The activation peptide of coagulation factor XIII is vital for its expression and stability.

BACKGROUND The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. OBJECTIVE To investigate the role of AP-FXIII in the expression and stability of FXIII. METHODS We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. RESULTS Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence (8) FGGR(12) R plays a substantial role in intersubunit interactions in FXIII-A2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. CONCLUSIONS We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A2 dimer.

Prevalence estimation of tick-borne encephalitis virus (TBEV) antibodies in dogs from Finland using novel dog anti-TBEV IgG MAb-capture and IgG immunofluorescence assay based on recombinant TBEV subviral particles

Tick-borne encephalitis (TBE) is one of the most dangerous human neurological infections occurring in Europe and Northern parts of Asia with thousands of cases and millions vaccinated against it. The risk of TBE might be assessed through analyses of the samples taken from wildlife or from animals which are in close contact with humans. Dogs have been shown to be a good sentinel species for these studies. Serological assays for diagnosis of TBE in dogs are mainly based on purified and inactivated TBEV antigens. Here we describe novel dog anti-TBEV IgG monoclonal antibody (MAb)-capture assay which is based on TBEV prME subviral particles expressed in mammalian cells from Semliki Forest virus (SFV) replicon as well as IgG immunofluorescence assay (IFA) which is based on Vero E6 cells transfected with the same SFV replicon. We further demonstrate their use in a small-scale TBEV seroprevalence study of dogs representing different regions of Finland. Altogether, 148 dog serum samples were tested by novel assays and results were compared to those obtained with a commercial IgG enzyme immunoassay (EIA), hemagglutination inhibition test and IgG IFA with TBEV infected cells. Compared to reference tests, the sensitivities of the developed assays were 90-100% and the specificities of the two assays were 100%. Analysis of the dog serum samples showed a seroprevalence of 40% on Åland Islands and 6% on Southwestern archipelago of Finland. In conclusion, a specific and sensitive EIA and IFA for the detection of IgG antibodies in canine sera were developed. Based on these assays the seroprevalence of IgG antibodies in dogs from different regions of Finland was assessed and was shown to parallel the known human disease burden as the Southwestern archipelago and Åland Islands in particular had considerable dog TBEV antibody prevalence and represent areas with high risk of TBE for humans.

The Role of Raf Kinase Inhibitor Protein in Cell Motility

Raf Kinase Inhibitor Protein (RKIP) has been identified as a phosphatidylethanolamine-binding protein capable of inhibiting Raf-1 kinase, an enzyme significant in cell proliferation and cancer development. When properly functioning, RKIP can mediate the expression of Raf-1 kinase and help prevent uncontrolled cell division. RKIP also has suggested, but unclear, roles in spindle fiber formation during mitosis, regulation of apoptosis, and cell motility. The Fenteany laboratory in the Chemistry Department identified a new small molecule, named Locostatin, as a cell migration inhibitor in mammalian cells, with RKIP as its primary molecular target. Dictyostelium discoideum possess two RKIP proteins, RKIP-A and RKIP-B. In order to begin to study the function of RKIP in D. discoideum and its role in cell motility, I created a mutant cell line which lacks a functional RKIP-A gene. In this paper, we show that removal of RKIP-A does not affect vegetative motility, but impairs chemotaxis and development in the presence of drug. Interestingly, RKIP-A knockout mutants appear more resistant to drug effects on vegetative motility than wild-type cells. More research is needed to reconcile these seemingly contrasting results, and to better develop a model for RKIP-A’s role in cell motility.

Investigations of stress responses in Saccharomyces cerevisiae: Transcriptional control and heat shock protein function

The baker's yeast, Saccharomyces cerevisiae responds to the cytotoxic effects of elevated temperature (37-42°C) by activating transcription of ∼150 genes, termed heat shock genes, collectively required to compensate for the abundance of misfolded and aggregated proteins and various physiological modifications necessary for the cell to survive and grow at heat shock temperatures. An intriguing facet of the yeast heat shock response is the remarkable similarity it shares with the global remodeling that occurs in mammalian cells in response to numerous pathophysiological conditions including cancer and cardiovascular disease and thus provides an ideal model system. I have therefore investigated several novel features of stress signaling, transcriptional regulation, and physiology. Initial work focused on the characterization of SYM1, a novel heat shock gene in yeast which was demonstrated to be required for growth on the nonfermentable carbon source ethanol at elevated temperature, and to be the functional ortholog of the mammalian kidney disease gene, Mpv17. Additional work addressed the role of two proteins, the Akt-related kinase, Sch9, and Sse1, the yeast Hsp110 protein chaperone homolog, in signaling by protein kinase A, establishing Sse1 as a critical negative regulator of this pathway. Furthermore, I have demonstrated a role for Sse1 in biogenesis and stability of the stress-response transcription factor, Msn2; a finding that has been extended to include a select subset of additional high molecular weight proteins, suggesting a more global role for this chaperone in stabilizing the cellular proteome. The final emphasis of my doctoral work has included the finding that celastrol, a compound isolated from the plant family Celasfraceae, a component of traditional Chinese herbal medicine, can activate heat shock transcription factor (Hsf1) in yeast and mammalian cells through an oxidative stress mechanism. Celastrol treatment simultaneously activates both heat shock and oxidative stress response pathways, resulting in increased cytoprotection. ^

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^

Hypoxia-induced transcriptional regulation through chromatin modifications and NC2 function

In response to tumor hypoxia, specific genes that promote angiogenesis, proliferation, and survival are induced. Globally, I find that hypoxia induces a mixed pattern of histone modifications that are typically associated with either transcriptional activation or repression. Furthermore, I find that selective activation of hypoxia-inducible genes occurs simultaneously with widespread repression of transcription. I analyzed histone modifications at the core promoters of hypoxia-repressed and -activated genes and find that distinct patterns of histone modifications correlate with transcriptional activity. Additionally, I discovered that trimethylated H3-K4, a modification generally associated with transcriptional activation, is induced at both hypoxia-activated and repressed genes, suggesting a novel pattern of histone modifications induced during hypoxia. ^ In order to determine the mechanism of hypoxia-induced widespread repression of transcription, I focused my studies on negative cofactor 2 (NC2). Previously, we found that hypoxia-induced repression of the alpha-fetoprotein (AFP) gene occurs during preinitiation complex (PIC) assembly, and I find that NC2, an inhibitor of PIC assembly, is induced during hypoxia. Moreover, I find that the beta subunit of NC2 is essential for hypoxia-mediated repression of AFP, as well as the widespread repression of transcription observed during hypoxia. Previous data in Drosophila and S. cerevisiae indicate that NC2 functions as either an activator or a repressor of transcription. The mechanism of NC2-mediated activation remains unclear; although, Drosophila NC2 function correlates with specific core promoter elements. I tested if NC2 activates transcription in mammalian cells using this core promoter-specific model as a guide. Utilizing site-specific mutagenesis, I find that NC2 function in mammalian cells is not dependent upon specific core promoter elements; however, I do find that mammalian NC2 does function in a gene-specific manner as either an activator or repressor of transcription during hypoxia. Furthermore, I find that binding of the alpha subunit of NC2 specifically correlates with NC2-mediated transcriptional activation. NC2α and NC2β are both required for NC2-mediated transcriptional activation; whereas, NC2β alone is required for hypoxia-induced transcriptional repression. Together, these data indicate that hypoxia mediates changes in gene expression through both chromatin modifications and NC2 function. ^

Identification and characterization of distinct classes of cAMP-pulse-induced genes in Dictyostelium discoideum development

The unicellular amoeba Dictyostelium discoideum embarks on a developmental program upon starvation. During development, extracellular oscillatory cAMP signaling orchestrates the chemotaxis-mediated aggregation of ∼105 amoebae and is required for optimal induction of so-called pulse-induced genes. This requirement for pulsatile CAMP reflects adaptation of the cAMP-receptor-mediated pathways that regulate these genes. Through examination of a collection of pulse-induced genes, we defined two distinct gene classes based on their induction kinetics and the impact of mutations that impair PKA signaling. The first class (represented by D2 and prtA) is highly dependent on PKA signaling, whereas the second class (represented by carA, gpaB, and acaA) is not. Analysis of expression kinetics revealed that these classes are sequentially expressed with the PKA-independent genes peaking in expression before the PKA-dependent class. Experiments with cycloheximide, an inhibitor of translation, demonstrated that the pulse induction of both classes depends on new protein synthesis early in development. carA and gpaB also exhibit pulse-independent, starvation-induced expression which, unlike their pulse induction, was found to be insensitive to cycloheximide added at the outset of starvation. This result indicates that the mechanism of starvation induction pre-exists in growing cells and is distinct from the pulse induction mechanism for these genes. In order to identify cis-acting elements that are critical for induction of carA, we constructed a GFP reporter controlled by a 914-base-pair portion of its promoter and verified that its expression was PKA-independent, pulse-inducible, and developmentally regulated like the endogenous carA gene. By a combination of truncation, internal deletion, and site-directed mutation, we defined several distinct functional elements within the carA promoter, including a 39-bp region required for pulse induction between base pairs -321 and -282 (relative to the transcription start site), a 131-bp region proximal to the start site that is sufficient for starvation induction, and two separate enhancer domains. Identification of factors that interact with these promoter elements and genetic approaches exploiting the GFP reporter described here should help complete our understanding of the mechanisms regulating these genes, including adaptation mechanisms that likely also govern chemotaxis of Dictyostelium and mammalian cells. ^

Mechanisms by which nonsense codons downregulate T-cell receptor transcripts

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). Two out of three T-cell receptor β (TCRβ) transcripts carry PTCs as a result of error-prone programmed rearrangements that occur at this locus during lymphocyte maturation. PTCs decrease TCRβ mRNA levels to a much greater extent than mRNAs transcribed from non-rearranging genes. This robust decrease in TCRβ mRNA levels is not a unique characteristic of the T-cell environment or the TCRβ promoter. The simplest explanation for this is that PTC-bearing TCRβ mRNAs elicit a stronger NMD response. An alternative explanation is NMD collaborates with another mechanism to dramatically decrease PTC-bearing TCRβ mRNA levels. ^ In my dissertation, I investigated the molecular mechanism behind the strong decrease in TCRβ mRNA levels triggered by PTCs. To determine the location of this response, I performed mRNA half-life analysis and found that PTCs elicited more rapid TCRβ mRNA decay in the nuclear fraction, not the cytoplasmic fraction. Although decay was restricted to the nuclear fraction, PTC-bearing TCRβ transcript levels were extremely low in the cytoplasm, a phenomenon that I named the nonsense-codon induced partitioning shift (NIPS). I established that NIPS shares several qualities with NMD, including its dependence on translation and NMD factors. Several lines of evidence suggested that NIPS results from PTCs eliciting retention of TCRβ transcripts in the nuclear fraction. This retention, as well as rapid TCRβ mRNA decay, most likely occurs in either the nucleoplasm or the outer nuclear membrane, based on analysis of nuclear and cytoplasmic markers in the highly purified nuclei I used for my studies. To further address the location of decay, I asked whether nuclear or cytoplasmic RNA decay factors mediated the destruction of PTC-bearing mRNAs. My results suggested that a nuclear component of the 3'-to-5' exosome, as well as an endonucleolytic activity, are involved in the destruction of PTC-containing TCRβ mRNAs. Individual endogenous NMD substrates had differential requirements for nuclear and cytoplasmic exonucleases. In summary, my results provide evidence that PTCs trigger multiple mechanisms involving multiple decay factors to remove and regulate mRNAs in mammalian cells. ^

Functional analysis of GNA -1, a Galphai superfamily member found in the filamentous fungus Neurospora crassa

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cAMP phosphodiesterase, phospolipase C-β, mitogen-activated protein kinases, and ion channels. ^ Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric Gα proteins (GNA-1–3) and one Gβ subunit (GNB-1). GNA-1 was the first microbial protein that could be classified in the Gαi superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. ^ Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The Gαi and Gαz members of the Gαi superfamily negatively regulate adenylyl cyclase activity in mammalian cells; therefore, adenylyl cyclase and cAMP levels were measured in Δgna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second Gα in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. ^ Genetic and biochemical epistasis experiments utilizing a Δ gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and crude membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Δgna-1 membrane fractions. These studies provide direct evidence for the involvement of Gα proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes. ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^

Proceedings of the Fifteenth Annual biochemical Engineering Symposium

This work represents the proceedings of the fifteenth symposium which convened at Colorado State University on May 24, 1985. The two day meeting was scheduled one month later than usual, i.e., after the spring semester, so that travelers from the Midwest (Iowa State University, Kansas State University and University of Missouri) could enjoy the unique mountain setting provided at Pingree Park. The background of the photograph on the cover depicts the beauty of the area. ContentsGreg Sinton and S.M. Leo, KSU. Models for the Biodegration of 2.4-D and Related Xenobiotic Compounds. V. Bringi, CSU. Intrinsic Kinetics from a Novel Immobilized Cell CSTR. Steve Birdsell, CU. Novel Microbial Separation Techniques. Mark Smith, MU. Kinetic Characterization of Growth of E. coli on Glucose. Michael M. Meagher, ISU. Kinetic Parameters of Di- and Trisaccharaide Hydrolysis by Glucoamylase II. G.T. Jones and A.K. Ghosh Hajra, KSU. Modeling and Simulation of Legume Modules with Reactive Cores and Inert Shells. S.A. Patel and C.H. Lee, KSU. Energetic Analysis and Liquid Circulation in an Airlift Fermenter. Rod R. Fisher, ISU. The Effects of Mixing during Acid Addition of Fractionally Precipitated Protein. Mark M. Paige, CSU. Fed-batch Fermentations of Clostridium acetobutylicum. Michael K. Dowd, ISU. A Nonequilibirium Thermodynamic Description of the Variation of Contractile Velocity and Energy Use in Muscle. David D. Drury, CSU. Analysis of Hollow Fiber Bioreactor Performance for MAmmalian Cells by On-Line MMR. H.Y. Lee, KSU. Process Analysis of Photosynthetic Continuous Culture Systems. C.J. Wang, MU. Kinetic Consideration in Fermentation of Cheese Whey to Ethanol.

Proceedings of the Twentieth Annual Biochemical Engineering Symposium

The 20th Annual Biochemical Engineering Symposium was held at Kansas State University on April 21,1990. The objectives of the symposium were to provide: (i) a forum for informal discussion of biochemical engineering research being conducted at the participating institutions and (ii) an opportunity for students to present and publish their work. Twenty-eight papers presented at the symposium are included in this proceedings. Some of the papers describe the progress of ongoing projects, and others contain the results of completed projects. Only brief summaries are given of the papers that will be published in full elsewhere. The program of the symposium and a list of the participants are included in the proceedings. ContentsCell Separations and Recycle Using an Inclined Settler, Ching-Yuan Lee, Robert H. Davis and Robert A. Sclafani Micromixing and Metabolism in Bioreactors: Characterization of a 14 L Fermenter, K.S. Wenger and E.H. Dunlop Production, Purification, and Hydrolysis Kinetics of Wild-Type and Mutant Glucoamylases from Aspergillus Awamori, Ufuk Bakir, Paul D. Oates, Hsiu-Mei Chen and Peter J. Reilly Dynamic Modeling of the Immune System, Barry Vant-Hull and Dhinakar S. Kompala Dynamic Modeling of Active Transport Across a Biological Cell: A Stochastic Approach, B.C. Shen, S.T. Chou, Y.Y. Chiu and L.T. Fan Electrokinetic Isolation of Bacterial Vesicles and Ribosomes, Debra T.L. Hawker, Robert H. Davis, Paul W. Todd, and Robert Lawson Application of Dynamic Programming for Fermentative Ethanol Production by Zymomonas mobilis, Sheyla L. Rivera and M. Nazmul Karim Biodegradation of PCP by Pseudomonas cepacia, R. Rayavarapu, S.K. Banerji, and R.K. Bajpai Modeling the Bioremediation of Contaminated Soil Aggregates: a Phenomenological Approach, S. Dhawan, L.E. Erickson and L.T. Fan Biospecific Adsorption of Glucoamylase-I from Aspergillus niger on Raw Starch, Bipin K. Dalmia and Zivko L. Nikolov Overexpression in Recombinant Mammalian Cells: Effect on Growth Rate and Genetic Instability, Jeffrey A. Kern and Dhinakar S. Kompala Structured Mathematical Modeling of Xylose Fermentation, A.K. Hilaly, M.N. Karim, I. C. Linden and S. Lastick A New Culture Medium for Carbon-limited Growth of Bacillus thuringiensis, W. -M. Liu and R.K. Bajpai Determination of Sugars and Sugar Alcohols by High Performance Ion Chromatography, T. J. Paskach, H.-P. Lieker, P.J. Reilly, and K. Thielecke Characterization of Poly-Asp Tailed B-Galactosidase, M.Q. Niederauer, C.E. Glatz, l.A. Suominen, C.F. Ford, and M.A. Rougvie Computation of Conformations and Energies of cr-Glucosyl Disaccharides, Jing Zepg, Michael K. Dowd, and Peter J. Reilly Pentachlorophenol Interactions with Soil, Shein-Ming Wei, Shankha K. Banerji, and Rakesh K. Bajpai Oxygen Transfer to Viscous Liquid Media in Three-Phase Fluidized Beds of Floating Bubble Freakers, Y. Kang, L.T. Fan, B.T. Min and S.D. Kim Studies on the Invitro Development of Chick Embryo, A. Venkatraman and T. Panda The Evolution of a Silicone Based Phase-Separated Gravity-Independent Bioreactor, Peter E. Villeneuve and Eric H. Dunlop Biodegradation of Diethyl Phthalate, Guorong Zhang, Kenneth F. Reardon and Vincent G. Murphy Microcosm Treatability of Soil Contaminated with Petroleum Hydrocarbons, P. Tuitemwong, S. Dhawan, B.M. Sly, L.E. Erickson and J.R. Schlup

Proceedings of the Twenty-Sixth Annual Biochemical Engineering Symposium

This volume contains the Proceedings of the Twenty-Sixth Annual Biochemical Engineering Symposium held at Kansas State University on September 21, 1996. The program included 10 oral presentations and 14 posters. Some of the papers describe the progress of ongoing projects, and others contain the results of completed projects. Only brief summaries are given of some of the papers; many of the papers will be published in full elsewhere. A listing of those who attended is given below. ContentsForeign Protein Production from SV40 Early Promoter in Continuous Cultures of Recombinant CHO Cells - Gautam Banik, Paul Todd, and Dhinakar Kampala Enhanced Cell Recruitment Due to Cell-Cell Interactions - Brad Farlow and Matthias Nollert The Recirculation of Hybridoma Suspension Cultures: Effects on Cell Death, Metabolism and Mab Productivity - Peng Jin and Carole A. Heath The Importance of Enzyme Inactivation and Self-Recovery in Cometabolic Biodegradation of Chlorinated Solvents - Xi-Hui Zhang, Shanka Banerji, and Rakesh Bajpai Phytoremediation of VOC contaminated Groundwater using Poplar Trees - Melissa Miller, Jason Dana, L.C. Davis, Murlidharan Narayanan, and L.E. Erickson Biological Treatment of Off-Gases from Aluminum Can Production: Experimental Results and Mathematical Modeling - Adeyma Y. Arroyo, Julio Zimbron, and Kenneth F. Reardon Inertial Migration Based Separation of Chlorella Microalgae in Branched Tubes - N.M. Poflee, A.L. Rakow, D.S. Dandy, M.L. Chappell, and M.N. Pons Contribution of Electrochemical Charge to Protein Partitioning in Aqueous Two-Phase Systems - Weiyu Fan and Charles C. Glatz Biodegradation of Some Commercial Surfactants Used in Bioremediation - Jun Gu, G.W. Preckshot, S.K. Banerji, and Rakesh Bajpai Modeling the Role of Biomass in Heavy Metal Transport Ln Vadose Zone - K.V. Nedunuri, L.E. Erickson, and R.S. Govindaraju Multivariable Statistical Methods for Monitoring Process Quality: Application to Bioinsecticide Production by 73 89 Bacillus Thuringiensis - c. Puente and M.N. Karim The Use of Polymeric Flocculants in Bacterial Lysate Streams - H. Graham, A.S. Cibulskas and E.H. Dunlop Effect of Water Content on transport of Trichloroethylene in a Chamber with Alfalfa Plants - Muralidharan Narayanan, Jiang Hu, Lawrence C. Davis, and Larry E. Erickson Detection of Specific Microorganisms using the Arbitrary Primed PCR in the Bacterial Community of Vegetated Soil - X. Wu and L.C. Davis Flux Enhancement Using Backpulsing - V.T. Kuberkar and R.H. Davis Chromatographic Purification of Oligonucleotides: Comparison with Electrophoresis - Stephen P. Cape, Ching-Yuan Lee, Kevin Petrini, Sean Foree, Micheal G. Sportiello and Paul Todd Determining Singular Arc Control Policies for Bioreactor Systems Using a Modified Iterative Dynamic Programming Algorithm - Arun Tholudur and W. Fred Ramirez Pressure Effect on Subtilisins Measured via FTIR, EPR and Activity Assays, and Its Impact on Crystallizations - J.N. Webb, R.Y. Waghmare, M.G. Bindewald, T.W. Randolph, J.F. Carpenter, C.E. Glatz Intercellular Calcium Changes in Endothelial Cells Exposed to Flow - Laura Worthen and Matthias Nollert Application of Liquid-Liquid Extraction in Propionic Acid Fermentation - Zhong Gu, Bonita A. Glatz, and Charles E. Glatz Purification of Recombinant T4 Lysozyme from E. Coli: Ion-Exchange Chromatography - Weiyu Fan, Matt L. Thatcher, and Charles E. Glatz Recovery and Purification of Recombinant Beta-Glucuronidase from Transgenic Corn - Ann R. Kusnadi, Roque Evangelista, Zivko L. Nikolov, and John Howard Effects of Auxins and cytokinins on Formation of Catharanthus Roseus G. Don Multiple Shoots - Ying-Jin Yuan, Yu-Min Yang, Tsung-Ting Hu, and Jiang Hu Fate and Effect of Trichloroethylene as Nonaqueous Phase Liquid in Chambers with Alfalfa - Qizhi Zhang, Brent Goplen, Sara Vanderhoof, Lawrence c. Davis, and Larry E. Erickson Oxygen Transport and Mixing Considerations for Microcarrier Culture of Mammalian Cells in an Airlift Reactor - Sridhar Sunderam, Frederick R. Souder, and Marylee Southard Effects of Cyclic Shear Stress on Mammalian Cells under Laminar Flow Conditions: Apparatus and Methods - M.L. Rigney, M.H. Liew, and M.Z. Southard

Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Interaction of SP100 with HP1 proteins: A link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment

The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARα hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.