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Biomedical subjects

A D Cox

Publications and source records attributed to A D Cox.

At least 73 records · Page 4Linked to original sources
PubMed1994

Characteristics of a novel low affinity complement C3dg-binding protein of human platelets.

The breakdown products of the complement protein C3 function in receptor-mediated immune clearance. The catabolism of the C3 molecule, mediated by factors I and H, results in the generation of the fragments C3c and C3dg. C3dg binds to human platelets in a specific and saturable manner. The direct interaction of platelets with soluble C3dg may contribute to immune-mediated platelet destruction. More importantly, platelets may interact with opsonized pathogens or complement-activating immune complexes via C3dg. In this report, we investigated the interaction of C3dg with platelets and calculated the Ka to be 3.2 x 10(6) M-1 with 1100 to 2200 specific binding sites/platelet. In the presence of 5 mM calcium, both the Ka and the number of specific binding sites were modestly decreased to Ka = 2.8 x 10(6) M-1 with 1400 to 2400 specific binding sites/platelet. The Scatchard plots demonstrated a curvilinear character. On labeling C3dg with peroxidase and visualizing platelet-bound C3dg by electron microscopy, it was shown that binding sites for C3dg were restricted to the platelet plasma membrane. Using a cell attachment assay, platelet adhesion to C3dg was readily detectable; attachment to C3dg-coated plates was not blocked by fibrinogen or fibronectin. We have characterized the C3dg-binding protein of platelets using the chemical cross-linkers, SASD and DSS, to cross-link C3dg to thrombin-stimulated platelets. Gel filtration of the 125I-labeled C3dg-platelet complex revealed the presence of a large protein complex that was absent when 125I-labeled C3dg alone was analyzed. SDS-PAGE of the radiolabeled cross-linked protein, followed by autoradiography, identified a 95-kDa membrane protein. The relationship of this C3dg-binding protein to other platelet membrane proteins has yet to be determined.

Blood Platelets↗
PubMed1994

Transfer RNA profiling: a new method for the identification of pathogenic Candida species.

A new molecular taxonomic method applicable to the identification of medically important Candida species and other yeast species has been developed. It is based on the electrophoretic pattern of total tRNA samples (a 'tRNA profile') isolated from Candida species and generated using high-resolution semi-denaturing urea-polyacrylamide gel electrophoresis and methylene blue staining. Species-specific tRNA profiles for the species C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii, C. glabrata and Pichia guilliermondii were obtained. Detailed studies with the major human pathogen of the Candida genus, C. albicans, demonstrated that the tRNA profile for a given species was both reproducible and strain-independent; seven different C. albicans strains generated identical tRNA profiles. Minor strain-specific heterogeneities in the tRNA profiles of C. guilliermondii and C. parapsilosis were detected, but in neither case did they significantly alter the species-specific diagnostic tRNA profile. The potential of this method in clarifying taxonomic anomalies was demonstrated by the finding that Type I and Type II strains of C. stellatoidea generate very different tRNA profiles, with that of a Type II strain being identical to the C. albicans tRNA profile. This method offers a number of advantages over current electrophoretic karyotype methods for species identification, both within the Candida genus and with yeast species in general.

Candida↗
PubMed1994

Transcriptional activation analysis of oncogene function.

Because no single assay provides a complete analysis of the transformed phenotype, transactivation assays complement the cell growth and tumorigenicity analyses of oncogene function. Transactivation of ORE-containing genes is such a common feature of a diverse variety of viral and cellular oncogenes that it can be considered one aspect of the oncogene-induced phenotype. After the initial identification of oncogenes that activate transcription, studies of the mechanisms of activation and the identification of the downstream target genes should lead to a better understanding of the events leading to cellular transformation. The fact that cell type specificity of transactivation and transformation can be similar means that the transactivation assay may be a useful tool in dissecting cell type-specific transformation. The transactivation assay of oncogene function also has the advantage that it is easy to perform and significantly more rapid than assays based on altered cell growth. This is of particular advantage when one wishes to examine the function of a large number of oncogene mutants generated in vitro. Overall, transactivation assays provide another tool for examining transforming potential and a starting point for the analysis of the downstream targets of oncogenes.

3T3 Cells↗
PubMed1994

Biological assays for cellular transformation.

A number of standard and widely applied procedures have been used to determine whether expression of a particular gene triggers the growth alterations that are characteristic of most oncogenes. The assays have been used extensively to evaluate the transforming potential of a wide variety of genes that encode tyrosine or serine/threonine kinases, small and heterotrimeric GTP-binding signal transduction regulators, and nuclear transcription factors, among others. Therefore, the growth-promoting characteristics of a particular gene can be compared with the properties of other genes that have been characterized by the same assays. The assays described do not represent a complete evaluation of the transforming activity of a gene. Failure to detect growth-promoting activity in any of the assays does not definitively eliminate the possibility that a particular gene is an oncogene. Specialized assays that use (nonfibroblast) recipient cells more closely approximating the likely environment of the gene of interest may provide better approaches for subsequent studies. Other biological assays for transforming potential include measurements of the adhesion properties of cells on different substrata, the ability to grow on confluent monolayers of normal cells, the ability to invade into various artificial tissue matrices, and transgenic animal models. Finally, more specific assays for biochemical alterations that reflect the transformed state can also be employed. For example, as discussed in [23] in this volume, one widely used biochemical measure of transforming potential employs transcriptional activation of genes whose promoters contain so-called oncogene-responsive elements. This, as well as other biochemical assays, can be applied to complement the biological studies described in this chapter.

3T3 Cells↗
PubMed1994

ADP causes partial degranulation of platelets in the absence of aggregation.

Whole blood flow cytometry has revealed that platelets undergo partial degranulation in response to ADP, in the absence of aggregation, as evidenced by the expression of the P-selectin and CD63 antigens of the alpha-granule and lysosomal membranes respectively. With maximum ADP (10(-5) M) fibrinogen bound to 76.1 +/- 7.2% of platelets but P-selectin and CD63 antigen were expressed on 26.9 +/- 9.8% and 8.6 +/- 3.5% of platelets respectively. Maximum fibrinogen binding, P-selectin and CD63 expression induced by alpha-thrombin were 96.1 +/- 1.4%, 92.8 +/- 2.3% and 77.6 +/- 9.7% respectively. beta-thromboglobulin release from the ADP-stimulated platelets correlated closely with the expression of P-selectin and CD63 (r = 0.98 +/- 0.02 for both antigens). No platelet aggregates were seen by flow cytometry and the absence of aggregation was confirmed by single cell counting. Addition of the GPIIb-IIIa antagonist echistatin, at concentrations that totally blocked fibrinogen binding to ADP-stimulated platelets, had no effect on the expression of the granule membrane antigens. The partial degranulation of normal platelets was independent of thrombin generation since it was not inhibited by hirudin (5 units/ml). In conclusion, ADP is capable of causing partial degranulation of platelets independently of aggregation, fibrinogen binding or thrombin generation. Thus release of potent procoagulant, vasoactive and mitogenic substances from the platelets could continue in the presence of thrombin inhibitors and GPIIb-IIIa antagonists.

Adenosine Diphosphate↗
PubMed1994

Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation.

Although the human Ras proteins are members of a large superfamily of Ras-related proteins, to date, only the proteins encoded by the three mammalian ras genes have been found to possess oncogenic potential. Among the known Ras-related proteins, TC21/R-Ras2 exhibits the most significant amino acid identity (55%) to Ras proteins. We have generated mutant forms of TC21 that possess amino acid substitutions analogous to those that activate Ras oncogenic potential [designated TC21(22V) and TC21(71L)] and compared the biological properties of TC21 with those of Ras proteins in NIH 3T3 and Rat-1 transformation assays. Whereas wild-type TC21 did not show any transforming potential in vitro, both TC21(22V) and TC21(71L) displayed surprisingly potent transforming activities that were comparable to the strong transforming activity of oncogenic Ras proteins. Like Ras-transformed cells, NIH 3T3 cells expressing mutant TC21 proteins formed foci of morphologically transformed cells in monolayer cultures, proliferated in low serum, formed colonies in soft agar, and developed progressive tumors in nude mice. Thus, TC21 is the first Ras-related protein to exhibit potent transforming activity equivalent to that of Ras. Furthermore, mutant TC21 proteins also stimulated constitutive activation of mitogen-activated protein kinases as well as transcriptional activation from Ras-responsive promoter elements (Ets/AP-1 and NF-kappa B). We conclude that aberrant TC21 function may trigger cellular transformation via a signal transduction pathway similar to that of oncogenic Ras and suggest that deregulated TC21 activity may contribute significantly to human oncogenesis.

3T3 Cells↗
PubMed1994

R-Ras induces malignant, but not morphologic, transformation of NIH3T3 cells.

Although previous studies have not identified transforming properties of the Ras-related protein R-Ras, two recent observations have prompted our further evaluation of R-Ras function. First, we observed that mutant forms of the closely related R-Ras2/TC21 protein (approximately 70% identity) exhibited the same potent transforming activity as oncogenic Ras proteins. Second, R-Ras association with Bcl-2 suggested a possible role for R-Ras in apoptotic growth control. Therefore, we have performed a detailed analysis of R-Ras transforming potential in NIH3T3 cells. Whereas expression of a mutant R-Ras protein (38V; analogous to the 12V activating Ras mutation) did not induce morphologic transformation of NIH3T3 cells, R-Ras(38V)-expressing cells proliferated in low serum, formed colonies in soft agar, and formed progressive tumors in nude mice. Like Ras-transformed cells, R-Ras(38V)-transformed cells exhibited constitutively activated mitogen activated protein kinases. Furthermore, R-Ras(38V) stimulated transcriptional activation of Ras-responsive promoter elements, and this activity (and transformation) was blocked by Raf dominant negative proteins. Finally, whereas co-expression of Bcl-2 did not cause significant alteration in wild type or mutant R-Ras transforming activity, coexpression of v-Myc and R-Ras(38V) induced a striking morphologic transformation of NIH3T3 cells. Taken together, these observations suggest that aberrant R-Ras function may stimulate malignant transformation, in the absence of morphologic transformation, via up-regulation of part of the Ras signal transduction pathway.

3T3 Cells↗
PubMed1993

The carboxyl-terminal CXXX sequence of Gi alpha, but not Rab5 or Rab11, supports Ras processing and transforming activity.

Although the heterotrimeric Gi alpha subunit terminates in an apparent CXXX prenylation signal (CGLF), it is not modified by isoprenylation. To determine if the Gi alpha CXXX sequence can signal prenylation when placed at the carboxyl termini of normally prenylated proteins, we have characterized the processing and biological activity of chimeric oncogenic Ras proteins that terminate in the Gi alpha CXXX sequence (Ras/Gi alpha). Surprisingly, these chimeras were prenylated both in vivo and in vitro, demonstrated significant membrane association, exhibited transforming activity, and induced transcriptional transactivation from Ras-responsive elements. We then extended these studies to determine if, unlike the CC or CXC carboxyl-terminal sequences of other Rab proteins, the carboxyl-terminal CXXX sequences of the Ras-related Rab5 and Rab11 proteins represent conventional CXXX prenylation signals that can support Ras processing and transforming activity. Unexpectedly, these Ras/Rab chimeras were nonprenylated, were cytosolic, and lacked detectable transforming or transcriptional transactivation activity. Taken together, these results suggest that the context within which a CXXX sequence occurs may also critically control the modification of a protein by prenylation, and that the Rab5 and Rab11 carboxyl termini do not possess conventional CXXX sequences. Instead, their CCXX and CCXXX motifs may represent additional classes of protein prenylation signals.

3T3 Cells↗
PubMed1993

Befriending young mothers.

The context of voluntary befriending schemes to help mothers of young children is outlined. There is evidence that two main UK voluntary befriending schemes to help mothers of young children engage families where there is significant psychosocial morbidity. It is concluded that well conducted befriending schemes can make a significant contribution to the mental health of mothers and children. Whether they do so will depend on their experience and training and the elements they incorporate.

Adolescent↗
PubMed1992

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.

3T3 Cells↗
PubMed1992

Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity.

We have introduced a variety of amino acid substitutions into carboxyl-terminal CA1A2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic [Val12]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the A1 position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (approximately 50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.

3T3 Cells↗
PubMed1992

Exploration of doctor and patient agendas in general practice consultations.

A method for the interactional analysis of doctor/patient consultations is described and applied to six naturally occurring general practice interviews by three raters. The method is reliable given sufficient training and satisfies the stringent criteria for any method of analysis for medical interviews proposed by Wasserman and Inui [1: Wasserman R.C. and Inui R.S. Med. Care 21, 279, 1983].

Communication↗
PubMed1992

Protein prenylation: more than just glue?

As with other lipid modifications of proteins, prenylation now appears to be critically important in the regulation of protein function. Recent research has led to an explosion of information concerning prenylation signals, prenyl transferase enzymes and the role of prenylation in protein-membrane interactions. Experiments have examined the role of prenylation in protein function and the results suggest that protein prenylation may be involved in facilitating proper subcellular localization, promoting protein-protein and protein-membrane interactions and regulating protein function.

Amino Acid Sequence↗
PubMed1992

Principle agendas of doctors and patients in general practice consultations.

Seventy-three general practice interviews were analysed according to the method described by Butler et al. Results for the three principal agenda types (physical, emotional and social) are presented in terms of the control exerted by either doctor or patient in determining the content of the interview. Both doctor and patient address physical agendas to a similar high degree. In contrast, patients present emotional agendas to a far greater extent than doctors address these concerns. The findings for social agendas are intermediate between those for physical and those for emotional agendas. It is argued that doctors can facilitate the expression of emotional and social agendas by giving explicit or implicit permission for their presence in the interview.

Attitude of Health Personnel↗
PubMed1992

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

3T3 Cells↗