Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

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 …

Lysosomal Enzyme Secretion In Tumor Cells And The Purification And Partial Characterization Of A 67 Kda Cathepsin B-Like Enzyme Secreted By B16 Melanoma Cells, Charles Achkar

Dissertations

No abstract provided.

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 …

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.

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

Scholarship

No abstract provided.

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 …

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 …

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 …

Intact Parathyroid Hormone: Performance And Clinical Utility Of An Automated Assay Based On High-Performance Immunoaffinity Chromatography And Chemiluminescence Detection, David S. Hage, Bob Taylor, Pai C. Kao

David Hage Publications

The performance and clinical utility of an automated assay of intact parathyroid hormone (parathyrin, PTH) are evaluated. The method is based on the extraction of PTH from plasma by an HPLC column containing immobilized anti-(44-68 PTH) antibodies. The PTH retained is detected with a postcolumn reactor and use of anti-(1--34 PTH) chemiluminescent-labeled antibodies. The total cycle time of the assay is 6.5 mm per injection after a 1-h incubation.The lower limit of detection for PTH in a 66-pL plasma sample was 0.5 pmol/L based on peak heights and 0.2 pmol/L based on peak areas. Mean analytical recovery for PTH added …

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

Dissertations

No abstract provided.

The Effects Of Selenate, Molybdate And Chromate On The Triiodothyronine Induced Enzyme Activity And Mrna Level For Both Fatty Acid Synthase And Malic Enzyme, Yuan Zhu

Masters Theses

In chick embryo hepatocytes in culture, triiodothyronine (T3) stimulates enzyme activity, mRNA level and transcription rate for both fatty acid synthase (FAS) and malic enzyme (ME). Recent evidence has demonstrated the effects of selenate, molybdate and chromate on various physiological processes. Little information, however, is available on the effects of these metal ions on the T3- regulated gene expression for FAS and ME.

In chick embryo hepatocytes incubated in a chemically defined medium, addition of sodium selenate (20 μM) coincident with T3 almost completely inhibited the T3-induced stimulation of FAS and ME activity and accumulation of their respective mRNA's. Neither …

Using Xenon As A Heavy Atom For Determining Phases In Sperm Whale Metmyoglobin, Jacqueline Vitali, Arthur H. Robbins, Steven C. Almo, Robert F. Tilton

Physics Faculty Publications

Xenon gas can be used as a heavy atom for determining phases in a protein. We demonstrate that an interpretable electron density map can be obtained for sperm whale metmyoglobin from a single xenon derivative using iterative single isomorphous replacement with the anomalous scattering method.

High Performance Liquid Chromatographic Separation Of Different Types Of Collagen, Yongjoo Chung

Chemistry & Biochemistry Theses & Dissertations

The objective of this research was to develop a method for the separation of different types of collagen using a large pore size (100 nm) reversed-phase C-8 column. Three different types of collagen (type I, III, and V) were sufficiently separated on the column using a mobile phase containing water-acetonitrile-trifluoroacetic acid. Collagens found in each HPLC peak were identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to assay the column effluents.

Trifluoroacetic acid (TFA) was used as an ion pair reagent. This compound improved the chromatographic profile for these proteins on a reversed-phase column. It was observed that with …

Expression Of Biologically Active Human Corticosteroid Binding Globulin By Insect Cells: Acquisition Of Function Requires Glycosylation And Transport, Jayasri Ghose-Dastidar, J. B. Alexander Ross, Reza Green

Chemistry and Biochemistry Faculty Publications

Human corticosteroid binding globulin (hCBG) is a 50- to 55-kDa serum glycoprotein that binds cortisol and progesterone with high affinity. To map the steroid-binding domain and to investigate the folding pathways of hCBG, we have established an expression system based on infection of insect cells with a recombinant baculovirus encoding hCBG. Infected Spodoptera frugiperda (Sf9) cells secrete immunoreactive hCBG at high levels (16-24 pmol per 10⁶ cells per 40 h), and the recombinant protein binds cortisol with an affinity and specificity equivalent to that of human serum-derived hCBG. Thus, this system has the potential to provide large amounts of …

Characterization Of Two Temperature-Sensitive Mutants Of Escherichia Coli Exhibiting An Altered L22 Ribosomal Protein, Bonnie A. Burnette-Vick

Electronic Theses and Dissertations

Analysis of E. coli strains SK1047 and SK1048 have shown them to be temperature-sensitive, protein-synthesis deficient. An alteration in ribosomal protein L22 was detected in both strains using two dimensional gel electrophoresis. Protein L22 was purified from both strains by reversed phase high performance liquid chromatography and from two dimensional electrophoretic gels. Purified ribosomal protein L22 was labeled by reductive methylation and used in 23S RNA binding assays with and without ribosomal protein L4. At the permissive temperature, protein L22 from SK1047 bound less efficiently than the control while protein L22 from SK1048 bound as efficiently as the control. At …

Purification And Characterization Of The Prostate Cancer-Associated Antigen: 7e11-C5, Qi Feng

Theses and Dissertations in Biomedical Sciences

A murine monoclonal antibody (MAb), 7E11-C5, recognizes a prostate organ-specific antigen. Under reducing conditions, the 7E11-C5 antigen exhibited major 100-Kd and minor 70-Kd bands, suggesting that the antigen comprises two peptide chains. The antigen was detected in pooled normal human seminal plasma, xenograft tissue extract of a prostate adenocarcinoma cell line, LNCaP, and tissue extracts of benign prostatic hyperplasia (BPH) and prostate carcinomas, but was not identified in any of the non-prostate tissue extracts tested. Antibodies of the three major prostate-associated antigens (prostatic acid phosphatase (PAP), prostate specific antigen (PSA) and prostate secretory protein (PSP)) were unable to block the …

Heterogeneity Of Methotrexate Binding In Human Colon Tumor Cells, M P. Iqbal, N Mehboobali, M A. Waqar, M Ahmed

Department of Biological & Biomedical Sciences

[3H]-methotrexate binding at pH 5.0 and pH 7.2 by the cytosol of tumor tissues and the surrounding normal areas of the gastrointestinal tract of patients suffering from colon or gastric cancer has been used to identify in these cells the presence of a binder of methotrexate having low-affinity for this drug in addition to the enzyme dihydrofolate reductase. Scatchard analysis of the [3H]-methotrexate binding data by a colon tumor sample also reveals that there are two binders of this drug present in the cytosol of these cells. The association constant (Kass) for one binder of methotrexate is = 5.6 x …

Aerotaxis In Halobacterium Halobium, Jemima Clara Lindbeck

Loma Linda University Electronic Theses, Dissertations & Projects

Aerotaxis, previously studied in eubacteria was investigated in the archaebacterium Halobacterium halobium. A quantitative temporal assay for aerotaxis of H. halobium was developed using computer assisted motion analysis. Aerotaxis was most pronounced in early log phase cultures. Peak aerotaxis was found to coincide with peak respiration consistent with a model in which respiration is required for aerotaxis. In a spatial assay a mutant strain lacking the proton motive force (pmf)-generating pigments, bacteriorhodopsin (bR) and halorhodopsin (hR), exhibited enhanced aerotaxis bands relative to the bR⁺hR⁺ parent. The enhanced aerotaxis in the mutant cells was not due solely to …

Cytolytic And Ion Channel Forming Properties Of The N-Terminus Of Lymphocyte Perforin, David M. Ojcius, Pedro M. Persechini, Li-Mou Zheng, Paulo C. Notaroberto, Sandro C. Adeodato, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Perforin lyses cells by binding to the target cell membrane, where it polymerizes into large nonspecific pores. It is shown here that the first 34 amino acids of the N-terminal region of either human or murine perforin are soluble in aqueous medium and spontaneously insert into membranes. The N-terminal peptides lyse liposomes and nucleated cells, and they form ion channels in planar bilayers, some of which are comparable to those previously described for perforin. The lytic activity of the N-terminal domains does not require calcium, is independent of the lipid headgroup composition, and can be inhibited by heparin. Tumor cells …

Molecular Genetics Of Endocrine Induced Regression Of Mouse Leydig Cell Tumors, Milton Rolando Drachenberg

Loma Linda University Electronic Theses, Dissertations & Projects

Chronic estrogen treatment results in malignant Leydig cell tumor formation in genetically susceptible strains of mice. The hypothesis that the mechanism of estrogen induced carcinogenesis includes a gene amplification event was tested using this in vivo endocrine carcinogenesis model. High molecular weight DNA isolated from liver, hyperplastic chryptorchid testes, primary Leydig cell tumors and transplanted tumor lines progressing to estrogen nondependence for growth, was studied using Southern blot analysis and the in-gel renaturation technique to identify amplification of characterized and anonymous sequences. No consistent gene amplification event could be demonstrated either in carcinogenesis or tumor progression to an estrogen nondependent …

Heterologous Expression And Characterization Of Three Active Recombinant Fungal Peroxidases From Phanerochaete Chrysosporium Using Baculovirus, Todd M. Johnson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

In a collaborative effort with Dr. Steven D. Aust's group at Utah State University, this research is the first to describe the heterologous expression of active peroxidases from the filamentous fungus Panerochaete chrysosporium. The cDNA clones λML-1, λML-6, λML-5 and λMP-1, which had been tentatively identified as fungal peroxidase clones, were subcloned into baculovirus transfer vectors and recombinant Autographa californica nuclear polyhedrosis viruses were constructed which contained copies of these potential peroxidase genes. The recombinant baculovirus clones were used to infect insect cells for the heterologous production of active recombinant enzymes. Three recombinant lignin peroxidases (H2, H6 and H8) …