Experimental linkage issues of petroleum site bioremediation

Bioremediation of petroleum-contaminated sites is expected to be a cost-effective remediation technology. However, many potential users of the technology expect the reliability of this technology to be similar to other candidate technologies for widespread consideration. In particular, candidate technologies should possess the property of reliable experimental linkage — there should be reasonable confidence that experiments done at one scale can be reliably related to another. An important example is bench-scale treatability studies that should result in linkages with commercial-scale operations. In this respect comparison of bioremediation to other candidate technologies reveals that bioremediation is in an early stage of its evolution. It is being pursued at a variety of sites and scales with practitioners from a variety of disciplines. Integration of activities between disciplines and an ability to quantitatively compare results at different sites and scales is proceeding. This paper addresses a number of physical, chemical, biological, analytical, and statistical issues regarding the successful comparison of results between experiments.     

An affinity label for alpha 2-adrenergic receptors in rat brain

Clonidine, a potent and highly selective alpha 2-adrenergic agonist of the central nervous system, was modified. Insertion of the strong alkylating isothiocyanate group (NCS) group, at its aromatic residue, makes clonidine a potential affinity label of the alpha 2-adrenergic receptors. In displacement of [3H]clonidine and p-[3H]aminoclonidine from rat brain membrane preparations, clonidine-NCS demonstrates high affinity for the alpha 2-adrenergic receptors (Kd = 50 mM). The covalent labelling of the central alpha 2-receptors requires higher concentrations of the irreversible ligand (1-70 microM), thus indicating possible non-productive interactions at the environment of the receptor site. Only partial protection of the receptors is observed with a reversible alpha 2-agonist. The new clonidine analog appears to be a general ligand for the alpha 2-adrenergic receptors and might serve as a potential affinity probe for these receptors.     

Cations residing at the selectivity filter of the voltage-gated Ca2+-channel modify fusion-pore kinetics

Strontium (Sr(2+)), Barium (Ba(2+)) and Lanthanum (La(3+)) can substitute for Ca(2+) in driving synaptic transmission during membrane depolarization. Ion recognition at the polyglutamate motif (EEEE), comprising the channel selectivity-filter, during voltage-driven transitions, controls the kinetics of the voltage-gated calcium channel (VGCC) and its interactions with the synaptic proteins. We tested the effect of different charge carriers on evoked-release, as a means of exploring the involvement of VGCC in the fusion pore configuration. Employing amperometry recordings in single bovine chromaffin cells we show that the size of the fusion pore, designated by the 'foot'-amplitude, was increased when Ba(2+) substituted for Ca(2+) and decreased, with La(3+). The fusion pore stability, indicated by 'foot'-width, was decreased in La(3+). Also, the mean open time of the fusion pore (tau(fp)) was significantly lower in Sr(2+) and La(3+) compared to Ba(2+) and Ca(2+). These cations when occupying the selectivity filter reduced the spike frequency in the order of Ca(2+) > Sr(2+) > Ba(2+) > La(3+), which is parallel to the reduction in total catecholamine release. The correlation between ion binding at the selectivity filter and fusion pore properties supports a model in which the Ca(2+) channel regulates secretion through a site at the selectivity filter, upstream to cation entry into the cell.     

Depolarization-evoked secretion requires two vicinal transmembrane cysteines of syntaxin 1A

Background

The interactions of the voltage-gated Ca²⁺ channel (VGCC) with syntaxin 1A (Sx 1A), Synaptosome-associated protein of 25 kD (SNAP-25), and synaptotagmin, couple electrical excitation to evoked secretion. Two vicinal Cys residues, Cys 271 and Cys 272 in the Sx 1A transmembrane domain, are highly conserved and participate in modulating channel kinetics. Each of the Sx1A Cys mutants, differently modify the kinetics of Cav1.2, and neuronal Cav2.2 calcium channel.

Methodology/Principle Findings

We examined the effects of various Sx1A Cys mutants and the syntaxin isoforms 2, 3, and 4 each of which lack vicinal Cys residues, on evoked secretion, monitoring capacitance transients in a functional release assay. Membrane capacitance in Xenopus oocytes co-expressing Cav1.2, Sx1A, SNAP-25 and synaptotagmin, which is Bot C- and Bot A-sensitive, was elicited by a double 500 ms depolarizing pulse to 0 mV. The evoked-release was obliterated when a single Cys Sx1A mutant or either one of the Sx isoforms were substituted for Sx 1A, demonstrating the essential role of vicinal Cys residues in the depolarization mediated process. Protein expression and confocal imaging established the level of the mutated proteins in the cell and their targeting to the plasma membrane.

Conclusions/Significance

We propose a model whereby the two adjacent transmembranal Cys residues of Sx 1A, lash two calcium channels. Consistent with the necessity of a minimal fusion complex termed the excitosome, each Sx1A is in a complex with SNAP-25, Syt1, and the Ca²⁺ channel. A Hill coefficient >2 imply that at least three excitosome complexes are required for generating a secreting hetero-oligomer protein complex. This working model suggests that a fusion pore that opens during membrane depolarization could be lined by alternating transmembrane segments of Sx1A and VGCC. The functional coupling of distinct amino acids of Sx 1A with VGCC appears to be essential for depolarization-evoked secretion.     

PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

Bioterrorism and biodefence research: changing the focus of microbiology

Fear that terrorists can use biological agents as weapons of mass destruction is significantly impacting the conduct of microbiological research. Abundant new funds are available for biodefence research, and many researchers are racing to enter the field. There are some concerns, however, that a large emphasis on this issue could skew the microbiology research agenda. Furthermore, new responsibilities for safely conducting research with biothreat agents and concern that information might be misused could drive some researchers away from the field.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Dihydrocytochalasin B. Biological effects and binding to 3T3 cells

Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function.