Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.     

Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation.

The IPL1 gene is required for high-fidelity chromosome segregation in the budding yeast Saccharomyces cerevisiae. Conditional ipl1ts mutants missegregate chromosomes severely at 37 degrees C. Here, we report that IPL1 encodes an essential putative protein kinase whose function is required during the later part of each cell cycle. At 26 degrees C, the permissive growth temperature, ipl1 mutant cells are defective in the recovery from a transient G2/M-phase arrest caused by the antimicrotubule drug nocodazole. In an effort to identify additional gene products that participate with the Ipl1 protein kinase in regulating chromosome segregation in yeast, a truncated version of the previously identified DIS2S1/GLC7 gene was isolated as a dosage-dependent suppressor of ipl1ts mutations. DIS2S1/GLC7 is predicted to encode a catalytic subunit (PP1C) of type 1 protein phosphatase. Overexpression of the full-length DIS2S1/GLC7 gene results in chromosome missegregation in wild-type cells and exacerbates the mutant phenotype in ipl1 cells. In addition, the glc7-1 mutation can partially suppress the ipl1-1 mutation. These results suggest that type 1 protein phosphatase acts in opposition to the Ipl1 protein kinase in vivo to ensure the high fidelity of chromosome segregation.     

[125I]CGP 42112 reveals a non-angiotensin II binding site in 1-methyl-4-phenylpyridine (MPP+)-induced brain injury

Summary 1. Intracerebral injection of the oxidative metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridine (MPP⁺), into the substantia nigra of adult rats resulted in a lesion at the injection site.2. Using autoradiography, we localized specific [¹²⁵]CGP 42112 binding that was not recognized by angiostensin II or angiotensin II AT₁ or AT₂ receptorselective ligands.3. Our results suggest that [¹²⁵I]CGP 42112 may be binding to activated microglia that appear at the lesion site.     

Carbohydrate binding activities ofBradyrhizobium japonicum: IV. Effect of lactose and flavones on the expression of the lectin,BJ38

BJ38 is a galactose/lactose-specific lectin (M _r 38000) found at one pole ofBradyrhizobium japonicum. It has been implicated in mediating the adhesion of the bacteria to soybean roots, leading to the establishment of a nitrogen-fixing symbiosis. When the ligand lactose is added to cultures of the bacteria for at least 1 h prior to harvesting the cells for BJ38 isolation, the yield of the protein was found to be elevated in a dose-dependent fashion. Half maximal stimulation was observed at 50 µm; the effect was saturated at 1mm, where a 10-fold higher yield of BJ38 was obtained. Saccharides with a lower affinity for BJ38 than lactose yielded a correspondingly smaller induction effect when compared at a concentration of 1mm. The higher level of BJ38 induced by lactose is also manifested by an elevated amount of BJ38 detectable at the cell surface and by a higher number ofB. japonicum cells adsorbed onto soybean cells. Surprisingly, the induction of BJ38 expression seen with lactose was also observed with certain, but not all, flavonoids that induce thenod genes of the bacteria; genistein mimicked the induction observed with lactose, whereas luteolin failed to stimulate BJ38 production.     

Desulfurization of dibenzothiophene to 2-hydroxybiphenyl by some newly isolated bacterial strains

Gram-positive, non-spore-forming, non-acid-fast, rod-shaped aerobic bacteria with the ability to desulfurize dibenzothiophene (DBT) or dibenzosulfone (DBTO₂) were isolated from soil samples contaminated with fossil fuels. Using a bioavailability method, cells with the desired DbtS⁺ phenotype were enriched. Modified fluorescence and colorimetric assays were used for the initial detection of 2-hydroxybiphenyl (OH-BP) in microtiter plates; subsequently, isolates were grown in wells of microtiter plates and screened for the production of desulfurization product. Fluorescence under UV light and the production of colored product in the phenol assay were used as presumptive indications of production of OH-BP. Confirmation of the presence of OH-BP was achieved with HPLC, UV-absorbance, and mass spectrometry. Nutrient utilization and fatty acid composition (as discerned with Biolog plates and gas chromatography, respectively) were used to identify presumptively the strains as Rhodococcus erythropolis; colony and cell morphology may not be consistent with the identification achieved by nutrient utilization and fatty acid composition. The desulfurization end product, OH-BP, can not be used as carbon source by the tested strain, N1-36.     

诺卡氏菌属GS-17耐热茁霉多糖酶的提纯和性质

诺卡氏菌属GS-17(Nocardia sp.GS-17)的耐热茁霉多糖酶(Pullulanase EC.3.2.1.41)的粗酶液经中空纤维柱超滤浓缩、羟基磷灰石柱层析和Pullulan-Sepharose 6B亲和层析,得到凝胶电泳均一的纯酶,比活提高264倍.酶作用最适温度为55℃,最适PH6.2,分子量140000,等电点pI为6.0.该酶水解茁霉多糖、支链淀粉和可溶性淀粉,但不水解糖原.酶在50℃作用于茁霉多糖的米氏常数K_m为0.90mg/ml,最大反应速度V_(max)为57μmol·min~(-1)·mg~(-1).Zn~(2 )、Fe~(3 )、Hg~(2 )、Cu~(2 )、Pb~(2 )和环状糊精对酶有抑制作用,Ca~(2 )对酶有激活作用.经蛋白质侧链化学修饰研究表明,色氨酸残基位于酶的活性位区.该酶是由1129个氨基酸残基组成的单肽链,酶的N末端序列经测定为:Ala-Gly-His-Gly-Pro-Asp-Val-Gln-Asp-Gly-