柠檬酸合酶的分子生物学研究进展

柠檬酸合酶（citrate synthase,CS）是细胞内多种重要代谢途径的关键酶。CS可催化草酰乙酸和乙酰辅酶A之间的缩合反应生成柠檬酸和辅酶A。通常革兰氏阳性细菌、古菌以及真核细胞的CS为同源二聚体,而革兰氏阴性细菌的CS为同源六聚体。根据其在细胞内的定位不同,CS可分为线粒体CS、乙醛酸循环体CS、过氧化物酶体CS。这些同工酶在能量代谢、植物脂肪的代谢、脂肪酸的氧化及细胞解毒过程中起着重要作用。不同来源的CS空间结构、催化机制和动力学性质十分相似。针对其生化特性、空间结构特点、催化机制以及分子进化等研究进展进行综述。     

Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.     

Mechanistic studies of melanogenesis: the influence of N-substitution on dopamine quinone cyclization

The influence of side-chain structure on the mode of reaction of ortho-quinone amines has been investigated with a view, ultimately, to developing potential methods of therapeutic intervention by manipulating the early stages of melanogenesis. Four N-substituted dopamine derivatives have been prepared and quinone formation studied using pulse radiolysis and tyrosinase-oximetry. Ortho-quinones with an amide or urea side chain were relatively stable, although evidence for slow formation of isomeric para-quinomethanes was observed. A thiourea derivative cyclized fairly rapidly (k = 1.7/s) to a product containing a seven-membered ring, whereas a related amidine gave more rapidly (k approximately 2.5 x 10(2)/s) a stable spirocyclic product. The results suggest that cyclization of amides, ureas and carbamates (NHCO-X; X = R, NHR or OR) does not occur and is not, therefore, a viable approach to the formation of tyrosinase-activated antimelanoma prodrugs. It is also concluded that for N-acetyldopamine spontaneous ortho-quinone to para-quinomethane isomerization is slow.     

Growth and regression of vasculature in healthy and diabetic mice after hindlimb ischemia

The formation of vascular networks during embryogenesis and early stages of development encompasses complex and tightly regulated growth of blood vessels, followed by maturation of some vessels, and spatially controlled disconnection and pruning of others. The adult vasculature, while more quiescent, is also capable of adapting to changing physiological conditions by remodeling blood vessels. Numerous studies have focused on understanding key factors that drive vessel growth in the adult in response to ischemic injury. However, little is known about the extent of vessel rarefaction and its potential contribution to the final outcome of vascular recovery. We addressed this topic by characterizing the endogenous phases of vascular repair in a mouse model of hindlimb ischemia. We showed that this process is biphasic. It encompasses an initial rapid phase of vessel growth, followed by a later phase of vessel rarefaction. In healthy mice, this process resulted in partial recovery of perfusion and completely restored the ability of mice to run voluntarily. Given that the ability to revascularize can be compromised by a cardiovascular risk factor such as diabetes, we also examined vascular repair in diabetic mice. We found that paradoxically both the initial growth and subsequent regression of collateral vessels were more pronounced in the setting of diabetes and resulted in impaired recovery of perfusion and impaired functional status. In conclusion, our findings demonstrate that the formation of functional collateral vessels in the hindlimb requires vessel growth and subsequent vessel rarefaction. In the setting of diabetes, the physiological defect was not in the initial formation of vessels but rather in the inability to sustain newly formed vessels.     

Methylcytosine and Normal Cytosine Deamination by the Foreign DNA Restriction Enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.     

Twenty-One Genome Sequences from Pseudomonas Species and 19 Genome Sequences from Diverse Bacteria Isolated from the Rhizosphere and Endosphere of Populus deltoides

To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.     

Draft genome sequence for Microbacterium laevaniformans strain OR221, a bacterium tolerant to metals, nitrate, and low pH

Microbacterium laevaniformans strain OR221 was isolated from subsurface sediments obtained from the Field Research Center (FRC) in Oak Ridge, TN. It was characterized as a bacterium tolerant to heavy metals, such as uranium, nickel, cobalt, and cadmium, as well as nitrate and low pH. We present its draft genome sequence.