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Biomedical subjects

F B Rudolph

Publications and source records attributed to F B Rudolph.

At least 37 records · Page 2Linked to original sources
PubMed1994

Amplification of an adenylosuccinate synthetase gene in alanosine-resistant murine T-lymphoma cells. Molecular cloning of a cDNA encoding the "non-muscle" isozyme.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of IMP to AMP. Two isoforms of this enzyme have been observed in vertebrates. A muscle isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle energy metabolism. The non-muscle isozyme, which is present at low levels in most tissues, likely functions in de novo AMP biosynthesis. The analysis of the non-muscle isozyme has been hampered by its low abundance and instability during purification. In this study a genetic selection scheme was used to generate a murine T-lymphoma cell line which was at least 100-fold enriched for the non-muscle isozyme, as a result of amplification of the non-muscle synthetase gene. This cell line made possible the purification of the non-muscle isozyme, and the subsequent isolation of isozyme-specific peptides. Based on peptide sequence information a degenerate oligonucleotide probe was designed and used to screen a mouse kidney cDNA library. A 1.5-kilobase cDNA encoding the non-muscle isozyme was cloned and found to contain an open reading frame of 1368 base pairs encoding 456 amino acids. Gene transfer experiments showed that the cDNA encoded a 50-kDa protein, the size expected for mammalian synthetases, that correlated with the presence of high levels of synthetase activity. The deduced amino acid sequence of the mouse non-muscle synthetase is approximately 75% identical to the previously reported mouse muscle synthetase. Southern blot analysis of mouse genomic DNA with the isozyme-specific cDNA probes revealed that the synthetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated muscle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases) are detected for the non-muscle isozyme which show a similar tissue distribution. The availability of a cDNA for the non-muscle isozyme of adenylosuccinate synthetase will facilitate further comparative analyses with the previously cloned muscle isozyme.

Adenylosuccinate Synthase↗
PubMed1994

The biochemistry and physiology of nucleotides.

Nucleotides are phosphate esters of nucleosides that contain a sugar linked through a glycosidic linkage with purine and pyrimidine bases. Purine and pyrimidine nucleotides are major components of the cells that make up the monomeric units of DNA and RNA, and they function in all cellular processes. Biosynthesis, interconversion, catabolism and other aspects of nucleotide metabolism, along with various cellular roles of nucleotides, will be discussed, and the possible use of dietary sources of preformed purines and pyrimidines will be considered.

Animals↗
PubMed1994

The role of nucleotides in adult nutrition.

Dietary nucleotides, found in normal diets, have been recently determined to be required for normal immune defenses. Rejection of cardiac transplants, graft-vs.-host disease, and delayed cutaneous hypersensitivity in animal models are all suppressed by a diet deficient in nucleotides. T lymphocytes seem to require dietary nucleotides for normal maturation and function. Host resistance to bacterial and fungal infections is decreased in mice on nucleotide free diets; addition of RNA or uracil prevents this vulnerability to infection. Dietary RNA is required to restore lost immune function after protein deprivation. Adequate calories and protein alone do not return immune function to normal. Dietary nucleotides can restore lost immune function even during protein starvation and weight loss. Because all parenteral and most enteral nutrient solutions are nucleotide free, clinical studies were undertaken comparing a new nucleotide containing diet (Impact) to a standard high protein enteral feeding. In two separate double blind clinical studies the patients fed the enteral diet containing nucleotides had improved immune function compared with patients receiving a nucleotide free diet. In addition, infectious complications and length of hospital stay were reduced in postoperative cancer patients fed Impact compared with a control group.

Adult↗
PubMed1994

The role of dietary sources of nucleotides in immune function: a review.

Dietary sources of preformed purines and pyrimidines seem to be important for optimal function of the cellular immune response. It was previously assumed that nucleotides were not needed for normal growth and development, but the results described in this review demonstrate a need for nucleotides in the response to immunological challenges. This effect is likely due to a requirement for preformed pyrimidines for proper development and activation of T cells. The need for sources of preformed nucleotides in defined formulas such as parenteral and enteral formulas and infant formulas is suggested by the studies reviewed below.

Animals↗
PubMed1994

Intracellular Concentrations of Coenzyme A and Its Derivatives from Clostridium acetobutylicum ATCC 824 and Their Roles in Enzyme Regulation.

Intracellular levels of coenzyme A (CoA) and its derivatives involved in the metabolic pathways for Clostridium acetobutylicum ATCC 824 were analyzed by using reverse-phase high-performance liquid chromatography (HPLC). During the shift from the acidogenic to the solventogenic or stationary growth phase, the concentration of butyryl-CoA increased rapidly and the concentrations of free CoA and acetyl-CoA decreased. These changes were accompanied by a rapid increase of the solvent pathway enzyme activity and a decrease of the acid pathway enzyme activity. Assays with several non-solvent-producing mutant strains were also carried out. Upon entry of the mutant strains to the stationary phase, the butyryl-CoA concentrations for these mutant strains were comparable to those for the wild type even though the mutants were deficient in solvent-producing enzymes. Levels of acetoacetyl-CoA, beta-hydroxy-butyryl-CoA, and crotonyl-CoA compounds in both wild-type and mutant extracts were below HPLC detection thresholds (<21 muM).

Journal Article↗
PubMed1993

The highest levels of purine catabolic enzymes in mice are present in the proximal small intestine.

Recent studies on the tissue distribution and developmental regulation of adenosine deaminase (ADA) activity in mice show that very high ADA levels exist in the murine alimentary tract (tongue, esophagus, forestomach, proximal small intestine) and at the fetal-maternal interface. To understand the role of ADA in these tissues, we measured the levels of three other enzymes involved in purine catabolism, purine nucleoside phosphorylase (PNP), guanine deaminase (GDA), and xanthine dehydrogenase (XDH), to see how their levels correlated with ADA activity. Our results show that the highest level of PNP, GDA, and XDH is present in the proximal small intestine. Levels of these purine catabolic enzymes are much lower in the tongue, esophagus, forestomach, and fetal-maternal interface in marked contrast to ADA distribution. We also determined mRNA levels encoding PNP, XDH, and ADA in a variety of tissues. Tissue-specific differences in PNP, XDH, and ADA activity correlated with RNA abundance, indicating that the regulation of gene expression is at the level of mRNA production. Thus, ADA is part of a purine catabolic pathway leading to the production of uric acid that is present at the highest known level in the proximal small intestine. ADA may have additional roles in other tissues.

Adenosine Deaminase↗
PubMed1992

Immunohemopoietic effects of dietary nucleotide restriction in mice.

The influence of dietary sources of nucleotides on host in vivo and in vitro immuno-hematologic responses in BALB/c (NCI) mice was studied. Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in popliteal lymph nodes undergoing proliferative response to syngeneic and allogeneic in vivo stimulation. Supplementation of a nucleotide-free (NF) diet with yeast RNA (NFR) or uracil (NFU) significantly enhanced the host PLN immune response as compared with NF and NF supplemented with adenine (NFA) diets. Levels of ADA and PNP enzymes in the PLNs increased with the alloimmune PLN response of host, and immunosuppression was associated with decreased ADA and PNP activities in lymphocytes following antigenic stimulation. The induction of these enzymes during immune response appears to require dietary sources of certain nucleotides. When bone marrow cells from control chow fed animals were cultured with supernatants (sups) from mitogen activated splenocytes of animals on each dietary group, NF sups significantly decreased (P less than 0.05) the BM proliferative response compared with the response observed with NFR sups, and similar to NFA or NFU sups. When stimulated with purified IL-3, NFR BM cells had higher levels of Thy1.2 or Lyt 1 surface markers as compared with other test groups. In the in vivo splenic colony formation-CFUs assay, spleens from NFR- and NFU-fed animals had a significantly higher number of colonies than spleens from NF- or NFA-fed mice. Thus, NF diet decreases both in vivo lymphoproliferation response to alloantigen and hemopoietic growth factor production, rendering the host splenic environment deficient for stem cell growth. These adverse effects are reversed by RNA supplementation of NF diet. These nutritional studies demonstrate a critical and regulatory role for dietary nucleotides in immunohemopoiesis.

Adenosine Deaminase↗
PubMed1991

Cloning of an NADH-dependent butanol dehydrogenase gene from Clostridium acetobutylicum.

The acetone-butanol fermentation of C. acetobutylicum is characterized by the unique shift from acid to solvent production. The mechanism of the solventogenic switch involves the induction of several enzymes, including NADH-dependent butanol dehydrogenase (BDH) at the onset of solventogenesis. This enzyme is responsible for the final conversion of butyraldehyde to butanol, and is distinct from the NADPH-dependent alcohol dehydrogenase (ADH) also present in the organism. To characterize the genetic control of this gene, we have cloned and expressed it in E. coli. A lambda EMBL3 phage library of C. acetobutylicum DNA was screened via plaque hybridization using a [32P]-radiolabeled, 32-fold degenerate, 62-mer oligonucleotide probe. The probe was designed by reverse translation of the NH2-terminal amino acid sequence of purified BDH II. Southern blot experiments indicate that the phage insert was of clostridial origin and had no homology with the previously cloned NADPH-dependent ADH. Subcloning of DNA from purified positive plaques has localized the gene to a 3.5-kb EcoRI fragment from which the enzyme is well expressed. The sequence of the 25 NH2-terminal amino acids for the cloned enzyme purified from E. coli was determined and found to be identical to that for the clostridial NADH-dependent BDH II. Maxicell analysis of [35S]-radiolabeled plasmid-encoded proteins identified a species encoded by the clostridial insert with the expected Mr of 42 kD.

Alcohol Oxidoreductases↗
PubMed1991

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.

Adenylosuccinate Synthase↗
PubMed1991

Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations.

The crystal structure of a murine adenosine deaminase complexed with 6-hydroxyl-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, has been determined and refined at 2.4 angstrom resolution. The structure is folded as an eight-stranded parallel alpha/beta barrel with a deep pocket at the beta-barrel COOH-terminal end wherein the inhibitor and a zinc are bound and completely sequestered. The presence of the zinc cofactor and the precise structure of the bound analog were not previously known. The 6R isomer of the analog is very tightly held in place by the coordination of the 6-hydroxyl to the zinc and the formation of nine hydrogen bonds. On the basis of the structure of the complex a stereoselective addition-elimination or SN2 mechanism of the enzyme is proposed with the zinc atom and the Glu and Asp residues playing key roles. A molecular explanation of a hereditary disease caused by several point mutations of an enzyme is also presented.

Adenosine Deaminase↗
PubMed1991

Conformation of NADP+ bound to a type II dihydrofolate reductase.

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for confering trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and its physical properties have been characterized. This enzyme has been shown to transfer the pro-R hydrogen of NADPH to its substrate, dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases [Brito, R. M. M., Reddick, R., Bennett, G. N., Rudolph, F. B., & Rosevear, P. R. (1990) Biochemistry 29,9825]. Two distinct binary RBG200.NADP+ complexes were detected. Addition of NADP+ to RBG200 DHFR results in formation of an initial binary complex, conformation I, which slowly interconverts to a second more stable binary complex, conformation II. The binding of NADP+ to RBG200 DHFR in the second binary complex was found to be weak, KD = 1.9 +/- 0.4 mM. Transferred NOEs were used to determine the conformation of NADP+ bound to RBG200 DHFR. The initial slope of the NOE buildup curves, measured from the intensity of the cross-peaks as a function of the mixing time in NOESY spectra, allowed interproton distances on enzyme-bound NADP+ to be estimated. The experimentally measured distances were used to define upper and lower bound distance constraints between proton pairs in distance geometry calculations. All NADP+ structures consistent with the experimental distance bounds were found to have a syn conformation about the nicotinamide-ribose (X = 94 +/- 26 degrees) and an anti conformation about the adenine-ribose (X = -92 +/- 32 degrees) glycosidic bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1991

Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824.

In Clostridium acetobutylicum, conversion of butyraldehyde to butanol is enzymatically achieved by butanol dehydrogenase (BDH). A C. acetobutylicum gene that encodes this protein was identified by using an oligonucleotide designed on the basis of the N-terminal amino acid sequence of purified C. acetobutylicum NADH-dependent BDH. Enzyme assays of cell extracts of Escherichia coli harboring the clostridial gene demonstrated 15-fold-higher NADH-dependent BDH activity than untransformed E. coli, as well as an additional NADPH-dependent BDH activity. Kinetic, sequence, and isoelectric focusing analyses suggest that the cloned clostridial DNA contains two or more distinct C. acetobutylicum enzymes with BDH activity.

Alcohol Dehydrogenase↗
PubMed1990

Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase.

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are different both in sequence and in structure from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for conferring trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and overproduced [Vermersch, P. S., Klass, M. R., & Bennett, G. N. (1986) Gene 41, 289]. With this cloned and overproduced protein, a rapid purification procedure has been developed that yields milligram quantities of apparently homogeneous RBG200 DHFR with a specific activity 1.5-fold greater than that previously reported for the purified R388 protein [Amyes, S. G. B., & Smith, J. T. (1976) Eur. J. Biochem. 61, 597]. The pH versus activity profile and the native molecular weight of RBG200 DHFR were found to be similar to those previously reported for other type II DHFRs but different from those of the known chromosomal DHFRs. Stereospecifically labeled [4(S)-2H,4(R)-1H]NADPH was synthesized and used to determine the stereospecificity of NADPH oxidation by RBG200 DHFR. RBG200 DHFR was found to specifically transfer the pro-R hydrogen of NADPH to dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases. Thus, although RBG200 DHFR is different both in sequence and in structure from known chromosomal enzymes, both enzymes catalyze identical hydrogen-transfer reactions. Two distinct binary RBG200 DHFR-NADP+ complexes were detected by monitoring the 1H NMR chemical shifts and line widths of the coenzyme in the presence of RBG200 DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Dietary nucleotides reverse malnutrition and starvation-induced immunosuppression.

The requirement of dietary nucleotide sources for maximal helper T-cell function has been demonstrated. The effect of dietary nucleotide restriction was tested during two forms of nutritional stress: starvation and protein malnutrition. In the starvation model, mice were fed chow diet, nucleotide free or nucleotide free supplemented with 0.25% yeast RNA, for at least 4 weeks. The animals were then starved for 5 days, at which time they were killed and mitogen assays were performed using spleen cells. Animals previously maintained on the nucleotide-free diet supplemented with RNA showed a significant increase in spontaneous concanavalin A and phytohemagglutinin-stimulated blastogenesis. Protein malnutrition was induced by feeding Balb/c mice a protein-free diet for 7 to 10 days. These mice then received either the protein-free diet, the nucleotide-free diet, or the nucleotide-free diet supplemented with 0.25% yeast RNA. Popliteal lymph node assays were then performed. The chow diet, nucleotide-free diet, and nucleotide-free diet supplemented with 0.25% yeast RNA led to a restoration of body weight, but only the chow and supplemented diets restored significant popliteal lymph node immune reactivity. These studies using starvation and protein-malnutrition models clearly indicate the nutritional role of nucleotides in the maintenance and restoration of the immune response.

Animals↗
PubMed1990

Developmental expression of adenosine deaminase in the upper alimentary tract of mice.

The distribution and localization of adenosine deaminase (ADA) was studied during postnatal development of the alimentary tract in mice. There was detectable enzyme activity in all organs examined, but a range of more than 10,000 fold in the relative levels of specific activity was observed among adult tissues. A comprehensive survey of multiple adult tissues revealed that the highest levels of ADA occur in the upper alimentary tract (tongue, esophagus, forestomach, proximal small intestine). Immunohistochemical analysis revealed that ADA was predominantly localized to the epithelial lining of the alimentary mucosa: the keratinized squamous epithelium that lines the forestomach, esophagus, and surface of the tongue; and the simple columnar epithelium of the proximal small intestine (duodenum, proximal jejunum). Biochemical analysis revealed that ADA was one of the most abundant proteins of these mucosal tissue layers, accounting for 5%-20% of the total soluble protein. Tissue-specific differences in ADA activity correlated both with levels of immunoreactive protein and RNA abundance. The level of ADA activity in the upper alimentary tissues was subject to pronounced developmental control, being low at birth and achieving very high levels within the first few weeks of postnatal life. The appearance in development of ADA-immunoreactivity coincided with maturation of the mucosal epithelium. These results suggest that ADA is subject to strong cell-specific developmental regulation during functional differentiation of certain foregut derivatives in mice.

Adenosine Deaminase↗