Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies

Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.     

Interactions among oscillatory pathways in NF-kappa B signaling

Background  

Sustained stimulation with tumour necrosis factor alpha (TNF-alpha) induces substantial oscillations—observed at both the single cell and population levels—in the nuclear factor kappa B (NF-kappa B) system. Although the mechanism has not yet been elucidated fully, a core system has been identified consisting of a negative feedback loop involving NF-kappa B (RelA:p50 hetero-dimer) and its inhibitor I-kappa B-alpha. Many authors have suggested that this core oscillator should couple to other oscillatory pathways.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4′-phosphopantetheine prosthetic group by holo-ACP synthase. The K_m value for NodF is 61 μM, as compared to 2 μM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for β-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E.coliacpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1–33 of AcpP fused to amino acids 43–93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes). Received: 22 September 1997 / Accepted: 31 October 1997     

Mitochondrial DNA structure and colony expansion dynamics of New Zealand fur seals (Arctocephalus forsteri) around Banks Peninsula

New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic.     

Effect of hydrocarbon concentration of pasture production (<i>Brachiaria humidicola</i>) in Texistepec,Veracruz

In this study, the relationship between the concentration of extra-heavy crude petroleum in a clayey material and the toxicity, field capacity, temperature, and growth of a tropical forage grass (Brachiara humidicola) was determined empirically. For this type of petroleum the acute toxicity (Microtox^®) was slight (CE₅₀ = 63200 - 76400 mg/kg) even at high hydrocarbon concentrations (29279 mg/kg). Nonetheless, serious impacts were encountered in terms of an increase in soil temperature (+ 1.3 °C), reduction in field capacity (-10.7%) and reduction in aerial biomass (-97%). The relationship between hydrocarbon concentration and biomass resulted in a typical dose-response curve (r = 0.99), where a concentration of 2626 mg/kg of hydrocarbons corresponds to a maintenance of 90% biomass. Furthermore, during the duration of this study (one year) the biodegradation was proportional to the pasture biomass production (r = 0.997) indicating a synergistic relationship between the petroleum biodegrading microorganisms in the rhizosphere and the pasture.     

Transcriptional response to alcohol exposure in Drosophila melanogaster

Background  

Alcoholism presents widespread social and human health problems. Alcohol sensitivity, the development of tolerance to alcohol and susceptibility to addiction vary in the population. Genetic factors that predispose to alcoholism remain largely unknown due to extensive genetic and environmental variation in human populations. Drosophila, however, allows studies on genetically identical individuals in controlled environments. Although addiction to alcohol has not been demonstrated in Drosophila, flies show responses to alcohol exposure that resemble human intoxication, including hyperactivity, loss of postural control, sedation, and exposure-dependent development of tolerance.