Effect of voluntary exercise on number and volume of cardiomyocytes and their mitochondria in the mouse left ventricle

Voluntary exercise (VE) has a beneficial influence on the heart and mean lifespan. The present study evaluates structural adaptations of cardiomyocytes and their mitochondria due to VE by new, unbiased stereological methods. Female, 7-9-week-old mice were randomly assigned to a control (CG, n = 7) or VE group (EG, n = 7). EG animals were housed in cages with free access to a running wheel and had a mean running distance of 6.7 (1.8) km per day. After 4 weeks, the hearts of all mice were processed for light and electron microscopy. We estimated the number and volume of cardiomyocytes by the disector method and the number and volume of mitochondria by estimation of the Euler number. In comparison to CG, VE did not have an effect on the myocardial volume of the left ventricle (CG: 93 (10), EG: 103 (17) (mm(3))), the number of cardiomyocytes (CG: 2.81 (0.27), EG: 2.82 (0.43) (x10(6))) and their number-weighted mean volume. However, the composition of the cardiomyocytes changed due to VE. The total volume of mitochondria (CG: 21.8 (4.9), EG: 32.2 (4.3) (mm(3)), P < 0.01) and the total number (CG: 3.76 (0.44), EG: 7.02 (1.13) (x10(10)), P < 0.001) were significantly higher in EG than in CG. The mean number-weighted mitochondrial volume was smaller in EG than in CG (P < 0.05). In summary, VE does not alter ventricular volume nor cardiomyocyte volume or number but the oxidative capacity of cardiomyocytes by an increased mitochondrial number and total volume in the left ventricle. These structural changes may participate in the beneficial effects of VE.

Transplantation of yeast-infected cardiac allografts: a report of 2 cases

For the first time in the literature to date, we report 2 cases of transplantation of yeast-infected cardiac allografts. In both cases, endocardial vegetations were observed before graft implantation. Microbiologic samples grew yeasts: Rhodotorula glutinis was found close to the left atrial appendage in the first case and Candida parapsilosis was identified in a vegetation located at the base of the tricuspid valve in the second case. We discuss the possible routes of donor organ infection and management of these 2 unusual cases.

Characterization of the sodium/hydrogen exchanger NHA2

Cation/proton exchange has been recognized for decades in mammalian mitochondria, but the exchanger proteins have eluded identification. In this study, a cDNA from a human brain library, previously designated NHA2 in the genome, was cloned and characterized. The NHA2 transcript bears more similarity to prokaryotic than known eukaryotic sodium/proton exchangers, but it was found to be expressed in multiple mammalian organs and cultured cells. A mAb to NHA2 was generated and found to label an approximately 55-kD native protein in multiple tissues and cell lines. The specificity of this antibody was confirmed by demonstrating the loss of the native NHA2 band on immunoblots when cultured cells were treated with NHA2-specific small interfering RNA. Although NHA2 protein was detected in multiple organs, within each, its expression was restricted to specific cell types. In the kidney, co-localization with calbindin 28k and reverse transcription-PCR of microdissected tubules revealed that NHA2 is limited to the distal convoluted tubule. In cell lines, native NHA2 was localized both to the plasma membrane and to the intracellular compartment; immunogold electron microscopy of rat distal convoluted tubule demonstrated NHA2 predominantly but not exclusively on the inner mitochondrial membrane. Furthermore, co-sedimentation of NHA2 antigen and mitochondrial membranes was observed with differential centrifugation, and two mitochondrial markers co-localized with NHA2 in cultured cells. Regarding function, human NHA2 reversed the sodium/hydrogen exchanger-null phenotype when expressed in sodium/hydrogen exchanger-deficient yeast and restored the ability to defend high salinity in the presence of acidic extracellular pH. In summary, NHA2 is a ubiquitous mammalian sodium proton/exchanger that is restricted to the distal convoluted tubule in the kidney.

Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.

Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis

Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.

Thermal plasticity of mitochondria: a latitudinal comparison between Southern Ocean molluscs

Mitochondrial volume density (Vv((mt,f))), cristae surface density (Sv((im,mt))), cristae surface area (Sv((im,f))) and citrate synthase (CS) activity were analysed as indicators of thermal acclimation in foot muscle of the limpet, Nacella concinna, and the clam, Laternula elliptica, collected from 4 locations within the Southern Ocean, South Georgia (54 degrees S, N. concinna only), Signy (60 degrees S), Jubany (L. elliptica only -62 degrees S) and Rothera (67 degrees S). Animals were acclimated to 0.0 degrees C whilst a sub-set of N. concinna (South Georgia, Signy and Rothera) and L. elliptica (Rothera) were acclimated to 3.0 degrees C. At 0.0 degrees C N. concinna had higher Vv((mt,f)), Sv((im,mt)), Sv((im,f)) and muscle fibre specific CS activity than L. elliptica which correlated with the more active life style of N. concinna. However, mitochondrial density was very low, 1-2% in both species, suggesting that low temperature compensation of mitochondrial density is not a universal evolutionary response of Antarctic marine ectotherms. Both Sv((im,mt)) and Sv((im,f)) were reduced by warm acclimation of N. concinna. South Georgia N. concinna maintained muscle fibre specific CS activity after acclimation, in contrast to N. concinna from Rothera and Signy and L. elliptica from Rothera, indicating that they have the physiological plasticity to respond to their warmer, more variable thermal environment.

Ctp1 and the MRN-complex are required for endonucleolytic Rec12 removal with release of a single class of oligonucleotides in fission yeast

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5' ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Delta and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal.

Mitochondrial Outer Membrane Proteome of Trypanosoma brucei Reveals Novel Factors Required to Maintain Mitochondrial Morphology

Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

Effect of Yeast Supplement on Performance of Steer Calves

The effect of Prosponse® yeast supplement fed to newly arrived steers was evaluated at the ISU Armstrong Research Farm near Lewis, Iowa. Four pens of approximately 32 head were fed similar diets with 2 pens receiving yeast supplement and 2 pens not. Calves were weighed on delivery to the feedlot and allocated on weight and sire. Calves were on test 34 days. There was no effect on daily gain or feed to gain. Dry matter intake trended higher for the yeast supplemented calves (P=.10). These data suggest that yeast supplement may improve dry matter intake in newly arrived feedlot calves.

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ~1mg of HAT from 2l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.