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Biomedical subjects

R S Brody

Publications and source records attributed to R S Brody.

At least 19 recordsLinked to original sources
PubMed1991

Nucleotide positions responsible for the processivity of the reaction of exonuclease I with oligodeoxyribonucleotides.

The processive hydrolysis of single-stranded oligodeoxyribonucleotides by exonuclease I from Escherichia coli has been investigated. Oligodeoxyribonucleotides and their analogues, which contain either an abasic site or a methylphosphonate internucleotide linkage, were partially hydrolyzed by exonuclease I. The relative dissociation constant for the enzyme and each oligomeric product was calculated from the concentration of that oligomer found in solution and hence released by the enzyme before complete hydrolysis. The results have led to a characterization of the two oligodeoxyribonucleotide domains that bind to exonuclease I. The first domain, which begins at the reactive 3'-terminal phosphodiester and extends to the 7th nucleoside base, requires both phosphodiester monoanions and base residues for its interaction with the enzyme. The second domain includes phosphodiester monoanions in positions 9-13 from the 3'-terminus but does not require nucleoside bases. Methylphosphonate substitutions indicate that only two or three of these phosphodiesters, in variable positions, must remain anionic in order to obtain full enzyme binding. The residues between the two binding domains do not play a significant role in the enzyme-oligomer interaction.

Base Sequence↗
PubMed1986

Galactose-1-phosphate uridylyltransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism.

A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described. It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage. Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50. The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme. The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P. The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP-galactose. The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration. In this paper we show that reaction of the enzyme with (RP)-[2-14C]UDP alpha S-glucose produces a [2-14C]uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-[2-14C]cUMPS. Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate. Therefore, formation of uridylyl alpha S-enzyme from (RP)-UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps. The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Radioisotopes↗
PubMed1986

Processivity and kinetics of the reaction of exonuclease I from Escherichia coli with polydeoxyribonucleotides.

The enzyme exonuclease I from Escherichia coli hydrolyzes successive nucleotides from the 3'-termini of single-stranded deoxyribonucleotide homopolymers. When the reaction is stopped after partial hydrolysis, only intact starting material and small oligomers can be isolated. The distribution of oligomeric products varies with the base composition of the polymer but the largest oligomer that can be isolated from the reaction of exonuclease I with homopolymers of deoxyadenylate, deoxythymidylate, or deoxycytidylate is a decamer. These results suggest a model in which exonuclease I possesses at least two nucleotide binding sites. When both sites are filled, with 11-mers and longer polymers, the enzyme does not dissociate from the polymer during hydrolysis. When, with smaller oligomers, only a single site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation. The kinetics of the reactions of exonuclease I with purified polydeoxyriboadenylates of defined size distributions have been investigated. The maximum rates of hydrolysis are nearly independent of polymer size while the apparent Michaelis constants are inversely proportional to the polymer size. A simple steady state model yields a kinetic equation that is consistent with our results. Competition experiments indicate that the rate at which exonuclease I associates with the 3'-terminus of a polydeoxyribonucleotide is independent of the polymer's chain length.

Binding Sites↗
PubMed1986

Fourier transform infrared studies of ribonuclease in H2O and 2H2O solutions.

Fourier transform infrared transmission spectra have been obtained of the enzyme ribonuclease in both H2O and 2H2O. The resolution of the spectra have been enhanced by Fourier self-deconvolution procedures. The infrared spectrum of ribonuclease changes during exchange of the enzyme's amide hydrogens for deuterium and the exchange has been followed in the amide I and amide II spectral regions. The amide I band shifts towards lower wavenumbers during both the fast and slow phases of hydrogen exchange and the interpretation of these shifts has aided the band assignments. In particular these studies have allowed an assignment to be made for the high frequency component of the beta-strand absorption that differs from that proposed previously. This paper represents the first example of the use of deconvoluted Fourier transform infrared spectra in conjunction with hydrogen-deuterium exchange in order to aid in the assignment of a protein's infrared bands.

Amides↗
PubMed1986

Comparative cost analysis of home and hospital treatment.

A comparative study evaluating home and hospital treatment for terminal cancer patients is reported. Home care was provided by a comprehensive home care program, the Don Monti Home Oncology Medical Extension (H.O.M.E.). A multispecialty team including an oncologist, oncology nurse, social worker, dietitian and technologist is transported to the home in a medically-equipped van to render treatment. Services provided include physical assessments, pain control, chemotherapy and transfusion administration, psychosocial support, nutrition education and bereavement counseling. Two-hundred eighteen patients were entered of which 174 were treated at home and 44 in the hospital. Patients were comparable in age, diagnosis, sites of metastases, prior treatment and Karnofsky status. Both groups received similar supportive care either at home or in the hospital. Cost analysis of home and hospital care revealed a per diem cost benefit of $256.00 for home treatment. Comprehensive home treatment provided by a multi-specialty team can deliver effective care with medical and financial benefits to terminal cancer patients.

Cost Control↗
PubMed1985

Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli.

Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 [Prasher, D. C., Conarro, L., & Kushner, S. R. (1983) J. Biol. Chem. 258, 6340-6343]. The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus. This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate. This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined.

Chemical Phenomena↗
PubMed1983

Assessment of colorectal cancer risk in patients with ulcerative colitis: experience from a private practice.

The cumulative risk of developing colon cancer in patients with ulcerative colitis has been stated to increase 10%-20% for every decade of duration of disease after 10 yr. We reviewed the clinical course of 673 patients with inflammatory bowel disease restricted to the colon who were seen by the authors since 1955. A subset of 258 patients with a diagnosis of ulcerative colitis established before 1970 and followed by the authors was studied by both the classical life table and a generalized approach to estimate the risk of colorectal carcinoma. Only nine instances of colorectal carcinoma occurred. Using the former method and eliminating 3 patients referred with known colorectal carcinoma, the actuarial risk for developing this complication for all patients, regardless of extent of disease, was computed to be only 6.6% at 26 yr and 11.4% at 32 yr following the onset of ulcerative colitis. The cumulative probability of colorectal carcinoma among patients with universal colitis at 26 yr was 19.7% by the generalized method and 11.6% using the standard life table. The generalized approach consistently gave higher risk estimates due to a smaller number of patients in the denominator of the risk calculation. Using an alternative method, we calculated cancer risk from the date first seen for patients with universal extent and a history of greater than or equal to 10 yr of disease (means = 17.4 yr). The magnitude of the resulting colorectal carcinoma risk was even less than previously reported in hospitalized patients. This method is more suited for colorectal carcinoma risk assessment of a large series of ulcerative colitis patients seen in private practice and the results should modify the fear of cancer development in these patients.

Actuarial Analysis↗
PubMed1982

Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase.

The bacteriophage T7 induced DNA polymerase, consisting of the phage specified gene 5 protein associated with Escherichia coli thioredoxin, catalyzes the copolymerization of SP-dATP alpha S with dTTP, producing the alternating of polymer poly[dTs-A)] by a mechanism involving inversion of configuration at P alpha. Degradation of poly[d(5s-A)] by the nucleolytic action of E. coli DNA polymerase produced the dinucleotide pdTps-dA, whose configuration at the phosphorothioate diester was assigned as R by comparison of the phosphorus-31 nuclear magnetic resonance chemical shift (55.0 ppm downfield from H3PO4) with that of an authentic sample. Further degradation by alkaline phosphatase to Rp-dTps-dA (55.6 ppm downfield from H3PO4) confirmed the configuration. The stereochemistry provides no evidence of a double displacement mechanism.

DNA-Directed DNA Polymerase↗
PubMed1981

Unambiguous determination of the stereochemistry of nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli.

Nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli proceeds with greater than 97% inversion of configuration at P alpha of the alpha-phosphorothioate analogue of dATP. This is shown by experiments in which dAMPS,18O2 is stereospecifically phosphorylated to (Sp)-dATP alpha S, alpha 18O2, which is then copolymerized with dTTP by DNA polymerase. The product of the polymerization is degraded to dAMPS,18O by methods that do not affect the configuration of the phosphorothioate. After the dAMPS,18O is stereospecifically phosphorylated, the resulting (Sp)-dATP alpha S, alpha 18O is copolymerized as before with dTTP. The 18O is found in the displaced pyrophosphate by mass spectral analysis and so must have been in the pyrophosphate bridge of (Sp)-dATP alpha S, alpha 18O. Since this 18O was originally non-bridging in (Sp)-dATP alpha S, alpha 18O2, the phosphorothioate configuration must have been inverted in the polymerization reaction. This confirms the determination of P. M. J. Burgers & F. Eckstein [(1979) J. Biol. Chem. 254, 6889-6893], who used kinetic correlations based on the stereoselectivity of snake venom phosphodiesterase to deduce the stereochemistry of this reaction.

Adenosine Triphosphate↗
PubMed1980

Inhibition of orotidine-5'-phosphate decarboxylase by 1-(5'-phospho-beta-d-ribofuranosyl)barbituric acid, 6-azauridine 5'-phosphate, and uridine 5'-phosphate.

1-(5'-Phospho-beta-D-ribofuranosyl)barbituric acid, an analogue of orotidylic acid, binds to orotidine-5'-phosphate decarboxylase about 100000 times as strongly as does the substrate. The Ki at pH 6 is 9 X 10(-12) M and the half-time for dissociation at 4 degrees C is about 10 h. The binding of the barbiturate analogue to the enzyme is thus one of the strongest interactions between small molecules and proteins that have been measured. The possibility that the inhibitor is a transition-state analogue is discussed.

Carboxy-Lyases↗
PubMed1979

The purification of orotidine-5'-phosphate decarboxylase from yeast by affinity chromatography.

We have prepared an affinity column for the purification of orotidine-5'-phosphate decarboxylase from yeast. The column effects a 3200-fold purification from yeast homogenate in one pass; simple additional steps produce enzyme that has been purified 6700-fold and is not contaminated by any other protein that can be detected by sodium dodecyl sulfate-acrylamide gel electrophoresis. Overall, 35% of the activity present in the yeast is recovered as pure enzyme. The resin for the column is synthesized by attaching the ethylenediamine amide of 5-(2-carboxyethyl)-6-azauridine 5'-phosphate to carboxymethyl-agarose.

Carboxy-Lyases↗
PubMed1977

Multiple primary cancer risk after therapy for Hodgkin's disease.

Forty-four antecedent, synchronous, and metachronous multiple primary cancers were identified among 41 patients who constituted 4.0% of 1028 patients initially treated for Hodgkin's disease during the years 1950-1954, 1960-1964, and 1968-1972. At 5 years post-therapy the cumulative probabilities of developing a multiple primary cancer for patients treated in 1950-1954, 1960-1964, and 1968-1972, were 1.14%, 1.48%, and 4.43%, respectively. At 10 years the cumulative probability of a multiple primary cancer was 2.54% for the 1950-1954 treatment group and 6.52% for the 1960-1964 treatment group. Among those patients 16-39 years of age, initially treated during the period 1960-1964, who had survived 6-10 years after receiving radiation plus single agent chemotherapy, we observed a significant 18-fold increase in the number of multiple primary cancers. A significant occurrence of two multiple primary cancers in a relatively small group of patients treated with chemotherapy only during the period 1968-1972 was also noted. Continued surveillance of patients extensively treated with combination chemotherapy and radiotherapy will enable assessment of the oncogenic potential of these modern therapeutic approaches to the management of Hodgkin's disease.

Adult↗