The molecular basis for genetic polymorphism of human deoxyribonuclease I: identification of the nucleotide substitution that generates the fourth allele

AbstractIn addition to the three alleles commonly responsible for the protein polymorphism of human deoxyribonuclease I, a mutation encoded by a fourth allele, DNASEI*4, was detected by isoelectric focusing. All 8 exons covering the entire open reading frame of the human DNase I gene were amplified by the polymerase chain reaction and subjected to direct sequencing. Only one nucleotide substitution, a C-to-G transition (CAG → GAG), in the codon for amino acid 9 of the mature enzyme was found. This substitution resulted in the replacement of Gln with Glu (Q9E)

Structural change of ribosomes during apoptosis: Degradation and externalization of ribosomal proteins in doxorubicin-treated Jurkat cells

金沢大学医薬保健研究域薬学系Changes in the amount and localization of human ribosomal proteins during apoptosis were determined. When total lysates of Jurkat cells undergoing apoptosis induced by doxorubicin were analyzed by Western blotting, degradation of three ribosomal proteins, S18, L5, and L14, was detected at 48 h after the induction of apoptosis. Decreases in the amounts of these three ribosomal proteins were also observed in ribosome-enriched fractions. These changes were partly abolished by the addition of the pan-caspase inhibitor z-VAD-fmk. Moreover, formation of the 80S ribosome complex appeared to be inhibited at 48 h after apoptosis induction. On the other hand, the rate of protein synthesis, assessed by measuring the incorporation of [35S]Met into bulk proteins, decreased as early as 12 h after the addition of doxorubicin. These results indicate that changes in the amount of ribosomal proteins and the overall structure of ribosomes in apoptosing cells occur after protein synthesis declines. Finally, analyses by flow cytometry, immunofluorescence, and Western blotting showed that six ribosomal proteins, S15, PO, L5, L6, L36a, and L41, were relocalized and expressed at the cell surface during apoptosis. The above results collectively indicate that ribosomes are structurally altered in apoptotic cells following inactivation of protein synthesis

Characterization of human Ccser2 as a protein tracking the plus-ends of microtubules

Abstract Objective Microtubules, which are closely related to cell proliferation, have been the promising therapeutic target of cancer. Therefore, it is necessary to understand the intracellular control mechanisms of microtubules, the whole picture of which is still unclear though. Intracellular dynamics of microtubules are regulated by various microtubule-associated proteins, one group of which is microtubule plus-end-tracking proteins (+ TIPs), localizing to the extending tips of microtubules. Here, we report the identification and analysis of Ccser2 as a new + TIP in human breast cancer MCF-7 cells. Results Ccser2 was found to be a member of + TIPs by microscopic observations including time-lapse imaging. The C-terminal region of Ccser2, including two SxIP motifs, was likely to be important for the tracking function. In MCF-7 cells, endogenous Ccser2 was mainly detected in the peripheral regions of microtubule fibers, suggesting that Ccser2 functions in cell projections

The murine Gcap14 gene encodes a novel microtubule binding and bundling protein

AbstractMicrotubules form flexible fibers, which are utilized in cell proliferation and differentiation. Although the flexibility of microtubules was shown to be regulated by various microtubule-associated proteins, this regulation is still far from complete understanding. Here, we report a new potential regulator of microtubules in mammals. Gcap14 colocalizes with microtubules in mammalian cells transfected with Gcap14 expression vector. Association of Gcap14 with microtubules was confirmed by biochemical subcellular fractionation. Recombinant Gcap14 protein cosedimented with pure microtubules, indicating a direct binding between the two. Furthermore, recombinant Gcap14 was shown to have the ability of inducing microtubule bundling in vitro.Structured summary of protein interactionsGcap14 physically interacts with Gcap14 by anti tag coimmunoprecipitation (View Interaction: 1, 2

Establishment of a mammary stromal fibroblastic cell line for in vitro studies in mice of mammary adipocyte differentiation

Mammary stromal adipose tissue remodeling is important for appropriate mammary gland development during pregnancy, lactation, and involution. However, the precise mechanisms underlying mammary stromal adipose tissue remodeling remain unclear. We have established a mammary stromal, fibroblastlike cell line (MSF) from primary mouse mammary culture by introducing a temperature-sensitive simian virus-40 large tumor antigen. Among several hormones related to mammary gland development, hydrocortisone was found to commit MSF cells to a preadipocyte lineage, whereas insulin was found to induce extracellular matrix-dependent adipogenic differentiation of the cells, as assessed by lipid accumulation and marker gene expression. Interestingly, such hormone-induced adipogenic differentiation of MSF cells, but not 3T3-L1 cells, was suppressed by prolactin through its receptor and downstream STAT5. Furthermore, coculture of MSF cells with mammary epithelial HC11 cells and culture in HC11-conditioned medium also suppressed adipogenic differentiation of MSF cells. We have demonstrated that adipogenic differentiation of at least some populations of mammary stromal cells is modulated by lactogenic hormones and humoral factors from epithelial cells, suggesting that the response of these mammary cells may differ from adipocytes at other sites. We believe that the MSF cell line will prove a useful model to elucidate mammary stromal adipose development in vitro as well as represent an important first step toward developing stable adipocyte cell lines that faithfully represent their site of origin