Biomedical subjects

G W Tregear

Publications and source records attributed to G W Tregear.

At least 55 records · Page 3Linked to original sources

PubMed1989

Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus.

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.

Amino Acid Sequence↗

PubMed1989

Structure of rhesus monkey relaxin predicted by analysis of the single-copy rhesus monkey relaxin gene.

The gene encoding rhesus monkey relaxin has been investigated. A cDNA library was prepared using corpus luteal RNA from a pregnant rhesus monkey, cDNA clones encoding relaxin were isolated and the nucleotide sequence was determined. The amino acid sequence of rhesus monkey preprorelaxin, predicted from the cDNA, demonstrates that the sequence has not been strongly conserved when compared with that of man, although features characteristic of the relaxin molecule have been maintained. This structural information will allow production of rhesus monkey relaxin, leading to studies investigating the bioactivity of relaxin in a homologous primate system. Southern blot analysis indicated that there is only one relaxin gene in the rhesus monkey and baboon genomes. In this respect these primate genomes are different from the human genome which contains two relaxin genes.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of the gene coding for ovine corticotropin-releasing factor and regulation of its mRNA levels by glucocorticoids.

The ovine gene CRF, coding for corticotropin-releasing factor, has been isolated and the nucleotide sequence determined. The degree of nucleotide sequence homology between the ovine and human CRF genes is unusual, in that the 5' flanking regions are more highly conserved than the protein-coding regions. This striking degree of homology would indicate that a strong selective pressure is being exerted over an extensive area of the 5' flanking region, which could include transcriptional control elements. The 5' flanking region of the ovine CRF gene contains five elements which share homology with the glucocorticoid receptor DNA binding sequence. Also Northern blot analysis indicates that hypothalamic CRF mRNA levels are negatively regulated by glucocorticoids. Dexamethasone treatment halves the CRF mRNA content of the hypothalamus, whereas adrenalectomy causes a three- to four-fold increase in CRF mRNA levels.

Animals↗

PubMed1988

Structure and chromosomal localization of the human renal kallikrein gene.

Glandular kallikreins are a family of proteases encoded by a variable number of genes in different mammalian species. In all species examined, however, one particular kallikrein is functionally conserved in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. This kallikrein is found in the kidney, pancreas, and salivary gland, showing a unique pattern of tissue-specific expression relative to other members of the family. We have isolated a genomic clone carrying the human renal kallikrein gene and compared the nucleotide sequence of its promoter region with those of the mouse renal kallikrein gene and another mouse kallikrein gene expressed in a distinct cell type. We find four sequence elements conserved between renal kallikrein genes from the two species. We have also shown that the human gene is localized to 19q13, a position analogous to that of the kallikrein gene family on mouse chromosome 7.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of UK bovine rotavirus segment 4: possible host restriction of VP3 genes.

The bovine UK and simian SA11 rotaviruses are commonly used VP7-type reference strains. Since the surface protein VP3 is a significant neutralization antigen, it is important to fully characterize the VP3 types associated with current reference strains. Here we present the complete nucleotide and predicted amino acid sequence of VP3 from UK rotavirus (VP7 type 6) and compare it to the published sequences of SA114fm and RV-5. We also compare the deduced amino acid sequence covering the trypsin cleavage region of UK VP3 to 25 other available sequences. The UK protein is clearly different from that of bovine NCDV (another commonly used VP7 type 6 strain) and represents a second VP3 type associated with bovine rotaviruses. Our SA11 sequence differs from that determined by Lopez et al. [1985, Virology 144, 11-19; later referred to as SA114fM by Lopez et al. (1986, Virology 154, 224-227], their sequence being very similar to the published sequence of NCDV VP3. The significance of these results with regard to virus serotypes is discussed. Finally, in analyzing the nucleotide sequence surrounding the initiation codon, a potential hairpin-loop structure was identified which may be involved in translational regulation.

Amino Acid Sequence↗

PubMed1988

Beta-subunit of baboon chorionic gonadotropin. Continuous flow Fmoc-polyamide synthesis of the C-terminal 37-peptide.

The synthesis of a 37-peptide fragment derived from the carboxyl terminal of the beta-subunit of baboon chorionic gonadotropin has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of N alpha-fluorenylmethoxy-carbonyl-glutamine with its sidechain protected by the 4,4'-dimethoxybenzhydryl group enabled successful incorporation of this residue onto a hydroxymethyl linkage agent without apparent side reaction.

Animals↗

PubMed1988

Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin.

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.

Amino Acid Sequence↗

PubMed1988

Minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin.

Synthetic peptides of increasing length and corresponding in sequence to the C-terminal end of the HA1 molecule of influenza virus were constructed and examined for their immunogenic and antigenic properties. Peptides containing at least the four C-terminal amino acids, when coupled to keyhole limpet hemocyanin, were capable of eliciting antibody in BALB/c mice that bound to the 24-residue parent peptide H3 HA1 (305 to 328). In the absence of a carrier, the C-terminal decapeptide was the shortest peptide capable of eliciting antibody. The specificity of this antibody was indistinguishable from that of a monoclonal antibody to the parent peptide which recognizes an epitope encompassed by the C-terminal seven residues. All peptides containing at least the C-terminal four residues were able to inhibit completely the binding of this monoclonal antibody to the parent peptide. Taken together, these results indicate that (i) the tetrapeptide is capable of eliciting specific antibody when coupled to a carrier, (ii) this tetrapeptide possesses all of the antigenic information necessary to occupy the paratope of a monoclonal antibody elicited by the longer parent peptide, and (iii) the decapeptide contains all of the information necessary to elicit a specific immune response and therefore carries an epitope recognized by T cells as well as one recognized by B cells.

Amino Acid Sequence↗

PubMed1988

Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin.

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.

Animals↗

PubMed1988

Human prostate-specific antigen (APS) is a member of the glandular kallikrein gene family at 19q13.

The amino acid sequence of human prostate-specific antigen (APS) suggests that it is a member of the glandular kallikrein subfamily of serine proteases. In the mouse, the kallikrein-like family is localized in a single locus on chromosome 7, while other serine proteases are distributed over a variety of different chromosomes. To investigate the physical relationship between the human kallikrein genes, we have used in situ hybridization and Southern analysis of a human x mouse somatic cell hybrid panel to map the APS gene to 19q13, concordant with the renal kallikrein KLK1 gene. This finding indicates that APS is a member of a human kallikrein-like gene family with analogous organization to that of the mouse.

Antigens, Neoplasm↗

PubMed1987

Hybridization histochemistry.

The location of gene expression by hybridization histochemistry is being applied in many areas of research and diagnosis. The aim of this technique is to detect specific mRNA in cells and tissues by hybridization with a complementary DNA or RNA probe. Requirements for optimal specificity, sensitivity, resolution and speed of detection may not all be encompassed in one simple technique suitable for all applications, thus appropriate procedures should be selected for specific objectives. With reference to published procedures and our own extensive experience, we have evaluated fixatives, probes, labels and other aspects of the technique critical to the preservation and hybridization in situ of mRNA and detection and quantitation of hybrids.

Animals↗

PubMed1987

Preparation of base-modified nucleosides suitable for non-radioactive label attachment and their incorporation into synthetic oligodeoxyribonucleotides.

A very mild and efficient procedure has been developed for the preparation of C-5 substituted deoxyuridines. The substituent carries a masked primary aliphatic amino group. These compounds are readily converted into their phosphoramidites and can be used to prepare oligonucleotides carrying one or more aliphatic amino groups. Fluorescein isothiocyanate coupled to these compounds gives oligonucleotide probes carrying multiple fluorescein labels. These compounds have a free 5'-hydroxy group enabling additional 5'- end radioactive labelling for evaluation of their hybridization characteristics. It was found that oligonucleotides carrying a long (11 atom) linker arm to the fluorescein hybridize more efficiently to mRNA than those carrying a short (4 atom) arm. The long linker arm derivatives are comparable to underivatized oligonucleotides in hybridizing to mRNA.

Animals↗

PubMed1987

Purification and cloning of a corpuscles of Stannius protein from Anguilla australis.

The kidneys of teleost fish are associated with tissues containing secretory granules--the corpuscles of Stannius (CS). Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function. In the present study, two-dimensional gel electrophoresis and electroelution was used to purify the major protein to homogeneity; it is approximately 32,000 Da in the reduced form and glycosylated. From the partial NH2-terminal sequence, a 75-mer oligonucleotide probe was synthesized and used to isolate a cDNA clone from which the complete amino acid sequence of the major CS protein was deduced. Polyclonal antibodies raised against CS homogenates were specific for the CS proteins (confirmed by immunohistochemistry). Hybridization histochemistry was used to confirm the location of the mRNA encoding the isolated protein. Incubation of CS homogenate with eel plasma or ovine renin substrate did not result in any angiotensin-like peptides whereas kidney homogenate did.

Amino Acid Sequence↗

PubMed1987

Alpha-inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin.

Inhibin is a glycoprotein hormone composed of two nonidentical subunits. It is produced by the ovary and testis and plays a vital role in gonadal function by inhibiting the secretion of FSH. More recently, additional activities associated with inhibin peptides have been identified. Inhibin heterodimers (alpha-beta) are reported to act directly on ovarian granulosa cells and inhibit estrogen production induced by FSH. Furthermore, homodimers of beta-inhibin subunits stimulate the secretion of FSH, an activity that is directly opposite to that of inhibin. Each of these inhibin-related activities are concerned with the hypothalamic-pituitary-gonadal axis. We have investigated further the complexity of inhibin activity by determining whether inhibin genes are expressed in nongonadal tissue. RNA hybridization experiments demonstrate that the alpha-inhibin gene is expressed in the sheep adrenal cortex and hybridization histochemistry shows that this gene is expressed in each of the functional zones within the cortex. Dot blot analysis showed that the level of alpha mRNA within the adrenal is influenced by ACTH, one of the major regulators of adrenal cortex function. These observations imply that there are inhibin-related peptides not directly associated with the gonads. beta-inhibin gene expression was not clearly detected in the adrenal and we conclude that if expression occurs then it does so at extremely low levels.

Adrenal Cortex↗

PubMed1987

Histochemical localization of IGF-I and -II mRNA in the developing rat embryo.

We describe the histological localization of embryonic and fetal tissues whose cells express the genes coding for insulin-like growth factors I and II (IGF-I and IGF-II) in the developing rat. Our studies span the period between early somite stages and full term. We have used oligodeoxyribonucleotide probes and obtained results which are both topographically precise and highly reproducible. The gene coding for IGF-II is predominant throughout development. It is strongly expressed in the liver and yolk sac. A variety of other tissues also expresses the IGF-II gene, especially many mesodermally derived structures in the process of differentiation. Many tissues do not express IGF genes. Thus no IGF mRNA was demonstrable in ectodermally derived structures, including the central and peripheral nervous systems as well as the skin and its derivatives.

Animals↗

PubMed1986

Mouse glandular kallikrein genes. Identification, structure, and expression of the renal kallikrein gene.

Glandular kallikreins are a highly homologous subfamily of serine proteases encoded by 25 genes in the mouse. Several of the gene products have been implicated as having specific roles in growth factor biosynthesis, suggesting a general role for members of this gene family in the generation of biologically active peptides (Mason, A.J., Evans, B.A., Cox, D.R., Shine, J., and Richards, R.I. (1983) Nature 303, 300-307). Here we describe the identification, structure, and expression of the renal kallikrein gene, the major kinin-generating member of the family. Kinins are potent vasodilators implicated in regulation of local blood flow and ion transport. A gene-specific oligodeoxyribonucleotide probe was designed and chemically synthesized from the renal kallikrein gene sequence to distinguish this gene's transcripts from those of other members of the gene family. Differential expression phenotypes were found between the renal kallikrein gene and other members of the kallikrein gene family.

Amino Acid Sequence↗

PubMed1986

Antigenic determinants of influenza virus hemagglutinin. XII. the epitopes of a synthetic peptide representing the C-terminus of HA1.

A synthetic peptide comprising the C-terminal 24 amino acids of the heavy chain (HA1) of influenza virus hemagglutinin was constructed and examined for antigenic and immunogenic activity. Monoclonal antibodies as well as polyclonal antisera raised against the synthetic peptide were able to bind to intact virus. This binding was greatly enhanced if the virus was first subjected to pH 5, suggesting that this treatment exposes the C-terminus of HA1. Using synthetic analogs of the native sequence it was shown that the epitope recognized by one of the monoclonal antibodies encompasses one or more of the C-terminal four amino acids of HA1 (residues 325-328), which are conserved within subtypes but differ between subtypes, while the other monoclonal antibody recognizes a different epitope which involves at least one of the five variable residues at positions 311-315.

Amino Acid Sequence↗

PubMed1986

Location of gene expression in CNS using hybridization histochemistry.

In this chapter we have placed heavy emphasis on our own recent work to lay out a workable recipe for hybridization histochemistry. Only a trickle of papers followed the initial benchmark excursions into in situ labeling of tissue sections. Our own entry into this field was as late starters in 1978, but since then a confluence of important questions and technical advances has served to make hybridization histochemistry much more attractive and universally applicable as a research tool. Hybridization histochemistry allows the location of anatomical sites of gene expression and viral replication with unique specificity and is able to solve some problems for which there is no other suitable technique available in the central nervous system. For example, allowing that peptides may enter neurons by a variety of mechanisms and then be christened neuroendocrine peptides, it has become a compelling issue to know which cells are manufacturing the peptide. Thus, much can be learned by the approach elegantly demonstrated by Gee et al., of locating mRNA and its peptide product within the same neuron. The intracellular location of specific mRNA for a neuropeptide in a cell body indicates a very high probability that the peptide is secreted as a neurotransmitter or a neuromodulator from sites associated with the cell body. Our introduction of the use of whole mouse sections and large sections of brain of large animals in hybridization histochemistry has great potential in locating hormonal, enzymatic, and growth factor gene expression. The technique has been applied most elegantly by others to developmental studies and for the examination of viral infection. Resolution down to a single cell in heterogeneous tissue was beyond the original expectation of the capability of 32P-labeled probes and single cells in sections shown in Fig. 2 is probably the limit of resolution with this isotope. There is no reason why other isotopes, fluorescent labels, or labels suitable for EM should not take the resolution of the technique to intracellular. The horizon of application is widened enormously by the successful application of synthetic oligonucleotide probes, and at the same time unshackles the procedure from dependence upon a fully functional molecular biology laboratory. Although hybridization is a valuable research tool which we have applied to location of neuropeptides in the brain, it should soon find a niche in many fields and in a short time should become a key diagnostic tool.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗