Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests

A detailed report is presented on the performance of the embryonic stem cell test (EST) in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the EST can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.     

Continuous measurements of plasma protein extravasation with microdialysis after various inflammatory challenges in rat and mouse skin

This study describes the use of microdialysis technique for continuous measurement of plasma protein extravasation (PPE) in rat and mouse skin with drug application either intravenously or via the microdialysis fiber. Hollow plasmapheresis fibers (3-cm length, 0.4-mm diameter, cutoff 3,000 kDa) were placed subcutaneously on the back of anesthetized mice and rats. Intravenous injection of dextran (Macrodex, 60 mg/ml) increased PPE by 355% from baseline within 30 min in rats with ligated kidneys (n = 6; P < 0.05) but not in animals with intact kidneys. Phalloidin (500 microg/kg iv 40 min before dextran, n = 6; P < 0.05) did not change the response to dextran in either group. Animals receiving PGE1, compound 48/80 (mice), paclitaxel, docetaxel, and cremophor EL via the microdialysis fiber were also provided with a control fiber receiving vehicle. Both rats and mice had constant PPE in the control fiber, and there was no change in PPE in the NaCl-treated groups (rats, n = 4; mice, n = 6). Application via the fiber of PGE1 (20 microg/ml), compound 48/80 (mice; 4 mg/ml), and docetaxel (0.5 mg/ml) increased PPE compared with baseline within 60 min by 139% (n = 6; P < 0.05), 273% (n = 6; P < 0.05), and 325% (n = 5; P < 0.05), respectively. Phalloidin alone did not increase PPE (n = 5; P < 0.05). Pretreatment with phalloidin did not inhibit the increase after PGE1 or compound 48/80 but inhibited that after docetaxel (n = 6). Paclitaxel (0.6 mg/ml, n = 5) or vehicle (Cremophor) (n = 5) gave no increase in PPE. The results demonstrate that microdialysis can be used to continuously measure changes in PPE after inflammatory challenges in skin of rats and mice.     

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath)

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.     

Human demineralised bone matrix as a bone substitute for reconstruction of cystic defects of the lower jaw

Ina retrospective study validated by a standardized clinical and radiologicalexamination, the bone regeneration in 90 patients with cystic mandibulardefectswas examined. In 50 patients bony defect reconstructions with humandemineralised bone matrix (HDBM) were carried out, while in a comparable groupof 40 patients the hollow pockets were left to regenerate bone spontaneously.The bone regeneration after the implantation of human demineralised bone matrix(HDBM) was subjected to a comparative validation. Osteoinductive proteinspresent in HDBM (bone morphogenetic proteins) can diffuse into the implant seatand induce new bone formation (osteoinduction). A markedly faster and morethorough bone regeneration was demonstrated after the surgical therapy ofcysticmandibular lesions with HDBM than without. HDBM also proved to be exceptionallybiocompatible.     

Licht- und elektronenmikroskopische Untersuchungen an Desmidiaceen

Zusammenfassung Die Zellwände vonMicrasterias rotata, Euastrum oblongum, Cosmarium spec. undHyalotheca dissiliens besitzen zu einem Netzwerk angeordnete Fibrillen, die vonClosterium moniliferum zeigen eine ausgesprochene Paralleltextur. Im Schnitt lassen die Zellwände einen lamellären Bau erkennen.BeiClosterium können deutlich zwei Zellwandschichten unterschieden werden, eine innere mit vorwiegend parallel zur Längsachse orientierten Fibrillen und eine äußere, deren Fibrillen senkrecht zur Längsachse verlaufen.Die Zellwände aller untersuchten Arten sind von Poren durchsetzt, die im elektronenmikroskopischen Bild eine Weite von 130–200 m aufweisen.In den Poren steckt ein in der Literatur als Porenapparat bezeichnetes Gebilde, das fürHyalotheca elektronenmikroskopisch abgebildet wird und einen Porenkanal erkennen läßt.Die Zusammensetzung der Hüllgallerte vonHyalotheca aus einzelnen Gallertprismen, die von den Poren ihren Ausgang nehmen, kann auch elektronenmikroskopisch nachgewiesen werden.Mit 32 Textabbildungen     

Drug target validation of the trypanothione pathway enzymes through metabolic modelling

A kinetic model of trypanothione [T(SH)(2)] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near-physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH)(2) synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH)(2) -dependent antioxidant/regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH)(2) synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by γ-glutamylcysteine synthetase (γECS) and trypanothione synthetase (TryS) (control coefficients of 0.58-0.7 and 0.49-0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH)(2)-dependent antioxidant machinery were determined. The concentration of reduced T(SH)(2) was controlled by TryR (0.98) and oxidative stress (-0.99); however, γECS and TryS also exerted control on the cellular level of T(SH(2)) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH)(2) synthesis flux by 50%, it is necessary to inhibit γECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of γECS and TryS appears to be a promising multi-target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites.     

Expression of integrins and Toll-like receptors in cervical cancer: effect of infectious agents

We hypothesized that development of cervical cancer is associated with alterations in the expression of innate immune receptors, i.e. integrins and TLRs, and that these alterations can be induced by infectious agents. We have studied the expression of these proteins in cervical biopsy tissues and cervical cancer-derived cell lines HeLa, CaSki, SiHa, C-33?A, and ME180. Immunohistochemistry analysis demonstrated an increase in integrin αv, β3, β4, and β6 expression in the epithelium during the development of cervical cancer. A clear trend towards higher expression of integrin β6 in cell lines harbouring human papillomavirus (HPV) genetic material, compared to HPV-negative C-33?A, was observed. To investigate whether bacterial infection can alter the expression of TLRs and integrins, we infected HeLa cells by two pathogens, Escherichia coli and Pseudomonas aeruginosa, using a common bacterium of the female genital tract, Lactobacillus reuteri, as a control. Infection with E. coli or P. aeruginosa, but not with L. reuteri, significantly altered the expression of TLR and integrins, particularly of TLR4 and integrin β6. Considering that both integrin β6 and TLR4 play important roles in tumorigenesis, our data suggest that bacterial infection may trigger cancer development in HPV-infected cervical epithelium.     

Skin and bones: the contribution of skin tone and facial structure to racial prototypicality ratings

Previous research reveals that a more 'African' appearance has significant social consequences, yielding more negative first impressions and harsher criminal sentencing of Black or White individuals. This study is the first to systematically assess the relative contribution of skin tone and facial metrics to White, Black, and Korean perceivers' ratings of the racial prototypicality of faces from the same three groups. Our results revealed that the relative contribution of metrics and skin tone depended on both perceiver race and face race. White perceivers' racial prototypicality ratings were less responsive to variations in skin tone than were Black or Korean perceivers' ratings. White perceivers ratings' also were more responsive to facial metrics than to skin tone, while the reverse was true for Black perceivers. Additionally, across all perceiver groups, skin tone had a more consistent impact than metrics on racial prototypicality ratings of White faces, with the reverse for Korean faces. For Black faces, the relative impact varied with perceiver race: skin tone had a more consistent impact than metrics for Black and Korean perceivers, with the reverse for White perceivers. These results have significant implications for predicting who will experience racial prototypicality biases and from whom.