Phenanthrene degradation by estuarine surface microlayer and bulk water microbial populations

Paired surface microlayer and bulk water samples from five sites in the Great Bay Estuary, New Hampshire, were examined with regard to numbers of bacteria,¹⁴C-phenanthrene biodegradation potentials, and organic and inorganic chemical characteristics. Microlayer samples were generally enriched in nutrients (N and P), dissolved organic matter, and culturable heterotrophic bacteria compared with their corresponding bulk waters. Microlayer samples from marina environments were also enriched in aromatic hydrocarbons, as determined by UV spectrophotometric and fluorometric analyses, and demonstrated substantial phenanthrene biodegradation activity in the assay employed. Biodegradation activity of marina bulk water samples ranged from nil to levels exceeding those exhibited by microlayer samples. No diminution of biodegradation activity was observed after filtration (1.2 m effective retention) of microlayer water, indicating that the responsible organisms were not particle-associated. Phenanthrene-degrading bacteria, enumerated by counting clearing zones in a crystalline phenanthrene overlay after colony development on a phenanthrene/toluene agar (PTA) medium, were superior to epifluorescence direct counts or standard plate counts on PTA or estuarine nutrient agar in predicting¹⁴C-phenanthrene biodegradative activity.     

The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex

The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3. Transformation of the Saccharomyces cerevisiae strain D273-10B/A/U produced a mutant unable to grow on glycerol medium. The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected. ATPase was rendered oligomycin-insensitive and the F1 sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of F0 revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0. It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex. Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes. Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type. In addition, a high percentage of spontaneous rho- mutants was detected.     

Expression of class I genes in the major histocompatibility complex: identification of eight distinct mRNAs in DBA/2 mouse liver

The mouse H-2 multigene family includes the genes coding for the major transplantation antigens and for genes located in the Qa-TIa region. We have studied a collection of class I cDNA clones made from liver mRNA of DBA/2 mice (H-2d haplotype) and found that at least six distinct class I genes are transcribed, including three genes of the Qa-TIa region. Two of these six genes each yield two distinct mRNAs, resulting from alternate splicing. Altogether, liver cells may express at least eight distinct class I polypeptides, of which three might be secreted, while one may be a new presumptive nonpolymorphic surface antigen.     

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

Understorey vegetation moderates climate in open forests: The role of the skirt-forming grass tree Xanthorrhoea semiplana F.Muell

Microsites are created by abiotic and biotic features of the landscape and may provide essential habitats for the persistence of biota. Forest canopies and understorey plants may moderate wind and solar radiation to create microclimatic conditions that differ considerably from regional climates. Skirt-forming plants, where senescent leaves create hut-like cavities around the stem, create microsites that are sheltered from ambient conditions and extreme weather events, constituting potential refuges for wildlife. We investigate day and night temperatures and humidity for four locations (grass tree cavities, soil, 20 cm above-ground, 1 m above-ground) in a South Australian forest with relatively open canopy of stringybark eucalypts (Eucalyptus baxteri, E. obliqua) and an understorey of skirt-forming grass trees (Xanthorrhoea semiplana) at 5, 10, 20, and 40 m from the forest edge. We also measured the percentage of canopy and understorey covers. Generally, temperature and humidity differed significantly between more sheltered (grass tree cavities, soil) and open-air microsites, with the former being cooler during the day and warmer and more humid during the night. Furthermore, our results suggest that canopy cover tends to decrease, and understorey cover tends to increase, the temperature of microsites. Distance to the edge was not significantly related to temperature for any microsite, suggesting that the edge effect did not extend beyond 10 m from the edge. Overall, grass trees influenced microclimatic conditions by forming a dense understorey and providing cavities that are relatively insulated. The capacity of grass tree cavities to buffer external conditions increased linearly with ambient temperatures, by 0.46°C per degree increase in maximum and 0.25°C per degree decrease in minimum temperatures, potentially offsetting climate warming and enabling persistence of fauna within their thermal limits. These climate moderation properties will make grass trees increasingly important refuges as extreme weather events become more common under anthropogenic climate change.     

The distribution of coastal fish eDNA sequences in the Anthropocene

Aim

Coastal fishes have a fundamental role in marine ecosystem functioning and contributions to people, but face increasing threats due to climate change, habitat degradation and overexploitation. The extent to which human pressures are impacting coastal fish biodiversity in comparison with geographic and environmental factors at large spatial scale is still under scrutiny. Here, we took advantage of environmental DNA (eDNA) metabarcoding to investigate the relationship between fish biodiversity, including taxonomic and genetic components, and environmental but also socio-economic factors.

Location

Tropical, temperate and polar coastal areas.

Time period

Present day.

Major taxa studied

Marine fishes.

Methods

We analysed fish eDNA in 263 stations (samples) in 68 sites distributed across polar, temperate and tropical regions. We modelled the effect of environmental, geographic and socio-economic factors on α- and β-diversity. We then computed the partial effect of each factor on several fish biodiversity components using taxonomic molecular units (MOTU) and genetic sequences. We also investigated the relationship between fish genetic α- and β-diversity measured from our barcodes, and phylogenetic but also functional diversity.

Results

We show that fish eDNA MOTU and sequence α- and β-diversity have the strongest correlation with environmental factors on coastal ecosystems worldwide. However, our models also reveal a negative correlation between biodiversity and human dependence on marine ecosystems. In areas with high dependence, diversity of all fish, cryptobenthic fish and large fish MOTUs declined steeply. Finally, we show that a sequence diversity index, accounting for genetic distance between pairs of MOTUs, within and between communities, is a reliable proxy of phylogenetic and functional diversity.

Main conclusions

Together, our results demonstrate that short eDNA sequences can be used to assess climate and direct human impacts on marine biodiversity at large scale in the Anthropocene and can further be extended to investigate biodiversity in its phylogenetic and functional dimensions.     

Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans.     

Relationship between root volatiles of some carrot cultivars and their resistance to the carrot fly, Psila rosae

Field experiments investigated the resistance of some carrot cultivars to Psila rosae. In addition, headspace vapour and steam distillate from the roots of resistant and susceptible varieties were compared by gas-liquid chromatography. The field data confirmed that resistance may operate by decreasing the numbers of eggs laid indicating a nonpreference by the female Psila. Root resistance to the larva was also confirmed but the mechanism was unclear. A new finding was that root resistance is independent of the effect of egg laying, some cultivars evincing one or the other effect and some such as Regulus Imperial displaying both. It was clear that root resistance to the larva is the crucial prerequisite in breeding resistant varieties.One consistent difference was detected by the chemical comparisons: intact roots of resistant varieties released substantially less volatiles. Specifically, Regulus released almost five times less of the volatiles already shown to positively influence host-finding behaviour by the larva.

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Résumé La résistance à P. rosae de quelques cultivars de carotte a été étudiée en plein champ. Parallèlement, les substances volatiles diffusées et celles extraites par la vapeur des racines de variétés résistantes et sensibles, ont été comparées en chromatographie gaz-liquide (GLC). Les résultats en champ ont confirmé que la résistance peut être due à une diminution du nombre d'oeufs pondus, révélant une absence d'attractivité pour les femelles de P. rosae. La résistance des racines aux larves a été aussi confirmée, mais les raisons n'en étaient pas claires. Un aspect nouveau est que la résistance des racines est indépendante de l'effet de la ponte, quelques cultivars présentant l'un ou l'autreeffet et certains, comme Regulus Imperial, manifestant les deux. Il est net que la résistance racinaire aux larves est la condition essentielle pour la sélection de variétés résistantes. Une différence importante a été mise en évidence par les comparaisons chimiques: les racines intactes de variétés résistantes libèrent nettement moins de substances volatiles. Précisément, Regulus a libéré 5 fois moins de substances volatiles déjà connues comme influençant positivement le comportement de découverte de l'hôte par la larve.

     

A new method for thawing frozen ram semen

The purpose of this work was to facilitate the on-farm use of frozen semen by initially thawing the straws in laboratory treated sperm (TS) rather than on-farm control sperm (CS), as is usually done. After thawing, TS was diluted, centrifuged, and extended in skim milk for storage at +15° C until utilized 3 to 6 hours later. $\text{In}$$\text{vitro}$: immediately after preparation and addition of skim milk for TS and thawing for CS, the percentage of stained cells and abnormal cells was higher (P < 0.01) in TS than in CS. In contrast, following a 3 hour incubation, TS and CS had the same proportion of motile cells. $\text{In}$$\text{vivo}$: fertility and prolificacy of FGA + PMSG-treated ewes were slightly higher following AI (1 AI/female) with TS than with CS: 52.4% $\text{vs}$ 44.2% and 155.0% $\text{vs}$ 148.0%, respectively. Fertility was also higher (P < 0.01) with fresh semen than with TS, but the difference was only 9.2 points (70.3% $\text{vs}$ 61.1% for the respective 798 and 242 ewes inseminated once). Prolificacy rates were similar (164.3% $\text{vs}$ 167.6%).     

Binding of adducts of NAD(P) and enolizable ketones to NAD(P)-dependent dehydrogenases

Carbonyl compounds such as alpha-ketoglutarate, pyruvate, oxaloacetate, butyraldehyde, acetaldehyde or acetone react with NAD or NADP to give adducts. Binding studies of adducts to dehydrogenases are performed by means of ultraviolet differential spectroscopy, circular dichroism and spectrofluorimetry. The dehydrogenases show a high degree of binding specificity toward the adducts which contain their specific oxidized substrate and their specific coenzyme. The high selectivity of the dehydrogenases for adducts is evidenced by binding studies of NAD(P)-pyruvate and NAD(P)-alpha-ketoglutarate adducts on glutamate dehydrogenase at pH 7.6 and 8.9. Evidence is presented showing that adducts bind to the active site of the enzymes.