Molecular phylogeny and biogeographic history of the piculets (Piciformes: Picumninae)

The subfamily Picumninae (piculets) includes 3 genera and 30 species of tiny and short-tailed woodpeckers with a pantropical distribution. Within the Picumninae, two cases of intercontinentally disrupted distributions at the genus level occur. The first one concerns the genus Sasia (one species in Africa and two in southeast Asia) while the second concerns Picumnus (one species in southeast Asia and 25 in South America). These disrupted distributions, as well as several morphological differences, have lead some authors to place the African representative of Sasia and the southeast Asian representative of Picumnus in their own monotypic genera ( Verreauxia and Vivia , respectively). To address the taxonomic status and biogeographic history of the piculets, we sequenced 2676 bp of DNA from one mitochondrial (ND2) and two nuclear markers (myoglobin intron 2 and β-fibrinogen intron 7). Monophyly of Picumninae could not be recovered with confidence, while monophyly of Sasia and Picumnus were always strongly supported. Molecular dating analyses revealed that the splits both between the African and Indo-Malayan Sasia and between the New World and Old World Picumnus occurred at ca 7.9 Myr BP. This time corresponds to the beginning of the formation of the northern Hemisphere ice sheets and the accompanying expansion of grasslands throughout the world. The spread of open areas in the northern parts of Eurasia and America prevented gene flow between tropical forest birds, such as the piculets, in Africa, southeast Asia and South America, respectively.     

Physical and chemical effects of litter on plant establishment in semi-natural grasslands

A field experiment was conducted in a semi-natural grassland to study the interspecific variation in the effect of litter on seedling emergence and establishment and separate physical from chemical effects. Seeds of seven forb species were sown in plots subjected to either litter amendment (0, 400 or 900 g m⁻²) or water extracts of litter (corresponding to 400 and 900 g litter m⁻²). In addition, an extract was treated with activated carbon to estimate the possible effects of secondary chemical compounds. The response to plant litter differed amongst species: negative, neutral and positive responses were observed. Anthriscus sylvestris was the only species with a strong positive response to litter. We found no consistent relation between seed size and response to plant litter. Physical effects of litter were generally stronger than chemical effects. However, water extract of litter inhibited emergence in three species. Activated carbon removed the negative effect of the litter extract, which suggests that the effect was caused by an inhibitory chemical compound rather than by increased competition in response to nutrients added via the extract. The balance between facilitative and inhibitory effects of litter depended on species identity and litter quantity. Facilitative effects dominated at low and intermediate quantities of litter, and inhibitory effects at high litter quantities. One species, Campanula rotundifolia, showed a switch from positive to negative responses with increasing quantities of litter. However, we found no general threshold for litter quantity valid across species.     

Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae

In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.     

Screening for weak monoclonal antibodies in hybridoma technology

As the interest in weak-affinity antibodies has been widened by their introduction to various analytical techniques such as HPLC, capillary electrophoresis and biosensors, there has been a need for new screening/monitoring methods. In this study, weak-affinity chromatography was adopted to screen/monitor directly for monoclonal antibodies in ascites. Monoclonal antibodies against a carbohydrate antigen (maltohexaose) were used to evaluate this approach. In short, malthohexaose was immobilized on an HPLC support in such a configuration to allow, during HPLC, retardation of weak monoclonal antibodies. Based on the retention, the affinity or the avidity, as determined by the presence of multiple binding of the monoclonal antibody towards antigen, can be estimated. In this way it is possible to select clones of hybridomas that produce desired weak monoclonal antibodies. Adjustments in temperature (10-20 degrees C) were used to moderate the retention and hence affinity of the weak monoclonal antibodies during chromatography.     

The effect of forest management operations on population performance of Vaccinium myrtillus on a landscape-scale

We have studied the variation in average performance of Vaccinium myrtillus L. among local populations within an intensively managed boreal forest landscape in southeast Norway. Our aim was to identify whether forest disturbance history was an important factor controlling the variation in average plant performance. We used three measures of forest maturity (maturity class, tree biomass and number of trees) as variables reflecting disturbance history. These variables were used in our analysis together with measured environmental conditions (soil moisture, pH, nitrogen, and elevation) a priori known to affect plant performance. To quantify plant performance, we used multiple measures of the local V. myrtillus populations, including percentage cover, average ramet age, height, diameter, biomass, growth, and sexual reproduction (number of fruits and number of mature seeds per fruit). Most of the explained variation in our performance variables could be related to our environmental variables, but disturbance history was also an important determinant of the performance of V. myrtillus. All performance variables were higher in younger, less mature stands, except ramet age, which was highest in mature stands. The increase in performance after clear cutting indicates that this period is of high importance for the population dynamics and persistence of V. myrtillus in intensively managed boreal forest landscapes.     

Phylogeny of the ovenbird genus Upucerthia: a case of independent adaptations for terrestrial life

In view of the amount of adaptive radiation among ovenbirds, Furnariinae, the traditional taxonomy needs scrutiny. We used nuclear DNA data to study relationships of the genus Upucerthia . Five species belong in the Furnariini, as traditionally assumed. The other species represent two independent cases of specialisation for terrestrial life, in response to formation of arid habitats in the southern Andes, within the paraphyletic assemblage of arboreal species referred to as Philydorini.     

Steroid transformation by living cells immobilized in calcium alginate

Summary Whole cells of Arthrobacter simplex were immobilized in a living state in calcium alginate gel. The bacteria showed steroid-¹-dehydrogenase activity and the production of prednisolone from cortisol was investigated. The ¹-dehydrogenase activity of the immobilized cells could be increased about ten-fold by incubation in nutrient media (e.g., containing 0.5% peptone abd 0.2% glucose). The reason for this activation was examined and it was found that the immobilized cells were capable of multiplying when supplied with nutrients. Furthermore, provided that an inducer, cortisol, was present, the steroid-¹-dehydrogenase activity increased in proportion to the increase in the number of cells and it was thus concluded that microbial growth was the cause of activation.Experiments on repeated, batch-wise pseudocrystallofermentation with immobilized A. simplex cells also showed that immobilized cells could be advantageously used for pseudocrystallofermentation of steroids.     

Synchronous intercontinental splits between assemblages of woodpeckers suggested by molecular data

The woodpeckers (Piciformes: Picinae) comprise a widely distributed and species-rich clade of birds that is strongly associated with trees for feeding, nesting, or both. Because of this association, woodpeckers provide a useful model for evaluating the impact of climatic and tectonic events on the diversification of forest birds during the Tertiary. In order to resolve the biogeographical history of the woodpeckers, we have analysed sequences from two nuclear introns and one mitochondrial gene using likelihood and Bayesian approaches. Our analyses favour a tropical Eurasian origin; divergences between African, Indo-Malayan and New World clades with subsequent colonizations of Africa and the New World occurred synchronously during the Middle Miocene, a period corresponding to the expansion of the C4 grasses and the uplift of the Himalayan-Tibetan plateau. The taxonomic diversification of woodpeckers at this time may be attributed to the fragmentation of forests in response to the drier climate, which in turn prevented gene flow between tropical stocks in Africa, Indo-Malaya and the New World. Our estimates of colonization times of South America predate the closure of the Panama Isthmus and support the hypothesis of a short-lived, terrestrial corridor at the end of the Miocene, 5.7 Myr BP.     

Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture studies (cell growth, viability and product formation) towards a number of target cells including myelomas, hybridomas, hepatocytes, fibroblasts and epithelial cells. In general the platelet lysate medium supported cell growth and maintained viabilities comparable or superior to fetal bovine serum. Productivity studies of antibodies (hybridomas) and transferrin (hepatocytes) showed similar or enhanced production in platelet-derived medium in comparison with FBS. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.