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171.
172.
地高辛标记反意RNA探针检测脑组织切片生长抑素mRNA 总被引:2,自引:0,他引:2
本实验采用含大鼠生长抑素基因的pSP65cDNA质位通过转化至噬菌体内大量扩增,经提取,纯化后,用限制性内切酶进行酶切使质枝线性化,并将其作为模板,用地高辛(DigoxigeninDig)作为标记物,体外转录合成生长抑素反意RNA(cRNA)探针。实验动物选用wistar新生大鼠。冰冻切片,端、间脑切片经杂交前用Dig-UTP标记的cRNA探针杂交,杂交后用抗Dig-碱性磷酸酶复合物进行酶联免疫反应。X-磷酸盐-NBT显色。结果显示新生大鼠脑内生长抑素mRNA神经元着紫蓝色。杂交反应物集中于核周的胞浆及短小的突起内。胞核不着色。胞体轮廓清晰,周围背底浅淡。结果表明Dig标记cRNA探针不仅具备非同位素标记探针的优点而且能快速和准确检测组织细胞内mRNA的表达。 相似文献
173.
Karen E. Mate Nechama S. Ksower Ian G. White John C. Rodger 《Molecular reproduction and development》1994,37(3):318-325
The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc. 相似文献
174.
Louis Faso Richard S. Trowbridge Wei Quan Xiu-Lan Yao Edmund C. Jenkins Alma Maciulis Thomas D. Bunch Henry M. Wisniewski 《In vitro cellular & developmental biology. Animal》1994,30(4):226-235
Summary A strain of cerebral endothelial cells was established from isolated cortical microvessels of caprine brain. These cells,
which are referred to as ECl cells, can be routinely subcultured to 32 passages without the loss of differentiated morphologic
and immunologic traits. The ability to routinely subculture ECl cells is an important asset, given that isolated cerebral
endothelial cells in mammals generally lose their differentiated traits after only 2 to 3 passages.
ECl cells were shown to contain Factor VIII-related antigen, which is a specific marker for cells of endothelial origin. ECl
cells morphologically demonstrated a scarcity of pinocytotic vesicles on their apical surfaces, a lack of trans-cytoplasmic
vesicles, and the ability to form in culture confluent monolayers with tight junctional complexes. Therefore, ECl cells possess
specific antigenic and ultrastructural features which classify them as being small vessel endothelial cells of the blood-brain
barrier type. Cytogenetic evaluation of ECl cells demonstrated a normal female goat 60,XX karyotype and confirmed the apparent
non-transformed nature of ECl cells due to the lack of chromosome abnormalities or rearrangements. Using scanning electron
microscopy, ECl cells were also shown to form confluent monolayers on mixed nitrocellulose filters, a feature that will enable
the development of an in vitro system to study trans-endothelial transport. Given that ECl cells are readily subcultured and
grow well on nitrocellulose filters, and that they resemble cerebral endothelium in vivo, it seems evident that ECl cells
can be used as a versatile model for the study of blood-brain barrier function, regulation, and pathology. 相似文献
175.
Karen Pardy 《Molecular biotechnology》1994,2(1):23-27
This article describes the use of three reporter enzymes used to study promoter activity in transgenic animals. Chloramphenicol acetyl transferase may be assayed by a nonchromatographic method that is rapid and sensitive. β-Galactosidase is measured by a photometric assay and luciferase is assayed by measuring the emission of light using a luminometer. The relative merits of each enzyme is discussed. Ths use of reporter enzymes provides a rapid and sensitive method for analysis of transgene expression. 相似文献
176.
Esterina Pascale Christine Liu Eulalia Valle Karen Usdin Anthony V. Furano 《Journal of molecular evolution》1993,36(1):9-20
Summary All modern mammals contain a distinctive, highly repeated (⩾50,000 members) family of long interspersed repeated DNA called
the L1 (LINE 1) family. While the modern L1 families were derived from a common ancestor that predated the mammalian radiation
∼80 million years ago, most of the members of these families were generated within the last 5 million years. However, recently
we demonstrated that modern murine (Old World rats and mice) genomes share an older long interspersed repeated DNA family
that we called Lx. Here we report our analysis of the DNA sequence of Lx family members and the relationship of this family
to the modern L1 families in mouse and rat. The extent of DNA sequence divergence between Lx members indicates that the Lx
amplification occurred about 12 million years ago, around the time of the murine radiation. Parsimony analysis revealed that
Lx elements were ancestral to both the modern rat and mouse L1 families. However, we found that few if any of the evolutionary
intermediates between the Lx and the modern L1 families were extensively amplified. Because the modern L1 families have evolved
under selective pressure, the evolutionary intermediates must have been capable of replication. Therefore, replicationcompetent
L1 elements can reside in genomes without undergoing extensive amplification. We discuss the bearing of our findings on the
evolution of L1 DNA elements and the mammalian genome. 相似文献
177.
Wayne A. Cass Nancy R. Zahniser Karen A. Flach Greg A. Gerhardt 《Journal of neurochemistry》1993,61(6):2269-2278
In vivo electrochemistry was used to investigate the mechanisms contributing to the clearance of locally applied dopamine in the dorsal striatum and nucleus accumbens of urethane-anesthetized rats. Chronoamperometric recordings were continuously made at 5 Hz using Nafion-coated carbon fiber electrodes. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible dopamine signals were detected. Substitution of L-a-methyldopamine, a substrate for the dopamine transporter but not for monoamine oxidase, for dopamine in the micropipette did not substantially alter the time course of the resulting signals. This indicates that metabolism of locally applied dopamine to 3,4-dihydroxyphenylacetic acid is not responsible for the decline in the dopamine signal. Similarly, changing the applied oxidation potential from ±0.45 to ±0.80 V, which allows for detection of 3-methoxytyramine formed from dopamine via catechol-O-methyltransferase, had little effect on signal amplitude or time course. In contrast, lesioning the dopamine terminals with 6-hydroxydopamine, or locally applying the dopamine uptake inhibitors cocaine or nomifensine before pressure ejection of dopamine, significantly increased the amplitude and time course of the dopamine signals in both regions. The effects of cocaine and nomifensine were greater in the nucleus accumbens than in the dorsal striatum. Local application of lidocaine and procaine had no effect on the dopamine signals. Initial attempts at modeling resulted in curves that were in qualitative agreement with our experimental findings. Taken together, these data indicate that (1) uptake of dopamine by the neuronal dopamine transporter, rather than metabolism or diffusion, is the major mechanism for clearing locally applied dopamine from the extracellular milieu of the dorsal striatum and nucleus accumbens, and (2) the nucleus accumbens is more sensitive to the effects of inhibitors of dopamine uptake than is the dorsal striatum. 相似文献
178.
Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development. 相似文献
179.
Dryas iulia appears to have undergone a mode of evolution different from that of other members of its subfamily (Heliconiinae). While other species constitute highly subdivided and inbred populations, those ofD. iulia are thought to be large and uniform. Analyzing six samples from Southern Brazil (state of Rio Grande do Sul) in relation to three enzyme systems (EST, LAP, and PGM) and their mtDNA RFLP patterns, we found that they are very similar at the molecular level. TheF statistics for enzyme polymorphism data revealed that inbreeding makes a great contribution to the population homozygosity, sinceF IS equals 0.1322 andF ST equals 0.0023. Since the chi-square test showed thatF ST is not significant, we conclude that all localities belong to the same population. The mtDNA differentiation was about 12 times greater than for nuclear genes;F ST was equivalent to 0.0265. We suggest that this difference is due to a higher dispersal of males, in relation to females. 相似文献
180.
Condition for the appearance of the metastable P beta' phase in fully hydrated phosphatidylcholines as studied by small-angle x-ray diffraction.
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In the ripple phase of fully hydrated multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), two kinds of small-angle x-ray diffraction profiles are observed on cooling through the main transition. One is a seemingly normal profile similar to that observed on heating and the other is the superposition of the diffraction profiles for the primary (normal) and the secondary ripple structures. We found that the profile obtained depended on the cooling rate. Increasing the cooling rate from 0.1 degrees C/min to 1 degrees C/min caused the peaks originating from the secondary ripple structure to diminish. After a cooling scan at 43 degrees C/min, the profile became similar to that of the normal ripple structure, although a trace of the secondary ripple structure remains. The results are interpreted in terms of the rise and fall of three-dimensional correlated domains composed of both primary and secondary ripple structures. At slow cooling rates, correlated domains of both kinds of ripple structures develop. As the cooling rate is increased, the domain of the primary ripple structure remains correlated, while that of the secondary ripple structure becomes less correlated. In addition, the multipeak profile appears even at rapid cooling rates, if the final low temperature lies just below the Tm for the main transition. This results suggests that formation of the correlated domains of the secondary ripple structure requires a certain time interval during which the DPPC vesicles experience the temperature just below the main transition. The secondary ripple structure takes place in phosphatidylcholines having more than 15 carbons in each hydrocarbon chain upon cooling through the main transition. 相似文献