In vitro incubation of golden (syrian) hamster ovarian oocytes and human sperm with a human oviduct specific glycoprotein

The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1-D SDS-PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre- and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct. © 1994 Wiley-Liss, Inc.     

An adherent mucus layer attenuates the genotoxic effect of colibactin

The human gastrointestinal tract is a complex ecosystem in which epithelial cells and microorganisms of the intestinal microbiota live in symbiosis. Certain members of the microbiota, in particular Escherichia coli strains of the B2 phylotype, carry the polyketide synthase‐island encoding the genotoxin colibactin. Colibactin is a nonribosomal peptide or polyketide‐nonribosomal peptide hybrid of still unsolved structure, which induces DNA double strand breaks (DSBs) in eukaryotic cells. However, direct contact between live bacteria and host cell is required in order to elicit these genotoxic effects. In this study, we used a variety of cell culture models, among them, a 3D cell culture approach based on decellularised small intestinal submucosa, to investigate whether the intestinal mucus layer has the potential to interfere with colibactin activity. We demonstrate that the expression of mucins and the formation of an adherent mucus layer significantly increased with increasing complexity of cell culture. Moreover, we show that the presence of an adherent mucus layer on epithelial cells attenuates the genotoxic activity of colibactin, by preventing the induction of DNA‐DSBs. Removal of the adherent mucus layer restored the occurrence of DNA‐DSBs.     

Dietary choices are influenced by genotype,mating status,and sex in Drosophila melanogaster

Mating causes many changes in physiology, behavior, and gene expression in a wide range of organisms. These changes are predicted to be sex specific, influenced by the divergent reproductive roles of the sexes. In female insects, mating is associated with an increase in egg production which requires high levels of nutritional input with direct consequences for the physiological needs of individual females. Consequently, females alter their nutritional acquisition in line with the physiological demands imposed by mating. Although much is known about the female mating‐induced nutritional response, far less is known about changes in males. In addition, it is unknown whether variation between genotypes translates into variation in dietary behavioral responses. Here we examine mating‐induced shifts in male and female dietary preferences across genotypes of Drosophila melanogaster. We find sex‐ and genotype‐specific effects on both the quantity and quality of the chosen diet. These results contribute to our understanding of sex‐specific metabolism and reveal genotypic variation that influences responses to physiological demands.     

Similar Organization of the Lipopolysaccharide-Binding Protein (LBP) and Phospholipid Transfer Protein (PLTP) Genes Suggests a Common Gene Family of Lipid-Binding Proteins

The transfer of lipids in aqueous environments such as serum has been attributed to a recently characterized class of proteins. Abnormal regulation of serum lipids by these proteins is thought to be a key event in the pathophysiology of cardiovascular diseases. Lipopolysaccharide (endotoxin) binding protein (LBP) was identified by virtue of its ability to bind bacterial lipid A. We have analyzed the exon–intron organization of the LBP gene and the nucleotide sequence of its approximately 20 kb spanning 5′- and 3′-untranslated regions. When comparing the genomic organization of LBP with that of two other genes coding for lipid transfer proteins, significant homologies were found. The LBP gene includes 15 exons, and the 2-kb promoter contains recognition elements of acute phase-typical reactants and a repetitive 12-mer motif with an as yet unknown protein-binding property. Detailed sequence comparison revealed a closer relatedness of LBP with PLTP than with CETP as demonstrated by an almost identical intron positioning. This high degree of similarity supports functional studies by others suggesting that like LBP, PLTP may also be able to bind and transport bacterial lipopolysaccharide.     

Cys359 of GrdD is the active-site thiol that catalyses the final step of acetyl phosphate formation by glycine reductase from Eubacterium acidaminophilum.

In the amino-acid-fermenting anaerobe Eubacterium acidaminophilum, acetyl phosphate is synthesized by protein C of glycine reductase from a selenoprotein A-bound carboxymethyl-selenoether. We investigated specific thiols present in protein C for responsibility for acetyl phosphate liberation. After cloning of the genes encoding the large and the small subunit (grdC1, grdD1), they were expressed separately in Escherichia coli and purified as Strep-tag proteins. GrdD was the only subunit that catalysed arsenate-dependent hydrolysis of acetyl phosphate (up to 274 U.mg-1), whereas GrdC was completely inactive. GrdD contained two cysteine residues that were exchanged by site-directed mutagenesis. The GrdD(C98S) mutant enzyme still catalysed the hydrolysis of acetyl phosphate, but the GrdD(C359A) mutant enzyme was completely inactive. Next, these thiols were analysed further by chemical modification. After iodoacetate treatment of GrdD, the enzyme activity was lost, but in the presence of acetyl phosphate enzyme activity was protected. Subsequently, the inactivated carboxymethylated enzyme and the protected enzyme were both denatured, and the remaining thiols were pyridylethylated. Peptides generated by proteolytic cleavage were separated and subjected to mass spectrometry. Cys98 was not accessible to carboxymethylation by iodoacetate in the native enzyme in the presence or absence of the substrate, but could be alkylated after denaturation. Cys359, in contrast, was protected from carboxymethylation in the presence of acetyl phosphate, but became accessible to pyridylethylation upon prior denaturation of the protein. This clearly confirmed the catalytic role of Cys359 as the active site thiol of GrdD responsible for liberation of acetyl phosphate.     

Fluorescence techniques in biotechnology

The high specificity and sensitivity of fluorescence techniques have made them important analytical tools in medicine and biotechnology. Besides monitoring and quantitative detection of biomolecules these methods can be used for controlling bacterial activities or for measuring physiological states of cells or tissues. Three topics of importance in biotechnology — immunoassays, photosynthesis and fermentation — are treated in detail.     

Eco-efficiency indicator framework implemented in the metallurgical industry: part 2—a case study from the copper industry

Purpose

Sustainability differentiation has become an important issue for companies throughout the value chain. There is thus a need for detailed and credible analyses, which show the current status and point out where improvements can be done and how. The study describes how a comprehensive product-centric eco-efficiency indicator framework can be used to evaluate, benchmark, and communicate the sustainability of a copper production value chain. The indicator framework, together with the suggested data collection and simulation methods, aims at evaluating the whole system, while still enabling a focus on scopes of different width. The status of the environment, current production technologies, location-specific and process-specific issues all play a role in achieving sustainable development.

Methods

Copper cathode production from copper ore was chosen to exemplify the developed framework. Data sets from a simulation tool were used when available and LCI databases and LCA software were utilized for the remaining steps. The value chain is analyzed and the benchmark for each indicator built according to the new Gaia Refiner indicator framework. This method enables analysis of specific production steps with a higher degree of accuracy.

Results and discussion

The case study shows how some important environmental sustainability issues in copper production can be analyzed and benchmarked within a product group. Benchmark data is collected and used in the analysis for the selected system scope. Data availability is still an issue and the example shows which areas require more information in this context so that products and value chains can be benchmarked in the future on a more consistent basis. The energy mix, chemical use, and land use contribute to potential environmental sustainability risks within the product benchmarking group, while emissions control shows competitive environmental sustainability advantages for the case study.

Conclusions

The methodology is shown to work well in highlighting the sustainability advantages and risks of value chains in copper production with the selected system scope in a visual manner through the Sustainability Indicator “Flower.” The importance of a baseline is clear. The effect of the metal ore grade on the results shows that the scalability of the analysis system is very important. Scaling the system scope up will show the differences in varying value chains and scaling the system scope down will show efficiency differences between more similar value chains, thus visualizing where innovation has the biggest impact.