Clara cell secretory protein is reduced in equine recurrent airway obstruction.

Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses.

Modifications of myelin basic protein in DM20 transgenic mice are similar to those in myelin basic protein from multiple sclerosis.

Transgenic mice containing different numbers of transgenes (2-70) of the myelin proteolipid protein DM20 were phenotypically normal up to 3 mo of age, after which the mice containing 70 copies of the transgene spontaneously demyelinated and died at 10-12 mo. Since we demonstrated that demyelination in multiple sclerosis involved specific chemical changes in myelin basic protein (MBP), we investigated the MBP in our transgenic line for similar changes. Both the total amount of MBP in brain and the MBP mRNA levels were unaffected at the different ages. All the isoforms (14-21 kD) of MBP were present, but the microheterogeneity (a posttranslational event) was changed resulting in a higher proportion of the less cationic components reminiscent of the changes in MBP found in multiple sclerosis. An increased amount of the citrullinated form of MBP was found by Western blot analysis. Immunogold labeling of cryosections of brain revealed a greater density of particles with the anticitrulline antibody at 10 mo and that the levels of peptidylarginine deiminase (which deiminates protein-bound arginine to citrulline) were increased. This stable transgenic line represents a useful animal model for the human disease multiple sclerosis.

Gene expression of islet cell antigen p69 in human, mouse, and rat.

Based on the detection of specific antibodies and T-cell sensitization in patients with IDDM, islet cell antigen p69 (ICAp69) has been suggested to be a target antigen of diabetic autoimmunity. The biological function, tissue expression, and developmental kinetics of ICAp69 are largely unknown. We analyzed ICAp69 expression at the gene transcription and protein level in human and rodent tissues. By using template-calibrated quantitative reverse transcriptase polymerase chain reaction (RT-PCR), high levels of ICAp69 mRNA were found in human pancreatic islets and brain. In mouse and rat, ICAp69 gene expression peaked in islet cell lines followed by testis, islets, and brain. ICAp69 mRNA was found at low levels in other organs by RT-PCR but not by Northern blot analysis. In mice, ICAp69 transcription becomes detectable in fetal life, and fetal and adult gene expression patterns are similar. Western blot analysis of human and mouse tissues showed high expression of ICAp69 in brain, testis, pancreatic tissue, and islet cell lines. In these organs, ICAp69 immunoreactivity is predominately localized at the blood brain barrier (capillary endothelium), at the blood testis barrier (Sertoli cells and spermatids), and in pancreatic islets (beta-cells). The subcellular localization of ICAp69 to endoplasmic reticulum, Golgi complex, and vesicles by immune electron microscopy suggests a role of this neuroendocrine molecule in cellular protein traffic and processing.off

Increased expression of CD44 on astrocytoma cells induced by binding myelin basic protein.

An astrocytoma cell line (HTB-14), expressing high amounts of a CD44 variant compared to other astrocytoma lines was shown to bind myelin basic protein to a greater extent than low expressing lines in a concentration-dependent manner. The CD44 variant expressed by HTB-14 cells was determined to migrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 100 kDa compared to that from white matter which had a molecular mass of 80 kDa. The most cationic component of myelin basic protein (MBP), (component 1) bound more avidly than the least cationic isomer (component 8). Internalization of MBP was demonstrated by immunogold electron microscopy and was localized to the perinuclear area with some gold particles in the cytoplasm but not near the plasma membrane. Colocalization with glial fibrillary acid protein suggested an interaction between these two molecules. Binding and internalization of MBP was accompanied by an increase in CD44 as determined by quantitation of gold particles and the measurement of CD44 by sandwich enzyme-linked immunosorbent assay. The implication of these studies for the mechanism of demyelination is discussed.

Identification of a mitogen-activated protein kinase site in human myelin basic protein in situ.

Ultrastructural localization of a specific phosphorylated isomer of myelin basic protein (MBP) has been achieved with a monoclonal antibody specific for human MBP sequence, 89-105, in which Thr98 was phosphorylated. Cryosections of human brain white matter revealed that gold particles were found localized almost exclusively to the major dense line demonstrating that threonine 98 in the sequence Thr-Pro-Arg-Thr-Pro-Pro-Pro, a mitogen-activated protein kinase-specific site, was phosphorylated in vivo. In two cases of multiple sclerosis, the density of gold particles in myelin was reduced by about 30%, in one case by 42%, and by 80% in a fourth case. However, gold labelling was seen in areas of demyelination suggesting that the phosphorylated threonyl peptide was protected from degradation.

Localization of CD44 (P80) on the external surface of a human astrocytoma cell.

A brain antigen, originally identified by a MAb 44D10, has been shown to be a glycoprotein with an Mr of 80 kDa. Cellular localization studies of sections of brain showed that the antigen was associated with the membrane of astrocytes. In the present study we demonstrate its localization to the membrane of an astrocytoma cell line by fluorescence and electron microscopic immunogold methods. Heavy labelling with immunogold was found along cellular processes. Labelling of the smooth surfaces of cells and the microvilli was also observed.

Clara cell secretory protein increases phagocytic and decreases oxidative activity of neutrophils.

Horses suffer from recurrent airway obstruction, an asthma-like condition induced by repeat inhalation of environmental substances present in barn air. Clara cell secretory protein (CCSP) is much reduced during active inflammation when neutrophils predominate in the airways, and in chronic asthmatics. We sought to investigate morphologic and functional interactions of CCSP with neutrophils. Bronchoalveolar and blood neutrophils from healthy control animals, and from animals with recurrent airway obstruction in remission and exacerbation, were evaluated by immuno-cytochemistry and immuno-electron microscopy for presence of CCSP. Blood neutrophil oxidative burst and phagocytic activities were determined in the presence of different concentrations of recombinant equine CCSP. Bronchoalveolar lavage neutrophils from horses with exacerbated lung inflammation, but not from control horses, and not blood neutrophils from either group of animal, contained abundant immunoreactive CCSP. On immuno-electron microscopy, CCSP localized to the cytoplasm and nucleus. Incubation of blood neutrophils with CCSP significantly reduced oxidative burst activity (P<0.0001) and increased phagocytosis (P<0.001) of neutrophils. These findings indicate that CCSP enters neutrophils in horses with active neutrophilic lung inflammation and alters the function of neutrophils in blood. Presence in the nucleus suggests a potential transcriptional role of CCSP in neutrophils.

Demonstration of oestrogens in developing pig trophectoderm and yolk sac endoderm between days 10 and 16.

Segments of individual blastocysts collected on Days 10, 12, 14 and 16 were examined microscopically to observe yolk-sac development and treated immunocytochemically to localize oestrogens in specific membranes. Mesoderm was present beneath the embryonic disc of ovoid blastocysts on Day 12. The mesoderm spread beyond 1 cm from the disc on Day 14, producing a splanchnic yolk-sac membrane extending across the blastocoelomic cavity, but no mesodermal cells had yet reached 5 cm. By Day 16, proliferation of mesoderm and development of the yolk sac had progressed beyond 20 cm from the disc in most of the specimens examined. Incubation of ultrathin sections with sheep antiserum to oestrone or oestradiol-17 beta followed by rabbit anti-ovine IgG-gold complex and subsequent counting of gold particles retained over the tissues gave a weakly positive reaction for oestrone in trophectodermal cells on Day 10. The most intense reaction for oestradiol-17 beta was also present in the trophectoderm and yolk-sac endoderm on Days 12, 14 and 16.

Structure-function relationships during preovulatory development of porcine follicles following equine chorionic gonadotropin stimulation.

Porcine follicular maturation begins by recruitment from a continually proliferating pool of small antral follicles; those receiving the appropriate stimulus differentiate rapidly through a series of structural and functional changes. Such ovarian activity can be induced in prepubertal gilts with a single injection of equine chorionic gonadotropin (eCG). Average follicular diameter in eCG treated females increased from approximately 2 mm before stimulation to 3.5 mm by 24 hr after injection, with subsequent growth to ovulatory size (8 or 9 mm) by 96 hr. Both theca and granulosa layers increased in thickness and complexity, and a prominent capillary bed evolved immediately outside the basement membrane separating the two layers. Cytoplasmic organelles associated with increased metabolic activity and steroidogenesis proliferated within the first 24 hr. Progressive changes included increasing amounts of lipid and rough and smooth endoplasmic reticulum, with the latter occurring in vesicular or lamellar forms and as lipid-associated whorls. Bizarre mitochondrial forms also appeared, often associated with lipids. The amount and proportion of rough and smooth endoplasmic reticulum shifted dramatically as follicles matured. By 24 hr, rough endoplasmic reticulum in thecal cells increased from 4.2 to 7% of cell volume, while the amount in granulosa cells increased from less than 3.5% to more than 10%; the quantity remained relatively constant in the theca but declined to prestimulation values in the granulosa layer. Rough endoplasmic reticulum predominated over smooth in the first 24 hr following stimulation but the proportions were then reversed, so that more than 10% of both layers was composed of smooth endoplasmic reticulum by the time ovulation was imminent. Some follicles had or were in the process of ovulating by 96 hr. Their walls were collapsed into prominent folds with the two cell types beginning to mix. Slight undulations and some regions of discontinuity were observed in basement membranes of large unovulated follicles at this time. In specimens collected at 96 hr poststimulation and processed for retention of lipid, lipid-like material was noticeable in the extracellular matrix surrounding cells that contained organelle configurations suggestive of steroidogenesis.

Localization of basic proteins in human myelin.

The myelin basic protein (MBPs) represent a family of proteins (charge isomers) which account for 35% of the total myelin protein. Localization studies have been inconclusive because MBP is not a single protein. Antibodies obtained by injection of MBP into animals recognized all members of the MBP family. In the studies reported here, we have fractionated the MBPs into specific components or charge isomers. One of these which contains citrulline accounts for about 20% of the total MBP. We report the localization of this single MBP to the intraperiod line of myelin by immunoelectron microscopy. For these studies several specific antibodies were used including antibodies raised against total MBP, specific MBP peptides, and against a tetracitrulline peptide. This latter antibody was specific for component 8 (C-8) of MBP. Since C-8 is the only MBP which contains citrulline it was used to localize this particular form of MBP principally to the intraperiod line by immunogold electron microscopy, while antibody against total MBP (consisting of all charge isomers C-1-->C-8) labelled both the major dense line and the intraperiod line. When the anti-citrulline antibody was used with a 3 nm gold conjugated Fab fragments prepared from the secondary antibody, 66.5% of the gold particles were localized to the intraperiod line, while 11.2% of gold particles were localized to the major dense line. On the other hand, with the monoclonal anti-MBP antibodies reactive with residues 69-74, 59.4% of the gold particles were localized to the major dense line and 23.6% of gold particles at the intraperiod line. Other supporting evidence includes increased labelling of myelin by 125I labelled anti-citrulline IgG when isolated myelin was swollen, a process known to take place at the intraperiod line. Gold particles were demonstrated at the intraperiod line in swollen and recompacted myelin. C-8 was shown to associate preferentially with lipids asymmetrically localized to the intraperiod line.

Tuberin expression in ganglioglioma.

The expression of tuberin in neurosurgically resected gangliogliomas was investigated. Neither neoplastic astrocytes nor abnormal, multipolar large neurons showed immunoreactivity for tuberin. The reduced immuno-histochemical staining for tuberin was consistent with the reduction observed in Western blot analysis. Eosinophilic granular bodies and Rosenthal fibers were strongly immunoreactive for tuberin. The accumulation of tuberin in astrocytes with intracellular degenerative changes suggests increased expression in reactive cells, and perhaps a broader role for tuberin in central nervous system disease.

The structure and organization of the bile canalicular cytoskeleton with special reference to actin and actin-binding proteins.

The distribution of actin filaments and actin-binding proteins in the bile canaliculus (BC) of normal human hepatocytes was determined as a means of establishing the structure and organization of the BC cytoskeleton. Immunoblots demonstrated that actin, and the actin-binding proteins, myosin II, tropomyosin, vinculin, alpha-actinin, villin, were present, as were the non-actin-related proteins beta-tubulin, and cytokeratins. Three actin filament regions were identified: microvillus core filaments, a membrane-associated microfilamentous network, and a circumferential pericanalicular actin filament band. Actin-binding proteins were nonrandomly associated with actin in these regions. In the case of the pericanalicular band, there was also association with the zonula adherens junction. Intermediate filaments inserted into desmosomes. The ultrastructural localization of the actin-binding proteins was fundamentally linked to the arrangement and organization of the major canaliculus-associated microfilament structures. Structural organization of the cytoskeleton was also linked to distinct components of the intercellular junctions. It is notable that tropomyosin and a-actinin, which in muscle cells are regulatory proteins of contractile activity, and myosin II are associated with the pericanalicular actin microfilament band; it is the BC counterpart of the contractile actin filament band found in the apical region of other secretory cells. The outer sheath of noncontractile intermediate filaments likely stabilizes the canalicular compartment.

Loss of the TSC2 product tuberin in subependymal giant-cell tumors.

We investigated immunocytochemically the expression of tuberin, the TSC2 gene product, in brain resections from children with and without tuberous sclerosis, to characterize the phenotype of balloon and tumor cells, and to elucidate the relationship between tuberin and formation of subependymal giant-cell tumors. In cortical tubers, tuberin was expressed in processes and cell bodies of balloon cells, which also showed consistent vimentin and nestin immunoreactivity, but no glial fibrillary acidic protein, neurofilament, or galactocerebroside positivity by immunofluorescence confocal microscopy. The majority of balloon cells in white matter showed weaker tuberin immunoreactivity than similar cells in cortical tubers. In subependymal giant-cell tumors, there was minimal to no immunoreactivity. Why tuberin is expressed in tubers but not in subependymal giant-cell tumors is not known. If tuberin plays a role in tumor suppression, then lack of tuberin might be a marker for deletion of both alleles, leading to enhanced cellular growth in subependymal giant-cell hamartomas, eventually of sufficient size to cause clinical symptoms.

Scanning electron, light optical, and transmission electron microscopy and plasma steroid values of porcine uterus at lactation.

Sows, while nursing litters for a period of 4 to 8 weeks, do not ovulate, thereby delaying the time sows can be rebred. Therefore, this study sought to determine how soon after parturition the morphological appearance of the uterus was such that blastocyst implantation could possibly proceed. Biopsy samples taken immediately post-partum and on days 1, 2, 3, 4 post-partum show that the endometrium was considerably damaged as evidenced by sloughing of the zona compacta and zona spongiosa and the presence of leukocytes around the surface epithelial (SE) cells. Around the 6th day post-partum the numbers of leukocytes were reduced and regeneration of the SE was evident. Around the 8th day post-partum, SE cells appeared similar to those of normal cyclic sows in the late luteal phase (i.e. 15 days post-estrum). Under normal breeding conditions, blastocyst implantation takes place during the late luteal phase. From 17 to 26 days post-partum the SE cells appeared similar to those of normal cyclic sows in the early follicular phase (i.e. 21 days post-estrum), and the subepithelial layers seemed edematous. Estradiol and progesterone concentrations dropped markedly at parturition and remained low up to the 26th day of lactation. Although there was considerable heterogeneity of samples between sows it was concluded, from the morphological results, that plasma steroid levels exert relatively little effect on the sow endometrium up to 26 days post-partum. If ovulation could be induced or ova transplant be performed in lactating sows the endometrium would appear ready for blastocyst implantation after day 8 post-partum.

Copper-induced myopathy in rabbits: ultrastructural observations.

Mature rabbits were injected intra-muscularly into the hamstring (HS) muscles with doses of 1.66 mg Cu/kg/day, as cupric acetate, in aqueous solution. Similar doses of Na acetate were injected into control rabbits. Skeletal (HS) and cardiac (MYO) muscles were examined by scanning electron microscopy, transmission electron microscopy and a microanalyzer and energy dispersive X-ray (MEDX). Na-injected rabbits showed slight myofibrillar lesions in HS after four injections, and mitochondrial inclusions in MYO were noted after two or seven injections. No Cu was found with MEDX. Cu-injected rabbits showed that the severity of lesions of myofibrillar degeneration and mitochondrial inclusions in HS increased with the number of injections and days the rabbits had been on experiment. After seven days on experiment signs of muscle regeneration were seen. Cu was found first to localize in the nucleus of HS after one day, thereafter it localized in the mitochondria after three and seven days. MYO lesions were found in the mitochondria after six or seven days after Cu-injection. With MEDX less Cu was found than had been found in HS. This Cu first localized in the nucleus of MYO after one day and then disappeared from the tissue altogether.

Cadmium encephalopathy: a report with elemental analysis and pathological findings.

We report a boy of East Indian origin, aged 2 years and 10 months, who died suddenly and unexpectedly. Autopsy findings showed marked cerebral swelling with herniation and histological evidence of marked cerebral edema with perivascular protein leakage, indicating blood-brain barrier disruption. Energy dispersive X-ray microprobe analysis of the brain demonstrated the presence of cadmium and a marked increase in sulfur, predominantly intracellular, both within neuroglial, and to a lesser degree endothelial, cells. Localization was predominantly in the nucleus. Analysis of the kidney showed cadmium deposition in renal tubules and in the basal lamina of podocytes within the glomerulus. Although the environmental source of cadmium remains unknown, we speculate that acute cadmium toxicity led to brain intracellular accumulation with resultant cellular dysfunction, blood-brain barrier disruption, and lethal cerebral edema.

Demyelination in a transgenic mouse: a model for multiple sclerosis.

A transgenic mouse containing 70 copies (ND4) of the transgene encoding DM20, a myelin proteolipid protein, appeared clinically normal up to 3 months of age. By 8-10 months, it showed tremors, unsteady gait, and died shortly thereafter. We concluded that the clinical symptoms correlated with demyelination based on the following criteria: 1) at 10 months of age only 17% of the amount of myelin obtained from normal mice was isolated from the ND4 mice; 2) astrogliosis, a prominent feature of demyelinating disease was minimal at 3 months of age but prominent by 10 months; 3) at the electron microscopic level disrupted myelin was seen at 8 months of age in the ND4 mice and ingested myelin debris was found in astrocytes; 4) lymphocytic infiltration in association with endothelial cells was observed routinely in the ND4 mice; 5) sections through optic nerves showed denuded and thinly myelinated axons in the 8 month old ND4 mice. Although the mechanism by which demyelination takes place is not fully understood, measurements of the amounts of PLP suggest it is down-regulated by the large amount of DM20. Since DM20 is a major proteolipid in the young but a minor one in the adult, the persistence of high levels in the adult results in improperly assembled myelin which is prone to disruption. Therefore demyelination in the ND4 mouse appears to result from the persistence of immature myelin into the adult.

The use of silver nitrate staining and backscattered electron imaging to visualize nematode sensory structures.

Parasitic nematodes of the species Cosmocercoides variabilis were stained with silver nitrate and examined with backscattered electron imaging (BEI). Sensory papillae were selectively highlighted in backscatter images. Silver stain deposited on papillae was located on the papillary surface as well as on the underlying dendritic process. Portions of the body cuticle were also stained. Some cuticular staining was attributed to non-specific deposition of silver but, consistent patterns of cuticular staining were noted in the anterior and posterior regions. This observation suggests that some staining of the cuticle was specific. Results of this preliminary work suggest that BEI is a technique useful to the study of nematode form.

Loss of myelin basic protein cationicity in DM20 transgenic mice is dosage dependent.

Demyelination in the transgenic mice depended on the dosage of the cDNA for DM20, in which low copy numbers (two to four and 17 copies of the minigene) showed no signs of demyelination. However when transgenic mice with 17 copies were made homozygous with 34 copies of the DM20 minigene (ND3A hm.) demyelination was observed at around 12 to 16 months compared with ND4 mice having 70 copies of the transgene which had an earlier onset of demyelinating symptoms at 3 months, demonstrating a transgene dosage effect. The process by which demyelination was initiated was associated with changes in myelin basic protein. An increased abundance of less cationic MBP (C-8) isomers occurred prior to demyelination. This increase was also associated with increased activity of peptidylarginine deiminase, the enzyme which converts arginine to citrulline in proteins, thereby providing a mechanism for generating less cationic forms of MBP. These data support a dosage effect of the DM20 transgene.