Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Specific eradication of micrometastases by transfer of tumour-immune T cells from major-histocompatibility-complex congenic mice

Summary DBA/2 (H-2^d) mice bearing a transplanted highly metastatic lymphoma (ESb) in a state of widely disseminated disease could be successfully treated by a combination of surgery (removal of the local tumour), irradiation (5 Gy) and adoptive immunotherapy. The immunotherapy was achieved by transfer of anti-ESb-immune spleen cells from B10.D2 mice, which express the same major histocompatibility complex (MHC) molecules as DBA/2. In contrast, anti-ESb-immune cells from MHC-disparate C57BL/6 mice did not confer protective immunity. The B10.D2 anti-ESb-immune T cells contain two types of cytolytic specificity as detected by limiting-dilution analysis: (1) clones with specificity for the ESb-tumour-associated transplantation antigen (TATA) (at low frequency), and (b) clones with specificity for minor DBA/2 histocompatibility (H) antigens (at high frequency). Immune B10.D2 cells raised against different tumour lines or against TATA^– ESb tumour variants did not confer the 100% protection seen with immune cells against ESb TATA⁺ cells. Finally we demonstrate that the allogeneic immune cells are more potent in terms of protective immunity than corresponding syngeneic immune cells. The data suggest that the strong graft-versus-leukemia effect with immune T cells from allogeneic MHC-identical but not from MHC-disparate mice was due to T cells with MHC-restricted specificity for an ESb-associated TATA. A graft-versus-host reactivity that developed much later and could not be prevented was most likely due to T cells sensitized against normal minor H antigens of the host. Our results are of potential relevance for allogeneic bone marrow transplantation and adoptive immunotherapy protocols.     

The glycoprotein 71 of ecotropic Friend murine leukemia virus. Structure of the oligosaccharides linked to asparagine-12

The glycoprotein from Friend murine leukemia virus was digested with protease from Staphylococcus aureus V8. A glycopeptide comprising the N-terminal glycosylation site (Asn-12) was isolated from the mixture of fragments and analyzed by amino acid sequencing and methylation-capillary gas chromatography-mass spectrometry before and after treatment with sialidase from Vibrio cholerae. Asn-12 was thus found to be substituted by a family of partially sialylated, fucosylated, and intersected glycoprotein N-glycans of the hybrid type.     

人白血病细胞癌性促凝物(CP)的分离纯化及其特性的初步研究

用三步纯化法从人M_3型白血病细胞中分离纯化出人类肿瘤癌性促凝物(CP)。促凝活性回收率为24％,CP纯化倍数为2481倍。纯化CP在SDS-PAGE上为单一区带,其理化和酶学特性类似于动物肿瘤CP,分子量约为70 000,PI为4.8,在FVⅡ缺乏血浆中以及在含有组织因子(TF)抑制剂情况下仍能激活FX。CP促凝活性能被半胱氨酸蛋白酶抑制剂HgCl_2抑制,纯化CP能与抗动物肿瘤CP抗体形成免疫沉淀反应。     

The extinction of slowly evolving dynamical systems

The time evolution of slowly evolving discrete dynamical systems x _{i + 1} = T(r _i ,x _i ), defined on an interval [0, L], where a parameter r _ichanges slowly with respect to i is considered. For certain transformations T, once r _i reaches a critical value the system faces a non-zero probability of extinction because some x _j [0, L]. Recent ergodic theory results of Ruelle, Pianigiani, and Lasota and Yorke are used to derive a simple expression for the probability of survival of these systems. The extinction process is illustrated with two examples. One is the quadratic map, T(r, x) = rx(1 – x), and the second is a simple model for the growth of a cellular population. The survival statistics for chronic myelogenous leukemia patients are discussed in light of these extinction processes. Two other dynamical processes of biological importance, to which our results are applicable, are mentioned.     

Effect of phospholipid vesicles on the activity of terminal transferase.

The terminal transferase activity is modified in the presence of lipid vesicles. A deep inhibitory effect takes place with phosphatidylserine and phosphatidylinositol, while some stimulation is present with sphingomyelin and almost no effect has been detected with phosphatidylethanolamine vesicles. These effects seem to be related to the charge properties of the lipid membranes.A possible involvement of phospholipids in the mechanism of action of the terminal transferase is suggested.     

Studies on the mechanism of action of hexamethylene bisacetamide, a potent inducer of erythroleukemic differentiation

Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up by the cell; an intracellular concentration equal to the extracellular concentration is achieved by 6–8 h. Commitment to differentiation is not detected until at least 10 h after equilibration. Both uptake and commitment to differentiate are concentration and temperature dependent. The majority of the compound is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in both cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, but acetylation of the diamine greatly increases inducing activity.     

Antiproliferative Activity and Mechanism of Action of Fatty Acid Derivatives of Arabinosylcytosine (ara‐C) in Leukemia and Solid Tumor Cell Lines

Resistance to, the hydrophilic drug ara‐C, might be meditated by decreased membrane transport. Lipophillic prodrugs were synthesized to facilitate uptake. These compounds were equally active as ara‐C, while the compounds with the shortest fatty‐acid group and highest number of double bonds were the more active. These compounds also show a better retention profile, their effect is retained longer than for ara‐C.