Response of harpacticoid copepods to habitat structure at a deep-sea site

Given the quiescent physical environment and the low rates of bioturbation in the deep sea, biologically produced structures, such as animals tubes, may play an important role in creating habitat heterogeneity. In San Diego Trough, the cirratulid polychaete Tharyx luticastellus builds and inhabits a robust mud concretion (a mudball). Mudballs are abundant and persist after the worm has died or abandoned them. Our analysis of twelve 6.6-cm-diameter cores from 32° 51.02 N, 117° 46.97 W, taken with the submersible SEA CLIFF, indicate that one of 29 species of harpacticoids common in San Diego Trough is associated with Tharyx mudballs. This species (of the genus Heteropsyllus) responded only to mudballs containing worms, suggesting that any effects of the inanimate structure were not the cause of the association. We could detect no difference in bacterial numbers in sediments between control and occupied-mudball samples. This result suggests that the response does not result from gross differences in food levels near a mudball, but the possibility that there are differences in some preferred food cannot be discounted. An univestigated possibility is that Heteropsyllus responds to occupied mudballs because the worm's presence deters some (presumably small) predator.     

A fast and simple genetic survey reveals the spread of poplar hybrids at a natural Elbe river site

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. × canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. × canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P.  × canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.     

A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site

A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product -aminobutyric acid (GABA) in fruit, are discussed.     

Isolation of matrices from maize leaf nuclei: identification of a matrix-binding site adjacent to theAdh1 gene

Nuclear matrices were isolated from maize leaves by the two conventional methods usually employed for the preparation of the corresponding structures of animal origin. It is demonstrated that functionally competent matrices, recognizing and specifically binding the MAR-containing DNA of the mousek-immunoglobulin gene may be prepared by both 2 M NaCl and LIS extractions of maize nuclei.A DNA region with a high affinity for the nuclear matrix was identified at the 5 end of the maizeAdh1-S gene, distal to the promoter region. The presence of sites of reported altered chromatin structure in this particular region is discussed. While the proximity and the cohabitation of MARs with different regulatory elements is a common feature of matrix association regions in animal systems, this is the first plant MAR identified in a region of known significance for gene regulation.     

Timing and site selection of spawning in a landlocked population of rainbow smelt in Wisconsin

To study the site selection and timing of spawning by rainbow smelt, Osmerus modax, in a freshwater system, smelt were sampled with fyke nets during the spawning run in spring 2002 and 2003. The sex ratio of smelt favored males in the early and late segments of the run, with over 90% of smelt caught being male. At the peak in the catch, females comprised 57% of the smelt collected. The gonadosomatic index of males and females declined significantly on the day of peak catch, signaling the peak in spawning activity. Higher catches of smelt occurred on cobble-dominated substrates during the peak of the run, but no site selection was apparent earlier or later. Groundwater outflow did not appear to affect catches. Our study suggests that smelt come inshore but do not select specific spawning substrates prior to or after the peak in spawning. Smelt do select cobble sites during the highest rates of spawning. The relation between the peak in spawning and site selection could have application in planning removal strategies for the species.     

Invasion of an exotic forb impacts reproductive success and site fidelity of a migratory songbird

Although exotic plant invasions threaten natural systems worldwide, we know little about the specific ecological impacts of invaders, including the magnitude of effects and underlying mechanisms. Exotic plants are likely to impact higher trophic levels when they overrun native plant communities, affecting habitat quality for breeding songbirds by altering food availability and/or nest predation levels. We studied chipping sparrows (Spizella passerina) breeding in savannas that were either dominated by native vegetation or invaded by spotted knapweed (Centaurea maculosa), an exotic forb that substantially reduces diversity and abundance of native herbaceous plant species. Chipping sparrows primarily nest in trees but forage on the ground, consuming seeds and arthropods. We found that predation rates did not differ between nests at knapweed and native sites. However, initiation of first nests was delayed at knapweed versus native sites, an effect frequently associated with low food availability. Our seasonal fecundity model indicated that breeding delays could translate to diminished fecundity, including dramatic declines in the incidence of double brooding. Site fidelity of breeding adults was also substantially reduced in knapweed compared to native habitats, as measured by return rates and shifts in territory locations between years. Declines in reproductive success and site fidelity were greater for yearling versus older birds, and knapweed invasion appeared to exacerbate differences between age classes. In addition, grasshoppers, which represent an important prey resource, were substantially reduced in knapweed versus native habitats. Our results strongly suggest that knapweed invasion can impact chipping sparrow populations by reducing food availability. Food chain effects may be an important mechanism by which strong plant invaders impact songbirds and other consumers.     

Aggregated seed dispersal by wreathed hornbills at a roost site in a moist evergreen forest of Thailand

Hornbills (Bucerotidae) are widely regarded as important seed dispersers in tropical forests in Africa and Asia. We investigated how the roosting behavior of wreathed hornbills (Aceros undulatus) influences seed deposition and seedling survival at a roost site in a moist evergreen forest of Khao Yai National Park, Thailand. Fallen fruits and seeds were collected in traps that were placed around a roosting site for 14 months, and seedlings were monitored in adjacent quadrats for 3 years. Seedfall and seedlings of species represented in the hornbill diet occurred at significantly higher densities in the traps and quadrats located beneath the crown of the roosting tree than in those located beyond the crown. With the exception of Cinnamomum subavenium, the seeds and seedlings of most diet species rarely survived beyond the first year. The quality of hornbill dispersal to this roosting site may be poor due to the highly concentrated seedfall, which results in high seed and seedling mortality. However, the number of seeds deposited by each hornbill each day at roosting sites is relatively low. Wreathed hornbills are primarily scatter dispersers during the day and probably serve as agents of seed dispersal in the moist evergreen forest of Khao Yai.     

A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

Summary Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 × g particulate fraction. Binding of [8-¹⁴C]-benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at pH 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (K_d) of 33 ± 6 nM. The number of binding sites found was 1,100 ± 120 fmol g^–1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [9 R] Z, [9 R] A.     

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Subcellular site of the biosynthesis ofO-acetylated sialic acids in bovine submandibular gland

Bovine submandibular glands were homogenized and fractionated under conditions which yielded subcellular fragments from mainly one cell type, the mucous acinar cell, as judged by morphological analysis of the glands before and after homogenization. The majorN-acetylneuraminate-9(7)-O-acetyltransferase activity was detected in the cytosolic fraction, a result supported by the high specific radioactivity of free sialic acids isolated after [¹⁴C]acetate-labelling experiments. Separation of membranes on a Ficoll density gradient gave six fractions which were analyzed biochemically and morphologically. The particulate activities of acetyltransferase and sialyltransferase were found in fractions containing smooth and mitochondrial membranes. MembraneO-acetyl sialic acids were present at the highest levels in these fractions and also had the highest specific radioactivity after [¹⁴C]acetate-labelling experiments. Significant amounts of theO-acetyltransferase activity also occur in the cytosol and are consistent with a model ofO-acetyl sialic acid biosynthesis involving both cytosolic and smooth membrane sites ofO-acetylation.     

Social influences on grooming site preferences among captive long-tailed macaques

Data on grooming in a colony of 38 captive Macaca fascicularis were collected over a period of 6 months. The goal was to investigate how five social parameters (age, kinship, sex, grooming frequency, and relative rank) influenced the choice of body part being groomed. Age and kinship did not have systematic effects of grooming site preferences. The sex composition of individual dyads and the frequency at which grooming occurred were the factors with the greatest effect on body sites groomed among adults. Relative to other dyad types, male-male dyads almost never groomed on the face, avoided the front side of the trunk, and preferred the tail and back. Dyads that groomed relatively infrequently also favored the tail and avoided the face, chest, and belly. Relative rank had an effect on the body sites groomed among adult females: a groomer ranking lower than her partner groomed more often on the face, chest, and belly than a groomer ranking higher than her partner. Several hypotheses are discussed in the context of these results. The only one that explains most of the major results is that recipients of grooming expose relatively invulnerable parts of their bodies (i.e., back and tail) to and avoid eye contact with groomers that are relatively dangerous.     

Arbuscular mycorrhizas on Athyrium yokoscense and A. niponicum grown at a lead-contaminated site

To investigate the occurrence of arbuscular mycorrhizas (AM) and their morphological types, four Athyrium yokoscense and five A. niponicum individuals were collected at a lead-contaminated site. Three of the A. yokoscense and four of the A. niponicum formed AM structures. However, the percentage of AM formation within both species differed significantly among individuals. Morphological types of most AM structures were the Paris-type, except for one A. yokoscense that formed both Paris-type and Arum-type-like structures in different parts of the same root section. These results demonstrate that the two Athyrium species form AM associations in lead-contaminated soil.     

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain

We have previously found that a peptide corresponding to residues 35–47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras–p21 peptide corresponding to residues 96–110 that blocks ras–jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35–47, but not the 96–110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35–47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras–p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35–47 and 96–110, act at two different critical downstream sites, the former at raf, the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.     

Chloroplast DNA restriction site mapping and the phylogeny ofRanunculus (Ranunculaceae)

A chloroplast DNA restriction site map forRanunculus sceleratus (Ranunculaceae) was constructed using 14 restriction endonucleases. The total size of the chloroplast genome is 152.4kb. No inversions were detected relative to the tobacco chloroplast DNA. Cladistic analyses of chloroplast DNA restriction site polymorphism were employed in order to elucidate the phylogeny among 76 species of the genusRanunculus in a wide sense and one species ofTrautvetteria. A total of 341 informative restriction site changes were detected. Parsimony jackknifing, bootstrapping and decay analysis were undertaken in order to evaluate the amount of support for the monophyletic groups. The results suggest that the analysed species ofRanunculus are divisible into two main clades. Only few of the traditional sections and subgenera ofRanunculus are monophyletic. The genusTrautvetteria is nested within a clade comprising, e.g.Ranunculus cymbalaria, R. andersonii, R. lapponicus andR. ficaria. SubgenusBatrachium lies within a larger clade containing, e.g.R. sceleratus andR. hyperboreus. Contractions of the inverted repeat due to parallel deletions of 200–300 bp close to the J_SB have occurred in many clades and the phylogenetic distribution of this size reduction was mapped among the species.     

Nest site selection by a neotropical swarm-founding wasp: seasonal alternation of nest orientation

Nest orientation in social insects has been intensively studied in warmer and cooler climates, particularly in the northern hemisphere. Previous studies have consistently shown that species subjected to these climatic conditions prefer to select mostly southern locations where the nests can gain direct sunlight. However, very little is known on nest orientation in tropical and subtropical social insects. We studied nest orientations initiated by swarms throughout a year in a Brazilian swarm-founding wasp, Polybia paulista von Ihering (Hymenoptera: Polistinae). Swarms selected various orientations as nest sites, but there was a particular trend in that swarms in the winter period (May–August) preferred to build northward-facing nests. This preference is opposite from that of social wasps observed in the northern hemisphere. Colonies of this species can potentially last for many years with continuous nesting, but nesting activities of colonies during the winter are severely limited due to cool temperature and a shortened day length. Northward-facing nests are warmer through the gain of direct solar heat during the winter period; consequently, choosing northward-facing sites may be advantageous for swarms in terms of a shortened brood development and shortened time needed to increase metabolic rates during warm-up for flight.     

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K_i 49 nM) compared to hDHFR (K_i 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K_i values of 593 and 617 nM, respectively; these K_i values are well above both the K_i for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.     

Contribution of the [Fe<Superscript>II</Superscript>(SCys)<Subscript>4</Subscript>] site to the thermostability of rubredoxins

The thermostabilities of Fe²⁺ ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH...S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)₄] site, and substitutions at this location affect the redox properties of the [Fe(SCys)₄] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V exchange mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)₄] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn²⁺, Cd²⁺) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and ¹H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [Fe^II(SCys)₄] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [Fe^II(SCys)₄] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)₄] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H⁺, Cd²⁺) on the Cys42 sulfur ligand at the [Fe(SCys)₄] site occurs through the V8 side and not the V44 side of the iron site.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0525-4Abbreviations BPS bathophenanthroline sulfonate, sodium salt - Cp Rd (Pf Rd, Dv Rd) recombinant rubredoxin from Clostridium pasteurianum (Pyrococcus furiosus, Desulfovibrio vulgaris) - HEPES hydroxyethylpiperazineethanesulfonic acid - MES morpholinoethanesulfonic acid - Tris tris(hydroxymethyl)aminomethane - wt wild-type - ZnRd recombinant rubredoxin containing a [Zn(SCys)₄] site