Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Genomic organization of the rat aromatic L-amino acid decarboxylase (AADC) locus: Partial analysis reveals divergence from the Drosophila dopa decarboxylase (DDC) gene structure

Aromatic L-amino acid decarboxylase (AADC) is responsible for the conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are important neurotransmitters. We characterized genomic clones derived from the rat AADC locus by Southern blot and nucleotide sequencing analyses to explore the exonal organization of the gene. Our results suggest that the rat AADC gene is relatively large, containing at least 12 exons and spanning at least 40 kb in the rat genome. In this study, nine exons corresponding to 71% of the published cDNA sequence were identified, the smallest of which was as short as 20 base pairs (bp). In the Drosophila dopa decarboxylase (DDC) gene, the sequences homologous to these nine exons are all present in the fourth exon. This implies that either multiple intron sequences have been added to the vertebrate AADC gene or alternatively, deleted from the invertebrate gene after the divergence of vertebrates and invertebrates during evolution.     

Maternal Transfer and Protective Role of the Alternative Complement Components in Zebrafish Danio rerio

Embryos of most fish develop externally and are exposed to an aquatic environment full of potential pathogens, whereas they have little or only limited ability to mount an efficient and protective response. How fish embryos survive pathogenic attacks remains poorly defined. Here we demonstrate that the maternal immunization of female zebrafish with formalin-killed Aeromonas hydrophila causes a significant increase in C3 and Bf contents in the mother, a corresponding rise in the offspring, and induces a remarkable increase in the hemolytic activities in both the mother and offspring. In addition, the embryos derived from the immunized mother are significantly more tolerant to A. hydrophila challenge than those from the unimmunized fish, and blocking C3 and Bf activities by injection of the antibodies against C3 and Bf into the embryos render them more susceptible to A. hydrophila. These results clearly show that the protection of zebrafish embryos against A. hydrophila can be achieved by the maternally-transferred immunity of the complement system operating via the alternative pathway. This appears to be the first report providing in vivo evidences for the protective role of the alternative complement components in the early embryos of zebrafish, paving the way for insights into the in vivo function of other maternally-transferred factors in fish.     

人体小卫星DNA探针的制备

根据人体小卫星DNA核心顺序,化学合成长23碱基寡核苷酸探针,筛选人体基因组文库,旨在获得能用作遗传分析探针的小卫星顺序。结果得到15个含小卫星的阳性重组子。随机取其一(C_(35.9))作探针,试做群体分析。所有个体均可检出多条杂交带。其中某些带具有多态性。在一定检测条件下,检出的DNA图谱在有限的个体内具有个体特异性。结果表明筛选文库得到的小卫星顺序可用于小卫星多态性的检测。其它小卫星探针的筛选和应用性研究正在进行。     

Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37.

A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.     

血友病患者血清中淋巴腺病病毒/人T细胞Ⅲ型病毒抗体检测

对浙江省1982～1984年注射了美国产浓缩Ⅶ因子制剂的18例血友病患者,用酶联免疫吸附法(ELTSA)检测了血清中淋巴腺病病毒/人T细胞Ⅲ型病毒(LAV/HTLV-Ⅲ)抗体,发现4例阳性,并经免疫荧光试验和Western印迹法证实。2例应用了国产浓缩Ⅷ因子者抗体阴性。一例从美国来华旅游死于艾滋病者,LAV/HTLV-Ⅲ抗体阳性。本研究证明,LAV/HTLVⅢ病毒巳通过美国生产的Ⅷ因子制剂传入中国。     

The effect of metabolic acidosis on the synthesis and turnover of rat renal phosphate-dependent glutaminase.

Regulation of the mitochondrial phosphate-dependent glutaminase activity is an essential component in the control of renal ammoniagenesis. Alterations in acid-base balance significantly affect the amount of the glutaminase that is present in rat kidney, but not in brain or small intestine. The relative rates of glutaminase synthesis were determined by comparing the amount of [35S]methionine incorporated into specific immunoprecipitates with that incorporated into total protein. In a normal animal, the rate of glutaminase synthesis constitutes 0.04% of the total protein synthesis. After 7 days of metabolic acidosis, the renal glutaminase activity is increased to a value that is 5-fold greater than normal. During onset of acidosis, the relative rate of synthesis increases more rapidly than the appearance of increased glutaminase activity. The increased rate of synthesis reaches a plateau within 5 days at a value that is 5.3-fold greater than normal. Recovery from chronic acidosis causes a rapid decrease in the relative rate of glutaminase synthesis, but a gradual decrease in glutaminase activity. The former returns to normal within 2 days, whereas the latter requires 11 days. The apparent half-time for glutaminase degradation was found to be 5.1 days and 4.7 days for normal and acidotic rats respectively. These results indicate that the increase in renal glutaminase activity associated with metabolic acidosis is due primarily to an increase in its rate of synthesis. From the decrease in activity that occurs upon recovery from acidosis, the true half-life for the glutaminase was estimated to be 3 days.