AIR synthetase in cowpea nodules: a single gene product targeted to two organelles?

A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.     

Vascular and cardiac effects of amlodipine in acute heart failure in dogs

BACKGROUND: Amlodipine improves exercise capacity in patients with chronic congestive heart failure (HF), but the mechanisms of this effect are unknown. OBJECTIVE: To test the hypothesis, in a canine model of acute, ischemic HF, that amlodipine increases vascular capacitance and reduces cardiac filling pressures. METHODS: Amlodipine was given to 13 anesthetized, splenectomized dogs (six controls and seven with HF). Aortic, left ventricular end-diastolic (LVEDP) and portal venous (Pportal) pressures, cardiac output, portal flow (ultrasonic probe) and intestinal blood volume (IBV, 99mTc blood-pool scintigraphy) were measured. Intestinal vascular conductance (= 1/resistance) and vascular capacitance (CAP) were measured before and 15 mins after repetitive 150 micrograms/kg dosages of amlodipine (maximum cumulative dosage, 1000 micrograms/kg). Pportal-IBV curves were obtained by impeding portal flow (pneumatic cuff), and change in CAP was defined by the change in IBV at Pportal = 7.5 mmHg. HF was induced by microsphere embolization of the left coronary artery. RESULTS: CAP increased in the control group (+ 28%, P < 0.01) but decreased (-9%, P < 0.05) in the HF group. Left ventricular stroke work increased in the control group (P < 0.05), while it decreased (P < 0.05) in the HF group, suggesting a negative inotropic effect. In the control group, LVEDP increased after amlodipine was given (P < 0.05) but did not change significantly in the HF group. CONCLUSIONS: In the acute experimental HF model, amlodipine failed to increase intestinal vascular CAP or decrease filling pressures, and may have had a negative inotropic effect. The experiment failed to demonstrate a beneficial hemodynamic effect of amlodipine in acute HF, and the mechanism of benefit of this agent in chronic HF remains unclear.     

Bcl-2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-xL

Bcr - Abl is the molecule responsible for both the transformation phenotype and the resistance to chemotherapeutic drugs found in chronic myelogenous leukemia (CML) cells. Wild-type HL-60, a transformed pro-myelocytic cell line, is very susceptible to apoptosis-inducing agents. We show here that expression of Bcr - Abl in HL-60 cells rendered them extremely resistant to apoptosis induced by a wide variety of agents. The anti-apoptotic effect of Bcr - Abl was found to be independent of the phase of the cell cycle. Treatment with antisense oligonucleotides directed to bcr decreased the expression of the ectopic bcr - abl and restored susceptibility to apoptosis. Double mutations affecting the autophosphorylation site and the phosphotyrosine-binding motif (FLVRES) have been previously shown to impair the transforming activity of Bcr - Abl in fibroblasts and hematopoietic cells, however HL-60 cells expressing this double mutant molecule exhibited the same level of resistance to apoptosis as those expressing the wild-type Bcr - Abl. Interestingly, wild type and mutant Bcr - Abl induced in HL-60 cells a dramatic down regulation of Bcl-2 and increased the levels of Bcl-xL. The level of Bax did not change in response to the presence of Bcr - Abl. Antisense oligonucleotides targeted to bcl-x downregulated the expression of Bcl-x, and increased the susceptibility of HL-60. Bcr - Abl cells to staurosporine. Importantly, HL-60 cells overexpressing Bcl-xL showed higher expression of Bcl-xL but lower resistance to apoptosis when compared to HL-60. Bcr - Abl cells. The results described here show that Bcr - Abl is a powerful mammalian anti-apoptotic molecule and can act independently of Bcl-2. Bcl-xL, however, seems to participate in part in Bcr - Abl-mediated resistance to apoptosis in HL-60 cells.     

Precursor-directed biosynthesis of 12-ethyl erythromycin

A precursor-directed method for the biosynthesis of novel 6-deoxyerythronolide B derivatives has been extended to allow alteration of the functionality at C-12. We recently described a simple and practical method for harnessing the biosynthetic potential of the erythromycin pathway to generate novel molecules of natural product-like complexity by feeding designed synthetic molecules to an engineered mutant strain having an altered 6-deoxyerythronolide B synthase (DEBS). Our initial applications of this technique focused on alteration of the ethyl side chain of 6-dEB (C14-C15). We now report the extension of this approach to modification of the C-12 substituent. An appropriately designed substrate is shown to incorporate into 6-dEB biosynthesis, yielding a 6-dEB analogue bearing a 12-ethyl group. This 6-dEB analogue is a substrate for post-polyketide tailoring enzymes, and is converted into the corresponding analogue of erythromycin C. These results show that many of the downstream active sites are tolerant of this unnatural functionality and suggest that a wide variety of erythromycin derivatives should be accessible by this approach or by total biosynthesis via genetic engineering.     

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.     

Electronic medical records for prenatal patients: challenges and solutions

A longstanding impediment to successful medical computing is resistance on the part of physicians. Interaction with many medical computing systems is difficult, requiring the physician to spend valuable time and energy trying to figure out how to get the machine to do what needs to be done. In developing encounter forms for use in prenatal medical records, we confronted the challenges involved in designing a computing system that provides an intuitive and physician-friendly method of recording clinical data. In trying to meet those challenges, we also learned about how to evaluate a medical computing system for flexibility and ease of use.     

一种基于蚁群算法的任务调度方法

任务调度是一个NP-hard问题,而且是并行与分布式计算中一个必不可少的组成部分,特别是在网格计算环境中任务调度更加复杂。文中结合蚁群算法的优点,提出了一种基于蚁群算法的任务调度方法,将算法应用于网格计算任务调度问题的求解之中。最后,实验结果这种算法优于普通算法。     

Design of TRIP Steel With High Welding and Galvanizing Performance in Light of Thermodynamics and Kinetics

 A new type of transformation induced plasticity (TRIP) steel with not only high strength and high ductility but also superior welding and galvanizing properties was designed and developed recently. Low carbon and low silicon content were preliminarily selected with the aim of meeting the requirements of superior quality in both welding and galvanizing. Phosphorus was chosen as one of the alloying elements, because it could reduce carbon activity in cementite and increase the stability of austenite. In addition, the possibility of phosphorus segregating at grain boundary was also discussed by thermodynamics as well as kinetics. Phase diagram was estimated at high temperature and the composition of the steel was then selected in the hyperperitectic range to avoid problems, which might occur in sheet steel continuous casting. Phase diagram in the inter critical temperature was estimated for the steel to obtain the starting temperature of fast cooling. For understanding the minimum rate of fast cooling, pearlite growth kinetics was calculated with self developed diffusion coefficients of elements in grain boundary. Overaging temperature was determined through the calculation of T0 temperature by both equilibrium and para equilibrium assumptions, which was different from the current determination, which is only based on an equilibrium estimation.