Elucidation and optimization of the medium constituents controlling antibiotic production by the cyanobacterium Nostoc muscorum.

A study has been made to determine which nutrient factors control antibiotic production by the cyanobacterium, Nostoc muscorum. A two-phase approach was employed using a factorial method to explore the response surface and a steepest ascent method to climb the response surface to the region of the optimum. It was found that nitrate and iron were the factors significantly affecting antibiotic production; 26.4 mM nitrate and 6 microM iron were the optimal concentrations for maximizing antibiotic production by N. muscorum.     

Effects of forskolin on contractile responses and protein phosphorylation in the isolated perfused rat heart.

Continuous perfusion of rat hearts with concentrations of forskolin between 0.1 and 12 microM resulted in transient increases in tension after 45 s, followed by a return to the control value after 5 min. In contrast, the content of cyclic AMP increased linearly with time over this period, reaching values up to 35 times control after 5 min. Increases in contractile force, intracellular cyclic AMP concentration and the proportion of phosphorylase in the a form were dependent on the concentration of forskolin when measured 45 s and 120 s after initiation of perfusion. In hearts perfused for 45 s with various concentrations of forskolin, the measured cyclic AMP-dependent protein kinase activity ratio and phosphorylase a content for a given measured intracellular cyclic AMP concentration were both much less than the corresponding values in hearts perfused for 30 s with various concentrations of isoprenaline. The phosphorylation of the contractile proteins troponin-I and C-protein also showed a concentration-dependent increase in hearts perfused with forskolin. There was a strong correlation between the cyclic AMP-dependent protein kinase activity ratios and the phosphorylation of the contractile proteins under all perfusion conditions. These results suggest that cyclic AMP is compartmented in perfused rat heart, and that much of the cyclic AMP produced in response to forskolin is unavailable to activate cyclic AMP-dependent protein kinase.     

Characterisation of the dihydrofolate reductase-thymidylate synthetase gene from human malaria parasites highly resistant to pyrimethamine

To investigate the genetic basis of drug resistance in human malaria parasites, we have sequenced the entire dihydrofolate reductase thymidylate synthetase DHFR-TS bifunctional gene from the highly pyrimethamine-resistant K1 isolate of Plasmodium falciparum. The protein is predicted to consist of 607 amino acids (aa), (71,685 Da), with an N-terminal methionine encoded by the second start codon of the open reading frame. Compared to the sequence from drug-sensitive parasites, there are two nucleotide changes in the coding region which bring about a substitution of Arg for Cys at aa position 59 and Asn for Thr at aa position 108. Both changes occur in regions of the DHFR domain involved in inhibitor and cofactor binding and are hence strongly implicated in drug resistance. The gene is present as a single copy in both K1 and drug-sensitive FCR3 isolates, and is assigned to chromosome 4. Codon usage follows the pattern observed in that of malarial surface antigen genes, with the exception fo codons corresponding to Val and Pro. The Asn and Lys contents of the predicted protein are exceptionally high, these residues being particularly concentrated in the DHFR and junction domains.     

Nematocysts of sea anemones (Actiniaria,Ceriantharia and Corallimorpharia: Cnidaria): nomenclature

A brief historical review of nematocyst terminology is given and three nomenclatural problems are discussed. It is proposed to combine the terms initiated by Weill (1934) with those of Schmidt (1969). A new mesobasic grade, intermediate between microbasic and macrobasic is proposed for amastigophores and p-mastigophores possessing a short Faltstück. A more liberal interpretation of Weill's (1934) terminology for nematocysts than that proposed by Cutress (1955) is suggested in respect of microbasic amastigophores and p-mastigophores. Basitrichs and b-mastigophores continue to be recognized as separate categories.deceased     

Isolation of a chlamydial agent from Rocky Mountain bighorn sheep

A total of 53 clinical specimens from both healthy and diseased Rocky Mountain bighorn sheep (Ovis canadensis) were examined for Chlamydia. An agent consistently lethal for chicken embryos was recovered from a nasal swab taken from a normal ewe. This agent, designated BHS-15, possesses antigens which fix complement in the presence of anti-chlamydial serum, is susceptible to chlortetracycline, and is resistant to sodium sulfadiazine and streptomycin. Attempts to culture the isolate in quality control media, including blood agar, thioglycolate broth, and PPLO broth and agar were unsuccessful. A recommendation is made for classification of agent BHS-15 as a member of the species Chlamydia psittaci. The possible relationship of the isolate to the pneumonia complex in bighorn sheep is discussed.     

Diaphragmatic work of breathing in premature human infants

Present methods of assessing the work of breathing in human infants do not account for the added load when intercostal muscle activity is lost and rib cage distortion occurs. We have developed a technique for assessing diaphragmatic work in this circumstance utilizing measurements of transdiaphragmatic pressure and abdominal volume displacement. Eleven preterm infants without evidence of lung disease were studied. During periods of minimal rib cage distortion, inspiratory diaphragmatic work averaged 5.9 g X cm X ml-1, increasing to an average of 12.4 g X cm X ml-1 with periods of paradoxical rib cage motion (P less than 0.01). Inspiratory work was strongly correlated with the electrical activity of the diaphragm as measured from its moving time average (P less than 0.05). Assuming a mechanical efficiency of 4% in these infants, the caloric cost of diaphragmatic work may reach 10% of their basal metabolic rate in periods with rib cage distortion. When lung disease is superimposed, the increased metabolic demands of the diaphragm may predispose preterm infants to fatigue and may contribute to a failure to grow.     

Understanding age-specific dispersal in fishes through hydrodynamic modelling, genetic simulations and microsatellite DNA analysis

Many marine species have vastly different capacities for dispersal during larval, juvenile and adult life stages, and this has the potential to complicate the identification of population boundaries and the implementation of effective management strategies such as marine protected areas. Genetic studies of population structure and dispersal rarely disentangle these differences and usually provide only lifetime-averaged information that can be considered by managers. We address this limitation by combining age-specific autocorrelation analysis of microsatellite genotypes, hydrodynamic modelling and genetic simulations to reveal changes in the extent of dispersal during the lifetime of a marine fish. We focus on an exploited coral reef species, Lethrinus nebulosus, which has a circum-tropical distribution and is a key component of a multispecies fishery in northwestern Australia. Conventional population genetic analyses revealed extensive gene flow in this species over vast distances (up to 1,500 km). Yet, when realistic adult dispersal behaviours were modelled, they could not account for these observations, implying adult dispersal does not dominate gene flow. Instead, hydrodynamic modelling showed that larval L. nebulosus are likely to be transported hundreds of kilometres, easily accounting for the observed gene flow. Despite the vast scale of larval transport, juvenile L. nebulosus exhibited fine-scale genetic autocorrelation, which declined with age. This implies both larval cohesion and extremely limited juvenile dispersal prior to maturity. The multidisciplinary approach adopted in this study provides a uniquely comprehensive insight into spatial processes in this marine fish.     

The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K(+) (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853-858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA(2) or the production of superoxide anion (O(2*)(-)). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn(2+) inhibition of NADPH oxidase activity assessed by H(2)O(2) production, thus validating previous studies showing that Zn(2+) inhibited O(2*)(-) production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.     

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4

The JNK‐interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP‐binding protein ARF6. The interaction of ARF6–GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin‐1 and dynactin. Here, we report the crystal structure of ARF6–GTP bound to the JIP4‐LZII at 1.9 Å resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6–(JIP4)₂–ARF6 configuration. Comparison of the ARF6–JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site‐directed mutagenesis and surface plasmon resonance, we further show that non‐conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure‐derived model of the association of the ARF6–JIP3/JIP4 complex with membranes shows that the JIP4‐LZII coiled‐coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6‐mediated motor switch regulatory function.