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Biomedical subjects

D A Mitchell

Publications and source records attributed to D A Mitchell.

At least 37 records · Page 2Linked to original sources
PubMed1999

Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides.

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.

Animals↗
PubMed1999

Solid-state fermentation in rotating drum bioreactors: operating variables affect performance through their effects on transport phenomena.

Aspergillus oryzae ACM 4996 was grown on an artificial gel-based substrate and on steamed wheat bran during solid-state fermentations in 18.7 L rotating drum bioreactors. For gel fermentations fungal growth decreased as rotational speed increased, presumably due to increased shear. For wheat bran fermentations fungal growth improved under agitated compared to static culture conditions, due to superior heat and mass transfer. We conclude that the effects of operational variables on the performance of SSF bioreactors are mediated by their effects on transport phenomena such as mixing, shear, heat transfer, and mass transfer within the substrate bed. In addition, the substrate characteristics affect the need for and the rates of these transport processes. Different transport phenomena may be rate limiting with different substrates. This work improves understanding of the effects of bioreactor operation on SSF performance.

Aspergillus oryzae↗
PubMed1999

Cutting edge: C1q protects against the development of glomerulonephritis independently of C3 activation.

C1q-deficient (C1qa-/-) mice develop antinuclear Abs and glomerulonephritis (GN) characterized by multiple apoptotic bodies. To explore the contribution of C3 activation to the induction of spontaneous GN, C1qa-/- mice were crossed with factor B- and C2-deficient (H2-Bf/C2-/-) mice. GN was present in 64% of the 45 C1qa/H2-Bf/C2-/- mice compared with 8% of the 65 H2-Bf/C2-/- mice and none of the 24 wild-type controls. IgG was detected in the glomeruli of diseased C1qa/H2-Bf/C2-/- kidneys. However, glomerular staining for C3 was absent. Increased numbers of glomerular apoptotic bodies were detected in undiseased C1qa/H2-Bf/C2-/- kidneys. These findings support the hypothesis that C1q may play a role in the clearance of apoptotic cells without the necessity for C3 activation and demonstrate that the activation of C3 is not essential for the development of GN in this spontaneous model of lupus-like disease.

Animals↗
PubMed1999

Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.

Animals↗
PubMed1999

Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae.

Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal CaaX motif (C is cysteine, a is any aliphatic residue, X is the carboxy terminal residue). To better understand the relationship between posttranslational processing and the subcellular localization of Ras, a yeast genetic screen was undertaken based on the loss of function of a palmitoylation-dependent RAS2 allele. Mutations were identified in an uncharacterized open reading frame (YLR246w) that we have designated ERF2 and a previously described suppressor of hyperactive Ras, SHR5. ERF2 encodes a 41-kDa protein with four predicted transmembrane (TM) segments and a motif consisting of the amino acids Asp-His-His-Cys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD. Mutations within the DHHC-CRD abolish Erf2 function. Subcellular fractionation and immunolocalization experiments reveal that Erf2 tagged with a triply iterated hemagglutinin epitope is an integral membrane protein that colocalizes with the yeast endoplasmic reticulum marker Kar2. Strains lacking ERF2 are viable, but they have a synthetic growth defect in the absence of RAS2 and partially suppress the heat shock sensitivity resulting from expression of the hyperactive RAS2(V19) allele. Ras2 proteins expressed in an erf2Delta strain have a reduced level of palmitoylation and are partially mislocalized to the vacuole. Based on these observations, we propose that Erf2 is a component of a previously uncharacterized Ras subcellular localization pathway. Putative members of an Erf2 family of proteins have been uncovered in yeast, plant, worm, insect, and mammalian genome databases, suggesting that Erf2 plays a role in Ras localization in all eucaryotes.

Acyltransferases↗
PubMed1998

Validation of a model describing two-dimensional heat transfer during solid-state fermentation in packed bed bioreactors.

A two-dimensional heat transfer model was validated against two experimental studies from the literature which describe the growth of Aspergillus niger during solid-state fermentation in packed bed bioreactors. With the same set of model parameters, the two-dimensional model was able to describe both radial temperature gradients, which dominated in one of the studies, and axial temperature gradients, which dominated in the other study. The sensitivity of the model predictions to the characteristics of the substrate and the microbe were explored. The temperatures reached in the column are most sensitive to parameters which affect the peak heat load, including the substrate packing density, the maximum specific growth rate, and the maximum biomass concentration. Even though the bed is assumed to be aerated with saturated air, the increase in temperature with bed height increases the water-carrying capacity of the air and therefore enables evaporation to contribute significantly to cooling. The model suggests that evaporation can remove as much as 78% of the heat from the bed during times of peak heat generation. Our model provides a tool which can guide the design and operation of packed bed bioreactors. However, further improvements are necessary to do this effectively, the most important of which is the incorporation of a water balance.

Aspergillus niger↗
PubMed1998

The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast.

Several end mutations that block the internalisation step of endocytosis in Saccharomyces cerevisiae also affect the cortical actin cytoskeleton [1]. END5 encodes a proline-rich protein (End5p or verprolin) required for a polarised cortical actin cytoskeleton and endocytosis [2,3]. End5p interacts with actin [4], but its exact function is not yet known. To help elucidate End5p function, we sought other End5p-interacting proteins and identified the LAS17/BEE1 gene (encoding the yeast homologue of the human Wiskott-Aldrich Syndrome protein, WASp) as a high-copy-number suppressor of the temperature-sensitive growth and endocytic defects of end5-1 cells (carrying a frameshift mutation affecting the last 213 residues of End5p). LAS17 is unable to suppress a full deletion of END5 (end5 delta), however, suggesting that the defective End5-1p in end5-1 mutants may be stabilised by Las17p. The amino terminus of Las17p interacts with the carboxyl terminus of End5p in the yeast two-hybrid system and similar interactions have been shown between WASp and a mammalian End5p homologue, WASp-interacting protein (WIP) [5]. As las17 delta deletion mutants are blocked in endocytosis, we conclude that Las17p and End5p interact and are essential for endocytosis.

Carrier Proteins↗
PubMed1998

Dendritic cell/macrophage precursors capture exogenous antigen for MHC class I presentation by dendritic cells.

Presentation of MHC class I antigens by professional antigen-presenting cells (APC) is an important pathway in priming cytotoxic T lymphocyte responses in vivo. This study sought to identify the nature of the professional APC responsible for indirect class I presentation by examining a special feature of professional APC, namely their ability to process exogenous forms of antigen for class I presentation. Incubation of highly purified bone marrow-derived precursor cells with chicken ovalbumin (OVA) led to the efficient presentation of the major class I-restricted OVA determinant by mature dendritic cells (DC), but not by macrophages (Mphi) derived from the precursor population. DC as well as macrophages were, however, able to mediate class II presentation of OVA, suggesting that macrophages were deficient in class I processing but not in capturing exogenous OVA. The majority of mature DC, i.e. over 80 %, generated from the precursor cells pulsed with OVA, presented the class I OVA epitope. Upon maturation, class I presentation of OVA by DC was greatly reduced, suggesting that class I processing of exogenous antigen is modulated during DC maturation in a manner similar to class II antigen processing. This study shows that bone marrow-derived DC/ME progenitors capture exogenous antigen for class I presentation, and that cells of the DC lineage can be functionally distinguished from cells of the macrophage lineage based on their ability to process exogenous antigen for class I presentation.

Amino Acid Sequence↗
PubMed1997

A multicentre audit of unilateral fractures of the mandibular condyle.

OBJECTIVE: To audit a series of isolated unilateral fractures of the mandibular condyle treated in a number of units in the United Kingdom. DESIGN: Prospective confidential multicentre audit with review by a single external examiner between July 1993 and January 1994. SETTING: Nine Maxillofacial units. Pro-formas were alphanumerically coded. Units are referred to by code letter. SUBJECTS: 142 patients with fractures of the mandibular condyle. MAIN OUTCOME MEASURES: Pain, crepitus, interincisal opening, mandibular deviation on movement, occlusion and posteroanterior and lateral radiographic analysis by a single examiner. RESULTS: Of the 142 patients, 135 over the age of 12 years had unilateral condylar fractures. Seventy-three attended appointments at both 6 and 12 weeks. Forty-one had identifiable symptoms attributable to the injury at 12 weeks, and most of these had fracture displacements or dislocations which had not been openly reduced. There was a 50-85% positive correlation between radiographic findings of the first clinician and the external auditor. Open reduction and internal fixation was uncommon. CONCLUSIONS: Follow-up studies of trauma patients are difficult because of poor attendance. Fractures of the mandibular condyle should be treated by specialists in the management of facial fractures. Accurate clinical and radiographic diagnosis is more difficult than commonly supposed. Fracture displacements and dislocations have a suboptimal outcome in nearly 30% of cases. More should be spent to improve care of these patients. A consensus on objective indicators of satisfactory outcome is needed to establish a 'gold standard' and close the audit loop.

Adolescent↗
PubMed1997

Comparative study of the ipsilateral full thickness forearm skin graft in closure of radial forearm flap donor site defects.

The early experience of our unit with the technique of ipsilateral full thickness forearm skin grafting of the radial forearm flap donor site defect is described. The technique provides the advantages of a full thickness skin graft whilst avoiding the need to harvest skin from a remote area. We have compared the results with that of split skin grafts used contemporaneously for the same purpose in our unit; the advantages and disadvantages are outlined. Continued success has encouraged us to utilize this technique routinely with certain modifications which we describe.

Cicatrix↗
PubMed1996

The effects of age, sex and time of testing on skin conductance activity.

An investigation of the electrodermal activity of 640 subjects was carried out in Mauritius. Results of a balanced factorial design with 5 age groups, sex, season (hot versus cold), time of day of testing (a.m./p.m.), as factors, suggested the need to take account of age and sex in studies using measures of electrodermal activity. However, season and time of day of testing are seen as important mainly in interaction with sex and give rise to the suggestion that females may be more responsive to environmental conditions than men. The results also provide data which show that a rate of electrodermal non-responsivity of about 25% appears to be constant over the age groups of 5-25 years used in the study.

Adolescent↗
PubMed1996

Case report. Two contrasting radiological presentations of prostatic adenocarcinoma in the jaws.

The contrasting radiological appearances of metastatic deposits in the mandible of prostatic adenocarcinoma in two patients are described. The clinical presentation was similar in that both presented with altered sensation of the lower lip. Radiologically, they differed in that one patient suffered from a large predominantly osteoblastic mass, while the other, who gave a history of previously treated prostatic adenocarcinoma, presented with a rather small osteolytic deposit. Investigations for bony metastatic disease usually include a bone scan which is a highly sensitive technique although non-specific. A skeletal survey can be useful although less sensitive than a bone scan. Blood investigations such as acid phosphatase and prostate specific antigen levels are also indicated in male patients where prostatic disease is suspected. Reasonable long term survival using relatively simple drug therapy without significant local surgery, highlights the need for accurate recognition and tissue diagnosis to differentiate this condition from osseous malignancy of the jaws, other metastatic disease or osteomyelitis.

Adenocarcinoma↗
PubMed1995

Farnesylation and proteolysis are sequential, but distinct steps in the CaaX box modification pathway.

Membrane localization of Ras proteins requires posttranslational modification of a conserved C-terminal sequence motif known as the CaaX box (C is Cys, a is any aliphatic amino acid, and X is the carboxyl terminal residue). The modification steps include farnesylation, removal of the three C-terminal amino acids, carboxyl-methylation, and palmitoylation. In yeast, the farnesyltransferase (FTase) is encoded by the RAM1(DPR1) and RAM2 genes, and the methyltransferase is the product of STE14. The gene encoding the protease(s) that is responsible for modification of the CaaX has not been identified. We have used in vitro-synthesized Ras2p and synthetic peptide substrates to investigate the relationship between farnesylation and proteolysis. Addition of yeast cytosolic extracts to rabbit reticulocyte extracts programmed to synthesize Ras2p led to prenylation of Ras2p and a change in electrophoretic mobility similar to that observed during Ras maturation in vivo. However, it was not possible to determine if the mobility shift is the result of prenylation, proteolysis or a combination of both steps. Therefore, we examined the relationship between farnesylation and proteolysis directly using extracts prepared from bacteria overexpressing the genes for the yeast FTase (RAM1 and RAM2) and synthetic CaaX box peptides. Extracts from bacteria expressing RAM1/RAM2 efficiently prenylate CaaX box peptides, but do not proteolyze the -aaX residues. However, addition of yeast extracts from wild type, ram1, or ste14 mutants resulted in the removal of the -aaX residues from prenylated CaaX box peptides.

Alkyl and Aryl Transferases↗