Bovine Adrenal Glands Research Articles

Isolation, Characterization, and Differentiation of Progenitor Cells from Human Adult Adrenal Medulla

Chromaffin cells, sympathetic neurons of the dorsal ganglia, and the intermediate small intensely fluorescent cells derive from a common neural crest progenitor cell. Contrary to the closely related sympathetic nervous system, within the adult adrenal medulla a subpopulation of undifferentiated progenitor cells persists, and recently, we established a method to isolate and differentiate these progenitor cells from adult bovine adrenals. However, no studies have elucidated the existence of adrenal progenitor cells within the human adrenal medulla. Here we describe the isolation, characterization, and differentiation of chromaffin progenitor cells obtained from adult human adrenals. Human chromaffin progenitor cells were cultured in low-attachment conditions for 10-12 days as free-floating spheres in the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor. These primary human chromosphere cultures were characterized by the expression of several progenitor markers, including nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox9, Sox10, Phox2b, and Ascl1 on the molecular level and of Sox9 on the immunohistochemical level. In opposition, phenylethanolamine N-methyltransferase (PNMT), a marker for differentiated chromaffin cells, significantly decreased after 12 days in culture. Moreover, when plated on poly-l-lysine/laminin-coated slides in the presence of FGF-2, human chromaffin progenitor cells were able to differentiate into two distinct neuron-like cell types, tyrosine hydroxylase (TH)(+)/β-3-tubulin(+) cells and TH(-)/β-3-tubulin(+) cells, and into chromaffin cells (TH(+)/PNMT(+)). This study demonstrates the presence of progenitor cells in the human adrenal medulla and reveals their potential use in regenerative medicine, especially in the treatment of neuroendocrine and neurodegenerative diseases.

Wars in the history of rheumatology

Some important discoveries in the history of rheumatology happened during war periods. It is well known that arthritis associated with conjunctivitis and urethritis, following dysenteric episodes, has been described during the First World War from the German Hans Reiter and, nearly contemporarily, from the French Nöel Fiessinger and Edgar Leroy. Less known is instead the fact that the first cases of sympathetic algoneurodystrophy have been reported by the American Silas Weir Mitchell in soldiers wounded by fire-arms, during the Civil War of Secession. Other war episodes have been crucial for the development of some drugs now abundantly applied to the care of rheumatic diseases. The discovery of therapeutic effects of immunosuppressive agents, in fact, happened as an indirect consequence of the use of poison gas, already during the First World War (mustard gas), but above all after an episode in the port of Bari in 1943, where an American cargo boat was sunk. It had been loaded with a quantity of cylinders containing a nitrogenous mustard, whose diffusion in the environment provoked more than 80 deaths owing to bone marrow aplasia.Moreover, the history of the cortisone shows a strict link to the Second World War, when Germany imported large quantities of bovine adrenal glands from Argentina, with the purpose of producing some gland extracts for the Luftwaffe aviators, in order to increase their performance ability.

Expression of urocortin and corticotropin-releasing hormone receptors in the bovine adrenal gland

Urocortin (UCN), a 40 amino acid peptide, is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated via two types of G protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The aim of the present study was to investigate the expression of UCN, CRHR1 and CRHR2 by immunohistochemistry, western blot and real-time RT-PCR in the bovine adrenal gland to clarify the mechanisms of the intra-adrenal CRH-based regulatory system. The results showed that UCN, CRHR1 and CRHR2 were expressed in both the cortex and medulla. Specifically, UCN-immunoreactivity (IR) was distributed in the outer part of the zona fasciculata and in the zona reticularis of the cortex and in the medulla. UCN and CRHR2 mRNA expression levels were higher in the cortex than in the medulla, while CRHR1 mRNA levels were undetectable in the cortex.These results suggest that UCN, CRHR1 and CRHR2 are expressed in the bovine adrenal gland and that UCN might play a role in the intra-adrenal CRH-based regulatory system through an autocrine mechanism.

The temperature dependence of [8-3H]adenosine labelling of the perfused bovine and canine adrenal gland.

The temperature dependence of [8-3H]adenosine labelling of the perfused bovine and canine adrenal gland.

1‐[(Benzofuran‐2‐yl) phenylmethyl] Imidazoles as Inhibitors of 17α‐Hydroxylase: 17, 20‐lyase (P450 17): Species and Tissue Differences

A series of 1-[(benzofuran-2-yl) phenylmethyl] imidazoles have been evaluated as inhibitors of microsomal 17α-hydroxylase: 17, 20-lyase (P450 17) from rat and human testes, and bovine adrenals. Ketoconazole has 110-fold selectivity for the human enzyme. This selectivity was mirrored in several equipotent imidazole derivatives. Some compounds, although potent inhibitors of the human enzyme, were less effective against the rat enzyme. This demonstrates the need to screen candidate inhibitors, intended as agents for the treatment of prostatic cancer, against the human enzyme.

G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D(2) dopamine receptor (DAR). Agonist activation of D(2) DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D(2) DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [(35)S]GTPgammaS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D(2) DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.

Carbon Isotope Ratio Analysis of Bovine Endogenous Ouabain and Commercial Sources of Plant Ouabain by High Resolution Mass Spectrometry

Endogenous ouabain (EO) and digoxin‐like materials (DILFs) have been identified in the circulation, brain, adrenal glands and urine of mammals. Although EO and DILF appear to be natural products of the mammalian adrenal cortex, their biosynthetic pathway(s) are unknown. Despite overwhelming evidence for EO, some have speculated that EO is a laboratory contaminant (Nicholls et al, J Hypertens 2009;27:3‐8). Arguments for the former view have been rebutted (Manunta et al, J Hypertens 2009;27:9‐18) and additional proof is provided by the present work. We isolated ~6 µg of EO from 6 Kg of bovine adrenal glands. The bioactivity of the bovine EO was followed through serial chromatography by a radioreceptor assay. Using high resolution mass spectrometry, we compared the exact mass and carbon isotope ratios in bovine EO with commercially available plant sources of ouabain (Sigma Chemical Co. and Calbiochem) that were the only other sources of ouabain in the laboratory environment. As expected, high resolution mass spectrometry showed identical exact mass to charge ratios for protonated molecular ions of EO and the two plant ouabains (M+H+ = 585.292 m/z). Crucially, the abundance of 13C in the bovine material (δ‐53.7‰) was significantly lower (p<0.001) than that in the plant ouabains (δ‐22.4‰, ‐16.6‰). We conclude that carbon isotope ratios distinguish bovine EO from that in ouabain synthesizing plants and exclude the possibility that bovine EO is a laboratory contaminant. As the depletion of 13C in bovine EO extends beyond that expected for plant metabolism (‐10 to ‐30‰), EO biosynthesis is likely to involve one or more enzymes with heightened preference for bonds with adjacent 12C atoms. Supported by NHLBI 078870/75584

Presence of ten isoforms of monocarboxylate transporter (MCT) family in the bovine adrenal gland

This study provides novel information regarding the existence and precise cellular localization of various monocarboxylate transporters (MCTs) in the mammalian adrenal gland. RT-PCR results revealed that 10 MCT isoforms, namely MCT1, MCT2, MCT3, MCT4, MCT5, MCT8, MCT9, MCT10, MCT13, and MCT14 are expressed in the bovine adrenal gland. MCTs (MCT1-MCT8) proteins were examined by Western blot analysis in the bovine adrenal gland. The precise cellular localization of six MCT isoforms (MCT1-MCT5 and MCT8) within the different zones of the adrenal gland has been determined by immunohistochemical and immunofluorescence confocal laser microscopy analyses. To gain insight on the species differences for the expression profiles of MCT isoforms in this vital organ, we also examined the expression and cellular localization of MCT1-MCT8 in the rat adrenal gland. Some discrepancies in MCTs profiles between cattle and rat have been observed in the different zones of the adrenal gland. The tissue distribution pattern of MCT isoforms in the steroid-secreting adrenal cortex and catecholamine-secreting adrenal medulla suggests that they may play distinct roles in the regulation of the different hormone biosynthesis in the adrenal gland. Also, it is possible that different MCT isoforms in adrenal gland can be differentially regulated under acute or chronic conditions. This report can form the basis for future research on the regulation of these transporters in the adrenal gland.

PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.

Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.

Primary culture of bovine chromaffin cells

This protocol describes the primary culture of individual chromaffin cells derived by enzymatic digestion from the adrenal medulla of the bovine adrenal gland. Since the late 1970s, such cells have provided a useful model system to study neurotransmitter biosynthesis, storage and release in the catecholaminergic system. The protocol can be divided into three stages: isolation of cells (4-6 h), determination of viable cell numbers (approximately 30 min) and growth in culture (3-7 d). An alternative procedure is to perform studies in a continuous chromaffin (pheochromocytoma) cell line, such as PC12, although such transformed cells are typically less highly differentiated than primary cells. The bovine chromaffin cell procedure should yield approximately 10-20 million cells, suitable for several experiments over the subsequent 3-7 d. Typical experiments involve transmitter biosynthesis, vesicular storage, exocytotic release, stimulus coupling (signal transduction) toward secretion or transcription, or morphology, including ultrastructure. The total time, from adrenal gland harvest until functional experiments, is typically 4-8 d.

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

Protein phosphorylation is a common regulator of enzyme activity. Chemical modification of a protein surface, including phosphorylation, could alter the function of biological electron-transfer reactions. However, the sensitivity of intermolecular electron-transfer kinetics to post-translational protein modifications has not been widely investigated. We have therefore combined experimental and computational studies to assess the potential role of phosphorylation in electron-transfer reactions. We investigated the steroid hydroxylating system from bovine adrenal glands, which consists of adrenodoxin (Adx), adrenodoxin reductase (AdR), and a cytochrome P450, CYP11A1. We focused on the phosphorylation of Adx at Thr-71, since this residue is located in the acidic interaction domain of Adx, and a recent study has demonstrated that this residue is phosphorylated by casein kinase 2 (CK2) in vitro.1 Optical biosensor experiments indicate that the presence of this phosphorylation slightly increases the binding affinity of oxidized Adx with CYP11A1ox but not AdRox. This tendency was confirmed by KA values extracted from Adx concentration-dependent stopped-flow experiments that characterize the interaction between AdRred and Adxox or between Adxred and CYP11A1ox. In addition, acceleration of the electron-transfer kinetics measured with stopped-flow is seen only for the phosphorylated Adx-CYP11A1 reaction. Biphasic reaction kinetics are observed only when Adx is phosphorylated at Thr-71, and the Brownian dynamics (BD) simulations suggest that this phosphorylation may enhance the formation of a secondary Adx-CYP11A1 binding complex that provides an additional electron-transfer pathway with enhanced coupling.

Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells

Secretoneurin (SN) and EM66 are two highly conserved peptides that derive from the processing of secretogranin II (SgII), one of the major constituents of chromaffin cell secretory vesicles. It has been shown that PACAP regulates SgII gene transcription and SN release in bovine adrenochromaffin cells. The aim of the present study was to localize and characterize EM66 in the bovine adrenal gland, and to examine the signaling pathways activated by PACAP to regulate the secretion of EM66 from cultured chromaffin cells. Double immunohistochemical labeling showed an intense EM66-immunoreactive (EM66-IR) signal in TH-positive medullary chromaffin cells of the adrenal gland. HPLC analysis combined with RIA detection revealed, in adrenal medulla extracts and cultured chromaffin cell media, the presence of a major EM66-IR peak co-eluting with the recombinant peptide. PACAP dose-dependently stimulated EM66 release from chromaffin cells (ED 50 = 4.8 nM). The effect of PACAP on EM66 secretion was observed after 6 h of treatment and increased to reach a 2.6-fold stimulation at 48 h. The nonselective calcium channel blocker NiCl 2, the cytosolic calcium chelator BAPTA-AM and the L-type calcium channel blocker nimodipine significantly inhibited the stimulatory effect of PACAP on EM66 release. The secretory response to PACAP was also significantly lowered by the protein kinase A inhibitor H89 and by the protein kinase C inhibitor chelerythrine. Concomitant administration of chelerythrine, H89, NiCl 2 and BAPTA totally abolished PACAP-stimulated EM66 secretion. The MAPK inhibitors U0126 and SB203580 respectively decreased by 63% and 72% PACAP-evoked EM66 release. These results indicate that, in bovine adrenal medulla, SgII is processed to generate the EM66 peptide and that PACAP activates multiple signaling pathways to regulate EM66 release from chromaffin cells, suggesting that EM66 may act downstream of the trans-synaptic stimulation of the adrenal medulla by neurocrine factors.

The Synthesis, Structural Characterization, and Receptor Specificity of the α-Conotoxin Vc1.1

The alpha-conotoxin Vc1.1 is a small disulfide-bonded peptide currently in development as a treatment for neuropathic pain. This study describes the synthesis, determination of the disulfide connectivity, and the determination of the three-dimensional structure of Vc1.1 using NMR spectroscopy. Vc1.1 was shown to inhibit nicotine-evoked membrane currents in isolated bovine chromaffin cells in a concentration-dependent manner and preferentially targets peripheral nicotinic acetylcholine receptor (nAChR) subtypes over central subtypes. Specifically, Vc1.1 is selective for alpha3-containing nAChR subtypes. The three-dimensional structure of Vc1.1 comprises a small alpha-helix spanning residues Pro6 to Asp11 and is braced by the I-III, II-IV disulfide connectivity seen in other alpha-conotoxins. A comparison of the structure of Vc1.1 with other alpha-conotoxins, taken together with nAChR selectivity data, suggests that the conserved proline at position 6 is important for binding, whereas a number of residues in the C-terminal portion of the peptide contribute toward the selectivity. The structure reported here should open new opportunities for further development of Vc1.1 or analogues as analgesic agents.

Identification of Morphine-6-glucuronide in Chromaffin Cell Secretory Granules

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

Ouabain-Like Compound Changes Rapidly on Physical Exercise in Humans and Dogs

Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by (86)Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5+/-0.5 to 86.0+/-27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3+/-1.7 to 129.8+/-51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1+/-1.2 nmol/L and at 30+/-1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7+/-0.8 to 166.9+/-91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the beta1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

Characterization of big stanniocalcin variants in mammalian adipocytes and adrenocortical cells

The hormone stanniocalcin (STC) is widely distributed, and in rodents the highest levels of expression are in the ovaries. In both cows and rodents, ovarian STC consists of three high-molecular-weight variants collectively known as big STC. In the ovary, big STC is made by theca cells and interstitial cells and is targeted to lipid storage droplets of nearby luteal cells to inhibit progesterone release. An endocrine pathway is operative during pregnancy and lactation. Whether or not big STC is made by tissues other than ovary has never been addressed. Therefore, the purpose of this study was to determine via a detailed characterization of adrenal glands and adipocytes whether big STC is present in other cells that store and release lipids. The results showed that STC was made in bovine and mouse adrenals, mainly in steroidogenic, adrenocortical cells. The majority of ligand and receptor were likewise confined to cortical zone cells. As in luteal cells, high levels of ligand and receptor were found in the adrenocortical cell lipid droplet fraction. However, adrenals made only the largest (135 kDa) of the three big STC variants. Nonetheless, adrenal STC had much greater receptor affinity than a mixture of the three big STC variants. Adipocytes contained all three big STC variants, and both ligand and receptor were heavily concentrated on the lipid droplets. Moreover, adipocyte lipid storage droplets had 50-fold more receptors than those in steroidogenic cells, indicating that big STC is heavily targeted to adipose cells. The findings collectively support the hypothesis that big STC is not unique to ovarian steroidogenic cells but is in fact common to cells with a role in lipid storage and release.

Metastatic Tumors to the Adrenal Glands in Domestic Animals

Although metastases to the adrenals are common in humans, they have not been thoroughly studied in animals. The purpose of this retrospective study was to document the types of malignant tumors that metastasize to canine, feline, equine, and bovine adrenals, and the rate at which they do so. The average rate of adrenal involvement in metastatic cancer was 112/534 (21.0%) in dogs, 12/81 (14.8%) in cats, 18/67 (26.9%) in horses, and 5/16 (31.3%) in cattle. In dogs, 26 different tumor types metastasized to the adrenals. Pulmonary, mammary, prostatic, gastric, and pancreatic carcinomas, and melanoma had the highest rates of metastasis to the adrenal glands in dogs. Hemangiosarcoma and melanoma had high rates of adrenal involvement in horses. In cats and cattle, relevant data were only available for lymphoma. Adrenal metastases usually occurred in the late stages of the disease. One dog had developed Addison's disease (hypoadrenocorticism) secondary to lymphoma. Metastatic lesions represented 126/472 (26.7%) of canine, 12/20 (60.0%) of feline, 21/80 (26.3%) of equine, and 5/9 (55.5%) of bovine adrenal neoplasms. This study shows that adrenal glands should be thoroughly examined during both clinical work-up and postmortems when disseminated neoplasia is suspected.

Identification of a Neural Cell Specific Variant of Microtubule-Associated Protein 4

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a region rich in proline and basic residues (proline-rich region). We searched the bovine adrenal gland for MAP4 isoforms, and identified a novel variant lacking 72 consecutive amino acid residues within the proline-rich region, as compared with the full-length MAP4. The amino acid sequence of the missing region was highly conserved (about 85% identity/similarity) among the corresponding regions of bovine, human, mouse, and rat MAP4, which suggested the functional significance of this region. A comparison of the genomic sequence with the cDNA sequence revealed that the missing region is encoded by a single exon. A MAP4 variant cDNA homologous to the bovine form was also detected in rat cells, suggesting that the new variant can be generated by alternative splicing, not only in bovine but also in other mammalian species. The mRNA expression of the novel isoform was restricted to the brain and the adrenal medulla, suggesting that this isoform is specific to a certain cell type. Using a bacterially expressed fragment corresponding to the microtubule-binding domain of the novel isoform, we analyzed its in vitro characteristics. The fragment induced microtubule assembly and bound to preformed microtubules, but the activities were slightly lower than those of the conventional MAP4 fragment, which carries the full-length proline-rich region. The microtubules assembled in the presence of the fragment failed to be bundled. Instead, a constant spacing between neighboring microtubules was observed.