Kinetic mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae

Kinetic data have been collected suggesting a preferred sequential ordered kinetic mechanism for the histidine-tagged homocitrate synthase (HCS) from Saccharomyces cerevisiae with alpha-ketoglutarate binding before AcCoA and CoA released before homocitrate. Oxaloacetate is also a substrate for HCS, but with lower affinity than alpha-ketoglutarate. In agreement with the ordered kinetic mechanism desulfo-CoA is uncompetitive and citrate is competitive vs alpha-ketoglutarate. Varying AcCoA, citrate is a noncompetitive inhibitor as predicted, but CoA is noncompetitive vs AcCoA suggesting binding of CoA to E:homocitrate and E:alpha-ketoglutarate. The product CoA behaves in a manner identical to the dead-end analogue desulfo-CoA, suggesting an E:alpha-ketoglutarate:CoA dead-end complex. Data further suggest an irreversible reaction overall, in agreement with the downhill nature of the reaction as a result of homocitryl-CoA hydrolysis. Fluorescence titration data generally agree with the steady state data, but show finite binding of CoA and AcCoA to free enzyme, suggesting that the mechanism may be random with a high degree of synergism of binding between the reactants.     

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

A molluscan model system in the search for the engram.

A 3-neuron central pattern generator, whose sufficiency and necessity has been directly demonstrated, mediates aerial respiratory behaviour in the pond snail, Lymnaea stagnalis. This behaviour can be operantly conditioned, and this associative learning is consolidated into long-lasting memory. Depending on the operant conditioning training procedure used the learning can be consolidated into intermediate term (ITM) or long-term memory (LTM). ITM persists for only 2-3 h, whilst LTM persists for days to weeks. LTM is dependent on both altered gene activity and new protein synthesis while ITM is only dependent on new protein synthesis. We have now directly established that one of the 3-CPG neurons, RPeD1, is a site of LTM formation and storage. We did this by ablating the soma of RPeD1 and leaving behind a functional primary neurite capable of mediating the necessary synaptic interactions to drive aerial respiratory behaviour by the 3-neuron CPG. However, following soma ablation the neuronal circuit is only capable of mediating learning and ITM. LTM can no longer be demonstrated. However, if RPeD1's soma is ablated after LTM consolidation memory is still present. Thus the soma is not needed for the retention of LTM. Using a similar strategy it may be possible to block forgetting.     

Establishment and characterization of an Ostrinia nubilalis cell line, and its response to ecdysone agonists

Summary A cell line derived from embryonic tissues of the European corn borer, Ostrinia nubilalis (UMC-OnE), was established in EX-CELL 401 medium containing 10% fetal bovine serum. The cells grew in suspension, and were mainly spherical in shape. The cell doubling times at the 17th and 79th passages were 56 and 36 h, respectively. DNA amplification fingerprinting showed that the DNA profile of the OnE cell line was different from that of the southwestern corn borer, Diatraea grandiosella (UMC-DgE), and that of the cotton bollworm, Helicoverpa zea (BCIRL-HZ-AM1). The OnE cell line was responsive to treatments of 20-hydroxyecdysone and the ecdysone agonists, methoxyfenozide (RH-2485) and tebufenozide (RH-5992). These compounds caused similar effects on the cells, which included cell clumping and decreased cell proliferation. The clumps were observed on the third day of incubation, and became larger after 7 d of incubation. After 168 h of incubation, methoxyfenozide and tebufenozide were 35 and 11 times more effective, respectively, in inhibiting proliferation of the OnE cells than was 20-hydroxyecdysone.     

Functional assessment of cryopreserved human aortas for pharmaceutical research

We evaluated the impact of standard cryopreservation on functional properties of human aortic homografts. From seven human donors, the thoracic descending aorta was obtained. Effects of cryopreservation on contractibility and endothelium function were tested. After cryopreservation no endothelium-dependent or endothelium-independent relaxation was found and the contractibility was strongly affected. Arteries showed no function and loss of endothelial integrity after cryopreservation and thawing.     

Sequencing of porcine enterovirus groups II and III reveals unique features of both virus groups

The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5'-cloverleaf structure.     

(31)P NMR spectroscopy senses the microenvironment of the 5'-phosphate group of enzyme-bound pyridoxal 5'-phosphate

In this review it is demonstrated that (31)P NMR spectroscopy can be used to elucidate information about the microenvironment around the phosphate group of enzyme-bound pyridoxal 5'-phosphate (PLP). The following information can be obtained for all PLP-dependent enzymes: 1) the protonation state of the 5'-phosphate and its exposure to solvent, and 2) tightness of binding of the 5'-phosphate. In addition, the 5-phosphate can report on the protonation state of the Schiff base lysine in some enzymes. Changes in the 5'-phosphate chemical shift can be used to determine changes in tightness of binding of the phosphate as the reaction pathway is traversed, providing information on the dynamics of the enzyme. (31)P NMR spectroscopy is thus an important probe of structure, dynamics and mechanism in native and site-directed mutations of PLP-dependent enzymes. Examples of all of the above are provided in this review. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.