Measurement of the reversibility of ATP binding to myosin in calcium-activated skinned fibers from rabbit skeletal muscle. Oxygen exchange between water and ATP released to the solution

We have measured the rate constant for ATP release from myosin heads of Ca2+-activated, demembranated muscle fibers using the technique of phosphate-water oxygen exchange. Single rabbit psoas fibers were held in an activating solution in [18O]water ([MgATP] = 8 mM, ionic strength = 0.2 M, pH = 7.0, 24 degrees C). After about 20% hydrolysis of ATP, product Pi and remaining ATP were isolated, and the distribution of 18O in both molecules was analyzed using a mass spectrometer. The exchange in Pi was similar to that previously reported (Hibberd, M. G., Webb, M. R., Goldman, Y. E., and Trentham, D. R. (1985) J. Biol. Chem. 260, 3496-3501). The amount of 18O in ATP gave a rate constant of about 4 s-1 for ATP release, if it is assumed that each rate constant in the pathway of ATP hydrolysis has the same value for all myosin ATPase sites. However, the distribution of 18O in both released Pi and ATP is not well explained by a single pathway for ATP hydrolysis. We present a model that indicates how such distributions could arise from a range of values for the rate constants for Pi and ATP release from actomyosin, and this range is determined by differences in the amounts of strain in attached crossbridges. The kinetic information obtained from these isotope exchange experiments is compared to show that they give a compatible set of rate constants for actomyosin in fibers.     

A low-spin iron complex in human melanoma and rat hepatoma cells and a high-spin iron(II) complex in rat hepatoma cells.

Human melanoma and rat hepatoma cells cultured in the presence of low concentrations (2.5 microM) of low-molecular-weight iron (Fe) chelates and Fe-transferrin complexes have been studied with 57Fe M?ssbauer spectroscopy. The spectra show that holoferritin is only a minor fraction of the total iron present in the cells. The major form of Fe was in a low-spin state unlike the high-spin Fe(III) found in ferritin. Only about 10% of the Fe could be attributed to ferritin. In addition, the hepatoma cells had a high-spin Fe(II) spectral component which made up about 20% of the Fe present.     

Effect of cell concentration on the rheology of glucoamylase fermentation broth

During the process optimisation of glucoamylase production by Aspergillus awamori, cell morphology was controlled at such a state that spore aggregation was completely prevented. Samples from five fermentations on complex media using either glucose or starch as carbon source were characterised with a Bohlin CS rheometer. The experimental data were conveniently described in terms of the power law model.     

Diet changes in the carbon and nitrogen content of the copepod Metridia lucens

Diel changes in the near-surface (0–50 m) abundance, prosomelength, and carbon and nitrogen content of the copepod Metridialucens were measured in Deep Cove, Doubtful Sound, New Zealand(45°27'S, 167°9'E) between 3 and 6 September 1996. Metridialucens showed maximal abundance a night, suggesting a patternof normal diel vertical migration (DVM). The change in abundancesuggested that the descent of the population occurred –1h prior to dawn and the ascent –1 h after dusk. However,a proportion of the population remained near the surface duringthe day. Although there was no diel pattern in the prosome lengthof M.lucens collected near the surface, there was a marked dielcycle in the measured carbon and nitrogen contents, with maximalvalues being measured towards the end of the night prior tothe downward migration. We suggest that this diet cycle in themeasured elemental content was caused by DVM occurring morestrongly in those animals which had a better body condition,i.e. a higher elemental content per unit length.     

Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Iron stress in open-ocean cyanobacteria (Synechococcus, Trichodesmium, and Crocosphaera spp.): identification of the IdiA protein.

Cyanobacteria are prominent constituents of the marine biosphere that account for a significant percentage of oceanic primary productivity. In an effort to resolve how open-ocean cyanobacteria persist in regions where the Fe concentration is thought to be limiting their productivity, we performed a number of Fe stress experiments on axenic cultures of marine Synechococcus spp., Crocosphaera sp., and Trichodesmium sp. Through this work, we determined that all of these marine cyanobacteria mount adaptive responses to Fe stress, which resulted in the induction and/or repression of several proteins. We have identified one of the Fe stress-induced proteins as an IdiA homologue. Genomic observations and laboratory data presented herein from open-ocean Synechococcus spp. are consistent with IdiA having a role in cellular Fe scavenging. Our data indicate that IdiA may make an excellent marker for Fe stress in open-ocean cyanobacterial field populations. By determining how these microorganisms respond to Fe stress, we will gain insight into how and when this important trace element can limit their growth in situ. This knowledge will greatly increase our understanding of how marine Fe cycling impacts oceanic processes, such as carbon and nitrogen fixation.     

Detecting Unidentified Changes

Does becoming aware of a change to a purely visual stimulus necessarily cause the observer to be able to identify or localise the change or can change detection occur in the absence of identification or localisation? Several theories of visual awareness stress that we are aware of more than just the few objects to which we attend. In particular, it is clear that to some extent we are also aware of the global properties of the scene, such as the mean luminance or the distribution of spatial frequencies. It follows that we may be able to detect a change to a visual scene by detecting a change to one or more of these global properties. However, detecting a change to global property may not supply us with enough information to accurately identify or localise which object in the scene has been changed. Thus, it may be possible to reliably detect the occurrence of changes without being able to identify or localise what has changed. Previous attempts to show that this can occur with natural images have produced mixed results. Here we use a novel analysis technique to provide additional evidence that changes can be detected in natural images without also being identified or localised. It is likely that this occurs by the observers monitoring the global properties of the scene.