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.

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.

Skin biopsy in Lafora disease: genotype-phenotype correlations and diagnostic pitfalls.

Lafora disease is characterized by pathognomonic inclusions, Lafora bodies (LB), in neurons and other cell types. In skin, LB have been reported in either eccrine sweat glands or in apocrine sweat glands. The disease is caused by mutations in either the EPM2A gene or in a second yet-unknown gene. Here the authors determine whether a genotype-phenotype correlation exists between the genetic form of the disease and the skin cell type affected by LB formation. Also is described an important source of false positivity in the use of axillary biopsies for disease diagnosis.

Laforin is a cell membrane and endoplasmic reticulum-associated protein tyrosine phosphatase.

Lafora disease (LD) is the only progressive myoclonus epilepsy with polyglucosan bodies. Among conditions with polyglucosan bodies, LD is unique for the subcellular location of its polyglucosans in neuronal perikarya and dendrites and not in axons. Here we report that the protein encoded by the EPM2A gene, which is mutated in LD, localizes at the plasma membrane and the endoplasmic reticulum and that it is a functional protein tyrosine phosphatase. The significance of these findings in the epilepsy of LD and in the origin and characteristic subcellular location of Lafora bodies is discussed.

Endogenous Zn(2+) is required for the induction of long-term potentiation at rat hippocampal mossy fiber-CA3 synapses.

The functional role of the abundant Zn(2+) found in some hippocampal synapses has been an enigma. We show here, using N-[6-methoxy-8-quinolyl]-P-toluenesulfonamide (TSQ) staining, that chelatable-Zn(2+) can be removed from hippocampal synaptic boutons using dietary depletion or with Zn(2+) chelators. A chronic dietary deficiency of bouton Zn(2+) resulted in the impairment of long-term potentiation (LTP) at mossy fiber-CA3 synapses. The averaged normalized fEPSP slope 30 min after tetanus was 209 +/- 28% of baseline value in control (mean +/- SEM, n = 10), and 118 +/- 12% in Zn(2+)-deficient rats (mean +/- SEM, n = 12, P < 0.01). In the deficient rats with Zn(2+) supplements, mossy fiber LTP returned to normal levels. The acute depletion of bouton Zn(2+) in the hippocampal slice with membrane-permeable Zn(2+) chelators, dithizone, or diethyldithiocarbamic acid (DEDTC) blocked the induction of mossy fiber LTP. The mean amplitudes of EPSCs after tetanus were 194 +/- 22% of baseline value in control (n = 5), compared to 108 +/- 14% in dithizone (n = 6) and 101 +/- 12% in DEDTC (n = 5). The averaged value of LTP, at the associational commisural fiber-CA3 synapses, was 193 +/- 20% in the control (n = 6), compared to 182 +/- 21% (n = 6, P > 0.1) in the presence of dithizone. The blockade of mossy fiber LTP by dithizone was reversible after washout. In addition, normal LTP could be induced by tetanus if exogenous Zn(2+) was applied immediately following dithizone. Our results indicate that the endogenous Zn(2+) is specifically required for LTP induction at the mossy fiber input into CA3 neurons.

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.

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.

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.

Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin.

Modification of synaptic strength in the mammalian central nervous system (CNS) occurs at both pre- and postsynaptic sites. However, because postsynaptic receptors are likely to be saturated by released transmitter, an increase in the number of active postsynaptic receptors may be a more efficient way of strengthening synaptic efficacy. But there has been no evidence for a rapid recruitment of neurotransmitter receptors to the postsynaptic membrane in the CNS. Here we report that insulin causes the type A gamma-aminobutyric acid (GABA[A]) receptor, the principal receptor that mediates synaptic inhibition in the CNS, to translocate rapidly from the intracellular compartment to the plasma membrane in transfected HEK 293 cells, and that this relocation requires the beta2 subunit of the GABA(A) receptor. In CNS neurons, insulin increases the expression of GABA(A) receptors on the postsynaptic and dendritic membranes. We found that insulin increases the number of functional postsynaptic GABA(A) receptors, thereby increasing the amplitude of the GABA(A)-receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) without altering their time course. These results provide evidence for a rapid recruitment of functional receptors to the postsynaptic plasma membrane, suggesting a fundamental mechanism for the generation of synaptic plasticity.

Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium.

Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Verotoxins (VTs) elaborated by these organisms produce cytopathic effects on a restricted number of cell types, including endothelial cells lining the microvasculature of the bowel and the kidney. Because human intestinal epithelial cells lack the globotriaosylceramide receptor for VT binding, it is unclear how the toxin moves across the intestinal mucosa to the systemic circulation. The aims of this study were to determine the effects of VT-1 on intestinal epithelial cell function and to characterize VT-1 translocation across monolayers of T84 cells, an intestinal epithelial cell line. VT-1 at concentrations up to 1 microgram/ml had no effect on the barrier function of T84 monolayers as assessed by measuring transmonolayer electrical resistance (102 +/- 8% of control monolayers). In contrast, both VT-positive and VT-negative VTEC bacterial strains lowered T84 transmonolayer resistance (45 +/- 7 and 38 +/- 6% of controls, respectively). Comparable amounts of toxin moved across monolayers of T84 cells, exhibiting high-resistance values, as monolayers with VTEC-induced decreases in barrier function, suggesting a transcellular mode of transport. Translocation of VT-1 across T84 monolayers paralleled the movement of a comparably sized protein, horseradish peroxidase. Immunoelectron microscopy confirmed transcellular transport of VT-1, since the toxin was observed within endosomes and associated with specific intracellular targets, including the Golgi network and endoplasmic reticulum. These data present a mode of VT-1 uptake by toxin-insensitive cells and suggest a general mechanism by which bacterial toxins lacking specific intestinal receptors can penetrate the intestinal epithelial barrier.

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.

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

Excess zinc associated with severe progressive cholestasis in Cree and Ojibwa-Cree children.

BACKGROUND: High hepatic copper concentrations have been reported in several liver disorders. We report six Native Canadian children with severe chronic cholestatic liver disease, who had excess hepatic copper and zinc. METHODS: The children, aged 22 months to 8 years, came from northern Ontario, Canada. All were referred for possible liver transplantation because of end-stage liver disease. We examined explanted liver samples (or liver biopsy material in one case) by scanning transmission electronmicroscopic (STEM) X-ray elemental microanalysis and atomic absorption spectrophotometry. Samples from four controls (two with no liver pathology, one with biliary atresia, and one with Wilson's disease) were also analysed by atomic absorption spectrophotometry. FINDINGS: The explanted livers showed similar distinctive signs of advanced biliary cirrhosis, and on electronmicroscopy there were dense deposits in enlarged lysosomes and in cytoplasm. Hepatic copper concentrations were many times higher in the five patients with measurements (47.6-56.9 microgram/g dry weight) than in two samples of normal control liver tissue (2.3 and 2.9 microgram/g). Similarly, hepatic zinc concentrations were many times higher in the patients than in controls (104-128 vs 1.9-3.2 microgram/g dry weight). INTERPRETATION: The excess copper may be due to chronic cholestasis but the excess zinc is unexplained. Since three of the patients are related (shared grandparents), a genetic disorder of metal metabolism is possible, but we cannot exclude environmental factors.

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.

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.

Localization and partial characterization of a 60 kDa citrulline-containing transport form of myelin basic protein from MO3-13 cells and human white matter.

The localization of myelin basic proteins (MBPs) in an immortalized human-human hybrid cell line (MO3-13) formed by fusion of rhabdomyosarcoma TE671-TG6 with primary human oligodendrocytes, cultured from surgical specimens, demonstrated an intracellular localization in vesicles and vacuoles with an intricate internal membranous network and to the external surface of the cell by immunogold electron microscopy. The availability of antibodies to one of the components of MBP, i.e., the citrulline containing component ("C-8"), permitted us to localize this component of MBP to intracellular vacuoles and also on the external surface of the MO3-13 cells. Since the apposition of the external surfaces of the oligodendrocyte is responsible for the intraperiod line of the myelin sheath, localization of C-8 to the external surface of non-permeabilized cells by immunogold scanning electron microscopy is consistent with our observations that C-8 is localized to the intraperiod line of myelin (McLaurin et al.: J Neurosci Res 35:618-628, 1993). Western blots of isolated MBP from MO3-13 cells, probed with an antibody reactive with residues 130-137 of MBP, recognized a protein in the 60 kDa range. No immunoreactivity was found in the 18.5 kDa range. This 60 kDa protein also reacted with a monoclonal antibody raised with residues 70-84 of MBP, 2 different polyclonals raised with whole bovine MBP, an antibody to human MBP raised in monkeys, and the anti-citrulline antibody. These data strongly suggested that the 60 kDa protein contained MBP sequences within its primary structure. A similar protein has been isolated from human myelin-containing fractions but not from compact myelin demonstrating that the 60 kDa protein from MO3-13 cells was not an artefact related to fusion. Sequence determination of peptides obtained from enzymic and chemical cleavages revealed that the 60 kDa protein contained MBP sequences and peptides with 55-60% homology with dynamin, a protein involved in intracellular transport. These data suggest that the externalization of MBP in this cell involves transport by fusion of MBP with another protein. By sequestering MBP in a larger protein, the possibility of inducing autoimmune disease by MBP released, due to cell death, is minimized.

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.

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.