Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria

Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

受体酪氨酸激酶依赖的信号转导在突触可塑性中的研究

突触可塑性对于脑发育过程中的神经环路重构以及学习记忆等脑的高级功能是非常重要的。许多受体酪氨酸激酶家族成员,包括TrkB、ErbB和Eph在神经连接的建立和重构过程中起到核心作用。比如,突触后EphB依赖的信号会导致树突棘的产生和神经递质受体的聚集,而ephrinA引起的EphA4激活可以导致树突棘的回缩。但是,目前对EphA4依赖的树突棘重组和对神经递质受体的调节背后的机制还知之甚少。本文将集中探讨EphA4及其下游的信号通路在神经肌肉接头和中枢神经的突触中,对神经递质受体的调节功能。     

Early cell evolution,eukaryotes, anoxia,sulfide, oxygen,fungi first (?), and a tree of genomes revisited

Genomes contain evidence for the history of life and furthermore contain evidence for lateral gene transfer, which was an important part of that history. The geological record also contains evidence for the history of life, and newer findings indicates that the Earth's oceans were largely anoxic and highly sulfidic up until about 0.6 billion years ago. Eukaryotes, which fossil data indicate to have been in existence for at least 1.5 billion years, must have therefore spent much of their evolutionary history in oxygen-poor and sulfide-rich environments. Many eukaryotes still inhabit such environments today. Among eukaryotes, organelles also contain evidence for the history of life and have preserved abundant traces of their anaerobic past in the form of energy metabolic pathways. New views of eukaryote phylogeny suggest that fungi may be among the earliest-branching eukaryotes. From the standpoint of the fungal feeding habit (osmotrophy rather than phagotrophy) and from the standpoint of the diversity in their ATP-producing pathways, a eukaryotic tree with fungi first would make sense. Because of lateral gene transfer and endosymbiosis, branches in the tree of genomes intermingle and occasionally fuse, but the overall contours of cell history nonetheless seem sketchable and roughly correlateable with geological time.     

Acute hypothyroidism slows the rate of left ventricular diastolic relaxation

The effect of acute thyroid hormone deficiency on left ventricular diastolic filling was studied by radionuclide ventriculography with simultaneous right heart catheterization in nine athyreotic patients without cardiovascular disease. The patients were studied when they were hypothyroid and when they were euthyroid on replacement therapy. Peak filling rate and the time to peak filling were used to characterize diastolic function. The time to peak filling was defined as the interval from end-systole on the radionuclide time-volume curve to the time of occurrence of peak filling. The peak filling rate was determined in absolute terms from the normalized radionuclide peak filling rate and from the end-diastolic volume, which was derived from the radionuclide ejection fraction and from the thermodilution stroke volume. In all patients, the values for peak filling rate were lower in the hypothyroid than in the euthyroid state (287 +/- 91 mL/s vs. 400 +/- 118 mL/s, delta = 41 +/- 13%, p less than 0.01). Peak filling always occurred during the first half of the diastolic interval. The time to peak filling was not significantly affected by the thyroid state (170 +/- 10 ms vs. 159 +/- 21 ms, delta = 7 +/- 10%). Left ventricular filling pressure as reflected by the pulmonary capillary wedge pressure and end-systolic volume were similar in both thyroid states (6 +/- 2 mmHg vs. 8 +/- 2 mmHg (1 mmHg = 133.32 Pa) and 32 +/- 11 mL vs. 32 +/- 7 mL, respectively). The data suggest that the rate of active diastolic relaxation is decreased in short-duration hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts

A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T₇ promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.Abbreviations RPE ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - PGK phosphoglycerate kinase - FBP fructose-1,6-bisphosphatase - SBP sedoheptulose-1,7-bisphosphatase - OPPP oxidative pentose phosphate pathway - Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphosphate aldolase - IPTG isopropyl -d-thiogalactoside - TPI triosephosphate isomerase     

New approach for the synthesis of [F]fluoroethyltyrosine for cancer imaging: Simple, fast, and high yielding automated synthesis

O-(2-[¹⁸F]fluoroethyl)-l-tyrosine ([¹⁸F]FET) is one of the first ¹⁸F-labeled amino acids for imaging amino acid metabolism in tumors. This tracer overcomes the disadvantages of [¹⁸F]fluorodeoxyglucose, [¹⁸F]FDG, and [¹¹C]methionine, [¹¹C]MET. Nevertheless, the various synthetic methods providing ¹⁸F[FET] exhibit a big disadvantage concerning the necessity of two purification steps during the synthesis including HPLC purification, which causes difficulties in the automation, moderate yields, and long synthesis times >60 min.A new approach for the synthesis of [¹⁸F]FET is developed starting from 2-bromoethyl triflate as precursor. After optimization of the synthesis parameters including the distillation step of [¹⁸F]-FCH₂CH₂Br combined with the final purification of [¹⁸F]FET using a simple solid phase extraction instead of an HPLC run the synthesis [¹⁸F]FET could be significantly simplified, shortened, and improved. The radiochemical yield (RCY) was about 45% (not decay corrected and calculated relative to [¹⁸F]F⁻ activity that was delivered from the cyclotron). Synthesis time was only 35 min from the end of bombardment (EOB) and the radiochemical purity was >99% at the end of synthesis (EOS). Thus, this simplified synthesis for [¹⁸F]FET offers a very good option for routine clinical use.     

Bacterial‐type oxygen detoxification and iron‐sulfur cluster assembly in amoebal relict mitochondria

The assembly of vital reactive iron‐sulfur (Fe‐S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial‐type Fe‐S cluster assembly proteins and possess instead an analogous bacterial‐type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial‐type Fe‐S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10‐fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe‐S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe‐S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe‐S protein‐mediated oxygen detoxification.