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.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.     

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。     

Keratinocyte Extracellular Matrix-Mediated Regulation of Normal Human Melanocyte Functions

Active roles of cell-cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell-cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation-associated protein (MEL-5) and proliferation-associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell-matrix interactions with those of direct melanocyte-keratinocyte, cell-cell contact in co-culture on melanocyte functions. Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs. Expression of pigmentation-associated protein detected by monoclonal antibody, MEL-5, was not changed on the kECM, while it was increased in melanocytes in co-culture with keratinocytes. Expression of PCNA/cyclin in melanocytes cultured on kECMs was generally downregulated on kECM and in co-culture compared to that in a control culture. We demonstrated that the kECMs play important roles in the melanocyte morphology and proliferation. These observations suggest that environmental (UVB) and physiological (Ca⁺⁺) stimuli can regulate melanocyte functions through the keratinocyte extracellular matrix in vivo.     

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.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.