Biomedical subjects

J M Green

Publications and source records attributed to J M Green.

At least 55 records · Page 3Linked to original sources

PubMed1998

Regulation of in vitro and in vivo T cell activation by CD43.

Accessory molecule interactions can be critical in determining the outcome of a T cell's encounter with antigen. Cell adhesion proteins may augment T cell responses by facilitating TCR engagement of the antigen-MHC complex, while co-stimulatory molecules may deliver distinct signals that modulate T cell responsiveness. CD43 (leukosialin, sialophorin) has been suggested to influence cell activation by steric hindrance based upon the large size and glycosylation of the protein, as well as the relative abundance of the protein on the cell surface. In this paper we examine both in vitro and in vivo T cell-dependent responses in CD43-deficient mice. We demonstrate that T cells from CD43-deficient mice are hyper-responsive following both in vivo and in vitro activation, and that this is observed in response to not only TCR-CD3-mediated stimulation, but also following receptor-independent activation. This data suggests that mechanisms other than non-specific steric hindrance are important in the regulation of T cell activation by CD43.

Animals↗

PubMed1998

Characterization of mutations that allow p-aminobenzoyl-glutamate utilization by Escherichia coli.

An Escherichia coli strain deficient in p-aminobenzoate synthesis was mutagenized, and derivatives were selected for growth on folic acid. Supplementation was shown to be due to p-aminobenzoyl-glutamate present as a breakdown product in commercial folic acid preparations. Two classes of mutations characterized by the minimum concentration of p-aminobenzoyl-glutamate that could support growth were obtained. Both classes of mutations were genetically and physically mapped to about 30 min on the E. coli chromosome. A cloned wild-type gene from this region, abgT (formerly ydaH) could confer a similar p-aminobenzoyl-glutamate utilization phenotype on the parental strain. Interruption of abgT on the plasmid or on the chromosome of the mutant strain resulted in a loss of the phenotype. abgT was the third gene in an apparent operon containing abgA, abgB, abgT, and possibly ogt and might be regulated by a divergently transcribed LysR-type regulator encoded by abgR. Two different single-base-pair mutations that gave rise to the p-aminobenzoyl-glutamate utilization phenotype lay in the abgR-abgA intercistronic region and appeared to allow the expression of abgT. The second class of mutation was due to a tandem duplication of abgB and abgT fused to fnr. The abgA and abgB gene products were homologous to one another and to a family of aminoacyl aminohydrolases. p-Aminobenzoyl-glutamate hydrolysis could be detected in extracts from several of the mutant strains, but intact abgA and abgB were not essential for p-aminobenzoyl-glutamate utilization when abgT was supplied in trans.

Amino Acid Sequence↗

PubMed1998

Role of epidermal growth factor receptor family members in growth and differentiation of breast carcinoma.

Members of the epidermal growth factor (EGF) family of tyrosine kinase receptors are involved in the regulation of cell growth and differentiation, and are found to be expressed in many types of cancers. Activation of these receptors can be elicited by multiple ligands, resulting in the formation of a spectrum of heterodimer complexes and a number of biological outcomes. A clear demonstration of biological activation by a single complex has been difficult to address because of the endogenous expression of HERs (human EGF-like receptors) in many cell lines. We have generated a collection of cell lines expressing all HERs alone or in all pairwise combinations in a clone of NIH 3T3 cells (3T3-7d) devoid of detectable EGF receptor family members. Transformation, as measured by growth in soft agar, only occurred in cells expressing two different HER family members. Transformation with activated Neu and the rate of in vivo tumour formation were also correlated with the expression of multiple HERs in the same cell. To further our understanding of the role of heterodimer signalling, we demonstrated that, within a breast carcinoma cell line, activation of HER-3 results in cellular differentiation, prolonged activation of extracellular-signal-related kinase 1 (ERK1) activity and an increase in p21CIP1/WAF1 nuclear staining. In contrast, activation of HER-4 is mitogenic, induces transient activation of ERK1 activity and decreases the nuclear staining of p21CIP1/WAF1. These differences in biochemical and biological responses are correlated with the contrasting abilities of HER-3 and HER-4 to be down-regulated from the cell surface. The cell-surface localization of HER-3 does not change in response to ligand, whereas activation of HER-4 results in a loss of cell-surface staining followed by accumulation into a perinuclear compartment.

3T3 Cells↗

PubMed1997

Role for a glycan phosphoinositol anchor in Fc gamma receptor synergy.

While many cell types express receptors for the Fc domain of IgG (Fc gamma R), only primate polymorphonuclear neutrophils (PMN) express an Fc gamma R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc gamma receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc gamma receptors. Jurkat T cells were stably transfected with cDNA encoding Fc gamma RIIA and/or Fc gamma RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either Fc gamma RIIA or Fc gamma RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fc gamma RIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca2+]i rise, as did crosslinking CD59 with Fc gamma RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc gamma receptors are not required for synergy. Replacement of the GPI anchor of Fc gamma RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc gamma RIIA cytoplasmic tail abolished synergy. While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB. These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.

Antigens, CD↗

PubMed1997

CD28 engagement and proinflammatory cytokines contribute to T cell expansion and long-term survival in vivo.

To mount a productive response to Ag, CD4+ T cells in mice must divide, differentiate, and survive at least until the Ag has been eliminated. It has been suggested that to accomplish this, T cells must receive two signals, one through their TCRs and a second through CD28. The second signal through CD28 has been thought to fulfill two roles, to stimulate T cell proliferation and to promote T cell survival. In this paper we confirm that CD28 engagement can contribute to vigorous T cell expansion in mice injected with superantigens. However, CD28 engagement does not protect T cells produced during a superantigen-specific proliferative response from undergoing subsequent deletion. Even if CD28 is bound, 4 days after superantigen exposure, the majority of T cells produced in response to superantigen exposure are eliminated in vivo. In contrast, this loss of superantigen-stimulated T cells can be prevented by the inflammatory stimuli created by injection of bacterial LPS. This protection does not require engagement of CD28 by its ligands, B7-1 and B7-2. These data suggest that productive T cell responses in mice involve a number of signals, including those initiated through TCR and CD28, which are primarily involved in the activation and expansion of T cells, and others delivered by proinflammatory cytokines that protect an activated T cell from subsequent deletion.

Abatacept↗

PubMed1997

Women's need for information before attending genetic counselling for familial breast or ovarian cancer: a questionnaire, interview, and observational study.

OBJECTIVES: To describe women's information needs prior to genetic counselling for familial breast or ovarian cancer. DESIGN: Prospective study including semistructured telephone interviews before genetic counselling, observations of consultations, completion of postal questionnaires, and face-to face interviews within two months of counselling. SUBJECTS: 46 women attending genetic counselling for familial breast or ovarian cancer. MAIN OUTCOME MEASURES: Subjects' understanding of process and content of genetic counselling before attending and attitudes about their preparation for the counselling session. RESULTS: Although all women interviewed before the clinic expected to discuss their risk of developing cancer and risk management options, there was evidence of a lack of knowledge about the process and content of genetic counselling, 17 (37%) women said they did not know what else would happen. Most women interviewed after counselling viewed it positively, but 26 (65%) felt they had been inadequately prepared and 11 (28%) felt that their lack of preparation meant that they could not be given an accurate estimation of their risk of cancer. CONCLUSIONS: Some women felt that they did not obtain optimum benefit from genetic counselling because they were inadequately prepared for it. We suggest that cancer family history clinics should provide women with written information about the process and content of genetic counselling before their clinic attendance.

Adult↗

PubMed1997

Preimplantation diagnosis and other reproductive options: attitudes of male and female carriers of recessive disorders.

This questionnaire-based study describes in detail the attitudes of 245 carriers of recessive disorders to preimplantation diagnosis (PID). These are compared to responses to other reproductive options for carriers, that is prenatal diagnosis (PND), adoption, donor insemination (DI) and egg donation. The positive attitudes of this sample to PID and corresponding negative attitudes to adoption, DI and egg donation are striking. Almost the entire sample indicated that they viewed early reassurance and the opportunity to avoid a termination of pregnancy as important advantages of PID. Disadvantageous aspects of PID were acknowledged, but did not result in many participants finding this approach unacceptable. The study revealed a number of differences between men and women, especially in their perceptions of the disadvantages of PID. Women saw the practical difficulties of PID as more important disadvantages than did the men, and were very much more concerned about the issues involving embryos. Men, conversely, rated the impact upon their partners as a more important disadvantage. These findings underline the importance of considering both sets of views. Despite support for PID, in an overall assessment it did not displace PND as the most useful reproductive option.

Adoption↗

PubMed1997

Who worries that something might be wrong with the baby? A prospective study of 1072 pregnant women.

BACKGROUND: Little is known about the normal range and pattern of pregnant women's worry about something being wrong with the baby, or how this relates to other factors. The objective of this study was to examine the extent to which women are worried about the possibility of something being wrong with the baby relative to other worries they may have, and to determine whether demographic, experiential, attitudinal, and personality characteristics are associated with this worry. METHODS: Longitudinal data were collected from 1072 pregnant women who completed postal questionnaires at 16, 22, and 35 weeks of pregnancy. Multivariate statistical techniques were used to determine which variables were independently related to worry about the baby. RESULTS: Worry that something might be wrong with the baby was one of the most prevalent worries at 16 weeks, although not as widespread a source of extreme worry as miscarriage or giving birth. Worry dropped in midpregnancy but rose again at 35 weeks. The most important factors related to this worry were perceived likelihood that something might be wrong with the baby and trait anxiety, although negative mood, previous pregnancy outcomes, and initial reactions to the current pregnancy all demonstrated significant, independent effects. CONCLUSIONS: Women who answered "Don't know" to "Have you any reason to think that your baby might be more likely than any other to have some sort of a problem?" had consistently high anxiety throughout pregnancy. We suggest that this question be asked routinely in early pregnancy to identify these potential worries and, it is to be hoped, reassure them at an early stage.

Adolescent↗

PubMed1996

HLA-DM is present in one-fifth the amount of HLA-DR in the class II peptide-loading compartment where it associates with leupeptin-induced peptide (LIP)-HLA-DR complexes.

HLA-DM has been shown in vitro to catalyze the release of invariant chain (Ii) derived peptides from the peptide-binding groove of class II molecules, thereby facilitating the binding of antigenic peptides. Previous studies showed that at steady state, the majority of DM resides in the class II peptide-loading compartment (IIPLC) where Ii dissociates from class II molecules and antigenic peptides are bound. Here we characterize the expression of DM in vivo in subcellular fractions containing the IIPLC. Using quantitative immunoblotting, we show that in the cell as a whole, class II molecules are expressed in 23-fold molar excess of DM. However, DM is concentrated in the IIPLC, where it is present in a considerably higher concentration relative to the class II molecules, in a molar ratio of 5DR:1 DM. This molar ratio of DM to DR in the IIPLC in vivo is consistent with the catalytic function proposed for DM from studies in vitro. We also provide both biochemical and genetic evidence that DM associates with complexes which contain Ii fragments and class II molecules in the IIPLC. Such complexes are only observed in leupeptin-treated cells in which Ii fails to be completely degraded and complexes containing the leupeptin-induced fragment of Ii (LIP) and class II molecules accumulate in the IIPLC. This observation is consistent with LIP-class II complexes being a substrate for DM in vivo and suggests that interactions of DM and LIP-class II are extremely transient under normal conditions.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1996

The relationship between human epidermal growth-like factor receptor expression and cellular transformation in NIH3T3 cells.

A collection of cell lines expressing each human epidermal growth factor receptor (HER) family member alone or in all pairwise combinations in a clone of NIH3T3 cells (3T3-7d) devoid of detectable epidermal growth factor receptor family members has been generated. Transformation, as measured by growth in soft agar, occurred only in the presence of appropriate ligand and only in cells expressing two different HER family members. Transfection of oncogenic neu (Tneu), conferred ligand-independent transformation only in cells which co-expressed HER1, HER3, or HER4, but not when expressed alone or with HER2. Cell lines were also tested for their ability to form tumors in animals. None of the cell lines expressing single HER family members was able to form tumors in animals with the exception of HER1, which was weakly tumorigenic. Although unable to form tumors when expressed alone, HER2 was tumorigenic when expressed with HER1 or HER3, but not HER4. Of all complexes analyzed, cells expressing HER1 + HER2 were the most aggressive. The relationship between HER1 activation, intracellular calcium fluxes, and phospholipase Cgamma1 activation is well established. We found that activation of HER1 was required for the induction of a calcium flux and the phosphorylation of phospholipase Cgamma1. These activities were independent of, and unaffected by, the co-expression of any other family member. Further, heregulin stimulation of all cell lines including those containing HER1 did not demonstrate any effect on intracellular calcium levels or phospholipase Cgamma1 phosphorylation. This demonstrates that heregulin induced cellular transformation by activating HER3- and HER4-containing complexes does not require the activation of either phospholipase Cgamma1 or the mobilization of intracellular calcium.

3T3 Cells↗

PubMed1996

Limited role of CD28-mediated signals in T helper subset differentiation.

The role of CD28-mediated signals in T helper cell maturation is not fully understood. We tested the requirement for costimulation through CD28 in several systems of CD4+, T cell differentiation. In vivo priming of mice with genetic disruption of CD28 (CD28-/-) yielded normal levels of antigen-specific interferon gamma production but markedly diminished levels of interleukin 4 (IL-4) after in vitro restimulation. In response to the pathogenic microbe, Leishman a major, C57BL6 CD28-/- mice were fully capable of controlling infection and exhibited a normal T helper 1 response. BALB/c CD28-/- mice unexpectedly exhibited normal susceptibility to L. major. BALB/c CD28-/- mice developed high levels of IL-4 mRNA and protein induction in the draining lymph nodes. In addition, susceptibility of BALB/c CD28-/- mice was reversed by neutralization of IL-4 in vivo. We also activated transgenic CD28-bearing T cells from the BALB and C57BL background in vitro in the presence of CTLA4Ig. BALB cells had greater IL-4 producing capacity than C57BL cells in the absence of costimulation. Diverse factors including costimulatory signals, genetic polymorphism, and the nature of the immunogen all influence T helper phenotype commitment, but these results provide evidence that CD28 is not an absolute requirement for generating either Th1 or Th2 responses.

Animals↗

PubMed1996

CD28 costimulation prevents cell death during primary T cell activation.

CD28 has been demonstrated to play an important role in augmenting T cell proliferation and effector function. Costimulation through CD28 has also been reported to enhance human T cell survival. in this report, we have further investigated the role of CD28 in regulating T cell survival by comparing the survival characteristics of T cells from wild-type and CD28-deficient mice. CD28 costimulation of anti-CD3-activated cells augmented the viability of T cells from wild-type but not from CD28-deficient mice. CTLA4Ig treatment reduced wild-type T cell viability to a level comparable with CD28-deficient T cells. The ability of CD28 to enhance survival during T cell activation correlated positively with its ability to up-regulate the protein product of the cell survival gene bcl-xL. No differences in the expression of either Bcl-2 or Fas were observed between wild-type and CD28-deficient T cells. The CD28-dependent enhancement of cell survival during in vitro activation was found to be independent of Fas expression, as CD28 costimulation enhanced T cell survival to comparable levels in both wild-type and lpr animals. Cell death in CD28-deficient animals and in wild-type animals treated with CTLA4Ig displayed the morphologic characteristics of apoptosis. Additionally, inhibitors of ICE proteases could reverse cell death induced by TCR engagement in the absence of CD28 costimulation. Thus, CD28 costimulation not only enhances the proliferative expansion of cells activated through the TCR but also increases the likelihood that individual cells survive during T cell activation.

Adjuvants, Immunologic↗

PubMed1996

Homotypic interactions mediated through LFA-1/ICAM-3 decrease the proliferative response of activated T cells.

T cell activation occurs when the T cell receptor (TCR) is engaged by an antigen-MHC complex on the surface of an antigen-presenting cell (APC). Additional signals provided by accessory molecules serve to modulate this response. Independent of TCR engagement, treatment of T lymphocytes with a combination of phorbol ester and CD28 ligation will result in a proliferative response. This also induces homotypic adhesion mediated by LFA-l/ICAM interactions. We demonstrate that the prevention of homotypic interactions between T cells resulted in a two- to fivefold increase in the proliferative response. This occurred whether the homotypic interactions were prevented by blockade of LFA-1, by the use of plate immobilized antibodies against other cell surface molecules, or by culture at low cell density. We further demonstrate that the increased proliferation was a result of interference with a negative signal delivered to the T cell as a result of ICAM-3-mediated events. These data demonstrate LFA-1/ICAM-3 interactions between T cells in turn regulate an LFA-1-independent pathway that results in homotypic adhesion and a downregulation of the proliferative response of activated T cells.

Adjuvants, Immunologic↗

PubMed1996

Escherichia coli aminodeoxychorismate synthase: analysis of pabB mutations affecting catalysis and subunit association.

p-Aminobenzoic acid (PABA), an essential component of the vitamin folic acid, is derived from the aromatic branch-point precursor chorismate in two steps. 4-Amino-4-deoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate, and is composed of two subunits, PabA and PabB. While various experiments have suggested that PabA and PabB act as a complex, attempts to isolate the intact complex have failed. We report here the first successful copurification of PabA and PabB by gel filtration chromatography. The association of PabA and PabB is greatly enhanced by the presence of 5 mM glutamine, and by preincubation at 37 degrees C. Conversely, the association is greatly reduced at cold temperatures. We also report the isolation and characterization of both chemically induced and site-directed mutations in PabB. Mutated PabB enzymes fall into three categories according to their properties: deficiency of chorismate amination coupled with failure to associate with PabA, deficiency of chorismate amination coupled with retention of PabA association, and competency of chorismate amination with failure of PabA association.

Amination↗

PubMed1996

Increased efficiency of alkaline phosphatase production levels in Escherichia coli using a degenerate PelB signal sequence.

To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels.

Alkaline Phosphatase↗

PubMed1996

HER4-mediated biological and biochemical properties in NIH 3T3 cells. Evidence for HER1-HER4 heterodimers.

The EGF receptor family of tyrosine kinase growth factor receptors is expressed in a variety of cell types and has been implicated in the progression of certain human adenocarcinomas. The most recent addition to this family of receptors, HER4, was expressed in NIH 3T3 cells to determine its biological and biochemical characteristics. Cells expressing HER4 were responsive to heregulin beta2 as demonstrated by an increase in HER4 tyrosine phosphorylation and ability to form foci on a cell monolayer. HER4 exhibited in vitro kinase activity and was able to phosphorylate the regulatory subunit of phosphatidylinositol 3-kinase and SHC. Peptide competition studies identified tyrosine 1056 of HER4 as the phosphatidylinositol 3-kinase binding site and tyrosines 1188 and 1242 as two potential SHC binding sites. Interestingly, transfection of HER4 into NIH 3T3 cells conferred responsiveness to EGF with respect to colony formation in soft agar. It was also found that in response to heregulin beta2, endogenous murine HER1 or transfected human HER1 became phosphorylated when HER4 was present. This demonstrates that HER1 and HER4 can exist in a heterodimer complex and likely activate each other by transphosphorylation.

3T3 Cells↗

PubMed1996

Proposal for a structured assessment of parenting based on attachment theory: theoretical background, description and initial clinical experience.

This paper proposes a structured clinical assessment of parenting and illustrates its use within child psychiatry practice. The aim was to develop a structured instrument based on current theoretical knowledge which was simple enough to be clinically viable while being precise and repeatable enough to enable quantification and research. Use is made of inpatient and daypatient resources but the assessment could be modified for outpatient practice. The assessment takes a "diagnostic" approach; concentrating on factors that have been shown in research to be good predictors of parenting dysfunction. These include parental personality, current mental state and degree of current social stress and support (including quality of marital relationship). Additionally, information regarding the adult's representation of early attachment relationships is elicited using the Adult Attachment Interview. Independent assessments of the child and the parent/child interaction are made. Initial clinical experience with this instrument is described and practical and theoretical issues raised by its use are explored.

Adult↗

PubMed1996

Class II antigen processing compartments and the function of HLA-DM.

The DM alpha and DM beta genes encode a nonpolymorphic, class II-like molecule which functions by an, as yet, undefined mechanism in the assembly of Major Histocompatibility Complex class II-peptide complexes. Indeed, mutant cells which express class II molecules but fail to express DM are unable to process and present native protein antigens. A striking phenotype of the mutation is class II molecules that contain almost exclusively a nested set of invariant chain peptides, termed CLIP, for class II associated Ii peptides, instead of the normal array of endogenously and exogenously derived peptides. Thus, DM appears to be required for the correct assembly of processed antigen-class II complexes. Recently, the subcellular compartments that contain DM and in which functional processed antigen-class II complexes are first formed have been described. Here, the evidence for the function of DM in the antigen-processing compartments is reviewed.

Antigen Presentation↗