Developmental regulation of NAD+ kinase in Neurospora crassa

The specific activity of NAD⁺ kinase (ATP:NAD⁺ 2-phosphotransferase, EC 2.7.1.23) from Neurospora crassa shows sharp peaks when the organism enters a new developmental stage of the asexual life cycle: the peaks are observed during hydration and germination of conidia, at the transition from exponential to stationary growth and at the photostimulated conidiation. As stimulation of NAD⁺ kinase activity by light in conidiating mycelium is not sensitive to translation inhibitors, the activiation of pre-existing molecules, rather than induction of protein synthesis de novo may be supposed. Enzyme electrophoresis revealed the presence of four forms of NAD⁺ kinase having different apparent molecular weights (I=333,000; II=306,000; III=229,000 and IV=203,000). Manifestation of the activity of individual forms of NAD⁺ kinase is developmentally controlled: form III is most abundant during vegetative growth, forms I and II prevail in conidia. At the conidial germination the increase of NAD⁺ kinase activity is associated with the activation of form III, whereas during photostimulation of conidiation form II is the most activated one. Therefore, certain molecular forms of the enzyme may be regarded as biochemical markers for different developmental stages of N. crassa.     

Intracellular aminopeptidases inStreptomyces lividans 66

Summary We have investigated the aminopeptidase activities present inStreptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.     

Alterations in the activity of phospholipases A2 in postmortem white matter from patients with multiple sclerosis

Activities toward arachidonyl-labelled phospholipase A₂ substrates were assayed in fractions of white matter and cerebral cortex from control subjects and in fractions of demyelinated plaque, normal-appearing white matter and cerebral cortex from subjects who died with multiple sclerosis. Membranous activity at pH 8.6 in the presence of Ca²⁺, characteristic of 14 kDa secretory phospholipase A₂, in either multiple sclerosis white matter or cortex did not differ from controls, whereas membranous activity at pH 4.5 in the absence of added Ca²⁺, characteristic of lysosomal enzymes was increased over controls in both plaque and normal-appearing white matter but not cerebral cortex. Activity in the cytosol fraction, at pH 8.6 in the presence of Ca²⁺ and glycerol characteristic of the cytosolic 85 kDa enzyme was decreased by greater than 50% in both white matter and cortex samples from multiple sclerosis subjects. Immuno-precipitation and-blotting confirmed that the deficient activity was largely attributable to the 85 kDa enzyme although the enzyme protein was not similarly reduced.Special issue dedicated to Dr. Leon S. Wolfe.     

Laboratory culture of Chironomus tentans for use in toxicity testing: optimum initial egg-stocking densities

The midge Chironomus tentans Fabricius is a commonly used freshwater invertebrate in sediment toxicity tests. Rigorous laboratory culturing techniques are needed to provide organisms of uniform quality and known age for use in testing and for the continuation of the culture itself. This study was conducted to determine the effect of initial culture stocking density on: (1) post-hatch (larval) dry weight, body length and head-capsule width at 10 and 20 days; (2) time to emergence; (3) number and sex of emergent adults; (4) number of larvae and pupae at test termination (day 42 post hatch); and (5) adult dry weight. Three egg stocking densities were used 690 (1.1 eggs cm^–2), 1043 (1.7 eggs cm^–2) and 1463 (2.4 eggs cm^–2). Mean weight of larvae at 10 days in high density tanks (0.13 mg/organism) was significantly higher (P=0.003) than both the medium and low density tanks (0.10 and 0.09 mg/organism, respectively). No significant differences between the three stocking densities were observed for the body length or head-capsule width at either 10 or 20 days post-hatch. Although not statistically significant, larval dry weight decreased with increased stocking density at day 20. A significantly (P=0.02) greater number of females (173±28) emerged from the low stocking density compared to both the medium and high stocking densities (123±45 and 118±54, respectively). Peak adult emergence for the low and medium stocking densities occurred between days 22 and 25 post-hatch, whereas peak adult emergence occurred between days 30 and 33 for the high stocking density. Survival relative to the initial number of eggs stocked was significantly greater (P=0.007) in the low density treatment compared to that in either the medium or the high density treatments. Mean adult weight exhibited an inverse relationship with initial stocking densities. At test end, there was not a significant difference in the mean number of organisms surviving and emerging in the three density levels. The central tendency for number of organisms surviving for all three treatments was 504 organisms per tank (0.82 organisms cm^–2). The results of this experiment suggest that an optimal egg stocking density of 1.0 egg cm^–2 (600 eggs/tank) be used with the feeding rate identified. This would ensure uniform larvae at the appropriate developmental stage (2nd–3rd instar) needed for toxicological research/testing (e.g. 10 days post-hatch), as well as producing sufficient emergence of males and females for future culture establishment.     

Indirect nontarget effects of host-specific biological control agents: Implications for biological control

Classical biological control of weeds currently operates under the assumption that biological control agents are safe (i.e., low risk) if they do not directly attack nontarget species. However, recent studies indicate that even highly host-specific biological control agents can impact nontarget species through indirect effects. This finding has profound implications for biological control. To better understand the causes of these interactions and their implications, we evaluate recent case studies of indirect nontarget effects of biological control agents in the context of theoretical work in community ecology. We find that although particular indirect nontarget effects are extremely difficult to predict, all indirect nontarget effects of host specific biological control agents derive from the nature and strength of the interaction between the biological control agent and the pest. Additionally, recent theoretical work suggests that the degree of impact of a biological control agent on nontarget species is proportional to the agent’s abundance, which will be highest for moderately successful control agents. Therefore, the key to safeguarding against indirect nontarget effects of host-specific biological control agents is to ensure the biological control agents are not only host specific, but also efficacious. Biological control agents that greatly reduce their target species while remaining host-specific will reduce their own populations through density-dependent feedbacks that minimize risks to nontarget species.     

Modification of Rifamycin Polyketide Backbone Leads to Improved Drug Activity against Rifampicin-resistant Mycobacterium tuberculosis

Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.