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Biomedical subjects

A McMichael

Publications and source records attributed to A McMichael.

At least 73 records · Page 4Linked to original sources
PubMed1995

Enantioselective inhibition of the epidermal growth factor receptor tyrosine kinase by 4-(alpha-phenethylamino)quinazolines.

4-Benzylaminoquinazolines can be potent reversible inhibitors of the EGFR tyrosine kinase at the ATP binding site. Examination of benzylic methylation reveals that an (R)-methyl group is four- to six-fold activating, with an optimal Ki of 630 pM for compound 11. In sharp contrast, (S)-methylation causes a > 30 to 500-fold loss of inhibitory activity, showing that the ATP-binding site of the receptor has very low tolerance for even moderate out-of-plane bulk in certain directions. It is suggested that the best of these inhibitors can induce a conformation of the kinase not available to poorer inhibitors.

Adenosine Triphosphate↗
PubMed1995

A mAb against HLA-A2 can be influenced both positively and negatively by the associated peptide.

Presentation of peptides by class I HLA molecules is essential for the development of a T cell-mediated immune response. TCRs have the ability to discriminate among large numbers of different HLA-peptide complexes. We have identified a mAb, MA2.1, that also discriminates among HLA-A2 associated with different peptides. A soluble form of HLA-A2 bound to single peptides was prepared and its serological reactivity was studied using four mAbs. Three antibodies, W6/32, BB7.2 and BBM.1, recognized all the complexes equally. MA2.1, however, recognized most of the complexes equally but showed markedly different reactivity to two peptides bound to HLA-A2. MA2.1 recognized HLA-A2 complexed with the HIV-1 p17 epitope (SLYNTVATL) at least 30 times more strongly than all other complexes studied and this enhanced reactivity was found to be sensitive to a point mutation of threonine to alanine at position 8 in the peptide. In addition, MA2.1 had a very low reactivity for HLA-A2 complexed with the peptide TLWVDPYEV. Previous studies have mapped the binding epitope of MA2.1 to the alpha 1 and alpha 2 helices of HLA-A2, suggesting two possible explanations for the ability of the bound peptide to influence MA2.1 reactivity. Either MA2.1 is sensitive to peptide-induced conformational changes of the helices, or it directly contacts certain peptides in the groove of HLA-A2.

Amino Acid Sequence↗
PubMed1995

Tropospheric ozone: respiratory effects and Australian air quality goals.

OBJECTIVE: To review the health effects of tropospheric ozone and discuss the implications for public health policy. DESIGN: Literature review and consultation with scientists in Australia and overseas. Papers in English or with English language abstracts were identified by Medline search from the international peer reviewed published reports. Those from the period 1980-93 were read systematically but selected earlier papers were also considered. Reports on ozone exposures were obtained from environmental agencies in the region. RESULTS: Exposure to ozone at concentrations below the current Australian air quality goal (0.12 ppm averaged over one hour) may cause impaired respiratory function. Inflammatory changes in the small airways and respiratory symptoms result from moderate to heavy exercise in the presence of ozone at levels of 0.08-0.12 ppm. The changes in respiratory function due to ozone are short lived, vary with the duration of exposure, may be modified by levels of other pollutants (such as sulphur dioxide and particulates), and differ appreciably between individuals. Bronchial lavage studies indicate that inflammation and other pathological changes may occur in the airways before reductions in air flow are detectable, and persist after respiratory function has returned to normal. It is not known whether exposures to ozone at low levels (0.08-0.12 ppm) cause lasting damage to the lung or, if such damage does occur, whether it is functionally significant. At present, it is not possible to identify confidently population subgroups with heightened susceptibility to ozone. People with asthma may be more susceptible to the effects of ozone than the general population but the evidence is not consistent. Recent reports suggest that ozone increases airway reactivity on subsequent challenge with allergens and other irritants. Animal studies are consistent with the findings in human populations. CONCLUSION: A new one hour air quality ozone goal of 0.08 ppm for Australia, and the introduction of a four hour goal of 0.06 ppm are recommended on health grounds.

Adaptation, Physiological↗
PubMed1995

Recognition of viral antigens at the cell surface.

There is now a very good understanding of the way in which epitope peptides are generated from virus proteins and how the peptides bind to the class I MHC molecules. This gives a framework in which to analyse the potentially protective cytotoxic T lymphocyte response in human immunodeficiency virus infection. A detailed understanding of the specificity of the responding T cells, the clonality of these T cells and the effects of virus variation on the CTL gives a possible explanation for the ultimate failure of the CTL response to control this virus infection.

Acquired Immunodeficiency Syndrome↗
PubMed1994

A specific inhibitor of the epidermal growth factor receptor tyrosine kinase.

A small molecule called PD 153035 inhibited the epidermal growth factor (EGF) receptor tyrosine kinase with a 5-pM inhibition constant. The inhibitor was specific for the EGF receptor tyrosine kinase and inhibited other purified tyrosine kinases only at micromolar or higher concentrations. PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low nanomolar concentrations in fibroblasts or in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression, and oncogenic transformation. PD 153035 demonstrates an increase in potency over that of other tyrosine kinase inhibitors of four to five orders of magnitude for inhibition of isolated EGF receptor tyrosine kinase and three to four orders of magnitude for inhibition of cellular phosphorylation.

3T3 Cells↗
PubMed1994

Analysis of mutant HLA-A2 molecules. Differential effects on peptide binding and CTL recognition.

Previous studies have identified several residues lining the groove of the HLA-A2.1 molecule that are critical for Ag presentation. However, it is not clear whether these residues are critical for binding of the peptide epitope per se or for determining the appropriate conformation of bound peptide. To distinguish between these possibilities, mutations at eight of these residues have been tested for their effects on the ability of the molecule to bind and present two known peptide epitopes--one derived from the influenza A matrix protein, the other from HIV pol. With only one exception, the mutations were found to affect the binding of the two peptides similarly. Most of the mutations resulted in intermediate deleterious effects on binding, with the B pocket mutant F9Y having the most dramatic negative effect on binding for both peptides. Two of the mutations significantly enhanced binding of both peptides and a peptide-specific effect on binding was seen with the substitution, Y99H, which enhanced binding of the matrix peptide yet diminished binding of the pol peptide. In contrast to the effects on binding, the effects of the mutations on presentation differed considerably for the two peptides. The most striking difference was seen with two alpha 2 alpha helix mutants that are fully recognized by pol peptide-specific CTL but not recognized by matrix peptide-specific CTL even though levels of binding were comparably diminished for the two peptides. These results suggest that some interactions, although not critical for binding per se, are critical for functional binding and the importance of these interactions differs among peptide epitopes.

Amino Acid Sequence↗
PubMed1994

Collagen-specific cytotoxic T lymphocyte responses in patients with ankylosing spondylitis and reactive arthritis.

Both ankylosing spondylitis (AS) and reactive arthritis (ReA) are strongly associated with HLA-B27 although the mechanism for this association is still unknown. Here we examine the hypothesis that B27-restricted, joint antigen-specific cytotoxic T lymphocytes (CTL) may be the driving force of AS and ReA. Type II and type XI procollagens (CII and CXI, respectively), expressed almost exclusively in the articular cartilage of the joints, were chosen as the possible targets of autoimmune CTL. Type I procollagen (CI), expressed in many different tissues, was also included as control. Nineteen nonamer peptides bearing appropriate HLA-B27 binding motifs from human CI, CII and CXI were identified and synthesized. When analyzed for binding affinity to HLA-B27 in assembly assays, four (two from CII, two from CXI) were found capable of binding to HLA-B27 with high affinity. These B27-binding collagen peptides were then used to stimulate peripheral blood lymphocytes from eight B27-positive AS and three ReA patients for identification of possible B27-restricted autoimmune CTL. HLA-B27-restricted CTL specific for one of the CII peptides, P109 were found in one of the ReA patients, but in none of the others.

Adult↗
PubMed1994

Design of a potent peptide inhibitor of the epidermal growth factor receptor tyrosine kinase utilizing sequences based on the natural phosphorylation sites of phospholipase C-gamma 1.

Peptides that possess primary sequences identical to segments surrounding the natural phosphorylation sites of phospholipase C-gamma 1 (i.e., tyrosines 472, 771, 783, and 1284) have been synthesized and evaluated with respect to substrate kinetics for the epidermal growth factor receptor tyrosine kinase. A peptide that was based on tyrosine 472 was the superior substrate in terms of lowest Km value at 37 microM and had the following amino acid sequence: Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr472-Glu-Glu-Val. This peptide sequence was used as a foundation to make amino acid substitutions and/or chemical modifications directed toward the synthesis of a potent peptide inhibitor. As a result, a nine amino acid peptide was synthesized having a K(i) of 10 microM.

Amino Acid Sequence↗
PubMed1993

Peptide selection by class I molecules of the major histocompatibility complex.

Class I molecules of the major histocompatibility complex (MHC) bind peptides derived from cytoplasmic proteins. Comparison of over 100 such peptides reveals the importance of the carboxy-terminal residue in selective binding. Recent evidence implicates the proteases and transporters of the processing pathway in providing peptides with the correct residues at the carboxyl terminus.

Journal Article↗
PubMed1993

A method for producing monoclonal antibodies to human T-cell-receptor beta-chain variable regions.

Study of the T-cell repertoire in humans has been hampered by the lack of monoclonal antibodies (mAbs) to the T-cell receptor (TCR) variable region (V) gene products. We describe a method for producing mAbs to the human TCR beta-chain V (V beta) gene products in which mice were immunized with a rat basophil cell line (RBL-2H3) transfected with the extracellular domain of the TCR heterodimer fused to the lambda chain of CD3. These cells acted as excellent immunogens for raising anti-TCR mAb and also formed the basis of a rapid screening assay. We generated mAbs against V beta protein of the TCR, showed that these mAbs stained approximately 1% of peripheral blood T cells, and further showed that the mAbs could stimulate proliferation of these T cells. We then characterized the mAbs by amplifying TCR cDNA derived from mAb-stimulated cells and sequencing the beta chain. All clones sequenced used the V beta 7.1 chain, proving conclusively that the mAbs generated were specific for V beta 7.1 subfamily. This method generates mAbs to human TCR V beta proteins efficiently and might allow production of a complete panel of mAbs directed against human TCR V beta proteins.

Amino Acid Sequence↗
PubMed1993

HIV-specific cytotoxic T-cell activity in an HIV-exposed but uninfected infant.

The factors necessary for protective immunity against HIV-1 are unknown. Important information about these factors should come from study of people at high risk of HIV infection who have not apparently become infected. Among these are the estimated 60-85% of children who may be exposed in utero or perinatally to HIV-1 but do not become infected. We observed the transient appearance of HIV-specific cytotoxic T-lymphocyte (CTL) activity in a baby born to HIV-1-infected parents, in whom all standard markers of infection remained negative. These findings suggest that HIV-specific CTLs may be a marker for recently exposed, but uninfected, individuals.

CD4-CD8 Ratio↗
PubMed1993

Different rates of HLA class I molecule assembly which are determined by amino acid sequence in the alpha 2 domain.

Assembly of HLA class I molecules was studied using pulse-chase labeling of B-lymphoblastoid cell lines with 35S-methionine, immunoprecipitation with antibodies detecting free or beta 2-microglobulin-associated heavy chain and isoelectric focusing. Marked differences between the products of different class I alleles were noted. HLA-B51 assembled very inefficiently, with considerable free heavy chain still detected in an unsialated form after a four hour chase. The closely related molecule HLA-B35 was in contrast rapidly assembled, all newly synthesized heavy chain being detected in a beta 2m-associated sialated form within 30 minutes. Analysis of naturally occurring variants related to HLA-B35 and HLA-B51 localized the region determining assembly efficiency to the alpha 2 domain, in which these molecules differ at eight amino acid residues. The effect was not due to a linked dominant gene, as both patterns of assembly were observed in a single cell line.

Amino Acid Sequence↗
PubMed1992

A critical role for conserved residues in the cleft of HLA-A2 in presentation of a nonapeptide to T cells.

The peptide binding cleft of the class I human histocompatibility antigen, HLA-A2, contains conserved amino acid residues clustered in the two ends of the cleft in pockets A and F as well as polymorphic residues. The function of two conserved tyrosines in the A pocket was investigated by mutating them to phenylalanines and of a conserved tyrosine and threonine in the F pocket by mutating them to phenylalanine and valine, respectively. Presentation of influenza virus peptides and of intact virus to cytolytic T lymphocytes (CTLs) was then examined. The magnitude of the reduction seen by the mutation of the two tyrosines in the A pocket suggests that hydrogen bonds involving them have a critical function in the binding of the NH2-terminal NH3+ of the peptide nonamer and possibly of all bound peptide nonamers. In contrast, the mutations in the F pocket had no effect on CTL recognition.

Amino Acid Sequence↗
PubMed1992

Loss of HLA class-I alleles, heavy chains and beta 2-microglobulin in colorectal cancer.

Using immunohistochemical methods, we have analysed colorectal biopsies of normal mucosa, metaplastic polyps (5 cases), adenomas (15 cases) and adenocarcinomas (70 cases) with 13 monoclonal antibodies (MAbs) to allelic products of the HLA-A, B, C loci. Nine of the 70 carcinomas showed total loss of HLA Class-I molecules due to an underlying defect regarding not only the expression of beta 2-microglobulin (beta 2-m), but also the heavy chains of HLA A, B and C loci, or both. Much commoner was a loss of one or more Class-I alleles as follows: A1/Aw36 (completely lost in 4 of 29 cases and focally lost in another 2), A2 (in 1 of 37 cases), A3 (in 2 of 14 cases), A1 1/28/31/33 (in 3 of 11 cases), B7 (in 3 of 13 and focally in 1 other case), B17 (in 1 case), Bw4 (in 8 of 45 and focally in another 6), Bw6 (in 9 of 62 and focally in another 3). Focal selective loss (Bw6 and a combined A1-Bw6), was observed in 2 adenomas. Normal colonic mucosa, as well as stromal and lymphoid cells present between the neoplastic glands, were studied in each case as a control. A particular allele was only considered to be lost by the malignant cells if it was still expressed on these adjacent tissues.

Alleles↗
PubMed1992

Genetic modulation of antigen presentation by HLA-B27 molecules.

In studies of antigenic peptide presentation, we have found a healthy volunteer whose lymphoblastoid cells were unable to present three different virus-derived epitopes to cytotoxic T lymphocytes (CTL) despite expressing the correct restricting HLA-B27 molecules on the cell surface. B cell lines were established from other members of the donor's family, including individuals suffering from ankylosing spondylitis and related diseases, and were tested for their ability to function as target cells in the same assay. None of the eight B cell lines that expressed HLA-B27 presented a known peptide epitope to CTL. However, cells from a family member that expressed HLA-B8 could present an epitope peptide restricted by that molecule. The B27 molecule in this family proved to be the B2702 subtype on isoelectric focusing gels, appearing in exactly the same position as B2702 from other cell lines that did present the peptide. To exclude mutations resulting in noncharged amino acid substitutions, cDNA coding for B2702 was cloned from the proband's cell line and sequenced. No coding changes were found. The cloned cDNA was transfected into HLA-A- and B-negative HMy/C1R cells, and the B2702 molecules generated in this environment rendered these cells, after incubation with peptide, susceptible to lysis by peptide-specific CTL. These data are compatible with the presence of a factor(s), possibly HLA linked, interfering with antigen presentation by otherwise normal B2702 molecules in this family.

Antigen-Presenting Cells↗
PubMed1992

Identification of the nonamer peptide from influenza A matrix protein and the role of pockets of HLA-A2 in its recognition by cytotoxic T lymphocytes.

Influenza matrix peptide 58-66 is shown to be the optimal nonamer for binding to HLA-A2 and presentation to cytotoxic T lymphocytes (CTL). If titered out to 2 x 10(-10) - 4 x 10(-10) M in CTL-mediated lysis assays and to 3 x 10(-9) M in an HLA-A2 assembly-stabilization assay in cell lysates. The peptide was shown to make probable contacts with its carboxy terminus close to residue 116 in the floor of the cleft of HLA-A2, close to the F pocket. The side chain of the amino-terminal amino acid was unimportant, but its free amino and carbonyl groups in the A pocket appeared important in optimizing peptide presentation. The B pocket probably accommodates the side chain of residue 2 (isoleucine) and was shown to be critical in peptide presentation.

Amino Acid Sequence↗