PHOTOSYNTHESIS AND PHOTOSYNTHESIS-COUPLED RESPIRATION IN NATURAL BIOFILMS QUANTIFIED WITH OXYGEN MICROSENSORS1

Photosynthesis and respiration were analyzed in natural biofilms by use of O₂ microsensors. Depth profiles of gross photosynthesis were obtained from the rate of decrease in O₂ concentration during the first few seconds following extinction of light, and net photosynthesis of the photic zone was calculated from O₂ concentration gradients measured at steady state. Respiration within the photic zone was calculated as the difference between gross and net photosynthesis. Two types of biofilms were investigated: one dominated by diatoms, and one dominated by cyanobacteria. High O₂/CO₂ ratios caused increased respiration especially within the diatom biofilm, which could indicate that photorespiration was a dominant O₂-consuming process. The rate of respiration was constant within both biofilms during the first 4.6 s following extinction of light, even when respiration was stimulated by high O₂/CO₂ ratio. The assumption of a constant rate of respiration during the dark period is an essential one for the determination of gross photosynthetic activity by use of O₂ microsensors. We here present the first evidence to substantiate this assumption. The results strongly suggest that gross photosynthesis as measured by use of O₂ microsensors may include carbon equivalents that are subsequently lost through photorespiration. Computer modeling of photosynthesis profiles measured after 1.1, 1.6, and 2.6 s of dark incubation illustrated how the actual photosynthesis profile could have appeared if it had been possible to do the determination at time 0. Diffusion of O₂ during the up to 4.6-s long dark incubations did not affect gross photosynthetic rate when integrated over all depths, but the apparent vertical distribution of the photosynthetic activity was strongly affected.     

Uniform selective pressures within redox zones drive gradual changes in microbial community composition in hadal sediments

Microbial communities in marine sediments are highly diverse, yet the processes that give rise to this complexity are unclear. It has been proposed that benthic microbial communities must be continuously re-seeded from the water column because dispersal within the sediment is severely limited. Previous studies consistently report that the composition of the microbial community gradually changes with sediment depth. However, the relative contributions of the processes that underlie these compositional gradients have not been determined, and it is unknown whether microbial dispersal is indeed too slow to outpace burial. Here, we applied ecological statistical frameworks to 16S rRNA gene amplicon-based community composition data from Atacama Trench sediments to investigate the links between biogeochemistry, burial, and microbial community assembly processes. We confirm that dispersal limitation affects microbial communities and find that gradual changes in community composition are driven by selective pressures that change abruptly across the discrete boundaries between redox zones rather than along continuous biogeochemical gradients, while selective pressures are uniform within each zone. The gradual changes in community composition over centimetres of depth within a zone hence reflects a decades-long response to the abruptly changing selective pressures.     

25-Hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes.

Using isotope dilution—mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D₃ when combined with NADPH-cytochrome P-450 reductase and a phospholipid. The rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D₃ metabolism.     

Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp

Various Naegleria strains were examined to determine the possible origin and significance of membrane-bound black bodies that were found in all exponentially growing cell populations. The bodies, 40–80 nm in diameter, were distributed randomly in the cytoplasm of Naegleria with ultrastructural features typical of trophozoites. No evidence was obtained that the contents of the black bodies were synthesized in the rough endoplasmic reticulum (ER) and packaged by membranous components, which could be a primitive “Golgi complex” in these amoebae. Examination of cells in various stages of encystment indicated that at least some of the cyst wall material was synthesized and packaged by the rough ER. After condensation into amorphous granules in the cisternae, the cyst wall material appeared in vesicles of the rough ER; these were frequently seen in close proximity to the cell membrane in the vicinity of developing cyst wall. Amorphous granules (～100 nm in diameter), which had variable densities and did not appear to be membrane bound, were seen in the cytoplasm of encysting cells. The substance of these granules also seemed to be incorporated into the cyst wall. The membrane-bound black bodies appeared to be destroyed in lysosomal elements during encystment. The membrane-bound black bodies were concluded to be characteristic of trophozoites and unrelated to encytment of Naegleria.     

Up-regulation of vascular endothelial growth factor (VEGF) in small-for-size liver grafts enhances macrophage activities through VEGF receptor 2-dependent pathway

This study aims to investigate the potential role of vascular endothelial growth factor (VEGF) and VEGF-R2 (fetal liver kinase (Flk)-1) in mediating macrophage activities in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed using either whole, 50, or 30% liver grafts (both 50 and 30% were regarded as small-for-size) in syngeneic or allogeneic combinations, respectively. Firstly, the mRNA and protein levels of VEGF and Flk-1 in liver grafts were detected by RT-PCR and Western blot, and the number of Flk-1(+) macrophages (labeled by ED1) was determined by flow cytometry. It was found that the small-for-size isografts and allografts presented higher levels of VEGF and Flk-1 expression than the whole isograft and allograft. In addition, a higher number of Flk-1(+)ED1(+) cells were detected in the small-for-size isografts and allografts than the whole isograft and allograft. Secondly, our study revealed that macrophage cell lines did not initially express detectable Flk-1, but could be induced by VEGF, and the inducible expression of Flk-1 in macrophages was related to their migration and proliferation activities. Finally, our study demonstrated that the induction of Flk-1 expression on macrophages by VEGF was associated with the expression of NF-kappaB and heat shock protein 90. In conclusion, the present study showed that the up-regulated expression of VEGF and its interaction with Flk-1 in small-for-size liver grafts might facilitate the activities of macrophages.     

Increased cyclin E expression may obviate the role of cyclin D1 during brain development in cyclin D1 knockout mice

Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild-type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild-type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.     

Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium

Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L(-1) selenomethionine, salts and trace metals, other amino acids including 10 mg L(-1) of methionine, vitamins except vitamin B12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take approximately 24 h. Culture growth in the auto-induction medium gave an average final optical density at 600 nm of approximately 6 and an average wet cell mass yield of approximately 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was approximately 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a approximately 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.     

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi

Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.     

Flocculation protein structure and cell–cell adhesion mechanism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cell–cell adhesion occurs in a broad spectrum of biological processes, of which yeast flocculation is an area of interest for evolutionary scientists to brewers and winemakers. The flocculation mechanism is based on a lectin-carbohydrate interaction but is not yet fully understood, although the first model dates back to the 1950s. This review will update the current understanding of the complex mechanism behind yeast flocculation. Moreover, modern technologies to measure the forces involved in single carbohydrate-lectin interactions, are discussed. The Flo1 protein has been extensively described as the protein responsible for strong flocculation. Recently, more research has been directed to the detailed analysis of this flocculin. Due to the advances in the field of bioinformatics, more information about Flo1p could be obtained via structurally or functionally related proteins. Here, we review the current knowledge of the Flo1 protein, with a strong emphasis towards its structure.