Biochemistry, Biophysics, and Structural Biology Commons^™

Endosome To Golgi Retrieval Of The Vacuolar Protein Sorting Receptor, Vps10p, Requires The Function Of The Vps29, Vps30, And Vps35 Gene Products, Matthew N. J. Seaman, Eric G. Marcusson, Joan Lin-Cereghino, Scott D. Emr

College of the Pacific Faculty Articles

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The …

Both An N-Terminal 65-Kda Domain And A C-Terminal 30-Kda Domain Of Seca Cycle Into The Membrane At Secyeg During Translocation, Jerry Eichler, William Wickner

Dartmouth Scholarship

SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane. SecA function and topology were studied with metabolically labeled [35S]SecA and with inner membrane vesicles from cells that overex- pressed SecYEGDFyajC, the integral domain of preprotein translocase. During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the mem- brane, as previously reported for a 30-kDa C-terminal domain [Economou, A. & Wickner, W. (1994) Cell 78, 835–843]. Insertion of both domains occurs at saturable SecYEGDFyajC sites …

Cγ- And Cα-Subunits Provide The Specificity For Camp/Pka Function, Wei Qing Zhang

Theses and Dissertations in Biomedical Sciences

The Cα and Cγ of the cAMP-dependent protein kinase (PKA) are two highly homologous (83% amino acid identity), yet functionally distinct isozymes in vitro for substrate and pseudosubstrate specificity, and in intact cells for cell phenotypes (Beebe, 1992).

To determine the molecular mechanisms underlying the distinct functions in cell phenotypes, in vitro experiments were designed to make a detailed comparison of Cγ and Cα for substrate and pseudosubstrate specificity. To this end, Cγ and Cα were expressed m mammalian cells, bacteria and Sf9 insect cells using baculovirus. Abundant expression of active enzyme was cell system specific. Cγ and Cα expressed …

I2b Is A Small Cytosolic Protein That Participates In Vacuole Fusion, Paul Slusarewicz, Zuoyu Xu, Kimberly Seefeld, Albert Haas, William T. Wickner

Dartmouth Scholarship

Saccharomyces cerevisiae vacuole inheritance requires two low molecular weight activities, LMA1 and LMA2. LMA1 is a heterodimer of thioredoxin and protease B inhibitor 2 (I^B2). Here we show that the second low molecular weight activity (LMA2) is monomeric I^B2. Though LMA2 / I^B2 was initially identified as a protease B inhibitor, this protease inhibitor activity is not related to its ability to promote vacuole fusion: ( i ) Low M r protease B inhibitors cannot substitute for LMA1 or LMA2, ( ii ) LMA1 and LMA2 promote the fusionof vacuoles from a strain that …

Characterization Of Recombinant Soybean Leghemoglobin A And Apolar Distal Histidine Mutants, Mark S. Hargrove, Jennifer K. Barry, Eric Allen Brucker, Michael B. Berry, George N. Phillips, Jr., John S. Olson, Raúl Arredondo-Peter, Jeanenne M. Dean, Robert V. Klucas, Gautam Sarath

Gautam Sarath Publications

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 Å. Rate constants for O₂, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37°C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold …

A Search For The Cause Of Excess Thiol Sensitivity In A Mutant Of Escherichia Coli, Hongying Zeng

Loma Linda University Electronic Theses, Dissertations & Projects

The subject of this study was a search for the biochemical basis of hypersensitivity to thiols in a mutant of Escherichia coli (IS16). A 1.3 kb chromosomal fragment from E. coli (introduced into the mutant on a plasmid) successfully abolished the hypersensitivity. This fragment contained only one intact gene, ubiX, which codes for polyprenyl-4-hydroxybenzoatelyase. The decarboxylation of octaprenyl-4- hydroxybenzoate in E. coli is catalyzed not only by the ubiX protein but also by the product of ubiD. The ubiquinone content of IS16 was only 2% of that of its parent strain, while the complementing plasmid increased it …

Salsalate Inhibition Of Thyroid Hormone Transport : A Model Of The Euthyroid Sick Syndrome, Ron Wang

Loma Linda University Electronic Theses, Dissertations & Projects

Multiple alterations of serum thyroid hormone metabolism have been noted in essentially all severe systemic nonthyroidal illnesses (Nil). This spectrum of abnormalities is collectively termed the “euthyroid sick syndrome”. One putative mechanism is the presence of circulating thyroid hormone binding inhibitors. Salsalate and salicylate are known to be such inhibitors. The objectives of this study are: to investigate the thyroid hormone changes induced in healthy people by salsalate, and to investigate the mechanisms of salsalate action, as a model for the euthyroid sick syndrome. This dissertation includes: (1) Study of the effect of salsalate on serum thyroid hormone levels in …

Ifn-Gamma, Igif, And Iddm., Nora Sarvetnick

Journal Articles: Regenerative Medicine

No abstract provided.

Docking Of Yeast Vacuoles Is Catalyzed By The Ras-Like Gtpase Ypt7p After Symmetric Priming By Sec18p (Nsf), Andreas Mayer, William Wickner

Dartmouth Scholarship

Vacuole inheritance in yeast involves the for- mation of tubular and vesicular “segregation struc- tures” which migrate into the bud and fuse there to es- tablish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and micro- scopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacu- oles must be primed for docking by the action of Sec17p ( a -SNAP) and Sec18p (NSF). Priming is neces- sary for both fusion …

A Heterodimer Of Thioredoxin And Ib2 Cooperates With Sec18p (Nsf) To Promote Yeast Vacuole Inheritance, Zuoyu Xu, Andreas Mayer, Eric Muller, William Wickner

Dartmouth Scholarship

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as IB2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo …

Identification Of The Adp-L-Glycero-D-Manno-Heptose-6-Epimerase (Rfad) And Heptosyltransferase Ii (Rfaf) Biosynthesis Genes From Nontypeable Haemophilus Influenzae 2019, W A. Nichols, B W. Gibson, William Melaugh, N G. Lee, M Sunshine, M A. Apicella

Chemistry Faculty Publications

Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core …

Characterization Of A Transposon Tn916-Generated Mutant Of Haemophilus Ducreyi 35000 Defective In Lipooligosaccharide Biosynthesis, B W. Gibson, A A. Campagnari, William Melaugh, M A. Apicella, S Grass, Jing Wang, Katherine L. Palmer, R S. Munson

Chemistry Faculty Publications

To define the role of the surface lipooligosaccharide (LOS) of Haemophilus ducreyi in the pathogenesis of chancroid, Tn916 mutants of H. ducreyi 35000 defective in expression of the murine monoclonal antibody (MAb) 3F11 epitope on H. ducreyi LOS were identified by immunologic screening. One mutant, designated 1381, has an LOS which lacks the MAb 3F11 epitope and migrates with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene disrupted by the Tn916 element in strain 1381 was identified by cloning the sequences flanking the Tn916 element. The sequences were then used to probe a lambda DASHII genomic library. …

Diet And Growth Of Larval And Juvenile Grass Pickerel, Esox Americanus Vermiculatus, And Central Mudminnow, Umbra Limi, In The Green Bottom Wildlife Management Area, Cabell County, West Virginia, Erica Midkiff

Theses, Dissertations and Capstones

The grass pickerel, Esox americanus vermiculatus, is listed as Undetermined on the Vertebrate Species List of Concern in West Virginia. The central mudminnow, Umbra limi, is a disjunct population in Green Bottom Swamp. The lentic, vegetated areas that are required for spawning for the grass pickerel and the central mudminnow have been reduced by residential, agricultural, and industrial developments. Green Bottom Swamp, a naturally occurring wetland of 58 ha, and a nearby mitigated wetland of 29 ha, provides spawning habitat for the grass pickerel and the mudminnow. Fishes were collected in beds of Potamogetan crispus and Ceratophyllum demursum …

Effects Of Creatine Supplementation On Muscular Strength And Power Development In Well-Trained Football Players, Josef D. Mcquain

Theses, Dissertations and Capstones

The purpose of this study was to determine the effects of creatine supplementation on strength and power performance in well-trained football players. Twenty-one subjects were randomly assigned to three treatment groups: 1) creatine monohydrate, 2) creatine monohydrate+dextrose, or 3) placebo utilizing a double-blind design. Prior to assignment, subjects were matched according to lean body mass. After a five day loading dose, a maintenance dose was administered for 12 days. Test protocols included three anaerobic work bouts: l) two 30 s Wingate bike tests interspersed with five minutes recovery were used to ascertain relative and absolute power, 2) two 100 yard …

An Immunomodulating Mycotoxin Interferes With The Development Of Autoimmune Diabetes In Diabetes-Prone Bb/Wor Rats, Honggang Liu

Theses, Dissertations and Capstones

Various fungal products have immunomodulating activity and some have been studied regarding prevention of transplantation rejection. Prior to this investigation, the mycotoxin, gliotoxin (GT), has never been investigated as an immunotherapeutic drug for autoimmune disease. GT is a fungal secondary metabolite and a member of the epipolythiodioxopiperazine (ETP) family which has been shown to inhibit phagocytosis, induction of cytolytic T cells and the proliferation of T cells following mitogen stimulation. GT also induces in vitro apoptosis in certain immune cell types. More importantly, GT exhibits selective activity towards cells of hemopoietic origin.

Autoimmune diseases are disorders caused by immune responses …

The Regulation Of Glucose Transport In Cultured Vascular Smooth Muscle Cells By Angiotensin Ii And Glucose, Leslie Ann Quinn

Theses, Dissertations and Capstones

Glucose transport was assessed in vascular smooth muscle (VSM) cells by measuring the uptake of a radiolabeled non-metabolizable glucose analog, [3H]-2-deoxglucose. VSM cells, isolated from rat aortae by enzymatic digestion, were maintained in culture in Dulbecco's Modified Eagle Medium supplemented with 10% newborn calf serum at 37°C with 5% CO2 and air. Angiotensin II (AU) increased glucose transport by 84%. Significant stimulation occurred by two hours of exposure with the maximum effect being observed between six and eight hours. All effects were concentration dependent with a threshold response being detected at 0.1 nM. All-stimulated transport was blocked by an AU …

Relationship Of Resting And Submaximal Cardiovascular Measures To Vo[2]Max In Untrained Children, Thomas A. Cappaert

Theses, Dissertations and Capstones

Participation by children in organized sport continues to increase. A 1994 survey of 10,000 households by the National Sporting Goods Association (1994) found that approximately 40,000 children between the ages of 7-17 years participate in an organized sport or recreational activity annually. In the realm of organized sports, especially endurance activities, there continues to be interest in how cardiovascular fitness is attained and how it can be maximized. Measurement of cardiovascular fitness is an important piece to that puzzle.

The most common and most descriptive measure for children and adults, is the maximal oxygen uptake or max (Astrand & Rodahl, …

Nerve Tissue-Specific Human Glutamate Dehydrogenase That Is Thermolabile And Highly Regulated By Adp / P. Shashidharan, Donald D. Clarke, Naveed Ahmed, Nicholas Moschonas, And Andreas Plaitakis Department Of Neurology, Mount Sinai School Of Medicine, New York; Department Of Chemistry, Fordham University, Bronx New York, Usa; And Department Of Biology And School Of Health Sciences, University Of Crete, Crete, Greece, P. Shashidharan, Donald Dudley Clarke Phd, Naveed Ahmed, Nicholas Moschonas

Chemistry Faculty Publications

Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a …

Complexes Between Nascent Polypeptides And Their Molecular Chaperones In The Cytosol Of Mammalian Cells, Daryl K. Eggers, W. J. Welch, W. J. Hansen

Faculty Publications, Chemistry

Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones. We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp70, CCT (TRiC), hsp40, p48 (Hip), and hsp90 were compared on the basis of their …

Rna Base-Amino Acid Interaction Strengths Derived From Structures And Sequences, Brooke Lustig, S Arora, R L. Jernigan

Faculty Publications, Chemistry

We investigate RNA base-amino acid interactions by counting their contacts in structures and their implicit contacts in various functional sequences where the structures can be assumed to be preserved. These frequencies are cast into equations to extract relative interaction energetics. Previously we used this approach in considering the major groove interactions of DNA, and here we apply it to the more diverse interactions observed in RNA. Structures considered are the three different tRNA synthetase complexes, the U1A spliceosomal protein with an RNA hairpin and the BIV TARTat complex. We use binding data for the base frequencies for the seryl, aspartyl …

Regulation Of The Expression Of Hmn-Sod At The Level Of The Mitochondrial Processing Of Precursor Hmn-Sod, Gary Leslie Wright

Theses, Dissertations and Capstones

The mitochondrion imports and processes the vast majority of the proteins which comprise its structural elements and metabolic pathways. Studies in the last decade have been successful at defining the mechanism and protein machinery responsible for recognizing, importing, and processing cytosolic precursor proteins into the mature mitochondrial proteins. Little is known, however, about physiological factors which might influence or compromise mitochondrial protein processing. The objective of this work was to develop a novel approach to study mitochondrial import and assembly of precursor proteins as a process within the context of the cellular environment. To these ends we employed the baculovirus …

In Vivo Incorporation Of Tritium To Measure Lipogenesis In Red Skeletal Muscle: Significance Of A Nonlipogenic Diet In La/N-Fa(K)("Corpulent") And Zucker Fa Rat Strains, Karin Traci Mann

Theses, Dissertations and Capstones

Skeletal muscle lipogenesis in rodents may equal that of liver but its importance in the obese animals is not known. We evaluated the rate of fatty acid and triglyceride synthesis and storage content in red skeletal muscles, liver, inguinal fat, and retroperitoneal fat of the LA/N-fa^k (“corpulent”) and Zucker fa obese rat strains. Obese-lean pairs of both rat strains, weaned at 32 days, fed a nonlipogenic diet (54% starch for 21 days) were either dosed with 2 µCi 3H-H² O per gram of body weight and tested after 60 minutes for whole body 3H-incorporation into fatty acids and …

Sodium Nitrite Alone Protects The Brain O _I_ Microsomal Ca -Atpase Against Potassium Cyanide-Induced Neurotoxicity In Rats, Odutayo O. Odunuge, G.. A. Adenuga

Faculty Publications

The effect of a short-term oral administration of potassium cyanide (KCN) (200 ppm in diet) with or without sodium nitrite (NaNO2) pretreatment on rat brain microsomal Ca2* ATPase was investigated. The specific activity value of the enzyme significantly decreased (p<0.05) by 50% compared with control and by 63% for KCN-treated rats compared with KCN-treated rats pretreated with NaNO;,. There was no significant difference at the h = 0.05 level between the values obtained for the control and KCN-treated rats pretreated with NaNO,. These results show both that feeding lowers brain microsomal Ca2f-ATPase activity and that NaNO, has a protective role (antidote function) in that respect.

Molecular Engineering Of Side-Chain Liquid Crystalline Polymers By Living Polymerizations, Coleen Pugh, Alan L. Kiste

Chemistry and Biochemistry

“Living” anionic, cationic, metalloporphyrin and ring-opening metathesis polymerizations have been used to prepare well-defined side-chain liquid crystalline homopolymers, block and graft copolymers and statistical copolymers. This paper analyzes their successes and failures by reviewing the mechanistic aspects and experimental conditions of each type of polymerization, and identifies other classes of mesogenic monomers that could be polymerized in a controlled manner in the future. The emerging structure/property relationships are then identified using well-defined SCLCPs in which only one structural feature is varied while all others remain constant.

The thermal transitions of liquid crystalline polymethacrylates, polynorbomenes and poly(viny1 ether)s reach their limiting …

Genetic Analysis Of Chlorophyll Biosynthesis, David Bollivar

Scholarship

No abstract provided.

The Measurement Of Angiotensin-Converting Enzyme In Human Serum, Andrew P. Kenny

Theses

The measurement of angiotensin converting enzyme using the substrate FAPGG was investigated with a view to optimising its performance. It was found that a 100 mnol/l triethanolamine-HCl buffer gave a significantly higher absorbance change then the existing TRIS-HCl in the method. As this buffer has not been previously described in the measurement of ACE it was decided to introduce this buffer into a kinetic procedure and evaluate the modified method. The reference interval for the triethanolamine Method was determined by measuring the ACE activity on 200 serum from blood donors and a significant difference (P < 0.05) in the SACE activity of male and female donors was observed.

The modified method was found …

A Study Of Biochemical And Haematological Variables With Performance In Showjumping Horses, Darren B. Simmons

Theses : Honours

This honours project examined the biochemical and haematological changes induced by exercise in the showjumping horse to determine whether a relationship exists between these values and performance. To complete this study 7 geldings and 1 stallion who were considered competition fit by their trainers were subjected to 2 forms of exercise. The first phase of this study involved an incremental exercise test performed at the Byford City Council horse training facility. The horses were cantered/galloped around a 1300m all weather training track at velocities of 6, 8, 10, and 12 m/sec for a duration of 2 minutes for each workload. …

Robert J. Boxer Papers, Zach S. Henderson Library Special Collections

Finding Aids

This collection consists of various drafts and the finished manuscript of Robert J. Boxer's 1997 textbook, Essentials of Organic Chemistry. Materials span 1988- 1997 and also include correspondence with publishers, including contracts.

Find this collection in the University Libraries' catalog.

The Drosophila Melanogaster Rad54 Homolog, Dmrad54, Is Involved In The Repair Of Radiation Damage And Recombination, Rolf Kooistra, José B. M. Zonneveld, Anja De Jong, Jan C. J. Eeken, Chris J. Osgood, Jean-Marie Buerstedde, Paul H. M. Lohman, Albert Pastink

Biological Sciences Faculty Publications

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females …

Phosphorylation Within The Amino-Terminal Acidic Domain I Of The Phosphoprotein Of Vesicular Stomatitis Virus Is Required For Transcription But Not For Replication, Asit K. Pattnaik, Leroy Hwang, Tong Le, Nathan Englund, Manjula Mathur, Tapas Das, Amiya Banerjee

School of Veterinary and Biomedical Sciences: Faculty Publications

Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support …