共查询到20条相似文献,搜索用时 31 毫秒
1.
Yanmei Lu Hong Xiang Peter Liu Raymond R Tong Ryan J Watts Alexander W Koch Wendy N Sandoval Lisa A Damico Wai Lee Wong Y Gloria Meng 《MABS-AUSTIN》2009,1(4):364-369
Neuropilin-1 (NRP1) acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factor and is an attractive angiogenesis target for cancer therapy. In addition to the transmembrane form, naturally occurring soluble NRP1 proteins containing part of the extracellular domain have been identified in tissues and a cell line. We developed ELISAs to study the existence of circulating NRP1 and to quantify it in serum. As measured by ELISAs, circulating NRP1 levels in mice, rats, monkeys and humans were 427 ± 77, 20 ± 3, 288 ± 86 and 322 ± 82 ng/ml (mean ± standard deviation; n ≥ 10), respectively. Anti-NRP1B, a human monoclonal antibody, has been selected from a synthetic phage library. A 4-fold increase in circulating NRP1 was observed in mice receiving a single dose of 10 mg/kg anti-NRP1B antibody. In rats and monkeys receiving single injections of anti-NRP1B at different dose levels, higher doses of antibody resulted in greater and more prolonged increases in circulating NRP1. Maximum increases were 56- and 7-fold for rats and monkeys receiving 50 mg/kg anti-NRP1B, respectively. In addition to the soluble NRP1 isoforms, for the first time, a ∼120 kDa circulating NRP1 protein containing the complete extracellular domain was detected in serum by western blot and mass spectrometry analysis. This protein increased more than the putative soluble NRP1 bands in anti-NRP1B treated mouse, rat and monkey sera compared with untreated controls, suggesting that anti-NRP1B induced membrane NRP1 shedding.Key words: angiogenesis, soluble neuropilin-1, circulating neuropilin-1, anti-neuropilin-1, ELISA, serum 相似文献
2.
Marcia R Saban Carole A Davis Antonio Avelino Francisco Cruz Julie Maier Dale E Bjorling Thomas J Sferra Robert E Hurst Ricardo Saban 《BMC physiology》2011,11(1):1-20
Background
This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity.Methods
Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder.Results
The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density. NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density.Conclusions
For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon. 相似文献3.
R. Grzanna M. F. Nelson R. M. Weinshilboum J. Dunnette J. T. Coyle 《Journal of neurochemistry》1979,33(4):913-921
Abstract– Rat serum dopamine-β-hydroxylase (DBH) activity decreased 5-7-fold between 15 and 60 days of age. Immunoprecipitation performed with homologous antibody (guinea-pig anti-rat adrenal DBH) showed that during this time period the quantity of antibody necessary to precipitate 50% of the enzymatic activity (AD50) decreased 5-fold from 0.25 to 0.05 μl/ml. The biochemical properties of rat serum DBH at 15 and 60 days of age were compared to test the hypothesis that there might be different biochemical forms of the enzyme in the blood of immature and adult rats. Thermal stability, apparent Km for tyramine, electrophoretic mobility, pH optima and elution profile on gel filtratioh chromatography were all found to be similar for rat serum DBH at both ages. On the basis of homospecific activity and multiple similarities in biochemical characteristics, it appears that differences in serum activity at the two ages reflect differences in the steady-state levels of enzyme. To determine the turnover of serum DBH in the two age groups, the recovery of enzyme activity was monitored after acute clearance of the circulating pool of DBH by treatment with the homologous antiserum. Immunotitration of DBH activity in vivo indicated that the total pool of serum enzyme was 4-fold greater in the mature rat than in 4-day-olds. After treatment of adult rats with 2μl of homologous antiserum, serum DBH activity was reduced by 85% with a half-life of recovery of 3.0 ± 0.6 days; the estimated fractional rate of degradation was 0.23 ± 0.06 day?1 and the rate of entrance was 2.3 ± 0.2 units/ml/day. After treatment of 4-day-old rats with 1 μl of homologous antiserum, serum DBH activity was reduced by 95% with a half-life of recovery of 3.3 ± 0.5 days: the estimated average fractional rate of degradation was 0.22 ± 0.06 day?1 and the average rate of entrance was 10.7 ± 1.6 units/ml/day. Thus, the several-fold difference in steady-state levels of serum DBH in rat pups as compared to adult rats appears to be due to greatly increased rates of entrance of the enzyme in the immature rats. 相似文献
4.
Neuropilin-1 is upregulated in Sj?gren’s syndrome and contributes to pathological neovascularization
Sisto M Lisi S Lofrumento DD D'Amore M Ribatti D 《Histochemistry and cell biology》2012,137(5):669-677
Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies
revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of
Sj?gren’s syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of
NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection
with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear
Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition
with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that
NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly
associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production
from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sj?gren’s syndrome. 相似文献
5.
H Q Miao S Soker L Feiner J L Alonso J A Raper M Klagsbrun 《The Journal of cell biology》1999,146(1):233-242
Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites. 相似文献
6.
VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding 总被引:19,自引:0,他引:19
Co-expression of NRP1 and (VEGFR-2) KDR on the surface of endothelial cells (EC) enhances VEGF165 binding to KDR and EC chemotaxis in response to VEGF165. Overexpression of NRP1 by prostate tumor cells in vivo results in increased tumor angiogenesis and growth. We investigated the molecular mechanisms underlying NRP1-mediated angiogenesis by analyzing the association of NRP1 and KDR. An intracellular complex containing NRP1 and KDR was immunoprecipitated from EC by anti-NRP1 antibodies only in the presence of VEGF165. In contrast, VEGF121, which does not bind to NRP1, did not support complex formation. Complexes containing VEGF165, NRP1, and KDR were also formed in an intercellular fashion by co-culture of EC expressing KDR only, with cells expressing NRP1 only, for example, breast carcinoma cells. VEGF165 also mediated the binding of a soluble NRP1 dimer to cells expressing KDR only, confirming the formation of such complexes. Furthermore, the formation of complexes containing KDR and NRP1 markedly increased 125I-VEGF165 binding to KDR. Our results suggest that formation of a ternary complex of VEGF165, KDR, and NRP1 potentiates VEGF165 binding to KDR. These complexes are formed on the surface of EC and in a juxtacrine manner via association of tumor cell NRP1 and EC KDR. 相似文献
7.
《Life sciences》1994,55(20):PL403-PL407
The potent and selective 5-HT1A antagonist WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide) was radiolabeled with 11C in high specific activity, and the in vivo properties of this radioligand were assessed in the brains of rats and monkeys. Following i.v. tail vein injection in rats, [11C]WAY 100635 rapidly penetrated into brain tissue and was retained over a 30–90 min time period in a manner consistent with the known distribution of 5-HT1A receptors. Pretreatment of rats with the selective 5-HT1A agonist (±)-8-OH-DPAT effectively blocked the retention of radioactivity in brain regions known to contain high densities of 5-HT1A receptors. The hippocampus-to-cerebellum radioactivity concentration ratio reached a maximum of 16:1 at 60 min post injection. Following i.v. injection of [11C]WAY 100635 in rhesus monkeys, the concentrations of radioactivity in brain regions were consistent with the reported distribution of 5-HT1A receptors in primates, and the frontal cortex-to-cerebellum ratio reached 5.5:1 at 80 min post injection. Pretreatment of the monkeys with (±)-8-OH-DPAT reduced this ratio to 1.4:1, and injection of (±)-8-OH-DPAT 20 min after the injection of [11C]WAY 100635 significantly displaced frontal cortex binding. The in vivo properties of [11C]WAY 100635 in rats and monkeys strongly support the future utility of this radioligand for imaging 5-HT1A receptors using positron emission tomography (PET). 相似文献
8.
Belal Chaudhary Yazan S. Khaled Basil J. Ammori Eyad Elkord 《Cancer immunology, immunotherapy : CII》2014,63(2):81-99
Neuropilin 1 (NRP1) is a transmembrane glycoprotein that acts as a co-receptor for a number of extracellular ligands including class III/IV semaphorins, certain isoforms of vascular endothelial growth factor and transforming growth factor beta. An exact understanding of the role of NRP1 in the immune system has been obscured by the differences in NRP1 expression observed between mice and humans. In mice, NRP1 is selectively expressed on thymic-derived Tregs and greatly enhances immunosuppressive function. In humans, NRP1 is expressed on plasmacytoid dendritic cells (pDCs) where it aids in priming immune responses and on a subset of T regulatory cells (Tregs) isolated from secondary lymph nodes. Preliminary studies that show NRP1 expression on T cells confers enhanced immunosuppressive activity. However, the mechanism by which this activity is mediated remains unclear. NRP1 expression has also been identified on activated T cells and Tregs isolated from inflammatory microenvironments, suggesting NRP1 might represent a novel T cell activation marker. Of clinical interest, NRP1 may enhance Treg tumour infiltration and a decrease in NRP1+ Tregs correlates with successful chemotherapy, suggesting a specific role for NRP1 in cancer pathology. As a therapeutic target, NRP1 allows simultaneous targeting of NRP1-expressing tumour vasculature, NRP1+ Tregs and pDCs. With the development of anti-NRP1 monoclonal antibodies and cell-penetrating peptides, NRP1 represents a promising new target for cancer therapies. This paper reviews current knowledge on the role and function of NRP1 in Tregs and pDCs, both in physiological and cancer settings, as well as its potential as a therapeutic target in cancer. 相似文献
9.
《Cell Adhesion & Migration》2013,7(2):56-61
It is now well established that neuropilins (NRP1 and NRP2), first described as mediators of neuronal guidance, are also mediators of angiogenesis and tumor progression. NRPs are receptors for the class-3 semaphorin (SEMA) family of axon guidance molecules and also for the vascular endothelial growth factor (VEGF) family of angiogenic factors. VEGF-NRP interactions promote developmental angiogenesis as shown in mouse knockout and zebrafish knockdown studies. There is also evidence that NRPs mediate tumor progression. For example, overexpression of NRP1 enhances tumor growth whereas NRP1 antagonists, such as soluble NRP1 and anti-NRP1 antibodies, inhibit tumor growth. Furthermore, some class-3 SEMAs acting via NRPs inhibit tumor angiogenesis, progression and metastasis. Clinical data suggest that high NRP levels correlate with poor prognosis and survival in a variety of cancer types. Taken together, these results suggest that NRPs are potentially valuable targets for new anti-cancer therapies. We analyze here the current knowledge on NRPs and their role in angiogenesis and tumor progression and enumerate strategies for targeting these receptors. 相似文献
10.
Abstract— Nicotine binds to homogenates of lobster walking leg nerve (Kd= 1.1 ± 0.3 μm , Bmax= 2.4 ± 0.5 nmol/g wet tissue), horseshoe crab leg nerve (Kd= 0.11 ± 0.06 μm , Bmax= 1.3 ± 0.6nmol/g), and kidney from 18-month-old rats (Kd= 0.8 ± 0.2 μm , Bmax= 23 ± 9 nmol/g). The pharmacological sensitivities of nicotine binding to lobster and horseshoe crab leg nerve homogenates are similar to that of the axonal cholinergic binding macromolecule (ACBM) (Denburg et al., 1972) of lobster leg. nerve membrane, while the binding to rat kidney is sensitive to α-bungarotoxin but not atropine or curare. There was no nicotine binding to rat heart or spleen, or to kidney from younger rats; little or no binding to blue crab nerve or to Torpedo electroplax motor nerve; and little binding (around 0.1 nmol/g) to rat liver. [3H]α-Bungarotoxin bound reversibly (0.17 nmol/g) to lobster leg nerve membrane The implications of these results for the distribution and function of the ACBM, and for the specificity of α-bungarotoxin, are discussed. 相似文献
11.
It is now well-established that neuropilins (NRP1 and NRP2), first described as mediators of neuronal guidance, are also mediators of angiogenesis and tumor progression. NRPs are receptors for the class-3 semaphorin (SEMA) family of axon guidance molecules and also for the vascular endothelial growth factor (VEGF) family of angiogenic factors. VEGF-NRP interactions promote developmental angiogenesis as shown in mouse knockout and zebrafish knockdown studies. There is also evidence that NRPs mediate tumor progression. For example, overexpression of NRP1 enhances tumor growth whereas NRP1 antagonists, such as soluble NRP1 and anti-NRP1 antibodies, inhibit tumor growth. Furthermore, some class-3 SEMAs acting via NRPs inhibit tumor angiogenesis, progression and metastasis. Clinical data suggest that high NRP levels correlate with poor prognosis and survival in a variety of cancer types. Taken together, these results suggest that NRPs are potentially valuable targets for new anti-cancer therapies. We analyze here the current knowledge on NRPs and their role in angiogenesis and tumor progression and enumerate strategies for targeting these receptors. 相似文献
12.
《MABS-AUSTIN》2013,5(2):567-575
RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with 89Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake. Studies with 89Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44+ or CD44-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44+, resp), PL45 (CD44+, non-resp) and HepG2 (CD44–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose. At 1, 2, 3, and 6 days after injection, 89Zr-RG7356 uptake in MDA-MB-231 (CD44+, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of 89Zr-RG7356 was similar in MDA-MB-231 (CD44+, resp) and PL45 (CD44+, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. 89Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.89Zr-RG7356 selectively targets CD44+ responsive and non-responsive tumors in mice and CD44+ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44+ tumors may not be sufficient in itself to drive an anti-tumor response. 相似文献
13.
Helene Larsen Barbara Muz Tak L Khong Marc Feldmann Ewa M Paleolog 《Arthritis research & therapy》2012,14(4):1-18
Introduction
Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.Methods
CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.Results
Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1B antibody significantly reduced disease severity and progression in CIA mice.Conclusions
Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA. 相似文献14.
Miguel Arredondo María José Mendiburo Sebastián Flores Steven T. Singleton Michael D. Garrick 《Biometals》2014,27(1):115-123
Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/?IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/?IRE construct. 64Cu1+ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. 64Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein?1 min?1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/?IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu1+. 相似文献
15.
Claudia Korneli Rebekka Biedendieck Florian David Dieter Jahn Christoph Wittmann 《Applied microbiology and biotechnology》2013,97(8):3343-3353
In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH?6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg?L?1) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U?mL?1 on fructose and 17.2 U?mL?1 on glycerol). This was further increased in high cell density fed-batch processes up to 55 U?mL?1, reflecting a levansucrase concentration of 0.52 g?L?1. This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts. 相似文献
16.
Seth R. Hootman 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,716(3):341-347
Chronic administration of DL-isoproterenol, a β-adrenergic agonist, to male Sprague-Dawley rats increased submandibular gland weights by 3 to 4-fold. This increase resulted from a combination of hyperplasia and hypertrophy of secretory cells. Possible effects of this drug regimen on submandibular gland muscarinic acetylcholine receptors were examined by analysis of the binding of the cholinergic antagonist, L-quinuclidinyl [3H]benzilate, to receptors in gland homogenates. Parallel investigations of receptors in exorbital lacrimal glands, an organ that is not grossly affected by chronic isoproterenol treatment, were also carried out. [3H]QNB bound to submandibular receptors with a Kd of 37.8±6.3 pM in control rats and 41.0±4.0 pM in isoproterenol-treated animals, a non-significant difference (P > 0.05). In contrast, the maximal binding level (Bmax) is isoproterenol-treated rats, 1.52±0.10 fmol/μg DNA, was depressed by approx. 30% (P<0.05) from that of 2.22±0.16 fmol/μg DNA in control animals. In lacrimal glands, both Kd (61.3±5.3 vs. 53.2±4.0 pM) and Bmax (1.74±0.24 vs. 1.78±0.17 fmol/μg DNA) were unchanged by isoproterenol treatment. The affinity of glandular muscarinic receptors for cholinergic agonists was also examined by competition experiments using carbachol. This agonist inhibited [3H]QNB binding to receptors in homogenates from both glands in a dose-dependent fashion. Inhibition constant (Ki) for this interaction were similar in control and isoproterenol-treated lacrimal glands; 53.6±5.4 μM and 66.6±7.9 μM, respectively (P>0.05). In submandibular glands, isoproterenol treatment elicited a highly significant (P < 0.01) shift in Ki from 17.3±1.4 μM to 68.3±5.2 μM. These results demonstrate that chronic administration of isoproterenol to rats results in a reduction in receptor numbers and a decrease in their sensitivity to cholinergic agonists in submandibular, but not lacrimal, glands. 相似文献
17.
Dr. D. Manor D. L. Rothman G. F. Mason F. Hyder O. A. C. Petroff K. L. Behar 《Neurochemical research》1996,21(9):1031-1041
Brain GABA levels rise and plateau following prolonged administration of the irreversible GABA-transaminase inhibitor vigabatrin
(γ-vinylGABA). Recently it has been shown that increased GABA levels reduces GAD67 protein, one of two major isoforms of glutamic acid decarboxylase (GAD). The effects of GABA elevation on GABA synthesis
were assessed in vivo using1H and13C-edited NMR spectroscopy. Rates of turnover of cortical glutamate and GABA from intravenously administered [1-13C]glucose were measured in α-chloralose anesthetized rats 24 hours after receiving vigabatrin (500 mg/kg, i.p.) and in non-treated
controls. GABA concentration was increased 2-fold at 24 hours (from 1.3±0.4 to 2.7±0.9 μmol/g) and GABA-T activity was inhibited
by 60%. Tricarboxylic acid cycle flux was not affected by vigabatrin treatment compared to non-treated rats (0.47±0.19 versus
0.52±0.18 μmol/g, respectively). GABA-C2 fractional enrichment (FE) measured in acid extracts rose more slowly in vigabatrin-treated
compared to nontreated rats, reaching >90% of the glutamate FE after 3 hours. In contrast, GABA FE≥glutamate FE in non-treated
rats. A metabolic model consisting of a single glutamate pool failed to account for the rapid labeling of GABA from glutamate.
Metabolic modelling analysis based on two (non-communicating) glutamate pools revealed a ∼70% decrease in the rate of GABA
synthesis following vigabatrin-treatment, from 0.14 (non-treated) to 0.04 μmol/g/min (vigabatrin-treated). These findings,
in conjunction with the previously reported differential effects of elevated GABA on the GAD isoforms, suggests that GAD67 may account for a major fraction of cortical GABA synthesis in the α-chloralose anesthetized rat brain in vivo.
Special issue dedicated to Dr. Herman Bachelard. 相似文献
18.
Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms 总被引:7,自引:0,他引:7
Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are both receptors for semaphorins, which regulate neuronal guidance, and vascular endothelial growth factor (VEGF), an angiogenic factor. The two human NRP1 and NRP2 genes were cloned, and the exon-intron boundaries were determined. The NRP1 and NRP2 genes span over 120 and 112 kb, respectively, and are composed of 17 exons. Five of the exons are identical in size in the two genes, suggesting that they arose by gene duplication. Both NRP genes are characterized by multiple alternatively spliced variants. Two NRP2 isoforms, NRP2a and NRP2b, were cloned. A striking feature of these two isoforms is that they have identical extracellular domains but have divergent transmembrane and cytoplasmic domains. In these domains, NRP2a is closer in sequence identity to NRP1 than to NRP2b. As determined by Northern blot analysis, both NRP2a and NRP2b are expressed in a variety of tissues, mostly in a nonoverlapping manner. Within NRP2a and NRP2b, there are several alternatively spliced species: NRP2a(17), NRP2a(22), NRP2b(0), and NRP2b(5). In addition to full-length NRPs, there are truncated NRPs as well, which contain only the extracellular a/CUB and b/coagulation factor domains. These genes encode proteins that are soluble (sNRP) and released by cells. In addition to s12NRP1, which was previously cloned, s11NRP1 and s9NRP2 have now been cloned. These sNRP molecules are characterized by having intron-derived sequences at their C-termini. Altogether, eight NRP isoforms are described in this report. It was concluded that there are multiple NRP1 and NRP2 isoforms including intact and soluble forms. Characterization of these isoforms should help to elucidate the function of NRPs in neuronal guidance and angiogenesis. 相似文献
19.
Neuropilin-1 (NRP1) is a coreceptor to a tyrosine kinase receptor for both the vascular endothelial growth factor (VEGF) family and semaphorin (Sema) family members. NRP1 plays versatile roles in angiogenesis, axon guidance, cell survival, migration, and invasion. NRP1 contains three distinct extracellular domains, a1a2, b1b2, and c. We report here the identification of two novel soluble human NRP1 isoforms, which we named sIIINRP1 and sIVNRP1. These soluble NRP1 isoforms were generated by alternative splicing of the NRP1 gene, a common regulatory mechanism occurring in cell surface receptor families. Both sIIINRP1 and sIVNRP1 contain a1a2 and b1b2 domains, but no c domain, and the rest of the NRP1 sequence. Additionally, sIIINRP1 is missing 48 amino acids within the C-terminus of the b2 domain. Both sIIINRP1 and sIVNRP1 are expressed in human cancerous and normal tissues. These molecules are capable of binding to VEGF165 and Sema3A. Furthermore, recombinant sIIINRP1 and sIVNRP1 proteins inhibit NRP1-mediated MDA-MB-231 breast cancer cell migration. These results indicate the multiple levels of regulation in NRP1 function and suggest that these two novel NRP1 isoforms are useful antagonists for NRP1-mediated cellular activities. 相似文献
20.
Xavier Khawaja Non Evans Yvonne Reilly Christine Ennis Michael C. W. Minchin 《Journal of neurochemistry》1995,64(6):2716-2726
Abstract: The specific binding of [3H]WAY-100635 {N-[2-[4-(2-[O-methyl-3H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [3H]WAY-100635 association (k+1 = 0.069 ± 0.015 nM?1 min?1) and dissociation (k?1 = 0.023 ± 0.001 min?1) followed monoexponential kinetics. Saturation binding isotherms of [3H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (Bmax) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [3H]WAY-100635 was ~36% higher compared with that of 8-hydroxy-2-(di-n-[3H]-propylamino)tetralin ([3H]8-OH-DPAT). The binding affinity of [3H]WAY-100635 was significantly lowered by the divalent cations CaCl2 (2.5-fold; p < 0.02) and MnCl2 (3.6-fold; p < 0.05), with no effect on Bmax. Guanyl nucleotides failed to influence the KD and Bmax parameters of [3H]WAY-100635 binding to 5-HT1A receptors. The pharmacological binding profile of [3H]WAY-100635 was closely correlated with that of [3H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine1A (5-HT1A) sites in rat hippocampus. [3H]WAY-100635 competition curves with 5-HT1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites. 相似文献