Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

植物细胞程序死亡的机理及其与发育的关系

细胞程序死亡（ＰＣＤ）是在植物体发育过程中普遍存在的,在发育的特定阶段发生的自然的细胞死亡过程,这一死亡过程是由某些特定基因编码的“死亡程序”控制的。ＰＣＤ的细胞分化的最后阶段。细胞分化的临界期就牌死亡程序执行中的某个阶段。ＰＣＤ包含启动期和清除期三个阶段,其间ＣＡＳＰＡＳＥ家族起着重要作用。ＰＣＤ在细胞和组织的平衡、特化,以及组织分化、器官建成和对病原体的反应等植物发育过程中起着重要作用。ＰＣＤ     

Regulation of branched-chain amino acid catabolism: glucose limitation enhances the component of isovalerylspiramycin for the bitespiramycin production

4′′-O-isovalerylspiramycins are the major components of bitespiramycin complex consisting of a group of 4′′-O-acylated spiramycins. The availability of isovaleryl group, usually in vivo derived from leucine, one of the branched-chain amino acids, affects the content of isovaleryispiramycin significantly. In this study, the effect of glucose on the activity of branched-chain α-keto acid dehydrogenase (BCKDH), which catalyzed the rate-limiting as well as the first irreversible reaction oxidative decarboxylation for branched-chain amino acids degradation, and isovaleryispiramycin biosynthesis was investigated. In the initial glucose concentration experiment, when the residual glucose concentration in the medium declined to 2–4 g/L, the BCKDH activity rose rapidly, and glucose deprivation and the summit of BCKDH activity appeared nearly at the same time. After a delay of about 6 h, the maximal isovalerylspiramycin content was observed. However, the shortage of glucose at the later production phase resulted in the marked decrease in BCKDH activity and isovaleryispiramycin content. In the fermentation in a 50 L fermentor, glucose feeding at the late production phase helped to maintain the residual glucose concentration between 0 and 1 g/L, leading to the high level of BCKDH activity and thus isovalerylspiramycin content. These suggested that glucose concentration could be used as a key parameter to regulate BCKDH activity and isovaleryispiramycin biosynthesis in the bitespiramycin production.     

Differential regulation of nuclear and mitochondrial Bcl-2 in T cell apoptosis

Activated T cells require anti-apoptotic cytokines for their survival. The anti-apoptotic effects of these factors are mediated by their influence on the balance of expression and localisation of pro- and anti-apoptotic members of the Bcl-2 family. Among the anti-apoptotic Bcl-2 family members, the expression level of Bcl-2 itself and its interaction with the pro-apoptotic protein Bim are now regarded as crucial for the regulation of survival in activated T cells. We studied the changes in Bcl-2 levels and its subcellular distribution in relation to mitochondrial depolarisation and caspase activation in survival factor deprived T cells. Intriguingly, the total Bcl-2 level appeared to remain stable, even after caspase 3 activation indicated entry into the execution phase of apoptosis. However, cell fractionation experiments showed that while the dominant nuclear pool of Bcl-2 remained stable during apoptosis, the level of the smaller mitochondrial pool was rapidly downregulated. Signals induced by anti-apoptotic cytokines continuously replenish the mitochondrial pool, but nuclear Bcl-2 is independent of such signals. Mitochondrial Bcl-2 is lost rapidly by a caspase independent mechanism in the absence of survival factors, in contrast only a small proportion of the nuclear pool of Bcl-2 is lost during the execution phase and this loss is a caspase dependent process. We conclude that these two intracellular pools of Bcl-2 are regulated through different mechanisms and only the cytokine-mediated regulation of the mitochondrial pool is relevant to the control of the initiation of apoptosis. D. Scheel-Toellner and K. Raza have contributed equally to this study.     

Effect of bacteriocin-induced cell damage on the branched-chain amino acid transamination by Lactococcus lactis

The effect of the bacteriocin lacticin 3147 on the branched-chain amino acid transamination by Lactococcus lactis IFPL359 was investigated. The bacteriocin provokes membrane permeabilisation of the cells, rendering them non-viable but metabolically active. Free diffusion of amino acids into the cell was facilitated. In addition, membrane permeabilisation promotes further cell lysis. Both facts render the enzymes more accessible to their substrates and hence increase branched-chain amino acid transamination. This research broadens the spectrum of technological applications of lacticin 3147 in the development of cheese flavour.     

A zinc-dependent nuclear endonuclease is responsible for DNA laddering during salt-induced programmed cell death in root tip cells of rice

DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20kDa (OsCyt20) and a nuclear of 37kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca(2+)/Mg(2+)-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn(2+)-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.     

Unusual regulation of the uptake system for branched-chain amino acids in Corynebacterium glutamicum

The uptake of branched-chain amino acids in threonine-dehydratase deficient mutants of Corynebacterium glutamicum is dependent on the presence of relatively high (>1 mM) intracellular concentrations of isoleucine, valine or leucine. This indicates that the respective uptake-system is induced by its substrate, i.e. branched-chain amino acids, at the internal side. This unusual regulation presumably is the reason for the failure to obtain mutants deficient in isoleucine uptake by use of a selection scheme which starts from isoleucine auxotroph mutants. The physiological meaning of this regulation is discussed with respect to isoleucine efflux and the cyclic retention hypothesis.Abbreviations amp ampicillin - dw dry weight - Km kanamycin - kb kilobase(s) - NMG N-methyl-N-nitro-N-nitrosoguanidine - ®, resistant resistance     

Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval-pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.     

Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures

Association of the availability of nutrients with the phenomenon of programmed cell death-apoptosis-was investigated using hybridoma cells cultured in protein-free medium under conditions of starvation, i.e., in RPMl-1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth-supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. (c) 1995 John Wiley & Sons, Inc.     

Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway

Inhibition of proteasome activity and the resulting protein accumulation are now known to be important events in the development of many neurological disorders, including Alzheimer’s and Parkinson’s diseases. Abnormal or over expressed proteins cause endoplasmic reticulum and oxidative stress leading to cell death, thus, normal proteasome function is critical for their removal. We have shown previously, with cultured SH-SY5Y neuroblastoma cells, that proteasome inhibition by the drug epoxomicin results in accumulation of ubiquitinated proteins. This causes obligatory loading of the mitochondria with calcium (Ca²⁺), resulting in mitochondrial damage and cytochrome c release, followed by programmed cell death (PCD). In the present study, we demonstrate that all-trans-retinoic acid (RA) pretreatment of SH-SY5Y cells protects them from PCD death after subsequent epoxomicin treatment which causes proteasome inhibition. Even though ubiquitinated protein aggregates are present, there is no evidence to suggest that autophagy is involved. We conclude that protection by RA is likely by mechanisms that interfere with cell stress-PCD pathway that otherwise would result from protein accumulation after proteasome inhibition. In addition, although RA activates both the AKT and ERK phosphorylation signaling pathways, only pretreatment with LY294002, an inhibitor of PI3-kinase in the AKT pathway, removed the protective effect of RA from the cells. This finding implies that RA activation of the AKT signaling cascade takes precedence over its activation of ERK1/2 phosphorylation, and that this selective effect of RA is key to its protection of epoxomicin-treated cells. Taken together, these findings suggest that RA treatment of cultured neuroblastoma cells sets up conditions under which proteasome inhibition, and the resultant accumulation of ubiquitinated proteins, loses its ability to kill the cells and may likely play a therapeutic role in neurodegenerative diseases.     

Cloning,expression, and nucleotide sequence of livR,the repressor for high-affinity branched-chain amino acid transport in Escherichia coli

The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106. The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations. Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment. Expression of the pANT plasmids in E. coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000. DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155. The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices. These results suggests that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.     

Evidence for an instructive role of apoptosis during the metamorphosis of Hydractinia echinata (Hydrozoa)

Apoptosis is a highly conserved mechanism of cell deletion that destroys redundant, dysfunctional, damaged, and diseased cells. Furthermore, apoptotic cell death is essential during the development of multicellular organisms. However, there are only a few examples where the occurrence of apoptosis has been shown to be a direct prerequisite for developmental processes. As described previously by our group, the degradation of larval tissue during the first half of the metamorphosis of Hydractinia echinata involves extensive cell death. A large number of cells are removed, and we observed several cellular features of apoptotic cell death in the dying tissue, e.g., nucleosomal DNA fragmentation and nuclear condensation. Furthermore, we showed that metamorphosis in the basal cnidarian H. echinata depends on the activity of caspases, the central enzymes of apoptosis. In the present study, we build on these previous investigations of apoptosis in H. echinata by characterising a caspase-3 sequence in this species and placing it in an evolutionary context by performing phylogenetic analyses. Furthermore, we report the successful knockdown of a caspase by RNAi and show that apoptosis plays a role as an instructive mechanism in the metamorphosis of H. echinata.     

Expression of programmed cell death 5 gene involves in regulation of apoptosis in gastric tumor cells

The protein of programmed cell death 5 (PDCD5) is believed to participate in regulation of apoptosis. Although PDCD5 is reducibly expressed in various human tumors, it is not clear which expression level of PDCD5 is in gastric cancer (GC). In this study, we have systematically employed the approaches of RT-PCR, Real- time PCR, Immunohistochemistry (IHC), Immunofluorescence staining (IFS) and Western blot to determine the PDCD5 expression in GC cells and primary tumors, at mRNA and protein level, respectively. Our data revealed that the positive rate of PDCD5 expression in the gastric tumor tissues was significantly less than that of the normal tissues (14 out of 102 vs 36 out of 51), whereas, the decreased expression of PDCD5 protein was well correlated with the up-regulated expression of Bcl-2 in these tissues, and the up-regulated expression and nuclear translocation of PDCD5 protein were verified in the apoptotic GC cells induced by Diallyl trisulfide (DATS). Furthermore, the survival curve has suggested that the more PDCD5 expressions were found in the patients, the longer the survival periods were. Therefore, our observations lay down a reasonable postulation that PDCD5 may play a key role to regulate the apoptotic processes in the GC cells and gastric tumors.     

The regulation of fatty acid and branched-chain amino acid oxidation in cancer cachectic rats: a proposed role for a cytokine, eicosanoid, and hormone trilogy

Some postulates of a hypothesis concerned with the deregulation of muscle turnover by the hypoketonemia of cachectic tumor-bearing rats were examined. Plasma concentrations of ketone bodies (D-(3)-hydroxybutyrate + acetoacetate) in rats bearing the Walker 256 carcinosarcoma were reduced by 45% (P less than 0.001) whereas the concentrations of triglyceride and free fatty acids were elevated by 223% (P less than 0.001) and 335% (P less than 0.001), respectively. Parallel with the changes in plasma, the livers of tumor-bearing animals showed decreased concentrations of KB by 35% (P less than 0.05) and increased concentrations of TG and FFA by 49% (NS) and 15% (NS), respectively. In comparison with values for the control liver (fed ad libitum), the perfused liver of animals bearing the Walker 256 tumor formed 42% (P less than 0.05) and 75% (P less than 0.05) less ketone bodies and CO2, respectively, from oleate, while TG formation was enhanced by 33% (P less than 0.001). There was two- to threefold (P less than 0.001) enhancement of [1-14C]leucine oxidation in vivo by the tumor-bearing animals. The activities of branched-chain amino acid aminotransferase and branched-chain keto acid dehydrogenase were elevated by 70% (P less than 0.001) and 560% (P less than 0.001) respectively in the gastrocnemius muscle of the tumor-bearing animals. The results of the investigation supported a second proposal of the hypothesis, namely, that cancer-induced cachexia resulted in the notable elevation in the concentration of arginine vasopressin that was accompanied by parallel increases in the plasma, urine, and muscle concentrations of prostaglandin E2. The proposals of the original hypothesis have been augmented to include roles for PGE2 and the cytokine cachectin/tumor necrosis factor which may engineer all of the events depicted in the original hypothesis.     

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.