Purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus

We have constructed the recombinant baculovirus which expresses the human immunodeficiency virus type 1 negative factor (nef) gene. Spodoptera frugiperda cells infected with the recombinant virus produced a 27-kDa protein which reacted with rabbit antisera raised against a carboxy-terminal synthetic peptide of the Nef protein by immunoblot analysis. Labeling experiment showed that the recombinant Nef protein was myristoylated. The recombinant Nef protein was purified to near homogeneity by DEAE-Sephacel, phenyl-Sepharose 4B, blue-Sepharose, and Sephadex G-150 column chromatography. No detectable GTP binding activity was observed in the purified recombinant Nef product.     

Ethical Reasoning Among Registered Nurses Experienced in Dementia Care

Abstract. Registered nurses regarded as “experienced and good” in dementia care were interviewed about the feeding of a severely demented patient who showed refusal-like feeding behaviour. Not one of the twenty nurses could see herself using force against her patients. Most interviewees justified their decisions to feed a severely demented patient and answered questions about whether they would change their minds if there were certain circumstances in terms of words that could be interpreted as referring to the ethical principle of beneficence. The nurses stressed the difficulty to understand the meaning of severely demented patients' feeding behaviour and decide when force-feeding occurs. When asked to rank ethical principles of importance for the decision, however, the most common answer was that they would give priority to the ethical principle of autonomy. The nurses did not see the ethical principles as separate entities, that could be applied one by one, but tried to integrate them into a whole. The findings of this study were interpreted as indicating that principled ethics is not an adequate model to describe experienced nurses' ethical reasoning.     

Phase I study of a recombinant gamma interferon (S-6810)

A phase I study of a recombinant gamma interferon (S-6810) was conducted in a cooperative study involving 11 institutions. S-6810 was administered at doses of 2, 4, 8, 12, 32 and 64 X 10(6) U/m2 by one-hour infusion for 5 consecutive days. A total of 40 courses were administered to 31 patients. High fever exceeding 38 degrees C with chills occurred in about 80% of patients. The incidences of other toxicities were fatigue in 50%, gastrointestinal toxicities in 30-40%, and changes in hepatic enzymes and hematologic toxicities in 20-30%. Dose-limiting factors were judged to be hypotension, leukopenia and central nervous toxicity. Maximum tolerated dose was 64 X 10(6) U/m2 and an optimal dose for phase II study was considered to be 6 X 10(6) U/m2 by daily chronic schedule. Blood concentration was highest at the end of infusion, and then decreased rapidly with a biphasic curve. The peak concentrations were elevated by escalation of doses. A partial response was observed in a patient with mycosis fungoides.     

Constitutive expression of the nef gene suppresses human immunodeficiency virus type 1 (HIV-1) replication in monocytic cell lines.

In order to study the effect of nef gene expression on viral replication in monocytic cells, we established monocytic (U937 and THP-1) cell transfectants constitutively expressing the human immunodeficiency virus type 1 nef gene. We constructed a plasmid expressing the nef gene derived from an infectious clone, NL432, under the control of SR alpha promoter which can drive a high level of gene expression. We found suppressed viral replication in nef-expressing monocytic cells, although a negative effect of nef was observed, with some variation depending on the virus strain and the cell. We also observed that the expression of the surface CD4 molecule is inversely related to the expression of the nef gene, especially in the U937 transfectants. These results indicate that the suppression of viral replication and the down-modulation of CD4 molecule by nef gene expression occur in monocytic cell lines as in T cell lines.     

Synergistic suppression of the clonogenicity of U937 leukemic cells by combinations of recombinant human interleukin 4 and granulocyte colony-stimulating factor.

The actions and interactions of purified recombinant human (rh) interleukin 4 (IL-4) and granulocyte colony-stimulating factor (G-CSF) on the clonogenicity of human leukemic cell line U937 were studied in vitro. Parameters analyzed were the suppression of stem cell generation using sequential clonal cultures, alterations of surface antigen expression, and morphological changes. IL-4 alone (10 U/ml) and G-CSF alone (1000 U/ml) only slightly reduced colony numbers (80% +/- 7% and 87% +/- 7% of control colonies, respectively). However, IL-4 interacted synergistically with G-CSF to further reduce the colony number (46% +/- 8% of control colonies) and suppress the self-renewal ability (clonogenicity) of U937 cells. This synergistic effect was not eliminated by cultures containing neutralizing concentrations of anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF), anti-interleukin 6 (anti-IL-6), anti-interferon-alpha (anti-IFN-alpha), anti-IFN-gamma, anti-transforming growth factor-beta (anti-TGF-beta) serum, and anti-tumor necrosis factor-alpha (anti-TNF-alpha) serum. The coexistence of IL-4 and G-CSF was required for at least 48 h to reveal the synergistic action as assessed by preincubation and delayed addition experiments. Combinations of IL-4 and G-CSF showed a significant increase in CD11b expression on U937 cells. This action was not observed with HL60, K562, ML-1, or KG-1 leukemic cell lines, and IL-4 did not show any synergistic suppression of clonogenicity of U937 leukemic cells in combination with other cytokines tested in this study. These results suggest that IL-4 in combination with G-CSF may have some capacity to synergistically suppress human leukemic cells of specific types with loss of clonogenicity.     

Assay of flow cytometry for the effect of cepharanthine on resistance to doxorubicin]

The biochemical activity of cepharanthine and the possible mechanism by which it reverses the resistance to doxorubicin in P388 leukemia cells were examined in vitro. The microfluorometric analysis of the cellular level of doxorubicin in drug-resistant cells showed that cepharanthine markedly enhanced the sensitivity of doxorubicin against resistant cells in the cellular level. Cepharanthine also enhanced the inhibitory effect of doxorubicin on the incorporation of thymidine into DNA in resistant cells. The analysis of DNA histogram obtained by flow cytometry showed that doxorubicin exerted its growth-inhibitory effect by blocking the cell cycle at the G2 phase in P388 cells. At higher concentrations, doxorubicin prolonged the S phase and inhibited cell cycle progression to the G2/M phase in cells. The treatment with cepharanthine potentiated these blocking effects induced by doxorubicin in cells. It seems that the modifications of the biological effect of doxorubicin by cepharanthine are due to the change of their ability to induce DNA damage in cells.     

人胎儿雪旺氏细胞的体外培养及其纯化研究

本实验用酶消化法从孕１４～１９周人胎儿周围神经中分离培养雪旺氏细胞，结合差速贴壁和阿糖胞苷处理进行纯化，根据Ｓ－１００蛋白免疫细胞化学染色和形态学特征，分析不同时期雪旺氏细胞的纯化程度。实验结果显示：培养４ｄ，倒置相差显微镜下观察，雪旺氏细胞呈典型的双极或三极形，端对端或旋涡状排列，Ｓ－１００蛋白免疫细胞化学染色呈阳性反应，其比例约占９８％；在２～３周内，雪旺氏细胞的纯度维持在８５％～９０％；培养３５ｄ，雪旺氏细胞约占８０％；单纯采用阿糖胞苷或差速贴壁处理，培养３５ｄ，雪旺氏细胞约占７０％；原代培养９２ｄ，传代１１代细胞生长良好。实验结果表明，多种方法联合使用较单一方法所得的纯度高。本方法快速、简便，能为雪旺氏细胞作为移植材料提供足够的来源。     

BUN/Cr ratio as an index of gastrointestinal bleeding mass in children.

Determining the site and severity of blood loss is important in the management of children with gastrointestinal (GI) bleeding. Blood urea nitrogen (BUN) and serum creatinine (Cr) were measured on the day of hospitalization and the ratio of BUN/Cr was calculated in 11 children with 16 episodes of upper GI bleeding and 49 with lower GI bleeding. There was a significant difference between the two GI bleeding groups with regard to BUN/Cr ratio (p less than 0.001). When the ratio was 30 or above, the specificity of upper GI bleeding was 98% with a sensitivity of 68.8%. A linear relationship was found between the BUN/Cr ratio and delta Hb (delta Hb = 0.08 x BUN/Cr +/- 0.8 g/dl) for bleeding originating from the upper GI tract. This study confirms that measurement of the BUN/Cr ratio is useful for localizing the source of bleeding to the upper GI tract and also demonstrates its usefulness as an estimation of the severity of blood loss from the upper GI tract.