The control of ionized calcium in squid axons

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.     

Membrane TNF confers protection to acute mycobacterial infection

Background

Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF).

Methods

C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice.

Results

While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice.

Conclusion

Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.     

Molecular basis for the dynamic strength of the integrin alpha4beta1/VCAM-1 interaction

Intercellular adhesion mediated by integrin alpha4beta1 and vascular cell adhesion molecule-1 (VCAM-1) plays a crucial role in both the rolling and firm attachment of leukocytes onto the vascular endothelium. Essential to the alpha4beta1/VCAM-1 interaction is its mechanical strength that allows the complex to resist the large shear forces imposed by the bloodstream. Herein we employed single-molecule dynamic force spectroscopy to investigate the dynamic strength of the alpha4beta1/VCAM-1 complex. Our force measurements revealed that the dissociation of the alpha4beta1/VCAM-1 complex involves overcoming at least two activation potential barriers: a steep inner barrier and a more elevated outer barrier. The inner barrier grants the complex the tensile strength to withstand large pulling forces (>50 pN) and was attributed to the ionic interaction between the chelated Mg2+ ion at the N-terminal A-domain of the beta1 subunit of alpha4beta1 and the carboxyl group of Asp-40 of VCAM-1 through the use of site-directed mutations. In general, additional mutations within the C-D loop of domain 1 of VCAM-1 suppressed both inner and outer barriers of the alpha4beta1/VCAM-1 complex, while a mutation at Asp-143 of domain 2 of VCAM-1 resulted in the suppression of the outer barrier, but not the inner barrier. In contrast, the outer barrier of alpha4beta1/VCAM-1 complex was stabilized by integrin activation. Together, these findings provide a molecular explanation for the functionally relevant kinetic properties of the alpha4beta1/VCAM-1 interaction.     

Transcriptional divergence of the duplicated oxidative stress-responsive genes in the Arabidopsis genome

Previous studies have indicated that Arabidopsis thaliana experienced a genome-wide duplication event shortly before its divergence from Brassica followed by extensive chromosomal rearrangements and deletions. While a large number of the duplicated genes have significantly diverged or lost their sister genes, we found 4222 pairs that are still highly conserved, and as a result had similar functional assignments during the annotation of the genome sequence. Using whole-genome DNA microarrays, we identified 906 duplicated gene pairs in which at least one member exhibited a significant response to oxidative stress. Among these, only 117 pairs were up- or down-regulated in both pairs and many of these exhibited dissimilar patterns of expression. Examination of the expression patterns of PAL1 and PAL2, ACD1 and ACD2, genes coding for two Hsp20s, various P450s, and electron transfer flavoproteins suggests Arabidopsis evolved a number of distinct oxidative stress response mechanisms using similar gene sets following the duplication of its genome.     

Structural basis of severe acute respiratory syndrome coronavirus ADP-ribose-1''-phosphate dephosphorylation by a conserved domain of nsP3

The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 A resolution. The structure of this "X" domain, seen in many single-stranded RNA viruses, reveals a three-layered alpha/beta/alpha core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1'-phosphate (Appr-1'-p). The SARS nsP3 domain readily removes the 1' phosphate group from Appr-1'-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1'-p.     

Life history and habitat preference in the Darling hardyhead,Craterocephalus amniculus (Teleostei,Atherinidae) in the northern Murray‐Darling Basin,Australia

The Darling hardyhead, Craterocephalus amniculus (Atherinidae), is a threatened fish species inhabiting upstream reaches of a number of northern Murray‐Darling Basin catchments. Little is known of its life history. Our goal was to determine patterns of seasonal size structure, interannual and spatial variation in diet, and habitat selection in this species across multiple sites and years in the upper Macintyre River, northern New South Wales. Preserved specimens from a separate study were used to obtain information on diet and size structure. Size structures suggested a single annual spawning season in late September or early October. Diets varied significantly, both between years at the downstream site and among the three sites although the underlying cause of this was untested. Dietary diversity increased with distance downstream. At the two upstream sites, aquatic invertebrates made up most of the diet while over half the gut contents at the downstream site was unidentified detritus. Preference was shown for pool habitats with a sand or cobble substrate, increased channel depth and width and distance from the bank, and reduced flow velocity. Overhanging exotic riparian vegetation and in‐stream woody debris were non‐preferred. This species may be vulnerable to further population decline in light of its restricted habitat preferences and narrow spawning season. However, comparable data from nearby catchments will be necessary to ascertain the species’ conservation status across its broader distribution.     

Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase

Fibroblast collagenase (MMP-1), a 169-residue protein with amolecular mass of 18.7 kDa, is a matrix metalloproteinase which has beenassociated with pathologies such as arthritis and cancer. The assignments ofthe ¹H, ¹⁵N, ¹³CO and¹³C resonances, determination of the secondary structure andanalysis of ¹⁵N relaxation data of the inhibitor-freecatalytic fragment of recombinant human fibroblast collagenase (MMP-1) arepresented. It is shown that MMP-1 is composed of a -sheet consistingof five -strands in a mixed parallel and antiparallel arrangement(residues 13–19, 48–53, 59–65, 82–85 and94–99) and three -helices (residues 27–43, 112–124and 150–160). This is nearly identical to the secondary structuredetermined from the refined X-ray crystal structures of inhibited MMP-1. Themajor difference observed between the NMR solution structure ofinhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is thedynamics of the active site. The 2D ¹⁵N-¹H HSQCspectra, the lack of information in the ¹⁵N-edited NOESYspectra, and the generalized order parameters (S²) determinedfrom ¹⁵N T₁, T₂ and NOE datasuggest a slow conformational exchange for residues comprising the activesite (helix B, zinc ligated histidines and the nearby loop region) and ahigh mobility for residues Pro¹³⁸-Gly¹⁴⁴ in thevicinity of the active site for inhibitor-free collagenase. In contrast tothe X-ray structures, only the slow conformational exchange is lost in thepresence of an inhibitor.