R-3 Repository :: Browsing by Author "Rudolph, Frederick B."

Browsing by Author "Rudolph, Frederick B."

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Additional insights into the structure and function of adenosine deaminase
(1998) Martinez, Jeannette Huczko; Rudolph, Frederick B.
Adenosine deaminase is a key enzyme in purine catabolism. The elucidation of the crystal structure and recent analysis of site-directed mutants have provided details on the structure and function of the enzyme. The studies described in this thesis provide new insights into ADA. The ADA mechanism is well defined except for the identification of the proton acceptor. Aspartate 295 is important to metal coordination (Wilson, 1991). This residue may be the catalytic base because of its position in the original crystal structure. Mutation of this residue to asparagine resulted in a 100,000 fold reduction in activity but retention of binding to substrates and inhibitors. UV difference spectra confirm that the tetrahedral intermediate is not formed during catalysis. These results suggest that Asp295 functions as the proton acceptor and in metal coordination. The crystal structure of mouse ADA provides a static picture of the enzyme in a single conformation. The sequestered nature of the active site indicates that the motion of one or more side chains is necessary for the proper functioning of the enzyme. Several mutants were created involving a flexible loop at residues 109-124. Complete excision, mutation to a hinge, or the replacement of the loop with the smaller loop found in E. coli ADA resulted in decreases in activity. Binding was not affected by any of these mutations. The 109-124 loop most likely acts in sequestering the active site from solvent during catalysis. An alanine to valine point mutation has been located in codon 329 of patients with SCID. Site directed mutagenesis was used to generate this mutation. Protein containing this change was generated and was found to be misfolded and catalytically inactive. The loop mutations, A329V, and D295N affect the rate of catalysis of ADA for three different reasons. The loop serves to shield the active site from solvent. Asp295 is the catalytic base. Ala329 is important to protein folding. These studies show how slight alterations in primary structure can affect the overall function of an enzyme.
Characterization of metabolites and genes in the fermentation pathway of Clostridium acetobutylicum ATCC824
(1996) Boynton, Zhuang Luo; Rudolph, Frederick B.
In Clostridium acetobutylicum, central-fermentation-pathway enzymes involved in butyryl-CoA synthesis play key roles in acid and solvent production, whereas the acetate production is an important element during acidogenesis and generates ATP. Genes encoding enzymes involved in acetate- and butyryl-CoA-synthesis were cloned, sequenced and expressed. CoA and its derivatives, which are intermediate metabolites for these pathways, were isolated and analyzed to identify regulatory factors for the enzymes involved in such fermentation. Five genes: crt, bcd, etfB, etfA and hbd, which encode the central clostridial pathway enzymes crotonase, butyryl-CoA dehydrogenase (BCD), putative electron-transfer flavoprotein (ETF) $\beta$- and $\alpha$-subunits, and $\beta$-hydroxybutyryl-CoA dehydrogenase, respectively, were cloned and shown to be clustered in the chromosome. These genes are co-transcribed and form an operon, suggesting that BCD in clostridia might interact with ETFs in its redox function. Cloning and sequencing studies reveal that pta and ack, encoding acetate-production-pathway enzymes phosphotransacetylase (PTA) and acetate kinase (AK), respectively, are adjacent in the chromosome. Primer extension analysis suggests an operon arrangement for these tandem genes. Overexpression of ack and pta in C. acetobutylicum shows that the final ratios of acetate to other major products were higher and also results in a greater proportion of two- versus four-carbon-derived products. Formation of a mutant strain by inactivation of the chromosomal pta gene decreased acetate formation. The PTA and AK activities of such a mutant were correspondingly reduced. Intracellular levels of CoA and its derivatives involved in the metabolic pathways were analyzed using reverse-phase high performance liquid chromatography. During the acidogenic-to-solventogenic shift, butyryl-CoA concentration increased rapidly, whereas those for free CoA and acetyl-CoA decreased. These observations were accompanied by a rapid increase of solvent-pathway enzyme activity and a decrease of acid-pathway enzyme activity. Levels of acetoacetyl-CoA, $\beta$-hydroxy-butyryl-CoA and crotonyl-CoA in crude cell extracts were below detectability. The possible roles of CoA and its derivatives in regulating specific enzyme activity were evaluated.
Cloning and expressing of rubredoxin oxidoreductase from Clostridium acetobutylicum in Escherichia coli
(1995) Gui, Lei; Rudolph, Frederick B.
A 550 bp DNA fragment which apparently contains part of the rubredoxin oxidoreductase gene has been amplified by a polymerase chain reaction employing genonic DNA as template. Oligonucleotides used in this amplification were designed based on the multiple alignment of rubredoxin oxidoreductase from related species. An EMBL3 $\lambda$ phage library of C. acetobutylicum genomic DNA was screened by an oligonucleotide hybridization method. The phages which hybridized with the radiolabeled PCR product were subcloned into pUC19 and pACYC184 vectors, with the recombinant plasmids being selected on the basis of white/blue color screening method and the insertional inactivation method, respectively. The probable identity of the pUC19 clone, which was designated RuNC51 and carried a 3.0 kb BglII fragment, was confirmed by rubredoxin oxidoreductase enzyme activity assay. The sequence of RuNC51 showed the same level of similarity to related genes. It also surprisingly showed a 53% similarity to PriA (protein n$\sp\prime$) which is involved in DNA replication.
Dietary nucleotide restriction and supplementation in mice: Influence on lymphocyte function, maturation and nucleotide metabolism
(1988) Fanslow, William Christian, III; Rudolph, Frederick B.
An observed phenomenon of cell mediated immunosuppression in mice caused by the lack of preformed purine and pyrimidines in the diet was characterized and the mechanism of action explored utilizing three different approaches. The three approaches employed were as follows: (1) measurement of T cell mediated immune response in vitro and in vivo; (2) analysis of lymphocyte phenotype involving surface marker immunofluorescence, measurement of the levels of purine and pyrimidine enzymes as predictors of lymphocyte maturation and evaluation of putative G$\sb1$ phase characteristics; and (3) examination of diet fed host effects on the in vivo out growth of syngeneic lymphoid tumors. Balb/c mice fed a nucleotide free (NF) diet exhibited significantly decreased T helper/inducer cell function and number relative to mice fed normal rodent chow (F) or NF plus RNA (NFR). Lymphoproliferative response in vitro and in vivo was significantly lower in mice fed the NF diet than the response exhibited by mice fed the control diets or NF plus uracil (NFU). This lack of proliferative response was accompanied by decreased induction of two purine enzymes important to the immune response: adenosine deaminase and purine nucleoside phosphorylase. Bone marrow, thymus and spleen obtained from NF diet fed mice contained significantly more T cells of an immature phenotype with high terminal deoxynucleotidyl transferase and adenosine deaminase levels and low purine nucleoside phosphorylase level enzyme profiles relative to that of similar lymphoid tissues obtained from mice fed the control diets. Levels of ecto 5$\sp\prime$ nucleotidase, another enzyme linked to optimal B cell function were not affected by the NF diet. Lymphocyte nucleotide pools were altered and the number of putative G$\sb1$ phase T lymphocytes were increased in mice fed the NF diet relative to mice fed the purine and pyrimidine supplemented diets. In vivo outgrowth of the syngeneic T cell lymphoma, 5F4, in Balb/c hosts fed the NF diet was significantly decreased compared to the 5F4 tumor growth observed in mice fed the NF diet supplemented with RNA, adenine or uracil. (Abstract shortened with permission of author.)
Effects of mechanical loading on osteoblast function using a three dimensional celijpolymer model
(1998) Jen, Anna Hsiao-Chieh; McIntire, Larry V.; Mikos, Antonios G.; Rudolph, Frederick B.; Gustin, Michael C.; Farach-Carson, Cindy
Mechanisms which trigger bone modeling/remodeling in response to changes in the mechanical environment are still unclear. In a three part study, effects of loading on osteoblast function were investigated using a three dimensional (3-D) ceWpolymer model. The 3-D model has advantages of cell culture while maintaining the natural matrix architecture of bone. Such celVpolymer constructs have been shown to form bone in vitro. Osteoblasts in 3-D ceWpolymer constructs were cyclically loaded (5% ). After five days, compressed constructs decreased in alkaline phosphatase activity, a marker of osteoblast maturation. After three weeks, loaded constructs showed lower alkaline phosphatase activity but higher RNA level of L-type calcium channels, involved in calcium signaling cascades. No difference was detected after twelve weeks. Results suggest osteoblasts sensed loading and altered functional activities in response. Use of the 3-D model to study other osteoblast functions under mechanical loading may increase understanding of regulated functional adaptation by bone.
Gas chromatography and gateway sensors for on-line state estimation of complex fermentations (butanol/acetone fermentations)
(1984) McLaughlin, Joseph K.; Papoutsakis, E. Terry; Rudolph, Frederick B.; McIntire, Larry V.
A bench-scale system for monitoring butanol/acetone fermentations was designed to demonstrate the use of on-line gas chromatography (GC) and the applicability of an "advanced" fermentation equation. Ultrafiltration was used to obtain sterilely a cell-free sample from a controlled fermentor for on-line GC analysis of ethanol, acetone, acetate, butanol, acetoin, and butyrate. The fermentor effluent gas was also analyzed by on-line GC for nitrogen, carbon monoxide, and carbon dioxide. Analytical data were automatically transmitted to a VAX 11/75 computer, for data archiving and fermentation equation calculations. The system was demonstrated in studies of pH and CO effects on the metabolism of Clostridium acetobutylicum. The on-line analytical systems provided excellent real time data. Also, three potentially useful gateway sensors were developed from the fermentation equation: glucose concentration, NADH2 from FdH2 and excess ATP. These demonstrated the usefulness of the fermentation equation for determination of intracellular and extracellular parameters in transient operation.
Heterologous expression of alcohol acetyltransferase genes in Escherichia coli and Clostridium acetobutylicum for the production of esters
(2004) Horton, Catherine Emily; Rudolph, Frederick B.; Bennett, George N.
This thesis focuses on the heterologous expression of alcohol acetyltransferase (AATase) genes in Escherichia coli and Clostridium acetobutylicum for the production of the esters isoamyl acetate, butyl acetate and butyl butyrate. Isoamyl acetate, butyl acetate and butyl butyrate are esters that confer a fruity aroma and taste to the materials in which they are found. AATases are a class of enzymes that have been found to enzymatically catalyze the reaction between alcohols and acyl-CoAs to produce the corresponding ester. Butanol, acetyl-CoA, and butyryl-CoA, the substrates necessary for butyl acetate and butyl butyrate production, are produced in high concentrations by C. acetobutylicum, making it an ideal host for the expression of genes for AATases. Previous studies have characterized AATases in yeast, and expressed them in E. coli, but this is the first report of ester production by AATase activity in C. acetobutylicum . The genes ATF1 and ATF2, encoding AATase I and AATase II from the yeast Saccharomyces cerevisiae, and SAAT encoding a strawberry alcohol acetyltransferase, Saatp, have previously been sequenced. Biochemical studies have shown them to possess AATase activity. For this thesis, ATF1, ATF2, and SAAT were subcloned and expressed in E. coli. Ester production was determined by head-space gas analysis on a gas chromatagraph. AATase I in E. coli cultures and cell-free extracts produced more ester than AATase II did with each alcohol substrate that was investigated. Saatp produced less ester than AATase I, but more than AATase II. The ester butyl butyrate was detected in cell-free extracts of E. coli expressing SAAT, but not in cell-free extracts expressing ATF1 or ATF2 . ATF2 was also expressed in C. acetobutylicum for the production of isoamyl acetate and butyl acetate. Ester concentrations in C. acetobutylicum strains expressing ATF2 were lower than ester concentrations in E. coli strains expressing ATF2. The expression of ATF2 in C. acetobutylicum demonstrates the viability of using this organism for natural ester production via microbial fermentation.
Immunosupportive drug sparing diet
(2004-03-16) Van Buren, Charles T.; Rudolph, Frederick B.; Board of Regents of the University of Texas System; Rice University; United States Patent and Trademark Office
The present invention is directed to immunosupportive, drug sparing diets and methods of using these diets. The compositions and methods disclosed herein increase the bioavailability of pharmaceutical compositions metabolized by the gut P450 isozymes.
Inhibition of rat liver pyruvate carboxylase by inert chromium-nucleotide coordination complexes
(1975) Armbruster, David Allyn; Rudolph, Frederick B.
Pyruvate carboxylase, Pyruvate:CO2 Ligase (ADP); EC 6.4.1.1, free of contaminating enzymatic activity and suitable for kinetic studies, was purified from rat liver. Protomers of the enzyme, rather than tetramers, were purified using agarose bead columns and the protomer molecular weight was found to be about 13,. CrATP and bidentate and monodentate CrADP were prepared and used along with other Cr-nucleotide complexes, which are substrate analogs of MgATP, as dead end inhibitors of pyruvate carboxylase. The Cr-nucleotides are effective initial inhibitors of pyruvate carboxylase but the inhibition becomes more severe with time. Finally, this tightening of the inhibition ceases and a linear time course is observed. Maximum inhibition of the enzyme occurs when it is incubated with the Cr-nucleotide in the presence of Mg and HCO^ prior to the initiation of the reaction. CrATP is a competitive inhibitor with respect to MgATP and a non-competitive inhibitor with respect to HCO-Bidentate and monodentate CrADP are both better inhibitors than CrATP and bidentate CrADP is a stronger inhibitor than monodentate CrADP. The amount of inhibition caused by various Cr-nucleotides may indicate the relative importance of the and "if* phosphate groups of MgATP in binding to pyruvate carboxylase. The results of the inhibition experiments are in agreement with the proposed mechanism of action of the enzyme which involves two partial reactions at separate catalytic sites, Possible explanations of the tightening of the inhibition phenomenon are discussed.
Investigation of the control of metabolic pathways in Clostridium acetobutylicum by the studies of glucose and non-glucose limitation, in vitro enzyme inhibition, and intermediary compound challenges in batch and continuous cultures
(1984) Roos, Joseph William; Papoutsakis, E. Terry; Rudolph, Frederick B.; McIntire, Larry V.
The fermentation of carbohydrates by Clostridium aoetobutylicum leads to the formation of acetate, acetoin, acetone, butanol, butyrate, ethanol, hydrogen, and carbon dioxide. The availability of ATP and NADH£ appears to be significant in determining the amount of products formed. Non-glucose limited, uncontrolled pH, batch cultures are capable of producing solvents. Increasing the ammonium to glucose ratio of these cultures resulted in an increase in acid formation and growth and a decrease in solvent yields. Maintaining a constant pH at 4.5 in batch cultures encouraged growth and glucose utilization resulting in glucose limited cultures. These ATP limited cultures gave high acid yields and small solvent yields. Butyraldehyde and acetoacetate influenced the metabolism of uncontrolled pH batch cultures. Their presence resulted in increased biomass formation but had mixed effects on acid and solvent production. A non-glucose limited continuous culture has been observed to produce butanol, acetone, butyrate and acetate, while under glucose limited conditions, continuous cultures produced primarily butyrate and acetate. In a glucose limited culture carbon monoxide was used to inhibit the hydrogenase activity. This inhibition resulted in an uptake of butyrate, a decrease in the rate of acetate production and a large increase in the specific rates of production of butanol, ethanol, and acetoin. Acetone was not affected.
Key enzymes in parasite sterol metabolism
(2002) Joubert, Bridget Mae; Rudolph, Frederick B.
The lanosterol synthases from Trypanosoma cruzi, Trypanosoma brucei and Pneumocystis carinii, the lanosterol 14-demethylases from Trypanosoma brucei and Trypanosoma cruzi, and the cytochrome P450 reductase from Trypanosoma brucei were cloned and characterized. The two trypanosome lanosterol synthases showed a novel difference in protein sequence identity from that of other lanosterol synthases, which could be exploited for the development of specific antitrypanosome inhibitors. Yeast strains for expressing lanosterol 14alpha-demethylases were also developed. The first strains developed by tetrad dissection were time-consuming to produce, therefore another expression system was developed. The new system involved transforming the existing yeast strains, BJY1[pTb14DM] or BJY5[pTb14DM], and selection on FOA medium. With the addition of the T. brucei P450 reductase characterized in this study, the trypanosome lanosterol 14alpha-demethylases were able to regenerate their catalytic activity more efficiently than the strains containing only the native yeast P450 reductase. The various yeast strains developed in this study should be useful for screening antiparasite drugs. The BJY1[pTb14DM] (without reductase) and BJY5[pTb14DM] (with reductase) strains would also be useful in creating expression strains for other 14DM genes. Hopefully, these will be cloned in the near future.
Mechanistic and structural studies of mouse adenosine deaminase
(1997) Sideraki, Vera; Rudolph, Frederick B.
Adenosine deaminase (ADA) catalyzes the irreversible deamination of (2$\sp\prime$-deoxy)adenosine to (2$\sp\prime$-deoxy)inosine. It is an indispensable enzyme, with a role in purine catabolism and in the development of a competent immune system. This work focuses on the study of the catalytic mechanism employed by the murine enzyme through the use of site-directed mutagenesis. A glutamate mutation at the conserved active site Asp 295 shows that this residue is necessary for the proper orientation and placement of the catalytic hydroxylate. An alanine and an asparagine mutant of Asp 296 show that this residue functions mainly by anchoring the substrate in the active site via hydrogen bonding and thus reducing the aromaticity of the purine ring. Alanine, glutamate and arginine mutations at the proposed base for the reaction, His 238, clearly show that it does not abstract the proton from the zinc-bound water, but rather promotes the formation of the hydroxylate through charge stabilization. Replacements of the conserved Cys 262 by alanine and serine clearly demonstrate that it is not directly involved in the reaction mechanism. Structural studies with the ADA apoenzyme reveal that chelation of zinc does not result in structural rearrangements of either the active site or the secondary and tertiary structures of the enzyme. Loss of zinc is accompanied by loss of activity, which can be restored upon stoichiometric re-addition of zinc or cobalt. A transition-state analog such as deoxycoformycin can bind the apoenzyme by inducing the same type of conformational change as it does when it binds the holoenzyme. Mutants such as D296A and D296N denature more slowly compared to the wild-type, probably due to the better packing of an Ala or Asn side chain compared to the native Asp in the part of the enzyme surrounding residue 296. By contrast, mutants such as D295E, H238A, and H238E destabilize the holoenzyme, and mutants H238R, C262A, and C262S destabilize the holoenzyme and may also impede the in vivo folding pathway. Removal of the metal cofactor from wild-type or mutant ADA generally increases the enzyme's rate of denaturation.
Method and compositions for promotion of wound treatment
(2002-01-29) Kulkarni, Anil D.; Van Buren, Charles T.; Rudolph, Frederick B.; Board of Regents of the University of Texas System; Rice University; United States Patent and Trademark Office
The present invention comprises compositions and preparations for the promotion of wound healing in an animal. Methods for preparing the compositions as well as methods for using the compositions to achieve the promotion of wound healing, are also provided. The composition may comprise a dietary regimen or a therapeutic agent. These compositions include a wound healing promoting concentration of nucleotides. By way of example, such nucleotides may comprise RNA, adenine, uracil or a mixture thereof. The compositions can be prepared as suitable for oral, parenteral, intravenous or topical administration. Methods for using the preparation as a treatment to enhance the healing of an already existing wound or for use as a pretreatment regimen for animals in anticipation of surgery, are also disclosed.
Ribonucleotide preparations and uses thereof
(1998-01-27) Kulkarni, Anil D.; Van Buren, Charles T.; Rudolph, Frederick B.; Board of Regents of the University of Texas System; Rice University; United States Patent and Trademark Office
The present invention comprises compositions and preparations for the promotion of wound healing in an animal. Methods for preparing the compositions as well as methods for using the compositions to achieve the promotion of wound healing are also provided. Methods for enhancing collagen production at a wound site are also disclosed. The composition may comprise a dietary regimen or a therapeutic agent. These compositions include a wound healing promoting concentration of ribonucleotides in a pharmaceutically acceptable carrier solution. By way of example, such ribonucleotides may comprise RNA, adenine, uracil or a mixture thereof. The compositions can be prepared as suitable for oral, parenteral, intravenous or topical administration. Methods for using the preparation as a treatment to enhance the healing of an already existing wound or for use as a pretreatment regimen for animals in anticipation of surgery, are also disclosed.
Synthesis of Phenanthroizidine Alkaloids and a Practical Total Synthesis of (208)-(+)-Camptothecin
(1997) Roschangar, Frank; Ciufolini, Marco A.; Engel, Paul S.; Rudolph, Frederick B.
Chapter I describes the total syntheses of the representative phenanthroizidine alkaloids tylophorine, antofine and of their seco congeners, septicine and julandine, which have been accomplished through the CiufoliniByrne pyridine synthesis. Chapter II discusses the evolution of our strategy for the synthesis of (208)-( + )-Camptothecin.
The effect of an oral or intravenous nucleotide-free diet on selected enzyme activities in purine metabolism in rats
(1982) Snyder, Sandra Lynn; Rudolph, Frederick B.; Berget, Susan M.; Schroepfer, George J.; Bennett, George; Storck, Roger L.
Interrelationships between purine metabolism and immunity, cancer, and the diet have been considered. In studying trends of metabolic changes which occur in response to changes in the purine content of the diet, it has been hypothesized that when purines are lacking from the diet, there is a general shift from a catabolic to an anabolic state. In the present investigation, the activities of selected enzymes on purine metabolism in rats were studied with respect to an oral or intravenous nucleotide-free diet compared to the activities in rats fed normal chow. The intravenous nucleotide-free diet caused a decrease in purine nucleoside phosphorylase activity, an increase in adenine phosphoribosyl transferase, and no change in the activity of hypoxanthine-guanine phosphoribosyl transferase, with respect to a normal control diet. The orally fed nucleotide-free diet caused a decrease in the activity of purine nucleoside phosphorylase and xanthine oxidase and increase in pancreatic ribonucléase and no change in adenine phosphoribosyl transferase or hyphoxanthine-guanine phosphoribosyl transferase, with respect to a normal control diet. These observations support the predicted shift from catabolism to anabolism. They also augment the growing awareness of the interrelationships between diet and cancer as well as diet and the immune response.

Browsing by Author "Rudolph, Frederick B."

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