Clathrin light chain b is capable of affecting potently a major protein phosphatase from microtubules (MT-PP1)

Clathrin light chain (CL) b purified from bovine brain postmicrotubule supernatant and identified by mass spectrometry potently inhibited a catalytic activity of a major protein phosphatase (PP) that was copurified with microtubules and recognized by antiPP1 antibodies. CLb similarly affected the catalytic subunit and holoenzyme of the PP, little inhibiting the activity of PP2A. Although the CLb from clathrin-coated vesicles was several hundredfold weaker than our purified CLb, the CLb in the postmicrotubule supernatant, independent of whether it was sedimentable or soluble, was as active as the purified CLb. Thus CLb may be a potent regulator of the PP.     

Possibility of transkingdom gene therapy for complex I diseases

Defects of complex I are involved in many human mitochondrial diseases, and therefore we have proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into mammalian cell lines. The expressed NDI1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species induced by rotenone. It was further shown that the NDI1 protein can be functionally expressed in tissues such as skeletal muscles and the brain of rodents, which scarcely induced an inflammatory response. The use of NDI1 as a potential molecular therapy for complex I-deficient diseases is briefly discussed, including the proposed animal model.     

Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii

Cryptococcus neoformans and Cryptococcus gattii are yeasts that cause meningoencephalitis, but that differ in host range and geographical distribution. Cryptococcus neoformans occurs world-wide and mostly infects immunocompromised patients, whereas C. gattii occurs mainly in (sub)tropical regions and infects healthy individuals. Anomalous C. neoformans strains were isolated from patients. These strains were found to be monokaryotic, and diploid or aneuploid. Amplified Fragment Length Polymorphism (AFLP) and sequence analyses indicated that AFLP genotypes 2 (C. neoformans) and 4 (C. gattii) were present. The strains were serologically BD. Mating- and serotype-specific PCR reactions showed that the strains were MATa-serotype D/MATalpha-serotype B. This study is the first to describe naturally occurring hybrids between C. neoformans and C. gattii.     

Disruption of structural and functional integrity of alpha 2-macroglobulin by cathepsin E.

alpha 2-Macroglobulin (alpha 2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, alpha 2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both alpha 2M and biological targets, little is known about the nature of alpha 2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of alpha 2M in the endolysosome system, we examined the capacity of alpha 2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. alpha 2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, alpha 2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that alpha 2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for alpha 2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808-Ala813 sequence of human alpha 2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of alpha 2M in the endolysosome system.     

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells

Background

Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils.

Amino Acid Sequence of Tremerogen A–10, a Peptidal Hormone,Inducing Conjugation Tube Formation in Tremella mesenterica Fr.

κ-Casein components having various carbohydrate contents were prepared by diethylaminoethyl-cellulose chromatography and the interactions of each κ-casein component both with α_s1-casein and with β-casein were examined by Sepharose 4B gel chromatography, ultra-centrifugal experiments and viscosity measurements. Each κ-casein component could form complex with α_s1- and β-casein in the absence and presence of CaCl₂. Molecular weight of complexes of unfractionated κ-casein both with α_s1-casein and with κ-casein were about 70 × 10⁴ at 37°C in the absence of CaCl₂, while those of complexes of each κ-casein component with α_s1 and β-casein were about 50 × 10⁴. Stokes radii of complexes increased with increasing calcium ion. While sedimentation coefficient at 37°C of complex with β-casein had almost the same value, those of complexes with α_s1-casein decreased with increase of carbohydrate content of κ-casein components. Intrinsic viscosity of complex of κ-casein component having much carbohydrate was almost the same among tested temperatures. It is suggested that heterogeneity of κ-casein is necessary to form large complex and that the carbohydrate moiety of κ-casein contributes the stability of casein complex.