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2.
AbstractIn dogs, it has been reported that acute ischemia or severe and terminal heart failure results in a selective reduction of myocardial α 3 isoform of Na, K-ATPase activity. The aim of this study was to evaluate if a similar change in the two canine digitalis receptor isoforms occurs following 4 weeks of rapid ventricular pacing-induced heart failure without profound necrosis. Heart failure was induced in dogs by rapid ventricular pacing (240 beats × min -1). Digitalis receptors were quantitated by [ 3H]-ouabain binding with isolated microsomal membranes from sham-operated (n = 3) and heart failure dogs (n = 4) and by Western blot analysis using specific α 1 and α 3 polyclonal antibodies. In kinetic studies, similar dissociation rates of 19 to 22 × 10 -4 s -1 and 1.3 to 2.4 10 -4 s -1 corresponding to high and low affinity sites respectively, were found in sham-operated and CHF dogs. Immunoblotting showed similar abundance of α 1 isoform in the two groups; however, levels of α 3 were increased by at least 50% in pacing-induced heart failure animals. In conclusion, heart failure selectively modulates the expression of cardiac α 3 isoform in dogs. 相似文献
3.
The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content
affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern
analysis of polyA +RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine
the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay.
Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid
animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T 3-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased
in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex
and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR
isoform mRNA levels returned to control values following T 4 intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005) 相似文献
4.
Peroxidase (POD) activity and isoform patterns were investigated during seedling growth (up to 20 days) of Ebenus cretica L. Seeds germinated to approx. 100% after a 24-h imbibition. Seedling growth proceeded smoothly, in both light and dark conditions. No seedling growth was noticed at 4°C. A positive effect of light and increasing temperature (4, 10, 16, 22 and 28°C) on seedlings growth, lignin content and POD activity was observed. Lignin content was 2.5 times higher in seedlings grown under light than in seedlings grown under darkness. Seedlingsȁ9 POD activity was higher in acid pH (5.5) in comparison to neutral pH (7.0). These activities were higher in seedlings grown under darkness than in those grown under light; since additional POD isoforms were expressed in dark conditions. The increase in POD activity was accompanied with the appearance of new POD isoforms correlated with the growth of the seedlings. Four soluble anionic POD isoforms (named A 1, A 2, A 3 and A 4) and three soluble cationic POD isoforms (named C 1, C 2 and C 3) were displayed depending on the treatment and the course of growth. POD isoforms were detected in gel after PAGE. The fast migrating (A 4) isoform, which appeared in the dark-grown seedlings as well as on day 20 at 28°C in the temperature treatment, was separated by DEAE–Sepharose column chromatography. A slow migrating C 1 isoform slightly appeared in both 4 and 10°C temperature treatments and could be related to the low temperature treatments, while A 1, A 2, A 3 and C 2 to the growth stage of seedlings. The expression of seven POD isoforms during seedling growth seems to be related to different developmental events of growth and could be used as useful biochemical markers in the analysis of metabolic regulation in seedling growth of Ebenus cretica. 相似文献
5.
ABSTRACTThis study aims to study the effects of adenosine A 2A receptor (A 2AR) on hippocampal cell apoptosis and the putative mechanisms in a mouse model of chronic hypoxic-hypercapnia. Wild-type (WT) or A 2AR knockout (A 2AR KO) mice were randomly divided into normal control (NC) groups and chronic hypoxic-hypercapnia (4HH) groups. Compared with their corresponding NC groups (WT-NC and KO-NC), the apoptosis index (AI), caspase-3 activity, Bax mRNA and P-p38 protein expression in the hippocampus of 4HH groups (WT-4HH and KO-4HH) were significantly increased, while Bcl2 mRNA expression was significantly decreased (P < 0.05). Moreover, A 2AR deficiency significantly rescued the effect of chronic hypoxic-hypercapnia on apoptosis when compared with the WT-4HH group (P < 0.05). A 2AR deficiency inhibits hippocampal cell apoptosis in mice exposed to chronic hypoxic-hypercapnia, which might be associated with dampened p38 MAPK activation and Bax mRNA expression, and augmented Bcl-2 mRNA expression. 相似文献
6.
Abstract In an in vitro screening for human carbonic anhydrase (hCA) inhibiting agents from higher plants, the petroleum ether and ethyl acetate extracts of Magydaris pastinacea seeds selectively inhibited hCA IX and hCA XII isoforms. The phytochemical investigation of the extracts led to the isolation of ten linear furocoumarins ( 1– 10), four simple coumarins ( 12– 15) and a new angular dihydrofurocoumarin ( 11). The structures of the isolated compounds were elucidated based on 1?D and 2?D NMR, MS, and ECD data analysis. All isolated compounds were inactive towards the ubiquitous cytosolic isoform hCA I and II ( K i?>?10,000?nM) while they were significantly active against the tumour-associated isoforms hCA IX and XII. Umbelliprenin was the most potent coumarin inhibiting hCA XII isoform with a K i of 5.7?nM. The cytotoxicity of the most interesting compounds on HeLa cancer cells was also investigated. 相似文献
7.
Human IgG2 consists of disulfide‐mediated structural isoforms, classified by the number of Fab arms disulfide‐linked to the heavy chain hinge. In the IgG2‐B isoform, both Fab arms are linked to the hinge region, and in IgG2‐A, neither Fab arm are linked to the hinge. IgG2‐A/B is a hybrid between these two forms, with only one Fab arm disulfide‐linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide‐linkage differences. Here we explored the structural basis for the A 1 and A 2 isoform subtypes. Whereas A 1 isoform converts into the A/B and B isoforms under mild redox conditions, A 2 does not. Characterization of the disulfide connectivities of A 2 isoform revealed a similar structure to A 1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A 2 isoform were resistant to reduction under conditions where A 1 isoform hinge disulfides became reduced and they required thermal treatment (>55°C) to obtain thiol‐dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A 2 isoform behavior. 1H NMR studies showed that the A 1 isoform Fc glycan was more dynamic than that on A 2 isoform and showed some other conformational differences. Results point to an IgG2‐A 2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG. 相似文献
8.
Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A1Rs, A2ARs, and A2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A1R?/?, A2AR?/?, and A2BR?/? rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A1Rs, A2ARs, and A2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A1R and A2AR constrained exosome production under normal conditions (p?=?0.0297; p?=?0.0409, respectively), and A1R, A2AR, and A2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p?=?0.0028) by the selective A2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A1R and A2BR agonists (p?=?0.0474). The selective A2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A2ARs suppress exosome release in all cell types examined, whereas effects of A1Rs and A2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A2AR antagonists may inadvertently increase exosome production from tumor cells. 相似文献
9.
AbstractContext: Asthma is multifaceted disease where many targets contribute towards its development and progression. Among these, adenosine receptor subtypes play a major role. Objective: MCD-KV-10, a novel thiazolo-thiophene was designed and evaluated pre-clinically for its implication in management of asthma. Materials and methods: This compound showed good affinity and selectivity towards A 2A/A 3 adenosine receptor (AR) subtypes. Furthermore, MCD-KV-10 was evaluated for in vitro lipoxygenase inhibition activity; in vivo mast cell stabilization potential and in vivo anti-asthmatic activity was done in ovalbumin-induced airway inflammation model in guinea pigs. Results: The compound showed good (>57%) inhibition of lipoxygenase enzyme and also effectively protected mast cell degranulation (>63%). The compound showed good anti-asthmatic activity as inferred from the in vivo studies. Discussion: These results indicate that MCD-KV-10 has an inhibitory effect on airway inflammation. Conclusion: Though, we have identified a potential candidate for management of asthma, further mechanistic studies are needed. 相似文献
10.
A new crotoxin B isoform PLA 2 (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin
B isoform PLA 2 (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA 2 (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit
of isoforms of PLA 2 (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3:
8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA 2 (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other
PLA 2s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region
with other myotoxic PLA 2s. The new crotoxin B isoform PLA 2 (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC
YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced
by crotoxin and new crotoxin B isoform PLA 2 (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin
was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA 2 (F6a) was clearly cytotoxic to these cells. 相似文献
11.
AbstractInsulin, when co-applied with GABA, can cause an inhibition of the induced current at GABA A receptors. This study investigated that inhibitory effect of insulin at a variety of receptor isoforms, concentrating on α1, α2 and α4 containing receptors. Various isoforms were expressed in Xenopus oocytes and currents determined using two-electrode voltage clamp. Submaximal GABA currents at all isoforms studied were inhibited by nanomolar concentrations of insulin. At α2 and α4 containing forms, insulin could inhibit maximal GABA currents. The ability to inhibit maximal currents, and the general potency and effects at submaximal currents paralleled the number of potential MAPK sites on the α subunits. The differences in insulin inhibition of GABA currents at different α containing GABA A receptors could be important in autocrine and paracrine control of hormone secretion in the pancreas, and in control of reward and food intake circuits of the brain. 相似文献
12.
Summary In all eukaryotes, myosin plays a major role in the maintenance of cell shape and in cellular movement; in association with actin and other contractile proteins it is also a major structural component of the muscle sarcomere. Several isoforms of myosin alkali light chain have been identified, associated with different muscle types. We have recently localized the gene encoding the fast skeletal muscle alkali light-chain isoforms MLC1 F and MLC3 F (HGM symbol, MYL1) to human chromosome 2q32.1-qter (Cohen-Haguenauer 1988). We present here the chromosomal assignment of two loci encoding the ventricular muscle isoform MLC1 V (equivalent to the slow skeletal muscle isoform MLC1 Sb) and the atrial muscle isoform MLC1 A (equivalent to the fetal isoform MLC1 emb) using a panel of 25 independent man-rodent somatic cell hybrids. The MLC1 V gene (HGM symbol, MYL3) was mapped to human chromosome 3 using a human full-length cDNA probe that hybridizes to a single major human TaqI 2.8-kb fragment. The MLC1 A probe (HGM symbol, MYL4) was a 360-bp mouse cDNA fragment that gave a distinct signal with human DNA using low stringency conditions of hybridization and washings and after presaturation of the Southern blots with rodent DNA. A single PstI 7.8-kb fragment gives an intense signal, and its presence correlates with the presence of chromosome 17 among the hybrids. These data are in keeping with the localizations of the MLC1 V gene to mouse chromosome 9, and of the MLC1 A gene to mouse chromosome 11, which share some markers in common with human chromosomes 3 and 17 respectively. 相似文献
14.
ABSTRACT Background : Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra. Research design and methods : The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies. Results : Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A 2 activity. Phospholipase A 2 and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs. Conclusions : Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms. 相似文献
16.
Abstract In a search for potent and selective adenosine agonists it has been found that 2-hexynyladenosine-5′-N-ethyluronamide (HENECA) displays high affinity at rat A 2A receptor combined with a good A 2A vs A 1 selectivity. The finding that HENECA shows good affinity also for A 3 receptors prompted us to investigate the effect of various substituents in different positions of this molecule. 相似文献
17.
AbstractBackground: Promoter methylation of tumour suppressor genes (TSGs) CDKN2A, CDKN2B and CDH13 has been reported in ovarian cancer. However, the clinicopathological characteristics and prognostic role of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian carcinoma remained unclear. Methods: The pooled odds ratio (OR) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated in this meta-analysis. The Cancer Genome Atlas data were obtained to confirm the role of CDKN2A, CDKN2B and CDH13 methylation in ovarian cancer. Results: CDKN2A, CDKN2B and CDH13 promoter methylation was higher in ovarian cancer than in normal ovarian tissues. CDH13 promoter methylation was correlated with tumour histology (serous vs. non-serous type: OR?=?0.33, p?=?0.031). CDKN2A promoter methylation was not linked to overall survival (OS), but it was correlated with a poor prognosis in progression-free survival (HR?=?1.55, p?=?0.004). TCGA data showed no correlation between CDKN2A, CDKN2B and CDH13 methylation and OS as well as disease-free survival (DFS). Conclusions: CDKN2A, CDKN2B and CDH13 promoter methylation may correlate with the increased risk of ovarian cancer. CDKN2A promoter methylation may be an independent prognostic biomarker for predicting progression-free survival. 相似文献
18.
In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL 50) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by
combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack
C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined
by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass
spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides,
including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein
from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH
CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation
and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These
activities were modulated by the presence of positively charged amino acid residues. The LD 50 of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route. 相似文献
19.
Background: Oxidative degradation of human recombinant erythropoietin (hrEPO) may occur in manufacturing process or therapeutic applications. This unfavorable alteration may render EPO inefficient or inactive. We investigated the effect of methionine/54 oxidative changes on the amino acid sequences, glycoform distribution and biological activity of hrEPO. Methods: Mass spectrometry was applied to verify the sequence and determine the methionine oxidation level of hrEPO. Isoform distribution was studied by capillary zone electrophoresis method. In vivo normocythemic mice assay was used to assess the biological activity of three different batches ( A, B, and C) of the proteins. Results: Nano-LC/ESI/MS/MS data analyses confirmed the amino acid sequences of all samples. The calculated area percent of three isoforms (2–4 of the 8 obtained isoforms) were decreased in samples of C, B, and A with 27.3, 16.7, and 6.8% of oxidation, respectively. Specific activities were estimated as 53671.54, 95826.47, and 112994.93?mg/mL for the samples of A, B, and C, respectively. Conclusion: The observed decrease in hrEPO biological activity, caused by increasing methionine oxidation levels, was rather independent of its amino acid structure and mainly associated with the higher contents of acidic isoforms. 相似文献
20.
AbstractIschemia-reperfusion (I/R) is a condition leading to serious complications due to death of cardiac myocytes. We used the cardiomyocyte-like cell line H9c2 to study the mechanism underlying cell damage. Exposure of the cells to simulated I/R lead to their apoptosis. Over-expression of Bcl-2 and Bcl-x L protected the cells from apoptosis while over-expression of Bax sensitized them to programmed cell death induction. Mitochondria-targeted coenzyme Q (mitoQ) and superoxide dismutase both inhibited accumulation of reactive oxygen species (ROS) and apoptosis induction. Notably, mtDNA-deficient cells responded to I/R by decreased ROS generation and apoptosis. Using both in situ and in vivo approaches, it was found that apoptosis occurred during reperfusion following ischemia, and recovery was enhanced when hearts from mice were supplemented with mitoQ. In conclusion, I/R results in apoptosis in cultured cardiac myocytes and heart tissue largely via generation of mitochondria-derived superoxide, with ensuing apoptosis during the reperfusion phase. 相似文献
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