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1.
利用在体注射实验和放射免疫测定法,研究了多巴胺能药物对性腺处于再发育期虎纹蛙的促性腺激素释放激素(GnRH)及促黄体激素(LH)分泌活动的影响。结果是:多巴胺(DA)及其激素剂阿扑吗啡(APO)可显著降低血浆LH水平;而多巴胺的拮抗剂-地欧酮(DOM)可显著增加垂体LH含量。DA对脑中cGnRH-Ⅱ的合成有抑制作用,而OM对其mGnRH的释放有一定的刺激作用。结果表明:DA可在脑及垂体水平分别抑制虎纹蛙GnRH和LH的释放,DA对LH释放的抑制作用很可能是通过D2受体实现的。 相似文献
2.
We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials. 相似文献
3.
Summary Gonadotropin releasing hormone (GnRH) and gonadotropic hormone (GTH) were identified by immunohistochemistry in the brains and pituitaries of neonate, juvenile and adult guppies. GTH was present in some cells of the pars intermedia (pi) and proximal pars distalis (ppd) of all animals. GnRH was found in the perikarya of the nucleus olfactoretinalis. In the pituitaries of juvenile 30-day-old guppies, GnRH-immunoreactive cells existed in a juvenile pattern, whereas in adult animals GnRH was recognized in only a few cells. GnRH-immunoreactive fibers were seen in the pituitaries of animals that were 30 days or older. In adult guppies, the ventral and lateral ppd (the gonadotropic region) contained a dense network of GnRH-immunoreactive fibers. Pituitary cells staining for either GnRH or GTH were located in different places. After immunohistochemical double staining of adult pituitaries, none of the GnRH-immunoreactive cells were LH-immunoreactive, although both cell types were often found in close proximity. After 20 days or more of ethinylestradiol treatment, less immunoreactive GnRH was detected in the pituitary cells of juvenile guppies, and fewer animals exhibited the juvenile pattern of GnRH-immunoreactive pituitary cells, when compared with untreated controls. The results indicate that GnRH-immunoreactive pituitary cells in the guppy are distinct from gonadotropes and that these cells are involved in regulatory processes along the juvenile brain-pituitary-gonad axis. 相似文献
4.
Interleukin-4 receptors (IL-4Rs) are expressed on a wide variety of human cancer cells, and therefore it may be a good option to treat IL-4R-bearing tumors with IL-4-fusing immunotoxins. In this study, the gene encoding human interleukin-4 mutein cpIL-4(13D) was obtained through overlapping polymerase chain reaction. A chimeric immunotoxin was constructed by genetically fusing the mutein cpIL-4(13D) to a modified version of Pseudomonas exotoxin A (PE38KDEL) and was expressed in Escherichia coli AD494 (DE3). The expression level of the fusion protein was about 30% of the total bacterial protein assessed by SDS-PAGE analysis. After purification by affinity chromatography and anion exchange chromatography, the chimeric protein was tested for its cytotoxicity. Our data show that cpIL-4(13D)-PE38KDEL has improved cytotoxicity on IL-4R-bearing tumor cells in comparison with other IL-4-fusing immunotoxins and might be useful in treating tumors with a large number of IL-4Rs. 相似文献
5.
嵌合重组caspase-3诱导表达促进肿瘤细胞凋亡 总被引:3,自引:0,他引:3
通过稳定转染人宫颈癌HeLa细胞,建立了野生型caspase-3(wt-casp3),大小亚基序列颠倒的重组caspase-3 (r-casp3),和N端融合绿脓杆菌外毒素(PE)转膜肽段的嵌合重组caspase-3 (cr-casp3)的诱导表达细胞系.蜕皮素诱导后细胞中检测到目的基因的表达,MTT检测和细胞计数结果表明,r-casp3和cr-casp3诱导表达后有效地导致HeLa细胞死亡,通过测定细胞中caspase-3活性,以及细胞周期检测、DNA梯状电泳条带检测(DNA ladder)、电镜观察等证实r-casp3和cr-casp3诱导表达后细胞发生了凋亡,且二者的促凋亡活性相当,而wt-casp3诱导表达细胞并未出现上述效应.结果表明,与野生型caspase-3活化需要上游分子的切割不同,重组caspase-3具有自发的促凋亡活性,而N端PE肽段的融合不影响这种活性,因此PE转膜结构域和重组caspase-3有望参与构建能转膜进入细胞内部,并杀伤细胞的新型肿瘤治疗分子. 相似文献
6.
Guilgur LG Ortí G Strobl-Mazzulla PH Fernandino JI Miranda LA Somoza GM 《Journal of molecular evolution》2007,64(6):614-627
Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number
of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms
expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of
these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses
(eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates
GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic
analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main
paralogous GnRH lineages with different function. A “forebrain lineage” separates evolutionarily from the “midbrain lineage”
as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated
before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein
sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically
rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from
chromosomal studies of synteny around the GnRH genes in vertebrates.
[Reviewing Editor: Dr. Axel Meyer] 相似文献
7.
Summary The double antibody immunofluorescence technique was applied to serial coronal and sagittal sections of the brains of the trout, Salmo gairdneri, using an antibody against mammalian LH-RH. Immunoreactive material was found in small perikarya, situated at both sides of the ventriculus communis in the area dorsalis pars medialis of the telencephalon.The axons of these perikarya, also containing immunoreactive material, do not form a tract, but run diffusely in a caudo-ventral direction towards the pituitary stalk. The ending of these fibres has not yet been established.The authors wish to thank Mr. E. van der Vlist and Mr. J.J. van der Vlis for preparing the illustrations 相似文献
8.
Microtubule-binding proteins from carrot 总被引:5,自引:0,他引:5
Microtubules (MTs) participate in several processes of fundamental importance to growth and development in higher plants, yet little is known about the proteins with which they associate. Information about these molecules is important because they probably play a role in mediating functional and structural differences between various MT arrays. As a first step in gaining insight into this problem, we have isolated, from suspension-cultured cells of carrot (Daucus carota L.), non-tubulin proteins which bind to and affect microtubules (MTs) in vitro. These proteins were isolated using taxol-stabilized neuronal MTs as an affinity substrate. They cause MT bundling at substoichiometric concentrations, support the assembly of tubulin in vitro, and at low concentrations, decorate single MTs in a periodic fashion. The bundled MTs formed in vitro share similarities with those seen in situ in a variety of plant cells, including a center-center spacing of 34 nm, cold stability, resistance to anti-microtubule drugs, and sensitivity to calcium. The bundling activity is specific; other cationic proteins, as well as poly-L-lysine, do not behave in a similar manner. The bundling activity is insensitive to ATP. By assaying bundling activity with dark-field microscopy and employing standard biochemical procedures, a small number of polypeptides involved in the bundling process were identified. Affinity-isolated antibodies to one of these polypeptides (Mr=76000) were found to co-localize with MTs in the cortical array of protoplasts. Our findings are discussed with reference to the importance of these proteins in the cell and to their relationship to microtubule-associated proteins in other eukaryotes.Abbreviations DEAE
diethylaminoethyl
- MAP(s)
microtubule-associated protein(s)
- MT(s)
microtubule(s)
- Mr
relative molecular mass
- OD
optical density
- PM
50 mM 1,4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM magnesium sulphate, 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis 相似文献
9.
Elena M. Glinka Alla S. Andryushchenko Alexander M. Sapozhnikov Olga V. Zatsepina 《Plasmid》2009,62(2):119-127
Tumor-targeted vectors encoding toxic protein genes are promising tools for treating malignant tumors. We used the pEGFP-N1 vector to construct a novel plasmid (pCMV-ETA-EGFP) for eukaryotic expression of a truncated Pseudomonas aeruginosa exotoxin A (ETA) that is known to inhibit protein synthesis, and subsequently induce cell death, by inactivation of elongation factor-2. ETA was linked to the enhanced green fluorescent protein (EGFP) gene, and ETA-EGFP gene expression was driven by the cytomegalovirus (CMV) promoter. The time-lapse effects of pCMV-ETA-EGFP expression were examined in transiently transfected HeLa cells. HeLa cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and рЕGFP-N1 showed lower fluorescence intensity than cells transfected with pEGFP-N1 alone. Analysis of the number of dead cells further confirmed the highly toxic effect of the ETA-EGFP fusion protein on cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and рЕGFP-N1. ETA-EGFP fusion protein induced apoptotic cell death through the caspase-3 activation. By using the antibody against a marker nucleolar antigen A3 [Grigoryev, A.A., Bulycheva, T.I., Sheval, E.V., Kalinina, I.A., Zatsepina, O.V., 2008. Cytological indicators of the overall suppression of protein synthesis revealed by staining with new monoclonal antibody. Cell Tissue Biol. 2, 191–199], the distribution of which changes when HeLa cells are treated with known translation inhibitors, we obtained evidence to support the idea that protein synthesis is inhibited in transfected cells in situ. ETA-EGFP fusion protein was identified in lysates of transfected cells using anti-GFP-BL antibodies. Collectively, our results indicate that HeLa cells transfected with pCMV-ETA-EGFP synthesize the ETA-EGFP fusion protein that efficiently inhibits protein synthesis, leading to massive cell death by an apoptosis-mediated pathway with a participation of caspase-3. The constructed vector can be used in suicidal gene therapy of cancer and may also be useful for investigating the general effects of translational downregulation in human cancer cells. We also suggest a novel approach for detecting the activity of new vectors in transfected cells, which is based on the redistribution of nucleolar proteins in transfected cells. 相似文献
10.
Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA
electrophoretic-mobility-shift assay
- FPLC
fast protein liquid chromatography
- HMG
high-mobility group
- kDa
kilodaltons
- PVDF
polyvinylidenedifluoride
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie. 相似文献
11.
Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine -lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells. 相似文献
12.
13.
Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A 总被引:6,自引:0,他引:6
Ann Koning Aubrey Jones JoAnne J. Fillatti Luca Comai Michael W. Lassner 《Plant molecular biology》1992,18(2):247-258
Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated -glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting -glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile. 相似文献
14.
When competition for sex-specific resources overlaps in time with offspring production and care, trade-offs can occur. Steroid hormones, particularly testosterone (T), play a crucial role in mediating such trade-offs in males, often increasing competitive behaviors while decreasing paternal behavior. Recent research has shown that females also face such trade-offs; however, we know little about the role of T in mediating female phenotypes in general, and the role of T in mediating trade-offs in females in particular. Here we examine the relationship between individual variation in maternal effort and endogenous T in the dark-eyed junco, a common songbird. Specifically, we measure circulating T before and after a physiological challenge (injection of gonadotropin releasing hormone, GnRH), and determine whether either measure is related to provisioning, brooding, or the amount of T sequestered in egg yolk. We found that females producing more T in response to a challenge spent less time brooding nestlings, but provisioned nestlings more frequently, and deposited more T in their eggs. These findings suggest that, while T is likely important in mediating maternal phenotypes and female life history tradeoffs, the direction of the relationships between T and phenotype may differ from what is generally observed in males, and that high levels of endogenous T are not necessarily as costly as previous work might suggest. 相似文献
15.
Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco 总被引:5,自引:0,他引:5
Gabriel Iturriaga Katharina Schneider Francesco Salamini Dorothea Bartels 《Plant molecular biology》1992,20(3):555-558
Three cDNAs encoding desiccation-induced proteins from the resurrection plant Craterostigma plantagineum were each ligated to a triplicated CaMV 35S promoter and a nopaline synthase 3-flanking region in an Agrobacterium vector and introduced into tobacco. Transgenic plants expressed the encoded Craterostigma proteins at high levels. This did not lead to changes in the phenotype, in the growth habit or in basic photosynthetic parameters. In tobacco, one protein was targeted to the chloroplast stroma which is its normal location in Craterostigma. These desiccation-related proteins are not sufficient per se to increase drought tolerance as measured by ion-leakage tests. 相似文献
16.
Maize (Zea mays L.) seedlings were grown in the presence or absence of an herbicide, norflurazon (4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-pyridazinone), which prevents the accumulation of colored carotenoids. In the absence of carotenoids, plants grown in high light incur extensive photooxidative damage to their plastids, but relatively little damage elsewhere. Growth in very low light minimizes chlorophyll photooxidation and allows chloroplast development to proceed. We have previously reported that mRNA encoding light-harvesting chlorophyll a/b protein (LHCP) fails to accumulate in high-light-grown carotenoid-deficient seedlings, but accumulates normally in carotenoid-deficient seedlings grown in low light. Here we extend these results by examining the levels of translatable mRNAs encoding seven additional nuclear-encoded chloroplast proteins. When norflurazon-treated seedlings were grown in low light for 8 d and then transferred to high light for 24 h, three cytosolic mRNAs (plastocyanin, Rieske Fe–S protein, and the 33-kdalton (kDa) subunit of the photosystem II O2-evolving complex) decreased to less than 1% the amount found in untreated seedlings. Two other mRNAs (NADP malic enzyme, EC 1.1.1.40, and the 23-kDa subunit of the photosystem II O2-evolving complex) decreased significantly but not to levels as low as the first three. Levels of translatable mRNA for two other chloroplast proteins (pyruvate orthophosphate dikinase, EC 2.7.9.1, and ferredoxin NADP oxidoreductase, EC 1.18.1.2) were not reduced in nonflurazon-treated seedlings after 24 h in high light, but did not show the normal light-induced increase found in untreated plants. Photooxidative damage in the chloroplast thus affects the accumulation of a number of cytosolic mRNAs encoding proteins destined for the chloroplast.Abbreviations Da
dalton
- FNR
ferredoxin NADP oxidoreductase
- LHCP
light-harvesting chlorophyll a/b-binding protein
- poly(A)RNA
polyadenylated RNA
- PPDK
pyruvate orthophosphate dikinase
- PSII
photosystem II
- SDSPAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SSu
small subunit (of ribulose-1,5-bisphosphate carboxylase) 相似文献
17.
Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust
Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation. 相似文献
18.
J C Zolman 《FEBS letters》1982,143(1):26-28
Aspartate, glutamate, serine and glycine all permeate the inner membrane of mitochondria isolated from both etiolated and green plant tissues. No significant difference was found in the transport characteristics shown by mitochondria from either tissue. Influx of each amino acid appears diffusional because substrate saturation was not observed and there was no indication of specific inhibition or a requirement for a compensatory or counter ion for uptake. In contrast, uptake of the keto acid pyruvate did appear saturable. Inhibition by α-cyano-4-hydroxycinnamate, mersalyl and FCCP, but not valinomycin, suggests a carrier and a ΔpH mediate pyruvate transport into the matrix. 相似文献
19.
20.
When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the
total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial
characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to
homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins.
Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular
masses (Mrs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous
on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products)
in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins
appear to be produced late in seed development but are capable of being induced early in development by culturing embryos
in vitro and are markedly enhanced by ABA. 相似文献