Biomedical subjects
G Shaw
Publications and source records attributed to G Shaw.
The pleckstrin homology domain: an intriguing multifunctional protein module.
Pleckstrin homology (PH) domains are a family of compact protein modules defined by sequences of roughly 100 amino acids. These domains are common in vertebrate, Drosophila, C. elegans and yeast proteins, suggesting an early origin and fundamental importance to eukaryotic biology. Many enzymes which have important regulatory functions contain PH domains, and mutant forms of several such proteins are implicated in oncogenesis and developmental disorders. Numerous recent studies show that PH domains bind various proteins and inositolphosphates. Here I discuss PH domains in detail and conclude that they form a versatile family of membrane binding and protein localization modules.
Localization of sites in the tail domain of the middle molecular mass neurofilament subunit phosphorylated by a neurofilament-associated kinase and by casein kinase I.
We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKl) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKl inhibitor CKl-7, (2) the similarity in substrate specificity of NFAK and authentic CKl, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKl under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKl. We have also identified Ser502, SER528, and Ser536 as phosphorylation sites by NFAK/CKl in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKl phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser502 nor Ser536 has been identified previously as NF-M phosphorylation sites.
The association of the C-terminal region of beta I sigma II spectrin to brain membranes is mediated by a PH domain, does not require membrane proteins, and coincides with a inositol-1,4,5 triphosphate binding site.
The beta spectrin genes each produce two alternate transcripts the longer of which has a approximately 210 amino acid C-terminal extension including a pleckstrin homology (PH) domain and also an uncharacterized membrane binding site. GST constructs including the entire or the N-terminal segment of the beta I sigma II spectrin PH domain bind to crude and extracted brain membranes, to protein free brain lipid and to vesicles containing phosphatidylinositol-4,5-bisphosphate. This PH domain also binds radiolabelled inositol-1,4,5-trisphosphate (IP3) and preincubation with IP3 inhibits binding to extracted brain membranes. We conclude that membrane binding of the beta I sigma II spectrin C-terminal region is by means of a direct interaction between the N-terminal region of the PH domain and membrane lipids and does not require membrane protein. The PH domain of the beta-adrenergic receptor kinase showed different binding properties in every assay employed, showing that different PH domains may have different membrane binding specificity.
Alpha 4 beta 1 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand.
alpha 4 beta 1 integrin (VLA-4) appears to be unique among the leukocyte integrins in that it can initiate the adhesion of circulating lymphocytes without cellular activation. It is not known how lymphocytes or other cell types maintain constitutive levels of alpha 4 beta 1 integrin activity. The current report describes a monoclonal antibody, 15/7, that recognizes a high affinity or ligand-occupied conformation of beta 1 integrin. Studies with 15/7 revealed that alpha 4 beta 1 integrin-dependent adhesion of leukocytic cell lines is mediated by a population of low affinity receptors that is conformationally responsive to ligand; the 15/7 epitope could be induced by nanomolar concentrations of soluble VCAM-1 or by micromolar concentrations of a peptide derived from the type III connecting segment domain of fibronectin (as ligands for alpha 4 beta 1 integrin). The same receptors were also responsive to adhesion activating reagents, such as Mn2+, activating anti-beta 1 integrin antibodies, and phorbol myristate acetate, which induced the 15/7 epitope directly and/or decreased the concentration of ligand required for epitope induction. In addition to the responsive receptor pool, cells expressed a second population of alpha 4 beta 1 integrin that was conformationally restrained, failing to respond to ligand or to any of the activating reagents. The relative size of the responsive and inactive receptor pools, as well as the affinity of the responsive receptors, represented a stable phenotype of different cell types and played important roles in defining the cells' adhesive capacity and ligand specificity. Similar receptor populations were measured on lymphocyte subsets in whole blood. These studies provide insight into how cells maintain different constitutive levels of alpha 4 beta 1 integrin activity, and how the activity of beta 1 integrin can be modulated by activators of cell adhesion.
The marsupial male: a role model for sexual development.
Sexual differentiation in male marsupials has many similarities with that of eutherians. Marsupials have an XX-XY sex determining mechanism, and have a homologue of the testis-determining SRY gene on their Y-chromosome. However, the development pattern of SRY gene expression is different from the mouse in that it is expressed for a much longer period. SRY is expressed in a range of non-gonadal tissues in male pouch young and adults which is similar to the human pattern, and raises questions as to its particular role(s) in sexual differentiation. Similarly Müllerian inhibiting substance (MIS) is produced in the developing testis over a longer period than in the mouse. Since ovaries cultured with MIS or transplanted into male recipient pouch young develop tubular structures, MIS may induce Sertoli cell formation. Testosterone is produced by the neonatal testis, and this stimulates Wolffian duct development to form the vas deferens and epididymis. Virilization of urogenital sinus is also androgen-dependent. However, virilization of the prostate and phallus occurs more than three weeks after the onset of testosterone production, suggesting that the timing of this may be regulated by delayed activation of the androgen receptor pathway. Unlike in eutherians, differentiation of the scrotum and mammary glands is not dependent on testicular hormones, but is independently regulated by an X-linked genetic mechanism. Clearly marsupials provide a unique perspective to help us clarify the mechanisms underlying sexual development in all mammals.
Binding of pleckstrin homology domains to WD40/beta-transducin repeat containing segments of the protein product of the Lis-1 gene.
In previous experiments we demonstrated an interaction between certain pleckstrin homology (PH) domains and regions containing the so-called WD40 (or beta-transducin) repeats of the beta subunit of trimeric G-proteins (G beta), a finding we here extend to the PH domains of the src-related tyrosine kinase TecIIa and the GTPase dynamin. To examine the possibility that WD40 repeats in molecules other than G beta might also bind PH domains we examined PAFAH-45, the protein product of the Lis-1 gene, which contains 7 WD40 repeats. We found that 1) Purified PAFAH-45 binds PH domain constructs in vitro. 2) Protein constructs expressing all 7 WD40 repeats of PAFAH-45 but lacking the N-terminal non WD40 region also bind PH domains of beta-adrenergic receptor kinase, beta-spectrin, TecIIa and dynamin but with a differing hierarchy of affinities than that seen with G beta. 3). PAFAH-45 WD40 repeats will reduce the binding of PH domains to brain G beta and brain G beta gamma will reduce the binding of PH domains to PAFAH-45. These data support the hypothesis that PH domain/WD40 interactions are involved in a wide variety of important protein/protein interactions.
The use of in vitro DNA adduct formation to estimate the genotoxicity of residues at contaminated sites.
Genotoxic carcinogens such as polycyclic aromatic hydrocarbons (PAHs) covalently bind to the bases in DNA to form adducts. The formation of DNA adducts is significant with respect to chemical carcinogenesis. Many contaminated sites contain quantities of carcinogens such as PAHs, and the evaluation of the genotoxicity of these soils has important implications for human risk assessment. DNA adducts can be formed using an in vitro system incorporating extracts from contaminated soils. The 32P-postlabelling assay is a sensitive technique for the detection of DNA adducts from complex mixtures of environmental carcinogens. These techniques have been used to form and detect DNA adducts using soils from a number of coal gasworks sites. The results show that the extent of adduct formation depends partially on the petroleum hydrocarbon content of samples, but also on other undetermined factors related to composition. While environmental weathering has been shown to effect the PAH composition of samples, this is not an important factor in controlling the genotoxicity of samples as estimated by DNA adduct formation.
Frequency of thyroid dysfunction in diabetic patients: value of annual screening.
A randomly selected group of 1310 adult diabetic patients attending a diabetic outpatient clinic received annual screening for thyroid disease, by estimating serum free thyroxine and TSH concentrations. The overall prevalence of thyroid disease was found to be 13.4%, and was highest (31.4%) in Type 1 diabetic females, and lowest in Type 2 diabetic males (6.9%). As a direct result of screening, new thyroid disease was diagnosed in 6.8% (89 patients) of the population screened; the commonest diagnosis was subclinical hypothyroidism (4.8%), followed by hypothyroidism (0.9%), hyperthyroidism 0.5%), and subclinical hyperthyroidism (0.5%). Female patients with Type 1 diabetes had the highest annual risk of developing thyroid disease (12.3%), but all patient groups had a higher incidence of thyroid dysfunction, compared to that reported in the general population. This study suggests that thyroid function should be screened annually in diabetic patients to detect asymptomatic thyroid dysfunction which is increased in frequency in a diabetic population.
Binding of PH domains of beta-adrenergic receptor kinase and beta-spectrin to WD40/beta-transducin repeat containing regions of the beta-subunit of trimeric G-proteins.
Pleckstrin homology (PH) domains are found in numerous proteins important in signal transduction and cytoskeletal function. Several PH domains are now known to contain a binding site for the beta gamma subunits of trimeric G-proteins (G beta gamma), a finding which naturally raises the question of where on the G beta gamma complex these PH domains bind. Here we demonstrate binding of the PH domains of beta-adrenergic receptor kinase and beta-spectrin to the G beta subunit and not the G gamma subunit in a nitrocellulose gel replica assay. Furthermore, the C-terminal tryptic fragment of G beta containing only 5 WD40/beta-transducin (WD40) repeats also binds these two PH domains. Finally, constructs containing only WD40 repeats of G beta were shown to bind to beta-ARK and beta-spectrin PH domains in solution. These findings suggest that WD40 repeats of G beta are ligands for PH domains and have interesting implications for other proteins containing WD40 sequences.
Added salmeterol versus higher-dose corticosteroid in asthma patients with symptoms on existing inhaled corticosteroid. Allen & Hanburys Limited UK Study Group.
Guidelines on asthma management recommend that in patients who still have symptoms on treatment with low-dose inhaled corticosteroids the first step should be an increase in inhaled corticosteroid dose. The addition of long-acting inhaled beta 2-adrenoceptor agonists is another option. We have compared these two strategies in a randomised, double-blind, parallel-group trial. We studied 429 adult asthmatic patients who still had symptoms despite maintenance treatment with 200 micrograms twice daily inhaled beclomethasone dipropionate (BDP). 3 did not provide verifiable data. Of the others, 220 were assigned salmeterol xinafoate (50 micrograms twice daily) plus BDP and 206 were assigned higher-dose BDP (500 micrograms twice daily) for 6 months. The mean morning peak expiratory flow increased from baseline in both groups, but the increase was greater in the salmeterol/BDP group than in the higher-dose BDP group at all time points (differences 16-21 L/min, p < 0.05). Mean evening PEF also increased with salmeterol/BDP but not with higher-dose BDP. There were significant differences in favour of salmeterol/BDP in diurnal variation of PEF (all time points) and in use of rescue bronchodilator (salbutamol) and daytime and night-time symptoms (some time points). There was no significant difference between the groups in adverse effects or exacerbations of asthma, indicating that in this group of patients regular beta 2-agonist therapy was not associated with any risk of deteriorating asthma control over 6 months. This study suggests a need for a flexible approach to asthma management.
Number and location of AUUUA motifs: role in regulating transiently expressed RNAs.
Many RNAs coding for either cytokines or oncogenes are unstable and have a short half-life (t1/2). The AUUUA motif is a highly conserved sequence and is repeated three or more times in the 3' untranslated region (3'UTR) of RNAs encoding many of these short-lived cytokines and oncogenes. These sequences can confer instability. In this study, we investigated the role of number and location of AUUUA motifs in stabilization of RNA. We introduced 1xATTTA, 2xATTTA, ATTTTTTTA (second adenosine of 2xATTTA was substituted with a thymidine), 3xATTTA, 5xATTTA, 7xATTTA [AT-rich sequence from granulocyte-macrophage colony-stimulating factor [GM-CSF] gene (AT-62)], and GC-62 (GC sequences were substituted for ATTTA sequences in the 7xATTTA) into the 3'UTR of rabbit beta-globin (R beta G) gene. This construct also contained the neomycin-resistance gene. These expression vectors were transfected into human lung fibroblasts (W138), which constitutively expressed low levels of GM-CSF mRNA. Stable transfectants were selected by growth in G418. Northern blot analysis of actinomycin D-treated, stably transfected cells demonstrated that the number of AUUUA sequences correlated with rapidity of turnover of the chimeric R beta G mRNA. The rank order of stability was GC-62 = 1xATTTA = 2xATTTA (no RNA decay at 4 hours) > 3xATTTA = 5xATTTA (t1/2, 4 hours) > 7xATTTA (t1/2, 2 hours). Stability of mRNA of R beta G also was reduced (t1/2, 2 to 4 hours) when AT-62 was introduced into the second exon of R beta G gene. In these same cells, the t1/2 of GM-CSF RNA was approximately 10 to 15 minutes, suggesting that the AUUUA motifs cannot alone account for the rapid degradation of this cytokine mRNA. Phorbol diesters, including 12-0-tetradecanoyl phorbol 13-acetate (TPA), stabilize a variety of transiently expressed RNAs, including GM-CSF RNA. We found that TPA markedly increased (> 30-fold) the accumulation of GM-CSF RNA. In contrast, TPA was unable to stimulate the levels of the chimeric R beta G when either 1x, 2x, 3x, or 5xATTTA motifs were fused to 3'UTR, or when either AT-62 or GC-62 control sequences were fused to the second exon. The chimeric beta-globin construct with either AT-62 or ATTTTTTTA in the 3'UTR had only an approximately twofold to threefold increase in accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Binding of G protein beta gamma-subunits to pleckstrin homology domains.
Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.
Distribution of plectin, an intermediate filament-associated protein, in the adult rat central nervous system.
Plectin is a high molecular weight protein originally identified and characterized as a major cytoskeletal component of the C6 rat glioma cell. Here we demonstrate by immunoblotting of crude intermediate filament (IF) protein preparations that plectin is a cytoskeleton-associated component of the rat spinal cord. We then used avidin-biotin peroxidase immunocytochemistry and indirect immunofluorescence to localize plectin within the adult rat central nervous system (CNS) and examine its distribution with respect to IF proteins. Plectin immunoreactivity is localized to all ependymal cells including the choroidal epithelial cells and tanycytes, Bergmann glial processes, radially oriented glial cells in the spinal cord, astrocytes in white matter, a subset of astrocytes in gray matter, a subset of motoneurons in the brainstem and spinal cord, and certain endothelial cells. Colocalization studies with neural IF proteins show that plectin has a unique distribution pattern which most closely resembles, but is distinct from, that of vimentin. The few plectin positive neurons invariably also contain the neurofilament triplet proteins and peripherin, so that the ability of plectin to bind to the triplet proteins in vitro may reflect an in vivo interaction. The predominance of plectin at the inner ventricular boundaries of the nervous system as well as at the blood-brain barrier is in line with the pattern of plectin expression in other tissues and suggests a general role for plectin in the maintenance of such junctional regions.
Cloning and characterization of a putative G-protein coupled receptor potentially involved in development.
Polymerase chain reaction techniques and medium stringency library screening were used to isolate a rat cDNA ("H218") which encodes a novel guanine nucleotide-binding protein coupled receptor homolog ("pH218"). Northern analysis revealed that brain H218 mRNA is preferentially expressed during embryogenesis. In addition, H218 mRNA is expressed in all developing tissues and rodent cell lines examined with highest levels detected in primitive, transformed cells. H218 mRNA expression in cell lines is rapidly increased by a tumor promoter and rapidly decreased by a differentiation-inducing growth factor. Finally, all of the sequence motifs characteristic of Src homology 2 domains are present in pH218 but in a unique arrangement. We conclude that pH218 may function as a growth factor receptor.
Evidence for the essential role of prostaglandins for parturition in a marsupial, Macropus eugenii.
Female tammar wallabies were treated prepartum with the prostaglandin synthase inhibitor indomethacin, with or without the dopamine agonist bromocriptine, to suppress the peripartum pulses of plasma prostaglandin and prolactin. The animals were observed continuously to detect birth, and a series of blood samples taken to define the hormonal profiles before and immediately after parturition. Birth was observed in ten of twelve control animals but not in the six animals treated with indomethacin alone or the six animals treated with indomethacin and bromocriptine. Indomethacin disrupted the normal profile of PGF2 alpha metabolite 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) concentrations, and in the females treated with bromocriptine plus indomethacin the pulse of prolactin normally seen at parturition was completely abolished. Plasma progesterone concentrations fell slowly in treated animals, whereas in control animals they fell steeply immediately after parturition. Postpartum oestrus was delayed or absent in treated and most control animals, suggesting that the frequent blood sampling and disturbances in the peripartum period interfered with these endocrine processes. We conclude that prostaglandin is essential for normal birth. Prolactin, in the apparent absence of a prostaglandin peak, does not induce birth or rapid luteolysis. Prostaglandin release may synchronize the rapid fall in progesterone concentrations associated with birth, but in the absence of this signal, the corpus luteum undergoes a less rapid, autonomous decline.
Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons.
Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen (T-ag), a neuronal cell line, RN46A, was cloned by serial dilution. At 33 degrees C, RN46A cells express nuclear T-ag immunoreactivity and divide with a doubling time of 9 hr. Undifferentiated RN46A cells express low levels of neuron-specific enolase (NSE) and low (NF-L)-and medium (NF-M)- but not high (NF-H)-molecular-weight neurofilament proteins. Under differentiation conditions, RN46A cells cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. Elevation of intracellular cAMP levels increases neurofilament protein expression, whereas activators of various other intracellular second messenger systems have no effect. Differentiated RN46A cells express low-affinity nerve growth factor (NGF) receptor (p75NGFR) and are immunoreactive using an antibody that recognizes the carboxy-terminal 13 amino acids of all three trk proteins (pan-trk). Both immunoreactivities could be potentiated by treatment with brain-derived neurotrophic factor (BDNF), NGF, and adrenocorticotropic hormone, fragment 4-10 (ACTH4-10). Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) immunoreactivity, which could be enhanced by treatment with ACTH4-10, BDNF, or NGF. Low levels of serotonin immunoreactivity are detected in differentiated RN46A cells, and this was potentiated by differentiating RN46A cells with BDNF for 8 d and 40 mM KCl for days 4-8. HPLC analysis confirmed these immunohistochemical data. RN46A cells should prove useful to elucidate intracellular mechanisms that control neurofilament assembly and 5-HT expression in differentiating raphe neurons.
A nosocomial outbreak of Mycobacterium tuberculosis.
BACKGROUND: The national incidence of tuberculosis (TB) is increasing, and hospitals are a site of transmission. We investigated a nosocomial outbreak of TB at a 160-bed community hospital in South Carolina that highlights the central role that primary care physicians must play to control this epidemic. METHODS: We reviewed medical records to identify potential source cases. We retrospectively evaluated exposures to suspected source patients and the subsequent tuberculin reactivity of the 38 hospital employees who had a previous negative tuberculin skin test and were assigned to the ward where the outbreak began. We also evaluated the out-of-hospital contacts of TB cases. RESULTS: A review of medical records identified one patient who had died of prostate cancer and chronic cavitary pneumonia but was never in isolation nor evaluated for TB. Ward employees who worked while this patient was hospitalized had an increased risk for skin-test conversion (43% [12 of 28] vs 0% [0 of 9]; relative risk undefined; P = .02). Among employees who worked with this patient, skin-test converters worked more shifts with (median, 10.5 vs 7), dispensed more medication to (median 7 doses vs 1), and wrote more notes on (median 18 vs 5) the index patient than did nonconverters. Five of 12 of the patient's close out-of-hospital contacts had newly recognized positive tuberculin skin tests. Among 20 casual contacts, there were no new skin-test conversions. CONCLUSIONS: A high index of suspicion, prompt isolation and diagnostic testing of potentially infectious hospitalized patients, and a thorough investigation of contacts of patients with TB are needed to prevent TB transmission.