The Aspergillus fumigatus Dihydroxyacid Dehydratase Ilv3A/IlvC Is Required for Full Virulence

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.     

In Vivo Availability of Pro-Resolving Lipid Mediators in Oxazolone Induced Dermal Inflammation in the Mouse

The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA₄ on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B₄ and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA₄ (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B₄ was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA₄. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.     

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.     

Mutations in algal and cyanobacterial Photosystem I that independently affect the yield of initial charge separation in the two electron transfer cofactor branches

In Photosystem I, light-induced electron transfer can occur in either of two symmetry-related branches of cofactors, each of which is composed of a pair of chlorophylls (ec2_A/ec3_A or ec2_B/ec3_B) and a phylloquinone (PhQ_A or PhQ_B). The axial ligand to the central Mg^2 + of the ec2_A and ec2_B chlorophylls is a water molecule that is also H-bonded to a nearby Asn residue. Here, we investigate the importance of this interaction for charge separation by converting each of the Asn residues to a Leu in the green alga, Chlamydomonas reinhardtii, and the cyanobacterium, Synechocystis sp. PCC6803, and studying the energy and electron transfer using time-resolved optical and EPR spectroscopy. Nanosecond transient absorbance measurements of the PhQ to F_X electron transfer show that in both species, the PsaA-N604L mutation (near ec2_B) results in a ~ 50% reduction in the amount of electron transfer in the B-branch, while the PsaB-N591L mutation (near ec2_A) results in a ~ 70% reduction in the amount of electron transfer in the A-branch. A diminished quantum yield of P₇₀₀⁺ PhQ^? is also observed in ultrafast optical experiments, but the lower yield does not appear to be a consequence of charge recombination in the nanosecond or microsecond timescales. The most significant finding is that the yield of electron transfer in the unaffected branch did not increase to compensate for the lower yield in the affected branch. Hence, each branch of the reaction center appears to operate independently of the other in carrying out light-induced charge separation.     

Mitotic Centromere–associated Kinesin Is Important for Anaphase Chromosome Segregation

Mitotic centromere–associated kinesin (MCAK) is recruited to the centromere at prophase and remains centromere associated until after telophase. MCAK is a homodimer that is encoded by a single gene and has no associated subunits. A motorless version of MCAK that binds centromeres but not microtubules disrupts chromosome segregation during anaphase. Antisense-induced depletion of MCAK results in the same defect. MCAK overexpression induces centromere-independent bundling and eventual loss of spindle microtubule polymer suggesting that centromere-associated bundling and/or depolymerization activity is required for anaphase. Live cell imaging indicates that MCAK may be required to coordinate the onset of sister centromere separation.     

Habitat selection by ice-associated pinnipeds near St. Lawrence Island,Alaska in March 2001

Aerial surveys of ice-associated pinnipeds were conducted south of St. Lawrence Island in March 2001. The observed distributions of bearded seals (Erignathus barbatus), ribbon seals (Phoca fasciata), ringed seals (P. hispida), spotted seals (P. largha), and walruses (Odobenus rosmarus) were compared to the distributions of ice habitat types and benthic communities. Randomization tests were used to investigate habitat selection for each species. Both ringed seals and walruses preferred large ice floes (>48 m in diameter) that were common in the interior ice pack. Spotted seals favored smaller ice floes (<20 m in diameter) common near the ice edge, and bearded seals avoided large floes and preferred transitional habitat between small and large floes. Ringed seals also seemed to prefer areas with greater than 90% sea ice coverage, and bearded seals preferred 70–90% sea ice coverage while avoiding areas with greater than 90% coverage. All species, except spotted seals, were seen most frequently in a region of high benthic biomass, and randomization tests suggested that bearded seals actively selected that region.     

Cuticular wax deposition in growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaves commences in relation to the point of emergence of epidermal cells from the sheaths of older leaves

In grasses, leaf cells divide and expand within the sheaths of older leaves, where the micro-environment differs from the open atmosphere. By the time epidermal cells are displaced into the atmosphere, they must have a functional cuticle to minimize uncontrolled water loss. In the present study, gas chromatography and scanning electron microscopy were used to follow cuticular wax deposition along the growing leaf three of barley (Hordeum vulgare L.). 1-Hexacosanol (C₂₆ alcohol) comprised more than 75% of extractable cuticular wax and was used as a marker for wax deposition. There was no detectable wax along the first 20 mm from the point of leaf insertion. Deposition started within the distal portion of the elongation zone (23–45 mm) and continued beyond the point of leaf emergence from the sheath of leaf two. The region where wax deposition commenced shifted towards more proximal (basal) positions when the point of leaf emergence was lowered by stripping back part of the sheath. When relative humidity in the shoot environment was elevated from 70% (standard growth conditions) to 92–96% for up to 4 days prior to analysis, wax deposition did not change significantly. The results show that cuticular waxes are deposited along the growing grass leaf independent of cell age or developmental stage. Instead, the reference point for wax deposition appears to be the point of emergence of cells into the atmosphere. The possibility of changes in relative humidity between enclosed and emerged leaf regions triggering wax deposition is discussed.     

Geographical variation in the breeding biology of the Purple Sandpiper Calidris maritima

Studies in Iceland (66°N) and Svalbard (78°N), combined with the results of previous work, allowed geographical comparisons of different aspects of the breeding biology of the Purple Sandpiper Calidris maritima , testing predictions that they may be related to latitude and altitude (here taken as surrogates for climatic severity). The breeding density was lowest in the polar deserts of Franz Josef Land (82°N) and highest in coastal Arctic Tern Sterna paradisaea colonies in Iceland where the terns perhaps afforded protection from predators. There was no difference in the percentages of 1-year-old (first-year) birds in the breeding populations in Svalbard and Iceland. Time of breeding was related to both latitude and altitude, being later at higher latitudes and altitudes. In Iceland, larger males and males over 1 year old nested earlier than small males and 1-year-old males, respectively. Relaying occurred in Iceland but it was not observed in Svalbard. There were no geographical differences in clutch size. Egg size was related to female size in Svalbard. There were also geographical differences in egg size associated with female size (wing length), with the largest eggs in Iceland and smallest in south Norway. Males usually attended broods and the rates at which females deserted their broods were similar in Iceland and Svalbard. Chick growth rates were similar for Iceland and Franz Josef Land. Thus, in breeding biology, Purple Sandpipers varied geographically only in breeding density, time of nesting and egg size, which was associated with female size.