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1.
Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey 总被引:21,自引:11,他引:10
The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain. 相似文献
2.
The isolated leaf proteins of lucerne (Medicago sativa L. and M. falcata L.) were fractionated by Sepharose 6B column chromatography. Analysis of fractionated proteins indicated that the 2nd peak component was almost entirely ribulose 1,5 bisphosphate carboxylase-oxygenase (Rubisco) which represented 57% of the total recovered protein.Rubisco yielded one large subunit (LSU) and one small subunit (SSU) polypeptide after SDS gel electrophoresis.Isoelectric focusing of the SSU of Rubisco from genotypes of M. sativa cv. Hunter River (HR), Hairy Peruvian (HP) and of M. falcata (MF) showed two SSU components for HR and HP, and three components for MF. Most components of genotypes were located in the alkaline region of the gel. While the pIs of the SSU components of HR and HP were identical they differed from those of the SSU of MF thus demonstrating heterogeneity for SSU in Medicago.It is suggested that the alkaline nature of SSU may have some adaptive physiological significance.Abbreviations Rubisco
ribulose bisphosphate 1,5-carboxylase-oxygenase
- LSU
large subunit
- SSU
small subunit
- HR
Hunter River
- HP
Hairy Peruvian
- MF
Medicago falcata
- SDS
Sodium dodecyl sulphate
- TCA
trichloracetic acid 相似文献
3.
Sheila McCormick Jeanne Niedermeyer Joyce Fry Arlene Barnason Robert Horsch Robert Fraley 《Plant cell reports》1986,5(2):81-84
The leaf disc transformation/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios. Several heterologous promoters function in tomato. A reduced efficiency of transformation was observed with binary T-DNA vectors as compared to co-integrate T-DNA vectors. The ease of the leaf disc method makes tomato a premier experimental organism for plant biotechnology.Abbreviations BA
benzyl adenine
- IAA
indole acetic acid
- LB
Luria Broth 相似文献
4.
An nmr spectral comparison of a model cyclic pentapeptide cyclo(Gly-Pro-Gly-D-Phe-Pro) with an analogous pseudopeptide has been made. The pseudopeptide contains a ψ[CH2S] amide bond replacement at the only amide linkage that, in the model, is not involved in an intramolecular hydrogen bond. Both proton and carbon-13 nmr spectral evidence confirms the retention of β- and γ-turns in the pseudopeptide in chloroform. Characteristic chemical shifts, temperature dependence, and glycine α-resonances support this interpretation. However, evidence of a more flexible conformation involving cis–trans proline isomerism is seen on addition of dimethylsulfoxide. 相似文献
5.
Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10–1 to 1.9×10–7. Highest numbers of transconjugants, 1.5×107 colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 103 colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system. 相似文献
6.
The following rodents and marsupials from the Western Highlands of Papua New Guinea have been examined for helminths: Anisomys imitator, Melomys spp., Pogonomelomys ruemmleri, Rattus spp., Echymipera kalubu and Peroryctes raffrayanus. Two new species and a number of new host records are reported. Echinostoma echymiperae n. sp., a digenean from the intestine of Echymipera kalubu, is characterised by the number of collar spines, the body armature and the shape and position of the gonads. Vampirolepis peroryctis n. sp., a cestode from the intestine of Peroryctes raffrayanus, is characterised by the length of the rostellar hooks, the shape of the ovary, the arrangement of the testes in a triangle and the extent of the cirrus-sac. Hymenolepis aklei, H. bradleyi, H. antechini, H. bettongiae, H. cercarteti, H. isoodontis and H. potoroi are transferred to Vampirolepis as new combinations. E. kalubu is a new host for Linstowia semoni and Pogonomelomys ruemmleri is a new host for Hymenolepis diminuta. V. peroryctis is the first platyhelminth to be reported from Peroryctes raffrayanus and Raillietina (Raillietina) sp. the second to be reported from the genus Melomys. 相似文献
7.
Arlene R. Wechezak Robert F. Viggers Daniel E. Coan Lester R. Sauvage 《Journal of cellular physiology》1994,159(1):83-91
An in vitro flow apparatus in combination with cultured endothelium was used to determine the effects of fluid-generated shear stress on cells undergoing mitosis and cytokinesis. Cell responses were recorded by time-lapse video microscopy under phase contrast or Hoffman modulation contrast optics. Completion of cell division in mitotic cells was dependent upon both the initial presence of intercellular attachments and the magnitude of fluid wall shear stress. In nonisolated populations, 95.3%, 69.5%, and 57.1% of the cells completed cell division as opposed to 66.6%, 20.4%, and 11.9% in the isolated cell groups at 2.8, 14.1, and 33 dynes/cm2, respectively. Prestressing cells for 14 h prior to monitoring failed to increase retention of isolated mitotic cells. The presence of neighboring cells facilitated replication by providing an anchoring attachment or a luminal surface for completion of division. Cell detachment most commonly occurred at the onset of cytokinesis when substrate contact areas were minimal and focal contacts were absent. A comparison between no flow controls and shear stress specimens indicated no significant differences in transit times for mitosis and cytokinesis. Thus, subconfluent endothelial cells may be more susceptible to detachment during cell division due to increases in shear stress, the absence of intercellular attachments, and reduced cell-substrate contacts. © 1994 wiley-Liss, Inc. 相似文献
8.
Arlene D. Acevedo Samuel S. Bowser Mary E. Gerritsen Rena Bizios 《Journal of cellular physiology》1993,157(3):603-614
Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5–15 cm H2O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: (1) Cells exposed to control (3 mm H2O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H2O for 7 days. (2) Conditioned medium from endothelial cells exposed to 10 cm H2O for 7 days contained at ansferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. (3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure. © 1993 Wiley-Liss, Inc. 相似文献
9.
Duration of Hydrogen Formation by Anabaena cylindrica B629 in Atmospheres of Argon, Air, and Nitrogen 总被引:3,自引:3,他引:0
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The time course of hydrogen formation by Anabaena cylindrica was followed beneath an argon atmosphere alone and also beneath atmospheres of argon, nitrogen, and air in the presence of carbon monoxide (0.2%) and acetylene (5%). Hydrogen production beneath argon alone was comparable in rate and duration (7 to 12 days) to that which occurred beneath air in the presence of carbon monoxide (0.2%) and acetylene (5%). However, much greater longevity (16 to 26 days) and improved rates of hydrogen formation were obtained when algae were incubated beneath argon and particularly nitrogen, each supplemented with carbon monoxide and acetylene. The total hydrogen produced by these cultures was up to three times as much as that released by cultures incubated beneath argon alone. Hydrogen-oxygen ratios for argon cultures either with or without carbon monoxide and acetylene were initially 1:5 but approximated 1:2 when measured over the entire incubation period. In each case oxygen production and nitrogenase activity (acetylene reduction) continued at reduced rates after hydrogen evolution had ceased. The effects of methionine sulfoximine (2 μM), ammonium ions (0.5 mM), or both on oxygen production were generally negligible, while effects on hydrogen production were variable depending on the atmosphere used; in most cases, eventual destabilization of the system occurred. A brief comparison was made of the time courses of anaerobic and aerobic hydrogen formation by the marine cyanobacterium Calothrix membranacea. It was found that shaking of cultures was beneficial for hydrogen production but not strictly necessary. It is concluded that hydrogen production by A. cylindrica in air and particularly nitrogen in the presence of carbon monoxide and acetylene offers the best potential of the atmospheres considered on the basis of four criteria: rates and longevity of hydrogen formation, practicality of the atmosphere used, and tolerance of hydrogen evolution to slight changes in composition of the atmosphere. 相似文献
10.
Richard Hammerschlag Charles Bakhit Arlene Y. Chiu Anant R. Dravid 《Developmental neurobiology》1977,8(5):439-451
Fast axonal transport of [3H]protein has been examined in bullfrog primary afferent neurons incubated in media supplemented with divalent cations that can act as agonists or antagonists of calcium ions. Incubation in calcium-free medium (CFM) had no effect on the rate of transport, but reduced the amount of transported [3H]protein by 40–60% relative to transport in the contralateral preparation maintained in normal medium. Preparations incubated in CFM supplemented with 1.8 mM SrCl2 (equimolar to the CaCl2 concentration in normal medium) carried out transport at control levels. Incubation conditions in which primary afferent somata were exposed to the Sr2+-medium while nerve trunks were maintained in CFM also supported normal transport. By contrast, selective exposure of nerve trunks to Sr2+-medium, and somata to CFM resulted in a reduced level of transport similar to that observed when the whole preparation was incubated in CFM. The depression of transport resulting from incubation in CFM was shown to be reversible when preparations were transferred from CFM to either Sr2+-supplemented CFM or to normal medium. By contrast to the effects of Sr2+, Ba2+ (up to 18 mM) did not substitute for Ca2+ in the transport process. When normal medium was supplemented with calciumantagonist cations, the amount of transport was depressed (Co2+ > Mn2+ >> Mg2+), with no concomitant effect on the rate of transport. Results of studies with Co2+, as well as those with Sr2+, suggest that a major locus of action of these cations is within the neuronal soma at a step subsequent to protein synthesis, and prior to the onset of protein translocation via the transport system. Thus, it is inferred that these divalent cations affect a calcium-dependent step that occurs during the initiation phase of fast axonal transport. 相似文献