Polyhydroxyalkanoate production in recombinant Escherichia coli

Abstract The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-β-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate- co -3-hydroxyvalerate (PHB- co -3-).     

Characterization of in vitro reactivity by BCG-treated guinea pigs to syngeneic line-10 hepatocarcinoma

Summary The purpose of this study was to characterize in vitro the systemic tumor immunity induced by a BCG-intratumoral injection in line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2). Macrophages from BCG-tumor-cured guinea pigs at effector to target cell ratios of 10:1 and 100:1 were cytotoxic in vitro to line-10 tumor cells, and this cytotoxicity was potentiated by autologous serum. Significant cytotoxicity of lymphocytes from BCG-tumor-cured guinea pigs could only be achieved at ratios of 10,000:1, and no effect of autologous serum could be demonstrated. Lymphocytes from both normal and BCG-tumor-cured (line-10 immune) guinea pigs had a significant cytotoxic effect on the highly antigenic line-1 cells at ratios of 1:10,000. Macrophages from both normal and line-10 immune guinea pigs were cytotoxic to line-1 target cells at ratios of 1:100. With respect to specific cytotoxicity (cytotoxicity above and beyond levels achieved with effector cells from normal animals), the only significant difference was demonstrated when line-10 served as target cells and the effector cells were isolated from BCG-tumor-cured (line-10 immune) guinea pigs. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; CMEM, complete minimum essential medium; cpm, counts per minute; HBSS, Hanks' balanced salt solution; i.d. intradermally; i.p., intraperitoneally; PEC, peritoneal exudate cells; SDA, superficial distal axillary; ¹²⁵IdUrd, [¹²⁵I]iododeoxyuridine.     

Testing for Independence of Binary Responses

In the context of experiments involving visual inspection of random dot patterns the problem of testing the null hypothesis of independence of binary responses is considered. A flexible model for dependence between binary responses is proposed. Two tests, optimal under different versions of the model, are derived. These two tests turn out to involve the same computations as the Wilcoxon two sample test and the runs test respectively.     

Dynamics of immunoglobulin sequence diversity in HIV-1 infected individuals

Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables such as viral load and CD4⁺ count. Although there are many potential explanations for this, we suggest that important factors include poor sampling resolution and complex B-cell dynamics that are difficult to summarize using simple summary statistics. Importantly, we find a significant association between observed Gini indices and sequencing read depth, and we conclude that more robust analytical methods and a closer integration of experimental and theoretical work is needed to further our understanding of B-cell repertoire diversity during viral infection.     

Syngeneic humoral immune responses to tumor-associated antigens expressed by K-1735 UV-induced melanoma and its metastases

Summary An enzyme-linked immunoassay (ELISA) was developed to study syngeneic humoral immune response to a primary tumor and its metastases in the K-1735 ultraviolet light (UV)-induced C3H murine melanoma system. Binding of sera from syngeneic animals previously immunized with primary tumor or metastatic tumor tissue (M-3, M-4) to corresponding 3 M KCl extracts of tumor was significantly greater than binding of control C3H mouse serum. Antibody binding was not significantly reduced by competitive binding with syngeneic murine muscle or liver extracts, indicating the presence of tumor antigen(s) not shared by normal murine tissue. Antibodies to the tumor-associated antigens were selectively removed by competitive binding with syngeneic K-1735 tumor extracts but not by the unrelated 102 murine sarcoma from C57BL/6. However, EL-4 extracts (C57BL/6) did inhibit antibody binding to the primary and both metastases. Further competitive binding studies demonstrated the presence of a common antigen(s) present on the primary tumor and both metastases. We conclude that the K-1735 UV-induced melanoma primary tumor and its metastases express serologically detectable shared antigenic determinate. Abbreviations used in this paper: CBI, competitive binding inhibition; CF, complement fixation; HI, hemagglutination inhibition; PBS, Dulbecco's phosphate-buffered saline; UV, ultraviolet light