Determination of the orientation of the axial ligands and of the magnetic properties of the haems in the tetrahaem ferricytochrome from Shewanella frigidimarina

The unambiguous assignment of the nuclear magnetic resonance (NMR) signals of the alpha-substituents of the haems in the tetrahaem cytochrome isolated from Shewanella frigidimarina NCIMB400, was made using a combination of homonuclear and heteronuclear experiments. The paramagnetic (13)C shifts of the nuclei directly bound to the porphyrin of each haem group were analysed in the framework of a model for the haem electronic structure. The analysis yields g-tensors for each haem, which allowed the assignment of some electron paramagnetic resonance (EPR) signals to specific haems, and the orientation of the magnetic axes relative to each haem to be established. The orientation of the axial ligands of the haems was determined semi-empirically from the NMR data, and the structural results were compared with those of the homologous tetrahaem cytochrome from Shewanella oneidensis MR-1 showing significant similarities between the two proteins.     

Analysis of the growth of rimu (Dacrydium cupressinum) in South Westland,New Zealand,using process-based simulation models

Two process-based models were used to identify the environmental variables limiting productivity in a pristine, mature forest dominated by rimu (Dacrydium cupressinum Sol. ex Lamb.) trees in South Westland, New Zealand. A model of canopy net carbon uptake, incorporating routines for radiation interception, photosynthesis and water balance was used to determine a value for quantum efficiency when climate variables were not limiting. The annual net carbon uptake by the canopy was estimated to be 1.1 kg C m(-2) and the quantum efficiency 22.6 mmol mol quanta(-1). This value of quantum efficiency, combined with other parameters obtainable from the literature, was then used in a model of forest productivity (3-PG), to simulate changes in net productivity and the allocation of carbon to tree components. The model was adjusted to match a measured stem increment of 10.6 Mg ha(-1) over a period of 13 years. To achieve this while maintaining a low, but stable value for leaf area index, it was necessary to set the site fertility rating very low and select high values for the parameters describing the proportional allocation of total carbon to roots. This approach highlighted nutrient availability as the principal constraint on productivity for the ecosystem and identified critical measurements that will be necessary for using the model to predict the effects of climate change on carbon sequestration. The low rates of carbon uptake and productivity are consistent with the low nutrient supply available from the highly leached, acid soils, most likely attributable to frequent saturation and a very shallow aerobic zone.     

Identification and characterization of the interaction between tuberin and 14-3-3zeta

Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called tuberin. Although it has been shown that hamartin and tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with tuberin. One of the proteins identified was 14-3-3zeta, a member of the 14-3-3 protein family. The interaction between tuberin and 14-3-3zeta was confirmed in vitro and by co-immunoprecipitation; multiple sites within tuberin for 14-3-3zeta binding were identified; and it was determined that 14-3-3zeta associated with the tuberin-hamartin complex. Finally, it was shown that the tuberin/14-3-3zeta interaction is regulated by Akt-mediated phosphorylation of tuberin, providing insight into how tuberin may regulate phosphorylation of S6.     

Fertilization success in marine invertebrates: the influence of gamete age

Gamete age has been postulated to be unimportant to the fertilization ecology of marine invertebrates. However, recent research suggests that, for some species at least, it may have a direct impact upon fertilization success. We present comparative data on the influence of gamete age on fertilization and development success in several marine invertebrates: the polychaetes Arenicola marina and Nereis virens and the asteroid echinoderm Asterias rubens. Oocytes are much longer lived in the polychaetes than in the echinoderm, with A. marina oocytes still capable of fertilizing and developing normally 96 h post-spawning. Developmental abnormalities and failure to reach blastula tend to occur well before the fertilizable life of the oocytes has expired. Sperm are similarly longer lived in the polychaetes; however, fertilizing capacity is markedly reduced following incubation in conspecific egg-conditioned seawater. These results are discussed in terms of the fertilization strategies of the three species. We further suggest that, for A. marina at least, longer-lived sperm and eggs are central to the fertilization strategy of this species.     

Transgenically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I

Perceptual completion can link widely separated contour fragments and interpolate illusory contours (ICs) between them. The mechanisms underlying such long-range linking are not well understood. Here we report that completion is much poorer when ICs cross the vertical meridian than when they reside entirely within the left or right visual hemifield. This deficit reflects limitations in cross-hemispheric integration. We also show that the sensitivity to the interhemispheric divide is unique to perceptual completion: a comparable task which did not require completion showed no across-meridian impairment. We propose that these findings support the existence of specialized completion mechanisms in early visual cortical areas (V1/V2), since those areas are likely to be more sensitive to the interhemispheric divide.     

Summation of Spatiotemporal Input Patterns in Leaky Integrate-and-Fire Neurons: Application to Neurons in the Cochlear Nucleus Receiving Converging Auditory Nerve Fiber Input

The response of leaky integrate-and-fire neurons is analyzed for periodic inputs whose phases vary with their spatial location. The model gives the relationship between the spatial summation distance and the degree of phase locking of the output spikes (i.e., locking to the periodic stochastic inputs, measured by the synchronization index). The synaptic inputs are modeled as an inhomogeneous Poisson process, and the analysis is carried out in the Gaussian approximation. The model has been applied to globular bushy cells of the cochlear nucleus, which receive converging inputs from auditory nerve fibers that originate at neighboring sites in the cochlea. The model elucidates the roles played by spatial summation and coincidence detection, showing how synchronization decreases with an increase in both frequency and spatial spread of inputs. It also shows under what conditions an enhancement of synchronization of the output relative to the input takes place.     

Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections

Colonization of the anterior nares in approximately 37% of the population is a major risk factor for severe Staphylococcus aureus infections. Here we show that wall teichoic acid (WTA), a surface-exposed staphylococcal polymer, is essential for nasal colonization and mediates interaction with human nasal epithelial cells. WTA-deficient mutants were impaired in their adherence to nasal cells, and were completely unable to colonize cotton rat nares. This study describes the first essential factor for S. aureus nasal colonization.     

delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.     

Stability,homodimerization, and calcium-binding properties of a single,variant betagamma-crystallin domain of the protein absent in melanoma 1 (AIM1)

AIM1 (absent in melanoma), a candidate suppressor of malignancy in melanoma, is a nonlens member of the betagamma-crystallin superfamily, which contains six predicted betagamma domains. The first betagamma-crystallin domain of AIM1 (AIM1-g1) diverges most in sequence from the superfamily consensus. To examine its ability to fold and behave like a normal betagamma domain, we cloned AIM1-g1 and overexpressed it in Escherichia coli as a recombinant protein. The recombinant domain was found to be a stable, soluble protein, similar to lens protein gammaBeta-crystallin in secondary structure. The tertiary structure of AIM1-g1 is dominated by the contribution of aromatic amino acids and cysteine. AIM1-g1 undergoes concentration-independent, noncovalent homodimerization with no trace of monomer, similar to a one-domain protein spherulin 3a. Since many betagamma domain proteins bind calcium, we have also investigated the calcium-binding properties of AIM1-g1 by various methods. AIM1-g1 binds the calcium-mimic dye Stains-all, the calcium probe terbium (with K(D) 170 microM), and (45)Ca when blotted on a membrane. AIM1-g1 binds calcium (K(D) 30 microM) with a comparatively higher affinity than bovine lens gamma-crystallin (90 microM). However, calcium binding does not induce significant change in the protein conformation in the near- and far-UV CD and in fluorescence. The AIM1-g1 domain is as stable as domains of betagamma-crystallins (betaB2- or gammaS-crystallins) as monitored by guanidinium chloride unfolding (midpoint of unfolding transition is 1.8 M GdmCl), and the stability of the protein is not altered upon binding calcium as evaluated by equilibrium unfolding. These results show that, despite the sequence variation, AIM1-g1 folds such as a betagamma domain, binds calcium and undergoes dimerization.     

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.