Cytoprotective peptide humanin binds and inhibits proapoptotic Bcl-2/Bax family protein BimEL

Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax-/-cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.     

Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and non-syndromic hearing loss

We report here the clinical, genetic, and molecular characterization of two Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects. Penetrances of hearing loss in BJ105 and BJ106 pedigrees are 67% and 33%, respectively. In particular, three of 10 affected matrilineal relatives of BJ105 pedigree had aminoglycoside-induced hearing loss, while seven affected matrilineal relatives in BJ105 pedigree and six affected matrilineal relatives in BJ106 pedigree did not have a history of exposure to aminoglycosides. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mtDNA variants belonging to haplogroups F3 and M7b. These variants showed no evolutionary conservation, implying that mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycosides and nuclear backgrounds appear to be major modifier factors for the phenotypic manifestation of the A1555G mutation in these Chinese families.     

Mechanistic studies on phosphopantothenoylcysteine decarboxylase: trapping of an enethiolate intermediate with a mechanism-based inactivating agent

Phosphopantothenoylcysteine decarboxylase (PPC-DC) catalyzes the decarboxylation of the cysteine moiety of 4'-phosphopantothenoylcysteine (PPC) to form 4'-phosphopantetheine (PPantSH); this reaction forms part of the biosynthesis of coenzyme A. The enzyme is a member of the larger family of cysteine decarboxylases including the lantibiotic-biosynthesizing enzymes EpiD and MrsD, all of which use a tightly bound flavin cofactor to oxidize the thiol moiety of the substrate to a thioaldehyde. The thioaldehyde serves to delocalize the charge that develops in the subsequent decarboxylation reaction. In the case of PPC-DC enzymes the resulting enethiol is reduced to a thiol giving net decarboxylation of cysteine, while in EpiD and MrsD it is released as the final product of the reaction. In this paper, we describe the characterization of the novel cyclopropyl-substituted product analogue 4'-phospho-N-(1-mercaptomethyl-cyclopropyl)-pantothenamide (PPanDeltaSH) as a mechanism-based inhibitor of the human PPC-DC enzyme. This inhibitor alkylates the enzyme on Cys(173), resulting in the trapping of a covalently bound enethiolate intermediate. When Cys(173) is exchanged for the weaker acid serine by site-directed mutagenesis the enethiolate reaction intermediate also accumulates. This suggests that Cys(173) serves as an active site acid in the protonation of the enethiolate intermediate in PPC-DC enzymes. We propose that this protonation step is the key mechanistic difference between the oxidative decarboxylases EpiD and MrsD (which have either serine or threonine at the corresponding position in their active sites) and PPC-DC enzymes, which also reduce the intermediate in an overall simple decarboxylation reaction.     

Nucleoside diphosphate kinase B knock-out mice have impaired activation of the K+ channel KCa3.1, resulting in defective T cell activation

Nucleoside diphosphate kinases (NDPKs) are encoded by the Nme (non-metastatic cell) gene family. Although they comprise a family of 10 genes, NDPK-A and -B are ubiquitously expressed and account for most of the NDPK activity. We previously showed that NDPK-B activates the K(+) channel KCa3.1 via histidine phosphorylation of the C terminus of KCa3.1, which is required for T cell receptor-stimulated Ca(2+) flux and proliferation of activated naive human CD4 T cells. We now report the phenotype of NDPK-B(-/-) mice. NDPK-B(-/-) mice are phenotypically normal at birth with a normal life span. Although T and B cell development is normal in NDPK-B(-/-) mice, KCa3.1 channel activity and cytokine production are markedly defective in T helper 1 (Th1) and Th2 cells, whereas Th17 function is normal. These findings phenocopy studies in the same cells isolated from KCa3.1(-/-) mice and thereby support genetically that NDPK-B functions upstream of KCa3.1. NDPK-A and -B have been linked to an astonishing array of disparate cellular and biochemical functions, few of which have been confirmed in vivo in physiological relevant systems. NDPK-B(-/-) mice will be an essential tool with which to definitively address the biological functions of NDPK-B. Our finding that NDPK-B is required for activation of Th1 and Th2 CD4 T cells, together with the normal overall phenotype of NDPK-B(-/-) mice, suggests that specific pharmacological inhibitors of NDPK-B may provide new opportunities to treat Th1- and Th2-mediated autoimmune diseases.     

CXCR3+CD4+ T cells mediate innate immune function in the pathophysiology of liver ischemia/reperfusion injury

Ischemia-reperfusion injury (IRI), an innate immune-dominated inflammatory response, develops in the absence of exogenous Ags. The recently highlighted role of T cells in IRI raises a question as to how T lymphocytes interact with the innate immune system and function with no Ag stimulation. This study dissected the mechanism of innate immune-induced T cell recruitment and activation in rat syngeneic orthotopic liver transplantation (OLT) model. Liver IRI was induced after cold storage (24-36 h) at 4 degrees C in University of Wisconsin solution. Gene products contributing to IRI were identified by cDNA microarray at 4-h posttransplant. IRI triggered increased intrahepatic expression of CXCL10, along with CXCL9 and 11. The significance of CXCR3 ligand induction was documented by the ability of neutralizing anti-CXCR3 Ab treatment to ameliorate hepatocellular damage and improve 14-day survival of 30-h cold-stored OLTs (95 vs 40% in controls; p < 0.01). Immunohistology analysis confirmed reduced CXCR3+ and CD4+ T cell infiltration in OLTs after treatment. Interestingly, anti-CXCR3 Ab did not suppress innate immune activation in the liver, as evidenced by increased levels of IL-1beta, IL-6, inducible NO synthase, and multiple neutrophil/monokine-targeted chemokine programs. In conclusion, this study demonstrates a novel mechanism of T cell recruitment and function in the absence of exogenous Ag stimulation. By documenting that the execution of innate immune function requires CXCR3+CD4+ T cells, it highlights the critical role of CXCR3 chemokine biology for the continuum of innate to adaptive immunity in the pathophysiology of liver IRI.     

Annually laminated lake sediments and environmental changes in Bashang Plateau, North China

Angulinuo Lake is the biggest lake on the Bashang Plateau, North China, and is 47.6 km² in area and 2-6 m in depth. A core from the inner part of Angulinuo Lake was sliced and the sediment was observed by Scanning Electronic Microscope (SEM). Annual laminations characterized by variable color and grain size were found and interpreted as recording the cyclic deposition of lacustrine clay and aeolian dust. The results of chemical analysis of coarse grains conducted by SEM-EDAX, and grain size analysis of modern aeolian dust in the ice on Angulinuo Lake, support an aeolian origin for the light coarse layers. Image analysis technique was used to calculate the size and number of coarse grains in each layer. The coarse grains were fractionated into four classes: > 42 μm, 14-42 μm, 14-4.2 μm and 1.4-4.2 μm. In general, the abundance of the four classes shows similar temporal variation patterns. Around Angulinuo Lake, the winter monsoon is strong and transports aeolian dust into the lake. When the winter monsoon is strong, the size and amount of coarse grains are expected to increase. We infer that the winter monsoon was weaker during 8430-5440 year BP, and was unstable in the later part of this period. From 5440 year BP, the winter monsoon became stronger, and then weaker from 3250 to 2490 year BP. During 2490-1170 year BP, the winter monsoon was slightly stronger, but since 1170 year BP, it has become weaker again. The changes of the winter monsoon intensity recorded in the annual laminations in Angulinuo Lake sediments correspond well to environmental changes in North China and to changes in sea level during the same period. Periods of weaker winter monsoon correspond to times of higher sea levels while the periods of stronger winter monsoon correspond to the Neoglaciation stage in China and the periods of lower sea levels.     

Influence of substituents on the structures of hybrid complexes constructed from tetranuclear copper(I) 1,2,4-triazolate clusters and octamolybdates

By changing the substituents on 1,2,4-triazole ring, six novel organic-inorganic hybrid complexes constructed from tetranuclear copper(I) 1,2,4-triazolate clusters and octamolybdates, [{Cu₄(L)_x}Mo₈O₂₆] (L = 3,5-diamino-1,2,4-triazole (datrz) and x = 4 for 1; L = 3-amino-1,2,4-triazole (3atrz) and x = 4 for 2; L = 3,5-dimethyl-1,2,4-triazole (dmtrz) and x = 4 for 3; L = 3,5-dimethyl-4-amino-1,2,4-triazole (dmatrz) and x = 6 for 4; L = 3,5-diethyl-4-amino-1,2,4-triazole (deatrz) and x = 4 for 5; L = 3,5-di(n-propyl)-4-amino-1,2,4-triazole (dpatrz) and x = 3 for 6), were obtained. The tetranuclear Cu(I) cluster in compound 1 acts as charge-compensating unit, which is the first polynuclear metal 1,2,4-triazole structure only with N1, N2 bridging mode. Compounds 2, 4, 5 and 6 are of polymeric 1D chains and 3 is of a 2D layer structure. In 2, three distinct Cu(I)-coordination geometries, distorted tetrahedral, T-shaped and V-shaped linear Cu(I), are observed in the same structure. The first extended hybrid structure constructed by δ-octamolybdates is founded in 4. A novel [Mo₈O₂₆]⁴⁻ anion is found in 5, which contains only three crystallographically independent Mo atoms. In compounds 5 and 6, terminal oxo groups of octamolybdate cluster act as μ₃-oxo bridges to link the copper(I) coordination complexes; such an unusual linking manner is unique in the coordination chemistry of octamolybdates with transition metal fragments. The influences of substituent on the structures of the tetranuclear units are also discussed in details.