Muscular involvement in lysosomal acid lipase deficiency in rats.

We investigated the pathological and biochemical changes of skeletal muscle in rats with lysosomal acid lipase deficiency, which is an animal counterpart of human Wolman's disease. In the affected rats, the acid lipase activity for three different substrates, 4-methylumbelliferyl-oleate (18.9% of the normal control level), [14C]cholesteryl oleate (23.5%), and [14C]triolein (26.9%), was similarly decreased in the lysosomal fraction of skeletal muscle which was obtained by differential centrifugation. Histochemical studies showed that acid phosphatase activity was high in the endomysium and perimysium and in some muscle fibers. Some fibers showed vacuolar degeneration resembling "rimmed vacuoles". Ultrastructural studies demonstrated many membrane-bound lipid droplets in the muscle fibers, especially in the subsarcolemmal space, indicating that a low density lipoprotein (LDL) uptake pathway apparently existed in the muscle cells. However, such lipid accumulation was much greater in the interstitial cells and the endothelial cells. This distribution also suggests that LDL/cholesterol is supplied to muscle cells predominantly through endothelial cells.     

Neurolysis of celiac plexus and splanchnic nerve under ultrasonographic guidance

Key words  intractable pain - celiac plexus neurolysis - ultrasonography     

Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease

Metal-catalyzed hydroxyl radicals are potent mediators of cellular injury, affecting every category of macromolecule, and are central to the oxidative injury hypothesis of Alzheimer disease (AD) pathogenesis. Studies on redox-competent copper and iron indicate that redox activity in AD resides exclusively within the neuronal cytosol and that chelation with deferoxamine, DTPA, or, more recently, iodochlorhydroxyquin, removes this activity. We have also found that while proteins that accumulate in AD possess metal-binding sites, metal-associated cellular redox activity is primarily dependent on metals associated with nucleic acid, specifically cytoplasmic RNA. These findings indicate aberrations in iron homeostasis that, we suspect, arise primarily from heme, since heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in AD, and mitochondria, since mitochondria turnover, mitochondrial DNA, and cytochrome C oxidative activity are all increased in AD. These findings, as well as studies demonstrating a reduction in microtubule density in AD neurons, suggest that mitochondrial dysfunction, acting in concert with cytoskeletal pathology, serves to increase redox-active heavy metals and initiates a cascade of abnormal events culminating in AD pathology.     

Modified Dynabeads method for enumerating CD4+ T-lymphocyte count for widespread use in resource-limited situations

The Dynabeads method showed the potential for enumerating CD4 T lymphocytes (CD4 count) in HIV-1-infected individuals. The large volume of Dynabeads required for 1 sample and complex procedure made the method expensive and not easy for use, however. To decrease the cost and simplify the procedure, we reduced the volume of the Dynabeads, added wash times, and skipped over the staining step so as to count the CD4 cells directly under an optical microscope. The CD4 count of 246 blood samples using our modified Dynabeads method (DynabeadsCD4) showed a significant correlation with that obtained by flow cytometry (FlowcytoCD4) (r = 0.91 [P < 0.0001]; slope = 1.03, intercept = -16). The sensitivity and specificity for a CD4 count less than 200 cells/microL were 79% and 94%, and for a CD4 count less than 350 cells/microL, the sensitivity and specificity were 95% and 88%, respectively. The positive and negative predictive values for a CD4 count less than 350 cells/microL were 97% and 83%, respectively. The systematic error was 8 cells/microL (95% confidence interval [CI]: 0.4-16). The cost of Dynabeads for 1 sample was less than $1.00; thus, the estimated cost per DynabeadsCD4 test is less than $3.00, including the cost of other disposable materials. Our modified method is simple, economic, and accurate enough to monitor antiretroviral therapy in resource-limited situations.     

Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway

Rat incisors grow continuously throughout life. Producing a variety of dental epithelial cells is performed by stem cells located in the cervical loop of the incisor apex. To study the mechanisms for cell differentiation, we established a dental epithelial cell line (HAT-7) originating from a cervical loop epithelium of a rat incisor. Immunochemical studies showed that HAT-7 produced the cells expressing amelogenin, ameloblastin, or alkaline phosphatase (ALP). To illustrate a role of Notch signaling in the determinant of the cell fate, we examined expression patterns of Notch1 and Jagged1 in HAT-7 density dependently. At lower cell density, Notch1- or Jagged1-expressing cells were not seen. However, when they were fully confluent, cells began to express Notch1 or Jagged1 strongly. Some ALP-positive cells were almost consistent with Notch1-expressing cells but not Jagged1-expressing cells. These results suggested that the determinant of direction of differentiation was associated with Notch signaling pathway.     

Priming effect of RANTES on eosinophil oxidative metabolism

Background RANTES has been shown to possess chemotactic activity for eosinophils, which have also been considered to play a role in allergic inflammation through reactive oxygen species. Thus, in this study, we examined the effect of RANTES on radical oxygen products from eosinophils.
Methods Purified eosinophils by CD16-negative selection or an eosinophilic cell line (EoL-1) were incubated with or without RANTES (2.5 x 10⁻⁶). To the mixture of eosinophils and luminol, calcium ionophore (A23187) or opsonized zymosan (OZ) was added, and radical oxygen products were determined by luminol-dependent chemiluminescence for 600 s.
Results Eosinophil-mediated radical oxygen products of untreated eosinophils produced with A23187 gave a peak value of 14.09 + 2.40 (mean±SE, n = 12) relative light units (RLU) and an integrated value of 3232.20 + 513.09 RLU. However, with treatment with RANTES, a peak value of 18.66 + 2.40 RLU and an integrated value of 5301.05 ±561.02 RLU were obtained. Eosinophil oxidative metabolism-induced A23187 or OZ was apparently augmented by the preincubation with RANTES. In addition, the radical oxygen products of EoL-1 showed similar results.
Conclusions Thus, we concluded that RANTES may play an important role the pathogenesis of allergic inflammation through its involvement in eosinophil activation, as evidenced by oxygen products, as well as in selective eosinophil infiltration as selective eosinophil chemoattractant.     

Glomerular expression of cell-cycle-regulatory proteins in human crescentic glomerulonephritis

To elucidate the mechanism underlying crescentic formation, we assessed the phenotypic characterization and cell-cycle protein expression in human crescentic glomerulonephritis (CRGN). Kidney tissue specimens taken from CRGN patients (10 patients with pauci-immune type rapidly progressive glomerulonephritis (RPGN), 2 patients with Henoch-Schönlein purpura nephritis, and 1 patient with IgA nephropathy) were examined immunohistochemically. Most of the cellular components of the crescents expressed cytokeratin, whereas few cells expressed PHM-5. CD68-positive cells were minor components of cellular crescents, indicating that the major principal cellular component of the crescents is made up of cells with the parietal glomerular epithelial cell (PEC) phenotype. Additionally, serial section analysis revealed that Ki-67-positive cells in the crescents were frequently cyclin-A positive and Bcl-2 positive, but seldom cyclin-B₁ positive. Moreover, the expression of cyclin-dependent kinase inhibitor p27^Kip1 was low in the cellular crescents, despite being exclusively positive in podocytes within the same section. We concluded that the major component of the cellular crescents is made up of PECs and that apparent expression of cyclins and Bcl-2 and restrained expression of p27^Kip1 may be synergistically associated with the development of cellular crescents in human CRGN.     

Demonstration of cholera toxin-related factor in cultures of Aeromonas species by enzyme-linked immunosorbent assay.

The production of toxins by Aeromonas species was examined by the suckling mouse test, the hemolysin test, and the enzyme-linked immunosorbent assay with anticholera enterotoxin. A factor that was immunologically related to cholera enterotoxin was produced by 5 of 14 strains of Aeromonas hydrophila and 4 of 15 strains of Aeromonas sobria. Analysis by these assays and by a test for heat stability suggested that the factor differed from hemolysin and from toxin that was active in the suckling mouse test.     

A mutant toxin of Vibrio parahaemolyticus thermostable direct hemolysin which has lost hemolytic activity but retains ability to bind to erythrocytes.

A mutant toxin, R7, of thermostable direct hemolysin (TDH) with a single amino acid substitution at glycine 62 was analyzed. The hemolytic activity of R7 decreased to less than 1/1,000 of that of wild-type TDH, and its mouse lethality was undetectable. This mutant toxin, however, showed a marked inhibitory effect on hemolysis by wild-type TDH. Enzyme immunoassay and flow cytometric analysis demonstrated that R7 retained approximately 50% of the ability to bind to erythrocytes compared with that of wild-type TDH, suggesting that its inhibition of hemolysis by wild-type TDH might be due to blocking the binding sites on the erythrocyte membrane. Wild-type TDH affected the erythrocyte membrane by causing an influx of calcium and propidium iodide, while R7 showed no detectable effects of these kinds. These results suggest that hemolysis by TDH consists of at least two steps, binding and postbinding, and that R7 is likely to be a postbinding activity-deficient mutant toxin of TDH.