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Biomedical subjects

D M Hinton

Publications and source records attributed to D M Hinton.

51 records · Page 3Linked to original sources
PubMed1985

Effects of cyclopiazonic acid on the ultrastructure of rat liver.

Groups of Sprague-Dawley rats were dosed per os for 4 consecutive days with 0.0, 0.2, 2.0 or 4.0 mg cyclopiazonic acid (CPA)/kg body weight/day, and killed on the fifth day. Sections of liver were prepared for electron microscopic examination. Dilatation of the rough endoplasmic reticulum was observed in all hepatocytes examined from the 2 highest dose groups, and in about 25% of liver cells from the 0.2 mg CPA/kg/day group. Vesiculation of the rough endoplasmic reticulum also occurred in these groups, an increasing amount of vesiculation being observed with increasing dosage. Control sections exhibited neither of these characteristics. No proliferation of smooth endoplasmic reticulum, or blockage of bile canaliculi was observed in any group. Lysing cells were present only in the 4.0 mg CPA/kg/day group; mitochondria in the 2.0 and 4.0 mg CPA/kg/day dose groups were swollen. Nuclei were ultrastructurally normal in all groups. The primary cellular effect of CPA was on the endoplasmic reticulum, even at relatively low doses. Possible interactions of CPA with other toxins likely to be produced by the same fungus, such as aflatoxin, are considered.

Animals↗
PubMed1983

Specific in vitro transcription of the insertion sequence IS2.

The insertion sequence IS2 is a small transposable element of Escherichia coli that lacks any known genetic markers. Insertion of this element in one orientation (I) within bacterial operons blocks expression of downstream genes. In the other orientation (II), IS2 has been associated with the constitutive expression of genes distal to its insertion, suggesting that IS2 might contain promoters directing transcription of IS2(II) into other genes. To test the transcription potential of IS2, we have transcribed in vitro DNA templates from gal3, a Gal- allele in which an IS2(I) is inserted between the gal promoter and the gal genes. We have detected two IS2-specific RNAs which initiate from promoters within IS2 and are transcribed in orientation II (away from the galETK genes). Though the presence and orientation of these promoters suggests that they could be responsible for the constitutive expression of genes adjacent to an IS2(II) element, an alternative role could be for transcription of IS2-encoded genes. Although IS2(I) insertions normally block expression of adjacent genes, certain altered (e.g. mutant) IS2(I) sequences lead to the constitutive expression of downstream genes. We have transcribed DNA templates from galwc5 and galc331, which are Galc alleles that contain altered IS2(I) insertions within the gal operon. For each allele, we have detected two gal-directed transcripts initiating within the IS2 sequence. These RNAs are not detected upon transcription of the unaltered IS2(I) DNA and the promoters arise as a direct consequence of the IS2(I) alterations. This result suggests that these promoters detected in vitro are responsible for the Galc phenotype of these alleles.

Autoradiography↗
PubMed1982

Transcription initiation sites within an IS2 insertion in a Gal-constitutive mutant of Escherichia coli.

Insertion of the insertion sequence Is2(I) directly before the galE gene of the galactose operon results in a Gal minus phenotype (1, 2). The Gal-constitutive allele galc200 (and its deletion derivative galc200 delta 31) arise from such a Gal minus mutant by the insertion of LS2(II) DNA within the LS2(I) sequence (3). We have transcribed in vitro a DNA template representing the IS2-galE region of galc200 delta 31. Gal-directed transcription initiates at two sites within the IS2(I) sequence, 51 and 52 bp from the IS2-galE junction. The promoter for these transcripts, Pgal200 delta 31, is composed of a novel joint between a -10 region from the IS2(I) DNA and a -35 region contributed by the IS2(II) insertion. No promoters intrinsic to the 121 bp of the IS2(II) sequence also present on the template were detected. The relevance of Pgal200 delta 31 to the Galc phenotype of galc200 and to general mechanisms for the constitutive expression of genes adjacent to IS2 is discussed.

DNA Restriction Enzymes↗
PubMed1982

The preparative synthesis of oligodeoxyribonucleotides using RNA ligase.

The synthesis of nmol quantities of defined sequences of oligodeoxyribonucleotides using T4 RNA ligase has been demonstrated. Reacting using from 18 to 200 nmol of substrates in which a single 2'-deoxyribonucleoside 3',5'-bisphosphate was added to an oligodeoxyribonucleotide resulted in yields from 13 to 95%. When two oligodeoxyribonucleotides were similarly joined using RNA ligase, the yields ranged from 10 to 50%. Although the reactions contained high concentrations of enzyme and were incubated from 5 to 21 days, there was little degradation of either substrates or products. We have also characterized an unusual product which arises when 3'-phosphate terminated oligodeoxyribonucleotides are incubated with RNA ligase and high concentrations of ATP. This product has an adenylyl group linked to the 3'-phosphate by an anhydride bond. The mechanistic and synthetic implications of forming this product are discussed.

Base Sequence↗
PubMed1980

T4 RNA ligase as a nucleic acid synthesis and modification reagent.

Oligodeoxyribonucleotides corresponding to portions of the recognition sequence and analogues thereof of the Eco RI restriction endonuclease have been synthesized using T4 RNA ligase. The successive addition of deoxyribonucleoside-3',5'-bisphosphates to preformed deoxyoligomers allowed stepwise oligodeoxyribonucleotide synthesis. Single strand deoxyoligomers were also joined to one another by the enzyme. In addition, biotin, and fluorophore tetramethylrhodamine, and hexylamine have been added to RNA via an ATP-independent RNA ligase reaction using their ADP adducts as substrates. When the beta-substituent on ADP is a good leaving group, e.g. p-nitrophenol or 4-methylumbelliferol, the RNA product is the 2'-(3')-cyclicphosphate derivative.

DNA Restriction Enzymes↗
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

Effects of intermittent exposures of aflatoxin B(1) on hepatic and testicular glutathione S-transferase in rats.

Glutathione S-transferase (GST) plays a major role in the detoxification of the potent hepatocarcinogen aflatoxin B(1) (AFB(1)). This study evaluated the effects of intermittent exposures to AFB(1) on hepatic and testicular GST in rats. Male Fischer 344 rats were fed diets containing AFB(1) (0, 0.01, 0.04, 0.4 and 1.6 ppm) intermittently at 4-week intervals up to 20 weeks. The control animals were fed an AFB(1)-free NIH-31 diet. Rats consuming diets with 0.01 ppm AFB(1) did not show the induction of hepatic or testicular GST activity. Intermittent exposures to AFB(1) at concentrations of 0.04-1.6 ppm significantly increased the GST activities. The increase of the enzyme activity was proportional to the dose and length of AFB(1) exposure.

Aflatoxin B1↗
PubMedDate not supplied

US FDA "Redbook II" immunotoxicity testing guidelines and research in immunotoxicity evaluations of food chemicals and new food proteins.

The rapid advances in the field of immunology and an understanding of the potential adverse effects of xenobiotics on the immune system have resulted in the development of a discipline in toxicology now referred to as immunotoxicology. This discipline has evolved steadily over the last 2 decades as a result of research in the national and international communities. Various US, European, and Japanese regulatory agencies have recognized a need to promulgate testing guidelines for immunotoxicity in support of the approval process involving toxicological testing. The US Food and Drug Administration "Redbook II" guidelines and some of the research conducted in support of the concepts and testing strategies are presented here. Concerns raised with regard to these guidelines are included, as are on-going initiatives in development of experimental approaches for assessing allergic potential and/or hypersensitivity responses to new foods and food constituents.

Animals↗
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↗