Life Sciences Commons^™

Circular Dichroism And Molecular Modeling Yield A Structure For The Complex Of Human Immunodeficiency Virus Type 1 Trans-Activation Response Rna And The Binding Region Of Tat, The Trans-Acting Transcriptional Activator, Erwann P. Loret, Philippe T. Georgel, W. Curtis Johnson Jr., Pui Shing Ho

Biological Sciences Faculty Research

Transcription in the human immunodeficiency virus type 1 (HIV-1) retrovirus is regulated by binding the viral Tat protein (trans-acting transcriptional activator) to the trans-activation response (TAR) RNA sequence. Here, vacuum UV circular dichroism (VUV-CD) is used to study the structure of TAR and its complex with two peptide fragments that are important for Tat binding to TAR. The VUV-CD spectrum of TAR is typical of A-form RNA and is minimally perturbed when bound to either the short or the long Tat peptide. The CD spectra ofthe complexes indicate an extended structure in the argnine-rich region of Tat from amino acid …

Structural Characterization Of Fucoidan And Its Role In The Hemizona Assay, Manish S. Patankar

Chemistry & Biochemistry Theses & Dissertations

Fucoidan is a polysaccharide commonly found in brown algae. It is a sulfated polymer of the monosaccharide L-fucose. Fucoidan is thought to function in water retention by algae. Commercially available fucoidan is extracted from a brown algae called Eurus vesiculosus.

This polysaccharide has immense potential as a biologically active agent: (i) it exhibits anticoagulant action, (ii) it has also been reported to block retroviral replication and (iii) it is capable of blocking sperm-egg binding in sea urchin, mouse and rabbit. Recently, fucoidan has also been shown to block the interaction of human sperm and egg in the hemizona assay …

The Structure Of A Complex Of Bovine Ɑ-Thrombin And Recombinant Hirudin At 2.8-Å Resolution, Jacqueline Vitali, Philip D. Martin, Michael G. Malkowski, William D. Robertson, Jerome B. Lazar, Richard C. Winant, Paul H. Johnson, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in …

The Structure Of A Complex Of Bovine &-Thrombin And Recombinant Hirudin At 2.8-A Resolution, Jacqueline Vitali, Philip D. Martin, Michael G. Malkowski, William D. Robertson, Jerome B. Lazar, Richard C. Winant, Paul H. Johnson, Brian F.P. Edwards

Physics Faculty Publications

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in …

Saccharomyces Cerevisiae Sec59 Cells Are Deficient In Dolichol Kinase Activity, Loree Heller, Peter Orlean, W. Lee Adair Jr.

Bioelectrics Publications

The temperature-sensitive Saccharomyces cerevisiae mutant sec59 accumulates inactive and incompletely glycosylated protein precursors in its endoplasmic reticulum at the restrictive temperature. O-mannosylation and glycosyl phosphatidylinositol membrane anchoring of protein are also abolished, consistent with a deficiency in dolichyl phosphate mannose. Membranes prepared from sec59 cells that had been shifted to the restrictive temperature, however, made normal amounts of dolichyl phosphate mannose when exogenous dolichyl phosphate was supplied, but dolichyl phosphate mannose synthesis was severely depressed in the absence of exogenous dolichyl phosphate. Quantitative measurements of dolichyl phosphate in sec59 cells showed that the levels were decreased to 48% of wild …

Appearance Of A Methotrexate Binding Plasma Protein During High Dose Methotrexate Therapy, M P. Iqbal, I Malik, N Mehboobali

Department of Biological & Biomedical Sciences

No abstract provided.

Isolation And Characterization Of The Triiodothyronine Regulatory Region Of The Fatty Acid Synthase Gene In Avian Species, Delfina Rivera Valencia

Masters Theses

Fatty acids are utilized for energy storage in the form of triglycerides or neutral fats. One way to regulate fatty acid biosynthesis is to regulate the activity of fatty acid synthase (FAS), the enzyme that catalyzes the reaction. Previous studies have shown that regulation of FAS activity and synthesis is regulated by the hormone triiodothyronine, T₃, at the level of transcription. Thus, isolation and characterization of the promoter region o f the FAS gene will enable the identification of the thyroid hormone response elements (TREs). This study could shed light on the mechanisms on how this gene can …

Biochemical Investigation Of Gout And Its Familial Incidences, Chin-Ling Hsu

Chemistry & Biochemistry Theses & Dissertations

Gout is a chronic metabolic disorder caused by deposition of uric acid on the joint. It is categorized into two types: primary and secondary. Primary gout is uric acid overproduction, caused by excessive synthesis of the purine precursors. Secondary gout occurs also as the result of overproduction or decreased renal excretion of uric acid resulting from other disease processes or medication.

The two purine bases, hypoxanthine and xanthine, liberated from ribosides by the action of nucleoside phosphorylase, are degraded to uric acid as the final excretory product in the purine degradation pathway. Hypoxanthine and xanthine are the physiological substrates of …

Kinetic Characterization Of A Recombinant C-Terminal Mutant Of Reverse Transcriptase From The Human Immunodeficiency Virus, Thomas S. Heard

Chemistry & Biochemistry Theses & Dissertations

The human immunodeficiency virus (HIV) reverse transcriptase (RT) (EC 2.7.7.49) is the central replication enzyme for HIV. In general, the kinetic mechanism for this and all other polymerases involves the ordered binding of two substrates: a primer-template (PT) followed by a deoxyribonucleoside triphosphate (dNTP). Previous investigations prompted this research when it was discovered that the substrate dNTP, in absence of PT, could protect a recombinant c-terminal mutant HIV-1 RT from inhibition by pyridoxal-5'-monophosphate (PLP), an active-site dNTP inhibitor. In contrast, the non-mutant recombinant HIV-1 RT required both substrates for protection from PLP inhibition. This investigation sought to determine if this …

Differential Regulation Of Collagenase Gene Expression By Retinoic Acid Receptors--Alpha, Beta And Gamma, Luying Pan, Stephen H. Chamberlain, David T. Auble, Constance E. Brinckerhoff

Dartmouth Scholarship

The mechanisms involved in retinoic acid (RA)-mediated regulation of the collagenase gene in a rabbit synovial fibroblast cell line (HIG82) were investigated. When HIG82 cells are cotransfected with expression vectors containing cDNAs for retinoic acid receptor (RAR) α1, β2, or γ1 and collagenase promoter-driven CAT reporter constructs, only RAR-γ1 represses basal CAT expression upon RA treatment, while RAR-α1, β2, and γ1 all suppress phorbol-induced CAT expression. Thus, transcriptional regulation of collagenase by RA is mediated by RARs in an RAR-type specific manner. Using mutatlonal and deletional analysis, we find that interaction between elements within 182 bp collagenase promoter plays an …

Evidence For Helical Structures In Poly(1-Olefin Sulfones) By Transmission Electron Microscopy, George C. Ruben, W H. Stockmayer

Dartmouth Scholarship

Transmission electron microscope images were obtained of fractions of poly(1-tetradecene sulfone) and poly(cyclohexene sulfone) cast from very dilute solutions (0.007%, wt/vol) and rapidly freeze-dried on a mica surface. The samples were then vertically platinum-carbon (Pt-C) replicated with 9 +/- 0.3-A Pt-C and held together with 128 A of electron-transparent evaporated carbon. The Pt-C coating enlarges the molecular chain diameters by approximately 5 A, so that a single polysulfone chain has an apparent diameter of 9-12 A in the transmission electron microscope. Poly(1-tetradecene sulfone) forms short helical regions that show irregular helical turns of pitch 7-18 A, two to eight turns …

Mobilization Of Iron Enhances The Iron-Dependent Biochemical Reactivity Of Asbestos In Vitro And Contributes To The Cytotoxicity Of Asbestos In Cultured Cells, Loren Glen Lund

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Asbestos related research began approximately 60 years ago, yet, the mechanism(s) by which asbestos exerts its biological effects is not well understood. The hypothesis upon which this dissertation is based is that mobilization of iron from asbestos enhances the iron-dependent biochemical reactivity of asbestos in vitro and contributes to asbestos-dependent cytotoxicity. The specific aims for this hypothesis were, 1) to determine whether iron was responsible for the biochemical reactivity of asbestos in vitro and asbestos-induced cytotoxicity in cultured cells, and 2) to determine whether mobilization of iron from asbestos enhanced the reactions catalyzed by asbestos in vitro and contributes to …

The Structure Of Residues 7-16 Of The Aɑ-Chain Of Human Fibrinogen Bound To Bovine Thrombin At 2.3 Å Resolution, Philip D. Martin, William Robertson, Dusan Turk, Robert Huber, Wolfram Bode, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active …

Methotrexate As An Alternative To Surgery In Ectopic Pregnancy: A New Role For An Old Drug, M P. Iqbal

Department of Biological & Biomedical Sciences

Abstract is not provided by the author/publisher

Biochemical Analysis Of Human Seminal Plasma, Jamshed Ayub

Chemistry & Biochemistry Theses & Dissertations

Presently there is no single biochemical criterion for differentiating between a potentially fertile male and an infertile male. However, for evaluating male fertility the biochemical analysis of seminal plasma is a very convenient and quantitative technique if it is combined with careful clinical examination.

In seminal plasma analysis, fructose and proteins are chemical indicators of seminal vesicle activity, citric acid and acid phosphatase indicate prostate function while glycerolphosphorylcholine indicates epididymis activity. In this research these five indicators are studied and an attempt is made to establish their normal ranges.

This study shows that the data obtained from the seminal plasma …

Estimation And Separation Of N-Acetyl-Beta-D-Glucosaminidase Isoenzymes In Urine, M H. Javed, A N. Hussain, M A. Waqar

Department of Biological & Biomedical Sciences

N-acetyl-beta-D-glucosaminidase is a lysosomal enzyme made up of two isoenzymes (A and B). It has been used extensively as a marker for kidney damage. However, its estimation in urine has not been standardized. We have established a method for the estimation and separation of NAG isoenzymes by ion-exchange chromatography. In 19 experiments done so far, this method has given reproducible results. The significance of this method is that with a single experiment, one can estimate total as well as individual isoenzyme activity. Furthermore, urine constituents do not appear to interfere in this assay.

Structural Changes That Accompany The Reduced Catalytic Efficiency Of Two Semisynthetic Ribonuclease Analogs, V. Srini J. De Mel, Philip D. Martin, Marilynn S. Doscher, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

The structures of two catalytically defective semi-synthetic RNases obtained by replacing aspartic acid 121 with asparagine or alanine have been determined and refined at a resolution of 2.0 A (R = 0.186 and 0.172, respectively). When these structures are compared with the refined 1.8-A structure (R = 0.204) of the fully active aspartic acid-containing enzyme (Martin, P.D., Doscher, M.S., and Edwards, B. F. P. (1987) J. Biol. Chem. 262, 15930-15938), numerous and widespread changes, much greater in number and magnitude than the small structural variations noted previously between the semisynthetic complex and RNase A, are found to have occurred. These …

Monoclonal Antibodies Specific For Mercuric Ions, Dwane E. Wylie, Di Lu, Larry D. Carlson, Randy Carlson, K. Funda Babacan, Sheldon M. Schuster, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Monoclonal antibodies (mAbs) that react with soluble mercuric ions have been produced by i 'ection of BALB/c mice with a hapten-carrier complex designed to maximize exposure of the metal to the immune system. Three hybridomas producing antibodies that reacted with bovine serum albumin (BSA)-glutathione-HgCI, but not with BSAglutathione, were isolated from the spleen of a mouse given multiple iqjections with glutathione-HgCl conjugated to keyhole limpet hemocyanin. Stable subclones were established from two of these antibodies, designated mAb 4A10 and mAb IF10. The binding of both antibodies to immobilized BSAglutathione- HgCl was inhibited by soluble HgCl₂, and dissociation constants …

Studies On Photoaffinity Labeling And Mass Spectrometry Of Bacteriorhodopsin, Charles John Beischel

MUSC Theses and Dissertations

The objective of these studies was to further define the amino acids of bacteriorhodopsin in close proximity to the ring end of retinal by labeling them using a retinal analog modified to contain a photoactivatable group. The sites of labeling were to be identified using mass spectrometry and tandem mass spectrometry. Eight potentially photoactivatable retinal analogs were characterized and thoroughly examined as bacteriorhodopsin analog pigments thus identifying those analogs that were most similar to the native retinal. A versatile flash photolysis apparatus was compared to a continuous wave light source on the basis of total photon output and on efficiency …

A New Family Of Heparin Binding Growth/Differentiation Factors: Differential Expression Of The Midkine (Mk) And Hb-Gam Genes During Mouse Development, Masaru Nakamoto, Shyuichiro Matsubara, Teruo Miyauchi, Hiroya Obama, Takashi Muramatsu

Biology Faculty Publications

MK (midkine) and HB-GAM (heparin-binding growth-associated molecule) constitute a new family of heparin-binding growth differentiation factors. The modes of expression of MK and HB-GAM during mouse development were quantitatively examined by mRNA hybridization. The following three distinct patterns of expression were observed in the brain/head region. On the 11th-13th days of gestation, MK was intensely, but HB-GAM relatively weakly expressed; on the 15th-19th days, both MK and HB-GAM expression became weaker; and in the neonatal period, HB-GAM was intensely expressed and MK expression increased slightly. The level of HB-GAM expression was lower than that of MK in the whole embryo …

The Cγ Subunit Is A Unique Isozyme Of The Camp-Dependent Protein Kinase, Stephen J. Beebe, Paul Salomonsky, Tore Jahnsen, Yixin Li

Bioelectrics Publications

There are at least three isozymes (Cα, Cβ, and Cγ) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the Cγ and Cα isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to Cα, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for Cγ. Furthermore, Cγ histone kinase activity was not inhibited by the …

Chloroplast Heteroplasmicity Is Stabilized By An Amber-Suppressor Tryptophan TrnaCua, Weizhu Yu, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

Photosynthesis-deficient mutants of the green alga Chlamydomonas reinhardtii were previously shown to arise from nonsense mutations within the chloroplast rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Photosynthesiscompetent revertants of these nonsense mutants have often been found to be stably heteroplasmic, giving rise to both mutant and revertant cells during mitotic or meiotic divisions under nonselective growth conditions. It was proposed that the stable heteroplasmicity might arise from a balanced polymorphism of suppressor and wild-type alleles of a tRNA gene maintained within the polyploid chloroplast genome. In the present study, we have focused on determining the …

Lack Of Types 1 And 2a Protein Serine(P)/Threonine(P) Phosphatase Activities In Chloroplasts, Gongqin Sun, John Markwell

Department of Biochemistry: Faculty Publications

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid lightharvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein …

Transition Metals In Legume Root Nodules: Iron-Dependent Free Radical Production Increases During Nodule Senescence, Manuel Becana, Robert V. Klucas

Department of Biochemistry: Faculty Publications

The cytosol from root nodules of soybean, bean, and cowpea contained Fe and Cu capable of catalyzing the formation of highly reactive free radicals. Specific and sensitive assays based on free radical-mediated DNA degradation revealed that most catalytic Fe and Cu were present as small chelates (300-600 Da). The involvement of catalytic Fe in free radical production during nodule senescence, which was induced by exposure of plants to continuous darkness for 2-4 days, was investigated. (i) Free heme remained at a constant and low concentration (1-4% of total nodule heme) during senescence, indicating that it is not an …

A Proposal For United States/Polish Collaboration On Energy And The Environment, Carole B. Szpunar

Chemistry: Faculty Publications and Other Works

Argonne National Laboratory, a U.S. Department of Energy facility that is operated by the University of Chicago and located southwest of Chicago, Illinois, has proposed a collaborative effort by the United States and Poland to conduct applied research on energy and environmental topics of common interest. Through this collaboration, evaluative studies on energy and environmental assessment issues could be developed and implemented by Argonne and one or more sister institutions in Poland, which could lead to active research, development, and demonstration joint activities. The objective of this initiative is to produce strategies and options for review by Poland's decision makers …

Nucleotide Sequence Of S-Adenosyl-L-Methionine: Magnesium Protoporphyrin Methyltransferase From Rhodobacter Capsulatus, David Bollivar

Scholarship

No abstract provided.

Light Activation Of Maize Phosphoenolpyruvate Carboxylase Protein-Serine Kinase Activity Is Inhibited By Mesophyll And Bundle Sheath-Directed Photosynthesis Inhibitors, Jin-An Jiao, Raymond Chollet

Department of Biochemistry: Faculty Publications

C₄ phosphoenolpyruvate carboxylase (PEPC) is post-translationally regulated by reversible phosphorylation of a specific N-terminal seryl residue in response to light/dark transitions of the parent leaf tissue. The protein-serine kinase (PEPC-PK) that phosphorylates/activates this mesophyll-cytoplasm target enzyme is slowly, but strikingly, activated by high light and inactivated in darkness in vivo by a mechanism involving cytoplasmic protein synthesis/degradation as a primary component. In this report, evidence is presented indicating that the inhibition of Calvin cycle activity by a variety of mesophyll (3-(3,4-dichlorophenyl)-1,1-dimethylurea, isocil, methyl viologen) and bundle sheath (DL-glyceraldehyde)-directed photosynthesis inhibitors blocks the light activation of maize (Zea mays …

The Effects Of Ethanol On Cultured Serotonergic Neurons By Denise Kay Lokhorst., Denise Kay Lokhorst

Dissertations

No abstract provided.

Association Of Tetrapyrrole Intermediates In The Bacteriochlorophyll A Biosynthetic Pathway With The Major Outer-Membrane Porin Protein Of Rhodobacter Capsulatus, David Bollivar

Scholarship

No abstract provided.

Improved Recovery Of A Radlolabeled Peptide With An Albumin-Treated Reversed-Phase Hplc Column, David S. Hage, Robert L. Taylor, Pai C. Kao

David Hage Publications

Reversed-phase high-performance liquid chromatography (RP-HPLC) is an important tool in the purification of radiolabeled peptides and proteins for immunoassay. However, for some proteins and peptides it is difficult to achieve reproducible behavior in RP-HPLC because of the low recovery of these compounds. Factors that can be varied to improve recovery include the strength or pH of the mobile phase, the chain length and spacing of groups on the reversed-phase support, and the flow rate or steepness of the elution gradient (1-5). ... In summary, we obtained better recovery and more reproducible chromatographic behavior for labeled 1-34 PTHrP with an albumin-pretreated …