Biomedical subjects

D M Hinton

Publications and source records attributed to D M Hinton.

8 recordsLinked to original sources

PubMed1992

Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage.

The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids. We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function. Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii. Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion. Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting. Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns. Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron.

Amino Acid Sequence↗

PubMed1992

Testing guidelines for evaluation of the immunotoxic potential of direct food additives.

Immunotoxicity testing is a new addition to the safety assessment guidelines for direct food and color additives. The approaches and philosophy for this area of specialized testing are consistent with the case-by-case strategy applied in the regulatory approval process, which is based on structure-activity relationships, preexisting knowledge, and projected exposure estimates. Specialized testing such as immunotoxicity is not part of the basic testing requirements, but would be applied when indicators are positive. Concepts for immunotoxicity testing have evolved, in part, from research for evaluating various testing methods as well as specific study designs. This research, conducted over the last decade, has focused mainly on the rat as the rodent species of choice. The miniature swine was evaluated as a nonrodent model. Testing is defined by type 1 and type 2 tests, which differ in that type 1 tests are performed on the same animals used in the core study design. Sets of type 1 and type 2 tests, with reference to the indicators, define various testing levels. Retrospective testing, expansion of basic testing (such as histopathology and serum chemistry profiles), and alternative study designs, which include satellite groups for evaluation of the functional capacity of the immune system, can be considered in the evaluation of immunotoxic potential.

Animals↗

PubMed1979

The synthesis of oligodeoxyribonucleotides using RNA ligase.

T4 RNA ligase catalyzes the addition of a single deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of oligodeoxyribonucleotides (Hinton et al. (1978) Biochemistry 17, 5091). We have determined improved conditions for this reaction which give yields equal to or greater than 85% when any of five common deoxyribonucleoside bisphosphate (pdAp, pdCp, pdGp, pdTp, or pdUp) are added to dA(PDA)4. A low ATP concentration, which is constantly maintained by a regeneration system composed of phosphocreatine, creatine kinase, and myokinase, contributes to the attainment of high yields. The addition of RNase A and spermine also enhances the rates and yields of the reactions. These conditions facilitate the use of RNA ligase as a reagent for the stepwise synthesis of DNA of defined sequence.

Base Sequence↗

PubMed1979

Pericardial-fluid complement: normal values.

Reports of low pericardial-fluid complement levels in systemic lupus erythematosus and rheumatoid arthritis have been difficult to interpret, as few data are available to describe complement concentrations in patients without pericardial disease. The authors therefore determined normal values under standardized conditions of collection, storage, and assay. The normal ranges for pericardial-fluid C3, C4, and total hemolytic complement were 35-127 mg/dl, 6.3-23 mg/dl, and 1.9-9.1 CH50 units, respectively. Storage at -20 C resulted in a 50% reduction in values. Hence, storage at -70 C is recommended. As the level of pericardial-fluid total hemolytic complement is normally low, caution is needed in interpreting its apparent reduction in various immunologic diseases.

Complement C3↗

PubMed1978

T4 RNA Ligase joins 2'-deoxyribonucleoside 3',5'-bisphosphates to oligodeoxyribonucleotides.

T4 RNA ligase catalyzes the ATP-dependent addition of a single 2'-deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of an oligodeoxyribonucleotide. The bisphosphate is joined to the deoxyoligomer by a 3' leads to 5' phosphodiester bond and the product, which is terminated by a 3'-phosphate, is one nucleotide longer than the substrate. Bisphosphates of dAdo, dCyd, dGuo, dThd, and dUrd are donors and oligodeoxyribonucleotides with dA, dC, dG, dT, or dU 3' termini act as acceptors. The preferred residue for both donor and acceptor is dCyd. Deoxyoligomers from 3 to 12 residues in length are active as acceptors. To obtain good yields, high concentration of enzyme, long incubation time at low temperature, and manganous rather than Mg(II) ion are required. Under optimal conditions, yields calculated with respect to deoxyoligomer converted to product vary from 40 to greater than 95%. The turnover number of the enzyme for DNA joining is extremely low but, because the preparation is nearly free of DNases, there is less than 3% degradation of substrate or product after 6 days of reaction. We anticipate that this reaction will serve as the basis for a method for the stepwise enzymatic synthesis of DNA of defined sequence.

Adenosine Triphosphate↗

PubMed1975

Ethidium binding affinity of circular lambda deoxyribonucleic acid determined fluorometrically.

Ethidium-binding isotherms for purified circular lambda DNA, isolated from a superinfected lysogen, and for linear lambda DNA, isolated from the purified phage, were constructed from fluorescence measurements of ethidium-DNA MIXTURES. The measurements were made in 0.01 M Tris-HC1-0.001 M EDTA,pH 7.1, buffer at 20 degrees and in the same buffer containing 0.1, 0.4, or 1.0 M NaC1. When NaC1 was present, differences in the binding affinity for supercoiled and linear DNA could be quantitated. As the ethidium concentration was increased, supercoiled lambda DNA molecules bound the intercalating dye first more and then less avidly than nonsupercoiled ones. The number of potential supercoils in a circular lambda DNA molecular in the absence of dye was calculated from the amount of dye bound when it exhibited the same affinity for dye as its linear counterpart. The point of equivalent affinity shifted from 0.053 mol of dye bound per mol of nucleotide in 0.1 M NaC1 to 0.067 mol in 1.0 M NaC1. This corresponds to the removal of 164 and 206 supercoiling turns per molecule and superhelix densities in the absence of dye equal to 0.036 and 0.045 superhelical turns per 10 base pairs. If this difference in the number of supercoils reflects a salt-dependent change in the average rotation angle between base pairs of the Watson-Crick helix the angle differs by 0.32% in the two ionic environments.

Binding Sites↗

PubMed1975

Purification of closed circular lambda deoxyribonucleic acid and its sedimentation properties as a function of Sodium chloride concentration and ethidium binding.

The sedimentation of circular lambda DNA suggests that the molecular undergoes significant changes in shape and super-coiling as the NaC1 concentration increases. Closed circular lambda DNA, species I, isolated and purified from superinfected immune bacteria, sediments in sucrose gradients of low ionic strength at a rate 2.0 times faster than linear lambda DNA, species III. The addition of ethidium causes the sedimentation rate of species I DNA to decrease until enough dye is bound to remove 121 supercoils per molecule. At this point, species I co-sediments with nicked and nonsupercoiled species II. Futher additions of ethidium cause the sedimentation rate to increase until the relative rate of species I is again at least twice that of species III. This classical behavior is altered when NaC1 is present in the buffer. In 1.0 M NaC1 the changes in S are complex. Initially, species I sediments 1.55 times faster than species III. Titration with ethidium caused a decrease in S to an early minimum value, than an increase to a first maximum, followed by a decrease to the S of species II. At this point enough dye has intercalated to remove 208 superhelical turns. Further additions of dye introduce supercoils and cause S to increase again. In 0.1 to 0.4 M NaC1 the relative S of species I is 1.69 and 1.59, respectively. If titrated with ethidium, S first increases to a maximum value then decreases to the minimum rate when enough dye is bound to remove 158 and 183 supercoils, respectively. The results indicate an increase in the superhelix density from 0.026 turns per 10 base pairs in buffer alone to 0.045 in the same buffer with 1.0 M NaC1. If this change in superhelix density results from a concomitant change in the average rotation angle between base pairs in the Watson-Crick helix, the addition of 1.0 M NaC1 alters the rotation angle by 0.68 degrees per base pair.

Binding Sites↗

PubMedDate not supplied

Evaluation of heat sterilization of commercial rat diets for use in FDA toxicological studies.

Certified commercial rat diets, control and fortified, in the form of pellets and meal, were evaluated in a simulated subchronic rat feeding study. The diets were analyzed before and after autoclaving to determine nutrient integrity and loss, as well as the efficiency of autoclaving for removal of microbiological contaminants. Sterilization reduced the level of heat-labile vitamins, but protein level was minimally reduced. Sterilization eliminated most of the bacterial contaminants and virtually all the mold and yeast colonies. Male and female Osborne-Mendel rats (3-4 wk old) were fed control or sterilized diet for 6 wk. Both males and females consumed more pelleted chow than meal chow. This apparent difference in consumption may be due to wastage of pellets, because there were no differences in male or female growth during the 6-wk study. At necropsy, no gross pathology was noted, and organ weights did not differ significantly among the groups for either sex. Testicular weights were also similar among the groups. Blood serum proteins were analyzed by electrophoresis to screen for possible effects on various target organs. Gamma globulin levels for female rats fed sterilized meal were significantly reduced compared to levels for rats fed the control diet. These results suggest that either nutritional factors or heat inactivation of the microbes affects basal levels of humoral immunity, possibly by reduction of gut-mediated immune responses.

Animal Feed↗