The Hansenula polymorpha PDD1 gene product,essential for the selective degradation of peroxisomes,is a homologue of Saccharomyces cerevisiae Vps34p

Via functional complementation we have isolated the Hansenula polymorpha PDD1 gene essential for selective, macroautophagic peroxisome degradation. HpPDD1 encodes a 116 kDa protein with high similarity (42% identity) to Saccharomyces cerevisiae Vps34p, which has been implicated in vacuolar protein sorting and endocytosis. Western blotting experiments revealed that HpPDD1 is expressed constitutively. In a H. polymorpha pdd1 disruption strain peroxisome degradation is fully impaired. Sequestered peroxisomes, typical for the first stage of peroxisome degradation in H. polymorpha, were never observed, suggesting that HpPdd1p plays a role in the tagging of redundant peroxisomes and/or sequestration of these organelles from the cytosol. Possibly, HpPdd1p is the functional homologue of ScVps34p, because—like S. cerevisiae vps34 mutants—H. polymorpha pdd1 mutants are temperature‐sensitive for growth and are impaired in the sorting of vacuolar carboxypeptidase Y. Moreover, HpPdd1p is associated to membranes, as was also observed for ScVps34p. Copyright © 1999 John Wiley & Sons, Ltd.     

Mitochondrial DNA enrichment for species identification and evolutionary analysis

 Nucleic acid-based species identification often targets the mitochondrial encoded cytb gene. However, polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) analysis using universal primers sometimes leads to ambiguous results, which are due to the presence of nuclear encoded pseudo-cytb genes. Such ambiguities were succesfully avoided using a newly developed method for the enrichment of mitochondrial DNA. In addition, a mitochondrial cytb-specific PCR system was designed allowing the unambiguous identification of game meat. Received: 24 February 1998     

Mitochondrial DNA enrichment for species identification and evolutionary analysis

 Nucleic acid-based species identification often targets the mitochondrial encoded cytb gene. However, polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) analysis using universal primers sometimes leads to ambiguous results, which are due to the presence of nuclear encoded pseudo-cytb genes. Such ambiguities were succesfully avoided using a newly developed method for the enrichment of mitochondrial DNA. In addition, a mitochondrial cytb-specific PCR system was designed allowing the unambiguous identification of game meat. Received: 24 February 1998     

SHM1: A multicopy suppressor of a temperature-sensitive null mutation in the HMG1-like abf2 gene

HM, an HMG1-like mitochondrial DNA-binding protein, is required for maintenance of the yeast mitochondrial genome when cells are grown in glucose. To better understand the role of HM in mitochondria, we have isolated several multicopy suppressors of the temperature-sensitive defect associated with an abf2 null mutation (lacking HM protein). One of these suppressors, SHM1, has been characterized at the molecular level and is described herein. SHM1 encodes a protein (SHM1p) that shares sequence similarity to a family of mitochondrial carrier proteins. On glycerol medium, where mitochondrial function is required for growth, shm1 deletion mutants are able to grow, whereas shm1 abf2 double mutants are severely inhibited. These results suggest that SHM1p plays an accessory role to HM in the mitochondrion. The GenBank Accession Number for the SHM1 sequence is U08352.     

Mam33p,an oligomeric,acidic protein in the mitochondrial matrix of Saccharomyces cerevisiae is related to the human complement receptor gC1q-R

Mam33p (mitochondrial acidic matrix protein) is a soluble protein, located in mitochondria of Saccharomyces cerevisiae. It is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is processed on import. Mam33p assembles to a homo-oligomeric complex in the mitochondrial matrix. It can bind to the sorting signal of cytochrome b₂ that directs this protein into the intermembrane space. Mam33p is encoded by an 801 bp open reading frame. Gene disruption did not result in a significant growth defect. Mam33p exhibits sequence similarity to gC1q-R, a human protein that has been implicated in the binding of complement factor C1q and kininogen. © 1998 John Wiley & Sons, Ltd.     

The Nucleotide Sequence of a 39 kb Segment of Yeast Chromosome IV: 12 New Open Reading Frames,Nine Known Genes and One Gene for Gly-tRNA

The complete nucleotide sequence of a 39 090 bp segment from the left arm of yeast chromosome IV was determined. Twenty-one open reading frames (ORFs) longer than 100 amino acids and a Gly-tRNA gene were discovered. Nine of the 21 ORFs (D0892, D1022, D1037, D1045, D1057, D1204, D1209, D1214, D1219) correspond to the previously sequenced Saccharomyces cerevisiae genes for the NAD-dependent glutamate dehydrogenase (GDH), the secretory component (SHR3), the GABA transport protein (UGA4), the high mobility group-like protein (NHP2), the hydroxymethylbilane synthase (HEM3), the methylated DNA protein-cysteine S-methyltransferase (MGT1), a putative sugar transport protein, the Shm1 protein (SHM1) and the anti-silencing protein (ASF2). The inferred amino acid sequences of 11 ORFs show significant similarity with known proteins from various organisms, whereas the remaining ORF does not share any similarity with known proteins. The nucleotide sequence has been entered in the EMBL data Library under the Accession Number X99000.©1997 John Wiley & Sons, Ltd.     

Resveratrol Improves Muscle Atrophy by Modulating Mitochondrial Quality Control in STZ‐Induced Diabetic Mice

1 Scope

In this study, we aim to determine the effects of resveratrol (RSV) on muscle atrophy in streptozocin‐induced diabetic mice and to explore mitochondrial quality control (MQC) as a possible mechanism.

2 Methods and results

The experimental mice were fed either a control diet or an identical diet containing 0.04% RSV for 8 weeks. Examinations were subsequently carried out, including the effects of RSV on muscle atrophy and muscle function, as well as on the signaling pathways related to protein degradation and MQC processes. The results show that RSV supplementation improves muscle atrophy and muscle function, attenuates the increase in ubiquitin and muscle RING‐finger protein‐1 (MuRF‐1), and simultaneously attenuates LC3‐II and cleaved caspase‐3 in the skeletal muscle of diabetic mice. Moreover, RSV treatment of diabetic mice results in an increase in mitochondrial biogenesis and inhibition of the activation of mitophagy in skeletal muscle. RSV also protects skeletal muscle against excess mitochondrial fusion and fission in the diabetic mice.

3 Conclusion

The results suggest that RSV ameliorates diabetes‐induced skeletal muscle atrophy by modulating MQC.     

The role of PaAAC1 encoding a mitochondrial ADP/ATP carrier in the biosynthesis of extracellular glycolipids,mannosylerythritol lipids,in the basidiomycetous yeast Pseudozyma antarctica

Pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (MEL), which show not only excellent surface‐active properties but also versatile biochemical actions. A gene homologous with a mitochondrial ADP/ATP carrier was dominantly expressed in P. antarctica under MEL‐producing conditions on the basis of previous gene expression analysis. The gene encoding the mitochondrial ADP/ATP carrier of P. antarctica (PaAAC1) contained a putative open reading frame of 954 bp and encodes a polypeptide of 317 amino acids. The deduced translation product shared high identity of 66%, 70%, 69%, 74%, 75% and 52% with the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae (AAC1), S. cerevisiae (AAC2), S. cerevisiae (AAC3), Kluyveromyces lactis (KlAAC), Neurospora crassa (NcAAC) and human (ANT1), respectively, and conserved the consensus sequences of all ADP/ATP carrier proteins. The gene expression by introducing a plasmid pUXV1‐PaAAC1 into the yeast cells increased the MEL production. In addition, the expression of PaAAC1 in which the conserved arginine and leucine required for ATP transport activity were replaced with isoleucine and serine, respectively, failed to increase MEL production. Accordingly, these results suggest that PaAAC1 encoding a mitochondrial ADP/ATP carrier should be involved in MEL biosynthesis in the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of Porcine Contamination in Laboratory Prepared dendeng Using Mitochondrial D-Loop686 and cyt b Gene Primers by Real Time Polymerase Chain Reaction

The identifications of species in meat products have created interests since these foods became the target of forgery and fraud in the market. The presence of pork in food products is not allowed for the Muslim community. Hence, an analysis is necessary to detect the presence of pork in processed meat products, such as in dendeng (dried meat) product. Real time polymerase chain reaction using mitochondrial displacement loop686 and cytochrome b (cytb) gene primers was used to identify specific pork DNA among other four types of DNA species; namely beef, chicken, goat, and horse. This method was also used to identify pork DNA in the laboratory processed pork-beef dendeng as well as commercial dendeng from market. The results showed that real time polymerase chain reaction using displacement loop686 and cytb gene primers were able specifically to distinguish between pork DNA and the other species. The lowest concentration of 0.5% of pork DNA in a mixture of pork-beef processed products of dendeng was able to be detected by both primers with the product amplification of 114 and 134 bp (base pair) for the displacement loop686 and 149 bp for cytb gene, respectively. High sensitivity was also obtained when both primers were applied with the lowest detection limit of 5 pg/µL pork DNA. The results of the six commercial dendeng amplification using both primers showed no amplified products present, meaning that these products do not contain porcine DNA.     

Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant‐Derived,Low‐Acid Foods,and Biomaterials

Cycle threshold (Ct) increase, quantifying plant‐derived DNA fragmentation, was evaluated for its utility as a time‐temperature integrator. This novel approach to monitoring thermal processing of fresh, plant‐based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time‐temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low‐acid product and simple linear regression, Ct value was highly correlated to time‐temperature treatment (R² = 0.87); the logarithmic reduction (log CFU/mL) of the spore‐forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R² = 0.87); and cumulative F‐value (min) in a canned retort process (R² = 0.88), all comparisons conducted at 121 °C. D₁₂₁ and z‐values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D₁₂₁ and z‐values for a 174‐bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof‐of‐concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low‐acid plant products.     

Yeast sequencing reports. Sequence of the AFG3 gene encoding a new member of the FtsH/Yme1/Tma subfamily of the AAA-protein family

A nuclear gene from Saccharomyces cerevisiae was cloned by genetic complementation of a temperature-sensitive respiratory-deficient mutant. DNA sequence analysis reveals that it encodes a protein with homology to Yme1, FtsH and Tma, proteins which belong to the AAA-protein family (ÃPases associated with diverse cellular activities). The members of this family are involved in very different biological processes. Yme1p, a yeast mitochondrial protein, affects the rate of DNA escape from mitochondria to the nucleus and the Escherichia coli FtsH protein is apparently involved in the post-translational processing of PBP3, a protein necessary for septation during cell division. This newly sequenced gene, which we have designated AFG3 for ÃPase family gene 3, encodes a putative mitochondrial protein of 760 amino acid residues that is closely related to FtsH, Tma (protein from Lactococcus lactis) and Yme1p with 58, 55 and 46% identity respectively. The sequence has been deposited in the EMBL data library under Accession Number X76643.     

Yeast PIG Genes: PIG1 Encodes a Putative Type 1 Phosphatase Subunit that Interacts with the Yeast Glycogen Synthase Gsy2p

The biosynthesis of glycogen involves multiple proteins that associate with each other and the glycogen macromolecule. In efforts to understand the nature of these proteins, a two-hybrid screen was undertaken to detect proteins able to interact with Gsy2p, a major form of glycogen synthase in Saccharomyces cerevisiae. Two positives expressed proteins derived from genes designated PIG1 and PIG2, on chromosomes XIIR and IXL respectively. PIG1 codes for a protein with 38% identity over a 230 residue segment to Gac1p, a protein thought to be a type 1 protein phosphatase targeting subunit whose loss impairs glycogen synthesis. Pig2p has 30% identity to the protein corresponding to an open reading frame, YER054, on chromosome V. Deletion of PIG1 on its own had little effect on glycogen storage but, in combination with loss of GAC1, caused a more severe glycogen-deficient phenotype than seen in gac1 mutants. This result is consistent with Pig1p being functionally related to Gac1p and we propose that Pig1p may be a type 1 phosphatase regulatory subunit. Delection of PIG2, YER054, or both genes together caused no detectable change in glycogen metabolism under the conditions tested. Gac1p, Pig1p, Pig2p and the YER054p are the only four proteins coded by the yeast genome that share a conserved segment of ∼25 residues, designated the GVNK motif, that is identifiable also in R_GI, the mammalian type 1 phosphatase targeting subunit. © 1997 by John Wiley & Sons, Ltd.     

The nutritive value for ruminants of thin stillage and distillers' grains derived from wheat,rye, triticale and barley

A study was conducted to determine nutrient degradabilities of thin stillages and distillers' grains derived from wheat‐, rye‐, triticale‐ and barley‐based ethanol production. In vitro protein degradabilities of wheat, rye, triticale and barley thin stillages were determined using a protease enzyme assay. One ruminally fistulated cow was used to determine ruminal nutrient degradabilities for wheat, rye, triticale and barley distillers' grains. Results of the in vitro study showed that the soluble protein fraction was highest for rye thin stillage and lowest for barley thin stillage. The degradation rate of the slowly degradable protein fraction was higher for wheat and triticale thin stillage than rye thin stillage and was higher for rye than barley thin stillage. Effective degradability of crude protein followed the order rye (659 g kg⁻¹) > triticale (632 g kg⁻¹) > wheat (608 g kg⁻¹) > barley (482 g kg⁻¹) thin stillage. Ruminal degradability of dry matter was highest for rye and lowest for barley distillers' grains. Ruminal degradability of dry matter was also higher for wheat than triticale distillers' grains. Crude protein from barley distillers' grains had a lower ruminal degradability relative to crude protein from wheat and rye distillers' grains. Ruminal degradability of neutral detergent fibre was highest for rye distillers' grains (470 g kg⁻¹), intermediate for wheat and triticale distillers' grains (average 445 g kg⁻¹) and lowest for barley distillers' grains (342 g kg⁻¹). It was concluded that thin stillage and distillers' grains derived from barley had a lower nutritive value for ruminants compared with those derived from wheat, rye and triticale. © 2000 Society of Chemical Industry