Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.     

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood‐ and mosquito‐stage development

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species.     

Primary structure of carboxypeptidase T: Delineation of functionally relevant features in Zn-carboxypeptidase family

The primary structure of carobxypeptidase T—a Zn-dependent extracellular enzyme ofThermoactinomyces vulgaris—was determined from the clonedcpT gene nucleotide sequence and compared to Zn-carboxypeptidases from various organisms. The compilation and analysis of multiple alignment accompanied by consideration of available tertiary structure data have shown that in the overall spatial structure and active site arrangement CpT is similar to other enzymes constituting the Zn-carboxypeptidase family. Nine of 16 amino acid residues found to be strictly invariant are presumably located close to the active site. The preservation of His69, Glu72, Asn144, Arg145, His196, Tyr248, and Glu270 identified previously as essential catalytic site participants implicates basically the same catalytic mechanism in the Zn-carboxy-peptidase family. It is proposed that Pro205 and Asp256 should play an important role in proper S₁-pocket spatial arrangement. The comparative analysis of amino acid variations in S₁-pocket enabled us to reveal structural determinants of the Zn-carboxypeptidase primary specificity. The relatively reduced size of the pocket and negative charge of Asp253 are supposed to contribute correspondingly to A- and B-type substrate preferences of carboxypeptidase T endowed with dual primary specificity.     

It's cheap to be colorful. Anthozoans show a slow turnover of GFP-like proteins

Pigments homologous to the green fluorescent protein (GFP) contribute up to approximately 14% of the soluble protein content of many anthozoans. Maintenance of such high tissue levels poses a severe energetic penalty to the animals if protein turnover is fast. To address this as yet unexplored issue, we established that the irreversible green-to-red conversion of the GFP-like pigments from the reef corals Montastrea cavernosa (mcavRFP) and Lobophyllia hemprichii (EosFP) is driven by violet-blue radiation in vivo and in situ. In the absence of photoconverting light, we subsequently tracked degradation of the red-converted forms of the two proteins in coral tissue using in vivo spectroscopy and immunochemical detection of the post-translational peptide backbone modification. The pigments displayed surprisingly slow decay rates, characterized by half-lives of approximately 20 days. The slow turnover of GFP-like proteins implies that the associated energetic costs for being colorful are comparatively low. Moreover, high in vivo stability makes GFP-like proteins suitable for functions requiring high pigment concentrations, such as photoprotection.     

Contributions of host and symbiont pigments to the coloration of reef corals

For a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange-emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein-like proteins that change their color from green to red upon irradiation with approximately 400 nm light. The optical absorption and emission properties of these proteins were characterized in detail. Their spectra were found to be similar to those of phycoerythrin from cyanobacterial symbionts. To unambiguously determine the molecular origin of the coloration, we performed immunochemical studies using double diffusion in gel analysis on tissue extracts, including also a third coral species, Montastrea cavernosa, which allowed us to attribute the red fluorescent coloration to green-to-red photoconvertible fluorescent proteins. The red fluorescent proteins are localized mainly in the ectodermal tissue and contribute up to 7.0% of the total soluble cellular proteins in these species. Distinct spatial distributions of green and cyan fluorescent proteins were observed for the tissues of M. cavernosa. This observation may suggest that differently colored green fluorescent protein-like proteins have different, specific functions. In addition to green fluorescent protein-like proteins, the pigments of zooxanthellae have a strong effect on the visual appearance of the latter species.     

In vivo assessment of mechanical properties during axolotl development and regeneration using confocal Brillouin microscopy

In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behaviour are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such an increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.     

Construction of a high-resolution genetic linkage map and comparative genome analysis for the reef-building coral Acropora millepora

Background  

Worldwide, coral reefs are in decline due to a range of anthropogenic disturbances, and are now also under threat from global climate change. Virtually nothing is currently known about the genetic factors that might determine whether corals adapt to the changing climate or continue to decline. Quantitative genetics studies aiming to identify the adaptively important genomic loci will require a high-resolution genetic linkage map. The phylogenetic position of corals also suggests important applications for a coral genetic map in studies of ancestral metazoan genome architecture.     

Urinary steroid profile after the completion of concentric and concentric/eccentric trials with the same total workload

High intensity strength training causes changes in steroid hormone concentrations. This could be altered by the muscular contraction type: eccentric or concentric. The aim of this study was to compare the effect of the completion of a short concentric (CON) and concentric/eccentric (CON/ECC) trial on the urinary steroid profile, both with the same total work. 18 males performed the trials on an isokinetic dynamometer (BIODEX III) exercising quadriceps muscles, right and left, on different days. Trial 1(CON): 4×10 Concentric knee extension + relax knee flexion, speed 60°/second; rest 90 seconds between each series and 4 minutes between each leg exercise. Trial 2(CON/ECC): 4×5 concentric knee extension + Eccentric knee flexion under similar conditions. Urine samples were taken before the exercise and one hour after finishing it. Androsterone, Etiocholanolone, DHEA, Androstenedione, Testosterone, Epitestosterone, Dehydrotestosterone, Estrone, B-Estradiol, Tetrahydrocortisone, Tetrahydrocortisol, Cortisone and Cortisol (free, glucoconjugated and sulfoconjugated) urinary values were determined using gas chromatography/mass spectrometry techniques. No significant differences were noted in Total Work and Average Peak Torque, although Maximum Peak Torque in the CON/ECC trial was higher than in the CON trial. These results demonstrate no changes in the steroid profile before and after trials, or when comparing CON to CON/ECC trials. The data suggest that eccentric contractions do not cause hormonal changes different to the ones produced by concentric contractions, when they are performed in strength short trials with the same total workload.     

High Motility Reduces Grazing Mortality of Planktonic Bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 μm s⁻¹ as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 μm s⁻¹) and a highly motile strain (>45 μm s⁻¹) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 μm³. Grazing mortality was lowest for cells of >0.5 μm³ and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (≤0.1 μm³, >50 μm s⁻¹) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.