Expression of the human retinoblastoma gene product pp110RB in insect cells using the baculovirus system

The product of the retinoblastoma susceptibility gene (RB) was overproduced in cultured insect cells using the baculovirus expression system. Upon insertion of the cloned human RB complementary DNA sequence into the viral genome downstream of the promoter of the polyhedrin gene, full-length RB protein with an apparent molecular weight of 110,000 was expressed in the insect cells. This protein was found to be phosphorylated, located in the nuclei of the infected cells, and immunologically indistinguishable from pp110RB of human cells as assayed by several anti-RB antibodies. Following cell disruption and a one-step immunoaffinity chromatographic purification, 6-12 mg of soluble pp110RB with approximately 95% purity were obtained per liter of infected suspension culture. Characterization of the two known biochemical properties of RB protein showed that this purified protein from insect cells behaved similarly to the authentic human pp110RB. First, it bound to DNA, and second, it could form a specific complex with SV40 T antigen in vitro. Prompt translocation of the protein from cytoplasm to nucleus after microinjection further indicated that the purified RB protein may be active. The availability of soluble, intact, and presumably active pp110RB in large quantity represents a significant advance for studying the biochemical and biophysical properties of the RB gene product as well as its potential biological function in cancer suppression.     

细小病毒非结构蛋白转基因小鼠模型建立

将小鼠乳腺癌病毒启动子控制的细小病毒非结构蛋白基因(长5.7kb)氯化铯超速离心,纯化透析后用显微注射法导入C57BL/SJL F_1小鼠受精卵雄核,植入假孕母鼠输卵管,得成活小鼠15只。抽取鼠尾DNA,对其中10只小鼠作PCR和southern blot鉴定,其中4只(40％)整合有目的基因。对首建者B_6()的8只子代小鼠鉴定,3只(37.5％)整合有目的基因。说明导入的目的基因能传代。     

RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

部分酶解酵母高效电击转化研究

以酵母质粒YCp50为外源DNA,电击转化部分酶解酵母宿主菌AB1380,转化效率稳定在10~6转化子/μg质粒DNA左右,比不酶解酵母或酵母原生质球作受体的电击转化效率高一个数量级以上,也比PEG介导的酵母原生质球转化高3～5倍,而且适合于大片段DNA如水稻YAC分子的转化。达最佳转化时的有关技术参数为:新接菌种通气培养至细胞密度1×10~8～1.5×10~8个/ml;转化时细胞密度控制在1×10~9～1.5×10~9个/ml;每毫升酶解缓冲液加15u溶菌酶(lyticase),30℃下处理酵母5min进行部分酶解;电击时,电场设置在6.25kV/cm、电容25μF,电击后直接铺板。     

Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc.     

Predictive models for phosphorus retention in wetlands

The potential of wetlands to efficiently remove (i.e., act as a nutrient sink) or to transform nutrients like phosphorus under high nutrient loading has resulted in their consideration as a cost-effective means of treating wastewater on the landscape. Few predictive models exist which can accurately assess P retention capacity. An analysis of the north American data base (NADB) allowed us to develop a mass loading model that can be used to predict P storage and effluent concentrations from wetlands. Phosphorus storage in wetlands is proportional to P loadings but the output total phosphorus (TP) concentrations increase exponentially after a P loading threshold is reached. The threshold P assimilative capacity based on the NADB and a test site in the Everglades is approximately 1 g m^–2 yr^–1. We hypothesize that once loadings exceed 1 g m^–2 yr^–1 and short-term mechanisms are saturated, that the mechanisms controlling the uptake and storage of P in wetlands are exceeded and effluent concentrations of TP rise exponentially. We propose a One Gram Rule for freshwater wetlands and contend that this loading is near the assimilative capacity of wetlands. Our analysis further suggests that P loadings must be reduced to 1 g m^–2 yr^–1 or lower within the wetland if maintaining long-term low P output concentrations from the wetlands is the central goal. A carbon based phosphorus retention model developed for peatlands and tested in the Everglades of Florida provided further evidence of the proposed One Gram Rule for wetlands. This model is based on data from the Everglades areas impacted by agricultural runoff during the past 30 years. Preliminary estimates indicate that these wetlands store P primarily as humic organic-P, insoluble P, and Ca bound P at 0.44 g m^–2 yr^–1 on average. Areas loaded with 4.0 g m^–2 yr^–1 (at water concentrations>150 g·L^–1 TP) stored 0.8 to 0.6 g m^–2 yr^–1 P, areas loaded with 3.3 g m^–2 yr^–1 P retained 0.6 to 0.4 g m^–2 yr^–1 P, and areas receiving 0.6 g m^–2 yr^–1 P retained 0.3 to 0.2 g m^–2 yr^–1. The TP water concentrations in the wetland did not drop below 50 g·L^–1 until loadings were below 1 g m² yr^–1 P.     

编码人疱疹病毒－6型核糖核苷酸还原酶和P41蛋白基因的克隆和序列测定

本文报道人疱疹病豢－６型（ＨＨＶ－６）ｐＳＴＹ２８ＤＮＡ片段的序列测定。应用分子克隆、缺损突变体（Ｄｃｌｅｔｉｏｎｍｕｔａｎｔ）制备和序列测定等技术,完成了３．９ｋｂＨＨＶ－６ｐＳＴＹ２８ＤＮＡ片段的全序列测定。经ＤＮＡＳＩＳ核酸蛋白软件分析,该片段含有两个开读框架（ＯＲＦ）核糖核苷酸还原酶（ＲＩＲ）ＯＲＦ有２４１４个核苷酸,可编码８０５个氨基酸;Ｐ４１蛋白由１１００个核苷酸组成。与其他疱疹病毒作氨基酸同源性比较,ＨＨＶ－６ＲｉＲ与人巨细胞病毒（ＨＣＭＶ）有高度同源性,最适记分（Ｏｐｔｉｍｉｚｅｄｓｃｏｒｅ）达４５９。实验结果支持Ｅｓｆｔａｔｈｉｏｕ提出的论点,ＨＨＶ－６属于β－疱疹病毒。     

Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells.

Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.     

5,7-Diphenyl-3-ureidohexahydroazepin-2-ones as Cholecystokinin-B receptor ligands

A series of 5,7-diphenyl-3-ureidohexahydroazepin-2-one cholecystokinin-B (CCK-B) receptor antagonists was synthesized using Beckmann ring expansion of a suitable 2,4-diphenylcyclohexanone as a key step. SAR studies revealed the importance of the 5-aryl group for high and selective CCK-B receptor affinity, as illustrated in compound (−)-10i (CCK-B IC₅₀ = 6.8 nM).