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Biomedical subjects

M A Mitchell

Publications and source records attributed to M A Mitchell.

At least 91 records · Page 5Linked to original sources
PubMed1990

Synthetic immunogens constructed from T-cell and B-cell stimulating peptides (T:B chimeras): preferential stimulation of unique T- and B-cell specificities is influenced by immunogen configuration.

In our effort to develop synthetic immunogens as vaccines, we have focused on the combination of a known T-cell stimulating peptide with putative B-cell stimulating peptide epitopes derived from the sequences of respiratory syncytial (RS) virus proteins. The T-cell stimulating peptide consists of residues 45 through 60 of the 1A protein of RS virus, and it also contains an overlapping antibody binding (B-cell) site. Herein, we have combined the 1A T-cell stimulating peptide with a putative B-cell peptide epitope derived from the viral G glycoprotein using linear synthesis or using chemical crosslinking. The chimeric immunogens were compared to each other and to free peptides for their T- and B-cell stimulating properties. Both chimeras had potent T-cell stimulating and antibody-inducing activity. However, T-cells primed to free peptide differentially recognized the two chimeras and immunization with the chimeras primed T-cells with different specificity. Most strikingly, the two chimeras had opposite antibody-inducing properties: The chimera constructed by linear synthesis overwhelmingly elicited antibody directed against the G peptide, whereas the chimera constructed by chemical crosslinking overwhelmingly elicited antibody directed against the 1A peptide. Competition blocking studies revealed that the chimeras adopted different configurations in solution. The resulting antibody response, and hence the B-cell clone elicited, was consistent with the antibody accessibility of the individual peptide epitope.

Amino Acid Sequence↗
PubMed1990

Effects of genetic selection on growth rate and intestinal structure in the domestic fowl (Gallus domesticus).

1. Pieces of small intestine taken from chickens subjected previously to continuous selection, relaxed selection or no selection for rapid growth were used to estimate villus surface area and microvillus development to determine what effects genetic selection might have on factors controlling intestinal function. 2. Crypt size and the rates at which enterocytes migrated out of crypts were also measured, after injection of tritiated thymidine, to determine the time course of microvillus elongation. 3. Differences in growth rates measured between highly selected, relaxed selected or unselected birds were found to be correlated with parallel changes in villus surface area. Selection for growth did not change the density, dimensions or pattern of development of enterocyte microvilli. Microvilli did, however, produce a maximal 20-fold increase in villus surface area under all conditions. 4. Crypt size and enterocyte migration rates did not vary significantly between tissue taken from unselected and relaxed selected chickens. Tissue taken from highly selected birds had a crypt size and enterocyte migration rate 40% higher than values found for the other two groups of chickens. 5. The possibility that early genetic selection increased growth potential by uncoupling diet-induced changes on crypt hyperplasia from secondary effects on villus structure, and that later selection increased growth potential by increasing appetite, is discussed.

Animals↗
PubMed1990

Problems associated with use of the benzyloxymethyl protecting group for histidines. Formaldehyde adducts formed during cleavage by hydrogen fluoride.

The use of N alpha-tert.-butyloxycarbonyl-N pi-benzyloxymethylhistidine in peptide synthesis resulted in significant levels of several different side products attributable to the generation of formaldehyde during the hydrogen fluoride cleavage reaction. Methylated impurities in a decapeptide were isolated and identified. These methylated impurities were attributed to the use of the benzyloxymethyl protecting group for the histidines, since the impurities did not form when the dinitrophenyl protecting group was used. Also, peptides containing benzyloxymethyl-protected histidines in addition to N-terminal cysteines quantitatively yielded their respective N-terminal thiazolidine derivatives upon isolation from standard hydrogen fluoride cleavage mixtures. Thiazolidine ring formation was circumvented by including in the cleavage reaction a formaldehyde scavenger such as cysteine hydrochloride or resorcinol.

Amino Acid Sequence↗
PubMed1990

Sequence and uptake specificity of cloned sonicated fragments of Haemophilus influenzae DNA.

Our data support the conclusion that all tight binding of DNA by competent cells of Haemophilus influenzae is due to 9 base pairs (bp) of the 11-bp motif 5'-AAGTGCGGTCA or its inverse complement, TGACCGCACTT. Sonicated chromosomal DNA of H. influenzae was absorbed and extracted from competent cells to prepare a subpopulation of uptake fragments enriched for single uptake (binding) sequences. This DNA was inserted into the plasmid pUC18 and cloned into Escherichia coli. Whole sonicated chromosomal DNA was also cloned in pUC18 as a population of control fragments. Seventy-one plasmids were labeled with 3H and tested for DNA binding, and the inserts of 28 of them were sequenced. The control plasmid pUC18 was absorbed to competent H. influenzae cells at low levels of 5 to 10% of DNA added. The plasmids containing uptake inserts were mostly absorbed at levels of 70 to 80%, but a significant number contained inserts with an intermediate level of binding, 20 to 30%. The inserts of 15 plasmids were excised and tested for DNA uptake to demonstrate good agreement between uptake of these plasmids and their insert fragments. Sequencing of inserts revealed that the presence of 9 bp of the 11-bp motif was associated with tight binding. Some inserts with intermediate levels of binding had no significant similarities to the 9-bp sequence. The 9-bp sequence appeared to account for most of the binding to competent cells, but appreciable binding occurred with fragments without 9-bp homology. About one-third of the 9-bp uptake sequences were found as inverted repeats that could form strong stem-loop structures. It has been suggested by Goodman and Scocca (Proc. Nal. Acad. Sci. USA 85:6982-6986, 1988) that in Neisseria gonorrhoeae, uptake sequences occur as palindromes and act as transcription terminators. Although consistent, the data are insufficient to make this conclusion for uptake sequence palindromes in H. influenzae.

Base Sequence↗
PubMed1990

Purification and biological activity of a single charge isomer of pituitary-derived chicken growth hormone.

The chicken pituitary gland contains a number of naturally occurring, developmentally regulated forms of GH which have identical molecular weights but differ in their isoelectric points. In order to characterize their biological properties, each must be separated from non-GH proteins and other forms of GH. Chickens GH (cGH) was separated from other pituitary proteins by immunoaffinity chromatography using an anti-GH monoclonal antibody covalently linked to Sepharose 4B. The cGH eluted from this column as a single peak and migrated as a single band during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), but showed multiple bands on isoelectric focussing. This material was chromatographed on a high-performance cation exchange column, and separation of charge isomers was monitored by a combination of isoelectric focussing and immunoblotting. Chicken GH eluted from this column in two distinct peaks. The minor peak (cGH P1) contained an isomer with an isoelectric point of 6.86 and the major peak (cGH P2) an isomer with an isoelectric point of 7.52. Each isomer migrated as a single band during isoelectric focussing and SDS-PAGE (Mr = 23,500), and as a single peak during high-performance gel permeation chromatography and reverse-phase high-performance liquid chromatography. Analysis of cGH P2 through 30 cycles in a gas-phase microsequencer gave an amino acid sequence identical to that predicted by translation of the GH complementary DNA nucleotide sequence. This single charge isomer increased the rate of lipolysis in chicken adipose tissue explants by about fourfold and was able to displace 125I-labelled cGH from binding sites in liver membranes with a dissociation constant of about 4 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1990

Pink spots of Hedley-Whyte in formaldehyde-fixed brains. The subject revisited.

In an article published in 1985, Hedley-Whyte described "pink spots," which she observed in gross sections of human brains fixed in formaldehyde solution. Hedley-White found that the spots in question were associated with the presence of bacteria within central nervous system blood vessels. We have recently encountered three cases of Hedley-Whyte-type pink spots during routine postmortem brain examination. In two of these cases, the patient's clinical history included a bacterial infection, which could account for the deposition of bacteria within the central nervous system blood vessels. In the third case, the patient had no clinical history of bacterial infection, and had a negative postmortem blood culture. On microscopic examination, all three cases showed central nervous system intravascular presence of bacteria within the macroscopically recognized pink spots. To test the hypothesis that pink spots result from bacterial fermentation products, at the time of postmortem removal of brains (different from the brains mentioned above), we perfused blood vessels in three brains with 100% ethanol. Subsequent macroscopic examination after fixation in formaldehyde revealed discoloration similar to the Hedley-Whyte spots.

Aged↗
PubMed1989

A 16-amino acid peptide of respiratory syncytial virus 1A protein contains two overlapping T cell-stimulating sites distinguishable by class II MHC restriction elements.

The 1A protein of respiratory syncytial (RS) virus is a small, 64-amino acid hydrophobic protein expressed in infected cells. We previously showed that the C-terminal domain of 1A contained a site for stimulation of RS virus-reactive Th lymphocytes in BALB/c and SJL/J mice. In this report we modeled a series of overlapping synthetic peptides of the 1A protein and we present evidence to suggest that the C-terminal domain of the 1A protein contains not one, but two, Th lymphocyte-stimulating sites. Although these sites are extensively overlapping they can be distinguished on the basis of presentation by distinct class II restriction elements of the murine MHC. Additionally, we present evidence to suggest that a 16-amino acid synthetic peptide which contains both of these T cell sites can stimulate T cell activity in mice of multiple different class II MHC haplotypes, perhaps representing a potential lead for designing a "universal" T cell stimulator.

Amino Acid Sequence↗
PubMed1989

Identification of specific binding proteins for a nuclear location sequence.

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.

Amino Acid Sequence↗
PubMed1989

Flight effects on plasma levels of lipid, glucagon and thyroid hormones in homing pigeons.

Significant increases in the concentration of plasma glucagon-like immunoreactivity (GLI) and plasma levels of free fatty acids (FFA) and triglycerides (TG) concomitant with decreases in circulating levels of thyroxine (T4) and triiodothyronine (T3) and T3/T4 ratio were observed in homing pigeons, untrained for 3 months, after a flight of 48 km lasting 90-160 min. The increased level of FFA is attributed to glucagon stimulated lipolysis. The elevation of TG levels may be due to altered partitioning and utilization of lipoprotein in adipose tissue and muscle. Reductions in plasma T4, T3 and T3/T4 ratio are probably due to inhibition of T4 secretion and 5'-monodeiodination with possible conversion of T4 to reverse T3 (rT3). These processes may represent a mechanism for regulation of thyroid hormone metabolism during strenuous and extended flight.

Animals↗
PubMed1988

Molecular basis for sequence-specific DNA alkylation by CC-1065.

CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5'-side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5'-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments, and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1988

The prevalence of macroscopic sarcocysts in New Zealand cattle at slaughter.

A survey carried out to determine the prevalence of visible Surcocystis spp. infection in 100 slaughtered cattle in a South Island abattoir revealed 64% infected. Although all ages, genders, breeds and export grades were infected, only host age had a statistically significant effect on the infection rate. Infections were most common in the rectus abdominis and the psoas muscles (47% and 41% respectively). Most infections were light, with less than ten macrocysts seen, though infections of more than 50 macrocysts occurred. The average macrocyst length was 4.WO.10 mm (SE) (n = 233), the average cyst wall thickness 5.3 +/- 0.2 microm (SE) (n = 45). It is thought that the actual prevalence of infection is higher than that recorded and reasons for this are discussed.

Journal Article↗
PubMed1988

Mapping an antibody-binding site and a T-cell-stimulating site on the 1A protein of respiratory syncytial virus.

A synthetic peptide modeled on residues 45 to 60 of the 1A protein of respiratory syncytial (RS) virus [1A(45-60)] was constructed and used for immunization of mice and rabbits. The immunoglobulin G fraction of the resulting rabbit antibody, purified on protein A-Sepharose, immunoprecipitated from RS-infected HEp-2 cells a protein with a molecular size of approximately 9.5 kilodaltons, which corresponds to the previously published molecular size of the 1A protein (Y. T. Huang, P. L. Collins, and G. W. Wertz, Virus Res. 2:157-173, 1985). To investigate the T-cell-inducing properties of 1A(45-60), six strains of mice were immunized and their popliteal lymph node cells were tested for proliferation upon restimulation with peptide in vitro. The lymph node cells of all six strains of mice were responsive to restimulation with 1A(45-60) and showed high- and low-responder strain variation. These peptide-primed lymph node cells also proliferated upon in vitro restimulation with RS virus-infected cells. Correlation of proliferation with interleukin 2 production suggested that the responding lymphocytes were T-helper cells. The antibody-binding and T-cell-stimulating sites of 1A were mapped by constructing a series of overlapping synthetic peptides and testing each for ability to react with antiserum prepared by immunization of BALB/C mice with free peptide 1A(45-60) or for ability to restimulate proliferation in 1A(45-60)-primed lymph node cells of BALB/C mice. Human antibody, obtained during confirmed RS virus infection, was similarly tested with the truncated peptides. Antibody-binding activity was reduced after truncation from the carboxy terminus, and a binding site was mapped to residues 51 through 60, the smallest peptide tested. T-cell-stimulating activity in mice was relatively resistant to truncation from the carboxy terminus and sensitive to truncation from the amino terminus. The smallest region which retained significant T-cell-stimulating activity mapped to residues 46 through 56. However, addition of the naturally occurring Cys at residue 45 and extension of the C terminus to residue 62 resulted in maximum T-cell-stimulating activity of the peptide. These data define both a T-cell epitope and a B-cell epitope of the 1A protein of RS virus and suggest that the carboxy terminus of 1A contains a B-cell epitope, involving residues 51 through 60, which is recognized during natural human infection.

Amino Acid Sequence↗
PubMed1987

Plasma glucagon and energy substrate responses of domestic fowl to treadmill exercise.

Exercise-induced alterations in the concentrations of plasma glucagon-like immunoreactivity (GLI), plasma free fatty acids (FFA) and blood glucose and lactate were measured in separate groups of male and female domestic fowl. There were only small changes in blood glucose and lactate concentrations but plasma FFA and GLI rose by up to 450 and 200% respectively. There was evidence that the GLI response was stronger at higher exercise intensities. It is suggested that the mobilization of FFA for use as energy substrates by the working muscles may be stimulated by the enhanced secretion of glucagon.

Animals↗
PubMed1986

The effects of ACTH and dexamethasone upon plasma thyroid hormone levels and heat production in the domestic fowl.

Treatment with long acting ACTH (20 IU kg-1) produces a large and sustained elevation of plasma corticosterone in the domestic fowl. Both ACTH treatment and administration of dexamethasone produce significant reductions in plasma concentrations of T4 and T3, and these changes are accompanied by a sustained hyperglycaemia. Despite the changes in circulating thyroid hormones only a small reduction in heat production (-14%) was induced by either treatment and mainly during the dark period. Whilst there may be some causal relationship between increased corticosterone secretion, decreased plasma thyroid hormone levels and reduced metabolic heat production it is unlikely that these responses alone account for the adjustments in energy expenditure observed in short term food deprivation.

Adrenocorticotropic Hormone↗
PubMed1986

The effects of glucagon and insulin on plasma thyroid hormone levels in fed and fasted domestic fowls.

Glucagon injection (50 micrograms kg-1) produced a biphasic response in plasma thyroxine (T4) level in both fed and fasted chickens. An initial inhibition was followed by an increase to levels above control value. Glucagon reduced plasma triidothyronine (T3) possibly as a consequence of inhibition of peripheral monodeiodination. This inhibition persisted in fasted animals despite a glucagon induced hyperglycaemia. Insulin injection (4 IU kg-1) decreased plasma T4 concomitant with a profound hypoglycaemia. These effects were more pronounced in fasted birds. Insulin induced hypoglycaemia was associated with decreased plasma T3 probably as a consequence of reduced thyroidal T4 secretion and reduced peripheral monodeiodination. Glucagon and insulin may play direct or indirect roles in the regulation of thyroid hormone secretion and metabolism in the domestic fowl.

Animals↗
PubMed1986

Silver acetate gum as a deterrent to smoking.

Silver acetate chewing gum, a nonprescription medication, produces an unpleasant metallic taste in the mouth of individuals who consume tobacco products in conjunction with this smoking deterrent. Use of the product leads to self-induced aversive conditioning. In the present double-blind controlled study, subjects using silver acetate for three weeks of treatment had a smoking cessation rate of 15 out of 136 (11 percent, p = 0.02). Placebo subjects had a smoking cessation rate of 6 out of 146 (4 percent, p = .102). Without further treatment, the group using silver acetate demonstrated a 7 percent nonsmoking rate at four months compared with a 3 percent nonsmoking rate for the placebo group. Silver acetate demonstrated a modest benefit over placebo as a smoking deterrent in a minimal intervention and highly cost-effective treatment setting.

Acetates↗
PubMed1985

Some new observations on the binding of thyroxine and triiodothyronine to plasma proteins and lipoproteins in the domestic fowl.

In addition to the previously recognised thyroid hormone-binding proteins, three plasma components possibly involved in T3 and T4 transport have been identified in chicken plasma using gel filtration at pH 7.4. In mature female, though not male and immature female birds, a significant amount of T3 (11%) is bound to very-low- and low-density lipoproteins. These could mediate transport of the hormone into the egg. Similarly vitellogenin appears to bind small quantities of both T3 and T4. Selective precipitation of the lipoproteins and vitellogenin from laying-hen plasma produces a binding profile identical to that observed in male and juvenile female birds. Treatment of adult male birds with oestrogen induces a binding profile for T3 similar to that observed in adult females. Also a low-molecular-mass protein (9 X 10(3) D) which preferentially binds thyroxine in substantial amounts (20-30%) was found in plasma from adult male and female and immature female birds. The technique employed allows detection of plasma thyroid hormone-binding proteins which have not previously been resolved by simple electrophoretic procedures.

Animals↗