Extant mammal body masses suggest punctuated equilibrium

Is gradual microevolutionary change within species simultaneously the source of macroevolutionary differentiation between species? Since its first publication, Darwin''s original idea that phenotypic differences between species develop gradually over time, as the accumulation of small selection-induced changes in successive generations has been challenged by palaeontologists claiming that, instead, new species quickly acquire their phenotypes to remain virtually unchanged until going extinct again. This controversy, widely known as the ‘punctuated equilibrium’ debate, remained unresolved, largely owing to the difficulty of distinguishing biological species from fossil remains. We analysed body masses of 2143 existing mammal species on a phylogeny comprising 4510 (i.e. nearly all) extant species to estimate rates of gradual (anagenetic) and speciational (cladogenetic) evolution. Our Bayesian estimates from mammals as well as separate sub-clades such as primates and carnivores suggest that gradual evolution is responsible for only a small part of body size variation between mammal species.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H2O2) through the plasma membrane decreases during adaptation to H2O2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H2O2 the anisotropy of the plasma membrane increases. Adaptation to H2O2 was studied at several times (15min up to 90min) by applying the steady-state H2O2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H2O2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H2O2 adaptation. H2O2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H2O2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H2O2 is mediated by modulation of the biophysical properties of the plasma membrane.     

Haploinsufficiency of cytochrome P450 17alpha-hydroxylase/17,20 lyase (CYP17) causes infertility in male mice

Cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17) is critical in determining cortisol and sex steroid biosynthesis. To investigate how CYP17 functions in vivo, we generated mice with a targeted deletion of CYP17. Although in chimeric mice Leydig cell CYP17 mRNA and intratesticular and circulating testosterone levels were dramatically reduced (80%), the remaining testosterone was sufficient to support spermatogenesis as evidenced by the generation of phenotypical black C57BL/6 mice. However, male chimeras consistently failed to generate heterozygous CYP17 mice and after five matings chimeric mice stopped mating indicating a change in sexual behavior. These results suggested that CYP17 deletion caused a primary phenotype (infertility), probably not due to the anticipated androgen imbalance and a secondary phenotype (change in sexual behavior) due to the androgen imbalance. Surprisingly, CYP17 mRNA was found in mature sperm, and serial analysis of gene expression identified CYP17 mRNA in other testicular germ cells. CYP17 mRNA levels were directly related to percent chimerism. Moreover, more than 50% of the sperm from high-percentage chimeric mice were morphologically abnormal, and half of them failed the swim test. Furthermore, 60% of swimming abnormal sperm was devoid of CYP17. These results suggest that CYP17, in addition to its role in steroidogenesis and androgen formation, is present in germ cells where it is essential for sperm function, and deletion of one allele prevents genetic transmission of mutant and wild-type alleles causing infertility followed by change in sexual behavior due to androgen imbalance.     

A statistical test of unbiased evolution of body size in birds

Abstract.— Of the approximately 9500 bird species, the vast majority is small-bodied. That is a general feature of evolutionary lineages, also observed for instance in mammals and plants. The avian interspecific body size distribution is right-skewed even on a logarithmic scale. That has previously been interpreted as evidence that body size evolution has been biased. However, a procedure to test for unbiased evolution from the shape of body size distributions was lacking. In the present paper unbiased body size evolution is defined precisely, and a statistical test is developed based on Monte Carlo simulation of unbiased evolution. Application of the test to birds suggests that it is highly unlikely that avian body size evolution has been unbiased as defined. Several possible explanations for this result are discussed. A plausible explanation is that the general model of unbiased evolution assumes that population size and generation time do not affect the evolutionary variability of body size; that is, that micro- and macroevolution are decoupled, which theory suggests is not likely to be the case.     

Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells

The active form of vitamin D(3) (1,25(OH)(2)D(3)) induces an increase in the intracellular free calcium ([Ca(2+)](i)) and caspase-independent cell death in human breast cancer cells. Here we show that the treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) or its chemotherapeutic analog, EB 1089, releases Ca(2+) from the endoplasmic reticulum. The increase in [Ca(2+)](i) was associated with the activation of a calcium-dependent cysteine protease, mu-calpain. Interestingly, ectopic expression of a calcium-binding protein, calbindin-D(28k), in MCF-7 cells not only attenuated the elevation in [Ca(2+)](i) and calpain activation, but also reduced death triggered by vitamin D compounds. Similarly, the inhibition of calpain activity by structurally unrelated chemical inhibitors increased the survival of the cells and reduces the amount of annexin V-positive cells. Despite the complete absence of effector caspase activation, transmission electron microscopy of MCF-7 cells treated with 1,25(OH)(2)D(3) or EB 1089 revealed apoptosis-like morphology characterized by the condensed cytoplasm, nuclei, and chromatin. Overall, these results suggest that calpain may take over the role of the major execution protease in apoptosis-like death induced by vitamin D compounds. Thus, these compounds may prove useful in the treatment of tumors resistant to therapeutic agents dependent on the classical caspase cascade.     

Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system

The major Escherichia coli multidrug efflux pump AcrAB-TolC expels a wide range of antibacterial agents. Using in vivo cross-linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate-induced assembly of the closely related TolC-dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross-linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo. Confirming this view, AcrA could be cross-linked independently to AcrB and TolC in vivo, and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C-terminal half of AcrA, but binding to TolC required interaction of N- and C-terminal polypeptides spanning the lipoyl-like domains predicted to present the intervening coiled-coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug-independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.     

Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis

The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore. Products were separated by HPLC and the amount of product was calculated by peak integration. Penta-N-acetylglucosamine (penta-nag) and hexa-N-acetylglucosamine (hexa-nag) were used as substrates. Hexa-nag was more efficiently converted than penta-nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra-N-acetylglucosamine (tetra-nag) and allosamidin were tested as inhibitors. Allosamidin was found to be a competitive inhibitor with a K(i) of 3.1 microM. Under the conditions tested, tetra-nag did not inhibit hevamine.     

Sensitivity enhancement in (HCA)CONH experiments

A novel sensitivity-enhancement technique is proposed for experiments which correlate protein backbone resonances and start with magnetization from ¹³C-¹H groups. The technique is based on replenishing magnetization lost by dipole-CSA cross-correlated relaxation of the ¹³C spin with ¹³C steady state magnetization. The principle is demonstrated for the (HCA)CONH experiment, resulting in 1.6-fold sensitivity enhancement compared to the HN(CA)CO experiment. Furthermore, other versions of the (HCA)CONH experiment were evaluated, including a novel experiment with spin-locking of transverse ¹³C-¹H two-spin coherence, and a cross-correlation compensated (CA)CONH experiment which starts from ¹³C rather than ¹H magnetization.