Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport

The p53 protein is rendered temperature-sensitive by a point mutation. Rat cells transformed by this mutant p53 and an activated ras oncogene grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C. Immunostaining demonstrates that the mutant p53 protein is in the nucleus of the arrested cells at 32 degrees C but in the cytoplasm of the growing cells at 37 degrees C. This is the first example of a protein which is temperature-sensitive for nuclear transport. The translocation from cytoplasm to nucleus and vice versa occurs 6 h after temperature shift and is coincident with the inhibition of DNA synthesis; transport from cytoplasm to nucleus does not require protein synthesis. Remarkably, inhibition of protein synthesis at 37 degrees C also results in the rapid appearance of mutant p53 in the cell nucleus. These results suggest the presence of a short-lived protein responsible for holding p53 in the cytoplasm at 37 degrees C but not at 32 degrees C. Analysis of a non-temperature-sensitive mutant p53 protein shows that its cytoplasmic location is sensitive to protein synthesis inhibitors but not to temperature.     

内质网应激反应与衰老相关疾病

内质网是真核细胞合成膜蛋白和分泌蛋白的主要场所,当细胞经历缺氧、钙离子稳态失衡、糖基化异常或内质网内蛋白合成急剧增加时,就会造成腔内未折叠蛋白聚集体的形成,引发细胞毒性.这时便会激活一系列信号通路,通过增加内质网中分子伴侣的数量、降低蛋白合成速率、加快未折叠蛋白降解来保护细胞,当刺激严重或时间过长则会引起细胞凋亡.这种反应就称为内质网应激反应,也叫未折叠蛋白反应.正常人体细胞随着分裂次数增加或受外界因素诱导逐渐进入一种不可逆的细胞周期阻滞,即细胞衰老.细胞衰老会伴随着各种生理生化的变化,如内质网的结果和功能的改变.内质网应激反应会随着细胞衰老而发生一些改变,与衰老相关疾病密切相关而备受关注.因此深入研究内质网应激反应对于揭示衰老及衰老相关疾病的分子机制具有重要的科学意义.     

Growth regulation of a cellular tumour antigen, p53, in nontransformed cells

Many transformed cells in culture have been found to express elevated levels of a cellular tumour antigen, termed p53. This protein has also been implicated in the regulation of cellular growth. For these reasons experiments were designed to examine the expression of p53 as quiescent cultures of nontransformed 3T3 fibroblasts were stimulated to reenter the cell cycle. Synchronous populations of cells were obtained by releasing a culture from density-dependent inhibition of growth with the addition of fresh serum. Steady-state levels of p53 protein and mRNA were measured as a function of time after addition of serum to quiescent cultures and the rate of synthesis of p53 protein was analysed at a number of time points. The results, reported here, demonstrate an increase in the synthesis and steady-state levels of p53 protein and mRNA prior to DNA synthesis in late G1, and suggest a role for p53 in the progression of cells from a growth-arrested state to an actively dividing state.     

实验性“脾虚”动物胰腺细胞代谢及功能变化的研究

用西宁西大黄水浸煎剂使雄性金黄地鼠泄泻致虚,再用“四君子汤”复健。注射[5-~3H]尿嘧啶核苷或D.L(4,5)~3H亮氨酸分别研究核酸或蛋白的合成功能。表明脾虚动物胰腺合成蛋白质和分泌消化酶功能的下降与脾虚症状的发展相关。“四君子汤”对其有显著改善作用。     

Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis

Leukotrienes, the biologically active metabolites of arachidonic acid, have been implicated in a variety of inflammatory responses, including asthma, arthritis and psoriasis. Recently a compound, MK-886, has been described that blocks the synthesis of leukotrienes in intact activated leukocytes, but has little or no effect on enzymes involved in leukotriene synthesis, including 5-lipoxygenase, in cell-free systems. A membrane protein with a high affinity for MK-886 and possibly representing the cellular target for MK-886 has been isolated from rat and human leukocytes. Here, we report the isolation of a complementary DNA clone encoding the MK-886-binding protein. We also demonstrate that the expression of both the MK-886-binding protein and 5-lipoxygenase is necessary for leukotriene synthesis in intact cells. Because the MK-886-binding protein seems to play a part in activating this enzyme in cells, it is termed the five-lipoxygenase activating protein (FLAP).     

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.     

Inhibition of mRNA binding to ribosomes by localized activation of dsRNA-dependent protein kinase

The initiation of protein synthesis can be regulated in mammalian cells by protein kinases which phosphorylate the alpha subunit of initiation factor eIF-2. This phosphorylation results in a block in the recycling of eIF-2 and in the inhibition of messenger RNA binding to 80S initiation complexes. After eIF-2 alpha is phosphorylated, the mRNA becomes associated with 48S complexes consisting of a 40S ribosomal subunit, eIF-2 (alpha P), GDP and Met-tRNAf. One of the eIF-2 alpha kinases is activated by low concentrations of double-stranded RNA (dsRNA). This kinase (PKds) is present at a basal level in all mammalian cells investigated and its synthesis is induced in cells treated with interferon. The PKds may be involved in the inhibition of translation of viral mRNA in interferon-treated cells infected with RNA viruses, as it is activated by viral replicative complexes. It is not known, however, if the activated PKds preferentially inhibits the translation of viral mRNA when cellular protein synthesis proceeds at a normal rate in infected cells. We now report that mRNA covalently linked to dsRNA is preferentially inhibited from binding to 80S complexes by a localized activation of PKds. This suggests that in interferon-treated cells the binding of some nascent viral mRNAs to functional initiation complexes may be preferentially inhibited by a similar mechanism.     

Competence for genetic transformation in pneumococcus depends on synthesis of a small set of proteins.

In bacterial genetic transformation the uptake of DNA and its integration into the resident chromosome is dependent on a special cellular state, termed competence. In those species where appearance of competence has been studied, specific (but often poorly defined) growth conditions lead to a simultaneous development of competence in a substantial fraction of the cells in a culture. In Bacillus subtilis, and in Haemophilus species, competence appears in the stationary phase of growth or in certain other growth-limiting conditions. Streptococcus pneumoniae (pneumococcus) is perhaps unusual in that virtually all cells of a culture become competent, for a short period at a specific cell density during logarithmic growth, without perturbing the growth rate. The synchronous appearance of competence in pneumococcal cultures results from an autocatalytic effect of a small protein released by the cells that induces competence. The response to competence factor has been shown to require protein synthesis. We report here additional information on the nature of competence in pneumococcus: pulse-labelling studies show that for the brief period of competence protein synthesis is restricted to a few specific polypeptides.     

Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta

Identification of the cellular proteins whose expression is regulated during the cell cycle in normal cells is essential for understanding the mechanisms involved in the control of cell proliferation. A nuclear protein called cyclin of relative molecular mass 36,000 (Mr 36K), whose synthesis correlates with the proliferative state of the cell, has been identified in several cell types of human, mouse, hamster and avian origin. The rate of cyclin synthesis is very low in quiescent cells and increases several fold after serum stimulation shortly before DNA synthesis. Immunofluorescence and autoradiography studies have shown that the nuclear staining patterns of cyclin during S phase have a sequential order of appearance and a clear correlation can be found between DNA synthesis and cyclin positive nuclei. The proliferating cell nuclear antigen (PCNA) and cyclin have many common properties and it has been shown that these two are identical. Recently a protein which is required by DNA polymerase-delta for its catalytic activity with templates having low primer/template ratios has been isolated from calf thymus. We report here that cyclin and the auxiliary protein of DNA polymerase-delta are identical.     

冠突伪尾柱虫接合生殖DNA,RNA和蛋白质的合成

利用放射自显影术对冠突伪尾柱虫接合生殖期间其体内大分子合成进行研究，结果表明，接合体内除第二次成熟分裂外，其它各次核分裂之前都须进行一次ＤＮＡ合成．在大核原基发育过程中，其ＤＮＡ合成明显分为前后两个阶段．在接合体的老大核内持续进行低度的ＲＮＡ合成，并于接合后体分离时突然终止．４ｄ以后，数枚新的大核才出现活跃的转录．第二次皮膜改组启动之前，细胞内的蛋白质合成速率较低，其后显著增强．早期合成的ＲＮＡ以及蛋白质都参与了大核原基的发育．     

Accumulation of a heat shock-like protein during differentiation of human erythroid cell line K562

The human erythroid cell line K562 provides a model system for studying erythroid differentiation and eukaryotic gene regulation. These cells express glycophorin A, spectrin and i antigen. They accumulate embryonic and fetal haemoglobins on induction of erythroid differentiation with haemin, sodium butyrate or hydroxyurea. In the present study, the protein composition of K562 cells during haemin-mediated induction of erythroid maturation was analysed by two-dimensional gel electrophoresis. Under conditions in which haemin did not effect cell viability and proliferation, a protein of approximately 70,000 molecular weight (MW) accumulated in the differentiated K562 cells. The accumulation appears to be due to an increase in the rate of RNA synthesis for this protein. The protein is related in sequence to a 70,000-MW heat shock protein. An antigenically related protein was also demonstrated in human bone marrow and accumulates at particular stages of human erythroid maturation.     

Identification of an actin-binding protein from Dictyostelium as elongation factor 1a

Indirect evidence has implicated an interaction between the cytoskeleton and the protein synthetic machinery. Two recent reports have linked the elongation factor 1a (EF-1a) which is involved in protein synthesis, with the microtubular cytoskeleton. In situ hybridization has, however, revealed that the messages for certain cytoskeletal proteins are preferentially associated with actin filaments. ABP-50 is an abundant actin filament bundling protein of native relative molecular mass 50,000 (50K) isolated from Dictyostelium discoideum. Immunofluorescence studies show that ABP-50 is present in filopodia and other cortical regions that contain actin filament bundles. In addition, ABP-50 binds to monomeric actin in the cytosol of unstimulated cells and the association of ABP-50 with the actin cytoskeleton is regulated during chemotaxis. Through complementary DNA sequencing and subsequent functional analysis, we have identified ABP-50 as D. discoideum EF-1a. The ability of EF-1a to bind reversibly to the actin cytoskeleton upon stimulation could provide a mechanism for spatially and temporally regulated protein synthesis in eukaryotic cells.     

基于生物细胞调控合成无机纳米材料的研究进展

细胞作为生物体结构和功能的基本单位,其内部可进行成千上万的生化反应.利用生物体细胞来调控合成纳米材料是一种新型的绿色合成方法,有着传统方法不可比拟的优点:如原料来源广、低毒、低能耗、反应温和可控、效率高,环境友好等.利用细胞调控合成得到的无机纳米材料富含蛋白质、脂类、多糖等生物物质,不仅可以避免颗粒间的团聚,而且具有独特的生物相容性.本文综述了生物细胞内外无机纳米材料的可控合成研究进展,并展望了基于细胞合成无机纳米材料的发展前景.     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.     

Formation of proinsulin by immobilized Bacillus subtilis

There has been an increasing interest in the use of immobilized cells for the production of pharmaceuticals as well as for products such as high fructose syrup or ethanol. Some of these compounds are now produced on an industrial scale whereby the cells are used in a resting or growing state or in a nonviable form as natural carriers of the enzyme(s) involved in the synthesis. The advantages of immobilized cell technology should also apply to microorganisms modified by recombinant DNA techniques to produce a variety of eukaryotic proteins such as hormones. We describe here the properties of immobilized Bacillus subtilis cells carrying plasmids encoding rat proinsulin. Cell proliferation normally coupled to DNA replication is undesirable in immobilized cell systems as "clogging' of the system occurs due to cells growing outside the beads. Therefore, different ways were investigated to inhibit cell division while allowing continued protein synthesis. We found that the addition of certain antibiotics in the growth medium, such as novobiocin which inhibits DNA replication, fulfills these requirements, allowing proinsulin synthesis and excretion to take place over a period of several days.     

Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.     

Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

Functional impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000, which inhibits replication of certain plant RNA viruses, and of herpes simplex virus, poliovirus and influenza virus. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CD5, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.