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Biomedical subjects

R Howes

Publications and source records attributed to R Howes.

15 recordsLinked to original sources
PubMed2001

Demonstration of biofilm in infectious crystalline keratopathy using ruthenium red and electron microscopy.

OBJECTIVE: Bacterial biofilm formation has been implicated in the pathogenesis of infectious crystalline keratopathy. Biofilm cannot be visualized by electron microscopy without the addition of a fixative that stabilizes the polysaccharide-rich bacterial extracellular matrix that surrounds the bacterial colonies in a biofilm. We used ruthenium red as a fixative to evaluate corneal biopsy specimens for the presence of bacterial biofilm in three cases of infectious crystalline keratopathy (ICK) and five cases of chronic microbial keratitis without crystalline changes. DESIGN: Case series with clinicopathologic correlation. PARTICIPANTS: Eight patients underwent corneal biopsy or therapeutic keratoplasty as part of their management for chronic unresponsive microbial keratitis. METHODS: The corneal specimens removed were trisected for microbiology, pathology, and transmission electron microscopy (TEM). The TEM specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.05% ruthenium red. MAIN OUTCOME MEASURES: Demonstration of bacterial biofilm with TEM. RESULTS: TEM demonstrated organisms with a surrounding extracellular matrix consistent with a bacterial biofilm in the three cases of ICK but not in the five other cases of chronic microbial keratitis. CONCLUSIONS: The presence of biofilm in ICK can be demonstrated with TEM with appropriate fixation techniques that stabilize the bacterial extracellular matrix. Biofilm stains intensely with periodic acid-Schiff because of the polysaccharide-rich extracellular matrix and weakly with Gram stain because of the high proportion of nonviable organisms. Biofilm formation occurs in ICK but probably not in chronic bacterial keratitis without crystalline changes. Secretion of an extracellular matrix by bacteria to form a biofilm is a response to a nutrient-deprived environment in which growth and replication is depressed. The extracellular matrix of the biofilm may mask bacterial antigens, explaining the relative lack of inflammatory response in these infections. It may also be one of the mechanisms explaining the resistance to in vivo antimicrobial therapy when in vitro sensitivities have been proven.

Aged↗
PubMed2000

Pattern formation: Wingless on the move.

Wingless is a key morphogen in Drosophila. Although it is evident that Wingless acts at a distance from its site of synthesis, there is considerable debate about how the protein travels across a field of cells. Recent studies have provided important new insights into this process, though the issue is still far from being resolved.

Animals↗
PubMed1999

Role of the EGF receptor pathway in growth and patterning of the Drosophila wing through the regulation of vestigial.

Growth and patterning of the Drosophila wing disc depends on the coordinated expression of the key regulatory gene vestigial both in the Dorsal-Ventral (D/V) boundary cells and in the wing pouch. We propose that a short-range signal originating from the core of the D/V boundary cells is responsible for activating EGFR in a zone of organizing cells on the edges of the D/V boundary. Using loss-of-function mutations and ectopic expression studies, we show that EGFR signaling is essential for vestigial transcription in these cells and for making them competent to undergo subsequent vestigial-mediated proliferation within the wing pouch.

Animals↗
PubMed1998

In vivo analysis of Argos structure-function. Sequence requirements for inhibition of the Drosophila epidermal growth factor receptor.

The Drosophila Argos protein is the only known extracellular inhibitor of the epidermal growth factor receptor (EGFR). It is structurally related to the activating ligands, in that it is a secreted protein with a single epidermal growth factor (EGF) domain. To understand the mechanism of Argos inhibition, we have investigated which regions of the protein are essential. A series of deletions were made and tested in vivo; furthermore, by analyzing chimeric proteins between Argos and the activating ligand, Spitz (a transforming growth factor-alpha-like factor), we have examined what makes one inhibitory and the other activating. Our results reveal that Argos has structural requirements that differ from all known EGFR activating ligands; domains flanking the EGF domain are essential for its function. We have also defined the important regions of the atypical Argos EGF domain. The extended B-loop is necessary, whereas the C-loop can be replaced with the equivalent Spitz region without substantially affecting Argos function. Comparison of the argos genes from Drosophila melanogaster and the housefly, Musca domestica, supports our structure-function analysis. These studies are a prerequisite for understanding how Argos inhibits the Drosophila EGFR and provide a basis for designing mammalian EGFR inhibitors.

Amino Acid Sequence↗
PubMed1995

Inhibition of Drosophila EGF receptor activation by the secreted protein Argos.

The Drosophila homologue of the mammalian epidermal growth factor (EGF) receptor (DER) is a receptor tyrosine kinase involved in many stages of fly development, including photoreceptor determination, and wing-vein formation. Its primary activating ligand is the Spitz protein, which is similar to mammalian TGF-alpha. Argos is a secreted protein that, like Spitz, contains a single EGF motif. It is a repressor of cell determination in the eye, and acts in other tissues, including the wing. Because Argos has the opposite effects to DER in the eye (the former blocks photoreceptor determination, the latter promotes it) we have tested whether it acts by blocking the DER pathway. We show that Argos does indeed repress this pathway in vivo and find that, in vitro, Argos protein can inhibit the activation of DER by Spitz. Thus the determination of cells by the DER pathway is regulated by a balance between extracellular activating and inhibiting signals. This is the first in vivo example of an extracellular inhibitor of a receptor tyrosine kinase.

Animals↗
PubMed1994

Pyruvate dehydrogenase deficiency. Clinical presentation and molecular genetic characterization of five new patients.

Fibroblast cultures from five patients with early onset severe encephalopathy and lactic acidosis were studied for evidence of pyruvate dehydrogenase (PDH) deficiency. Three males had significantly reduced activity (0.29-0.45 nmol/mg protein/min versus normal controls 0.7-1.1 nmol/mg protein/min); two females had PDH activity within the normal range. However, as the majority of cases of PDH deficiency result from defects in the X-linked E1 alpha subunit and both females had biased patterns of X-inactivation (making it impossible to rule out the possibility that they were heterozygous for an E1 alpha gene defect) molecular genetic studies were performed. cDNA from the male patients was sequenced and mis-sense mutations found: Y243N (T-->A) in exon 7, D315A (G-->A) in exon 10 and R378H (G-->A) in exon 11. Single-strand conformation polymorphism analysis of amplified genomic DNA fragments and sequencing revealed a mis-sense mutation M282L (A-->C) in one female and a frameshift mutation caused by insertion of T (R288ins) in the other. Adding to recent descriptions of new mutations, this report emphasizes the allelic heterogeneity of the condition. The identification of mutations in females with a suggestive clinical phenotype, even when peripheral fibroblasts do not show deficient PDH activity, illustrates the importance of molecular analysis of this disease.

Base Sequence↗
PubMed1994

The blood-retinal barrier in experimental autoimmune uveoretinitis. Leukocyte interactions and functional damage.

BACKGROUND: In posterior uveitis the blood-retinal barrier (BRB) plays an important role in the pathogenesis of the disease. However, the morphologic correlate of BRB breakdown and the route of leukocyte migration remain poorly defined. EXPERIMENTAL DESIGN: Using an experimental model of autoimmune uveoretinitis in the rat, we have examined the ultrastructural alterations and leukocyte interactions occurring at the BRB. By employing electron-dense tracers, the development of BRB breakdown, and the route of extravasation were investigated. RESULTS: No increase in BRB permeability was found before lymphocytic infiltration. At day 10 postimmunization with retinal-soluble antigen and beyond, inflammatory cells could be seen within the retina that was quickly followed by an extensive increase in the permeability of the retinal vasculature to lanthanum and horseradish peroxidase. Occasionally, horseradish peroxidase reaction product could be seen extending throughout the 'tight junctions' of the retinal endothelia, but not those of the retinal pigment epithelia. Inflammatory cells, particularly mononuclear cells, were seen forming perivascular cuffs and extending posteriorly towards the outer retina. Retinal damage was initially restricted to the outer nuclear and photoreceptor layers that were in close proximity to these vessels. Leukocytes could be seen adhering to the retinal vessels and penetrating the endothelial cell cytoplasm close to tight junctions, but were never seen probing the junctions directly. At the retinal pigment epithelium, however, there was little evidence of migration into the retina during the early stages of the disease, even though the choroid often became packed with inflammatory cells. At later stages, occasional inflammatory cells could be seen between, and apparently within, retinal pigment epithelium cells in areas overlying sites of severe choroidal infiltration. CONCLUSIONS: The prime site of leukocyte infiltration and damage to the BRB in autoimmune uveoretinitis occurred at the level of the vascular endothelia and that diapedesis takes place primarily via an intraendothelial process.

Animals↗
PubMed1988

Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation.

Subcutaneous implantation of demineralized bone matrix induces the local formation of cartilage and bone. In this study we have investigated the influence of adding various growth factors to the implant. Cartilage formation was monitored by measuring collagen II mRNA levels, and bone formation in the implant was assessed from alkaline phosphatase activity and calcium content. Supplements of the platelet-derived growth factor to implants in older rats increased and production of mRNA for collagen II, alkaline phosphatase activity, and the calcium content of the implant, whereas the other growth factors tested were without effect. The data suggest that under some conditions bone induction is submaximal and can be increased by local supplement of platelet-derived growth factor (PDGF). The present observations may have important therapeutic implications in the treatment of nonunions of fractures and impaired bone formation in the aged.

Animals↗
PubMed1978

Lateral subluxation of the atlanto-axial joint in rheumatoid arthritis.

Involvement of the cervical spine by rheumatoid disease is common, but lateral subluxation at the atlanto-axial level has not been recorded previously. The condition is due to asymmetrical erosion of the lateral atlanto-axial facet joint, and may be complicated by collapse of the lateral mass of the axis. The condition should be suspected in patients with rheumatoid arthritis (RA) who present with occipital, auricular, and/or facial pain.

Adult↗