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21.
High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses 总被引:1,自引:1,他引:0
Bender FL Fischer M Funk N Orel N Rethwilm A Sendtner M 《Histochemistry and cell biology》2007,127(4):439-448
Primary neurons are a common tool for investigating gene function for survival and morphological and functional differentiation.
Gene transfer techniques play an important role in this context. However, the efficacy of conventional gene transfer techniques,
in particular for primary motoneurons is low so that it is not possible to distinguish whether the observed effects are representative
for all neurons or only for the small subpopulation that expresses the transfected cDNA. In order to develop techniques that
allow high gene transfer rates, we have optimized lentiviral-based gene transfer for cultured motoneurons by using a replication-defective
viral vector system. These techniques result in transduction efficacies higher than 50%, as judged by EGFP expression under
the control of SFFV or CMV promoters. Under the same conditions, survival and morphology of the cultured motoneurons was not
altered, at least not when virus titers did not exceed a multiplicity of infection of 100. Under the same cell culture conditions,
electroporation resulted in less than 5% transfected motoneurons and reduced survival. Therefore we consider this lentivirus-based
gene transfer protocol as a suitable tool to study the effects of gene transfer on motoneuron survival, differentiation and
function. 相似文献
22.
Two species of Camellia L. (Theaceae) are described here as new: C. curryana and C. longii. The new species were discovered in the southern Annamite Mountains of Vietnam and are endemic to tropical rainforest remnants that occur at altitudes between 1500 and 1700 m a.s.l. Camellia curryana possesses almost sessile, unevenly circular, two whorled, white‐cream and proximally yellowish flowers, proximally joined outer stamens, three styles and mature fruit that dehisces into three parts, or longitudinally into two halves. Camellia longii has campanulate or almost campanulate, intensely dark orange to red flowers with uneven whitish margins, 5–6 petals and 3–2 petaloids, filaments that are united with the petals and one another, a diamond shaped ovary and compound 5–6 styles that are connate at the base. 相似文献
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Guy Kamnesky Orel Hirschhorn Hadassa Shaked Jingfei Chen Lishan Yao Jordan H Chill 《Protein science : a publication of the Protein Society》2014,23(10):1403-1416
The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels. 相似文献
25.
The neurokinin-1 receptor (NK1R) is a G-protein coupled receptor found in the central and peripheral nervous systems of vertebrates, and is responsible for many physiological processes. The C-terminus domain seems to be essential for coupling to the corresponding G-protein and β-arrestin, and is important for receptor desensitization, internalization and recycling. We have focused our study on expression of the human NK1R (hNK1R) C-terminus in Escherichia coli, and its purification and characterization, in order to elucidate its structural properties. CD and Fourier transform infrared spectroscopy showed that the hNK1R C-terminus, rather than having a random structure, has well-defined secondary-structure patterns. The presence of three tyrosine residues in the primary sequence of the hNK1R C-terminus facilitated the use of UV and fluorescence spectroscopy techniques which revealed tyrosine fluorescence and UV absorption at anomalous wavelengths. In their entirety, the results show that the hNK1R C-terminus has clearly defined secondary (25% α-helix, 27% unordered structure and 48% β-sheets and β-turns) and tertiary structures which, it is believed, are tightly related to its multiple functions. 相似文献
26.
Rad5 is the key component in the Rad5-dependent error-free branch of postreplication repair in yeast (Saccharomyces cerevisiae). Rad5 is a member of the Snf2 ATPase/helicase family, possessing as a characteristic feature, a RING-finger domain embedded in the Snf2-helicase domain and a HIRAN domain. Yeast mutants are sensitive to DNA-damaging agents and reveal differences in homologous recombination. By sequence comparisons we were able to identify two homologs (AtRAD5a and AtRAD5b) in the Arabidopsis thaliana genome, sharing about 30% identity and 45% similarity to yeast Rad5. AtRad5a and AtRad5b have the same kind of domain organization with a higher degree of similarity to each other than to ScRad5. Surprisingly, both genes differ in function: whereas two independent mutants of Atrad5a are hypersensitive to the cross-linking agents mitomycin C and cis-platin and to a lesser extent to the methylating agent, methyl methane sulfonate, the Atrad5b mutants did not exhibit any sensitivity to all DNA-damaging agents tested. An Atrad5a/Atrad5b double mutant resembles the sensitivity phenotype of the Atrad5a single mutants. Moreover, in contrast to Atrad5b, the two Atrad5a mutants are deficient in homologous recombination after treatment with the double-strand break-inducing agent bleomycin. Our results suggest that the RAD5-dependent error-free branch of postreplication repair is conserved between yeast and plants, and that AtRad5a might be functionally homologous to ScRad5. 相似文献
27.
Shinitzky Meir Nudelman Fabio Barda Yaniv Haimovitz Rachel Chen Effie Deamer David W. 《Origins of life and evolution of the biosphere》2002,32(4):285-297
We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 °C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization. 相似文献
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Fer is a nuclear and cytoplasmic tyrosine kinase that is ubiquitously expressed in mammalian cells. Herein we show that Fer sustains a key signaling step in hypoxic cells. Knock-down of the Fer protein using a specific siRNA decreased the production of VEGF by the hypoxic cells. Conversely, ectopic expression of this kinase led to an elevated production of VEGF under hypoxia. At the molecular level, Fer was found to associate with ERK1/2 and this interaction was intensified under hypoxia. Moreover, Fer increased the activation levels of ERK1/2, and reducing the level of Fer, impaired the activation of ERK1/2 in hypoxic cells. Blocking the MEK-ERK1/2 signaling pathway with the MEK inhibitors U0126, or PD98059 led to the abrogation of ERK1/2 activity in hypoxic cells, an effect that was counteracted by Fer. Hence, Fer sustains the activation of ERK1/2 and increases the production of VEGF in hypoxic cells, without affecting the MEK-ERK signaling pathway. 相似文献
30.