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.     

Receptor binding and internalization of immobilized transcobalamin II by mouse leukaemia cells

Membrane transport of vitamin B12 (cyanocobalamin; Cbl) into mammalian cells is mediated by the serum protein transcobalamin II (TCII). In mouse leukaemia L1210 cells, TCII-Cbl binds to membrane receptors in a rapid, temperature-independent step and is internalized by a slow, temperature-dependent process. To delineate the location of receptors on these cells, we have constructed a visual probe by covalently coupling purified TCII-Cbl to submicrometre latex particles (minibeads). We report here that when L1210 cells are incubated with minibeads containing TCII-Cbl at 4 degrees C and examined by scanning electron microscopy (SEM), the particles are found attached predominantly to microvilli. Incubation of the cells at 37 degrees C results in the internalization of the minibeads. As visualized by transmission electron microscopy (TEM), this endocytotic process seems to occur in clathrin-coated pits and vesicles at the cell surface.     

Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine

The Abelson murine leukaemia virus protein (P120) can become phosphorylated in vitro by [gamma-32P]ATP. The protein has been purified from cell membranes to the point that in specific conditions virtually all of the incorporated 32P is in P120. The reaction is stimulated by Mn2+ and Mg2+ but not Ca2+ and is very rapid even at 0 degrees C. The phosphate is linked to P120 at tyrosine, a linkage not previously reported for a phosphorylation reaction. Phosphorylation may be involved in the transforming activity of viruses that cause leukaemia as well as sarcomas.     

Cell-cycle, cell-shape mutant with features of the Go state.

A cold-sensitive mutant of CHO cells has features of "reverse transformation" at the non-premissive temperature of 33 degrees C. Cells accumulate at G1 with altered morphology and remain viable and quiescent for more than 40 d. Such cultures are synchronised by a temperature shift back to the permissive 39 degrees C.     

Expression of endogenous murine leukaemia viruses in AKR/J streaker mice

MICE of the AKR/J strain show high rates of spontaneous thymic lymphoma and are chronically infected with murine leukaemia viruses. Total thymectomy in young mice of this strain results in a marked decrease in the incidence of leukaemia. The finding of athymic mutant mice on the AKR/J background (referred to as streaker mice) is of importance in the study of genetic factors which influence virus expression and leukaemogenesis. Here we report that mice of both normal and mutant genotypes vary in the expression of a particular class of murine leukaemia virus. This lower virus expression in conjunction with the absence of a thymus leads to a lower tumour incidence in streaker mice.     

Empty MHC class I molecules come out in the cold

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.     

BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.     

Effect of temperature on batch elastase production by Bacillus sp. EL31410

The production of elastase by Bacillus sp. EL31410 at various temperatures was investigated. In order to study the effect of temperature on elastase fermentation, different cultivation temperatures, ranging from 39℃ to 28℃, were evaluated in shake flask. The result indicated that 37℃ was best for cell growth at earlier stage; while maximum elastase activity was obtained when the cells were cultivated at 30℃. This result was verified by batch fermentation in 5-L bioreactor under 37 ℃ and 30 ℃ temperature, respectively. The specific cell growth rate at 37 ~C was higher than that at 30 ℃ during earlier stage of cultivation. The maximum value [5.5 U/(h-g DCW)] of elastase formation rate occurred at 24 h at 30℃ compared to 4.6 U/(h-g DCW) at 30 h at 37℃. Based on these results, two-stage temperature shift strategy and oscillatory temperature cultivation mode were evaluated in the next study. When compared to single temperature of 37 ℃ or 30℃, both two-stage temperature shift strategy and oscillatory temperature strategy improved biomass but did not yield the same result as expected for elastase production. The maximum biomass (both 8.6 g/L) was achieved at 30 h at 37℃, but at 42 h using two-stage temperature cultivation strategy. The highest elastase production (652 U/ml) was observed at 30℃ in batch process. It was concluded that cultivation at constant temperature of 30℃ was appropriate for elastase production by Bacillus sp. EL31410.     

Interhemispheric climate links revealed by late-glacial cooling episode in southern Chile

Understanding the relative timings of climate events in the Northern and Southern hemispheres is a prerequisite for determining the causes of abrupt climate changes. But climate records from the Patagonian Andes and New Zealand for the period of transition from glacial to interglacial conditions--about 14.6-10 kyr before present, as determined by radiocarbon dating--show varying degrees of correlation with similar records from the Northern Hemisphere. It is necessary to resolve these apparent discrepancies in order to be able to assess the relative roles of Northern Hemisphere ice sheets and oceanic, atmospheric and astronomical influences in initiating climate change in the late-glacial period. Here we report pollen records from three sites in the Lake District of southern Chile (41 degrees S) from which we infer conditions similar to modern climate between about 13 and 12.2 14C kyr before present (BP), followed by cooling events at about 12.2 and 11.4 14C kyr BP, and then by a warming at about 9.8 14C kyr BP. These events were nearly synchronous with important palaeoclimate changes recorded in the North Atlantic region, supporting the idea that interhemispheric linkage through the atmosphere was the primary control on climate during the last deglaciation. In other regions of the Southern Hemisphere, where climate events are not in phase with those in the Northern Hemisphere, local oceanic influences may have counteracted the effects that propagated through the atmosphere.     

谷子抽穗期突变体jt1242-14b的遗传分析和定位

为鉴定控制谷子抽穗的关键基因,通过甲基磺酸乙酯(ethyl methyl sulfonate,EMS)诱变谷子参考基因组测序品种豫谷1号,获得遗传稳定的谷子超早抽穗突变体jt1242-14b.与豫谷1号相比,jt1242-14b抽穗期明显提前,茎秆变细,叶片变窄、变短.遗传分析表明,突变性状受隐性单基因控制.以SSR41(父本)、jt1242-14b(母本)和F₂群体进行突变基因定位,结果表明,该基因位于第9号染色体,在分子标记In4746和In9-11之间的4 447 kb之内.进一步测序比对分析发现突变位点位于PHYB基因内部,因此,PHYB很可能是该早熟突变体的目标基因.本研究为谷子早抽穗基因克隆及PHYB基因功能研究提供材料基础.     

一株谷氨酰胺合成酶高产菌株的酶学分析及其基因的克隆分析

对谷氨酰胺合成酶菌球形节杆菌(Arthrobacter globiformis)突变菌株Y3进行分析.其谷氨酰胺合成酶摇瓶发酵单位是原始菌株的2.81倍;最适反应温度为40℃;最适pH为6.5.以突变菌株和原始菌株基因组为模板,通过同源克隆策略,成功地获得了球形节杆菌谷氨酰胺合成酶基因gln片段.通过序列比对,突变菌株gln基因在第409 bp处产生T→C的碱基的突变,该处突变使酪氨酸(Tyr409)被组氨酸(His409)取代,该突变解决了谷氨酰胺合成酶腺苷酰基化问题,保证了其在高浓度铵盐条件下保持酶活性.     

Change in the body temperature of healthy term infant over the first 72 hours of life

OBJECTIVE: To determine the range of body temperature in a group of healthy Chinese term neonates over the first 72 hours of life and to assess the influence of body weight, gestational age and route of delivery.METHOD: All 200 consecutive cases of neonates delivered at our hospital from March to August 2001 were included in this retrospective study. Temperatures were measured immediately after delivery, after 30 minutes, 1 hour, 2 hours, 8 hours and 15 hours and on the 2nd and 3rd day. Axillary temperatures ranging from 36.5 degrees C to 37 degrees C were regarded as normal. No cases of maternal fever or systemic infection of the newborns were discovered. All infants were discharged in good general condition. RESULTS: The mean rectal temperature at birth was 37.19 degrees C. The lowest average temperature was reached at 1 hour after delivery (36.54 degrees C) with a significant difference between natural delivery (36.48 degrees C) and section (36.59 degrees C) (P<0.05). Temperature subsequently rose to 36.70 degrees C at 8 hours and 36.78 degrees C at 15 hours (P<0.05). Hypothermia was seen in 51.8% and hypothermia in 42.5% of the patients. On the 3rd day after delivery, 96% of all temperatures were in the normal range. A significant relation was found between hypothermia and both low birth weight (P<0.001) and low gestational age (P<0.05). CONCLUSION: The reference range presently used did not include all physiological temperatures in the first 72 hours of life. Considering other factors, such as birth weight, route of delivery, gestational age and body temperature on the 2nd and 3rd day of life, may help to correctly assess the significance of temperatures beyond the reference range.     

人血清核磁共振谱在白血病临床研究上的应用

运用核磁共振波谱（NMRS）研究血清中磷脂、脂蛋白及乳酸的代谢变化，并探讨该法在白血病临床研究上的应用和可行性。实验采用MSL-300MHz型超导核磁共振谱仪测量白血病人及健康人血清标本，通过^31P-NMRS分析比较血清中磷脂酰胆碱（PC）信号及磷脂酰乙醇胺和（神经）鞘磷脂（PE SM）信号相对积分面积，了解各自血清中的磷脂含量水平。并对血清^1H-NMRS中甲基和亚甲基峰进行线性拟合，从而分析血清中各种脂蛋白及乳酸含量的变化。白血病人血清中的磷脂信号强度相对正常人明显更低，反映了对应的PC及（PE SM）含量相对正常人更低，尤其对初发未治疗的病人，差别更加明显。病人血清中各种脂蛋白含量也与正常人存在差异，且乳酸水平明显高于正常人。该方法较好地从分子水平反映了白血病人与正常人血清中磷脂、脂蛋白及乳酸水平的差异，为白血病临床研究提供了一个简便、直观的新方法，值得进一步研究。     

Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.     

Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor

Retrovirus protease is an enzyme that cleaves gag and gag-pol precursor polyproteins into the functional proteins of mature virus particles. The correct processing of precursor polyproteins is necessary for the infectivity of virus particles: in vitro mutagenesis which introduces deletions into the murine leukaemia virus genome produces a protease-defective virus of immature core form and lacking infectivity. A therapeutic drug effective against disease caused by retrovirus proliferation could likewise interfere with virus maturation. The primary structure has so far been determined for the protease of avian myeloblastosis virus, and of murine, feline and bovine leukaemia viruses. Amino acid sequencing of the retrovirus proteases, either after their purification or from prediction from the nucleotide sequence, shows that they possess the Asp-Thr-Gly sequence characteristic of the aspartyl proteinases. In this report we show that retrovirus proteases belong to the aspartyl proteinase group and demonstrate an inhibition by the aspartyl proteinase-specific inhibitor, pepstatin A, on the activity of bovine leukaemia, Moloney murine leukaemia and human T-cell leukaemia virus proteases.     

Serine phosphorylation-independent downregulation of cell-surface CD4 by nef

A decline in the T-cell population usually marks the onset of progressive immunological disease in individuals infected with the human immunodeficiency virus (HIV). Because CD4+ cells help to coordinate efficient immune responses, some of the defects in the immune function in advanced cases of AIDS may be explained by the disappearance of these cells. Therefore, an understanding of the mechanisms used by HIV to induce the reduction of CD4+ cells is important. Here we use a Moloney murine leukaemia virus-based retroviral vector in order to express the nef gene of HIV-1 in three lymphocytic cell lines expressing CD4. In all cases we find that cell-surface CD4 expression is inversely related to nef expression. However, nef does not alter steady-state levels of CD4 RNA or CD4 protein. Also, nef can downregulate a CD4 triple mutant (Ser----Ala) that is neither phosphorylated nor down-regulated by phorbol esters, indicating that nef is acting by a different mechanism.