Heterogeneity of β2-Microglobulin in Human Urine

Purification and characterization of β₂-microglobulin from human urine was performed. The yield was 30.1%, and 150.4 mg of β₂-microglobulin was obtained. The final preparation of β₂-microglobulin obtained showed three bands on disc gel electrophoresis at pH 9.5, and all of them have immunological activity. However, these three bands migrated as a single band on disc gel electrophoresis at pH 4.3. It is concluded that the three bands observed on disc gel electrophoresis at pH 9.5 were charge isomers. The isoelectric points of isomers were determined by isotachophoresis and two of them were 5.4 and 5.9 respectively, while the other one was not determined.     

Tissue distribution of constitutive proteasomes, immunoproteasomes, and PA28 in rats

We investigated the expression of standard proteasomes, immunoproteasomes, and their regulators, PA28, and PA700, in rat tissues. Immunoproteasomes (with subunits LMP2, LMP7, and MECL1) were abundant in the spleen but almost absent in the brain. In contrast, standard proteasomes (with X, Y, and Z) were highly expressed in the brain but not in the spleen. Both proteasome types were present in the lung and the liver. PA700 subunits (p112, S5a, and p45) were found in all tissues. PA28alpha, PA28beta, and PA28gamma were also expressed in all tissues, except for the brain which contained very little PA28beta. The results did not depend on rat sex or age. The cleavage specificity for peptide substrates differed greatly between brain and spleen proteasomes. Hybrid proteasomes, containing both PA28alphabeta and PA700, were not present in the brain but in all other tissues examined.     

Contribution of potential organic matter sources to the macrobenthos nutrition in the Natori estuarine tidal flats,Japan

In this investigation, we used stable isotope and fatty acid biomarker analyses to estimate and compare the potential food sources that support macrobenthos (Nuttallia olivacea, Corbicula japonica, and Hediste sp.) in the Natori estuarine tidal flats of Japan. The δ¹³C and δ¹⁵N mean values for the sediment organic matter (SOM) were ?23.6‰ and 6.1‰, respectively, which were due to the contribution of terrestrial and/or aquatic vascular plant particulate organic matter (POM) from upper stream river or surrounding areas. Furthermore, from the results of the IsoSource mixing model, the contributions of estuarine POM to the diets of Hediste sp., C. japonica, and N. olivacea were 85.1%, 74.9%, and 48.9%, respectively. Moreover, essential fatty acids such as 20:5ω3, 18:2ω6 and 18:3ω3 highly contributed to the diets of macrobenthos from benthic diatoms, terrestrial and/or aquatic vascular plants. The contents of fatty acid markers of terrestrial OM (e.g., long chain fatty acids [LCFAs]) in the 3 species of macrobenthos were low in comparison to those of other food sources. Overall, the marine POM dietary contribution was minimal, while terrestrial OM, bacteria, and benthic diatoms constituted a significant portion of the macrobenthos diet, although the contribution varied among the 3 species of macrobenthos.     

Effects of hemagglutinin-neuraminidase protein mutations on cell-cell fusion mediated by human parainfluenza type 2 virus

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.     

Properties and repeated use of a reversibly soluble-insoluble yeast lytic enzyme

Summary A yeast lytic enzyme was covalently immobilized on an enteric coating polymer, Eudragit S, that is reversibly soluble and insoluble (S-IS) depending on the pH of the reaction medium. The yeast lytic enzyme immobilized on Eudragit S (Y-E) showed a sharp response of solubility to slight changes in pH without decrease in enzymatic activity. The specific activity per amount of enzyme protein of Y-E for dry yeast cells was about two-thirds that of the native enzyme. In both lysis reactions of dry and pressed baker's yeast cells, changing the pH of the reaction medium from 7.0 to 4.8 at an appropriate interval allows the insoluble Y-E and the reaction products (soluble protein for dry yeast cells and invertase and soluble protein for pressed baker's yeast cells) to be repeatedly separated. The reaction method using a reversible S-IS enzyme is a promising procedure for repeated use of the enzyme in a heterogeneous reaction system containing yeast cells as a substrate.     

ATP-Dependent Inactivation and Sequestration of Ornithine Decarboxylase by the 26S Proteasome Are Prerequisites for Degradation

The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.     

Establishment and characterization of clonal cell lines from the vagina of p53-deficient young mice

Clonal cell lines have been established from vagina of prepubertal female p53(-/-) mice. Because the mouse vagina has a dual origin (the cranial three-fifths derived from the Müllerian duct and the caudal two-fifths derived from the urogenital sinus), both parts were separately subjected to cloning. Sixteen epithelial and two fibroblastic cell lines were established from the cranial three-fifths (Müllerian vagina group), and four epithelial and three fibroblastic cell lines were established from the caudal two-fifths (sinus vagina group). They were maintained in Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 containing 10% fetal calf serum and 17 beta-estradiol at 10(-8) M. Two cell lines (one epithelial and one fibroblastic) were examined using soft agar assay, but no colonies were formed. The doubling time of the cell lines was approximately 24 h, and all of them divided more than 200 times without crisis, suggesting that they were immortalized. All epithelial cell lines expressed cytokeratin 8. However, the epithelial cell lines expressed cytokeratin 14 and cytokeratin 10 when exposed to medium containing different concentrations of Ca(2+). Fibroblastic cell lines expressed vimentin. All epithelial and fibroblastic cell lines expressed estrogen receptor-alpha protein. This is the first successful establishment of clonal cell lines from the normal mouse vagina, and these lines may provide good models in vitro of the vagina for the study of the mechanism of estrogen action.