The cytochromes P450 (P450s) are important enzymes involved in metabolic pathways, which use exogenous and endogenous substances as their substrate for various enzymatic reactions. These enzymes can also use precarcinogens as their substrate and activate them into carcinogens, which leads to a cancer development. If the P450s are induced, the cancer risk increases. Some chemopreventive compounds may induce the P450s and thus be harmful to the human body. Therefore it is necessary to pay enough attention to a study of the mechanism of action of P450s and the influence of the chemopreventive compounds on the activity of cytochromes P450. mRNA expression of most of the P450s isoforms is detected in a number of healthy (nontransformed) tissues, viz. liver, brain, heart, colon, kidney or placenta. Nevertheless there are a few P450s isoforms which mRNAs are expressed at relatively low levels in the nontransformed tissues, whereas the expression in the transformed tissues is significantly higher. One of these P450s is CYP2W1, which can be used as a prognostic marker for colorectal cancer - therefore it is useful to be able to detect a presence of this enzyme in various tissues. A detection of P450s can be accomplished by using a method Western blot. In this method, the immunodetection is achieved by using...

ABSTRACT OBJECTIVE: To determine the prevalence and clinical correlates of anti-phospholipid antibodies (aPL), including anti-cardiolipin antibodies (aCL), lupus anti-coagulant (LA), anti- β2-glycoprotein 1 (aβ2GP1) and anti-prothrombin (aPT) antibodies, in Black South African patients with systemic lupus erythematosus (SLE) METHODS: A cross-sectional study of 100 SLE patients in whom clinical characteristics, including features of the anti-phospholipid syndrome (APS), disease activity, and damage were documented, and sera tested for aCL, aβ2GP, and aPT of all isotypes, and LA. RESULTS: Positive aCL, aβ2GP1 and aPT and LA were found in 53, 84, 20, and 2 patients, respectively. Immunoglobulin (Ig)A aCL and IgG aβ2GP1 were the commonest aCL (49.1%) and aβ2GP1 (47%) isotypes, respectively. IgA aβ2GP1 were associated with both a history of thrombosis alone (p<0.05) and a history of any clinical feature, thrombosis and/or spontaneous abortion of the APS (p<0.05); IgA aCL were associated with a history of any clinical APS event (p<0.05); and aβ2GPI of any isotype were associated with a history of arthritis (p<0.001). CONCLUSION: My findings provide further evidence that the screening for aβ2GP1 and IgA aCL isotype may improve the risk assessment for APS in SLE patients of African extraction. Further prospective studies are warranted to determine the clinical utility of these tests and to elucidate the genetic basis for increased IgA aPL response in SLE patients of African extraction.

Glicoconjugados são abundantes na superfície de Trypanosoma cruzi e têm sido bastante estudados por diferentes grupos. A degradação dessas moléculas, no entanto, tem sido alvo de pouco interesse. O objetivo deste trabalho foi determinar a atividade de alfa-L-fucosidase em T.cruzi uma vez que trabalhos anteriores haviam concluído que várias hidrolases, entre elas a alfa-L-fucosidase estavam ausentes em epimastigota (AVILA, et al., 1979). Empregando-se p-nitrofenilfucopiranosídeo como substrato e extrato de formas epimastigotas, verificou-se que a enzima apresenta praticamente a mesma atividade em um intervalo de pH entre 6,0 e 7,5, caindo drasticamente em pHs mais ácidos. A incubação prévia da enzima a 28°C em pH 7,0 leva à perda de aproximadamente 30% de sua atividade após 1h 30mim e à perda de 100% após 4 horas de incubação. O efeito de íons na atividade da enzima foi estudado,verificando-se que Zn +2 inibe 90% sua atividade, enquanto que outros, como o Ca +2 praticamente não tem efeito. A enzima é parcialmente encontrada na fração particulada, podendo ser solubilizada parcialmente com 1% de Triton X-100 ou com NaCl 1 M. As tentativas feitas de purificar a enzima foram infrutíferas, uma vez que não se encontraram condições para manter a proteína ativa por longos períodos de tempo. A alfa-L-fucosidase está presente não só em pimastigotas, mas também em tripomastigotas, embora parentemente com diferentes atividades específicas, sendo maior em epimastigotas. Mesmo nos epimastigotas, grandes variações de atividade específica foram detectadas ao longo deste trabalho (de 0,03 a 0,23 unidades). Anticorpos preparados contra alfa-L-fucosidase comercial de epidídimo bovino imunoprecipitaram de extratos de epimastigotas previamente marcados com 35 S-metionina, um polipeptídeo em torno de 50 kDa após eletroforese em gel desnaturante e uma banda de 130-150 kDa em gel não desnaturante, sugerindo que a enzima em T.cruzi pode ser dimérica, a exemplo de outras alfa-L-fucosidases descritas na literatura. A imunoprecipitação de extrato de epimastigotas marcados com 35 S-metionina na presença de tunicamicina, com o anticorpo anti-alfa-L-fucosidase revelou um polipeptídeo de 45 kDa, mostrando que a enzima é glicosilada. A glicosilação daenzima também foi observada pelo emprego de corantes comerciais. Além disso, os anticorpos anti-alfa-L-fucosidase imunoprecipitam moléculas com atividade de alfa-L-fucosidase,embora não se tenha observado aumento da atividade, possivelmente devido à perda de atividade da enzima nas condições empregadas durante a imunoprecipitação. Os anticorpos anti-alfa-L-fucosidase reconhecem, por imunofluorescência indireta, tanto as formas epimastigotas como tripomastigotas de cultura de tecido. A análise por microscopia de transmissão mostra a reatividade intensa do anticorpo com uma região membranar localizada na região posterior do epimastigota. No caso do tripomastigota, a reatividade é menos pronunciada mostrando uma leve marcação no interior do parasita. / Alpha-L-fucose is a component of glycoproteins, inc1uding glycoproteins isolated from Tcruzi. a-L fucosidases have been isolated from different sources, but earlier studies were unable to detect this enzyme in T. cruzi epimastigotes (AVILA et al., 1979). In this work immunocytochemical and biochemical techniques have been used to localize and characterize a membrane-associated, neutral-pH-optimum alpha-L fucosidase from Trypanosoma cruzi epimastigotes. Light and electron microscopy specifically localized the alpha-L fucosidase on membranes in the posterior region of the epimastigotes and on the parasite surface. Immunoreactivity for alpha-L-fucosidase, a1though less intense, was also detected on the surface of trypomastigotes. Fractionation of epimastigotes homogenates indicated that over 50% of the a-Lfucosidase activity was associated with the 80 000 g pellet. This pellet-associated activity could be solubilized with 1 M NaCl or with 1% Triton X-I 00, suggesting that alpha-L-fucosidase is peripherally associated with membranes. Analysis of alpha-L-fucosidase on epimastigote extracts indicated that the enzyme had a pH-activity curve (with an optimum near 7) which was comparable to other alpha-L-fucosidases reported in the literature. A higher specific activity (in units/mg) was found in epimastigotes as compared to the other differentiation stages of the parasite: 0.028 for epimastigotes, 0.002 for metacyc1ic trypomastigotes and 0.015 for tissue - cultured trypomastigotes. SDS/PAGE and Westem blotting analysis indicated that epimastigotes have a protein band of 50 kDa which was immunoreactive with anti-alpha-L-fucosidase antibodies.

by Chiu-lun Fok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / List of Abbreviations --- p.i / Abstract --- p.iii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Properties of Oxalysine --- p.1 / Chapter 1.1.1 --- Chemical Structure and Properties --- p.1 / Chapter 1.1.2 --- Biological Properties --- p.2 / Chapter 1.1.2.1 --- Antimicrobial Activity --- p.2 / Chapter 1.1.2.2 --- Antitumor Activity --- p.2 / Chapter 1.1.2.3 --- Immunomodulatory Activity --- p.5 / Chapter 1.1.2.4 --- Other Biological Properties --- p.5 / Chapter 1.1.3 --- Pharmacokinetics and Toxicity --- p.6 / Chapter 1.2 --- General Properties of Ribosome-Inactivating and Abortifacient Proteins --- p.8 / Chapter 1.2.1 --- Research History --- p.8 / Chapter 1.2.1.1 --- Ribosome-Inactivating Proteins --- p.8 / Chapter 1.2.1.2 --- Abortifacient Proteins --- p.9 / Chapter 1.2.2 --- Relationship between Ribosome- Inactivating Proteins and Abortifacient Proteins --- p.10 / Chapter 1.2.3 --- Distribution --- p.11 / Chapter 1.2.4 --- Physicochemical Characteristics --- p.12 / Chapter 1.2.5 --- Biological Properties --- p.13 / Chapter 1.2.5.1 --- Effect on Protein Synthesis --- p.13 / Chapter 1.2.5.2 --- Effect on the Immune System --- p.14 / Chapter 1.2.5.3 --- Cytotoxic and Antitumor Activities --- p.16 / Chapter 1.2.5.4 --- Termination of Pregnancy --- p.17 / Chapter 1.2.5.5 --- Antiviral Activity --- p.18 / Chapter 1.2.6 --- The Study on Luffaculin --- p.19 / Chapter 1.3 --- Aim of the Present Study --- p.20 / Chapter 1.3.1 --- Oxalysine --- p.20 / Chapter 1.3.2 --- Luffaculin --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials --- p.22 / Chapter 2.2 --- Methods --- p.30 / Chapter 2.2.1 --- In Vivo Drug Treatment --- p.30 / Chapter 2.2.2 --- Isolation and Preparation of Cells --- p.30 / Chapter 2.2.2.1 --- Peritoneal Exudate Cells --- p.30 / Chapter 2.2.2.2 --- Spleen Cells --- p.30 / Chapter 2.2.2.3 --- Ficoll-Paque Separation of Lymphocytes --- p.31 / Chapter 2.2.2.4 --- Congo Red-Stained Yeast Cells --- p.31 / Chapter 2.2.3 --- Lymphocyte Transformation --- p.32 / Chapter 2.2.4 --- Haemolytic Plaque Assay --- p.33 / Chapter 2.2.5 --- Phagocytic Activity --- p.33 / Chapter 2.2.6 --- Macrophage-Mediated Cytostatic Activity --- p.34 / Chapter 2.2.7 --- Delayed Type Hypersensitivity (DTH) --- p.35 / Chapter 2.2.8 --- Production of and Assay for Interleukin-2(IL-2) --- p.36 / Chapter 2.2.9 --- Cytotoxicity of the Drugs on Various Cell Lines --- p.38 / Chapter 2.2.9.1 --- Trypan Blue Exclusion Method --- p.38 / Chapter 2.2.9.2 --- Neutral Red Uptake Method --- p.38 / Chapter 2.2.10 --- Cytostatic Effect of the Drugs on Various Cell Lines --- p.39 / Chapter 2.2.11 --- Evaluation of Antitumor Activity (In Vivo ) --- p.40 / Chapter 2.2.11.1 --- Tumor Size --- p.40 / Chapter 2.2.11.2 --- Survival Study --- p.40 / Chapter 2.2.12 --- TLC Analysis --- p.40 / Chapter 2.2.13 --- Preparation of Ribosome-Inactivating and Abortifacient Proteins --- p.41 / Chapter 2.2.13.1 --- Trichosanthin (TCS) --- p.41 / Chapter 2.2.13.2 --- Luffaculin (LFC) --- p.42 / Chapter 2.2.14 --- Protein Determination --- p.42 / Chapter 2.2.15 --- Statistical Analysis --- p.43 / Chapter Chapter 3 --- The Immunomodulatory and Antitumor Activities of Oxalysine (OXL) --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- The Immunomodulatory Activity of Oxalysine --- p.46 / Results --- p.46 / Chapter 3.2.1 --- Effect of Oxalysine on the Proliferation of Mouse Splenocytes --- p.46 / Chapter 3.2.2 --- Effect of In Vitro Oxalysine Exposure on the Response of Murine Splenocytes to Mitogens --- p.46 / Chapter 3.2.3 --- Effect of In Vivo Oxalysine Treatment on the Response of Murine Splenocytes to Mitogens --- p.49 / Chapter 3.2.4 --- Effect of Oxalysine on Delayed Type Hypersensitivity (DTH) Response --- p.51 / Chapter 3.2.5 --- Effect of Oxalysine on the In Vitro Phagocytic Activity of Mouse Peritoneal Macrophages --- p.51 / Chapter 3.2.6 --- Effect of Oxalysine on Macrophage- Mediated Cytostatic Activity --- p.53 / Chapter 3.2.7 --- Effect of Oxalysine on the Humoral Response to SRBC --- p.55 / Discussion --- p.59 / Chapter 3.3 --- Mechanistic Studies on Inhibition of Mitogen´ؤ Induced Lymphocyte Transformation by Oxalysine --- p.62 / Results --- p.62 / Chapter 3.3.1 --- Lack of Direct Cytotoxic Effect of Oxalysine on Mouse Splenocytes In Vitro --- p.62 / Chapter 3.3.2 --- Effect of Oxalysine on the Kinetics of Con A-Induced Lymphoproliferative Response --- p.62 / Chapter 3.3.3 --- Time Course Studies on the Effect of Oxalysine on Mitogen-Induced Lymphocyte Transformation --- p.64 / Chapter 3.3.3.1 --- Preincubation of Oxalysine --- p.64 / Chapter 3.3.3.2 --- Delayed Addition of Oxalysine --- p.67 / Chapter 3.3.4 --- Effect of Exogenous IL-2 on the Oxalysine-Mediated Suppression of Lymphocyte Blastogenesis --- p.69 / Chapter 3.3.5 --- Effect of Oxalysine on the Activity of IL-2 Containing Medium to Maintain the Proliferation of the IL´ؤ2 Dependent CTLL-2 Cells --- p.73 / Chapter 3.3.6 --- Production of IL-2 from Splenocytes of Oxalysine´ؤTreated Mice --- p.75 / Chapter 3.3.7 --- The In Vitro Effect of Oxalysine on the Production of IL-2 from Con A-Activated Mouse Splenocytes --- p.75 / Discussion --- p.79 / Chapter 3.4 --- The Antitumor Activity of Oxalysine --- p.83 / Results --- p.83 / Chapter 3.4.1 --- Cytotoxicity of Oxalysine on Various Tumor Cell Lines --- p.83 / Chapter 3.4.2 --- Cytostatic Effect of Oxalysine on Various Tumor Cell Lines --- p.85 / Chapter 3.4.3 --- Effect of Oxalysine on the Survival of Tumor-Bearing Mice --- p.93 / Chapter 3.4.4 --- Effect of Oxalysine on the Growth of Transplantable Tumor Cells In Vivo --- p.95 / Discussion --- p.100 / Chapter 3.5 --- General Discussion --- p.102 / Chapter Chapter 4 --- The Immunomodulatory and Cytotoxic Properties of Luffaculin (LFC) --- p.104 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- The Immunomodulatory Activity of Luffaculin --- p.106 / Results --- p.106 / Chapter 4.2.1 --- Lack of Direct Cytotoxic Effect of LFC on Mouse Splenocytes In Vitro --- p.106 / Chapter 4.2.2 --- Effect of Luffaculin on the Proliferation of Mouse Splenocytes --- p.108 / Chapter 4.2.3 --- Inhibition of the Mitogen-Induced Lymphocyte Transformation by Luffaculin --- p.108 / Chapter 4.2.4 --- Effect of Luffaculin on Delayed Type Hypersensitivity (DTH) Response --- p.114 / Chapter 4.2.5 --- Primary Humoral Immune Response to SRBC in Luffaculin-Treated Mice --- p.114 / Chapter 4.2.6 --- Effect of Luffaculin on Phagocytosis of Macrophages In Vitro --- p.117 / Chapter 4.2.7 --- Effect of Luffaculin on Macrophage- Mediated Cytostatic Activity --- p.117 / Chapter 4.2.8 --- Production of Interleukin´ؤ2 from Splenocytes of Luffaculin-Treated Mice --- p.119 / Discussion --- p.122 / Chapter 4.3 --- The Cytotoxic and Cytostatic Effects of Luffaculin on Various Tumor Cell Lines --- p.125 / Results --- p.125 / Chapter 4.3.1 --- Cytotoxicity of Luffaculin on Various Tumor Cell Lines --- p.125 / Chapter 4.3.2 --- Cytostatic Effect of Luffaculin on Various Tumor Cell Lines --- p.127 / Discussion --- p.138 / Chapter 4.4 --- General Discussion --- p.140 / References --- p.143

by Yeung Shing On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1989 / Bibliography: leaves 91-112.

While the last century of medical discoveries has made a significant impact on improving the lives of human populations across the globe, a perfect solution to the yearly infection cycle from the influenza virus has yet to be discovered. Although vaccines stand the best chance at targeting yearly epidemics, new treatment options must be created to combat the arrival of rapidly mutating and antiviral-resistant strains of the virus that could lead to another pandemic such as the 1918 Spanish flu that killed millions worldwide. We describe a method to create functionally enhanced monoclonal antibodies targeting influenza via genetic engineering of fragment crystallizable glycan structures. Muscle and liver cell lines were lentivirally-transduced to produce the broadly neutralizing antibody, Fi6v3, while also overexpressing a critical glycosylation enzyme, B-1,4-N-acetyl-glucosaminyltransferase III. Secreted antibodies were tested for effector functionality using a Natural Killer cell degranulation assay and an antibody-dependent cellular phagocytosis assay. Results conclude that modified antibodies from both muscle and liver cells lines exhibit enhanced function in comparison to their unmodified counterparts, providing support to the future creation of an influenza prophylactic or treatment option using antibodies with the ability to more effectively activate innate immune killing mechanisms.

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability to perform correct post-translational modifications. A major issue for use of CHO cells lines for the production of recombinant proteins is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval. Protein production is complex and is influenced by cell growth, transcription, translation, protein folding and post-translational processing and secretory events, which may interact to determine stability of expression during prolonged culture. This thesis aims to identify features associated with stability/instability of recombinant protein expression and methods to improve protein production, with the addition of chemically defined (CD) feed and chemicals. Two exemplar CHO cell lines, which secrete the same recombinant antibody were characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and 51.69) exhibited unstable protein production over LTC, with a loss in final antibody titres and specific productivity (Qp). The instability observed within the exemplar cell lines was not due to decreased recombinant gene copy numbers or mRNA expression but was associated with lower viable cell densities, increased ER stress (GADD153 and spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during the decline phase of batch culture). Improvement of recombinant protein expression in response to feed or DMSO addition was associated with lower expression of ER stress markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein level) and alterations to the metabolic activity of the cultures (prevention of alanine and lactate re-utilisation, and greater glucose utilisation between the stationary and decline phase of batch culture).Although feed or DMSO addition improved recombinant protein production, these additions did not reverse the appearance or progression of instability for cells after LTC. ER stress expression was not abolished as a consequence of feed or DMSO addition. Expression of stress markers at earlier time points may be the factor that limits antibody production and secretion. The consequences of the presence of feed and DMSO addition on ER stress markers and antibody production serves to highlight approaches that may be utilised for engineering more productive or stable protein production phenotypes in parental cell lines.

No description available.

Kwok Leung Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 149-170). / Acknowledgments --- p.i / Abstract --- p.ii -v / Abbreviations --- p.vi-vii / Chapter Chapter 1 : --- General Introduction --- p.1 / Chapter Chapter 2 : --- Development of Polyclonal Antibodies Against 6-B-hydroxycortisol （6-B-OHC) And Its Applications / Chapter 2.1 : --- Introduction --- p.40 / Chapter 2.2 : --- Materials and methods --- p.43 / Chapter 2.3: --- Results --- p.55 / Chapter 2.4: --- Discussion --- p.73 / Chapter Chapter 3 : --- Development of Monoclonal Antibody-Based ELISA Against 6-B-hydroxycortisol (6-B-OHC) And Its Applications / Chapter 3.1 : --- Introduction --- p.76 / Chapter 3.2 : --- Materials and methods --- p.89 / Chapter 3.3: --- Results --- p.108 / Chapter 3.4: --- Discussion --- p.135 / Chapter Chapter 4 --- : General Conclusion --- p.141 / References --- p.149

An antibody microarray consists of antibody bound to a surface. Antibody microarrays have great potential in many fields, particularly as a tool to detect antigens. Unfortunately, antibodies suffer from poor performance. A greater understanding of how antibodies interact with surfaces would improve microarray design and performance, but experimental methods fall short of being able to observe these interactions. Therefore molecular simulation has emerged as the primary method to study protein/surface interactions.The simulations here were coarse grain simulations performed using the model of Karanicolas and Brooks. Additionally, an advanced surface model was used that allows for different surface chemistries. PyMBAR analysis was used to find heat capacities and determine relative stabilities of different linkers and tethering sites for the antibody/surface system.The actual work looked at how 24 different tethering sites affect antibody stability on two different surfaces and examined nine linkers varying in length and rigidity. Ultimately the findings were that antibody stability is a function of tethering position when tethered to a hydrophobic surface, but not when tethered to a hydrophilic surface. Furthermore, the length and rigidity of the linkers do not have a significant impact on stability.