Osteochondroma on the cranial aspect of the distal radial metaphysis causing tenosynovitis of the extensor carpi radialis tendon sheath in a horse.

CASE REPORT: A 3-year-old Thoroughbred gelding was presented with a grade 3/5 lameness of the right forelimb and effusion of the extensor carpi radialis tendon sheath (ECRTS). Radiographic and ultrasonographic examinations revealed an osteochondroma on the cranial aspect of the distal radius projecting into the fibrous lining of the ECRTS. An open surgical approach was used to remove the osteochondroma and some of the proliferative synovial tissue. CONCLUSION: Six months after surgery the clinical signs had resolved and the horse raced successfully.

Dynamic ventrorostral displacement of the dorsal laryngeal mucosa in horses.

The objectives of this report were to describe the occurrence and features of dynamic ventrorostral displacement of the dorsal laryngeal mucosa (VRDDLM) in a group of Thoroughbred horses presented for investigation of poor performance and/or abnormal respiratory noise. Records from 600, dynamic, endoscopic examinations of the upper respiratory tract of horses were reviewed. Horses with VRDDLM were identified as those in which the dorsal laryngeal mucosa progressively obscured the interarytenoid notch and dorsoaxial portion of the corniculate processes of the arytenoid cartilages during high-speed exercise. The condition was recognised in 12 horses. Concurrent abnormalities of the respiratory tract of eight horses were also identified and included, axial deviation of the aryepiglottic folds, vocal cord prolapse, unilateral and bilateral ventromedial luxation of the apex of the corniculate process of the arytenoid cartilage, and intermittent dorsal displacement of the soft palate. VRDDLM is a rare abnormality of the upper portion of the respiratory tract of horses that may be associated with abnormal respiratory noise and potentially poor performance. The significance of the condition is not known, but the presence of this condition in combination with other, obstructive diseases of the equine airway warrants further investigation.

The development and activity-dependent expression of aggrecan in the cat visual cortex.

The Cat-301 monoclonal antibody identifies aggrecan, a chondroitin sulfate proteoglycan in the cat visual cortex and dorsal lateral geniculate nucleus (dLGN). During development, aggrecan expression increases in the dLGN with a time course that matches the decline in plasticity. Moreover, examination of tissue from selectively visually deprived cats shows that expression is activity dependent, suggesting a role for aggrecan in the termination of the sensitive period. Here, we demonstrate for the first time that the onset of aggrecan expression in area 17 also correlates with the decline in experience-dependent plasticity in visual cortex and that this expression is experience dependent. Dark rearing until 15 weeks of age dramatically reduced the density of aggrecan-positive neurons in the extragranular layers, but not in layer IV. This effect was reversible as dark-reared animals that were subsequently exposed to light showed normal numbers of Cat-301-positive cells. The reduction in aggrecan following certain early deprivation regimens is the first biochemical correlate of the functional changes to the γ-aminobutyric acidergic system that have been reported following early deprivation in cats.

Evaluation of a modification of the McKinnon technique to correct urine pooling in mares.

The urethral fold of 30 mares was split transversely into dorsal and ventral shelves, and the ventral shelf was used to help create a urethral extension. The dorsal shelf was stretched caudally and sutured to the roof of the extension so that it covered at least the cranial half of the extension. For 20 mares, a relaxing, vaginal incision was created cranial to the external urethral orifice to enable the dorsal shelf to be retracted further caudally. Ten of the 30 mares (33.3 per cent) developed a defect, but none developed a defect in that portion covered by the dorsal shelf of the urethral fold. Two of the 30 mares (6.7 per cent) developed a defect so small that the defect could be detected only by inserting a dye, under pressure, into the tunnel. The total number of mares that developed only a grossly visible and palpable defect was eight of 30 (26.6 per cent). Four of the 10 mares that did not receive the relief incision and six of 20 mares that did receive the relief incision developed a defect in the extension. Modifying the McKinnon technique by transversely splitting the urethral fold and retracting the dorsal half helps prevent a defect from forming in the cranial portion of the extension. The dorsal shelf can be retracted further caudally by creating a relief incision on the floor of the vagina.

Brain enriched hyaluronan binding (BEHAB)/brevican increases aggressiveness of CNS-1 gliomas in Lewis rats.

Gliomas are the most common primary intracranial tumors. One extracellular matrix component that has been implicated in glial tumor biology is brain enriched hyaluronan binding (BEHAB)/brevican. In this study, the CNS-1 rat glioma cell line was transfected with a vector containing either a full-length BEHAB/brevican cDNA, a 5' insert encoding the NH(2)-terminal BEHAB/brevican cleavage product, or a 3' insert encoding the COOH-terminal cleavage product. As a control, CNS-1 cells were transfected with green fluorescent protein. Rats with intracranial grafts of BEHAB/brevican-transfected CNS-1 cells displayed significantly shorter survival times than did rats with CNS-green fluorescent protein intracranial grafts (P < 0.001). Histological examination showed that the BEHAB/brevican-transfected tumors were just as, if not more, aggressive than control tumors, even though the BEHAB/brevican tumors had been growing for only approximately two-thirds the time as long as control tumors. These data suggest that up-regulation and proteolytic cleavage of BEHAB/brevican increase significantly the aggressiveness of glial tumors. It will be important to investigate the effect of inhibiting cleavage of BEHAB/brevican in these cells and to determine the therapeutic potential of inhibiting BEHAB/brevican cleavage in gliomas.

Ran binding protein RanBP1 in zebrafish embryonic development.

Ran, which functions in nucleocytoplasmic transport and mitosis, binds to and is regulated in part by RanBP1. We have identified a zebrafish RanBP1 cDNA and report that it encodes for a polypeptide of 233 amino acids with considerable similarity to human and Xenopus RanBP1, despite the fact that it is 10% longer due to an extension at its carboxy terminus. RanBP1 mRNA is present as a maternal transcript and is expressed ubiquitously throughout the developing embryo. At the protein level, RanBP1 is present at all embryonic stages. Surprisingly, the ectopic overexpression of the protein had no obvious effect on embryogenesis. Attempts were also made to down-regulate RanBP1 activity by RNA interference. Injecting double-stranded RNA augmented both the mortality rate and the frequency of induced defects. Specific defects accompanied by changes in RanBP1 expression were not seen, leading us to propose that RNAi is not a reliable method for deregulating the activity of constitutively expressed genes, like RanBP1, in zebrafish. Mol. Reprod. Dev. 59:235-248, 2001.

Differential distribution of the G protein gamma3 subunit in the developing zebrafish nervous system.

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein alpha subunit was the sole regulator of intracellular molecules. The G protein betagamma complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein gamma subunit. The 666 nucleotides of the zebrafish G protein gamma subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse gamma subunits. BLAST search analysis of GenBank revealed that the zebrafish gamma subunit is 93% identical and 97% similar to the mammalian gamma3 subunit. The gamma3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected gamma3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish gamma3 subunit to form a signaling heterodimeric complex with a beta subunit was tested using a human beta2 subunit. The gamma3 formed a heterodimer with beta2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the beta2gamma3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a betagamma heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.

New thoughts on the role of the beta-gamma subunit in G-protein signal transduction.

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound alpha subunit and a beta-gamma heterodimer. The beta-gamma dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of beta-gamma signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by beta-gamma and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on beta-gamma signaling in embryogenesis.

Blockade of pilocarpine-induced cerebellar phosphoinositide hydrolysis with metabotropic glutamate antagonists: evidence for an indirect control of granule cell glutamate release by muscarinic agonists.

The ability in vivo of the muscarinic agonist, pilocarpine, to increase phosphoinositol (PI) hydrolysis in lithium pretreated rats was investigated by measuring the accumulation of [(3)H]inositol phosphates (IP). As expected, 20 mg/kg s.c. pilocarpine, a muscarinic agonist, increased PI hydrolysis in the striatum, frontal cortex and hippocampus. Somewhat surprisingly, an increase in IP was also found in the cerebellar homogenates. In all four tissues the pilocarpine-induced effect could be completely inhibited by pretreatment with the muscarinic antagonist scopolamine (1.2 mg/kg i. p.). It was also found that the cerebellar but not the hippocampal pilocarpine-induced rise in PI hydrolysis could be blocked by the metabotropic glutamate (mGlu) receptor antagonist, LY341495 (100 nmol, i.c.v.). The same dose of LY341495 was found to also block both the cerebellar and hippocampal increase in IP formed by stimulation with the group I mGlu receptor agonist 3, 5-dihydroxyphenylglycine (1 micromol, i.c.v.). Given this data and the current information on the distribution of muscarinic and mGlu receptors in the cerebellum, it is suggested that these results may be a reflection of pilocarpine acting at M(2) receptors to indirectly increase glutamate release from parallel fibers by inhibition of gamma-aminobutyric acid-releasing Golgi cells.

The Translocon-Associated Protein beta (TRAPbeta) in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors.

The normal translocation of nascent polypeptides into the lumen of the endoplasmic reticulum (ER) is thought to be aided in part by a translocon-associated protein (TRAP) complex consisting of 4 protein subunits. The association of mature proteins with the ER and Golgi, or other intracellular locales, such as lysosomes, depends on the initial targeting of the nascent polypeptide to the ER membrane. A similar scenario must also exist for proteins destined for secretion. We have identified a member of the TRAP complex using a two hybrid screen to isolate proteins that bind to zebrafish (Danio) Ran binding protein 1. The polypeptide predicted from the largest open reading frame contains 183 amino acids with a 86 and 87% sequence identity to the TRAPbeta subunits in human and chicken, respectively. Sequence analysis identified a cleavable amino-terminal signal peptide in the zebrafish TRAPbeta subunit and a region of the protein spans the membrane of the endoplasmic reticulum. A reverse transcriptase-polymerase chain reaction assay showed that TRAPbeta mRNA is expressed in the developing zebrafish embryo. TRAPbeta mRNA is maternally supplied to the egg and is expressed constitutively throughout development and in the adult. This pattern of expression indicates that the message encoding part of the machinery targeting nascent polypeptides to the ER lumen is available at the onset of embryogenesis when the rate of translation increases exponentially over that occurring in the oocyte. In situ hybridization was used to test whether or not TRAPbeta transcripts might become localized and/or enriched in the developing embryo. Homogeneous staining is seen in the blastula and early gastrula stages. At mid-to-late gastrula stages, however, the message becomes enriched in the developing notochord and polster, or hatching gland rudiment. The TRAPbeta gene, mapped using the LN54 mouse-zebrafish radiation hybrid panel to linkage group 19, resides next to a gene (Z15451) which has sequence homology to notch2 and vascular endothelial growth factor. TRAPbeta, however, does not appear to belong to a group of genes which are syntenic with orthologues or paralogues on human chromosomes.

Intracranial injury acutely induces the expression of the secreted isoform of the CNS-specific hyaluronan-binding protein BEHAB/brevican.

Hyaluronan (HA) plays an important role in tissue reorganization in response to injury. The mechanisms by which HA participates in these processes are likely to include HA-binding proteins. Previously, we reported the cloning and initial characterization of a central nervous system (CNS)-specific HA-binding protein, BEHAB (brain enriched hyaluronan binding), which was independently cloned in another laboratory and named brevican. BEHAB/brevican mRNA is expressed in the ventricular zone coincident with the initial proliferation and migration of glial cells and in surgical samples of human glioma, where glial-derived cells proliferate and migrate. To determine whether BEHAB/brevican is also expressed during the cellular proliferation and migration associated with CNS injury, we have examined BEHAB/brevican expression during reactive gliosis. BEHAB/brevican occurs as secreted and cell-surface, glycosylphosphatidylinositol (GPI)-anchored, isoforms. The secreted, but not the GPI-anchored, isoform is up-regulated in response to a stab wound to the adult rat brain. The temporal regulation and spatial distribution of BEHAB/brevican expression parallel the gliotic response and the expression of the intermediate filament protein nestin. The up-regulation of BEHAB/brevican in response to CNS injury suggests a role for this extracellular matrix molecule in reactive gliosis. Glial process extension, a central element in the glial response to injury, may require the reexpression of both cytoskeletal and matrix elements that are normally expressed during the glial motility seen in the immature brain.

Phosphoinositide hydrolysis in vivo with group I metabotropic glutamate receptor agonists.

The present report describes the effect of mGluR agonists and antagonists administration on phospholipase C activation by measuring accumulation of [3H] inositol monophosphates (IP) in rats pre-labeled with [3H]myo-inositol (i.c.v. 24 h pre-treatment). The levels of accumulated [3H]IP were then determined from clarified tissue homogenates using ion-exchange chromotography. Following lithium chloride treatment (10 mg/kg, s.c.), (R/S)-3, 5-dihydroxyphenylglycine (DHPG), a selective group I mGluR agonist was found to dose-dependently cause a maximal increase in the levels of [3H]IP at 0.3 to 3 micromol/8 microliter i.c.v. with lower doses resulting in less efficacious or no responses. This effect was temporal-dependent reaching a plateau at 2 h. The DHPG-induced increases in [3H]IP were most pronounced in the hippocampus where a 3- to 5-fold increase above vehicle was consistently found, but significant approximately 2-fold increases were also seen in the cerebellum, striatum and frontal cortex. The mixed group I and II agonist, (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (1S, 3R-t-ACPD), similarly resulted in dose-dependent increases in [3H]IP levels with doses of 1 to 3 micromol i.c.v. Furthermore, this effect was enantiomer specific since the less active 1R,3S-t-ACPD failed to alter phosphoinositol hydrolysis. Administration of the selective mGluR5 agonist (R/S)-2-chloro-5-hydroxyphenyl-glycine (CHPG) resulted in a dose-dependent increase in hippocampal but not cerebellar levels of [3H]IP, consistent with the receptor distribution of the two group I mGluRs. The Group II agonist LY354740 (1S,2S,5R,6S-2-aminobicycl[3.1.0]hexane-2,6-dicarboxylate monohydrate) and the group III agonist L-AP4 (L-(+)-2-amino-4-phosphonobutyric acid) failed to alter the levels of [3H]IP. LY341495 (2S-2-amino-2-(1S, 2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid) is a nM potent Group II antagonist. However, LY341495 has also been found to have microM potency in inhibiting mGluR1 and 5. The stimulation of [3H]PI hydrolysis by 1 micromol DHPG was dose-dependently blocked by co-administration of the mGluR antagonists, LY341495 at doses that are constant with an interaction at Group I mGluR's. Taken together these results suggest that stimulation of group I mGluRs results in measurable increases in PI hydrolysis in vivo. This method could be quite useful in determining the doses and routes of administration of agonists and antagonists that are required to interact with group I mGluRs.

BEHAB/brevican: a brain-specific lectican implicated in gliomas and glial cell motility.

Several recent findings have advanced our understanding of the composition and function of the brain extracellular matrix (ECM). BEHAB/brevican, a recently identified CNS-specific proteoglycan, is a component of the brain ECM and is upregulated during glial cell motility. It is expressed at high levels during development, in response to injury, and in primary brain tumors. Cleavage of the BEHAB/brevican protein may increase invasion of tumor cells.

Phospholipase C-beta1 is present in the botrysome, an intermediate compartment-like organelle, and Is regulated by visual experience in cat visual cortex.

Monoclonal antibody Cat-307 identifies a 165 kDa neuronal protein expressed in the cat visual cortex during the period of sensitivity to alterations in visual experience (). Dark-rearing, which prolongs the sensitive period, also prolongs the expression of the Cat-307 protein. The Cat-307 protein localizes to an organelle, here called the botrysome (from the Greek botrys, cluster of grapes), that is located between the endoplasmic reticulum (ER) and Golgi apparatus. The botrysome is composed of small ring-shaped profiles with electron-dense coats. The size and morphology of the rings and their coats are similar to those described for ER to Golgi transport vesicles. Biochemically, the Cat-307 protein cofractionates with microsomes and partitions with subunits of the coatomer proteins that coat ER-to-Golgi transport vesicles. Partial amino acid sequencing reveals that the Cat-307 protein is phospholipase C-beta1, the G-protein-dependent phosphodiesterase that hydrolyses phosphatidylinositol 4,5 biphosphate into inositol 1,4,5 triphosphate and diacylglycerol after the stimulation of a variety of neurotransmitter receptors at the cell surface. These results suggest a role for phospholipase C-beta1 and the botrysome in developmental plasticity and provide a possible link between receptor activation at the cell surface and protein transport during neuronal development.

The zebrafish's swim to fame as an experimental model in biology.

The zebrafish has long been the favorite organism in many scientific disciplines. Although its attributes as a model were expounded for many years and thus were no secret, the zebrafish sat in the wings while other more popular vertebrates such as chick, amphibians, and mouse were examined at length. We cannot say there was a resurgence in popularity, but more an explosion of research utilizing the zebrafish beginning in the late 1970s when investigators at the University of Oregon began using it as their model in neuroscience. Prior to this reawakening, the zebrafish was one of the significant organisms in the study of teratology and toxicology, development, and, to some extent, behavior. Recently, however, the field of zebrafish genetics has gained immense popularity and success, in part owing to the fact that zebrafish are diploid and are amenable to genetic manipulations. Here we present an overview of the multidisciplinary research that has laid some of the foundation of our present understanding of the biochemical, cell biological, and molecular genetic events accompanying zebrafish development.

Characterization of a cDNA encoding a novel band 4.1-like protein in zebrafish.

Membrane skeleton protein 4.1 and other members of a family of proteins that link the cytoskeleton to the plasma membrane may play an integral role in cell communication during development. The polymerase chain reaction and degenerate oligodeoxynucleotide primers to consensus sequences in the putative membrane-binding domain of the protein 4.1 superfamily were used to isolate cDNAs encoding members of the zebrafish protein 4.1 family. Zebrafish stage- and tissue-specific first strand cDNA was used in the PCR. After the reaction, amplicons of the predicted size were sequenced to confirm their relationship to the protein 4.1 superfamily. One cDNA, with a high degree of similarity to a mouse novel band 4.1-like cDNA, was used to probe a zebrafish adult brain library. A 2.4-kb cDNA was isolated and found to encode a 619 amino acid polypeptide homologous to mouse novel band 4.1-like protein 4. Zebrafish nbl4 mRNA is maternally supplied and is expressed throughout embryogenesis. In adults, nbl4 is found in the ovary, eye, heart, and brain, but not in gut or skeletal muscle. When synthetic nbl4 mRNA is translated in vitro it binds calmodulin in a calcium-dependent manner. These data indicate that zebrafish nbl4 is a maternal transcript owing to its presence before the midblastula transition, and it is present later on in specific adult structures. The ability to bind calmodulin would suggest that the function of nbl4 protein may be potentially regulated via a calcium-calmodulin dependent mechanism.

Identification and characterization of novel developmentally regulated proteins in rat spinal cord.

We previously used 2-dimensional (2-D) gel electrophoresis to identify novel proteins that may be involved in the genesis of the mammalian nervous system [1]. Several novel proteins that were up- or down-regulated coincident with neurogenesis and neuronal migration in rat neocortex were identified. To further investigate the expression of some of these developmentally regulated proteins during a comparable period in spinal cord development, 2-D electrophoresis is used to study their regulation in the crude membrane and soluble fractions of spinal cord at embryonic day 12 (E12) and embryonic day 21 (E21). This analysis indicates that 7 of the proteins that exhibited large changes in their synthesis in cerebral cortex between embryonic day 14 (E14) and embryonic day 21 (E21) demonstrate similar up- or down-regulation during spinal cord neurogenesis. However, two proteins are restricted in their expression or developmental regulation. One of these, 667-800, appears cortex-specific, while the up-regulation of protein SC.1 appears to be spinal cord specific. Several of these proteins also appear to be enriched in both the cortex and spinal cord relative to non-neural tissues (117, 162, 182, 310 [TOAD-64], 667-800) and may be neural specific. To further characterize its expression, one of these neural-specific, up-regulated proteins, TOAD-64 (protein 310) [2-4], is studied throughout embryonic and postnatal spinal cord development using peptide-specific polyclonal antibodies. As suggested by the 2-D gel analysis and the previously reported expression pattern in cerebral cortex [3], TOAD-64 is transiently expressed in postmitotic spinal cord neurons early in their development and sharply down-regulated after the second postnatal week. In the adult spinal cord, TOAD-64 expression is remarkably restricted to a subset of primary afferents to the spinal cord. This expression pattern, coupled with its recently discovered homology to two proteins implicated in axon pathfinding in the chick and nematode [5,3], suggests that TOAD-64 may have a fundamental role in axon pathfinding.

BEHAB (brain enriched hyaluronan binding) is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cell lines.

Malignant gliomas aggressively invade the surrounding normal brain, whereas brain metastases of nonglial tumors do not. The invasive behavior of gliomas may be mediated by tissue- or tumor-specific extracellular proteins. mRNA for the brain-specific extracellular brain enriched hyaluronan-binding protein (BEHAB) is not detectable in normal adult human cortex or in any nonglioma tumor examined. BEHAB is consistently expressed in surgical samples of glioma (n = 27). Glioma cell lines maintained under standard cell culture conditions or grown as s.c. tumors do not express BEHAB. When grown as intracranial grafts, glioma cell lines that invade the brain express BEHAB, whereas noninvasive cell lines do not. BEHAB is a unique and selective marker for glioma and may play a role in tumor invasion.