Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Solar‐induced chlorophyll fluorescence is strongly correlated with terrestrial photosynthesis for a wide variety of biomes: First global analysis based on OCO‐2 and flux tower observations

Solar‐induced chlorophyll fluorescence (SIF) has been increasingly used as a proxy for terrestrial gross primary productivity (GPP). Previous work mainly evaluated the relationship between satellite‐observed SIF and gridded GPP products both based on coarse spatial resolutions. Finer resolution SIF (1.3 km × 2.25 km) measured from the Orbiting Carbon Observatory‐2 (OCO‐2) provides the first opportunity to examine the SIF–GPP relationship at the ecosystem scale using flux tower GPP data. However, it remains unclear how strong the relationship is for each biome and whether a robust, universal relationship exists across a variety of biomes. Here we conducted the first global analysis of the relationship between OCO‐2 SIF and tower GPP for a total of 64 flux sites across the globe encompassing eight major biomes. OCO‐2 SIF showed strong correlations with tower GPP at both midday and daily timescales, with the strongest relationship observed for daily SIF at the 757 nm (R² = 0.72, p < 0.0001). Strong linear relationships between SIF and GPP were consistently found for all biomes (R² = 0.57–0.79, p < 0.0001) except evergreen broadleaf forests (R² = 0.16, p < 0.05) at the daily timescale. A higher slope was found for C₄ grasslands and croplands than for C₃ ecosystems. The generally consistent slope of the relationship among biomes suggests a nearly universal rather than biome‐specific SIF–GPP relationship, and this finding is an important distinction and simplification compared to previous results. SIF was mainly driven by absorbed photosynthetically active radiation and was also influenced by environmental stresses (temperature and water stresses) that determine photosynthetic light use efficiency. OCO‐2 SIF generally had a better performance for predicting GPP than satellite‐derived vegetation indices and a light use efficiency model. The universal SIF–GPP relationship can potentially lead to more accurate GPP estimates regionally or globally. Our findings revealed the remarkable ability of finer resolution SIF observations from OCO‐2 and other new or future missions (e.g., TROPOMI, FLEX) for estimating terrestrial photosynthesis across a wide variety of biomes and identified their potential and limitations for ecosystem functioning and carbon cycle studies.     

Involvement of the yeast metacaspase Yca1 in ubp10Delta-programmed cell death

UBP10 encodes a deubiquitinating enzyme of Saccharomyces cerevisiae. Its inactivation results in a complex phenotype characterized by a subpopulation of cells that exhibits the typical cellular markers of apoptosis. Here, we show that additional deletion of YCA1, coding for the yeast metacaspase, suppressed the ubp10 disruptant phenotype. Moreover, YCA1 overexpression, without any external stimulus, had a detrimental effect on growth and viability of ubp10 cells accompanied by an increase of apoptotic cells. This response was completely abrogated by ascorbic acid addition. We also observed that cells lacking UBP10 had an endogenous caspase activity, revealed by incubation in vivo with FITC-labeled VAD-fmk. All these results argue in favour of an involvement of the yeast metacaspase in the active cell death triggered by loss of UBP10 function.     

Glycation and post-translational processing of human interferon-gamma expressed in Escherichia coli

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-gamma during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140/Arg141, Phe137/Arg138, Met135/Leu136, and Lys131/Arg132) and two minor (Lys109/Ala110 and Arg90/Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-gamma originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-gamma, whereas covalent dimerization completely abolished it.     

Synthesis and anti-inflammatory activity of new arylidene-thiazolidine-2,4-diones as PPARγ ligands

Eight new 5-arylidene-3-benzyl-thiazolidine-2,4-diones with halide groups on their benzyl rings were synthesized and assayed in vivo to investigate their anti-inflammatory activities. These compounds showed considerable biological efficacy when compared to rosiglitazone, a potent and well-known agonist of PPARγ, which was used as a reference drug. This suggests that the substituted 5-arylidene and 3-benzylidene groups play important roles in the anti-inflammatory properties of this class of compounds. Docking studies with these compounds indicated that they exhibit specific interactions with key residues located in the site of the PPARγ structure, which corroborates the hypothesis that these molecules are potential ligands of PPARγ. In addition, competition binding assays showed that four of these compounds bound directly to the ligand-binding domain of PPARγ, with reduced affinity when compared to rosiglitazone. An important trend was observed between the docking scores and the anti-inflammatory activities of this set of molecules. The analysis of the docking results, which takes into account the hydrophilic and hydrophobic interactions between the ligands and the target, explained why the 3-(2-bromo-benzyl)-5-(4-methanesulfonyl-benzylidene)-thiazolidine-2,4-dione compound had the best activity and the best docking score. Almost all of the stronger hydrophilic interactions occurred between the substituted 5-arylidene group of this compound and the residues of the binding site.     

Histopathology of the pterygium in population on Croatian Island Rab

In the 1975-2004 period, 130 cases of pterygium were estimated, 83 males and 47 females, on a small island Rab. Island Rab is located in the north part of the Adriatic sea, which has very high index of sun radiation. Pterygium is usually histopathologically defined as a hyperplasia of conjunctival tissue, elastoid degeneration of subepithelieum and fragmentation of Bowman's membrane. Our histopathological findings in 73 eyes are following: conjunctiva with neovascularisation, leukocyte margination and subepithelial basophilic degeneration, proliferation of conjunctival tissue, acanthosis and squamous metaplasia, and mucinous elements, focaly present plasma cells, focuses of increased pigmentisation in basal epithelial layers, tenon capsule with edema, diffuse neovascularisation and fragmentation of fibrils. These findings suggest that in severe cases of pterygium histopathologically exists precancerosis. In conclusion, on the basis of these histopathological findings, especially in the Tenon capsule, we suggest that for surgical procedure of pterygium the excision of the Tenon capsule, extensively under pterygium, is necessary.     

Synthesis and biological activity of dialkylphosphocholines

A series of dialkylphosphocholines were prepared and evaluated for their biological activity. The antiprotozoal activity was determined against Acanthamoeba lugdunensis. Compound 15 exhibited excellent trophocidal activity. None of the tested dialkylphosphocholines exhibited better fungicidal activity against Candida albicans than miltefosine. The antineoplastic activity was determined against HeLa. The most cytotoxic was compound 10, which was more active against tumor cells as against normal cells.