Genetic variation of Mimetes hirtus and Mimetes fimbriifolius (Proteaceae) – a comparative analysis of two closely related fynbos species

We investigated the influence of differing life history traits on the genetic structure of the related species Mimetes fimbriifolius and Mimetes hirtus (Proteaceae), which occur in the South African fynbos. Both species are bird‐pollinated and ant‐dispersed, but differ in rarity, longevity, ecological strategy and the fragmentation of their distribution area. We used AFLPs to study genetic variation within and between 21 populations of these two species across their distribution range. AFLP analysis revealed significantly higher genetic variation within populations of M. fimbriifolius than within M. hirtus. While M. fimbriifolius clearly lacked any significant genetic differentiation between populations, a distinct geographic pattern was observed for M. hirtus. Differentiation was, however, stronger at the regional (Φ_PT = 0.57) than at the local scale (Φ_PT = 0.08). Our results clearly indicate that even closely related species that share the same mode of pollination and seed dispersal can differ in their genetic structure, depending on the magnitude of fragmentation, longevity of individuals and ecological strategy.     

The turnover of medium-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by a wide range of bacteria, including Pseudomonads. These polymers are accumulated in the cytoplasm as carbon and energy storage materials when culture conditions are unbalanced and hence, they have been classically considered to act as sinks for carbon and reducing equivalents when nutrients are limited. Bacteria facing carbon excess and nutrient limitation store the extra carbon as PHAs through the PHA polymerase (PhaC). Thereafter, under starvation conditions, PHA depolymerase (PhaZ) degrades PHA and releases R -hydroxyalkanoic acids, which can be used as carbon and energy sources. To study the influence of a deficient PHA metabolism in the growth of Pseudomonas putida KT2442 we have constructed two mutant strains defective in PHA polymerase ( phaC1 )- and PHA depolymerase ( phaZ )-coding genes respectively. By using these mutants we have demonstrated that PHAs play a fundamental role in balancing the stored carbon/biomass/number of cells as function of carbon availability, suggesting that PHA metabolism allows P. putida to adapt the carbon flux of hydroxyacyl-CoAs to cellular demand. Furthermore, we have established that the coordination of PHA synthesis and mobilization pathways configures a functional PHA turnover cycle in P. putida KT2442. Finally, a new strain able to secrete enantiomerically pure R -hydroxyalkanoic acids to the culture medium during cell growth has been engineering by redirecting the PHA cycle to biopolymer hydrolysis.     

Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo

Previous studies suggest that the n-3 polyunsaturated fatty acids (PUFAs) eicosapenteinoic acid (EPA) and docosahexaenoic acid (DHA), constituents of fish oil, exert chemopreventive activity in colon cancer. One of the mechanisms involved is the facilitation of apoptosis. While a pro-apoptotic potential of n-3 PUFAs has been suggested, it is still unclear whether additional consumption of fish will also lead to comparable results. The aim of this study was to assess EPA- and DHA-mediated effects on endpoints of apoptosis and to use a novel biomarker-approach to measure modulation of apoptosis by consumption of fish. LT97 human colon adenoma and HT29 human colon adenocarcinoma cells were used to investigate modulation of apoptosis by EPA, DHA or linoleic acid (LA) using a set of endpoints, namely phosphatidylserine staining with Annexin-V (flow cytometry), Bcl-2 expression (Real-time RT–PCR), and Bid, caspase 3, 8 and 9 expression as well as PARP cleavage (Western Blot). Furthermore, faecal water (FW) of volunteers (n = 89) from a human trial intervening with fish was used to investigate changes in apoptosis by flow cytometry. DHA was more effective at inducing apoptosis than EPA. LT97 cells were more prone to DHA and EPA induced apoptosis than HT29 cells. Treatment of LT97 cells with FW from volunteers consuming fish did not result in any changes in apoptosis. Taken together, our results show that adenoma cells are highly susceptible to n-3 PUFA-induced apoptosis. By using a biomarker-approach (FW) to measure apoptosis-induction ex vivo no change in apoptosis after additional fish consumption was detectable.     

DNA triplex stabilization by a delta-carboline derivative tethered to third strand oligonucleotides

A delta-carboline derivative was covalently coupled to a 7 mer oligonucleotide at its 5'- or 3'-end. The stability of triplexes formed from the conjugates and a double-helical target was studied by UV melting experiments. Compared to the unmodified control triple helices, triplexes with the conjugate exhibit a significantly higher stability. However, the degree of stabilization depends on the particular triplex structure formed.     

Long-term stability and effective population size in North Sea and Baltic Sea cod (Gadus morhua)

DNA from archived otoliths was used to explore the temporal stability of the genetic composition of two cod populations, the Moray Firth (North Sea) sampled in 1965 and 2002, and the Bornholm Basin (Baltic Sea) sampled in 1928 and 1997. We found no significant changes in the allele frequencies for the Moray Firth population, while subtle but significant genetic changes over time were detected for the Bornholm Basin population. Estimates of the effective population size ( N _e ) generally exceeded 500 for both populations when employing a number of varieties of the temporal genetic method. However, confidence intervals were very wide and N _e 's most likely range in the thousands. There was no apparent loss of genetic variability and no evidence of a genetic bottleneck for either of the populations. Calculations of the expected levels of genetic variability under different scenarios of N _e showed that the number of alleles commonly reported at microsatellite loci in Atlantic cod is best explained by N _e 's exceeding thousand. Recent fishery-induced bottlenecks can, however, not be ruled out as an explanation for the apparent discrepancy between high levels of variability and recently reported estimates of N _e  << 1000. From life history traits and estimates of survival rates in the wild, we evaluate the compatibility of the species' biology and extremely low N _e / N ratios. Our data suggest that very small N _e 's are not likely to be of general concern for cod populations and, accordingly, most populations do not face any severe threat of losing evolutionary potential due to genetic drift.     

Antikinetoplastid antimitotic activity and metabolic stability of dinitroaniline sulfonamides and benzamides

N(1)-Phenyl-3,5-dinitro-N(4),N(4)-di-n-propylsulfanilamide (1) and N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (2) show potent in vitro antimitotic activity against kinetoplastid parasites but display poor in vivo activity. Seventeen new dinitroaniline sulfonamide and eleven new benzamide analogs of these leads are reported here. Nine of the sulfonamides display in vitro IC(50) values under 500 nM against African trypanosomes, and the most active antikinetoplastid compounds also inhibit the in vitro assembly of purified leishmanial tubulin with potencies similar to that of 2. While several of the potent compounds are rapidly degraded by rat liver S9 fractions in vitro, N(1)-(3-hydroxy)phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (21) displays an IC(50) value of 260 nM against African trypanosomes in vitro and is more stable than 2 in the in vitro metabolism assay.     

Gain of function in an ERV/ALR sulfhydryl oxidase by molecular engineering of the shuttle disulfide

The ERV/ALR sulfhydryl oxidase domain is a versatile module adapted for catalysis of disulfide bond formation in various organelles and biological settings. Its four-helix bundle structure juxtaposes a Cys-X-X-Cys dithiol/disulfide motif with a bound flavin adenine dinucleotide (FAD) cofactor, enabling transfer of electrons from thiol substrates to non-thiol electron acceptors. ERV/ALR family members contain an additional di-cysteine motif outside the four-helix-bundle core. Although the location and context of this "shuttle" disulfide differs among family members, it is proposed to perform the same basic function of mediating electron transfer from substrate to the enzyme active site. We have determined by X-ray crystallography the structure of AtErv1, an ERV/ALR enzyme that contains a Cys-X4-Cys shuttle disulfide and oxidizes thioredoxin in vitro, and compared it to ScErv2, which has a Cys-X-Cys shuttle and does not oxidize thioredoxin at an appreciable rate. The AtErv1 shuttle disulfide is in a region of the structure that is disordered and thus apparently mobile and exposed. This feature may facilitate access of protein substrates to the shuttle disulfide. To test whether the shuttle disulfide region is modular and can confer on other enzymes oxidase activity toward new substrates, we generated chimeric enzyme variants combining shuttle disulfide and core elements from AtErv1 and ScErv2 and monitored oxidation of thioredoxin by the chimeras. We found that the AtErv1 shuttle disulfide region could indeed confer thioredoxin oxidase activity on the ScErv2 core. Remarkably, various chimeras containing the ScErv2 Cys-X-Cys shuttle disulfide were found to function efficiently as well. Since neither the ScErv2 core nor the Cys-X-Cys motif is therefore incapable of participating in oxidation of thioredoxin, we conclude that wild-type ScErv2 has evolved to repress activity on substrates of this type, perhaps in favor of a different, as yet unknown, substrate.     

Nerve growth factor-induced phosphorylation of amphiphysin-1 by casein kinase 2 regulates clathrin-amphiphysin interactions

Amphiphysins interact directly with clathrin and have a function in clathrin-mediated synaptic vesicle recycling and clathrin-mediated endocytosis. The neuronal isoform amphiphysin-1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Rephosphorylation was stimulated by nerve growth factor. We analysed the regulation of amphiphysin-clathrin interactions by phosphorylation. The N-terminal domain of clathrin bound to unphosphorylated amphiphysin-1, but not to the phosphorylated protein. A search for possible phosphorylation sites revealed two casein kinase 2 consensus motifs in close proximity to the clathrin binding sites in amphiphysin-1 and -2. We mutagenized these residues (T350 and T387) to glutamate, mimicking a constitutive phosphorylation. The double mutant showed a strong reduction in clathrin binding. The assumption that casein kinase 2 phosphorylates amphiphysin-1 at T350 and T387 was corroborated by experiments showing that: (i) casein kinase 2 phosphorylated these residues directly in vitro, (ii) when expressed in HeLa cells, the glutamate mutant showed reduced phosphorylation, and (iii) casein kinase 2 inhibitors blocked nerve growth factor-induced phosphorylation of endogenous amphiphysin-1 in PC12 cells. These observations are consistent with the hypothesis that, upon activation by nerve growth factor, casein kinase 2 phosphorylates amphiphysin-1 and thereby regulates the endocytosis of clathrin-coated vesicles via the interaction between clathrin and amphiphysin.     

Structural stabilization of CNS synapses during postnatal development in rat cortex

CNS synapses are produced rapidly upon pre- and post-synaptic recruitment. However, their composition is known to change during development and we reasoned that this may be reflected in the gross biochemical properties of synapses. We found synaptic structure in adult cortical synaptosomes to be resistant to digestion with trypsin in the presence and absence of calcium ions, contrasting with previous observations. We evaluated the divalent cation dependence and trypsin sensitivities of synapses using synaptosomes from different developmental stages. In contrast to adult synapses, at postnatal day (P) 10 EDTA treatment eliminated approximately 60% of the synapses, and trypsin and EDTA, together, eliminated all junctions. Trypsinization in the presence of calcium eliminated approximately 60% of the junctions at P10. By P35, all synapses were calcium independent, whereas full trypsin resistance was not attained until P49. To compare the calcium dependence and trypsin sensitivity of synapses in another region of the adult brain, we examined synapses from adult (P50) hippocampus. Adult hippocampus maintained a population of synapses that resembled that of P35 cortex. Our results show that synapses are modified over a long time period in the developing cortex. We propose a model in which the addition of synergistic calcium-dependent and -independent adhesive systems stabilize synapses.