4NQO- or MNNG-resistant variants established from a human cell line, RSb, with high sensitivity to both agents

From a human cell line, RSb, with high sensitivity to the killing effects of 4-nitroquinoline 1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 254-nm ultraviolet light, a 4NQO-resistant variant, Qr-10, and an MNNG-resistant one, Gr-10, were established using ethyl methanesulfonate as the mutagen. Cell proliferation studies and colony-formation assays revealed that Qr-10 and Gr-10 cells actively proliferated under conditions where RSb cell proliferation was greatly inhibited by 4NQO and MNNG, respectively. Total cellular DNA synthesis, as estimated by [Me-3H]thymidine uptake into acid-insoluble cell materials, was depressed in 4NQO-treated Qr-10 and MNNG-treated Gr-10 cells as it was in chemical-treated RSb cells, but recovered more markedly from such inhibition in the variants. 4NQO- and MNNG-induced DNA-repair replication synthesis was enhanced to a greater extent in Qr-10 and Gr-10 cells, respectively, than in RSb cells. The Qr-10 and Gr-10 cells showed the same respective susceptibility to the effects of MNNG and 4NQO, on cell growth and DNA synthesis and DNA-repair synthesis as did the parent cells. But, Qr-10 cells had more resistance to UV-killing and higher levels of UV-induced DNA-repair synthesis than did RSb cells, while UV-susceptibility of Gr-10 cells was the same as that of the latter.     

A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line

When cells of a human clonal cell line, RSa, with high sensitivity to UV lethality, were treated with the mutagen, ethyl methanesulfonate, a variant cell strain, UVr-1, was established as a mutant resistant to 254-nm far-ultraviolet radiation (UV). Cell proliferation studies showed that UVr-1 cells survived and actively proliferated at doses of UV-irradiation that greatly suppressed the proliferation of RSa cells. Colony-formation assays also confirmed the increased resistance of UVr-1 cells to UV. The recovery from a UV-induced inhibition in DNA synthesis, as [methyl-3H]thymidine uptake into cellular DNA, was more pronounced in UVr-1 cells than in RSa cells. Nevertheless, there was no significant difference in the activity of UV-induced DNA repair synthesis in either cell line, as estimated by the extent of unscheduled DNA synthesis and DNA repair replication. UVr-1 cells were also more refractory to 4-nitroquinoline 1-oxide (4NQO), but the activity of DNA repair synthesis induced by 4NQO in UVr-1 cells was much the same as in the RSa cells. Both N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) sensitivity and MNNG-induced DNA repair synthesis activity in UVr-1 cells were similar to that of RSa cells. These characteristics of UVr-1 cells are discussed in the light of a previously reported UV-resistant variant, UVr-10, which had an increased DNA repair synthesis activity.     

Characterization of plasminogen activator produced by an established cell line from human ovary

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro-UK), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high-performance liquid chromatography with a Zn chelate-5PW column and with a p-amino-benzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro-UK. An immunological study demonstrated that OC-1-PA cross-reacted with anti-UK IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the UK type, but its structure differs from that of UK.     

Characterization of a B cell-derived growth-enhancing factor produced by a human B cell line established from a patient with rheumatoid arthritis

A human B cell line, TKS-1, which was established from the peripheral blood of a patient with rheumatoid arthritis, was found to spontaneously produce a factor which enhances the activity of interleukin 1 (IL-1). This factor, designated B cell-derived growth-enhancing factor (BGEF), enhanced IL-1-induced proliferation of peanut agglutinin nonagglutinated thymocytes. BGEF also enhanced IL-1-induced production of interleukin 2 (IL-2) by both thymocytes and a human T cell clone, HSB.2 C5B2. BGEF alone did not induce the production of IL-2. BGEF failed to induce proliferation of the IL-2-dependent T cell clone, and did not enhance its response to IL-2. The activity of BGEF was not blocked by antisera against human IL-1-alpha or human IL-1-beta. Gel filtration analysis revealed that BGEF has a m.w. of 60,000 to 65,000 in its native state. We concluded that BGEF differed from IL-1 and IL-2, but is a novel factor produced by TKS-1 cells. In addition, we found that partially purified B cells from patients with rheumatoid arthritis produced factors which enhanced the activity of IL-1.     

Some properties of alkaline phosphatase from a human cell strain and from a clonal derivative with low activity

A comparative study of some physico-chemical properties of alkaline phosphatase of a human cell line, the EUE, with high level of enzyme and one of its clonal derivatives the E6D, with low activity, has been carried out. Electrophoretic analysis reveals a multiple banding pattern within each line and qualitative differences between the two lines. The alkaline phosphatase activity of the E6D cell extracts is almost completely inhibited by 5 × 10^?2 M inorganic phosphate while in the EUE the enzymic activity is reduced to one third under these conditions. The enzymes of the two lines show also a different thermostability which is not referable to extrinsic factors, as demonstrated by mixing experiments. The time course of heat inactivation at 70°C suggests molecular heterogeneity in each line, and a prevalence of a thermostable fraction in the cells with low activity and a thermolabile one in those with high enzymic levels. A rough estimate of inactivation constants does not rule out the possibility that the molecular species in the two lines are the same but in different proportions. The cytological analysis confirms the relationship between the number of small acrocentric chromosomes and alkaline phosphatase levels. The significance of the biochemical data in relation to the proposed model of a gene dosage effect is discussed.     

Glucocorticoid receptors and cortico-sensitivity in a human clonal monocytic cell line, CM-SM

CM-SM is a clonal line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Untreated CM-SM cells contain single class, high-affinity (KD = 4.0 X 10(-9) M) glucocorticoid-specific receptor sites (approximately 60,000 per cell), as measured by a whole cell assay, at 37 degrees C, using [3H]triamcinolone acetonide (TA). Exposure of CM-SM to dexamethasone (DEX) produced a progressive, dose- and time-related series of changes in CM-SM cell growth, saturation density, morphology, and functional properties, with half-maximal effects at about 10(-9) M for DEX. TA-receptor sites rapidly decreased (about 70%) after DEX treatment, without any apparent change in steroid specificity and affinity. After 5 days in culture with a saturating concentration (3.6 X 10(-8) M) of hormone, the cells reached a saturation density of about 9.0 X 10(6) viable cells/ml (about 4.0 X 10(6) viable cells/ml in the controls), while the modal volume of the resulting cell population was approximately 60%, as compared to the volume of untreated cells. DEX-treated cells appeared less differentiated than controls, as assessed by combined morphologic, antigenic, and cytoenzymatic analyses. DEX almost completely inhibited TPA activation of the following macrophage functions: adherency to the culture plate, expression of lysosomal enzymes, Fc and C3 receptors, and stimulation of phagocytosis. After removal of DEX, the cells, within a few passages, returned to a state apparently identical to the untreated controls and could be induced to macrophage differentiation in response to TPA.     

Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo

The lifespan of the tooth is influenced by the periodontal ligament (PDL), a specialized connective tissue that connects the cementum with the tooth socket bone. Generation of a cell line from PDL progenitor/stem cells would allow development of tissue engineering-based regenerative PDL therapy. However, little is known about the characteristics of PDL progenitor/stem cells because PDL tissue consists of a heterogeneous cell population and there are no pure PDL cell lines. Recently, we succeeded in immortalizing primary human PDL fibroblasts (HPLFs) by transfecting them with SV40 T-antigen and hTERT (Cell Tissue Res 2006; 324: 117-125). In this study, we isolated three clonal cell lines from these immortalized cells (lines 1-4, 1-11, and 1-24) that express RUNX-2, Col I, ALP, OPN, OCN, RANKL, OPG, scleraxis, periostin, Col XII, and alpha-SMA mRNA. Immunocytochemical analysis demonstrated that CD146 was expressed in cell lines 1-4 and 1-11 and that STRO-1 was expressed in lines 1-11 and 1-24. Lines 1-4 and 1-11 differentiated into osteoblastic cells and adipocytes when cultured in lineage-specific differentiation media. Four weeks after transplanting cell line 1-11 into immunodeficient mice with beta-tricalcium phosphate (beta-TCP), the transplant produced cementum/bone-like tissues around the beta-TCP. Eight weeks after transplantation, the 1-11 cell transplant formed PDL-like structures on the surface of the beta-TCP. These data suggest that cell line 1-11 was derived from a progenitor/stem cell present in the PDL and should be very useful for studying the biology and regeneration of human periodontium.     

Production of fibronectin by HUH6 C15 cell line established from a human hepatoblastoma

Fibronectin was detected by indirect immunofluorescence on the cell surfaces of HUH6 C15 cells, established from a human hepatoblastoma and maintained with serum-free RPMI 1640 medium. Fibronectin synthesized by HUH6 Cl5 was purified by gelatin-Sepharose affinity chromatography and compared with human plasma fibronectin in respect to molecular weight, electrophoretic mobility and antigenicity. Fibronectin synthesized by this cell line was proved to be identical with human plasma fibronectin.     

Effects of cholesterol and alfa-2-recombinant interferon on cell growth and cell antigen production in the HT 29 cells, an established cell line of human colon adenocarcinoma

HT 29 cells, an established cell line of human colon adenocarcinoma, were grown in RPMI 1640 medium without or with cholesterol at 25, 50, 100 micrograms/ml concentrations. In some experiments 100 or 200 U/ml alfa-2-A recombinant Interferon were added to the medium. Only in the case of the highest cholesterol concentration there was a reduced number of cells at confluence. Moreover, only the production of CEA increased in the presence of cholesterol. Interferon did not affect cell growth appreciably but stimulated CEA release into the medium during the first three days of culture. Morphological analysis of cells in the presence of cholesterol seems to indicate an attempt of the cells to differentiate.     

A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival

Repair of injured peripheral nerve is thought to play important roles in tissue homeostasis and regeneration. Recent experiments have demonstrated enhanced functional recovery of damaged neurons by some types of somatic stem cells. It remains unclear, however, if periodontal ligament (PDL) stem cells possess such functions. We recently developed a multipotent clonal human PDL cell line, termed cell line 1-17. Here, we investigated the effects of this cell line on neurocytic differentiation, migration, and survival. This cell line expressed the neural crest cell marker genes Slug, SOX10, Nestin, p75NTR, and CD49d and mesenchymal stem cell-related markers CD13, CD29, CD44, CD71, CD90, CD105, and CD166. Rat adrenal pheochromocytoma cells (PC12 cells) underwent neurocytic differentiation when co-cultured with cell line 1-17 or in conditioned medium from cell line 1-17 (1-17CM). ELISA analysis revealed that 1-17CM contained approximately 50 pg/ml nerve growth factor (NGF). Cell line 1-17-induced migration of PC12 cells, which was inhibited by a neutralizing antibody against NGF. Furthermore, 1-17CM exerted antiapoptotic effects on differentiated PC12 cells as evidenced by inhibition of neurite retraction, reduction in annexin V and caspase-3/7 staining, and induction of Bcl-2 and Bcl-xL mRNA expression. Thus, cell line 1-17 promoted neurocytic differentiation, migration, and survival through secretion of NGF and possibly synergistic factors. PDL stem cells may play a role in peripheral nerve reinnervation during PDL regeneration.     

淀粉酶高产菌株的筛选、紫外诱变及产酶条件优化

【背景】提高淀粉酶的稳定性进而适应多变的工业生产条件,是淀粉酶开发的重要方向。【目的】淀粉酶在食品加工、布料退浆、酿酒制造、养殖等领域都有广泛的应用,但目前生产用的淀粉酶来源单一,在溶液中的稳定性较差,需要扩大淀粉酶来源,增强酶的稳定性,使其进一步适应复杂多变的工业生产环境。【方法】利用选择培养基在三门峡黄河湿地表层土样中筛选得到20株具有淀粉酶活性的菌株,采用杯碟法检测粗酶液的活性并对其中活性最高的3株菌进行鉴定,对其中酶活性最高的菌株运用紫外照射的方式进行诱变并测定致死率,选择突变后酶活最高的菌株,优化培养条件,并测定突变后淀粉酶的活性和作用条件范围。利用3,5-二硝基水杨酸（3,5-dinitrosalicylic acid,DNS）法测定诱变前后酶活并进行活性对比。【结果】在三门峡黄河湿地表层土壤中筛选得到3株淀粉酶活性较高的菌株并编号S03、S08和S17。根据16S rRNA基因序列比对、革兰氏染色形态观察结合生理生化分析显示,菌株S03、S08和S17分别为Aeromonas、Exiguobacterium和Bacillus,其淀粉酶最适NaCl浓度是10%-12%,最适...     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

A lymphoblastoid cell line with high phagocytic activity established from peripheral blood of a patient with chronic myelocytic leukemia in blast crisis

A human hematopoietic cell line (K-23-M) was established from a patient with chronic myelocytic leukemia in blast crisis. Morphologically, the cultured cells were lymphoblastoid cells that produced IgA and were Epstein-Barr viral nuclear antigen positive. But they showed high phagocytic activity to glutaraldehyde-treated sheep red cells and had properties of a monocyte or macrophage that included surface Fc receptors, alpha-naphthyl butyrate esterase positivity blocked by NaF, migration in soft agar and the ability to attach to a glass surface. Lysozyme secretion was absent, and chromosomes were diploid and Ph1 negative. This cell line is unique in that it has strong phagocytic activity. Its existence shows that lymphoblastoid cell line may be a more important cell line for the study of human hematopoietic cells than previously has been believed.     

Ocular avirulence of a herpes simplex virus type 1 strain is associated with heightened sensitivity to alpha/beta interferon.

BALB/c mice infected on the scarified cornea with herpes simplex virus type 1 strain 35 [HSV-1(35)] rarely developed ocular disease even at challenge doses as high as 10(7) PFU per eye. In contrast, HSV-1(RE) consistently induced stromal keratitis at an inoculum of 2 x 10(4) PFU. The goal of this study was to determine the reason for the difference in virulence between the two HSV strains. Both HSV-1 strains replicated to similar titers in excised corneal "buttons." However, after in vivo infection of the cornea, the growth of strain 35 was evident only during the first 24 h postinfection, whereas the replication of strain RE persisted for at least 4 days. In vitro tests revealed that HSV-1(35) was greater than 10 times more sensitive to alpha/beta interferon (IFN-alpha/beta) than HSV-1(RE). Both strains induced comparable serum levels of IFN after intraperitoneal inoculation. The kinetics of HSV-1(35) clearance from the eye was markedly altered by treatment with rabbit anti-IFN-alpha/beta. Virus titers exceeding 10(4) PFU per eye could be demonstrated 4 to 5 days postinfection in mice given a single inoculation of antiserum 1 h after infection. Furthermore, anti-IFN treatment in 3-week-old mice infected with HSV-1(35) led to the development of clinically apparent corneal disease which subsequently progressed to stromal keratitis in the majority of recipients. These results indicate that the striking difference in the capacity of HSV-1(35) and HSV-1(RE) to induce corneal disease was related to the inherently greater sensitivity of strain 35 to IFN-alpha/beta produced by the host in response to infection.     

Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line

We previously reported that a single DNA double-strand break (DSB) near a telomere in mouse embryonic stem cells can result in chromosome instability. We have observed this same type of instability as a result of spontaneous telomere loss in human tumor cell lines, suggesting that a deficiency in the repair of DSBs near telomeres has a role in chromosome instability in human cancer. We have now investigated the frequency of the chromosome instability resulting from DSBs near telomeres in the EJ-30 human bladder carcinoma cell line to determine whether subtelomeric regions are sensitive to DSBs, as previously reported in yeast. These studies involved determining the frequency of large deletions, chromosome rearrangements, and chromosome instability resulting from I-SceI endonuclease-induced DSBs at interstitial and telomeric sites. As an internal control, we also analyzed the frequency of small deletions, which have been shown to be the most common type of mutation resulting from I-SceI-induced DSBs at interstitial sites. The results demonstrate that although the frequency of small deletions is similar at interstitial and telomeric DSBs, the frequency of large deletions and chromosome rearrangements is much greater at telomeric DSBs. DSB-induced chromosome rearrangements at telomeric sites also resulted in prolonged periods of chromosome instability. Telomeric regions in mammalian cells are therefore highly sensitive to DSBs, suggesting that spontaneous or ionizing radiation-induced DSBs at these locations may be responsible for many of the chromosome rearrangements that are associated with human cancer.     

Characterization and isolation of a C-reactive protein receptor from the human monocytic cell line U-937

Human C-reactive protein (CRP) is an acute phase reactant that is opsonic and an activator of macrophage tumoricidal function. CRP also activates the classical C cascade. These activities suggest that CRP might interact with monocytes/macrophages via specific receptors in a manner analogous to the interaction of IgG with FcR. With the use of radio-labeled human CRP, we have observed specific binding of CRP to human blood monocytes and the human monocytic cell line U-937. Binding was saturable at a pathophysiologic concentration of CRP, with an estimated KD of 9.5 x 10(-8) M and 3.6 x 10(5) binding sites/cell. Specific binding was inhibited by polyclonal human IgG as well as an IgG1 myeloma. In the converse experiment, CRP failed to inhibit specific [125I]IgG binding. The mAb IV.3, which inhibits binding of IgG immune complexes to FcRII, did not inhibit CRP binding. A 100-fold excess of phosphorylcholine or the phosphorylcholine binding peptide of CRP (residues 47-63) failed to inhibit binding. Although human rIFN-gamma and PMA increased FcRI expression, these reagents had no affect on CRP receptor expression. A single membrane protein of 38 to 41 kDa from U-937 cells was chemically cross-linked to [125I]CRP; the cross-linking was inhibited by human IgG1 but not the IV.3 mAb. Furthermore, two membrane proteins with a Mr of 38 to 40 kDa and 58 to 60 kDa were isolated by CRP ligand-affinity chromatography. These proteins were of a distinct size from those isolated for FcRI from an IgG ligand matrix. These studies demonstrate specific binding of human CRP to a human monocytic cell line via receptors that are distinct from the IgG FcR and implicate CRP in nonspecific, preimmune host defense reaction mediated by cells of the monocytic lineage.