Molecular clocks and geological dates: cytochrome b of Anolis extremus substantially contradicts dating of Barbados emergence

Even though molecular clocks vary in rate to some extent, they are widely used and very important in a range of evolutionary studies, not least in interpreting cause and colonization in phylogeography. Evolutionists may use island age and emergence to give the earliest possible date for colonization by a species and hence give the lower limit in a molecular clock calibration. The geology of the Lesser Antilles is well studied and Barbados, although composed of some ancient rocks, is thought to have emerged only about 1 million years ago (Ma). The cytochrome b mitochondrial gene is the most widely used gene in vertebrate phylogeography, and generally evolves at a rate of 1-2% per million years (Myr) for poikilothermic vertebrates. Divergence measured across almost all of this gene in the endemic anole (Anolis extremus) reveals a mean patristic distance of approximately 8.3% between this clade and its sister, together with distinct divergence and phylogeographical structure within Barbados. The divergence time, estimated by a range of procedures using four calibration points, is not in the least compatible with the proposed geological time of emergence of Barbados. Hence, either the molecular clock rate does not apply to the Barbadian anole population, or the geological dating of the emergence of Barbados is erroneous. The compatibility of geological times and molecular divergence of this complex on Martinique, together with relative rates tests comparing the rates on Barbados and Martinique, do not suggest atypical clock rates. The question of whether Barbados emerged much earlier than is currently thought, or whether the molecular clock assumptions are inappropriate, remains open.     

Structure-based design of a fluorimetric redox active peptide probe

Structure-based iterative design was used to prepare a disulfide-containing nonapeptide as a fluorimetric probe for chemical and biochemical disulfide forming and breaking reactions. The peptide is composed entirely of natural amino acids and exhibits a marked (42%) change in fluorescence between its oxidized and its reduced states. The probe is easily synthesized and highly water soluble and exhibits well-behaved kinetics on reduction with the reductant tris-carboxyethylphosphine. The reduced peptide is an excellent substrate of the enzyme quiescin-sulfhydryl oxidase and may find utility in the characterization of other disulfide oxidoreductases.     

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.     

Synthesis and pharmacology of a novel pyrrolo[2,1,5-cd] indolizine (NNC 45-0095), a high affinity non-steroidal agonist for the estrogen receptor

1-Ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine (NNC 45-0095) is a novel compound which represents the parent pharmacophore structure of a series of pyrrolo[2,1,5-cd]indolizine derivatives with mixed estrogen agonist/antagonist properties. NNC 45-0095 binds with high affinity to the estrogen receptor (IC50=9.5 nM) and exhibits full protection of bone loss in the ovariectomized mouse model for post-menopausal osteoporosis.     

Pre-pleistocene refugia and differentiation between populations of the caucasian salamander (Mertensiella caucasica)

A 350-bp fragment of the mitochondrial cytochrome-b gene was sequenced in the Caucasian salamander, Mertensiella caucasica, representing 10 populations from across its range along the Black Sea coast. Five haplotypes were discovered among 65 fragments analyzed, differing at 2-50 positions. The highest differentiation between haplotypes was observed in animals from the eastern part of the species' range (Borjomi) compared to those from the remainder of the species' range. Randomly amplified nuclear DNA revealed a pattern of spatial genetic variation similar to that of the mitochondrial genome. M. caucasica, as currently known, represents two evolutionary lineages that evolved independently, perhaps since the lower Pliocene. These lineages represent taxa, possibly to be described as species, distributed in the Borjomi area in central Georgia and in southwestern Georgia and northeastern Turkey. The multivariate analysis of morphological data did not reveal significant differences between the taxa. However, substantial morphological differentiation was observed within both lineages, showing parallel patterns in body proportions and coloration patterns. This variation is possibly associated with extant ecological conditions. Salamanders with reduced pigmentation from southwestern Georgia were not genetically distinguishable from neighboring populations.     

Phosphatidylserine externalization and membrane blebbing are involved in the nonclassical export of FGF1

The mechanisms of nonclassical export of signal peptide-less proteins remain insufficiently understood. Here, we demonstrate that stress-induced unconventional export of FGF1, a potent and ubiquitously expressed mitogenic and proangiogenic protein, is associated with and dependent on the formation of membrane blebs and localized cell surface exposure of phosphatidylserine (PS). In addition, we found that the differentiation of promonocytic cells results in massive FGF1 release, which also correlates with membrane blebbing and exposure of PS. These findings indicate that the externalization of acidic phospholipids could be used as a pharmacological target to regulate the availability of FGF1 in the organism.     

Strain differences in a high response-cost daily time-place learning task

Previous research has shown that rats, unlike birds, do not readily demonstrate daily time-place learning (TPL). It has been suggested, however, that rats are more successful at these tasks if the response cost (RC) is increased. Widman et al. (2000) found that female Sprague Dawley (SD) rats learned a daily TPL task in which they were required to climb different towers depending on the time of day to find a food reward. Using a similar apparatus, we found that male SD rats learned the task, while male Long Evans rats did not. While all rats quickly learned to restrict the majority of their searching to the two towers that provided food, only the SD rats learned to go to the correct location at the correct time of day. Thus, there appears to be a strain difference in the effectiveness of a high RC task to promote learning. Tests of the timing strategies used revealed individual differences with one rat using a circadian strategy and another using an ordinal strategy. Post criterion decrements in performance did not allow sufficient testing to determine the timing strategies of the remaining rats. Possible interactions between strain, response cost, species typical behaviors and dependent measures are discussed.     

Oxidant-induced cell-cycle delay in Saccharomyces cerevisiae: the involvement of the SWI6 transcription factor

Cells treated with low doses of linoleic acid hydroperoxide (LoaOOH) exhibit a cell-cycle delay that may provide a mechanism to overcome oxidative stress. Strains sensitive to LoaOOH from the genome-wide deletion collection were screened to identify deletants in which the cell-cycle delay phenotype was reduced. Forty-seven deletants were identified that were unable to mount the normal delay response, implicating the product of the deleted gene in the oxidant-mediated cell-cycle delay of the wild-type. Of these genes, SWI6 was of particular interest due to its role in cell-cycle progression through Start. The swi6 deletant strain was delayed on entry into the cell cycle in the absence of an oxidant, and oxidant addition caused no further delay. Transforming the swi6 deletant with SWI6 on a plasmid restored the G1 arrest in response to LoaOOH, indicating that Swi6p is involved in oxidant sensing leading to cell division delay. Micro-array studies identified genes whose expression in response to LoaOOH depended on SWI6. The screening identified 77 genes that were upregulated in the wild-type strain and concurrently downregulated in the swi6 deletant treated with LoaOOH. These data show that functions such as heat shock response, and glucose transport are involved in the response.     

Acyl-CoA dehydrogenases. A mechanistic overview.

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the alpha,beta-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of alpha,beta-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of alpha,beta-dehydrogenation is viewed as a process in which the substrate alphaC-H and betaC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO- as an H+, the second being transferred as a hydride to the flavin N(5) position. Hereby the pKa of the substrate alphaC-H is lowered from > 20 to approximately 8 by the effect of specific hydrogen bonds. Concomitantly, the pKa of Glu376-COO- is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.