Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.     

Effects of methylation and ring size on mutagenicity of cyclic nitrosamines in Drosophila melanogaster

The mutagenic activity of 7 nitrosopiperazines, 2 nitropyrrolidines, and 3 nitrosomorpholines was examined in the X-linked recessive-lethal assay of Drosophila melanogaster. Mutagenicity is also reported for a series of cyclic nitrosamines that differ in structure only in the number of carbon atoms in the ring. Of the 18 compounds tested, 6 (nitrosopiperazine; 2,3,5,6-tetramethyl-dinitrosopiperazine; nitrosoproline; 2,5-dimethylnitrosopyrrolidine; nitrosothiomorpholine; and nitrosooctamethyleneimine) were nonmutagenic. As we reported earlier in investigations with the nitrosopiperidines, substitutions with methyl groups at all of the α-carbon atoms reduce or eliminate the mutagenic activity of dinitrosopiperazine and nitrosopyrrolidine.     

Medical Students Learn How to Read

Over a six-year period, courses in effective reading have been conducted for first-year students in the Faculty of Medicine at McGill University. Approximately one-third (about 30 students) had reading rates below 300 words per minute. Classes for this group met for two periods a week for five weeks (first term) during which time emphasis was placed on improving reading rate and comprehension, skimming, scanning and the development of sound study habits. Although no formal research has been conducted on the results of these classes, students report better study habits, less anxiety about achievement, better recall of material, maintenance of comparable academic standing with less study time, and satisfaction in knowing how to increase reading speed after formal academic training.     

RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS

Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.     

Quantifying observer heterogeneity in bird counts

An essential pilot study was designed to quantify observer heterogeneity and to compare observation methods for the detectability of forest birds in stands of Eucalyptus and Pinus radiata forest as a basis for a major research project on habitat fragmentation near Tumut, southern New South Wales. Twelve experienced observers participated in the investigation. Point interval counts, zig-zag walks and strip transects were used to count birds in both eucalypt and pine forests. The 65 species of birds recorded in the study were assigned to one of nine groups classified by a set of attributes that characterized bird detection by field observers (e.g. body size, colour and calling patterns). Observer heterogeneity varied between groups of birds and was most apparent for small birds foraging in low shrubs (species such as the white-browed scrub wren, assigned to group 2), frequent calling, active birds (species such as the golden whistler, assigned to group 7), and midstorey, undercanopy foragers with distinctive behaviour (species such as the grey fantail assigned to group 4). For bird groups 2, 4 and 7, additional variability due to observer differences resulted in an average increase of ~ 40% in the width of a 95% confidence interval for the logarithm of bird abundance generated from a 20 minute count. Our analysis shows that taking the average of counts obtained by two or more observers would negate the increase in variance of counts due to observer heterogeneity. Few differences between methods of field observation were found. However, for frequent calling, active birds (group 7) there was evidence that more birds were heard using the point interval count method. Our study clearly demonstrated a need to either control for observer differences or to assign at least two observers to individual sites when designing bird surveys for comparative studies. Failure to do so will result in a decrease in precision of bird counts.     

Calcium Binding-Mediated Sustained Release of Minocycline from Hydrophilic Multilayer Coatings Targeting Infection and Inflammation

Infection and inflammation are common complications that seriously affect the functionality and longevity of implanted medical implants. Systemic administration of antibiotics and anti-inflammatory drugs often cannot achieve sufficient local concentration to be effective, and elicits serious side effects. Local delivery of therapeutics from drug-eluting coatings presents a promising solution. However, hydrophobic and thick coatings are commonly used to ensure sufficient drug loading and sustained release, which may limit tissue integration and tissue device communications. A calcium-mediated drug delivery mechanism was developed and characterized in this study. This novel mechanism allows controlled, sustained release of minocycline, an effective antibiotic and anti-inflammatory drug, from nanoscale thin hydrophilic polyelectrolyte multilayers for over 35 days at physiologically relevant concentrations. pH-responsive minocycline release was observed as the chelation between minocycline and Ca²⁺ is less stable at acidic pH, enabling ‘smart’ drug delivery in response to infection and/or inflammation-induced tissue acidosis. The release kinetics of minocycline can be controlled by varying initial loading, Ca²⁺ concentration, and Ca²⁺ incorporation into different layers, enabling facile development of implant coatings with versatile release kinetics. This drug delivery platform can potentially be used for releasing any drug that has high Ca²⁺ binding affinity, enabling its use in a variety of biomedical applications.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

A genetic screen identifies genes essential for development of myelinated axons in zebrafish

The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin.     

Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.