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.     

The effect of fatty acids on the developmental direction of Strongyloides ratti first-stage larvae

The effect of fatty acids was studied on the developmental direction of Strongyloides ratti first-stage larvae (L1). The proportion of third-stage infective larvae increased markedly when L1 were cultured in faeces with added fatty acids such as palmitic (C16), stearic (C18), oleic (C18:1) and linoleic (C18:2) acids. Unsaturated fatty acids were more effective than saturated ones. Moreover, the proportion of infective larvae increased with quantity of linoleic acid but the triacylglycerols of any fatty acid had no effect. These results suggest that these free fatty acids cause physiological changes that determine the developmental course of L1 of S. ratti in nature.     

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.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Proteolytic Activity against Model Peptides of the Cell Wall Lytic Enzyme from Chlamydomonas

A cell wall lytic enzyme (gamete wall-autolysin) from Chlamydomonasreinhardtii specifically cleaved several synthetic model peptides,-neo-endorphin, dynorphin (1–13), neurotensin and mastoparan,at the peptide bonds between consecutive hydrophobic amino-acidresidues. The cleavage was not significantly affected by high-saltconditions which are known to inhibit digestion of the cellwall. (Received December 14, 1989; Accepted April 5, 1990)