Molecular phylogeny of two lineages of Leuciscinae cyprinids (Telestes and Scardinius) from the peri-Mediterranean area based on cytochrome b data

We examined phylogenetic relationships in two lineages of Leuciscinae cyprinid fishes based on the sequence data of the complete mitochondrial DNA region coding for the cytochrome b gene (1140 bp). Telestes includes obligate riverine, moderately cold water-adapted species whereas Scardinius comprises warm-adapted species living in lowland lakes and still waters of rivers and streams. We also analysed selected representatives of Leuciscus and Phoxinellus because the taxonomic status of some species belonging to these genera is dubious and they could be placed in the genus Telestes. The study includes 18 species, 43 populations, and 111 individuals from 9 of the 14 peri-Mediterranean ichthyogeographic districts. Clades recovered from the phylogenetic analyses do not support previous taxonomic assumptions based on morphology. Telestes, Leuciscus, and Phoxinellus do not form monophyletic assemblages; phylogenetic analyses suggest that L. polylepis, L. turskyi, P. croaticus, and P. metohiensis should be included in Telestes. Similarly, populations of Scardinius erythrophthalmus do not cluster together and the endangered S. scardafa, endemic to central Italy and surviving in a single locality, is nested within them. The radiations of Telestes and Scardinius occurred in different time periods. A major diversification of Telestes is consistent with a sea dispersal during the freshwater Messinian "Lago Mare" phase of the Mediterranean Sea. Cladogenetic events within Scardinius are likely related to the extension and confluence of river drainages in lowlands following multiple lowering of the sea level during the Quaternary glaciations.     

Low-temperature micronization of a peptide drug in fluid propellant: Case study cetrorelix

Aim of this study was to elaborate an efficient method for the micronization of the decapeptide cetrorelix (a GnRH-antagonist), in order to obtain a microsuspension as basis for other pharmaceutical preparations, such as e.g. inhalation aerosols. A modified pearl-mill coupled with a cryostat was used for the micronization of cetrorelix in fluid propellant and operated under different conditions. The obtained cetrorelix suspensions were analyzed for particle size distribution, purity of cetrorelix, and for metal contamination through abrasion from parts of the mill. The method allowed an effective micronization of cetrorelix. The mean particle size of the initial cetrorelix lyophilizate bulk ware was reduced from 52.5 μ (Volume Mean Diameter, VMD) down to 14.9, 6.1 and 3.1 μm, respectively, respectively. The HPLC analysis of all cetrorelix suspensions after micronization did not show signs of decomposition as compared to the initial product. The elementary analysis of the suspensions performed by inductively coupled plasma mass spectrometry revealed a negligible amount of contaminants in the suspension (Zr=max. 0.6 ppm; Fe, Cr, Ni, Ba, below limit of quantification, i.e.<0.14 ppm). The only appreciable contaminant. Aluminum (Al=1.1 ppm), was derived from the mechanical capping of aluminum canisters prior to analysis. The Zr determination in the suspension of 0.6 ppm, is still considered to be negligible as compared to the legally tolerated limit of air contamination. By low-temperature micronization in fluid propellant, fine drug suspensions of cetrorelix for pMDIs can be directly manufactured in one-step procedure without destruction of the peptide structure and without appreciable product contamination. Published: July 12, 2001.     

Chemoenzymatic synthesis of peptidyl 3,4-dihydroxyphenylalanine for structure-activity relationships in marine invertebrate polypeptides

An improved method for hydroxylating tyrosine-containing sequences in polypeptides to peptidyl 3,4-dihydroxyphenylalanine (DOPA) using mushroom tyrosinase at relatively high enzyme-to-substrate ratios is described. The new method involves incorporating borate into the reaction mixture to stop formation of the unwanted side product 3,4,5-trihydroxyphenylalanine. Using this method, a model for the palindromic central sequence for the antimicrobial peptide family, the styelins, Y*Y*KHKY*Y* (where Y* is DOPA), was successfully synthesized in high yield from YYKHKYY. This sequence represents a particularly challenging target because of the cluster of four precursor tyrosine residues are in close proximity. The method should be readily applied to larger polypeptides produced by either solid-phase synthesis or recombinant techniques and give greater insight into the roles of this unusual posttranslational modification in marine invertebrates such as mussels and ascidians.     

A partial skeleton of Prodeinotherium bavaricum (Proboscidea, Mammalia) from the Middle Miocene of Unterzolling (Upper Freshwater Molasse, Germany)

Here, a partial skeleton of Prodeinotherium bavaricum from Unterzolling (Southern Germany) is documented. The following elements are preserved and described for the first time: cervical vertebrae 1-2 and 5-7, the first thoracic vertebra, one lumbar vertebra, trapezium, metacarpals 1-5, tibia, calcaneus, endo- and mesocuneiform, cuboid, the fourth metatarsal, and some phalanges. Comparisons with the skeletons of P. bavaricum from Franzensbad (Czech Republic) and Deinotherium giganteum from Eserovo (Bulgaria) show osteological differences that are described and discussed.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Myxobolus desaequalis n. sp. (Myxozoa,Myxosporea), parasite of the Amazonian freshwater fish,Apteronotus albifrons (Teleostei,Apteronotidae)

Myxobolus desaequalis n. sp. is described from the gill lamellae of the freshwater fish Apteronotus albifrons, collected in the Amazon River, near the city of Salvaterra, Brazil. Large spherical plasmodia filled with disporic pansporoblasts and spores were observed. Ellipsoidal to pyriform spores are 18.3 microm length x 11.2 microm width x 4.4 microm thickness. The anterior end of the spores contain two extremely unequal pyriform polar capsules measuring: (larger): 11.2 microm length, 4.9 microm width, and an isofilar polar filament with 11 to 12 turns obliquely to the longitudinal axis; (smaller): 4.6 microm length, 2.8 microm width, and an isofilar polar filament with 4 to 5 turns, obliquely to the longitudinal axis.     

Acidification of freshwaters by red soil in a subtropical silicate rock area,Okinawa, Japan

Two samples of red soil, one from Gushikawa Recreation Center (GRC) and one from Okinawa Royal Golf Club (ORGC), were examined for particle size distribution, textures, minerals, and chemical compositions. The effects of particle size and grinding of clay minerals on pH, electrical conductivity (EC), and dissolved chemical species were studied in deionized water and river water. The results of red soil solutions were compared with those of acidic waters found in red soil dominated areas. The minimum pH values of soil solutions extracted by deionized water were 4.38–5.36 and 5.16–5.89 and the maximum values of EC were 4.91–16.98mSm^–1 and 3.54–11.23mSm^–1 for GRC and ORGC, respectively. In the river water samples equilibrated with red soils, the minimum pH values were 4.48–5.10 and 4.77–5.91 and the maximum EC values were 19.6–34.2mSm^–1 and 17.5–25.0mSm^–1 for GRC and ORGC, respectively. The values of pH and EC varied with the soil–solution ratio and the particle size. The chemical composition of river water without mixing with red soil shows Na⁺K⁺ and Ca²⁺Mg²⁺. After mixing with red soil, the trend of the concentrations changed to Na⁺K⁺ and Mg²⁺Ca²⁺, which is the same as that of soil solutions in deionized water as well as that of acidic waters found in the red soil area. The pH of the acidic waters was 4.95–5.81 and EC was 7.76–30.0mSm^–1. Laboratory experimental results agreed well with those found in the field in terms of trend of concentrations of the chemical species and pH. Therefore, the results of this study suggest that the low pH and trend of the concentrations of chemical species of the acidic waters found in the red soil dominated areas were the result of the interaction of natural water and red soil.     

Reduced byssal thread production and movement by the intertidal mussel<Emphasis Type="Italic"> Hormomya mutabilis</Emphasis> in response to effluent from predators

Laboratory experiments were carried out to investigate byssal thread production by the intertidal mytilid mussel Hormomya mutabilis in response to effluent from the predatory crab Eriphia smithii and the starfish Coscinasterias acutispina. During the early period of the experiment, large H. mutabilis exposed to crab effluent produced a significantly smaller number of functional byssal threads than mussels in crab-free water. No significant difference in the diameter of threads produced in the two treatments was detected. The number of functional byssal threads produced by small H. mutabilis exposed to crab effluent did not differ significantly from that of mussels in crab-free water. However, small H. mutabilis exposed to crab effluent tended to discard fewer byssal bundles, that is, they shifted their attaching sites less frequently than similar mussels in crab-free water. In the presence of waterborne cues from the crab, H. mutabilis tended to reduce both the secretion of byssal threads and movement across the substratum. No significant differences in behaviour were observed between large mussels exposed to effluent from the starfish and those unexposed. The adaptive significance of the responses shown by H. mutabilis is discussed in terms of protection against predators differing in foraging behaviour. Electronic Publication     

Freshwater ecology: changes, requirements, and future demands

The past development and evolution of limnology as a discipline has demonstrated that experimentally controlled disturbances of parts of aquatic ecosystems are essential for quantitative evaluation of causal mechanisms governing their operation. Correlative analyses and modeling only establish hypotheses, not causality, and allow only therapeutic management applications. Rather than constantly searching for differences, commonality must be sought. Among the large diversity of species, communities, and biogeochemical processes controlling growth and reproduction, commonality emerges at the levels of regulation of metabolism. Five areas of current and future limnological research are discussed in relation to greatest needs and promise to yield insights into material and energy flows in freshwater ecosystems and their effective management: (1) coupled metabolic mutualism in the physiological ecology of microbes (viruses, bacteria, fungi, and protists) and their biogeochemical, especially organic, couplings with the environment; (2) biochemical regulation of collective metabolism, recycling, and bioavailability of nutrients and growth regulators; (3) application of genetic and molecular techniques to addressing biogeochemical, evolutionary, and pollution remediation problems; (4) recognition that the metabolism within lakes and streams is dependent upon and regulated to a major extent by organic matter of the drainage basin and especially by the land-water interface biogeochemistry; and (5) recognition that food-web alterations ("biomanipulation") are short-term, expensive therapeutic tools that may minimize effects of eutrophication but will not solve or control eutrophication. Received: October 30, 1999 / Accepted: December 6, 1999     

Identification and characterization of ecologically significant prokaryotes in the sediment of freshwater lakes: molecular and cultivation studies

The aim of this review is to interpret recent studies in which molecular methods were used to identify and characterize prokaryotes in lake sediments and related habitats. In the first part studies based on the phylogenetic diversity of prokaryotes found in lacustrine habitats are summarized. The application of various cultivation-independent methods for the characterization of distinct groups of sediment bacteria is exemplified with morphologically conspicuous, colorless sulfur bacteria in the second part of this review. Finally, traditional and recently developed methods are described which could be used for linking the function of microbial populations with their identification. The potential of these approaches for the study of lake sediments is discussed in order to give a perspective for future studies in this habitat.