长双歧杆菌NCC2705葡萄糖与果糖代谢的比较蛋白质组学

为揭示长双歧杆菌NCC2705 (Bifidobacterium longum NCC2705)果糖代谢途径, 建立其果糖发酵模型。以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础, 进行了果糖和葡萄糖生长的菌体比较蛋白质组学研究, 利用MALDI-TOF和ESI-MS/MS鉴定差异蛋白, 进一步通过半定量RT-PCR验证二者显著差异表达蛋白。果糖生长的菌体蛋白中鉴定到了所有葡萄糖降解途径中的酶和蛋白质, 另外鉴定到3倍以上差异蛋白点9个, 其对应的5个蛋白在果糖发酵中上调。半定量RT-PCR验证显著差异蛋白, 显示在果糖发酵中具有高水平表达是ABC 转运系统的果糖特异性-结合蛋白BL0033和ATP结合蛋白BL0034。果糖的发酵时间和浓度梯度试验显示诱导时间越长、浓度越高, BL0033的表达量越高。第一, 比较蛋白谱证明果糖和葡萄糖以相同途径降解。第二, BL0033的表达是受果糖诱导的, 果糖的吸收可能是通过一个特殊的转运系统, 即ABC转运系统将果糖从胞外转运到胞内, 其中BL0033和BL0034共同作为系统元件扮演了重要角色。     

Serine Catabolism Feeds NADH when Respiration Is Impaired

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Design,synthesis, and in vitro evaluation of an activity-based protein profiling (ABPP) probe targeting agmatine deiminases

Agmatine deiminases (AgDs) belong to a family of enzymes known as guanidinium group modifying enzymes (GMEs). Many pathogenic bacteria encode an AgD that participates in the catabolism of agmatine (decarboxylated arginine). This catabolism may confer a competitive survival advantage, by virtue of energy production and increased acid tolerance, making this sub-family of enzymes a potential therapeutic target that warrants further study. Herein we report the development of an activity-based protein profiling (ABPP) probe that selectively targets the AgD from Streptococcus mutans. Due to the selectivity and covalent nature of the modification, this probe could prove to be a valuable tool for the study of other AgD family members.     

Incomplete proline catabolism drives premature sperm aging

Infertility is an increasingly common health issue, with rising prevalence in advanced parental age. Environmental stress has established negative effects on reproductive health, however, the impact of altering cellular metabolism and its endogenous reactive oxygen species (ROS) on fertility remains unclear. Here, we demonstrate the loss of proline dehydrogenase, the first committed step in proline catabolism, is relatively benign. In contrast, disruption of alh‐6, which facilitates the second step of proline catabolism by converting 1‐pyrroline‐5‐carboxylate (P5C) to glutamate, results in premature reproductive senescence, specifically in males. The premature reproductive senescence in alh‐6 mutant males is caused by aberrant ROS homeostasis, which can be countered by genetically limiting the first committed step of proline catabolism that functions upstream of ALH‐6 or by pharmacological treatment with antioxidants. Taken together, our work uncovers proline metabolism as a critical component of normal sperm function that can alter the rate of aging in the male reproductive system.     

Presence of indole-3-acetic acid in chloroplasts ofNicotiana tabacum andPinus sylvestris

The compartmentation and metabolism of indole-3-acetic acid (IAA) was examined in protoplasts derived from needles ofPinus sylvestris L., leaves of normal plants ofNicotiana tabacum L., leaves ofN. tabacum plants carrying the T-DNA gene 1 (rG1 plants) and leaves ofN. tabacum plants carrying the T-DNA gene 2 (rG2 plants) by using a rapid cell-fractionation method. In all tissues, 30%–40% of the IAA pool was located in the chloroplast, while the remainder was found in the cytosol. Quantitative analysis of indole-3-ethanol (IEt) showed that in bothPinus andNicotiana the IEt pool was located exclusively in the cytosol. The only plant that contained endogenous indoleacetamide (IAAm) was therG1-mutant ofN. tabacum, expressing theAgrobacterium tumefaciens T-DNA gene 1. Cellular fractionation of protoplasts from this transgenic plant showed that the entire IAAm pool was located in the cytosol. Feeding experiments utilizing [5-³H]tryptophan, [5-³H]IEt, [1′-¹⁴C] and [2′-¹⁴C]IAA demonstrated that the biosynthesis and catabolism of IAA occurred in the cytosol in bothPinus and in the wild type and the different mutants ofNicotiana. Furthermore, the biosynthesis of IAAm in therG1 plants was also shown to be localized in the cytosol.     

Gene transfer in Acinetobacter calcoaceticus NCIB8250

Abstract Filter matings of mutant strains of Acinetobacter calcoaceticus NCIB8250 showed that catabolic and auxotrophic markers were transferred in the absence of a conjugative plasmid. There were no specific 'donor' or 'recipient' strains. Deoxyribonuclease had no effect on the mating system. Some crosses appeared to be highly polarized towards certain parental strains whilst others showed a two-way transfer of genetic markers. There was a high frequency of transfer of the ability to utilize L(+)-mandelate from a mutant of A. calcoaceticus NCIB8250 to certain strains of a second wild-type, EBF65/65, but there was no evidence that the recombinants had acquired another plasmid. This process, whose mechanism is not clear, has some potential in the construction of novel strains but is not likely to be generally useful for mapping purposes and may even prove to be a hazard in the interpretation of results from other genetic techniques.     

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized.Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N¹-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N¹-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N¹-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N¹-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N¹-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes.In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.     

Metabolic reconstructions identify plant 3‐methylglutaconyl‐CoA hydratase that is crucial for branched‐chain amino acid catabolism in mitochondria

The proteinogenic branched‐chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant‐prokaryote comparative genomics detected candidates for 3‐methylglutaconyl‐CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non‐homologous N‐terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein‐fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3‐hydroxymethylglutaryl‐CoA into 3‐methylglutaconyl‐CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark‐induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3‐methylglutaconyl‐CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.