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Jurgen Markl - One of the best experts on this subject based on the ideXlab platform.
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the refined structure of functional unit h of Keyhole Limpet Hemocyanin klh1 h reveals disulfide bridges
Iubmb Life, 2011Co-Authors: Elmar Jaenicke, Jurgen Markl, Kay Buchler, Heinz Decker, Gunnar F SchroderAbstract:Hemocyanins are multimeric oxygen-transport proteins in the hemolymph of many arthropods and mollusks. The overall molecular architecture of arthropod and molluscan Hemocyanin is very different, although they possess a similar binuclear type 3 copper center to bind oxygen in a side-on conformation. Gastropod Hemocyanin is a 35 nm cylindrical didecamer (2 × 10-mer) based on a 400 kDa subunit. The latter is subdivided into eight paralogous “functional units” (FU-a to FU-h), each with an active site. FU-a to FU-f contribute to the cylinder wall, whereas FU-g and FU-h form the internal collar complex. Atomic structures of FU-e and FU-g, and a 9 A cryoEM structure of the 8 MDa didecamer are available. Recently, the structure of Keyhole Limpet Hemocyanin FU-h (KLH1-h) was presented as a Cα-trace at 4 A resolution. Unlike the other seven FU types, FU-h contains an additional C-terminal domain with a cupredoxin-like fold. Because of the resolution limit of 4 A, in some loops, the course of the protein backbone could not be established with high certainty yet. Here, we present a refined atomic structure of FU-h (KLH1-h) obtained from low-resolution refinement, which unambiguously establishes the course of the polypeptide backbone and reveals the disulfide bridges as well as the orientation of bulky amino acids. © 2011 IUBMB IUBMB Life, 63(3): 183–187, 2011
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Keyhole Limpet Hemocyanin 9 a cryoem structure and molecular model of the klh1 didecamer reveal the interfaces and intricate topology of the 160 functional units
Journal of Molecular Biology, 2009Co-Authors: Christos Gatsogiannis, Jurgen MarklAbstract:Abstract Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan Hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-A cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod Hemocyanin decamer [ Nautilus pompilius Hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five “arcs” (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod Hemocyanin didecamer assembled from two asymmetric decamers [isoform Keyhole Limpet Hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five “slabs” (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-A data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod Hemocyanin and essentially solve the gastropod Hemocyanin structure.
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haliotis tuberculata Hemocyanin hth analysis of oligomeric stability of hth1 and hth2 and comparison with Keyhole Limpet Hemocyanin klh1 and klh2
Micron, 2000Co-Authors: J. R. Harris, Wolfgang Gebauer, Dirk Scheffler, R Lehnert, Jurgen MarklAbstract:Abstract The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata Hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from Keyhole Limpet Hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole Limpet Hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31–41; Harris, J.R., Gebauer, W., Sohngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole Limpet Hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43–56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole Limpet Hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329–339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8 MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100 mM concentrations of calcium and magnesium ions in Tris–HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100 mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100 mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two Hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan Hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.
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Keyhole Limpet Hemocyanin molecular structure of a potent marine immunoactivator a review
European Urology, 2000Co-Authors: Robin J Harris, Jurgen MarklAbstract:Objectives: In this short review we present a survey of the available biochemical and electron microscopic data on Keyhole Limpet Hemocyanin (KLH). Results: The b
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Keyhole Limpet Hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h.
Journal of structural biology, 1999Co-Authors: Wolfgang Gebauer, J. Robin Harris, Gaby Geisthardt, Jurgen MarklAbstract:Keyhole Limpet Hemocyanin (KLH) is a mixture of two Hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan Hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.
Philip O. Livingston - One of the best experts on this subject based on the ideXlab platform.
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Keyhole Limpet Hemocyanin conjugate vaccines against cancer the memorial sloan kettering experience
Journal of Cancer Research and Clinical Oncology, 2001Co-Authors: Cristina Musselli, Philip O. Livingston, Govind RagupathiAbstract:Passively administered and actively induced antibodies have been associated with the eradication of circulating tumor cells and micrometastases in mice and humans. We have identified a series of cell surface carbohydrate and peptide antigens on melanomas, sarcomas, and cancer of the breast, prostate, ovary, and lung tissues. We found that breaking tolerance toward these tumor antigens was best achieved using vaccines containing antigens chemically conjugated to Keyhole Limpet Hemocyanin (KLH) plus a potent immunological adjuvant (QS-21). To date, by using this approach to vaccination, antibodies have been induced in patients against glycolipid . antigens GM2, GD2, GD3, Fucosy1GM1, Globo H, and Lewis Y, and glycoprotein (mucin) antigens Tn, sialyl Tn, TF, and MUCL More recently, in a comparative study we investigated the T cell response induced by MUC1-KLH conjugates. Although a MUC1-specific T cell response was not consistently detected in any patient, the role of KLH in orienting the cytokine environment was crucial. We were able to confirm that KLH in these conjugate vaccines induces a Th1 T cell response as demonstrated by the high anti-KLH INF-7 secretion and the IgG1 and IgG3 subclasses of this high titer IgG antibodies induced. Clinical trials using KLH conjugated glycolipid and glycoprotein vaccines, are currently ongoing. These range from phase I/II single antigens trials with glycosilated MUC1, polysialic acid, synthetic Fucosyl GM1 and GD2, to phase II trials with a polyvalent vaccine containing six or seven antigens. Randomized phase 11 trials with polyvalent vaccines are planned for initiation in 2001–2002 in patients with ovarian, breast, and prostate cancer.
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vaccination with a bivalent gm2 and gd2 ganglioside conjugate vaccine a trial comparing doses of gd2 Keyhole Limpet Hemocyanin
Clinical Cancer Research, 2000Co-Authors: Paul B Chapman, R J Israel, Katherine S. Panageas, Donna Morrisey, Linda Williams, Jonathan J Lewis, Bradley W Hamilton, Philip O. LivingstonAbstract:Immunization with GMK vaccine (G(M2) ganglioside conjugated to Keyhole Limpet Hemocyanin mixed with QS-21 adjuvant) induces anti-G(M2) antibodies in close to 100% of patients. We found previously that anti-G(D2) antibodies could be induced in some patients using G(D2)-Keyhole Limpet Hemocyanin + QS-21 (GDK). In this trial, we wished: (a) to determine whether immunization with both GMK and GDK vaccines could induce antibodies against both G(M2) and G(D2); and (b) to determine the optimal dose of GDK. Thirty-one patients with melanoma or sarcoma who had no evidence of disease after complete surgical resection were immunized with both GMK (30 microg of G(M2)) and GDK on weeks 1, 2, 3, 4, 12, 24, and 36. Patients were assigned to one of five GDK dose levels (3, 10, 30, 70, or 130 microg of G(D2)). Anti-G(M2) IgM or IgG were induced in 97% of patients. The dose of GDK did not affect the anti-G(M2) response, although at the highest GDK dose level, 3 of 7 patients did not make anti-G(M2) IgG. GDK was less immunogenic; overall 45% of patients developed either IgM or IgG against G(D2). At GDK doses of 30 or 70 microg, 8 of 11 patients (73%) made either IgM or IgG anti-G(D2) antibodies. We conclude that both GMK and GDK vaccines can induce antibodies against G(M2) and G(D2) in a majority of patients and are safe. The optimal dose of GDK appears to be either 30 or 70 microg when administered with GMK vaccine.
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vaccination of high risk breast cancer patients with mucin 1 muc1 Keyhole Limpet Hemocyanin conjugate plus qs 21
Clinical Cancer Research, 2000Co-Authors: Teresa Gilewski, Sucharita Adluri, Shengle Zhang, Mary Ellen Moynahan, Katherine S. Panageas, Alan N. Houghton, Larry Norton, Philip O. LivingstonAbstract:Our objective was to determine whether an immune response can be generated against MUC1 peptide and against tumor cell MUC1 after vaccination with MUC1-Keyhole Limpet Hemocyanin (KLH) conjugate plus QS-21 in breast cancer patients. Nine patients with a history of breast cancer but without evidence of disease were treated with MUC1-KLH conjugate plus QS-21, containing 100μ g of MUC1 and 100 μg of QS-21. s.c. vaccinations were administered at weeks 1, 2, 3, 7, and 19. Peripheral blood was drawn at frequent intervals to assess antibody titers. Skin tests were placed at weeks 1, 3, 9, and 21 to determine delayed type hypersensitivity reactions. Common toxicities included a local skin reaction at the site of the vaccine, usually of 4–5 days’ duration, and mild flu-like symptoms usually of 1–2 days’ duration. High IgM and IgG antibody titers against synthetic MUC1 were detected. IgG antibody titers remain elevated from a minimum of 106–137 weeks after the first vaccination. Binding of IgM antibody to MCF-7 tumor cells was observed in seven patients, although there was minimal binding of IgG antibody. Two patients developed significant antibody titers post-high-dose chemotherapy and stem cell reinfusion. There was no evidence of T cell activation. This MUC1-KLH conjugate plus QS-21 was immunogenic and well tolerated in breast cancer patients. Additional trials are ongoing to determine the optimal MUC1 peptide for use in larger clinical trials. Further investigation of vaccine therapy in high-risk breast cancer is warranted.
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induction of antibodies against gm2 ganglioside by immunizing melanoma patients using gm2 Keyhole Limpet Hemocyanin qs21 vaccine a dose response study
Clinical Cancer Research, 2000Co-Authors: Paul B Chapman, D M Morrissey, W B Hamilton, Alicia N Destro, R J Israel, C. Zhan, Katherine S. Panageas, Lawrence J. Williams, Philip O. LivingstonAbstract:In a previous randomized Phase III trial (P. O. Livingston et al. , J. Clin. Oncol., 12: 1036–1044, 1994), we demonstrated that immunization with GM2 and bacille Calmette-Guerin reduced the risk of relapse in stage III melanoma patients who were free of disease after surgical resection and who had no preexisting anti-GM2 antibodies. That vaccine formulation induced IgM anti-GM2 antibodies in 74% but induced IgG anti-GM2 antibodies in only 10% of the patients. To optimize the immune response against GM2, a reformulated vaccine was produced conjugating GM2 to Keyhole Limpet Hemocyanin (KLH) and using the adjuvant QS21 (GM2-KLH/QS21). In pilot studies, 70 μg of vaccine induced IgG anti-GM2 antibodies in 76% of the patients. We wished to define the lowest vaccine dose that induced consistent, high-titer IgM and IgG antibodies against GM2. Fifty-two melanoma patients who were free of disease after resection but at high risk for relapse were immunized with GM2-KLH/QS21 vaccine at GM2 doses of 1, 3, 10, 30, or 70μ g on weeks 1, 2, 3, 4, 12, 24, and 36. Serum collected at frequent and defined intervals was tested for anti-GM2 antibodies. Overall, 88% of the patients developed IgM anti-GM2 antibodies; 71% also developed IgG anti-GM2 antibodies. GM2-KLH doses of 3–70 μg seemed to be equivalent in terms of peak titers and induction of anti-GM2 antibodies. At the 30-μg dose level, 50% of the patients developed complement fixing anti-GM2 antibodies detectable at a serum dilution of 1:10. We conclude that the GM2-KLH/QS21 formulation is more immunogenic than our previous formulation and that 3 μg is the lowest dose that induces consistent, high-titer IgM and IgG antibodies against GM2.
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Immunization of mice with fucosyl-GM1 conjugated with Keyhole Limpet Hemocyanin results in antibodies against human small-cell lung cancer cells
Cancer Immunology Immunotherapy, 1999Co-Authors: Sarah Cappello, Cristina Musselli, Fred-thomas Brezicka, Philip O. Livingston, Govind RagupathiAbstract:Fucosyl-GM1 (Fuc-GM1) [Fucα1 → 2Galβ1 → 3GalNAcβ1 → 4(NeuAcα2-3)Galβ1 → 4Glcβ1 → O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein Keyhole Limpet Hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC.
Donald Y M Leung - One of the best experts on this subject based on the ideXlab platform.
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response to cutaneous immunization with low molecular weight subunit Keyhole Limpet Hemocyanin
International Archives of Allergy and Immunology, 2012Co-Authors: Henry Milgrom, Karen Kesler, Margie Byron, Ronald J Harbeck, Robert Holliday, Donald Y M LeungAbstract:Background: This study was carried out to determine whether humoral and cellular immune responses would be provoked by cutaneous administration of Keyhole Limpet Hemocyanin (KLH) and in particular by scarification of the skin (SS). Methods: This was an unblinded, single-center, 8-week pilot study in healthy young adults. Twenty-four subjects assigned to 4 groups completed the study. Each group was immunized twice, with a 3-week interval, either by SS or intradermally (ID), with an SS dose of 50 or 250 µg and an ID dose of 100 or 250 µg. Serum was collected for antibody assays at baseline and 3 weeks after both the first and second immunizations. Delayed-type hypersensitivity (DTH) testing was performed before the first immunization and 3 weeks after the second. Results: In the 250-µg SS group, there was a significant increase from day 0 to day 47 in anti-KLH IgG (p = 0.02; day 0: 3.46 ± 5.49 mg/dl, day 47: 7.54 ± 8.87 mg/dl) and anti-KLH IgA (p = 0.04; day 0: 4.78 ± 9.15 mg/dl, day 47: 11.42 ± 13.62 mg/dl). One subject in each treatment group showed a positive DTH test result representing 20% (50-µg SS), 10% (250-µg SS), 25% (100-µg ID) and 20% (250-µg ID) of the subjects. Conclusions: It was possible to induce both humoral and cellular immune responses by SS administration despite the limited antigenic potency of the low-molecular-weight KLH preparation. This approach may be useful for studying the mechanisms of immune response in allergic skin diseases such as atopic dermatitis.
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response to cutaneous immunization with low molecular weight subunit Keyhole Limpet Hemocyanin klh
The Journal of Allergy and Clinical Immunology, 2011Co-Authors: Henry Milgrom, Karen Kesler, Margie Byron, Ronald J Harbeck, Robert Holliday, Donald Y M LeungAbstract:Background: This study was carried out to determine whether humoral and cellular immune responses would be provoked by cutaneous administration of Keyhole Limpet Hemocyanin (KLH) and in particular by scarification of the skin (SS). Methods: This was an unblinded, single-center, 8-week pilot study in healthy young adults. Twenty-four subjects assigned to 4 groups completed the study. Each group was immunized twice, with a 3-week interval, either by SS or intradermally (ID), with an SS dose of 50 or 250 g and an ID dose of 100 or 250 g. Serum was collected for antibody assays at baseline and 3 weeks after both the first and second immunizations. Delayed-type hypersensitivity (DTH) testing was performed before the first immunization and 3 weeks after the second. Results: In the 250- g SS group, there was a significant increase from day 0 to day 47 in anti-KLH IgG (p = 0.02; day 0: 3.46 8 5.49 mg/dl, day 47: 7.54 8 8.87 mg/dl) and anti-KLH IgA (p = 0.04; day 0: 4.78 8 9.15 mg/dl, day 47: 11.42 8 13.62 mg/dl). One subject in each treatment group showed a positive DTH test result representing 20% (50- g SS), 10% (250- g SS), 25% (100- g ID) and 20% (250- g ID) of the sub
Dale R. Riggs - One of the best experts on this subject based on the ideXlab platform.
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Keyhole Limpet Hemocyanin : an effective adjunct against melanoma in vivo
American journal of surgery, 2007Co-Authors: Irfan A. Rizvi, Barbara Jackson, Dale R. Riggs, David W. McfaddenAbstract:Abstract Background We have previously demonstrated the potent in vitro antiproliferative effects of Keyhole Limpet Hemocyanin (KLH) against melanoma. Our prior studies directed us to hypothesize that KLH would be effective in vivo against melanoma, alone and in combination with conventional immunotherapy. Methods Mice were inoculated with 2 × 10 7 HTB68 cells and randomized to 6 groups. Treatment groups consisted of control, KLH 200 μg, alpha interferon (AIFN) 1000 IU, interleukin-2 (IL-2) 5000 IU, KLH + AIFN, and KLH + IL-2. Results KLH + IL-2 exhibited the greatest reduction in tumor volume (30%) as compared to control ( P = .014), followed by KLH + AIFN (28%, P = .031). Singly treated animals had less tumor inhibition: IL-2 (30%, P = .022), KLH (18%, not significant), and AIFN (16%, not significant). Conclusions KLH augments the effects of AIFN, one of the standard immunotherapeutic agents against melanoma in vivo. Further in vivo and early clinical studies into the effects of KLH as both a single and combined agent are warranted.
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Keyhole Limpet Hemocyanin potentiates standard immunotherapy for melanoma.
American journal of surgery, 2007Co-Authors: David W. Mcfadden, Dale R. Riggs, Barbara Jackson, Cynthia CunninghamAbstract:Abstract Introduction Our hypothesis was that Keyhole Limpet Hemocyanin (KLH) would augment the effects of standard immunotherapies for melanoma including interferon-alpha (AIFN) and interleukin (IL)-2. Methods The HTB68 melanoma cell line was treated with KLH, AIFN, and IL-2 as single and combined agents. Cell viability, apoptotic activity, and vascular endothelial growth factor levels were all evaluated. Results Cell growth was reduced with KLH (28%), AIFN (54%), and IL-2 (29%) (all P P P Conclusions The additive effects exhibited by the combination of KLH with AIFN or IL-2 are encouraging and support combination therapy as an effective treatment for this aggressive disease.
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in vitro effects of Keyhole Limpet Hemocyanin in breast and pancreatic cancer in regards to cell growth cytokine production and apoptosis
American Journal of Surgery, 2005Co-Authors: Dale R. Riggs, Barbara Jackson, Linda Vonadavis, Ankesh Nigam, David W. McfaddenAbstract:Abstract Background We have previously shown the inhibitory effects of Keyhole Limpet Hemocyanin (KLH) against breast and pancreatic cancer in vitro. We hypothesize that its actions in breast and pancreas cancer cells are via apoptotic or cytokine pathways. Methods Two breast cancer cell lines, ZR75-1 and MCF-7, and one pancreas cancer cell line, PANC-1, were treated with KLH at 500 μg, 250 μg, and 250 ng/mL. Cell viability, cytokine production, and apoptosis were measured. Results Significant growth inhibition was observed in all cell lines at all KLH concentrations tested. Significant changes in cytokine production were observed in all cell lines. An increase in early and late apoptotic activity was observed in the MCF-7, whereas a reduction in late apoptotic activity was observed in the ZR75-1 cells. Conclusions KLH directly inhibits the growth of human breast and pancreas cancer in vitro by apoptotic and nonapoptotic mechanisms.
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Proteomic analysis of SEG-1 human Barrett's-associated esophageal adenocarcinoma cells treated with Keyhole Limpet Hemocyanin.
Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract, 2004Co-Authors: Linda Vona-davis, Dale R. Riggs, Barbara Jackson, Sara Zulfiqar, Timothy S Vincent, David W. McfaddenAbstract:Keyhole Limpet Hemocyanin (KLH) is an immune stimulant derived from a circulating glycoprotein of the marine mollusk Megathura crenulata. We previously reported that KLH inhibited the growth of human Barrett’s-associated esophageal adenocarcinoma in vitro via apoptotic and nonapoptotic mechanisms. We hypothesize that KLH reduces the growth of Barrett’s cancer cells by altering protein expression profiles. A cell line (SEG-1) derived from Barrett’s-associated adenocarcinomas of the distal esophagus was selected. Cells were administered KLH (500 µg/ml) or vehicle. After 24 hours, cytosolic fractions were separated through two-dimensional gel electrophoresis. Statistical analysis was performed with Evolution Pro software to identify spots that were differentially expressed between the KLH and control groups. Proteins displaying a twofold or greater change in expression levels were selected for identification. In a total of 420 spots, 31 were differentially expressed between the KLH and control groups. In all, 12 were upregulated and 19 were downregulated. Of the 31, 17 were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteomic evaluation shows downregulation of proteins associated with metabolic processes (glycolysis, protein synthesis). KLH also induced proteins indicative of oxidative stress (heat shock 70 family and UDP-glucose 6-dydrogenase). Our results indicate that growth arrest by KLH is accompanied by a cellular stress response and attenuation of metabolic processes. The use of KLH as adjuvant or topical therapy for Barrett’s adenocarcinoma provides a promising development in the treatment of this disease.
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Keyhole Limpet Hemocyanin, a novel immune stimulant with promising anticancer activity in Barrett's esophageal adenocarcinoma.
American journal of surgery, 2003Co-Authors: David W. Mcfadden, Dale R. Riggs, Barbara Jackson, Linda Vona-davisAbstract:Abstract Background Keyhole Limpet Hemocyanin (KLH) is a recently described immune stimulant and hapten carrier derived from a circulating glycoprotein of the marine mollusk Megathura crenulata. We previously reported that KLH has significant antiproliferative effects in vitro against breast, pancreas, and prostate cancers. We hypothesized that KLH would be effective against Barrett's esophageal adenocarcinoma in an in vitro model. Methods Barrett's esophageal adenocarcinoma cell lines (SEG-1 and BIC-1) were cultured using standard techniques. Cells were plated at 1 × 10 5 and KLH was added at concentrations ranging from 400ng to 100μg/well. After 24 and 72h incubation, cells were assayed for viability using the MTT technique. Statistical analysis was performed using ANOVA. Apoptosis was evaluated using a cell death detection kit after 16 hours of incubation with KLH. Results KLH treatment significantly (p Conclusions KLH directly inhibits the growth of human Barrett's esophageal cancer in vitro by apoptotic and nonapoptotic mechanisms.
Robin J Harris - One of the best experts on this subject based on the ideXlab platform.
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Keyhole Limpet Hemocyanin molecular structure of a potent marine immunoactivator a review
European Urology, 2000Co-Authors: Robin J Harris, Jurgen MarklAbstract:Objectives: In this short review we present a survey of the available biochemical and electron microscopic data on Keyhole Limpet Hemocyanin (KLH). Results: The b
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Keyhole Limpet Hemocyanin klh slow in vitro reassociation of klh1 and klh2 from immucothel
Micron, 1998Co-Authors: Robin J Harris, Wolfgang Gebauer, Marc Adrian, Jurgen MarklAbstract:Abstract Following our in vitro reassociation of Keyhole Limpet Hemocyanin subunits in the presence of high concentrations (100 mM each) of calcium and magnesium chloride (Harris et al., 1997a, Micron 28, 31–41; 1997b, Micron 28, 43–56), we have now extended our investigations by using a buffer system containing a lower concentration of the two divalent cations (10 mM each). Reassociation of mixed KLH subunits present in the commercially available product Immucothel® was performed using a standardized buffer solution containing 50 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl2 and 10 mM MgCl2 (pH 7.4) over a minimum period of one week, at 4°C. This solution was selected as being close to our KLH stabilizing buffer (used routinely for the 4°C storage of native KLH), but with a slightly elevated concentration of both divalent cations (i.e. 10 mM instead of the usual 5 mM) to potentiate KLH reassociation. The reformation of the higher molecular mass oligomeric and polymeric forms of KLH was monitored throughout from air-dried negatively stained specimens prepared on continuous carbon support films and across the small holes of holey carbon support films, and also by cryo-negative staining. Compared to our previous studies, KLH reassociation has been found to be much slower, but a high percent recovery of total KLH (ca. 87%, produced by centrifugal pelleting), was achieved after a period of seven–ten days reassociation at 4°C. In vitro production of naturally occurring oligomeric forms of KLH1 and KLH2 now predominates, and the proportion of smaller diameter tubular polymeric forms is much less. After reassociation of the mixed KLH1 and KLH2 subunits present in Immucothel®, separation of intact KLH1 from dissociated KLH2 was achieved by gel filtration chromatography after dialysis against 1% ammonium molybdate–0.2% PEG (Mr 1000) at pH 5.7 and monitored by native PAGE. This purification enabled the reassociation characteristics of the two purified KLH subunits (from a 130 mM glycine–NaOH buffer solution at pH 9.6) to be investigated in our 10 mM divalent cation-supplemented Tris–saline buffer. In both cases, the second reassociation, performed over a period of one–two weeks at 4°C, led to the production of the characteristic decameric oligomeric forms; for KLH1 the didecamer predominates, and for KLH2 didecamers and short multidecamers predominate, but in both cases a small quantity of the previously described helical/tubular polymers is also present. Subsequent centrifugal pelleting of this reassociated KLH1 and KLH2, with resuspension and storage at −70°C in stabilizing buffer containing 10% (w/v) trehalose, has been found to provide a biochemically and structurally characterized stock of purified KLH oligomers. The prolonged time-periods required by the overall reassociation and purification procedure do not appear to detract from the quality or stability of the high molecular mass forms of KLH1 and KLH2 so produced.
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mass determination subunit organization and control of oligomerization states of Keyhole Limpet Hemocyanin klh
FEBS Journal, 1997Co-Authors: Sabine M Sohngen, Robin J Harris, Alexandra Stahlmann, Shirley A Muller, Andreas K Engel, Jurgen MarklAbstract:Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of Keyhole Limpet Hemocyanin (KLH; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45–60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain 1h, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5 mM MgCl2, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.
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structure of Keyhole Limpet Hemocyanin type 1 klh1 at 15 a resolution by electron cryomicroscopy and angular reconstitution
Journal of Molecular Biology, 1997Co-Authors: Jurgen Markl, Robin J Harris, Elena V Orlova, Prakash Dube, Erich Beckman, F Zemlin, Marin Van HeelAbstract:Abstract A three-dimensional reconstruction of Keyhole Limpet Hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to ∼45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan Hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan Hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod Hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan Hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the “functional units” within the dimeric “morphological units” of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical Hemocyanin. Keyhole Limpet Hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this Hemocyanin may be associated with its intricate hierarchy of local-symmetry components.
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Keyhole Limpet Hemocyanin klh i reassociation from immucothel followed by separation of klh1 and klh2
Micron, 1997Co-Authors: Robin J Harris, Wolfgang Gebauer, Franziska U M Guderian, Jurgen MarklAbstract:Abstract Studies of Keyhole Limpet Hemocyanin (KLH) normally require purification of functional complexes directly from living animals. An alternative procedure is described wherein a commercial preparation of KLH which is fully dissociated into its subunits (Immucothel®, biosyn Arzneimittel GmbH) is reassociated in the presence of a high concentration of calcium and magnesium. The reassociation products, when observed by electron microscopy, consist of didecamers, multidecamers and flexible tubules of varying length. The two forms of KLH described previously and designated KLH1 and KLH2, are present in the reassocated mixture as homo-oligomers/polymers and can be separated by selective dissociation of the KLH2 by treatment with 1% ammonium molybdate-0.2% PEG at pH 5.7, followed by gel filtration chromatography in this solution. In addition to discrete elution peaks containing didecameric KLH1 and dissociated subunits of KLH2, a leading peak contains a tubular/polymeric form of KLH1, not previously described. Under negative staining conditions designed specifically for the creation of 2-dimensional crystals on mica (the negative staining-carbon film procedure), this tubular form of KLH1 can be transformed into a larger diameter multidecameric form, again not previously described for KLH1. The purified KLH2 peak is indistinguishable from subunit material prepared from living animals. Thus, Immucothel® appears to provide a standardized source of subunits suitable for biochemical and structural studies on the two types of KLH.
