Proceedings of the Sixth Annual Biochemical Engineering Symposium

This symposium is the sixth of an annual series conducted so that results of biochemical engineering research can be exchanged by the researchers who actually carry it out. The first four meetings were held alternately at Kansas State University and the University of Nebraska–Lincoln for attendees from those two schools. The fifth and sixth involved participants from Kansas State University and Iowa State University; this was the first meeting away from a university campus. Contents"Mathematical Model of Oxygen Transfer in Airlift Fermentors," Chester S. Ho, Kansas State University "Effect of Column Height on Oxygen Transfer in Airlift Systems," Mark E. Orazem, Kansas State University "Mixing Studies in an Oil-Water Airlift System with Motionless 15 Mixers", J. R. Gutierrez, Kansas State University "Purification and Properties of (3-Xylosidase," Gbekeloluwa B. Oguntimein, Iowa State University "Immobilization of Invertase to Cellulose with Cyanuric Chloride," William J. Smith, Iowa State University "Purification and Properties of Dextransucrase," Yah Eric Chen and Hossein Kaboli, Iowa State University "Properties of Immobilized (3-Amylase," Clarence C. Ron, Iowa State University

Proceedings of the Seventh Annual Biochemical Engineering Symposium

This is the seventh in a series of symposia devoted to talks by students on their biochemical engineering research. The first four meetings were held alternately at Kansas State University and the University of Nebraska–Lincoln, with participants from those two schools. The next two took place at Kansas State and then in conjunction with the 8lst American Institute of Chemical Engineers National Meeting in Kansas City, with attendees from Kansas State and Iowa State Universities. This meeting, at Iowa State, was the first to include participation from the University of Missouri–Columbia. Contents"Properties of Soluble and In:anoblized Dextransucrase," Hossein Kaboli and Yah Eric Chen, Iowa State University "Growth of Lipid-Producing Organisms on Formic and Acetic Acid-Containing Waste Waters," Lin-Chang Chiang, University of Missouri–Columbia "Design of an Automated Alkaline Copper Reducing Sugar Assay," Alfred R. Fratzke and James R. Frederick, Iowa State University "Determination of Oxygen Transfer Coefficients in Hydrocarbon Fermentations Using a Material Balance Method," Sarafin N. Sanchez and J. R. Gutierrez, Kansas State University "Oxygen Transfer Characteristics in One Stage and Two Stage Air-Lift Towers," Mark E. Orazem, Kansas State University "A Comparison of Biological Digestibility Tests for Cellulose," Dou-Houng Hwang, University of Missouri–Columbia "Mechanism of Enzymatic Hydrolysis of Cellulose," L. T. Fan, Yong-Hyun Lee, and Liang-Shih Fan, Kansas State University "Purification of Xylan-Hydrolyzing Enzymes," James R. Frederick, Alfred R. Fratzke, and Mary M. Frederick, Iowa State University "Cellulase Production from Bagasse and Pith," A. Ferrer, Y. Alroy, and I. Brito, Kansas State University

Proceedings of the Ninth Annual Biochemical Engineering Symposium

This report presents the proceedings of the Biochemical Engineering Symposium held at Kansas State University, April 28, 1979. Since a number of the contributions will be published in detail elsewhere, only brief reports of each contribution are included here. Requests for further information on work at Iowa State University should be directed to Dr. Peter J. Reilly; at Colorado State University to Drs. V. G. Murphy and A. R. Moreira, and at Kansas State University to Drs. L. T. Fan and L. E. Erickson. ContentProperties of a Homogeneous Xylobiohydrolase from Aspergillus niger, Mary M. Frederick, Iowa State University Kinetic Studies on the Enzymatic Hydrolysis of Cellulose–Absorption and Desorption of Cellulase onto Cellulose and the Behavior of Absorbed Cellulase, Yong-Hyun Lee and L. T. Fan, Kansas State University Properties of a Homogeneous Endo-Xylanase from Aspergillus niger, Ricardo Fournier A., Iowa State University Solid State Fermentation of Manure Fibers, D. C. Ulmer, Colorado State University Analysis and Consistency of Experimental Data for Microbial Growth on Renewable Resources, B. 0. Solomon, Kansas State University Biochemical Mechanisms of Enzyme Regulation, Frederick A. Blum, Colorado State University An Evaluation of Cellulose Pretreatments for Enzymatic Hydrolysis, David H. Beardmore, Kansas State University Use of Immobilized 8-Amylase/Glucoamylase Mixtures to Produce High Maltose Syrups, Carol G. Bohnenkamp, Iowa State University Effect of Viscosity on Bubble Behavior in an Airlift Fermentor, Vasanti Deshpande, Kansas State University

Proceedings of the Eighth Annual Biochemical Engineering Symposium

Contents"Effects of Swell-Shear Treatment on the Digestibility of Cellulosics", Dou-Houng Hwang, UMC "Application of Material and Energy Balance Regularities to Biomass Production from Cellulosic Substrates", Y.H. Lee, KSU "Immobilization of Aspergillus niger beta-Xylosidase", Gbekeloluwa B. Oguntimein, ISU "The Effect of the Major Structural Parameters of Cellulose on Enzymatic Hydrolysis", David H. Beardmore, Y.H. Lee, and L.T. Fan, KSU "Purification of a High Molecular Weight Hemicellulase", Ricardo A. Fournier, ISU "Aerobic Fermentation of Banana Pulp by Aspergillus Fumigatus", Stephen Lorbert, UMC "Purification and Properties of Two Very Small Xylanases", Chih-hen Kiang, ISU "Testing Theoretical Models for Cellulose Enzymatic Hydrolysis", Lin-Chang Chiang, UMC "Utilization of Material and Energy Balances in Hydrocarbon Fermentation", Alexis Ferrer, KSU "Purification of a Series of Closely Related Xylanases", Mary M. Frederick, ISU

Proceedings of the Twelfth Annual Biochemical Engineering Symposium

This work presents the proceedings of the twelfth symposium which was held at Kansas State University on April 24, 1982. Since a number of the contributions will be published in detail elsewhere, only brief reports are included here. Some of the reports describe current progress with respect to ongoing projects. Requests for further information should be directed to Dr. Peter Reilly at Iowa State University, Dr. V. G. Murphy at Colorado State University, Dr. Rakesh Bajpai at University of Missouri, Dr. Ed Clausen at University of Arkansas, Dr. L. T. Fan and Dr. L. E. Erickson at Kansas State University. ContentsA Kinetic Analysis of Oleaginous Yeast Fermentation by Candida curvata on Whey Permeate, B.D. Brown and K.H. Hsu, Iowa State University Kinetics of Biofouling in Simulated Water Distribution Systems Using CSTR, T.M. Prakash, University of Missouri Kinetics of Gas Production by C. acetobutylicum, Michael Doremus, Colorado State University Large Scale Production of Methane from Agricultural Residues, O.P. Doyle, G.C. Magruder, E.C. Clausen, and J.L. Gaddy, University of Arkansas The Optimal Process Design for Enzymatic Hydrolysis of Wheat Straw, M.M Gharpuray and L.T. Fan, Kansas State University Extractive Butanol Fermentation, Michael Sierks, Colorado State University Yields Associated with Ethyl Alcohol Production, M.D. Oner, Kansas State University Estimation of Growth Yield and Maintenance Parameters for Microbial Growth on Corn Dust, B.O. Solomon, Kansas State University Milling of Ensiled Corn, Andrzej Neryng, Iowa State University Protein Extraction from Alfalfa, Ravidranath Joshi, Colorado State University Analysis of Disaccharides by Capillary Gas Chromatography, Z.L. Nikolov, Iowa State University Characterization of High Viscosity Fermentations in Tower Fermentors, S.A. Patel and C.H. Lee, Kansas State University Utilization of Sugars in Sorghum Molasses by Clostridium acetobutylicum B. Hong, K.C. Shin, and L.T. Fan, Kansas State University

Proceedings of the Eleventh Annual Biochemical Engineering Symposium

This is the eleventh in a series of symposia devoted to talks by students on their biochemical engineering research. The first, third, fifth, and ninth were at Kansas State University in Manhattan; the second and fourth were at the University of Nebraska–Lincoln; the sixth was in Kansas City in conjunction with the 8lst American Institute of Chemical Engineers National Meeting; the seventh and the tenth were at Iowa State University in Ames; and the eighth was held at the University of Missouri–Columbia. ContentsPretreatment of Wheat Straw for Cellulose Hydrolysis, M. M. Gharpuray, Yong-Hyun Lee, and L. T. Fan, Kansas State University Sugar Production During Autohydrolysis of Wheat Straw, Robert A. Lewis, Colorado State University An Alkaline Copper Reagent for Use in Automated Analysis, Alfred R. Fratzke, Iowa State University Sugars Produced During Extrusion Processing of Corn, Ruth S. Korn, Colorado State University Characterization and Comparison of Renewable Energy Resources, Snehal A. Patel, Kansas State University Anaerobic Digestion of Alcohol Stillage, Laureen K. Binder, Colorado State University Estimation of Growth Yield and Maintenance Parameters, Bamidele 0. Solomon and Mehmet D. Oner, Kansas State University Immobilization of Glucoamylase Using TiCl4 and Organic Titanates, Robert E. Lesch, Iowa State University Solvent Toxicity in the Acetone-Butanol Fermentation, Jeanine M. Costa, Colorado State University

Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels.

Early changes in biochemical markers of bone formation during teriparatide therapy correlate with improvements in vertebral strength in men with glucocorticoid-induced osteoporosis

Summary Changes of the bone formation marker PINP correlated positively with improvements in vertebral strength in men with glucocorticoid-induced osteoporosis (GIO) who received 18-month treatment with teriparatide, but not with risedronate. These results support the use of PINP as a surrogate marker of bone strength in GIO patients treated with teriparatide. Introduction To investigate the correlations between biochemical markers of bone turnover and vertebral strength estimated by finite element analysis (FEA) in men with GIO. Methods A total of 92 men with GIO were included in an 18-month, randomized, open-label trial of teriparatide (20 μg/day, n = 45) and risedronate (35 mg/week, n = 47). High-resolution quantitative computed tomography images of the 12th thoracic vertebra obtained at baseline, 6 and 18 months were converted into digital nonlinear FE models and subjected to anterior bending, axial compression and torsion. Stiffness and strength were computed for each model and loading mode. Serum biochemical markers of bone formation (amino-terminal-propeptide of type I collagen [PINP]) and bone resorption (type I collagen cross-linked C-telopeptide degradation fragments [CTx]) were measured at baseline, 3 months, 6 months and 18 months. A mixed-model of repeated measures analysed changes from baseline and between-group differences. Spearman correlations assessed the relationship between changes from baseline of bone markers with FEA variables. Results PINP and CTx levels increased in the teriparatide group and decreased in the risedronate group. FEA-derived parameters increased in both groups, but were significantly higher at 18 months in the teriparatide group. Significant positive correlations were found between changes from baseline of PINP at 3, 6 and 18 months with changes in FE strength in the teriparatide-treated group, but not in the risedronate group. Conclusions Positive correlations between changes in a biochemical marker of bone formation and improvement of biomechanical properties support the use of PINP as a surrogate marker of bone strength in teriparatide-treated GIO patients.

Extended kalman filter for estimation of parameters in nonlinear state-space models of biochemical networks.

It is system dynamics that determines the function of cells, tissues and organisms. To develop mathematical models and estimate their parameters are an essential issue for studying dynamic behaviors of biological systems which include metabolic networks, genetic regulatory networks and signal transduction pathways, under perturbation of external stimuli. In general, biological dynamic systems are partially observed. Therefore, a natural way to model dynamic biological systems is to employ nonlinear state-space equations. Although statistical methods for parameter estimation of linear models in biological dynamic systems have been developed intensively in the recent years, the estimation of both states and parameters of nonlinear dynamic systems remains a challenging task. In this report, we apply extended Kalman Filter (EKF) to the estimation of both states and parameters of nonlinear state-space models. To evaluate the performance of the EKF for parameter estimation, we apply the EKF to a simulation dataset and two real datasets: JAK-STAT signal transduction pathway and Ras/Raf/MEK/ERK signaling transduction pathways datasets. The preliminary results show that EKF can accurately estimate the parameters and predict states in nonlinear state-space equations for modeling dynamic biochemical networks.

Bioavailability of silver nanoparticles and ions: from a chemical and biochemical perspective

Owing to their antimicrobial properties, silver nanoparticles (NPs) are the most commonly used engineered nanomaterial for use in a wide array of consumer and medical applications. Many discussions are currently ongoing as to whether or not exposure of silver NPs to the ecosystem (i.e. plants and animals) may be conceived as harmful or not. Metallic silver, if released into the environment, can undergo chemical and biochemical conversion which strongly influence its availability towards any biological system. During this process, in the presence of moisture, silver can be oxidized resulting in the release of silver ions. To date, it is still debatable as to whether any biological impact of nanosized silver is relative to either its size, or to its ionic constitution. The aim of this review therefore is to provide a comprehensive, interdisciplinary overview--for biologists, chemists, toxicologists as well as physicists--regarding the production of silver NPs, its (as well as in their ionic form) chemical and biochemical behaviours towards/within a multitude of relative and realistic biological environments and also how such interactions may be correlated across a plethora of different biological organisms.

Molecular and biochemical analysis of the {\it Drosophila\/} segmentation gene {\it runt\/}

Genetic evidence has indicated that the segmentation gene runt plays a key role in regulating gene expression of the pair-rule genes hairy, even-skipped, and fushi tarazu. In contrast to other pair-rule genes, sequence data of the runt open reading frame did not reveal homologies to DNA-binding motifs of known transcriptional regulatory proteins. This thesis project examined several properties of the runt gene based on the sequence of the transcription unit, including the subcellular localization of the protein in vivo, its ability to bind DNA, and the functionality of a putative nucleotide binding domain.^ A runt-specific antibody was generated and used to demonstrate that runt is localized in the nucleus. Since the precise overlap of the pair-rule stripes is thought to be critical for the determination of cellular identity along the anterior-posterior axis, phasing of early runt expression in the blastoderm was examined with regard to the segmentation genes hairy, even-skipped, and fushi tarazu. runt was also expressed at later stages of embryogenesis, including expression in neuroblasts, and ganglion mother cells of the developing nervous system. Expression at this stage was required for the subsequent formation of specific neurons and runt was extensively expressed in the central and peripheral nervous systems.^ Several experiments were done to address the biochemical function of the runt protein. A direct interaction of runt with DNA was first examined. Although bacterial expressed runt was found to bind dsDNA-cellulose, subsequent experiments failed to detect sequence-specific interactions with DNA. Inter-species conservation of the putative nucleotide binding domain suggested that this region was functionally important, and runt protein bound a labeled ATP analog with high affinity in vitro. Finally, the effect of substitution of a critical residue of the nucleotide binding domain on runt activity was examined in vivo. Ectopic expression of the mutant protein indicated that this conserved substitution altered, but did not eliminate, runt activity as evaluated by segmentation phenotype and viability. ^

Biochemical characterization of PICK1, a PKC binding protein

Protein kinase C (PKC) is a family of serine-threonine kinases that are activated by a wide variety of hormones, neurotransmitters and growth factors. A single cell type contains multiple isoforms that are translocated to distinct and different subcellular sites upon mitogenic stimulus. Many different cellular responses are attributed to PKC activity though relatively few substrates or binding proteins have been definitively characterized. We used the hinge and catalytic domain of PKC$\alpha$ (PKC7) in a yeast two-hybrid screen to clone proteins that interact with C-kinase (PICKs). One protein which we have termed PICK1 may be involved in PKC$\alpha$-specific function at the level of the nuclear membrane after activation. Binding of PICK1 to PKC$\alpha$ has been shown to be isoform specific as it does not bind to PKC$\beta$II or PKC$\alpha$ in the yeast two-hybrid system. PICK1 mRNA expression level is highest in testis and brain with lower levels of expression in skeletal muscle, heart, kidney, lung and liver. PICK1 protein contains five PKC consensus phosphorylation sites and serves as an in vitro substrate for PKC. The PICK1 protein also contains a P-Loop motif that has been shown to bind ATP or GTP in the Ras family of oncoproteins as well as the G-Protein family. Proteins which bind ATP or GTP using this motif all have some sort of catalytic function although none has been identified for PICK1 as yet. PICK1 contains a DHR/GLGF motif at the N-terminus of the protein. The DHR/GLGF motif is contained in a number of recently described proteins and has been shown to mediate protein-protein interactions at the level of membranes and cytoskeleton. When both PKC$\alpha$ and PICK1 are co-expressed in Cos1 cells the two proteins co-localize to the perinucleus in immunoflouresence studies and co-immunoprecipitate. The binding site for PKC7 has been localized to amino acids 1-358 on PICK1 which contains the DHR/GLGF motif. Binding of PICK1 to PKC$\alpha$ requires the hinge and C-terminal domains of PKC$\alpha$. In vitro, PICK1 binds to PKC$\alpha$ and inhibits its activity as assayed by myelin basic protein phosphorylation. PICK1 also binds to TIS21, a primary response gene that is expressed in response to phorbol ester and growth factor treatment. The Caenorhabditis elegans homologue of PICK1 has been cloned and sequenced revealing a high degree of conservation in the DHR/GLGF motif. A more C-terminal region also shows a high degree of conservation, and the C. elegans PICK1 homologue binds to PKC7 suggesting a conservation of function. Taken together these results suggest that PICK1 may be involved in a PKC$\alpha$-specific function at the level of the nuclear membrane. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^