Contrasting patterns of radiation in African and Australian Restionaceae

The floras of the Mediterranean-climate areas of southern Africa and southwestern Australia are remarkably species rich. Because the two areas are at similar latitudes and in similar positions on their respective continents, they have probably had similar Cenozoic climatic histories. Here we test the prediction that the evolution of the species richness in the two areas followed a similar temporal progression by comparing the rates of lineage accumulation for African and Australian Restionaceae. Restionaceae (Poales) are typical and often dominant elements in the fynbos vegetation of the Cape Floristic Region of southern Africa and the kwongan vegetation of the Southwestern Floristic Province of Western Australia. The phylogeny of the family was estimated from combined datasets for rbcL and trnL-F sequences and a large morphological dataset; these datasets are largely congruent. The monophyly of Restionaceae is supported and a basal division into an African clade (approximately 350 species) and an Australian clade (146 species) corroborated. There is also support for a futher subdivision of these two large sister-clades, but the terminal resolution within the African clade is very weak. Fossil pollen records provided a minimum age of the common ancestor of Australian and African Restionaceae as 64-71 million years ago, and this date was used to calibrate a molecular clock. A molecular clock was rejected by a likelihood ratio test; therefore, rate changes between the lineages were smoothed using nonparametric rate smoothing. The rate-corrected ages were used to construct a plot of lineages through time. During the Palaeogene the Australian lineage diversity increased consistent with the predictions of the constant birthrate model, while the African lineage diversity showed a dramatic increase in diversification rate in the Miocene. Incomplete sampling obscures the patterns in the Neogene, but extending the trends to the modern extant diversity suggests that this acceleration in the speciation rate continued in the African clade, whereas the Australian clade retained a constant diversification rate. The substantial morphological and anatomical similarity between the African and Australian Restionaceae appear to preclude morphological innovations as possible explanations for the intercontinental differences. Most likely these differences are due to the greater geographical extent and ecological variation in temperate Australia than temperate Africa, which might have provided refugia for basal Restionaceae lineages, whereas the more mountainous terrain of southern Africa might have provided the selective regimes for a more rapid, recent speciation.     

Metabolic efficiency of liver mitochondria in rats with decreased thermogenesis

We have studied changes in hepatic mitochondrial efficiency induced by 24-h fasting or acclimation at 29 degrees C, two conditions of reduced thermogenesis. Basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, are not affected after 24-h fasting, when serum free triiodothyronine decreases significantly and serum free fatty acids increase significantly. In rats at 29 degrees C, in which serum free triiodothyronine and fatty acids decrease significantly, basal proton leak increases significantly, while no variation is found in palmitate-induced proton leak. The present results indicate that mitochondrial efficiency in the liver is not related to a physiological decrease in whole body thermogenesis.     

Combinatorial redesign of the DNA binding specificity of a prokaryotic helix-turn-helix repressor

Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences. Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences. Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a lambda bacteriophage-specific repressor were identified. The libraries contained variations within the HTH sequence at only three positions. In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences. For the best 434 Cro variant identified, an apparent K(d) for lambda O(R)3 of 1 nM was observed. In competition experiments, this Cro variant was observed to be highly selective. We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix. Important characteristics of the screening system responsible for the successful identifications are discussed. Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes.     

Endostatin inhibits human tongue carcinoma cell invasion and intravasation and blocks the activation of matrix metalloprotease-2, -9, and -13

Endostatin, a 20-kDa collagen XVIII fragment, inhibits angiogenesis and tumor growth in vivo, but the mechanisms are still unclear. Matrix metalloproteases (MMPs), a family of extracellular and membrane-associated endopeptidases, collectively digest almost all extracellular matrix and basement membrane components, and thus play an important role in tumor progression. We studied the effects of recombinant human endostatin on human MMP-2, -9, -8, and -13. We found that endostatin inhibited the activation and catalytic activity of pro-MMP-9 and -13 as well as recombinant pro-MMP-2. It prevented the fragmentation of pro-MMP-2 that was associated with reduction of catalytic activity. Endostatin had no effect on MMP-8 as shown by collagenase activity assays. An in vitro migration assay and an in vivo chicken chorioallantoic membrane intravasation assay with the human tongue squamous cell carcinoma cell line HSC-3 revealed the biphasic nature of endostatin; low endostatin concentrations inhibited intravasation and migration of these cells in a dose-dependent manner, but at increased concentrations, the inhibitory effect was far less efficient. The results show that endostatin blocks the activation and activities of certain tumor-associated pro-MMPs, such as pro-MMP-2, -9, and -13, which may explain, at least in part, the antitumor effect of endostatin. Our results also suggest that endostatin inhibits tumor progression by directly affecting the tumor cells and not just acting via endothelial cells and blockage of angiogenesis.     

Identification of metabolites from type III F2-isoprostane diastereoisomers by mass spectrometry

F(2)-isoprostanes (F(2)-iPs) are prostaglandin (PG)-like products of non-enzymatic free radical-catalyzed peroxidation of arachidonic acid that are now widely used as indices of lipid peroxidation in vivo. Knowledge of the metabolic fate of F(2)-iPs in vivo is still scant, despite its importance for defining their overall formation and biological effects in vivo. Type III F(2)-iPs, which are diastereoisomers of cyclooxygenase-derived PGF(2alpha), may be metabolized through the pathways of PG metabolism. We therefore studied the in vitro metabolism of eight synthetic Type III F(2)-iP diastereoisomers in comparison with PGF(2alpha). We used gas chromatography-mass spectrometry and high performance liquid chromatography-electrospray-tandem mass spectrometry for structural identification of metabolites formed after incubation of the various compounds with isolated rat hepatocytes. PGF(2alpha) was metabolized to several known products, resulting from a combination of beta-oxidation, reduction of Delta(5) and/or Delta(13) double bonds, and 15-OH oxidation, plus other novel products deriving from conjugation with taurine of PGF(2alpha) and its metabolites. Of the eight F(2)-iP diastereoisomers, some were processed similarly to PGF(2alpha), whereas others showed peculiar metabolic profiles according to specific stereochemical configurations.These data represent the first evidence of biodegradation of selected Type III F(2)-iP isomers other than 8-epi-PGF(2alpha), through known and novel pathways of PGF(2alpha) metabolism. The analytical characterization of these products may serve as a basis for identifying the most significant products formed in vivo.     

Diversity in and evidence for selection on the mitochondrial genome of Phytophthora infestans

Two extant nomenclature systems were reconciled to relate six mitochondrial DNA (mtDNA) haplotypes of Phytophthora infestans, the oomycete pathogen causing late blight disease on potato and tomato. Carter's haplotypes I-a and I-b were included in Goodwin's haplotype A, while Carter's haplotypes II-a and II-b were included in Goodwin's haplotype B. In addition, haplotypes E and F were included in Carter's haplotype I-b. The mutational differences separating the various haplotypes were determined, and we propose that either haplotype I-b(A) or haplotype I-a(A) is the putative ancestral mtDNA of P. infestans, because either can center all the other haplotypes in a logical stepwise network of mutational changes. The occurrence of the six haplotypes in 548 isolates worldwide was determined. Haplotypes I-a and II-a were associated with diverse genotypes worldwide. As previously suggested, haplotype I-b was found only in the US-1 clonal lineage and its variants (n = 99 isolates from 16 countries on 5 continents), and haplotype II-b was limited to the US-6 clonal lineage and its derivatives (n = 36). In a confirmation of a previous suggestion, the randomly mating population in the Toluca Valley of central Mexico (n = 78) was monomorphic for mtDNA haplotype I-a(A). We hypothesize that selection there may be driving the dominance of that single mtDNA haplotype.     

The role of the amygdala in signaling prospective outcome of choice

Can brain activity reveal a covert choice? Making a choice often evokes distinct emotions that accompany decision processes. Amygdala has been implicated in choice behavior that is guided by a prospective negative outcome. However, its specific involvement in emotional versus cognitive processing of choice behavior has been a subject of controversy. In this study, the human amygdala was monitored by functional magnetic resonance imaging (fMRI) while subjects were playing in a naturalistic choice paradigm against the experimenter. In order to win, players had to occasionally choose to bluff their opponent, risk "getting caught," and suffer a loss. A critical period, when choice has been made but outcome was still unknown, activated the amygdala preferentially following the choice that entailed risk of loss. Thus, the response of the amygdala differentiated between subject's covert choice of either playing fair or foul. These results support a role of the amygdala in choice behavior, both in the appraisal of inherent value of choice and the signaling of prospective negative outcomes.     

Separation methods for catechol O-methyltransferase activity assay: physiological and pathophysiological relevance

Catechol O-methyltransferase (COMT) transfers a methyl group from S-adenosyl-L-methionine to the catechol substrate in the presence of magnesium. After the characterisation of COMT more than four decades ago, a wide variety of COMT enzyme assays have been introduced. COMT activity analysis usually consists of the handling of the sample and incubation followed by separation and detection of the reaction products. Several of these assays are validated, reliable and sensitive. Besides the studies of the basic properties of COMT, the activity assay has also been applied to explore the relation of COMT to various disease states or disorders. In addition, COMT activity analysis has been applied clinically since COMT inhibitors have been introduced as adjuvant drugs in the treatment of Parkinson's disease.     

Monitoring the effects of drug treatment in rat models of disease by serum protein analysis

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.