Expression of human factor X with normal biological activity in human embryonic kidney cells

Summary Human embryonic kidney cells (293) were transfected with a construct containing human factor X cDNA and selected for G418 resistance. The level of expression of recombinant factor X in serum-free medium was 4 to 5 g/ml. Purified recombinant factor X had a molecular size identical to that of normal plasma factor X. Amino-terminal sequencing revealed normal processing cleavages. The -carboxy Glu and -OH Asp content of the recombinant factor X was close to 90% of the expected levels of these post-translational residues. The specific activity of recombinant factor X was about 95% of that of plasma factor X in three plasma-based clotting assays. This report demonstrates that 293 cells can produce a high level of biologically active factor X and describes a visual criterion for verifying the transfection process.Abbreviations FX factor X - rFX recombinant factor X - DMEM Dulbecco's modified Eagle's medium - RVV-X Russell's viper venom - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - Gla -carboxy glutamic acid     

Chirospecific synthesis of D and Lp-chlorohomophenylalanine N-t-BOC DCHA salts

Summary The chirospecific conversions of D-glucosamine hydrochloride and D-mannosamine hydrochloride to the configurationally stable L and D isomers of N-t-butyloxycarbonylserinal were carried out byt-butylcarbonylation followed by sodium borohydride reduction and sodium meta-periodate oxidation. Reaction of the L and D aldehydes with the Wittig reagent prepared from 4-chlorobenzyltriphenylphosphonium chloride and butyl lithium followed by catalytic hydrogenation, Jones oxidation and salt formation with dicyclohexylamine gave the DCHA salts of the D and L isomers ofp-chlorohomophenylalanine N-t-Boc in high enatiomeric excess. The optical purity of the title compounds was established by hydrolysis to the respective free amino acids, followed by chiral derivatization and HPLC analysis.This was presented at the Fifth International Kyoto Conference on new Aspects of Organic Chemistry, Kyoto, Japan, November 11–15, 1991. Abstract #GO-13.     

MHC antigen expression by melanomas recovered from mice treated with allogeneic mouse fibroblasts genetically modified for interleukin-2 secretion and the expression of melanoma-associated antigens

Recent approaches toward the immunotherapy of neoplastic disease involve the introduction of expression-competent genes for interleukin-2 (IL-2) into autologous malignant cells. Treatment of tumor-bearing experimental animals with the IL-2-secreting cells successfully induces partial and at times complete remissions. In most instances, however, although delayed, progressive tumor growth continues. Here, certain of the characteristic of B16 melanomas (H-2^b) persisting in C57BL/6 mice (H-2^b) treated with an IL-2-secreting, melanoma-antigen-positive cellular immunogen (RLBA-IL-2 cells) are described. Unlike the melanoma cells first injected, B16 cells recovered from mice treated with RLBA-IL-2 cells were deficient in the experssion of MHC class I, but not class II determinants. Deficient MHC class I expression correlated with the cells' resistance to cytotoxic T lymphocytes (CTL) from the spleens of mice immunized with RLBA-IL-2 cells. Melanomas persisting in mice treated with non-IL-2-secreting, melanoma-antigen-positive cell constructs (RLBA-ZipNeo cells) were also deficient in the expression of MHC class I determinants, and the melanoma cells were resistant to CTL from mice immunized with RLBA-ZipNeo cells. Thus, the expression of melanoma-associated antigens rather than IL-2-secretion correlated with deficient MHC class I expression by the persistent melanomas. This point was substantiated by the expression of MHC class I antigens by melanomas persisting in mice treated with IL-2-secreting, melanoma-antigen-negative LM cells (LM-IL-2); it was equivalent to that of melanomas in untreated mice. The involvement of MHC class I antigens in the immune resistance of persistent melanoma cells from mice treated with the melanoma-autigen-positive immunogens was indicated by the effect of interferon (IFN) orN-methyl-N-nitro-N-nitrosoguanidine (MNNG) on the susceptibility of the cells to anti-melanoma CTL. Treatment of the resistant melanomas with IFN or MNNG stimulated MHC class I antigen expression and restored the cells' sensitivity to CTL from mice immunized with IL-2-secreting or nonsecreting, melanoma-antigen-positive cellular immunogens. Prior treatment of the treated cells with antibodies to MHC class I determinants inhibited the cells' susceptibility to CTL from mice immunized with RLBA-IL-2 cells.     

Production and initial characterisation of the xylan-degrading system of Phanerochaete chrysosporium

We report the optimum conditions for the degradation of oat spelt arabinoxylan and a preliminary characterisation of the inducible xylan-degrading system of the lignin-degrading white-rot fungus Phanerochaete chrysosporium. Xylanase activity was optimal at pH 5.0 and 50°C; see attached sheet the maximum reaction velocity (V_max) of the system was 3.86 units (U) mg^–1 protein with arabinoxylan as substrate and the substrate concentration giving half V_max (S_0.5) was 0.52 mg ml^–1. At concentrations of arabinoxylan greater than 15 mg ml^–1 excess substrate inhibition was observed. Xylose at 0.9 mm inhibited activity to the extent of 50%. Xylanase activity increased as a function of the dilution of the enzyme preparation prior to assay. It was resolved into four peaks by using a DEAE-Biogel column; the material in these peaks differed with respect to xylan solubilisation and the formation of reducing sugars. Electrofocusing gels allowed visualisation of several bands of activity corresponding to each peak. The arabinoxylan degradation system of P. chrysosporium is therefore composed of multiple components. Correspondence to: P. Broda     

Z → B transition in poly[d(G-m5C)2] induced by interaction with 4′,6-diamidino-2-phenylindole

The Z form of poly[d(G-m⁵C)₂], in presence of Mg²⁺ ion, is found to be transformed into B form upon interaction with 4′,6-diamidino-2-phenylindole (DAPI). The Z → B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6–7 base pairs. An interaction between DAPI and poly[d(G-m⁵C)₂] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time-resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature. © 1993 John Wiley & Sons, Inc.     

ANNUAL DIFFERENCES IN FEMALE REPRODUCTIVE SUCCESS AFFECT SPATIAL AND COHORT-SPECIFIC GENOTYPIC HETEROGENEITY IN PAINTED TURTLES

Long-term ecological data were used to evaluate the relative importance of movements, breeding structure, and reproductive ecological factors to the degree of spatial and age-specific variation in genetic characteristics of painted turtles (Chrysemys picta) on the E. S. George Reserve in southeastern Michigan. Estimates of the degree of spatial genetic structuring were based on the proportion of total genotypic variance partitioned within and between subpopulations (inferred from hierarchical F-statistics based on variation at 18 protein loci), and in terms of gene correlations (co-ancestry among individuals derived from reproductive data on full-sib families of females nesting at specific nesting areas). Little variation in allele frequency was observed among turtles from different marshes (F_mt = 0.003), though significant variation was observed among turtles from different nesting areas associated with each marsh (F_nm = 0.046). Gene correlations among individuals within nesting areas varied greatly over years (0.032-0.171; mean = 0.069) and were negatively correlated to the proportion of females that successfully nested during each year. General concordance between independent estimates of genotypic correlations (i.e., F_nm derived from protein electrophoretic variation vs. mean co-ancestry) suggests that allozyme data, when collected over spatial scales consistent with species behavioral characteristics and reproductive ecology, may accurately reflect the apportionment of gene diversity within and among subpopulations. The magnitude and patterning of allelic variation among nesting areas and individuals appears to be primarily a function of gametic correlations among members of full-sib families, irrespective of the degree of gene flow or female nesting-site fidelity. Comparisons of genetic characteristics among 11 cohorts (1974-1984) revealed that heterozygosity (H) and inbreeding coefficients (F) varied greatly. Cohort estimates of H and F were correlated to female nesting success and to estimates of co-ancestry for the same years. Results clearly reflect the concomitant importance of ecological factors (principally the proportion of the female population that successfully produce offspring during each year) in determining the magnitude and patterning of gene correlations within and among groups, and to the genotypic composition of offspring born during each year.     

Cytological and molecular characterization of a chromosome interchange and addition lines in Cadet involving chromosome 5B of wheat and 6Ag of Lophopyrum ponticum

Efforts to transfer wheat curl mite (Eriophyes tulipae Keifer) resistance from Lophopyrum ponticum 10X (Podb.) Love to bread wheat (Triticum aestivum L.) have resulted in the production of a number of cytogenetic stocks, including an addition line of 6Ag, a ditelo addition line, and a wheat-Lophopyrum translocation line. Characterization of these lines with C-banding, in situ hybridization with a Lophopyrum species-specific repetitive DNA probe (pLeUCD2), and Southern blotting with pLeUCD2 and a 5S ribosomal DNA probe (pScT7) confirmed that the distal portion of the short arm of 6Ag was translocated onto the distal portion of 5BS (5BL. 5BS-6AgS). It was also determined that the ditelo addition was an acrocentric chromosome of 6AgS.     

Model-based control of feed rate and illuminance in a photosynthetic fed-batch reactor for H2S removal

Traditional application of computer to fermentation processes has focused on the measurement and control of parameters such as temperature, pH, vessel pressure, sparge rate, dissolved oxygen, substrate concentration, and product concentration. In a fed-batch reactor with the photosynthetic green sulfur bacterium Chlorobium thiosulfatophilum which converts hydrogen sulfide to elementary sulfur or sulfate, separate measurement of cell mass concentration and sulfur particle concentration turbidimetrically was difficult due to their combined contributions to the total turbidity. Instead of on-line measurement of many process variables, a model-based control of feed rate and illuminance was designed. Optimal operation condition relating feed rate vs. light intensity was obtained to suppress the accumulation of sulfate and sulfide, and to save light energy in a 4-1 photosynthetic fed-batch reactor. This relation was correlated with the inreasing cell mass concentration. A model which describes the cell growth by considering the light attenuation effects due to scattering and absorption, and to crowding effect of the cells, was established beforehand with the results from the experiments. Based on these optimal operating conditions and the cell growth model, automatic controls of feed rate and illuminance were carried out alternatively to the traditional application of computer to fermentation with on-line measurement, realtime response and adjustment of process variables.List of Symbols F ml/min Flow rate of gas mixture - hV lux Average illuminance - Q mmol/(l h) Removal rate of hydrogen sulfide - X mg protein/l Cell mass concentration as protein - X ₀ mg protein/l Initial cell mass concentration - X _m mg protein/l Maximum cell mass concentration - _a h^–1 Apparent specific growth rate     

Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.