Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与HSA相互作用的~(23)(Na)-NMR研究

 本文应用~23Na-NMR波谱技术,研究了Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与人体血清白蛋白(HSA)的相互作用。在实验基础上,通过引入两位快交换模型,拟合计算获得了Na~(+)与HSA相互作用的结合常数和处于结合状态Na~(+)的相关时间;实验表明Ca~(2+)能与Na~(+)竞争同HSA结合,拟合计算获得了两者与HSA相互作用结合常数的比值,棕榈酸钠能增强Ca~(2+)同Na~(+)竞争与HSA结合的能力;从实验上未能观察到Cu~(2+)、Zn~(2+)能同Na~(+)竞争与HSA相互作用的证据。     

Ultrastructural localization of silver-staining nuclear proteins at the onset of transcription in early bovine embryos.

Argyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four-, eight-, and 16-cell bovine embryo blastomere nuclei using two different silver-staining procedures. Within the eight-cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full-capacity ribosome-producing machinery is established. In the four-cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus-precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear-cut localization of argyrophilic proteins was detected during the eight-cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus-associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16-cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver-staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.     

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs ARS1 and CENIII, showed that the HSC80 5MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5 MAR can outcompete the 3 MAR and not vice versa. Last, we observed that the interaction of the 3 MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.     

蓝氏贾第虫核被膜缺口的电镜观寨

双滴虫类是迄今所知的现存最原始的真核生物类群。以蓝氏贾第虫作为双滴虫类的代表,对其细胞核进行了电镜观察。除了未见有核仁外,还发现其核被膜的横切面上存在有缺口。在缺口的边缘处,核内膜与校外膜是相互连接着的,表明并非切片时所造成的假象。核被膜缺口处常有一核纤层样的薄层分隔核质与细胞质。用高锰酸钾固定细胞以求只保存膜结构时,核被膜缺口仍然可见,上述的薄层即未见到。核被膜缺口的发现证实了李靖炎（１９７９）的核被膜起源假说所作出的推断。     

31P nuclear magnetic resonance study on changes in phosphocreatine and the intracellular pH in rat skeletal muscle during exercise at various inspired oxygen contents

We measured ATP, phosphocreatine (PCr), inorganic phosphate (P_i), and the intracellular pH in rat hindlimb muscles during submaximal isometric exercise with various O₂ deliveries using³¹P nuclear magnetic resonance spectroscopy (³¹P NMR) to evaluate changes in energy metabolism in relation to O₂ availability. Delivery of O₂ to muscles was altered by controlling the fractional concentration of inspired oxygen (F _IO₂) at 0.50, 0.28, 0.21, 0.11 and 0.08 with monitoring partial pressure of oxygen and carbon dioxide, and bicarbonate at the femoral artery. The steady-state ratio of PCr : (PCr + P_i) during exercise decreased as a function ofF _IO₂ even at 0.21. Significant acidification of the intracellular pH during exercise occurred at 0.08F _IO₂. Change in the PCr : (PCr + P_i) ratio demonstrated that the oxidative capacity, i.e. the maximal rate of the oxidative phosphorylation reaction, in muscle was not limited by O₂ delivery at 0.50F _IO₂, but was significantly limited at 0.21F _IO₂ or below. Change in the intracellular pH at 0.08F _IO₂ could be interpreted as an increase in lactate, suggesting activation of glycolysis. Correlation between the PCr : (PCr + P_i) ratio and the intracellular pH revealed the existence of a critical PCr : (PCr + P_i) ratio and pH for glycolysis activation at around 0.4 and 6.7, respectively.     

Phylogenetic reticulation in subtribe Helianthinae

Incongruence between phylogenetic estimates based on nuclear and chloroplast DNA (cpDNA) markers was used to infer that there have been at least two instances of chloroplast transfer, presumably through wide hybridization, in subtribe Helianthinae. One instance involved Simsia dombeyana, which exhibited a cpDNA restriction site phenotype that was markedly divergent from all of the other species of the genus that were surveyed but that matched the restriction site pattern previously reported for South American species of Viguiera. In contrast, analysis of sequence data from the nuclear ribosomal DNA internal transcribed spacer (ITS) region showed Simsia to be entirely monophyletic and placed samples of S. dombeyana as the sister group to the relatively derived S. foetida, a result concordant with morphological information. A sample of a South American species of Viguiera was placed by ITS sequence data as the sister group to a member of V. subg. Amphilepis, which was consistent with cpDNA restriction site data. Samples of Tithonia formed a single monophyletic clade based on ITS sequence data, whereas they were split between two divergent clades based on cpDNA restriction site analysis. The results suggested that cpDNA transfer has occurred between taxa diverged to the level of morphologically distinct genera, and highlight the need for careful and complete assessment of molecular data as a source of phylogenetic information.     

重组人GM—CSF基因在昆虫细胞中的表达

利用苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）带β－Ｇａｌａｃｔｏｓｉｄａｓｅ基因标记的非融合蛋白基因转移载体ｐＢｌｕｅＢａｃ将人粒细胞巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）基因成功地插入病毒ＡｃＮＰＶ的基因组中．ｈＧＭ－ＣＳＦ基因在感染重组病毒的草地夜蛾（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）培养细胞Ｓｆ９中得到表达,感染后的Ｓｆ９细胞培养液能刺激人骨髓细胞在体外形成典型的集落,表达水平可达２．７×１０５５ＣＦＵ／ｍｌ。以ｈＧＭ－ＣＳＦ单抗所作的ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ表明,表达的ｈＧＭ－ＣＳＦ对是３种糖基化程度不同的产物,分子量分别约为１５ｋｄ,１８ｋｄ和２０ｋｄ。     

In Vitro Culture of Phytomonas Sp. Isolated from Euphorbia characias. Metabolic Studies by 1H NMR

ABSTRACT. We describe the in vitro culture of Phytomonas species isolated from Euphorbia characias . The best choice between tested media was SDM-79, in which promastigotes, after 6 days of culture, reached cell densities as high as 4 × 10⁷ cells/ml. Cells growing in LIT or MTL medium showed longer division times and lower cell densities. We succeeded in obtaining Phytomonas sp. amastigote and spheromastigote forms in modified GRACE's medium, yielding transformation rates of up to 70%. Electron microscopy studies were performed in order to characterize the ultrastructural features of these forms obtained in vitro. On the other hand, metabolic studies based on qualitative (nuclear magnetic resonance spectroscopy) and quantitative metabolic methods (enzymatic assays) showed that promastigote forms secreted mainly ethanol, acetate, glycine, glycerol, piruvate and succinate in SDM-79 medium, whereas the major metabolites found after transformation in modified Grace's medium were ethanol, acetate, glycine, piruvate and smaller amounts of glycerol.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.