Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure

Dilated cardiomyopathy, a disease of unknown etiology and pathogenesis, is associated with heart failure and compensatory hypertrophy. Although cell and animal models suggest a role for altered gene expression in the transition to heart failure, there is a paucity of data derived from the study of human heart tissue. In this study, we used DNA microarray profiling to investigate changes in the expression of genes involved in apoptosis that occur in human idiopathic dilated cardiomyopathic hearts that had progressed to heart failure. We observed altered gene expression consistent with a proapoptotic shift in the TNF-alpha signaling pathway. Specifically, we found decreased expression of TNF-alpha- and NF-kappaB-induced antiapoptotic genes such as growth arrest and DNA damage-inducible (GADD)45beta, Flice inhibitory protein (FLIP), and TNF-induced protein 3 (A20). Consistent with a role for apoptosis in heart failure, we also observed a significant decrease in phosphorylation of BAD at Ser-112. This study identifies several pathways that are altered in human heart failure and provides new targets for therapy.     

Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.

In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R-S(*)) in the substrate binding subunit R1. We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by Ce(IV)/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines. In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals. The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1. In aerobic R1 samples without spin trap that were frozen after treatment with Ce(IV)/NTA or laser photolysis, we observed sulfinyl radicals (R-S(*)=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one beta-proton. Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used.     

Shared protein components of SINE RNPs

The heterogeneous, short RNAs produced from the high, copy, short mobile elements (SINEs) interact with proteins to form RNA-protein (RNP) complexes. In particular, the BC1 RNA, which is transcribed to high levels specifically in brain and testis from one locus of the ID SINE family, exists as a discrete RNP complex. We expressed a series of altered BC1, and other SINE-related RNAs, in several cell lines and tested for the mobility of the resulting RNP complexes in a native PAGE assay to determine which portions of these SINE RNAs contribute to protein binding. When different SINE RNAs were substituted for the BC1 ID sequence, the resulting RNPs exhibited the same mobility as BC1. This indicates that the protein(s) binding to the ID portion of BC1 is not sequence specific and may be more dependent upon the secondary structure of the RNA. It also suggests that all SINE RNAs may bind a similar set of cellular proteins. Deletion of the A-rich region of BC1 RNA has a marked effect on the mobility of the RNP. Rodent cell lines exhibit a slightly different mobility for this shifted complex when compared to human cell lines, reflecting evolutionary differences in one or more of the protein components. On the basis of mobility change observed in RNP complexes when the A-rich region is removed, we decided to examine poly(A) binding protein (PABP) as a candidate member of the RNP. An antibody against the C terminus of PABP is able to immunoprecipitate BC1 RNA, confirming PABP's presence in the BC1 RNP. Given the ubiquitous role of poly(A) regions in the retrotransposition process, these data suggest that PABP may contribute to the SINE retrotransposition process.     

Effect of light quality on sulfide photo-oxidation and growth in an artificial biofilm of the green sulfur bacterium Prosthecochloris aestuarii

We have succeeded in culturing an axenic biofilm of the green sulfur bacterium Prosthecochloris aestuarii strain CE 2404 in an artificial sandy sediment under visible light (400–700 nm). This simulates the conditions of deep submerged sediments. A five-week incubation period, using a 16-hour light / 8-hour dark regime, was applied in the benthic gradient chamber (BGC). The biofilm was located below the oxygen penetration depth of 1.2 mm, namely between 1.5 and 2.5 mm and the biomass peak was at 2.1 mm depth. This is much shallower compared to previously described artificial mats of P. aestuarii, which were grown in the BGC under near infrared (NIR)-rich light. High resolution time courses of photosynthesis were measured as sulfide photo-oxidation rates and studied under visible light and visible light amended with NIR to assess the effect of light quality. Sulfide photo-oxidation rates were rather low under visible light and strongly stimulated at most depths under full light conditions. However, under the latter conditions the rates decelerated after a maximum rate was reached at 8–10 min, apparently due to diffusional limitation of sulfide supply. It was concluded that the top of the mat was not limited by the photon flux density, while the biomass peak and the bottom of the biofilm were severely light limited under the culture conditions. These results support the hypothesis that a biofilm of P. aestuarii can develop in deep submerged sediments, when the oxygen penetration depth is very shallow. Nevertheless, the addition of NIR light strongly enhances the potential of P. aestuarii to grow deeper in the sediment.This revised version was published online in October 2005 with corrections to the Cover Date.     

Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions

Proteolytic processing of mutant huntingtin (mhtt) is regarded as a key event in the pathogenesis of Huntington's disease (HD). Mhtt fragments containing a polyglutamine expansion form intracellular inclusions and are more cytotoxic than full-length mhtt. Here, we report that two distinct mhtt fragments, termed cp-A and cp-B, differentially build up nuclear and cytoplasmic inclusions in HD brain and in a cellular model for HD. Cp-A is released by cleavage of htt in a 10 amino acid domain and is the major fragment that aggregates in the nucleus. Furthermore, we provide evidence that cp-A and cp-B are most likely generated by aspartic endopeptidases acting in concert with the proteasome to ensure the normal turnover of htt. These proteolytic processes are thus potential targets for therapeutic intervention in HD.     

Mutational analysis reveals a role for the C terminus of the proteasome subunit Rpt4p in spindle pole body duplication in Saccharomyces cerevisiae

The ubiquitin/proteasome pathway plays a key role in regulating cell cycle progression. Previously, we reported that a conditional mutation in the Saccharomyces cerevisiae gene RPT4/PCS1, which encodes one of six ATPases in the proteasome 19S cap complex/regulatory particle (RP), causes failure of spindle pole body (SPB) duplication. To improve our understanding of Rpt4p, we created 58 new mutations, 53 of which convert clustered, charged residues to alanine. Virtually all mutations that affect the N-terminal region, which contains a putative nuclear localization signal and coiled-coil motif, result in a wild-type phenotype. Nine mutations that affect the central ATPase domain and the C-terminal region confer recessive lethality. The two conditional mutations identified, rpt4-145 and rpt4-150, affect the C terminus. After shift to high temperature, these mutations generally cause cells to progress slowly through the first cell cycle and to arrest in the second cycle with large buds, a G2 content of DNA, and monopolar spindles, although this phenotype can vary depending on the medium. Additionally, we describe a genetic interaction between RPT4 and the naturally polymorphic gene SSD1, which in wild-type form modifies the rpt4-145 phenotype such that cells arrest in G2 of the first cycle with complete bipolar spindles.