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Biomedical subjects

C J Murphy

Publications and source records attributed to C J Murphy.

At least 19 recordsLinked to original sources
PubMed2006

Characterizing a rat Brca2 knockout model.

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2-/- rats are 100% viable and the vast majority live to over 1 year of age. Brca2-/- rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2-/- rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

Animals↗
PubMed2006

The scale of substratum topographic features modulates proliferation of corneal epithelial cells and corneal fibroblasts.

The cornea is a complex tissue composed of different cell types, including corneal epithelial cells and keratocytes. Each of these cell types are directly exposed to rich nanoscale topography from the basement membrane or surrounding extracellular matrix. Nanoscale topography has been shown to influence cell behaviors, including orientation, alignment, differentiation, migration, and proliferation. We investigated whether proliferation of SV40-transformed human corneal epithelial cells (SV40-HCECs), primary human corneal epithelial cells (HCECs), and primary corneal fibroblasts is influenced by the scale of topographic features of the substratum. Using basement membrane feature sizes as our guide and the known dimensions of collagen fibrils of the corneal stroma (20-60 nm), we fabricated polyurethane molded substrates, which contain anisotropic feature sizes ranging from 200-2000 nm on pitches ranging from 400 to 4000 nm (pitch = ridge width + groove width). The planar regions separating each of the six patterned regions served as control surfaces. Primary corneal and SV40-HCEC proliferation decreased in direct response to decreasing nanoscale topographies down to 200 nm. In contrast to corneal epithelial cells, corneal fibroblasts did not exhibit significantly different response to any of the topographies when compared with planar controls at 5 days. However, decreased proliferation was observed on the smallest feature sizes after 14 days in culture. Results from these experiments are relevant in understanding the potential mechanisms involved in the control of proliferation and differentiation of cells within the cornea.

Biocompatible Materials↗
PubMed2006

The in vitro activity of selected defensins against an isolate of Pseudomonas in the presence of human tears.

BACKGROUND/AIMS: Pseudomonas aeruginosa is a major cause of severe bacterial keratitis and remains a difficult clinical entity to treat successfully with the current arsenal of antimicrobial agents. Defensins are small cationic peptides with broad in vitro antimicrobial activity and are potential ocular therapeutic agents. The authors characterised the in vitro activity of defensins NP-1 and NP-3a against P aeruginosa in the presence of human tears. METHODS: A clinical Pseudomonas isolate was grown to mid-log phase, and 1 x 10(6 )colony forming units were exposed to the peptides (200 microg/ml) for up to 2 hours in the presence of varying concentrations (10-70%) of human tears. RESULTS: For both peptides in the presence of 10% tears, >3 log units of killing was achieved within 30 minutes. In 70% tears, NP-1 produced >1 log unit of killing at 2 hours, indicating that, although reduced, its activity remained significant. In 20% tears, NP-3a demonstrated 2 log units of killing at 2 hours; however, the antimicrobial activity of this defensin was completely inhibited in the presence of 70% tears. CONCLUSION: These in vitro data suggest that while the microbicidal activity of some defensins may be diminished at the ocular surface in vivo, significant activity is still possible with certain peptides.

Animals↗
PubMed2005

Nanoscale topography modulates corneal epithelial cell migration.

The purpose of this study was to evaluate the effect of surface topographic features that mimic the corneal epithelial basement membrane on cell migration. We used electron-beam and X-ray lithography and reactive ion etching to pattern silicon wafers with pitches (groove width plus ridge width) of nano- and microscale dimensions (pitches ranged from 400 to 4000 nm). Additionally, polyurethane patterned surfaces were created by replication molding techniques to allow for real-time imaging of migrating cells. Individual SV40-transformed human corneal epithelial cells frequently aligned with respect to the underlying surface patterns and migrated almost exclusively along grooves and ridges of all pitches. Direction of migration of individual cells on smooth surfaces was random. In cell dispersion assays, colonies of cells migrated out from initially circular zones predominantly along grooves and ridges, although there was some migration perpendicular to the ridges. On smooth surfaces, cells migrated radially, equally in all directions, maintaining circular colony shapes. We conclude that substratum features resembling the native basement membrane modulate corneal epithelial cell migration. These findings have relevance to the maintenance of corneal homeostasis and wound healing, as well as to the evolution of strategies in tissue engineering, corneal prosthesis development, and cell culture material fabrication.

Cell Line, Transformed↗
PubMed2005

Protective ocular mechanisms in woodpeckers.

AIMS: Woodpeckers possess mechanisms protecting the eye from shaking/impact. Mechanisms available to woodpeckers but not humans may help explain some eye injuries in Shaken Baby syndrome (SBS). METHODS: Gross dissection and histologic examination of eyes and orbits of seven woodpeckers. RESULTS: All birds showed restricted axial globe movement due to the tight fit within the orbit and fascial connections between the orbital rim and sclera. The sclera was reinforced with cartilage and bone, the optic nerve lacked redundancy, and the vitreous lacked attachments to the posterior pole retina. CONCLUSIONS: Woodpecker eyes differ from human infants by an inability of the globe to move axially in the orbit, the sclera to deform, and the vitreous to shear the retina. These findings support current hypotheses that abusive acceleration-deceleration-induced ocular injury in human infants may be related to translation of vitreous within the globe and the globe within the orbit. The woodpecker presents a natural model resistant to mechanical forces that have some similarity to SBS.

Animals↗
PubMed2005

Thermal decomposition of generation-4 polyamidoamine dendrimer films: decomposition catalyzed by dendrimer-encapsulated Pt particles.

The thermal decomposition of hydroxyl-terminated generation-4 polyamidoamine dendrimer (G4OH) films deposited on Au surfaces has been compared with decomposition of the same dendrimer encapsulating an approximately 40-atom Pt particle (Pt-G4OH). Infrared absorption reflection spectroscopy studies showed that, when the films were heated in air to various temperatures up to 275 degrees C, the disappearance of the amide vibrational modes occurred at lower temperature for the Pt-G4OH film. Dendrimer decomposition was also investigated by thermogravimetric analysis (TGA) in both air and argon atmospheres. For the G4OH dendrimer, complete decomposition was achieved in air at 500 degrees C, while decomposition of the Pt-G4OH dendrimer was completed at 400 degrees C, leaving only platinum metal behind. In a nonoxidizing argon atmosphere, a greater fraction of the G4OH decomposed below 300 degrees C, but all of the dendrimer fragments were not removed until heating above 550 degrees C. In contrast, Pt-G4OH decomposition in argon was similar to that in air, except that decomposition occurred at temperatures approximately 15 degrees C higher. Thermal decomposition of the dendrimer films on Au surfaces was also studied by temperature programmed desorption (TPD) and X-ray photoelectron spectroscopy (XPS) under ultrahigh vacuum conditions. Heating the G4OH films to 250 degrees C during the TPD experiment induced the desorption of large dendrimer fragments at 55, 72, 84, 97, 127, 146, and 261 amu. For the Pt-G4OH films, mass fragments above 98 amu were not observed at any temperature, but much greater intensities for H(2) desorption were detected compared to that of the G4OH film. XPS studies of the G4OH films demonstrated that significant bond breaking in the dendrimer did not occur until temperatures above 250 degrees C and heating to 450 degrees C caused dissociation of C=O, C-O, and C-N bonds. For the Pt-G4OH dendrimer films, carbon-oxygen and carbon-nitrogen bond scission was observed at room temperature, and further decomposition to atomic species occurred after heating to 450 degrees C. All of these results are consistent with the fact that the Pt particles inside the G4OH dendrimer catalyze thermal decomposition, allowing dendrimer decomposition to occur at lower temperatures. However, the Pt particles also catalyze bond scission within the dendrimer fragments so that decomposition of the dendrimer to gaseous hydrogen is the dominant reaction pathway compared to desorption of the larger dendrimer fragments observed in the absence of Pt particles.

Journal Article↗
PubMed2001

Cloning, expression, and function of BLAME, a novel member of the CD2 family.

The CD2 family is a growing family of Ig domain-containing cell surface proteins involved in lymphocyte activation. Here we describe the cloning and expression analysis of a novel member of this family, B lymphocyte activator macrophage expressed (BLAME). BLAME shares the structural features of the CD2 family containing an IgV and IgC2 domain and clusters with the other family members on chromosome 1q21. Quantitative PCR and Northern blot analysis show BLAME to be expressed in lymphoid tissue and, more specifically, in some populations of professional APCs, activated monocytes, and DCS: Retroviral forced expression of BLAME in hematopoietic cells of transplanted mice showed an increase in B1 cells in the peripheral blood, spleen, lymph nodes, and, most strikingly, in the peritoneal cavity. These cells do not express CD5 and are CD23(low)Mac1(low), characteristics of the B1b subset. BLAME may therefore play a role in B lineage commitment and/or modulation of signal through the B cell receptor.

Amino Acid Sequence↗
PubMed2001

Oligonucleotide-directed assembly of materials: defined oligomers.

A nano-architectural system that has high variability while maintaining component specificity is described. Tetraphenylcyclobutadiene(cyclopentadienyl)cobalt complexes and phenyleneethynylene trimers were synthesized and subsequently modified with oligonucleotides utilizing standard phosphoramidite chemistry. The resulting oligonucleotide modified organics (OMOs) were characterized by UV-vis spectroscopy, fluorescence spectroscopy, and phosphate analysis. Hybridization of these OMOs resulted in a series of self-assembled oligomeric hybrids of varying length and topology. These hybrids were characterized by melting temperature, polyacrylamide gel electrophoresis, and fluorescence spectroscopy. This model system demonstrates the power of DNA to self-assemble modules of interest-independent of the module itself.

DNA↗
PubMed2001

Refractive state and accommodation in the eyes of free-swimming versus restrained juvenile lemon sharks (Negaprion brevirostris).

Optical measurements of the refractive state of the eyes of various shark species typically have depicted sharks as hyperopic (far-sighted) with little evidence of accommodation (i.e. the ability to change focus for visualizing objects at different distances from the eye). In this study, we used infrared video retinoscopy to measure the refractive state in juvenile lemon sharks (Negaprion brevirostris). This technique allows dynamic measurement of refractive state in free-swimming animals as they pass by an aquarium window. We found that unrestrained lemon sharks are focused emmetropically relative to a 1-m distant photorefractor for the lateral visual field. However, when restrained either right side up or upside down (the latter inducing tonic immobility), the sharks become increasingly hyperopic, an artifact also reported in some other vertebrates. In addition, unrestrained lemon sharks display small amplitude accommodative excursions. Thus, refractive state measurements on restrained sharks in general may not reflect the natural, resting state of the shark eye, but rather, an induced hyperopia and lack of accommodative function. Such an artifact may be present in other vertebrate species, underscoring the need to obtain measurements of refractive state in unrestrained animals.

Accommodation, Ocular↗
PubMed2001

A case of Peters' anomaly in a springer spaniel.

An 8-week-old springer spaniel presented with a large central corneal opacity of the left globe, which was accompanied by cords of tissue spanning from the iris collarette to the posterior cornea. A posterior cortical cataract was noted in the right eye. At the owner's request the puppy was humanely destroyed, and a necropsy was performed. Upon sectioning the left globe in the vertical plane, a circle of pigmented strands of tissue was observed spanning the anterior chamber from the iris to the posterior aspect of the cornea. The right globe appeared normal when inspected grossly. Histologically, a membrane of pigmented tissue covered the posterior aspect of the broad central corneal leukoma of the left globe. This membrane and the cords traversing the anterior chamber were composed of vascular uveal tissue. Descemet's membrane and the corneal endothelium were reduced or absent in the zone of corneal opacity. Other than the changes associated with cataract, the right globe was histologically normal. The clinical and histological findings in the left globe were identical with those described for Peters>> anomaly in human beings.

Animals↗
PubMed2001

Recognition of hypermethylated triplet repeats in vitro by cationic nanoparticles.

Genomic DNA contains many higher-order structural deviations from the Watson-Crick global average. The massive expansion and hypermethylation of the duplex triplet repeat (CCG)(n)(CGG)(n) has characteristic higher-order structures that are associated with the fragile X syndrome. We have used luminescent mineral nanoparticles of protein-sized cadmium sulfide in optical assays to detect anomalous DNA structures. The photoluminescence of these particles is sensitive to the presence and nature of adsorbates. We previously found that our nanoparticles bind the fragile X repeat well but do not bind to normal double-helical DNA. In this study, we have determined that these particles are also able to detect the hypermethylated forms of these triplet repeats. Therefore, these nanoparticles may form the basis for future optical assays of higher-order DNA structures, especially those associated with human disease.

Base Sequence↗
PubMed2001

Photopigment basis for dichromatic color vision in the horse.

Horses, like other ungulates, are active in the day, at dusk, dawn, and night; and, they have eyes designed to have both high sensitivity for vision in dim light and good visual acuity under higher light levels (Walls, 1942). Typically, daytime activity is associated with the presence of multiple cone classes and color-vision capacity (Jacobs, 1993). Previous studies in other ungulates, such as pigs, goats, cows, sheep and deer, have shown that they have two spectrally different cone types, and hence, at least the photopigment basis for dichromatic color vision (Neitz & Jacobs, 1989; Jacobs, Deegan II, Neitz, Murphy, Miller, & Marchinton, 1994; Jacobs, Deegan II, & Neitz, 1998). Here, electroretinogram flicker photometry was used to measure the spectral sensitivities of the cones in the domestic horse (Equus caballus). Two distinct spectral mechanisms were identified and are consistent with the presence of a short-wavelength-sensitive (S) and a middle-to-long-wavelength-sensitive (M/L) cone. The spectral sensitivity of the S cone was estimated to have a peak of 428 nm, while the M/L cone had a peak of 539 nm. These two cone types would provide the basis for dichromatic color vision consistent with recent results from behavioral testing of horses (Macuda & Timney, 1999; Macuda & Timney, 2000; Timney & Macuda, 2001). The spectral peak of the M/L cone photopigment measured here, in vivo, is similar to that obtained when the gene was sequenced, cloned, and expressed in vitro (Yokoyama & Radlwimmer, 1999). Of the ungulates that have been studied to date, all have the photopigment basis for dichromatic color vision; however, they differ considerably from one another in the spectral tuning of their cone pigments. These differences may represent adaptations to the different visual requirements of different species.

Adaptation, Physiological↗
PubMed2001

Morphology and neurochemistry of canine corneal innervation.

PURPOSE: To determine the architectural pattern and neuropeptide content of canine corneal innervation. METHODS: Corneal nerve fibers in normal dog eyes were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH). Relative innervation densities and distribution patterns for each fiber population were assessed qualitatively by serial line-drawing reconstructions and quantitatively by computer-assisted analyses. RESULTS: More than 99% of all corneal PGP-9.5-immunoreactive (IR) nerves contained both CGRP and SP, approximately 30% contained TH, and none contained VIP. Distribution patterns of corneal PGP-9.5-, CGRP-, SP-, and TH-IR nerves were indistinguishable, except that TH-IR fibers were absent from the corneal epithelium. Morphologically, canine corneal innervation consisted of a rich anterior stromal plexus, divided on the basis of morphologic criteria into anterior and posterior levels, and a rich epithelial innervation, characterized by large numbers of horizontally oriented, basal epithelial "leash" formations. Leash axons in all quadrants of the corneal epithelium oriented preferentially toward a common locus in the perilimbal cornea. CONCLUSIONS: The results of this study demonstrate for the first time the detailed architectural features, distinctive basal epithelial leash orientations, and peptidergic content of canine corneal innervation. The normal innervation pattern described in this study will provide other investigators with essential baseline data for assessing corneal nerve alterations in canine patients with spontaneous chronic corneal epithelial defects (SCCED) and other ocular diseases or injuries.

Animals↗
PubMed2001

Spontaneous chronic corneal epithelial defects (SCCED) in dogs: clinical features, innervation, and effect of topical SP, with or without IGF-1.

PURPOSE: To delineate the clinical features and alterations in innervation and substance P (SP) content in spontaneous chronic corneal epithelial defects (SCCED) in dogs and to conduct a preliminary investigation evaluating the efficacy of topical SP, with or without insulin-like growth factor (IGF)-1, in the treatment of this disorder. METHODS: Complete ophthalmic examinations, including Cochet-Bonnet aesthesiometry, were performed in 45 canine patients that had spontaneous corneal epithelial defects of at least 3 weeks' duration and with no identifiable cause. Eighteen patients had superficial keratectomies performed, and the corneal nerves were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, SP, vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH). Relative fiber densities were assessed qualitatively and quantitatively. Corneal epithelial cell and tear SP contents were determined in affected and normal dogs by an enzyme immunoassay. A preliminary open-label treatment trial of topical SP, with and without IGF-1, was conducted in 21 dogs. RESULTS: The duration of the erosion before admittance into the study was a mean of 9.22 weeks (range, 3-52). The average patient was middle aged (mean, 9.25 +/- 1.85 years [SD]); no sex predisposition of the disease was identified. Boxers, golden retrievers, and keeshonds were overrepresented when compared with the normal hospital population. Corneal sensation was normal. Marked alterations in corneal innervation were identified in affected dogs with abnormal increased SP and calcitonin gene-related peptide (CGRP)-immunoreactive nerve plexuses identified surrounding the periphery of the epithelial defect. The SP content of epithelial cells surrounding the defect increased, whereas the tear SP content remained unchanged. Of the canine patients treated with SP, with or without IGF-1, 70% to 75% had complete healing of the defect. CONCLUSIONS: This idiopathic spontaneous corneal disease in dogs shares clinical features with chronic epithelial defects in humans. The presence of marked alterations in peptidergic innervation and positive response to topical therapy with SP suggest that SP plays a critical role in corneal wound-healing processes.

Administration, Topical↗
PubMed2001

Morphology and immunohistochemistry of spontaneous chronic corneal epithelial defects (SCCED) in dogs.

PURPOSE: To determine the morphologic features of the epithelium and extracellular matrix in spontaneous chronic corneal epithelial defects (SCCED) in dogs. METHODS: Forty-eight superficial keratectomy specimens were obtained after confirmation of the presence of a superficial corneal erosion for longer than 3 weeks with no discernible underlying cause. Histologic samples were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Immunolocalization of laminin, collagen IV, fibronectin, and collagen VII was performed. RESULTS: Epithelial cells adjacent to the defect were poorly attached to the underlying extracellular matrix. A prominent superficial stromal hyaline acellular zone composed of collagen fibrils in the area of the erosion was present in most specimens. Samples exhibited a varying degree of fibroplasia, vascularization, and leukocytic infiltrate. Laminin, collagen IV, and collagen VII were usually either not present or were present only in discontinuous segments on the surface of the erosion. Fibronectin usually coated the surface of the erosion, either as a continuous sheet or in discontinuous segments. Transmission electron microscopy of 15 samples revealed that the basement membrane was either absent in the area of the erosion or was present only in discontinuous segments. Scanning electron microscopy of eight of nine samples confirmed the absence of continuous basement membrane. Epithelial and extracellular matrix components in the peripheral cornea appeared normal. CONCLUSIONS: Most canine patients with spontaneous chronic corneal epithelial defects do not have a normal basement membrane structure in the region of the epithelial defect and have other abnormalities in the subjacent extracellular matrix that may reflect a part of the underlying pathophysiology of chronic and recurrent erosions.

Animals↗
PubMed2000

Intrinsic bending in GGCC tracts as probed by fluorescence resonance energy transfer.

Double-stranded oligonucleotides containing the sequence 5'-GGCC-3' can be intrinsically bent, according to X-ray crystallography and gel electrophoresis mobility studies. We have performed fluorescence resonance energy transfer (FRET) experiments with dye-labeled oligonucleotides to further investigate the solution structure of this sequence. We find that 5'-GGCC-3'-containing oligonucleotides bring 5'-attached donor and acceptor dyes much closer together than a comparable "straight" sequence that contains 5'-GCGC-3'. The bend angle for the 5'-GGCC-3' sequence is estimated to be approximately 70 degrees, much larger than the crystallographically observed 23 degrees kink but in agreement with other FRET work. In contrast to gel electrophoresis studies, divalent metal ions do not promote increased kinking in 5'-GGCC-3' above background as judged by FRET. Thus, sequence-dependent structural effects in DNA may be a complicating feature of FRET experiments.

Chromatography, High Pressure Liquid↗