Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Isolation of duplicated human c-src genes located on chromosomes 1 and 20.

The oncogene (v-src) of Rous sarcoma virus apparently arose by transduction of the chicken gene known as c-src(chicken). We isolated DNA fragments representative of two src-related loci from recombinant DNA bacteriophage libraries of the human genome. One of these loci, c-src1(human), appeared to direct the synthesis of a 5-kilobase polyadenylated RNA that presumably encodes pp60c-src(human). Probes specific for the other locus, c-src2(human), did not hybridize to polyadenylated RNA prepared from a variety of human cell lines. Partial nucleotide sequence determinations of the loci demonstrated that c-src1(human) is highly related to chicken c-src and that c-src2(human) is slightly more divergent. The sequences imply that the final two coding exons of each human locus are identical in length to those of chicken c-src and that the location of an amber stop codon is unchanged in all three loci. c-src1(human) has been mapped to chromosome 20, and the second locus is located on chromosome 1. We conclude that c-src1(human) is the analog of c-src(chicken) and that the duplicated locus, c-src2(human), may also be expressed.     

Human brain natriuretic peptide-like immunoreactivity in human brain.

The presence of immunoreactive human brain natriuretic peptide in the human brain was studied with a specific radioimmunoassay for human brain natriuretic peptide-32. This assay showed no significant cross-reaction with human alpha atrial natriuretic peptide, porcine brain natriuretic peptide or rat brain natriuretic peptide. Immunoreactive human brain natriuretic peptide was found in all 5 regions of human brain examined (cerebral cortex, thalamus, cerebellum, pons and hypothalamus) (0.6-6.7 pmol/g wet weight, n = 3). These values were comparable to the concentrations of immunoreactive alpha atrial natriuretic peptide in human brain (0.5-10.1 pmol/g wet weight). However, Sephadex G-50 column chromatography showed that the immunoreactive human brain natriuretic peptide in the human brain eluted earlier than synthetic human brain natriuretic peptide-32. These findings suggest that human brain natriuretic peptide is present in the human brain mainly as larger molecular weight forms.     

A comparative study of the neutrophil stimulatory activity in vitro and pro-inflammatory properties in vivo of 72 amino acid and 77 amino acid IL-8.

IL-8, a potent neutrophil-activating protein, can be produced by many cell types including monocytes, lymphocytes, fibroblasts, neutrophils, and endothelial cells. Depending on the cell source, the N-terminal amino acid sequence of IL-8 displays heterogeneity that has been shown to confer differences in its neutrophil stimulatory activity in vitro. Despite these observations the relative potency of different IL-8 molecules in vivo is unknown. To address this question we have investigated the biologic activity of the two predominant forms of IL-8, the 72 and the 77 amino acid proteins, in vitro and in vivo. In vitro, human rIL-8(72) and human rIL-8(77) dose dependently induced adherence of rabbit peritoneal neutrophils and human neutrophils to laminin-coated plates and elevated cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2 loaded neutrophils. In these in vitro assays human rIL-8(72) was more potent than human rIL-8(77) while inducing comparable responses to human rC5a. With respect to enhancing [Ca2+]i, neutrophils desensitized to human rIL-8(72) failed to respond to human rIL-8(77). However, neutrophils fully desensitized to human rIL-8(77) could exhibit a partial response to human rIL-8(72). Further, human rIL-8(72) was approximately 10-fold more effective than human rIL-8(77) in displacing human [125I]rIL-8(72) from rabbit peritoneal neutrophils in a receptor-binding assay. In vivo, intradermally administered human rIL-8(72) and human rIL-8(77) induced 111In-neutrophil accumulation and edema formation in rabbit skin. In contrast to the in vitro studies, the two forms of IL-8 gave identical responses in vivo although they were less potent than human rC5a. Our results demonstrate that, in vitro, human rIL-8(72) is more potent than human rIL-8(77) in stimulating neutrophils. It may be that IL-8)72) has a greater affinity and/or efficacy for the neutrophil IL-8 cell-surface receptors. One possibility for the observation that both forms of IL-8 are equipotent in inducing inflammatory responses in vivo is that the extended form is proteolytically cleaved to the more biologically active IL-8(72).     

Production of antigen-specific human monoclonal antibodies: comparison of mice carrying IgH/kappa or IgH/kappa/lambda transloci

Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/IgK loci and harbor human IgM + Igkappa(BABkappa) or human IgM + Igkappa + IgA transloci (BABkappa,lambda). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BABkappa comprise human mu chains that are associated with mouse lambda chains. In contrast, BABkappa,lambda mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Iglambda translocus (in addition to the human IgH + Igkappa transloci) not only diminishes problems of endogenous mouse Iglambda expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.     

Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1

Here we show that transplantation of autologous human hematopoietic fetal liver CD34+ cells into NOD/SCID mice previously implanted with human fetal thymic and liver tissues results in long-term, systemic human T-cell homeostasis. In addition, these mice show systemic repopulation with human B cells, monocytes and macrophages, and dendritic cells (DCs). T cells in these mice generate human major histocompatibility complex class I- and class II-restricted adaptive immune responses to Epstein-Barr virus (EBV) infection and are activated by human DCs to mount a potent T-cell immune response to superantigens. Administration of the superantigen toxic shock syndrome toxin 1 (TSST-1) results in the specific systemic expansion of human Vbeta2+ T cells, release of human proinflammatory cytokines and localized, specific activation and maturation of human CD11c+ dendritic cells. This represents the first demonstration of long-term systemic human T-cell reconstitution in vivo allowing for the manifestation of the differential response by human DCs to TSST-1.     

Ergological viewpoint in real-life study

I think that real-life studies on human work must be done, if possible, not objectively, but subjectively from an ergological viewpoint. Essentially, I consider ergology as the biology of human living and work, along German biologist Haeckel's idea. He considered ergology a field of anthropology. The instinctive (genetic) element and the learned (environmental) element constitute human behavior. It is conceivable that the adaptation by human learnability has today brought about high-tech society, and genetic human attributes (human nature) have been adapted to the living environment of hunters and gatherers, which has been long-lasting in human history. Hunters-gatherers form a band consisting of about 50 (30-100) persons and move in a more or less vaguely defined territory (1,000-3,000 km2). The unsuitableness of human nature for modern scientific civilization has caused various problems. Always keeping human nature in mind when considering human living and work is the philosophical background of human ergology. Two real-life working conditions in agriculture, the packing of leeks and promoting a system for securing employees in strawberry production, are presented.     

Assignment of the gene for acid beta-glucosidase to human chromosome 1.

The structural gene for human acid beta-glucosidase (GBA) has been assigned to chromosome 1 using somatic cell hybridization techniques for gene mapping. The human enzyme was detected in mouse RAG cell-human fibroblast cell hybrids by a sensitive double antibody immunoprecipitation assay using a mouse antihuman GBA antibody. No cross-reactivity between mouse beta-glucosidase and human GBA or neutral beta-glucosidase (GBN) was observed. Fifty-two primary, secondary, and tertiary manmouse hybrid lines, derived from three separate fusion experiments, were analyzed for human GBA and enzyme markers for the human chromosomes. Without exception, the presence of human GBA in these hybrid clones was correlated with the presence of human chromosome 1 or its enzymatic markers, phosphoglucomutase 1 (PGM1), and fumarate hydratase (FH). All other human chromosomes were eliminated by the independent segregation of GBA and their respective enzyme markers and/or chromosomes. Using a RAG X human fibroblast line with a mouse-human rearrangement of human chromosome 1, the locus for GBA was limited to the region 1p11 to 1qter.     

Degenerative change in tumor cells caused by human fibroblasts and its enhancement by human interferons

The target cells (KB, HeLa, FL, human hepatoma and murine L929) were cocultured with human embryonic fibroblasts in the Petri dish. The degenerative changes of the target cells except L929 cells by the human fibroblasts were found. Human leukocyte interferon (HuIFN-alpha) and human fibroblast interferon (HuIFN-beta) enhanced these changes, but mouse IFN (MuIFN-alpha, beta) did not. The other human fibroblasts also caused the degenerative changes of the target cell, and HuIFN-alpha enhanced these changes. It was concluded that human fibroblasts play a certain role in the suppression of the human tumor cell.     

Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common gamma-chain deficient NOD/LtSz-scid (NOD-scid IL2Rgamma(null)) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rgamma(null) mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rgamma(null) mice with human HSC generate 6-fold higher percentages of human CD45(+) cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rgamma(null) mice contain human Ig(+) B cells and lower numbers of human CD3(+) T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4(+)CD8(+) thymocytes as well human CD4(+)CD8(-) and CD4(-)CD8(+) peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rgamma(null) mice was validated by 1) high levels of TCR excision circles, 2) complex TCRbeta repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rgamma(null) mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.     

Localization of human variable and constant region immunoglobulin heavy chain genes on subtelomeric band q32 of chromosome 14

Analysis of a group of human/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human variable region (VH), junctional (JH), and constant region (C epsilon) heavy chain immunoglobulin genes indicates that all of these IgH genes are localized on the subtelomeric (q32) band of chromosome 14. Somatic cell hybrids were isolated in selective medium after fusing human fibroblasts with hprt- Chinese hamster cells. The human parental cells contained two translocation chromosomes representing a reciprocal translocation between chromosomes X and 14. Only those hybrid cell lines retaining a complete human autosome 14 or the X/14 translocation chromosome (i.e. containing band 14q32) retained the human IgH genes. Retention of these genes did not correlate with the presence of the other translocation chromosome, 14/X. These results indicate that all human IgH genes (VH, JH, and CH) map to the same chromosomal band (14q32) which is commonly involved in reciprocal translocations with human chromosome 8 (8q24) in B-cell neoplasms.     

槲寄生中的化学成分及其抗肿瘤活性(Ⅰ)

从槲寄生75%乙醇提取物中分离得到4个化合物,应用波谱学方法鉴定它们的结构为尼克酰胺(1),乙酰胺(2),齐墩果酸(3),β-香树脂醇乙酸酯(4).其中,化合物1和2为首次从槲寄生属植物中分离得到.采用MIT法对化合物1～4进行体外抗肿瘤活性研究,结果显示化合物1、2和4对HO-8910人卵巢浆液性腺癌、SMMC7721人肝癌、T24人膀胱癌、HepG2人肝癌和SHG人神经胶质瘤细胞系没有细胞毒活性,化合物3则对这五种肿瘤细胞系有显著的抑制作用.     

Isolation of a human X chromosome-linked gene essential for progression from G1 to S phase of the cell cycle

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21/13, cannot progress into S phase at 39.5 degrees C, following the release from isoleucine deprivation. The mutant cells were transfected with high molecular weight (HMW) DNA from human KB cells, and several human DNA bands were found to be conserved through three cycles of ts+ transformation. Conserved human DNA was isolated from the cosmid library of the secondary ts+ transformant (K-1-1), using 32P-labelled total human DNA as a probe. The isolated human DNA covers about 70 kb of human DNA flanked with hamster DNA, and originates from the human X chromosome. The middle part (56 kb) of the isolated human DNA was conserved through the primary, secondary and tertiary ts+ transformation, without gross rearrangement.     

Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells.

Hemopoietic stem cell factor (SCF), which is the ligand for the proto-oncogene c-kit receptor (allelic with W locus) and the product of Sl locus of the mouse, has recently been cloned. The human homologue has also been cloned, and recombinant protein (human rSCF) expressed and purified to homogeneity. To determine the effect of human rSCF in the presence or absence of human rIL-3 on human bone marrow-derived mast cells and basophils, human CD34+ pluripotent progenitor cells, highly enriched (greater than 99%) from bone marrow mononuclear cells, were cultured over agarose surfaces (interphase cultures) in the presence of human rIL-3, human rIL-3 and increasing concentrations of human rSCF, or human rSCF alone. Over 3 to 4 wk, human rSCF acted synergistically with human rIL-3 at all concentrations, producing a three- to fivefold increase in total, mast cell, and basophil numbers over human rIL-3 alone when used at 100 ng/ml. The percentage of cell types in the human rIL-3 and human rIL-3 plus human rSCF cultures, however, remained the same, with basophils constituting 18 to 35% of the final cultured cells, and mast cells 3% or less of the final cell number. In the presence of human rSCF alone, the combined total percentage of mast cells and basophils was 0 to 1.0%, the majority of cells being macrophages. Mast cells cultured in human rIL-3 plus human rSCF, but not human rIL-3 alone, were berberine sulfate positive, suggesting the presence of heparin proteoglycans within granules. Electron microscopic examination of cultures supplemented with human rIL-3 and rSCF, but not human rIL-3 alone, revealed that after 3 wk in culture, mast cell granules contained tryptase and exhibited scroll, reticular, and homogeneous patterns as seen previously in CD34+/3T3 fibroblast cocultures. Thus, CD34+ cells cultured in the presence of both human rIL-3 and rSCF give rise to cultures containing increased numbers of basophils and mast cells, with the mast cells by ultrastructural studies showing evidence of maturation although the percentages of basophils and mast cells arising in these cultures remained unchanged.     

自然和热凝固的人肝组织对KTP/YAG激光的光学特性的比较研究

本文采用双积分球测量系统和Inverse Add ing-Doub ling方法,研究了自然和热凝固的人肝组织对532nm的KTP激光和1 064 nm的Nd:YAG激光的光学特性。结果表明:热凝固的人肝组织对532 nm的KTP激光的吸收系数较自然的肝组织的吸收系数增大了23.5%(P<0.05),热凝固的肝组织对1 064 nm的Nd:YAG激光的吸收系数较自然的肝组织的吸收系数减小了34.3%(P<0.05)。热凝固的人肝组织对532 nm的KTP激光的散射系数较自然的肝组织的散射系数增大了4.50倍(P<0.05),热凝固的肝组织对1 064 nm的Nd:YAG激光的散射系数较自然的肝组织的散射系数增大了6.41倍(P<0.05)。热凝固的人肝组织对532 nm的KTP激光的各向异性因子较自然的肝组织的各向异性因子减小了5.47%,热凝固的肝组织对1 064 nm的Nd:YAG激光的各向异性因子较自然的肝组织的各向异性因子减小了1.95%。     

Construction and expression of human/bovine P45017 alpha chimeric proteins: evidence for distinct tertiary structures in the same P450 from two different species

In the human and bovine adrenal cortex, 17 alpha-hydroxylase (P45017 alpha) catalyzes reactions involved in the production of C21-glucocorticoids (17 alpha-hydroxylation) and C19-androgens (17,20-lyase). The bovine and human forms of P45017 alpha share 71% primary sequence identity. Using naturally occurring restriction sites common to cDNAs encoding both human and bovine P45017 alpha, we have constructed bovine/human (bovine amino terminus and human carboxy terminus) and human/bovine (human amino terminus and bovine carboxy terminus) cDNAs that have been expressed in COS 1 cells, and the enzymatic properties of the resultant chimeric proteins have been examined. The three bovine/human chimeras studied have 17 alpha-hydroxylase activities intermediate between those of the wild-type bovine and wild-type human enzymes, although the 17,20-lyase activity of these chimeras is significantly lower than that of either of the wild-type enzymes. Surprisingly, the opposite chimeras (those containing a human amino-terminal sequene and a bovine carboxy-terminal sequence) are all virtually inactive, even though they appear to be expressed at normal levels. These results indicate that the folding of P45017 alpha initiated by the bovine amino terminus can accommodate human P45017 alpha sequences of various lengths to produce a relatively normal 17 alpha-hydroxylase having decreased 17,20-lyase activity. On the other hand, folding initiated by the human P45017 alpha amino terminus does not easily accommodate bovine carboxy-terminal sequences to produce a functional enzyme. Presumably this difference arises from the fact that the tertiary structures of the bovine and human forms of P45017 alpha are sufficiently different so that interchanging sequences will not lead to functional enzymes in a predictable fashion.     

抗人CD3单链抗体四聚体基因的构建及表达

为构建和表达抗人CD3单链抗体 (scFv) 人p5 3四聚功能域融合基因 ,选用人IgG3上游铰链区作为抗人CD3scFv和人p5 3四聚功能域之间连接的linker .利用递归PCR法扩增人IgG3上游铰链区与人p5 3四聚功能域融合基因 ,克隆入pUC18载体中构建pUC18 IgG3 p5 3克隆载体 .将抗人CD3scFv克隆入pUC18 IgG3 p5 3载体中 ,构建抗人CD3scFv 人p5 3四聚功能域融合基因 .经酶切鉴定及序列测定证实后 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后利用流式细胞仪进行亲和活性测定 .获得了抗人CD3scFv 人p5 3四聚功能域融合基因 ,基因全长 882bp ,可编码 2 94个氨基酸 ,与已发表的抗人CD3scFv、人IgG3上游铰链区和人p5 3四聚功能域基因cDNA序列一致 .表达产物经SDS PAGE和Western印迹实验证实为约 35kD的特异蛋白条带 ,纯化后经流式细胞仪检测可以特异性地结合人外周血单个核细胞 (PBMC)细胞 ,亲和力高于scFv ,为进一步临床应用奠定基础     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.