The effect of salts on the kinetics of iron release from N-terminal and C-terminal monoferrictransferrins

The kinetics of iron release from N-terminal and C-terminal monoferric human transferrins has been studied using EDTA as the accepting chelate. In the absence of added salts iron release from the N-terminal site is more facile but the relative lability can be reversed by the addition of NaClO₄, NaCl and LiCl. The results indicate that both anions and cations can affect the lability of the two sites. Since the relative lability of the two monoferrictransferrins is affected by fairly moderate concentrations of NaCl and NaClO₄ we suggest that the ionic composition serum may play an important role in determining the observed distribution of iron among the sites. A new method for the preparation of N-terminal monoferrictransferrin is described.     

The Jungle Cat <Emphasis Type="Italic">Felis chaus</Emphasis> in Indochina: a threatened population of a widespread and adaptable species

The Jungle Cat Felis chaus is widespread in India and neighbouring countries but is known by only one historical specimen from Cambodia, Laos or Vietnam (Indochina), widely published as from Vietnam, but in fact from Cambodia. All but two of the recent Indochinese records come from extensive natural lowland habitat dominated by deciduous dipterocarp forest in northeast Cambodia. The species probably occurred more widely in Indochina, largely through additional use of secondary habitats, where hunting pressure is now very heavy. Suggestions of decline in Indochina are corroborated by more conclusive evidence from Thailand. In Indochina, all other small and medium-sized cats are recorded much more frequently than Jungle Cat: closed evergreen forest supports source populations of them, but there is no evidence that Jungle Cat uses extensively such forest. The open forests of northern and eastern Cambodia are highly significant for conserving threatened biodiversity, notably large waterbirds, vultures and ungulates, groups where species formerly widespread across Indochina have contracted in range and declined steeply. These taxa were better collected than small cats and it seems plausible that Jungle Cat showed a similar change. Jungle Cat conservation in Indochina needs extensive habitat retention with intensive anti-poaching activities, because suitable habitat is easily accessible to hunters. The habitat adaptability shown elsewhere by Jungle Cat could allow a much healthier regional conservation status if hunting (including trapping) can be greatly reduced in scrub and agricultural habitats, but changing culturally ingrained hunting practices will take a long time.     

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.      

Structural parameters of the Pf1 gene 5 protein-DNA complex in solution by neutron scattering

Neutron-scattering experiments have been performed on the intracellular complex formed by the gene 5 protein and single-stranded DNA in cells infected by filamentous bacteriophage Pf1. The contrast matched point of the complex (37% 2H2O) is lower than expected and implies that a substantial fraction of potentially labile hydrogen atoms are unable to exchange with the solvent. The mass/length ratio of the complex (3270 daltons/A) indicates an axial subunit repeat of 5.1 A, a value much larger than the subunit repeat previously determined in fibres. The measured value of the cross-sectional radius of gyration at infinite contrast (Rc = 43.3 A) indicates an outer radius of 60 to 63 A for the complex. The variation in Rc with contrast shows that regions of higher scattering density are located, on average, towards the outside of the complex. The high-angle region of the intensity curve (measured in 2H2O) reveals a clear subsidiary maximum at 0.105 A-1 arising from the 60 A helical pitch of the nucleoprotein complex. The structural parameters of the Pf1 gene 5 protein-DNA complex in solution are compared with those of the fd gene 5 protein-DNA complex.     

Distribution and abundance of young fish in the St. Clair River and associated waters,Ontario

Fish larvae were sampled in 1986 in the St. Clair River, and adjacent waters. Species richness (9 taxa as larvae; 4 others as juveniles) and abundance was lowest in the river, where many larvae (e.g., burbot, rainbow smelt, and yellow perch) were in transit from Lake Huron. The most abundant, and localized, species was gizzard shad, which reached a peak mean density of 4600 larvae 100 m^-3 in an agricultural canal. Adjacent waters contribute greatly to the fish communities of the river and adjoining Lakes Huron and Erie, especially in terms of the number and quantity of forage species.     

Shift-down in growth rate rather than high cell density induces toxic shock syndrome toxin-1 gene expression in Staphylococcus aureus

A luciferase-based reporter system for the expression of the toxic shock syndrome toxin-1 gene (tst) of Staphylococcus aureus FRI 1187 was used in continuous culture to determine whether high cell density on transient shift-down or shift-up of specific growth rate (mu) induced expression of tst. Little expression occurred at steady state at a low dilution rate (D) and in a transient period of increasing mu. However, a rapid and approximately 130-fold increase in expression occurred during a transient shift-down of mu. These findings suggest reduction of mu is a key element in the control of tst expression.     

Microarray analysis and functional characterization of the nitrosative stress response in nonmucoid and mucoid Pseudomonas aeruginosa

The type strain of Pseudomonas aeruginosa, PAO1, showed great upregulation of many nitrosative defense genes upon treatment with S-nitrosoglutathione, while the mucoid strain PAO578II showed no further upregulation above its constitutive upregulation of nor and fhp. NO* consumption however, showed that both strains mount functional, protein synthesis-dependent NO*-consumptive responses.     

Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering.

Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation.Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.