Granulosa cell tumour in a harbour porpoise (Phocoena phocoena) from German waters

Neoplastic diseases in harbour porpoises Phocoena phocoena have rarely been described, and there are no reported gonadal stromal tumours. A 12 yr old female harbour porpoise was stranded on the North Sea coast of Schleswig-Holstein, Germany. Necropsy findings included a severe granulomatous pneumonia, pregnancy and a left ovarian tumour. Respiratory insufficiency was the likely cause of death. There was a multinodular mass composed of cords with peripherally palisading cells within the left ovary. The histological and cytological appearance of the neoplasm was suggestive of a granulosa cell tumour; supportive immunohistochemical stains, including those for vimentin, cytokeration, carcinoembryonic antigen, c-kit, chromogranin and α-smooth muscle action, were negative.     

The challenge of continuous exogenous glucocorticoid administration in mice

Background

Chronic administration of exogenous glucocorticoids is often required in experimental research. We compared the efficacy and reliability of three different methods of continuous glucocorticoid administration in mice.

Materials and methods

Male CD1 Swiss White mice aged 7-9 weeks received corticosterone (CS) or carrier by either subcutaneous (s.c.) injection (n = 15), s.c. implantation of micro-osmotic pumps (n = 20) or s.c. implantation of slow-release pellets (n = 20). Serial blood samples were taken for the measurement of plasma CS and osteocalcin (OC). Bone structural parameters were analysed by micro-computed tomography (μ-CT) in animals treated via slow-release pellets for 4 weeks.

Results

Injection of CS (10 mg/kg) resulted in peak plasma CS levels of up to 2600 μg/L after 1 h, with levels returning to baseline within 4 h post-injection. Micro-osmotic pumps failed to consistently alter plasma CS levels and had variable effects on plasma OC levels. Implantation of 10 mg CS pellets induced hypercorticosteronemia within 24 h but levels returned to baseline within 7 days. Plasma OC levels fell rapidly on day 1 and remained suppressed until day 7. Weekly replacement of pellets maintained elevated plasma CS and suppressed plasma OC concentrations, and resulted in significant bone loss at the tibia and spine after 28 days.

Conclusion

Once-weekly s.c. implantation of slow-release pellets to mice appears to result in relatively consistent plasma CS and OC levels with significant biological effects. However, at least in our hands, no method delivered CS at a constant rate and variability in plasma CS levels was pronounced.     

Recombinant production of an Aspergillus nidulans class I hydrophobin (DewA) in Hypocrea jecorina (Trichoderma reesei) is promoter-dependent

Fungal hydrophobins have potential for several applications because of their abilities to change the hydrophobicity of different surfaces. Yet because of their tendency for aggregation and attachment to interfacial areas only few production processes have so far been reported. Towards the development of a heterologous production system, we report here the expression of a class I hydrophobin DewA of Aspergillus nidulans in Hypocrea jecorina (Trichoderma reesei). Using the H. jecorina hfb2 (class II hydrophobin-encoding) promoter and lactose as a carbon source, only a minor fraction of the DewA remained cell-wall-bound and the majority of it secreted into the medium with up to 15% of the total secreted protein. N-terminal amino acid sequencing showed that it was correctly processed. In contrast, no DewA was produced under the cel7A (cellobiohydrolase I) promoter, although its mRNA was abundantly detected in the cells. This lack of secretion is not due to trapping in the cell wall or to its degradation because of the unfolded protein response. Recombinant DewA could be conveniently precipitated from the culture filtrate, and its bioactivity proven by its ability to stably bind to hydrophilic and hydrophobic surfaces (glass and Teflon, respectively). We thus consider H. jecorina as a promising host for further optimization of DewA production.     

Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: toward tailor-made polymer and oligosaccharide products

Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency.     

Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.     

Generation of ρ0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (ρ⁰ cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. ρ⁰ cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of ρ⁰ cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ⁰ cell lines within 3–5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK⁻ the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat ρ⁰ cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK⁻ ρ⁰ cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ⁰ mitochondria.     

The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity

P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.