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141.
S基因在甘蓝EDFs上的高分辨率荧光原位杂交定位 总被引:1,自引:0,他引:1
植物细胞壁及细胞质的存在和植物染色体所具有的高浓缩特性,限制高效率原位杂交定位在植物细胞内的进行。针对小型染色体芸薹属植物采用常规方法DNA制备纤维的效果不佳的特点,新建制备方法:利用减数分裂前期的染色体为材料,在硅化的玻片上先后通过蛋白酶解和乙醇:乙酸(3:1)的适当处理,采用移动界而法制备EDFs。制备的EDFs比未经伸长处理的染色体在经向和横向方面分别取得较高程度的伸长与膨胀,长度可达到89-257μm,比相应地中期染色体增长30107倍,分辨率可达42.853.0kb。利用SRK和SCR两种探针同时在甘蓝粗线期染色体和EDFs上进行了原位杂交,首次鉴定了S基因座在其单倍体基因组中单拷贝性。在杂交信号检测中尽管未经过信号放大,但仍然可以观察到清晰的绿色信号;经荧光显微镜观察,在单一的EDF上发现两个相距1μm的SCR和SRK的信号点,由此得出局部分辨率为4kb的最高伸长度。 相似文献
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144.
Vermeulen W Van Troys M Bourry D Dewitte D Rossenu S Goethals M Borremans FA Vandekerckhove J Martins JC Ampe C 《Journal of molecular biology》2006,359(5):1277-1292
The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability. 相似文献
145.
Bhasmas are unique Ayurvedic metallic preparations with herbal juices/fruits, known in the Indian subcontinent since the seventh
century BC and widely recommended for treatment of a variety of chronic ailments. Twenty bhasmas based on calcium, iron, zinc,
mercury, silver, potassium, arsenic, copper, tin, and gemstones were analyzed for up to 18 elements by instrumental neutron
activation analysis, including their C, H, N, and S contents. In addition to the major constituent element found at % level,
several other essential elements such as Na, K, Ca, Mg, V, Mn, Fe, Cu, and Zn have also been found in μg/g amounts and ultratrace
(ng/g) amounts of Au and Co. These seem to remain chelated with organic ligands derived from medicinal herbs. The bhasmas
are biologically produced nanoparticles and are taken along with milk, butter, honey, or ghee (a preparation from milk), thus,
this makes these elements easily assimilable, eliminating their harmful effects and enhancing their biocompatibility. Siddha
Makaradhwaja, a mercury preparation is found to be stoichiometrically HgS without any traces of any other element. Similarly,
Swet Parpati is stoichiometrically KNO3 but is found to have Mn, Cu, Zn, Na, P, and Cl as well. An attempt has been made to correlate the metallic contents with
their medicinal importance. Na and K, the two electrolytic elements, seem to be well correlated, although K/Na varies in a
wide range from 0.06 to 95, with specifically low values for Ca-, Fe-, and Zn-based hasmas. K/P also varies in a wide range
from 0.23 to 12, although for most bhasmas (n=12)., it is 2.3±1.2. Further, Fe/Mn is linearly correlated (r=0.96) with Fe in nine noniron bhasmas. 相似文献
146.
Gussakovsky EE Ionov MV Giller YE Ratner K Aripov TF Shahak Y 《Photosynthesis research》2006,87(3):253-265
Circularly polarized chlorophyll luminescence (CPL) may serve as a measure of chiral macroaggregates of the light-harvesting chlorophyll-protein complexes (LHC II) in both isolated chloroplasts and intact leaves (Gussakovsky et al (2000) Photosynth Res 65: 83–92). In the present work, we applied the CPL approach to study the effect of fast (1–2 min) thermal impacts on LHC II macroaggregates. The results revealed unexpected temperature-response kinetics, composed of initial bell-shaped changes in the CPL signal, followed by degradation down to a steady state (equilibrium). The bell-shape effect was dependent upon illumination, and vanished in the dark. A mathematical analysis of the temperature-response kinetics uniquely indicated that LHC II chiral macroaggregates may persist in both left- and right-handed forms. These forms differ in their response to high temperatures. Both forms are more thermostable in leaves than in isolated chloroplasts. The cooperative degradation of LHC II macroaggregates, which is induced by the thermal impact, is irreversible. It is therefore suggested that the native LHC II macroaggregates are stable, stationary, non-equilibrium, spatially heterogeneous (dissipative) structures. The dissipative properties probably allow the interconversion between left- and right-handed forms under perturbation by certain factors. Illumination probably serves as one such perturbation factor, initiating the interconversion of dark-adapted, left-handed to light-dependent, right-handed LHC II macroaggregates. The chiral heterogeneity of the LHC II macroaggregates is a newly revealed aspect which needs to be taken into consideration in future circular dichroism or CPL studies. 相似文献
147.
Plasmid yield from Acidiphilium strains always had been poor following various standard methods. We adopted some simple modifications in the alkaline lysis procedure to get a better yield of plasmid from these bacteria. An approximately 10- to 20-fold increase in the plasmid yield was achieved when harvested Acidiphilium cells were preincubated 16-20 h at pH 6 in nitrogen-free medium. Another independent approach showed that freezing (-18 to -20 degrees C) of the harvested cells initially and at two subsequent steps in the alkaline lysis procedure of plasmid DNA extraction improved the yield further by 1.5- to 3-fold. The combination of these changes yielded at least 15- to 30-fold more plasmid from various Acidiphilium strains as compared with standard methods. 相似文献
148.
Prelovsek PM Batista U Bulog B 《In vitro cellular & developmental biology. Animal》2006,42(8-9):255-262
Summary In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the
common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron
microscopy and biochemical analyses. A typical perfusion yielded 1.7×105 viable hepatocytes per gram body weight with an average viability of 86±5%. The majority of isolated cells remained in suspension
and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration
and incubation temperature. Viability was best at 8°C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural
characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron
microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics
to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes,
and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity,
as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical
analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture
for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates. 相似文献
149.
Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents. 相似文献
150.
Chia-Chih Lin Nan-Kuang Hsieh Huey Ling Liou Hsing I Chen 《Journal of biomedical science》2012,19(1):27