Differential regulation of protein phosphatase-1(I) by neurabin.
ABSTRACT: Neurabin is a brain-specific actin and protein phosphatase-1 (PP-1) binding protein that inhibits the purified catalytic subunit of protein phosphatase-1 (PP-1(C)). However, endogenous PP-1 exists primarily as multimeric complexes of PP-1(C) bound to various regulatory proteins that determine its activity, substrate specificity, subcellular localization and function. The major form of endogenous PP-1 in brain is protein phosphatase-1(I) (PP-1(I)), a Mg(2+)/ATP-dependent form of PP-1 that consists of PP-1(C), the inhibitor-2 regulatory subunit, an activating protein kinase and other unidentified proteins. We have identified four PP-1(I) holoenzyme fractions (PP-1(IA), PP-1(IB), PP-1(IC), and PP-1(ID)) in freshly harvested pig brain separable by poly-L-lysine chromatography. Purified recombinant neurabin (amino acid residues 1-485) inhibited PP-1(IB) (IC(50)=1.1 microM), PP-1(IC) (IC(50)=0.1 microM), and PP-1(ID) (IC(50)=0.2 microM), but activated PP-1(IA) by up to threefold (EC(50)=40 nM). The PP-1(IA) activation domain was localized to neurabin(1-210). Our results indicate a novel mechanism of PP-1 regulation by neurabin as both an inhibitor and an activator of distinct forms of PP-1(I) in brain.
Project description:Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to hypoxia. To gain insight into the structural and functional evolution of the HIF family, we characterized the HIF? gene from amphioxus, an invertebrate chordate, and identified several alternatively spliced HIF? isoforms. Whereas HIF? Ia, the full-length isoform, contained a complete oxygen-dependent degradation (ODD) domain, the isoforms Ib, Ic, and Id had 1 or 2 deletions in the ODD domain. When tagged with GFP and tested in mammalian cells, the amphioxus HIF? Ia protein level increased in response to hypoxia or CoCl2 treatment, whereas HIF? Ib, Ic, and Id showed reduced or no hypoxia regulation. Deletion of the ODD sequence in HIF? Ia up-regulated the HIF? Ia levels under normoxia. Gene expression analysis revealed HIF? Ic to be the predominant isoform in embryos and larvae, whereas isoform Ia was the most abundant form in the adult stage. The expression levels of Ib and Id were very low. Hypoxia treatment of adults had no effect on the mRNA levels of these HIF? isoforms. Functional analyses in mammalian cells showed all 4 HIF? isoforms capable of entering the nucleus and activating hypoxia response element-dependent reporter gene expression. The functional nuclear location signal (NLS) mapped to 3 clusters of basic residues. (775)KKARL functioned as the primary NLS, but (737)KRK and (754)KK also contributed to the nuclear localization. All amphioxus HIF? isoforms had 2 functional transactivation domains (TADs). Its C-terminal transactivation (C-TAD) shared high sequence identity with the human HIF-1? and HIF-2? C-TAD. This domain contained a conserved asparagine, and its mutation resulted in an increase in transcriptional activity. These findings reveal many ancient features of the HIF? family and provide novel insights into the evolution of the HIF? family.
Project description:Enterococci are nosocomial pathogen with multiple-drug resistance by intrinsic and extrinsic mechanisms. Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. 178 enterococcal isolates were analyzed in this study. E. faecalis is identified to be the predominant Enterococcus species, along with E. faecium, E. avium, E. hirae, E. durans, E. dispar and E. gallinarum. High level aminoglycoside resistance (HLAR) by MIC for gentamicin (GM), streptomycin (SM) and both (GM + SM) antibiotics was found to be 42.7%, 29.8%, and 21.9%, respectively. Detection of aminoglycoside modifying enzyme encoding genes (AME) in enterococci was identified by multiplex PCR for aac(6')-Ie-aph(2'')-Ia; aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id and aph(3')-IIIa genes. 38.2% isolates carried aac(6')-Ie-aph(2'')-Ia gene and 40.4% isolates carried aph(3')-IIIa gene. aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id were not detected among our study isolates. aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa genes were also observed in HLAR E. durans, E. avium, E. hirae, and E. gallinarum isolates. This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.
Project description:The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.
Project description:In all, 15 aryl-containing phosphonates have been synthesized and tested for their effect on protein-tyrosine phosphatase (PTPase) activity. Two compounds, (naphth-2-yl) difluoromethylphosphonic acid (12) and (napthy-1-yl) difluoromethylphosphonic acid (13) have been found to inhibit dephosphorylation of [32P]insulin receptors by PTP-1B, a protein tyrosine phosphatase (PTPase), with IC50 values of 40-50 microM. Compound 12 competitively inhibited insulin-receptor dephosphorylation by PTP-1B. Compound 12 also inhibited PTP-1B-catalysed dephosphorylation of a synthetic tyrosine phosphorylated substrate poly(Glu80-Tyr20) at the same potency, indicating that 12 acted via interaction with the PTPase. Additionally, 12 inhibited insulin-receptor PTPase(s) and epridermal-growth-factor-receptor PTPase(s) present in solubilized membranes from CHO (Chinese-hamster ovary)/HIRc and A431 cells respectively. IC50 values of 40-50 microM were obtained in all cases with compound 12. Of note is the fact that these compounds did not have any effect on insulin-receptor autophosphorylation. Nine out of the 15 compounds potently inhibited serine/threonine phosphatase PP-2A activity without any effect on serine/threonine phosphatase PP-1 when tested at a concentration as high as 675 microM. The most potent compounds acting toward PP-2A had IC50 values of 45-50 microM. These PP-2A inhibitors could be useful tools for studying serine/threonine-phosphatase-mediated signal transduction. Two compounds, 12 and 13, inhibited both tyrosine phosphatase PTP-1B and serine/threonine phosphatase PP-2A with similar potency; IC50 values being 40-50 microM in both cases. Details of the synthesis of compounds 10, 11 and 13 are given in Supplementary Publication SUP 50177 (6 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1995) 305, 9.
Project description:OBJECTIVES:The purpose of this study was to examine the diversity of the G and P types of human rotavirus strains isolated in South Korea during 2000 to 2004. METHODS:We selected 38 Group A rotavirus isolates among 652 fecal samples, which were collected from infants and children < 5 years of age with acute gastroenteritis or diarrhea admitted in 8 hospitals representative of five provinces of South Korea between 2000 and 2004. Rotavirus P- and G-genotypes were determined by nucleotide sequencing and phylogenetic analysis was performed. RESULTS:One G1P consisted G1-Id-P-V; one G1P consisted G1-Id-P-Ia; nine G1P consisted G1-Ib-P-Ia (n=3), G1-Ic-P-Ia (n=1), and G1-Id-P-Ia (n=5); 13 G2P consisted G2-V-P-V; two G3P consisted G3-IIId-P-V; five G3P consisted G3-IIId-P-Ia; four G4P consisted G4-Ie-P-Ia; two G4P consisted G4-Ie-P-II; one G9P consisted G9-III-P-Ia. CONCLUSIONS:A considerable amount of rotavirus genotypic diversity was detected in South Korea from 2000 to 2004. These findings are important to develop the effective vaccines and to undertake epidemiologic studies.
Project description:1. The inhibition of the cardiac 'rapid' delayed rectifier current (I(Kr)) and its cloned equivalent HERG mediate QT interval prolonging effects of a wide range of clinically used drugs. In this study, we investigated the effects of the Class Ic antiarrhythmic agent flecainide (FLEC) on ionic current (I(HERG)) mediated by cloned HERG channels at 37 degrees C. We also compared the inhibitory potency of FLEC with other Class I agents: quinidine (QUIN, Class Ia); lignocaine (LIG, Class Ib) and propafenone (PROPAF, Class Ic). 2. Whole cell voltage clamp recordings of I(HERG) were made from an HEK293 cell line stably expressing HERG. FLEC inhibited I(HERG) 'tails' following test pulses to +30 mV with an IC(50) of 3.91+/-0.68 microM (mean+/-s.e.mean) and a Hill co-efficient close to 1 (0.76+/-0.09). 3. In experiments in which I(HERG) tails were monitored following voltage commands to a range of test potentials, I(HERG) inhibition by FLEC was observed to be voltage-dependent and to be associated with a approximately -5 mV shift of the activation curve for the current. Voltage-dependence of inhibition was greatest over the range of potentials corresponding to the steep portion of the I(HERG) activation curve. The time-course of I(HERG) tail deactivation was not significantly altered by FLEC. 4. In experiments in which 10 s depolarizing pulses were applied from -80 to 0 mV, the level of current inhibition by FLEC did not increase between 1 and 10 s. Some time-dependence of inhibition was observed during the first 200 - 300 ms of depolarization. This observation and the voltage-dependence of inhibition are collectively consistent with FLEC exerting a rapid open channel state inhibition of I(HERG). 5. Under similar recording conditions QUIN inhibited I(HERG) with an IC(50) of 0.41+/-0.04 microM and PROPAF inhibited I(HERG) with an IC(50) of 0.44+/-0.07 microM. Similar to FLEC, both QUIN and PROPAF showed voltage-dependence of inhibition and blockade developed rapidly during a sustained depolarization. 6. LIG showed little effect on I(HERG) at low micromolar concentrations, but could inhibit the current at higher concentrations; the observed IC(50) was 262.90+/-22.40 microM. 7. Our data are consistent with FLEC, PROPAF and QUIN exerting I(HERG) blockade at clinically relevant concentrations. The rank potency as HERG blockers of the Class I drugs tested in this study was QUIN=PROPAF>FLEC>>LIG.
Project description:Glycogen-storage diseases type I (GSD type I) are due to a deficiency in glucose-6-phosphatase, an enzymatic system present in the endoplasmic reticulum that plays a crucial role in blood glucose homeostasis. Unlike GSD type Ia, types Ib and Ic are not due to mutations in the phosphohydrolase gene and are clinically characterized by the presence of associated neutropenia and neutrophil dysfunction. Biochemical evidence indicates the presence of a defect in glucose-6-phosphate (GSD type Ib) or inorganic phosphate (Pi) (GSD type Ic) transport in the microsomes. We have recently cloned a cDNA encoding a putative glucose-6-phosphate translocase. We have now localized the corresponding gene on chromosome 11q23, the region where GSD types Ib and Ic have been mapped. Using SSCP analysis and sequencing, we have screened this gene, for mutations in genomic DNA, from patients from 22 different families who have GSD types Ib and Ic. Of 20 mutations found, 11 result in truncated proteins that are probably nonfunctional. Most other mutations result in substitutions of conserved or semiconserved residues. The two most common mutations (Gly339Cys and 1211-1212 delCT) together constitute approximately 40% of the disease alleles. The fact that the same mutations are found in GSD types Ib and Ic could indicate either that Pi and glucose-6-phosphate are transported in microsomes by the same transporter or that the biochemical assays used to differentiate Pi and glucose-6-phosphate transport defects are not reliable.
Project description:In aquatic animals, synthesis of the metal-binding protein metallothionein (MT) can be induced through exposure to elevated levels of metals in food or water. Whether the different routes of exposure lead to expression of different metallothionein isoforms in different tissues in unknown. In this study we examined the induction of metallothionein isoforms in the hepatopancreas and gills of the blue crab Callinectes sapidus. When blue crabs are exposed to cadmium in their diet, the metal accumulates in the hepatopancreas. Size-exclusion and anion-exchange chromatography show the presence of five low-molecular-mass cadmium-binding proteins. All of the observed cadmium-binding proteins belong to the class I MT family. They are designated as MT-Ia, MT-Ib, MT-Ic, MT-IIa and MT-IIb. All purified proteins run as single peaks upon rechromatography on anion-exchange HPLC, except for MT-Ic, which segregates into two peaks corresponding to MT-Ia and MT-Ic. The amino acid sequence of MT-Ia and MT-Ic is identical. MT-Ib differs from MT-Ia and MT-Ic only in having an extra N-terminal methionine. The 18 cysteine residues in MT-Ia and MT-IIa occur in identical positions; however, of the remaining 40 amino acids, 15 are found to be different. MT-IIb is identical with MT-IIa, except for an extra methionine residue at its N-terminal position. It appears therefore that, of the five observed CdMTs, only two are the products of distinct genes. CdMT-Ia and -IIa are posttranslationally modified forms of Ib and IIb, respectively, and CdMT-Ia and -Ic appear to be conformational isomers. Cadmium-induced expression of the two genes is tissue-specific. When crabs are exposed to cadmium in water, the metal accumulates in the gills, where it is bound to MT-II. MT-I is virtually absent.
Project description:Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.
Project description:Novel benzene polyphosphates were synthesised as inositol polyphosphate mimics and evaluated against type-I inositol 1,4,5-trisphosphate 5-phosphatase, which only binds soluble inositol polyphosphates, and against the PH domain of protein kinase Balpha (PKBalpha), which can bind both soluble inositol polyphosphates and inositol phospholipids. The most potent trisphosphate 5-phosphatase inhibitor is benzene 1,2,4-trisphosphate (2, IC(50) of 14 microM), a potential mimic of D-myo-inositol 1,4,5-trisphosphate, whereas the most potent tetrakisphosphate Ins(1,4,5)P(3) 5-phosphatase inhibitor is benzene 1,2,4,5-tetrakisphosphate, with an IC(50) of 4 microM. Biphenyl 2,3',4,5',6-pentakisphosphate (4) was the most potent inhibitor evaluated against type I Ins(1,4,5)P(3) 5-phosphatase (IC(50) of 1 microM). All new benzene polyphosphates are resistant to dephosphorylation by type I Ins(1,4,5)P(3) 5-phosphatase. Unexpectedly, all benzene polyphosphates studied bind to the PH domain of PKBalpha with apparent higher affinity than to type I Ins(1,4,5)P(3) 5-phosphatase. The most potent ligand for the PKBalpha PH domain, measured by inhibition of biotinylated diC(8)-PtdIns(3,4)P(2) binding, is biphenyl 2,3',4,5',6-pentakisphosphate (4, K(i)=27 nm). The approximately 80-fold enhancement of binding relative to parent benzene trisphosphate is explained by the involvement of a cation-pi interaction. These new molecular tools will be of potential use in structural and cell signalling studies.