMP-induced secretion of Na+ and Cl- at high [Na+]o and [Cl-]o.
Amiloride↗
Neonatal nasal deformities secondary to nasal continuous positive airway pressure.
Nasal continuous positive airway pressure (CPAP) is being used more frequently in neonatal intensive care units to avoid endotracheal intubation and its attendant complications. Nasal deformities secondary to the nares-occluding prongs employed to deliver the CPAP in former preterm infants have been recognized, and prevention and surgical treatment of these deformities are discussed. An awareness of the potential for these deformities is important to all pediatric otolaryngologists as nasal CPAP is gaining increasing favor as a primary treatment for respiratory disease of the newborn.
Child, Preschool↗
Human insulin receptor mutated at threonine 1336 functions normally in Chinese hamster ovary cells.
Phosphorylation of threonine 1336 of the human insulin receptor (HIR) is stimulated by insulin or 4 beta-phorbol 12-myristate 13-acetate in Chinese hamster ovary (CHO) transfectant cells expressing the wild type receptor (CHO/HIR). To examine the role of this phosphorylation in insulin signal transduction, a mutant human insulin receptor, in which threonine 1336 was replaced with asparagine, has been stably expressed in CHO cells (CHO/HIRT1336N). CHO cell lines expressing equivalent numbers of the wild type or the mutant receptor were developed, which bound 125I-insulin comparably (Kd = 0.1 nM). After stimulation of CHO/HIR or CHO/HIRT1336N cells with insulin, the wild type and mutant receptors internalized the hormone and were down-regulated with similar rates. Hormone stimulation of the receptor tyrosine kinase activity was also unaffected by the mutation. Metabolic and mitotic effects of insulin were also unimpaired by the mutation. Thus, insulin stimulated phosphatidylinositol 3-kinase activity, glycogen synthesis, and thymidine incorporation into DNA similarly in CHO/HIR and CHO/HIRT1336N cells. These data suggest that by itself phosphorylation of threonine 1336 has no significant effect on insulin binding, regulation of insulin receptor expression, or insulin signal transduction.
Animals↗
Origin of a soluble truncated transferrin receptor.
It has recently become evident that elevation of reticulocytes in the circulation of several species, including humans, leads to the formation of a noncellular transferrin receptor (TFR). In humans, the majority of the released receptor is in truncated form (Shih et al: J Biol Chem 265:19077, 1990). In other species (sheep, rat, chicken) the receptor is associated with a vesicle (exosome) and is full length (Johnstone et al: J Cell Physiol 147:27, 1991). In this report we show that in sheep reticulocytes incubated in vitro, the majority (approximately 75%) of the released receptor is of native size and is exosome associated. A fraction (approximately 25%) is a truncated form of approximately 80 Kd corresponding to the exofacial domain of the TFR. Herein we also address the question of whether the truncated receptor originates by proteolytic cleavage directly from the cell surface or by cleavage from exosomes. Using surface 125I-labeled sheep reticulocytes as the experimental model, we show that during in vitro maturation, 125I-TFR of native size appears in exosomes before the soluble, truncated, exofacial domain of the receptor is detected in the medium. Because cleavage and release of the exofacial domain would likely leave the truncated cytoplasmic and transmembrane domains in the originating membrane (plasma membranes or exosomes), both fractions were probed with antibodies specifically generated against the cytoplasmic domain of the receptor. Only exosomes, not plasma membranes, show the presence of a approximately 17-Kd peptide recognized by the antibody to the cytoplasmic domain of the transferrin receptor. Thus, it is concluded that the truncated, soluble receptor originates from exosomes in sheep. A 17-Kd cytoplasmic domain of the TFR was also detected in exosomes from the reticulocytes of an anemic man, suggesting that the truncated receptor in man may also originate from exosomes. Using in vitro cultures of surface 125I-labeled sheep reticulocytes, it is concluded that exosome formation is the principal route for maturation-associated loss of the TFR. A similar conclusion was made earlier (Johnstone et al: J Cell Physiol 147:27, 1991) for the nucleoside transporter of maturing sheep reticulocytes.
Animals↗
Interaction of the human insulin receptor tyrosine kinase from the baculovirus expression system with protein kinase C in a cell-free system.
The cytoplasmic domain of the human insulin receptor (HIR) from the baculovirus expression system (BIRK) is a soluble, constitutively activated protein-tyrosine kinase. In a cell-free system, BIRK is phosphorylated on serine and threonine residues by protein kinase C (PKC) purified from rat brain. Two-dimensional tryptic phosphopeptide mapping of PKC-phosphorylated BIRK identified one phosphothreonine and three phosphoserine peptides, which were also in tryptic digests of insulin receptors from insulin- or PMA-treated Chinese hamster ovary (CHO) cells transfected with the HIR. After Lys-C proteolysis of PKC-phosphorylated BIRK, radioactive phosphopeptides were purified on a C8 reverse-phase high pressure liquid chromatography column. Amino acid sequence analysis identified a phosphothreonine peptide corresponding to amino acids 1331-1340 of the HIR. This peptide contains only one threonine, amino acid 1336, which is identified as a site for PKC phosphorylation in BIRK. CHO cells transfected with the wild type (CHO/HIR) or a mutant human insulin receptor (CHO/HIRT1336N), in which threonine 1336 was substituted with asparagine, were 32P labeled and then stimulated with insulin or phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide analysis of the HIR revealed that phosphorylation of phosphothreonine peptide T, shown to be in PKC-phosphorylated BIRK, was increased by insulin or PMA. However, the corresponding peptide was not in the mutant receptor. Therefore, the present study directly identifies threonine 1336 in the HIR as a phosphorylation site for insulin and PMA. These data also show that BIRK can be used as a model for the study of the regulation of the insulin receptor kinase.
Animals↗
Naloxone-responsive encephalopathy in end-stage renal disease.
We report the case of a 61-year-old diabetic woman with end-stage renal disease who was on hemodialysis and who developed an encephalopathy and episodes of hypotension and hypoventilation, all of which showed rapid and dramatic responses on multiple occasions to the administration of the opiate antagonist naloxone. Improvement in encephalopathy was confirmed by electroencephalography. The patient had received no exogenous opiates and had a normal beta-endorphin level. She subsequently developed myoclonus and was treated for possible aluminum overload that was of borderline magnitude. We conclude that this patient had an encephalopathy that responded to opiate receptor blockade. Because of cerebrovascular disease, episodes of diminished blood pressure due to a state of increased opiate receptor stimulation may have unmasked this underlying encephalopathy. These effects may have been secondary to increased opiate-binding sites or to elevated central nervous system levels of endogenous opiates.
Brain Diseases↗