Bimodal variations in subcellular structures of rat thyroid follicular cells during 24 hours: fine structural and morphometric studies

To clarify 24-hr variations in rat thyroid follicular cells under physiological conditions, their subcellular structures were examined at six evenly spaced times during 24 hr by using a morphometric technique. The volume, surface, and numerical densities of subcellular structures varied distinctly over each 24-hr period, with a bimodal pattern. The cellular and nuclear volumes varied also bimodally over 24 hr. A decrease in the surface density of the apical plasmalemma at 1200 and 0000 hr coincided with an increase in volume density of cytoplasmic granules representing colloid droplets and dense bodies. Most granules (colloid droplets) appearing at these times were reduced in electron density. At other times, especially at 1600 and 0400 hr, morphometric parameters of rough endoplasmic reticulum (rER), Golgi complex, and subapical vesicles were prominently increased, although values for rER did not peak at 1600 hr. At these times, the volume densities of cytoplasmic granules, most of which were heterogeneous and of homogeneous electron density, were decreased. These findings coincided with immediate and subsequent reactions of follicular cells after injection of thyroid-stimulating hormone (TSH). From the evidence, it seems likely that variations in follicular cells over a 24-hr period reflect variations in blood TSH concentration. The total membrane areas of membrane components in follicular cells were calculated from the morphometric measurements. These areas fluctuated unimodally during 24 hr over a 65% range. This suggests that the membranes in follicular cells are subjected to cyclic degradation and regeneration during each 24-hr period.     

Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.     

Hydropiperoside,a novel coumaryl glycoside from the root of Polygonum hydropiper

From the methanol extract of the root of Polygonum hydropiper, a novel coumaryl glycoside hydropiperoside was isolated together with anthraquinone, ellagic acid 3,3′-di-O-methyl ether, gallic acid, two quercetin glycosides and an unidentified aromatic δ-lactone possessing antifertility activity. The structure of hydropiperoside was established as β-d-(1,3,6-tri-p-coumaryl)-fructofuranosyl-α-d-glucopyranoside by combination of extensive ¹H NMR and ¹³C NMR spectra, and the FD/MS spectrum.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Activation of murine T cells by streptococcal pyrogenic exotoxin type A. Requirement for MHC class II molecules on accessory cells and identification of V beta elements in T cell receptor of toxin-reactive T cells

We investigated the mechanisms of murine T cell activation by streptococcal pyrogenic exotoxin type A (SPE A), focusing on the role of MHC class II molecules on accessory cells (AC) and V beta usage in alpha beta TCR of SPE A-reactive T cells in comparison with staphylococcal enterotoxin B-reactive T cells. L cells transfected with I-Ab genes functioned as effective AC for SPE A-induced responses by C57BL/6 T cells, proliferation, and IL-2 production, but control L cells were not effective AC. Anti-I-Ab mAb inhibited the SPE A-induced responses. Staphylococcal enterotoxin B-induced C57BL/6 T cell blasts were composed of cells bearing V beta 3, members of the V beta 8 family, and V beta 11. Most of the SPE A-induced T cell blasts (about 80%) bore V beta 8.2. mAb reactive to V beta 8.2 markedly inhibited SPE A-induced T cell responses. Apparently, SPE A activates mainly T cells bearing V beta 8.2 in physical association with MHC class II molecules expressed on AC. We also discuss the pathogenic activities of SPE A in relation to toxic shock syndrome.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

Activation of murine T cells by toxic shock syndrome toxin-1. The toxin-binding structures expressed on murine accessory cells are MHC class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding structures present on murine lymphoid tissues were investigated by using 125I-TSST-1. T-depleted C57BL/6 spleen cells incubated with TSST-1 for 3 h at 0 degree C were mitogenic to splenic T cells, indicating that the former cells bind and present TSST-1 to T cells. TSST-1-binding activity was observed in C57BL/6 splenic B cells and L cells transfected with I-Ab genes, but not in splenic T cells and control L cells. Scatchard plot analysis showed that these B cells and transfectants bound TSST-1 with similar binding affinity. SDS-PAGE analysis showed that lysates of C57BL/6 spleen cells and the I-Ab-positive transfectants contain a single band which bound TSST-1 and comigrated with I-Ab heterodimers. TSST-1-binding activity observed clearly in C57BL/6. BALB/c, and C3H/HeN spleen cells and L cells transfected with I-Ab or I-Ak genes was not reduced by paraformaldehyde fixation. Binding of 125I-TSST-1 to the three spleen cells was markedly reduced by anti-I-A antibodies, but not by anti-I-E antibodies. C57BL/6, C3H/HeN, and (C3H/HeN x C57BL/6) F1 T cells were activated by TSST-1 to proliferate and produce IL-2 in the presence of FT6.2 cells, LT1-30-3 cells and either of them, respectively, but not in the presence of control L cells. These results indicate that I-A molecules function as the structures via that accessory cells directly bind TSST-1 on the cell surface and present a triggering signal of TSST-1 to T cells.