Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.     

Enhanced secretion of Escherichia coli beta-lactamase by a spontaneous erythromycin-resistant mutant of Bacillus subtilis

An extracellular-protease-deficient mutant, ME142, was isolated from Bacillus subtilis as a spontaneous erythromycin-resistant (Eryr) clone. This mutant showed conditional sporulation and only sporulated normally in the absence of erythromycin. In the presence of the antibiotic, sporulation was greatly reduced. Production of extracellular proteases by ME142 also exhibited conditional deficiency, possibly due to pleiotropic effects of the sporulation deficiency. The production of protease was 2-10% that of the wild-type level in the presence of erythromycin. ME142 showed poor competence for transformation even in the absence of erythromycin; however, derivatives of ME142 were isolated which had the same Eryr phenotype but which exhibited normal competence. One such mutant, ME162, was used as a host for the secretion of Escherichia coli beta-lactamase. The amount of beta-lactamase in the culture supernatants of ME162 increased significantly when the cells were cultured with erythromycin, suggesting that proteolysis of the beta-lactamase in the supernatants of ME162 was greatly reduced as compared to that in the supernatants of the wild-type strain.     

Inhibitory effect of anti-class II antibody on the spontaneous activation of B cells in patients with systemic lupus erythematosus. Analysis with IL-1 production and IL-1 receptor expression

The mechanism of the spontaneous activation of B cells in patients with SLE was analyzed from the standpoint of the production of IL-1 from B cells and the expression of IL-1R on B cells. SLE B cells spontaneously produced IL-1-like factors which stimulated murine thymocyte proliferative responses. Their m.w. was about 17,000 and their isoelectric point was 4.8. The IL-1-like activity produced by B cells was absorbed with rabbit anti-IL-1 alpha antibody, but not with anti-IL-1 beta antibody. The differentiation of SLE B cells was enhanced by rIL-1 alpha, beta or IL-1-like factors produced by SLE B cells in a concentration-dependent manner. SLE B cells expressed large number of IL-1R detected by FITC-conjugated IL-1 alpha. By a Percoll gradient density centrifugation, IL-1-producing cells and B cells responsive to IL-1 were enriched in a higher density fraction, but were reduced in a lower density fraction. IL-1R-positive B cells were enriched in the lower density fraction, but were depleted in the higher density fraction. However, the expression of IL-1R on the lower density B cells was reduced by 2-day culture. The expression of IL-1R on the higher density B cells was increased during a 2-day culture. Anti-class II antibody inhibited the production of IL-1R on the higher density B cells. These results suggest that the cellular interaction among B precursor cells mediated by class II Ag induces the production of IL-1 and the expression of its receptors on their surface and the interaction between IL-1 and its receptors stimulates B precursor cells to spontaneously differentiate into Ig-producing cells as an autocrine mechanism in patients with SLE.     

An electrophoretic polymorphism in salivary amylases (Amy-1) of mastomys (Praomys coucha).

An electrophoretic polymorphism of salivary amylases (Amy-1) in mastomys (Praomys coucha) (MWC, MRJ and MCC strains) was detected. Amylase in MWC or MRJ saliva, which migrated fast toward the anode, was designated as AMY-1A, and that in MCC saliva migrating slowly as AMY-1B. Salivary amylases are controlled by a pair of codominant alleles at a single autosomal locus (Amy-1). No polymorphism was seen in pancreatic amylases (Amy-2). The frequencies of these phenotypes did not differ between the sexes. Some isoamylases were observed and these were different from those in mouse or rat.     

Changes in phospholipid composition of the mitochondrial membrane after orthotopic liver transplantation

Changes in phospholipids and their fatty acid composition in liver mitochondria obtained from allogenic rats with orthotopic liver transplants were measured with and without immunosuppressive treatment. In untreated allogenic rats, mitochondrial phosphorylation activity was severely deteriorated at 8 days after transplantation. A significant change was also found in the amount of cardiolipin compared with other classes of phospholipids. Namely, cardiolipin decreased, and lysodiphosphatidylglycerol and phosphatidylglycerol increased concomitantly. Furthermore, the percentage of linoleic acid in cardiolipin decreased dramatically. Decrease in cardiolipin and changes in its fatty acid composition may be attributed to the deterioration of mitochondrial function upon acute rejection.     

Hepatocyte growth factor rapidly induces the tyrosine phosphorylation of 41-kDa and 43-kDa proteins in mouse keratinocytes.

We have examined the hepatocyte growth factor (HGF)-mediated changes in protein-tyrosine phosphorylation in mouse keratinocytes (PAM-212) and canine kidney epithelial cells (MDCK). In PAM-212 cells HGF and epidermal growth factor, both of which stimulated the DNA synthesis, rapidly induced the tyrosine phosphorylation of two 41-kDa and two 43-kDa proteins: increased tyrosine phosphorylation of those proteins has been commonly observed when quiescent fibroblasts are stimulated with a variety of mitogenic agents. In contrast, HGF did not stimulate the DNA synthesis but induced cell dissociation in MDCK cells; under this condition, increased tyrosine phosphorylation of the 41-kDa and 43-kDa protein was not observed. A possible role of the increased tyrosine phosphorylation of 41-kDa and 43-kDa protein in the signaling pathway of HGF is discussed.     

Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin.

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.     

Viscometric and spectroscopic studies on the solution behaviour of hydrophobically modified cellulosic polymers

The solution properties of hydroxyethyl cellulose (HEC) and hydrophobically modified hydroxyethyl cellulose (HM-HEC) have been investigated by means of viscometric and spectroscopic techniques involving free radical and fluorescent probes. The greater viscosity of HM-HEC solutions above a critical polymer concentration (C_p) of approximately 0·2% has been interpreted in terms of the formation of a three-dimensional network structure in which the polymer chains are effectively crosslinked by the intermolecular association of neighbouring hydrophobic side chains. C_p is considerably less than the predicted polymer coil overlap concentration (C*) of approximately 1%.

The interaction of the polymers with an anionic surfactant, sodium dodecyl sulphate (SDS) has also been investigated. A mechanism involving the interaction of free surfactant with the regions of intermolecular hydrophobic association is suggested to account for the considerable differences in the rheological behaviour of the polymers in the presence of SDS.