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Biomedical subjects

G J Clark

Publications and source records attributed to G J Clark.

At least 19 recordsLinked to original sources
PubMed2002

Growth factors, cytokines and dendritic cell development.

Dendritic cells (DC) initiate tumor specific immune responses in animal studies and initial human trials suggest that certain tumor-antigen loaded DC preparations generate clinical responses. DC may be obtained from blood or generated in vitro from precursor cells. In vitro generation of DC from precursor cells, under the influence of cytokines, has been favoured to date as a source because of the greater numbers of DC produced. However, the different cytokine combinations and serum or plasma component(s) used, differentiate precursor cells into DC with different physiological properties and ultimate immunogenicity. Thus, the quality of in vitro cytokine derived DC may have a profound influence on clinical outcomes. The administration of certain growth factors, which increase the number of circulating blood DC, may provide an alternative source of DC for use in clinical trials. Although clinical trials in prostate cancer, melanoma and metastatic renal carcinoma patients are encouraging, some data suggest certain DC preparations and administration protocols are sub optimal, even potentially tumor enhancing. As basic scientific studies establish how to provide DC with stable phenotype, resistance to tumour inhibitory factors and high migratory capacity, the technology for producing cytokine derived DC in vitro using Good Manufacturing Practise (GMP) conditions needs to be developed. Future DC vaccination protocols will require careful control of the DC used for tumor-antigen loading and repetitive long term DC vaccination may be necessary to maintain effective anti-tumor immune responses.

Animals↗
PubMed2001

Thermal capsulorrhaphy for acute or chronic shoulder instability.

During the past two decades, arthroscopic procedures have been replacing traditional, more invasive orthopedic surgical procedures. As technology becomes more advanced, the opportunity to provide a greater number of minimally invasive surgical interventions continues to improve. Thermal modification of joint capsule and ligamentus tissues, a recent introduction to medical science, has been investigated extensively during the past six years. Arthroscopic thermal capsulorrhaphy is one such procedure, and it is performed on individuals with a history of joint instability. These patients now can be treated surgically without large incisions and significant shoulder joint trauma. The thermal unit in both monopolar and bipolar models has similar properties to those of the basic electrosurgical unit. Relatively low-temperature heat is directed to the supportive structures of the shoulder joint causing the tissues to expand. This tightens a previously stretched and attenuated shoulder capsule.

Arthroscopy↗
PubMed2001

The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family.

The immunoregulatory signaling (IRS) family includes several molecules, which play major roles in the regulation of the immune response. The CMRF-35A and CMRF-35H molecules are two new members of the IRS family of molecules, that are found on a wide variety of haemopoietic lineages. The extracellular functional interactions of these molecules is presently unknown, although CMRF-35H can initiate an inhibitory signal and is internalized when cross-linked. In this paper, we described the gene structure for the CMRF-35A gene and its localization to human chromosome 17. The gene consists of four exons spanning approximately 4.5 kb. Exon 1 encodes the 5' untranslated region and leader sequence, exon 2 encodes the immunoglobulin (Ig)-like domain, exon 3 encodes the membrane proximal region and exon 4 encodes the transmembrane region, the cytoplasmic tail and the 3' untranslated region. A region in the 5' flanking sequence of the CMRF-35A gene, that promoted expression of a reporter gene was identified. The genes for the CMRF-35A and CMRF-35H molecules are closely linked on chromosome 17. Similarity between the Ig-like exons and the preceding intron of the two genes suggests exon duplication was involved in their evolution. We also identified a further member of the CMRF-35 family, the CMRF-35J pseudogene. This gene appears to have arisen by gene duplication of the CMRF-35A gene. These three loci - the CMRF-35A, CMRF-35J and CMRF-35H genes-form a new complex of IRS genes on chromosome 17.

3' Untranslated Regions↗
PubMed2000

Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis.

Although activated Ras proteins are usually associated with driving growth and transformation, they may also induce senescence, apoptosis, and terminal differentiation. The subversion of these anti-neoplastic effects during Ras-dependent tumor development may be as important as the acquisition of the pro-neoplastic effects. None of the currently identified potential Ras effector proteins can satisfactorily explain the apoptotic action of Ras. Consequently, we have sought to identify novel Ras effectors that may be responsible for apoptosis induction. By examining the EST data base, we identified a potential Ras association domain in the tumor suppressor RASSF1. We now show that RASSF1 binds Ras in a GTP-dependent manner, both in vivo and directly in vitro. Moreover, activated Ras enhances and dominant negative Ras inhibits the cell death induced by transient transfection of RASSF1 into 293-T cells. This cell death appears to be apoptotic in nature, as RASSF1-transfected 293-T cells exhibit membrane blebbing and can be rescued by the addition of a caspase inhibitor. Thus, the RASSF1 tumor suppressor may serve as a novel Ras effector that mediates the apoptotic effects of oncogenic Ras.

3T3 Cells↗
PubMed2000

The Ras/p120 GTPase-activating protein (GAP) interaction is regulated by the p120 GAP pleckstrin homology domain.

Pleckstrin homology domains are structurally conserved functional domains that can undergo both protein/protein and protein/lipid interactions. Pleckstrin homology domains can mediate inter- and intra-molecular binding events to regulate enzyme activity. They occur in numerous proteins including many that interact with Ras superfamily members, such as p120 GAP. The pleckstrin homology domain of p120 GAP is located in the NH(2)-terminal, noncatalytic region of p120 GAP. Overexpression of the noncatalytic domains of p120 GAP may modulate Ras signal transduction pathways. Here, we demonstrate that expression of the isolated pleckstrin homology domain of p120 GAP specifically inhibits Ras-mediated signaling and transformation but not normal cellular growth. Furthermore, we show that the pleckstrin homology domain binds the catalytic domain of p120 GAP and interferes with the Ras/GAP interaction. Thus, we suggest that the pleckstrin homology domain of p120 GAP may specifically regulate the interaction of Ras with p120 GAP via competitive intra-molecular binding.

3T3 Cells↗
PubMed2000

Elucidation of binding determinants and functional consequences of Ras/Raf-cysteine-rich domain interactions.

Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been implicated in regulating Raf kinase activity. While recognition elements that promote Ras.RBS1 complex formation have been characterized, relatively little is known about Ras/Raf-CRD interactions. In this study, we have characterized interactions important for Ras binding to the Raf-CRD. Reconciling conflicting reports, we found that these interactions are essentially independent of the guanine nucleotide bound state, but instead, are enhanced by post-translational modification of Ras. Specifically, our findings indicate that Ras farnesylation is sufficient for stable association of Ras with the Raf-CRD. Furthermore, we have also identified a Raf-CRD variant that is impaired specifically in its interactions with Ras. NMR data also suggests that residues proximal to this mutation site on the Raf-CRD form contacts with Ras. This Raf-CRD mutant impairs the ability of Ras to activate Raf kinase, thereby providing additional support that Ras interactions with the Raf-CRD are important for Ras-mediated activation of Raf-1.

Animals↗
PubMed2000

Analysis of function and regulation of proteins that mediate signal transduction by use of lipid-modified plasma membrane-targeting sequences.

It is now established that the function of many signaling molecules is controlled, in part, by regulation of subcellular localization. For example, the dynamic recruitment of normally cytosolic proteins to the plasma membrane, by activated Ras or activated receptor tyrosine kinases, facilitates their interaction with other membrane-associated components that participate in their full activation (e.g., Raf-1). Therefore, the creation of chimeric proteins that contain lipid-modified signaling sequences that direct membrane localization allows the generation of constitutively activated variants of such proteins. The amino-terminal myristoylation signal sequence of Src family proteins and the carboxy-terminal prenylation signal sequence of Ras proteins have been widely used to achieve this goal. Such membrane-targeted variants have proved to be valuable reagents in the study of the biochemical and biological properties of many signaling molecules.

Amino Acid Sequence↗
PubMed2000

The role of dendritic cells in the innate immune system.

Dendritic cells (DCs) are bone-marrow-derived leucocytes that are specialised antigen-presenting cells capable of stimulating a primary T-lymphocyte response to specific antigen. In this chapter we discuss the role DCs play in the innate response acting as a critical link with the adaptive response and the influence of the innate response on dendritic cells.

Animals↗
PubMed2000

The CMRF-35H gene structure predicts for an independently expressed member of an ITIM/ITAM pair of molecules localized to human chromosome 17.

The CMRF-35 monoclonal antibody recognizes an epitope found on at least two cell surface molecules, differentially expressed by many leukocytes. These molecules, the CMRF-35H (9) and CMRF-35A (CMRF-35) antigens are both members of the immunoglobulin (Ig) superfamily with a single V-like Ig domain. The function of these molecules is unknown, however the presence of putative immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic domain of the CMRF-35H molecule suggests that this molecule may play a regulatory role in leukocyte function. The CMRF-35H and CMRF-35A molecules show several similarities to the family of molecules containing ITIM or immunoreceptor tyrosine-based activatory motifs (ITAM) suggesting that CMRF-35H/CMRF-35A may be new members of this family. This would further indicate that, like other ITIM/ITAM containing molecules, CMRF-35H/CMRF-35A will also play an important role in the immune response. To further characterize these molecules, we have isolated genomic clones for the CMRF-35H gene and determined its intron-exon organization. The gene spans approximately 12 kb and consists of seven exons. Furthermore, this gene has been mapped to chromosome 17 and thus is not linked to the known human ITIM containing genes which map to human chromosome 19 or the recently characterized molecule, NKp44, localized to human chromosome 6.

3' Untranslated Regions↗
PubMed1999

Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis.

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuated 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

Animals↗
PubMed1999

Expression of the RelB transcription factor correlates with the activation of human dendritic cells.

The RelB gene product is a member of the nuclear factor (NF)-kappaB family of transcription factors. It has been identified recently within mouse antigen-presenting cells and human monocyte-derived dendritic cells (DC). Disruption of the mouse RelB gene is accompanied, amongst other phenotypes, by abnormalities in the antigen-presenting cell lineages. In order to define RelB expression during human DC differentiation, we have analysed RelB mRNA by reverse transcriptase-polymerase chain reaction and RelB protein by intracellular staining in CD34+ precursors and different types of DC preparations. RelB mRNA was not detected in CD34+ precursor populations. Fresh blood DC (lineage-human leucocyte antigen-DR+ (lin-HLA-DR+)) lacked RelB mRNA and cytoplasmic RelB protein but a period of in vitro culture induced RelB expression in blood DC. Purified Langerhans' cells (LC) (CD1a+ HLA-DR+) failed to express RelB mRNA. Immunocytochemical staining identified RelB protein in human skin epithelium. RelB protein was expressed in a very few CD1a+, CD83+ or CMRF-44+ dermal DC but was not present in CD1a+ LC. Tonsil DC (lin-HLA-DR+ CMRF-44+) were positive for RelB mRNA and RelB protein. Intestinal DC (HLA-DR+) also lacked immunoreactive RelB protein. The majority of interdigitating CD83+, CMRF-44+, CMRF-56+ or p55+ DC located in paracortical T-lymphocyte areas of lymph node and tonsil contained RelB protein. The expression of RelB mRNA and RelB protein correlates with the activated phase of blood DC and the postmigration cell (activated) stage of tissue DC development.

Antigens, CD34↗
PubMed1998

Increasing complexity of Ras signaling.

The initial discovery that ras genes endowed retroviruses with potent carcinogenic properties and the subsequent determination that mutated ras genes were present in a wide variety of human cancers, prompted a strong suspicion that the growth-promoting actions of mutated Ras proteins contribute to their aberrant regulation of growth stimulatory signaling pathways. In 1993, a remarkable convergence of experimental observations from genetic analyses of Drosophila, S. cerevisiae and C. elegans as well as biochemical and biological studies in mammalian cells came together to define a clear role for Ras in signal transduction. What emerged was an elegant linear signaling pathway where Ras functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases that included the mitogen-activated protein kinases (MAPKs). Activated MAPKs in turn regulated the activities of nuclear transcription factors. Thus, a signaling cascade where every component between the cell surface and the nucleus was defined and conserved in worms, flies and man. This was a remarkable achievement in our efforts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell. However, the identification of this pathway has proven to be just the beginning, rather than the culmination, of our understanding of Ras in signal transduction. Instead, we now appreciate that this simple linear pathway represents but a minor component of a very complex signaling circuitry. Ras signaling has emerged to involve a complex array of signaling pathways, where cross-talk, feedback loops, branch points and multi-component signaling complexes are recurring themes. The simplest concept of a signaling cascade, where each component simply relays the same message to the next, is clearly not the case. In this review, we summarize our current understanding of Ras signal transduction with an emphasis on new complexities associated with the recognition and/or activation of cellular effectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple effectors.

Amino Acid Sequence↗
PubMed1998

cDNA cloning of human DEC-205, a putative antigen-uptake receptor on dendritic cells.

Dendritic cells (DC) are specialist antigen presenting cells which capture antigens in the periphery, migrate centrally, and present the processed antigens in the context of major histocompatibility complex and appropriate co-stimulatory molecules to T lymphocytes for the initiation of an immune response. DEC-205 has been identified as a putative antigen-uptake receptor, which is expressed abundantly on mouse DC. The recently cloned mouse DEC-205 cDNA predicts a molecular structure which has a marked similarity to the macrophage mannose receptor. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA library screening, we obtained the full coding region of human DEC-205 cDNA from the Hodgkin's disease-derived L428 cell line. The predicted protein structure is a type I transmembrane protein of 1722 amino acids consisting of a signal peptide, cysteine-rich domain, fibronectin type II domain, ten carbohydrate recognition-like domains, transmembrane domain, and a cytoplasmic tail. Human DEC-205 is 77% identical to the mouse protein with completely conserved cysteines. The DEC-205 gene (LY75) was mapped to chromosome band 2q24 by somatic cell hybrid panel analysis and fluorescent in situ hybridization. Northern blot analysis detected 7.8 and 9.5 kilobase DEC-205 transcripts in myeloid, B lymphoid, and Hodgkin's disease-derived cell lines. RT-PCR analysis indicated that immature blood DC contain a barely detectable amount of DEC-205 transcripts but these were markedly increased upon differentiation/activation.

Amino Acid Sequence↗
PubMed1998

The CMRF-35 mAb recognizes a second leukocyte membrane molecule with a domain similar to the poly Ig receptor.

The CMRF-35 mAb recognizes an antigen found on most leukocytes including monocytes, neutrophils, macrophages, dendritic cells, and subpopulations of lymphocytes and bone marrow cells. A cDNA expressing the CMRF-35 epitope was isolated by expression cloning and this predicts for a type I cell surface glycoprotein belonging to the Ig superfamily. Here we demonstrate that the CMRF-35 mAb recognizes an epitope more widely distributed on hemopoietic cells and cell lines than suggested by expression analysis of the CMRF-35 mRNA. Furthermore, we have isolated a novel cDNA (CMRF-35-H9) that encodes a protein product also recognized by the CMRF-35 mAb. This cDNA product is a type I cell surface glycoprotein with a single Ig V-like domain. Although the sequences of the extracellular V-like domains of the two molecules are very similar, there is little similarity between the remainder of their sequences. The two transcripts are expressed independently of each other, and their presence accounts for the discrepancy between CMRF-35 mAb binding and mRNA analysis. The cytoplasmic tail of CMRF-35-H9 contains motifs similar to the inhibitory motifs found in some leukocyte surface receptors. Their expression in hemopoietic cells suggests that these two molecules may play distinct but related roles in the regulation of leukocyte function.

Amino Acid Sequence↗
PubMed1997

14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

14-3-3 Proteins↗
PubMed1997

The Ras-related protein Rheb is farnesylated and antagonizes Ras signaling and transformation.

Presently, nothing is known about the function of the Ras-related protein Rheb. Since Rheb shares significant sequence identity with the core effector domains of Ras and KRev-1/Rap1A, it may share functional similarities with these two structurally related, yet functionally distinct, small GTPases. Furthermore, since like Ras, Rheb terminates with a COOH terminus that is likely to signal for farnesylation, it may be a target for the farnesyltransferase inhibitors that block Ras processing and function. To compare Rheb function with those of Ras and KRev-1, we introduced mutations into Rheb that generate constitutively active or dominant negative forms of Ras and Ras-related proteins and were designated Rheb(64L) and Rheb(20N), respectively. Expression of wild type or mutant Rheb did not alter the morphology or growth properties of NIH 3T3 cells. Thus, aberrant Rheb function is distinct from that of Ras and fails to cause cellular transformation. Instead, similar to KRev-1, co-expression of Rheb antagonized oncogenic Ras transformation and signaling. In vitro and in vivo analyses showed that like Ras, Rheb proteins are farnesylated and are sensitive to farnesyltransferase inhibition. Thus, it is possible that Rheb function may be inhibited by farnesyltransferase inhibitors treatment and, consequently, may contribute to the ability of these inhibitors to impair Ras transformation.

3T3 Cells↗