Functional capacities of marsupial hearts: size and mitochondrial parameters indicate higher aerobic capabilities than generally seen in placental mammals

This study of marsupial hearts explored the aerobic capacities of this group of mammals; recent information suggests that marsupials possess higher aerobic abilities than previously accepted. Characteristics such as heart mass, mitochondrial features and capillary parameters were examined. A comprehensive study of the heart of red kangaroos was included because of the high maximum oxygen consumption of this species. Goats were also included as a reference placental mammal. Marsupials have a heart that is generally larger than that of placentals. The allometric equation for the relationship between heart mass and body mass for marsupials was M_h=7.5M_b^0.944 (M_h in g and M_b in kg); the equivalent equation for placental mammals was M_h=6.0M_b^0.97. Mitochondrial volume density and inner mitochondrial surface density do not differ between the two mammal groups; although capillary parameters indicated a lower capillary volume in marsupials. Heart size appears to be the major difference between the two groups. The overall pattern seen in marsupials is similar to that of "athletic" placentals and indicates a relatively high aerobic potential.Abbreviations BMR basal metabolic rate - c(K,0) tortuosity factor - Jv(c,f) capillary length density - M_b body mass - M_h heart mass - N_A(c,f) numerical capillary density - r_c mean capillary radius - S(im,m) total surface area of inner mitochondrial membranes in the heart - Sv(im,m) surface density of the inner mitochondrial membranes - Sv(im,mt) surface density of inner mitochondrial membranes per unit volume of mitochondria - TEM transmission electron microscope - O₂max maximum aerobic capacity - V(mt,m) total mitochondrial volume - Vv(f,m) volume fraction of muscle occupied by muscle fibres - Vv(mt,f) mitochondrial volume densityCommunicated by I.D. Hume     

New high-resolution mass spectrometric approach for the measurement of polychlorinated biphenyls and organochlorine pesticides in human serum

To increase our analytical throughput for measuring polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides without sacrificing data quality, we have developed and validated a combined PCB/OC pesticide gas chromatography-high-resolution mass spectrometry (GC-HRMS) analysis. In a single GC-HRMS analysis, both selected PCBs and OC pesticides are detected and quantified. Previously, this has been difficult, if not impossible, because of the major difference in masses of the most abundant electron-impact ions. However, we have identified slightly less abundant ions to monitor that allow us to successfully combine these analytes into a single analysis without sacrificing any analytical sensitivity or instrument reliability. Consequently, we have been able to double our analytical throughput by modification of mass spectrometric parameters alone. Our new methodology has been validated against our current GC-HRMS method, which entails using two separate injections, one for PCB analysis and one for OC pesticide analysis. The two methods differ by less than 4% overall, with no systematic bias. We used this method to analyze approximately 350 serum samples over a period of several months. We found that our new method was as reliable in automated, overnight runs as our current method.     

A multi-analyte method for the quantification of contemporary pesticides in human serum and plasma using high-resolution mass spectrometry

We have developed a sensitive and accurate analytical method for quantifying 29 contemporary pesticides in human serum or plasma. These pesticides include organophosphates, carbamates, chloroacetanilides, and synthetic pyrethroids among others and include pesticides used in agricultural and residential settings. Our method employs a simple solid-phase extraction followed by a highly selective analysis using isotope dilution gas chromatography-high-resolution mass spectrometry. Our method is very accurate, has limits of detection in the low pg/g range and coefficients of variation of typically less than 20% at the low pg/g end of the method linear range. We have used this method to measure plasma pesticide concentrations in females living in an urban area. We found detectable concentrations of carbaryl/naphthalene, propoxur, bendiocarb, chlorpyrifos, diazinon, dicloran, captan and folpet or their metabolites in more than 20% of the plasma samples tested.     

Hit-to-lead studies: the discovery of potent, orally active, thiophenecarboxamide IKK-2 inhibitors

A hit-to-lead optimisation programme was carried out on the thiophenecarboxamide high throughput screening hits 1 and 2 resulting in the discovery of the potent and orally bioavailable IKK-2 inhibitor 22.     

Genomic affinities in Arachis section Arachis (Fabaceae): molecular and cytogenetic evidence

Section Arachis is the largest of nine sections in the genus Arachis and includes domesticated peanut, A. hypogaea L. Most species are diploids (x=10) with two tetraploids and a few aneuploids. Three genome types have been recognized in this section (A, B and D), but the genomes are not well characterized and relationships of several newly described species are uncertain. To clarify genomic relationships in section Arachis, cytogenetic information and molecular data from amplified fragment length polymorphism (AFLP) and the trnT-F plastid region were used to provide an additional insight into genome composition and species relationships. Cytogenetic information supports earlier observations on genome types of A. cruziana, A. herzogii, A. kempff-mercadoi and A. kuhlmannii but was inconclusive about the genome composition of A. benensis, A. hoehnei, A. ipaensis, A. palustris, A. praecox and A. williamsii. An AFLP dendrogram resolved species into four major clusters and showed A. hypogaea grouping closely with A. ipaensis and A. williamsii. Sequence data of the trnT-F region provided genome-specific information and showed for the first time that the B and D genomes are more closely related to each other than to the A genome. Integration of information from cytogenetics and biparentally and maternally inherited genomic regions show promise in understanding genome types and relationships in Arachis.     

The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans

Genomic DNA from a wide variety of prokaryotic and eukaryotic organisms has been assayed for the simple repeat sequence poly(dT-dG).poly(dC-dA) by Southern blotting and DNA slot blot hybridizations. Consistent with findings of others, we have found the simple alternating sequence to be present in multiple copies in all organisms in the animal kingdom (e.g., mammals, reptiles, amphibians, fish, crustaceans, insects, jellyfish, nematodes). The TG element was also found in lower eukaryotes (Saccharomyces cerevisiae, Neurospora crassa, and Dictyostelium discoideum) and at a much lower frequency in protozoans (Oxytricha fallux and Tetrahymena thermophila). The sequence was also repeated in high copy number in a higher plant (Zea mays) as well as at very high levels in a unicellular green alga (Chlamydomonas reinhardi). Although the copy number of the repeat per haploid genome was generally proportional to genome size, there was a greater-than-1,000-fold variation in the number of (TG)25/100-kb genomic DNA. By contrast, no eu-or archaebacterium--including Myxococcus xanthus, whose life cycle is very similar to that of the slime mold Dictyostelium discoideum, and Halobacter volcanii, whose genome contains other repeated sequences-- was found whose genomic DNA contained this sequence in detectable amounts. A computer search also failed to find the TG element in human mitochondrial DNA.      

Characterization of ixodid tick salivary-gland gene products,using recombinant DNA technology

Ticks secrete an array of lesion-maintenance factors into the host via the salivary glands while feeding, some of which elicit an immune response by the host that adversely affects the ability of the tick to feed and reproduce. Our approach to characterizing these factors has been to make expression libraries from mRNA of salivary glands (from unfed and 3-day-feedingAmblyomma americanum females) which will serve as sources of the genes (clones) that code for them. Thus far, we have detected 10 positive clones in primary screens using polyspecific antiserum from rabbits hyperimmunized to 3-day-feeding tick salivary glands. We also report making a cDNA library from whole unfed females, and a genomic library from whole unfed ticks, which will serve as additional sources of genetic information for characterizing salivary-gland secretory products. Immunoblots of salivary glands fromA. americanum females feeding for various intervals (unfed, and 12, 48, 72, and 96 h) revealed the presence of several prominent polypeptides (90 & 45 kDa) when probed with the same rabbit antiserum that was used to screen the expression library.Ixodes dammini had several immunogens in common withA. americanum at 96 h (90, 45, 43 and 23 kDa). We plan to use monospecific antiserum raised to antigens detected in our immunoblots (e.g. 90 kDa) to further screen the expression libraries, in addition to using the polyspecific antiserum already in hand. We discuss the future use of the salivary-gland genes for characterizing secretory products which facilitate attachment to the host (cement) and maintain the lesion during the lengthy feeding interval.     

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

Protein evolution in different cellular environments: cytochrome b in sharks and mammals

DNA sequences for the mitochondrial cytochrome b gene were determined for 13 species of sharks. Rates and patterns of amino acid replacement are compared for sharks and mammals. Absolute rates of cytochrome b evolution are six times slower in sharks than in mammals. Bivariate plots of the number of nonsynonymous and silent transversions are indistinguishable in the two groups, however, suggesting that the differences in amino acid replacement rates are due primarily to differences in DNA substitution rates. Patterns of amino acid replacement are also similar in the two groups. Conserved and variable regions occur in the same parts of the cytochrome b gene, and there is little evidence that the types of amino acid changes are significantly different between the groups. Similarity in the relative rates and patterns of protein change between the two groups prevails despite dramatic differences in the cellular environments of sharks and mammals. Poor penetrance of physiological differences through to rates of protein evolution provides support for the neutral theory and suggests that, for cytochrome b, patterns of evolution have been relatively constant throughout much of vertebrate history.