Biomedical subjects
F R Burns
Publications and source records attributed to F R Burns.
Identification of a structural constituent and one possible site of postembryonic formation of a teleost otolithic membrane.
A gelatinous otolithic membrane (OM) couples a single calcified otolith to the sensory epithelium in the bluegill sunfish (Lepomis macrochirus) saccule, one of the otolithic organs in the inner ear. Though the OM is an integral part of the anatomic network of endorgan structures that result in vestibular function in the inner ear, the identity of the proteins that make up this sensory accessory membrane in teleosts, or in any vertebrate, is not fully known. Previously, we identified a cDNA from the sunfish saccular otolithic organ that encoded a new member of the collagen family of structural proteins. In this study, we examined biochemical features and the localization of the saccular collagen (SC) protein in vivo using polyclonal antisera that recognize the noncollagenous domains of the SC protein. The SC protein, in vivo, was identified as a 95-kDa glycoprotein in sunfish whole-saccule lysate and in homogenates of microdissected saccular OMs. Immunohistochemical analyses demonstrated that the SC protein was localized within one of the two distinct layers of the sunfish saccular OM. The SC protein was also detected within the cytoplasm of supporting cells at the edges of the saccular sensory epithelium, indicating that these cells are a primary site for the synthesis of this structural protein. Further studies of the organization of this matrix molecule in the OM may help clarify the role of this sensory accessory membrane in vestibular sensory function.
Molecular cloning and characterization of an inner ear-specific structural protein.
Molecular biological studies of the mammalian inner ear have been limited by the relatively small size of the sensory endorgans contained within. The saccular otolithic organ in teleostian fish is structurally similar to its mammalian counterpart but can contain an order of magnitude more sensory cells. The prospect of the evolutionary conservation of proteins utilized in the vertebrate inner ear and the relative abundance of teleostian saccular sensory tissue made this an attractive system for molecular biological studies. A complementary DNA obtained by differential screening of a saccular complementary DNA library was identified that encodes an inner ear-specific collagen molecule.
Use of the teleost saccule to identify genes involved in inner ear function.
The vertebrate inner ear sensory epithelia contain different types of hair cells and supporting cells. The teleost saccule is anatomically similar to the mammalian saccule and is primarily involved in the detection of translational acceleration and orientation with respect to gravity. To facilitate molecular studies of the teleost saccule cDNA libraries were constructed from microdissected Lepomis macrochirus (bluegill sunfish) saccular maculae. To our knowledge, this is the first report of cDNA libraries constructed from the saccule. In one instance, a non-polymerase chain reaction-based method of amplifying a mRNA population from limited amounts of starting tissue was employed that allowed construction of cDNA libraries from nanogram amounts of tissue mRNA. Conventional cDNA libraries were constructed from the sunfish saccular maculae as well. These cDNA libraries enriched in hair cell and supporting cell transcripts should facilitate molecular biological studies of inner ear sensory epithelia. As an example of their utility, efforts to identify tyrosine kinases expressed in the saccular endorgan using low-stringency hybridization screening of these cDNA libraries and the partial sequence of a cDNA found to encode an erbB-2-related tyrosine kinase are also reported.
Organization of the mouse GP42/Basigin gene: a member of the Ig superfamily.
We have mapped and sequenced the GP42/Basigin gene isolated from a Balb/C mouse genomic library. The genomic organization and upstream, putative regulatory, regions of this gene have not been previously reported. Our data show that exon 5 of the GP42/Basigin gene encodes the carboxy proximal half of the second Ig-like domain, the highly conserved transmembrane region and a portion of the cytoplasmic tail. This inclusion of Ig-like and other functional domains in a single exon is unusual. Splice junction analysis indicates that two reported alternate GP42/Basigin cDNA isoforms are likely due to cloning artifacts. In addition, we find that GP42/Basigin is polymorphic in mice. Our data also support the proposal that the transmembrane domain and portions of the cytoplasmic region of GP42/Basigin have been evolutionarily conserved.
Regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) and the putative adhesion molecule Basigin on murine cerebral endothelial cells by MHV-4 (JHM).
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The effect of a synthetic metalloproteinase inhibitor on corneal ulceration in alkali burns and pseudomonas keratitis.
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DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension.
We have identified a 95 kd cell surface protein, DM-GRASP, that is expressed on a restricted population of axons. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons. We have cloned and sequenced the cDNA corresponding to this protein and find that it is a new member of the immunoglobulin superfamily of adhesion molecules. Consequently we have named this protein DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein that is expressed in the dorsal funiculus and ventral midline of the chick spinal cord.
Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide.
Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P. aeruginosa infections. Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea. This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P. aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin. A synthetic metalloproteinase inhibitor, HSCH2 (DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, which we previously showed to be a potent inhibitor of corneal collagenase and alkali-induced corneal ulceration, was tested as a potential inhibitor of P. aeruginosa elastase. Inhibition constants (Kis) for the resolved diastereomers were determined with the chromogenic substrate furylacryloyl-glycyl-L-leucyl-L-alanine. One isomer had a Ki of 0.3 microM, while the other had a Ki of 0.4 microM. The more potent diastereomer was evaluated in vivo in experimentally induced Pseudomonas keratitis in rabbits. Following inoculation of one cornea of each rabbit, topical treatment with a 1 mM solution of the inhibitor significantly delayed the onset of corneal melting and perforation, as compared with the results for the control and gentamicin-treated groups. This protective effect suggests that the inhibitor may have a therapeutic application by delaying the progression of corneal destruction in Pseudomonas keratitis.
Inhibition of alkali-induced corneal ulceration and perforation by a thiol peptide.
Corneal ulceration and perforation following a severe alkali burn occur as a consequence of collagen destruction by locally released enzymes. A thiol peptide, which recently was shown to be a potent inhibitor of corneal collagenase in vitro, was tested in alkali-burned rabbit corneas to determine its effectiveness in inhibiting corneal ulceration. Following a standard alkali burn to one eye of each rabbit, ten animals were treated topically six times daily and subconjunctivally one time daily with a 1 mM solution of the peptide for a period of 3 weeks. A control group of ten rabbits was administered vehicle only using the same regimen as the experimental group. Corneal ulceration occurred in ten out of ten of the control eyes and seven out of ten progressed to perforation. The experimental group demonstrated ulcerations in four out of nine animals, only one of which was deep (one of nine), and no perforations. There was no significant difference when comparing the onset of ulceration between the two groups, but the difference was significant when comparing the total number of ulcerations (0.02 less than P less than 0.05), deep ulcerations (0.01 less than P less than 0.02) and perforations (0.001 less than P less than 0.01) between the two groups. Histologic examination of the corneas after 3 weeks of treatment revealed that the experimental, thiol-treated corneas that did not ulcerate contained relatively few PMNs, whereas the control corneas demonstrated a marked inflammatory infiltrate in the form of PMNs, most notably at sites of corneal ulceration. These findings demonstrate that a synthetic thiol peptide inhibits alkali-induced corneal ulceration and perforation in vivo.
T cell determinants of myelin basic protein include a unique encephalitogenic I-E-restricted epitope for Lewis rats.
The major encephalitogenic epitope for Lewis rats is the 72-89 sequence of guinea pig basic protein (GP-BP) or rat basic protein (Rt-BP). T cells responsive to this epitope are I-A restricted and preferentially express the V alpha 2:V beta 8 gene combination in their TCR. In this work, we describe for the first time the delayed appearance of T cells specific for additional discrete determinant of BP, the nonencephalitogenic 55-68 sequence of GP-BP restricted by I-A, and the encephalitogenic 87-99 sequence of Rt-BP restricted by I-E. The TCR V alpha 2:V beta 8 gene combination was expressed by both encephalitogenic GP-BP S72-89 and Rt-BP S87-99 T cell specificities but not by GP-BP 44-68-specific T cells. This is the first demonstration of I-E-restricted encephalitogenic T cells in Lewis rats and supports the conclusion that the I-E class II locus is involved in autoimmune diseases.
Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V alpha and V beta chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different.
Prospects for specific immune intervention in T cell-mediated autoimmune disease via anti-idiotypic regulation depend on the degree of diversity of the responder cell antigen receptor repertoire. A highly heterogenous response against self epitopes offers little chance for such regulation. We report here that the Lewis rat autoimmune disease experimental allergic encephalomyelitis, generally considered to be a model of human multiple sclerosis, is caused by T cells that use a limited set of TCR V genes. We have cloned the rat TCR alpha and beta chain cDNAs from the Lewis rat x mouse T cell hybridoma 510, which retains the rat specificity for the encephalitogenic determinant of myelin basic protein (MBP). Using Northern blot analysis of T cell RNA with the cloned V region probes, we have found a specific, and near perfect, correlation between expression of TCR message hybridizing to the V alpha 510 and VB510 probes and specificity for the encephalitogenic determinant of MBP in both T cell hybridomas and encephalitogenic T cell clones. This restricted V gene usage provides a basis for observed idiotypic regulation of auto-reactive T cells, and possible therapy for autoimmune disease. A curious and unexplained observation is that the Lewis rat V alpha/V beta combination that dominates the encephalitogenic response to the 68-88 peptide of MBP is precisely the same V alpha/V beta combination used by the B10.PL mouse response to the encephalitogenic response to the 1-9 peptide of MBP.
Inhibition of purified collagenase from alkali-burned rabbit corneas.
The inhibitory potency of four classes of compounds that inhibit corneal ulceration (thiols, tetracyclines, sodium citrate and sodium ascorbate) was assessed with collagenase purified from culture medium of alkali-burned rabbit corneas. The most potent inhibitor, a beta-mercaptomethyl tripeptide HSCH2(DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, exhibited 50% inhibition (IC50) at approximately 10 nM using the synthetic metalloproteinase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2. The inhibitor was somewhat less potent with type 1 collagen as substrate (IC50 between 1 and 3 microM), possibly because autooxidation of the essential - SH moiety of the inhibitor occurred during the longer time required for assay with the natural substrate. An N-carboxyalkyl tripeptide, CH3(CH2)2(DL)CH-(COOH)-Leu-Phe-Ala-NH2, was less potent (IC50 = 25 microM) than the thiol peptide. N-acetylcysteine, which is used to treat corneal ulceration, gave IC50 values of 2.7 mM and less than 10 mM with the synthetic and natural substrates, respectively. The IC50 values for the tetracyclines using the synthetic substrate were 15, 190 and 350 microM for doxycycline, minocycline and tetracycline, respectively. Inhibition by sodium citrate, but not the tetracyclines, could be reversed by excess Ca2+. Sodium ascorbate did not inhibit collagenase-mediated hydrolysis of either collagen or the synthetic substrate, thus indicating that the mechanism by which this agent inhibits corneal ulceration is not related to inhibition of collagen degradation by collagenase.
Prompt irrigation of chemical eye injuries may avert severe damage.
Chemical burns to the eye are among the most urgent of ocular emergencies. The clinical outcome of the injury is directly related to the expediency with which treatment is begun. Copious irrigation is the most important emergency treatment of the chemically-burned eye. This irrigation should begin immediately at the scene of the accident with any non-toxic liquid. Removal of any particulate matter must be done to prevent further ocular damage. The subsequent therapy is directed at the treatment of secondary sequelae and at preserving the globe to surgically rehabilitate the eye. Many of the treatments which are used in the intermediate and late phases of the injury are used to prevent corneal ulceration and perforation. These are the most difficult sequelae to threat in alkali injuries; thus, preventing the progression to this stage is of the utmost importance. Again the immediate and continuous irrigation of the eye may help accomplish this goal. The availability of emergency eye-wash equipment dispensing a safe, preserved, pH-balanced, physiologically correct solution in the industrial, agricultural and even the home setting is a necessity. The education of employees and family members in the proper technique of irrigating the eye following a chemical burn is also of extreme importance. Immediate irrigation of the eye, continued during rapid transport to a medical care facility, minimizes the damage to the eye and enhances the eventual clinical outcome.