Collagens, modifying enzymes and their mutations in humans, flies and worms

Collagens and proteins with collagen-like domains form large superfamilies in various species, and the numbers of known family members are increasing constantly. Vertebrates have at least 27 collagen types with 42 distinct polypeptide chains, >20 additional proteins with collagen-like domains and approximately 20 isoenzymes of various collagen-modifying enzymes. Caenorhabditis elegans has approximately 175 cuticle collagen polypeptides and two basement membrane collagens. Drosophila melanogaster has far fewer collagens than many other species but has approximately 20 polypeptides similar to the catalytic subunits of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. More than 1300 mutations have so far been characterized in 23 of the 42 human collagen genes in various diseases, and many mouse models and C. elegans mutants are also available to analyse the collagen gene family and their modifying enzymes.     

Development of a sensory neuronal cell model for the estimation of mild eye irritation

In an attempt to improve the in vitro test strategy for the estimation of eye irritation, a neuronal cell model has been developed, with cells expressing vanilloid receptor type 1 (VR1) nociceptors. The currently accepted method for measuring eye irritancy is the ethically and scientifically criticised Draize rabbit eye test, despite the fact that alternative in vitro methods are available which have proved to be reliable and reproducible for predicting severe ocular toxicity. However, no alternative tests for measuring neuronal stimulation have yet been developed, and the prediction of eye irritation in the mild range is therefore insufficient. VR1 is a nociceptor localised in C-fibre neurons innervating the cornea and the surrounding tissue, and it responds to potentially damaging stimuli by releasing Ca2+ into the cytoplasm. As a sensory endpoint, [Ca2+]i was measured in VR1 transfected cells, as well as in control cells. Short-term cell cytotoxicity studies (cell membrane rupture and morphological divergence) were used to determine the non-corrosive concentrations of the test chemicals. Preliminary results indicated that hygiene products used daily may induce eye irritation via VR1 nociceptors. The lowest toxic concentration (0.025%) of liquid hand soap, as determined by morphologic divergences of cells, generated an 80% increase in [Ca2+]i over the basal [Ca2+]i in VR1 transfected cells, whereas the non-specific [Ca2+]i increased by 33%. Furthermore, all the endpoints studied indicated that shampoo for children was less active than shampoo for adults. If this method is successfully validated with standardised reference chemicals, the model could complete the test battery of in vitro alternatives, resulting in the saving of thousands of laboratory animals.     

Target-biased scoring approaches and expert systems in structure-based virtual screening

Structure-based virtual screening followed by selection of a top fraction of the rank-ordered result list suffers from many false positives and false negatives because the general scoring functions are not accurate enough. Many approaches have emerged to address this problem by including knowledge about the specific target in the scoring and selection steps. This target bias can include requirements for critical interactions, use of pharmacophore patterns or interaction patterns found in known co-crystal structures, and similarity to known ligands. Such biases are implemented in methods that vary from filtering tools for pre- or post-processing, to expert systems, quantitative (re)scoring functions, and docking tools that generate target-biased poses.     

Process considerations and economic evaluation of two-step steam pretreatment for production of fuel ethanol from softwood

To increase the overall ethanol yield from softwood, the steam pretreatment stage can be carried out in two steps. The two-step pretreatment process was evaluated from a techno-economic standpoint and compared with the one-step pretreatment process. The production plants considered were designed to utilize spruce as raw material and have a capacity of 200,000 tons/year. The two-step process resulted in a higher ethanol yield and a lower requirement for enzymes. However, the two-step process is more capital-intensive and has a higher energy requirement. The estimated ethanol production cost was the same, 4.13 SEK/L (55.1 cent /L) for both alternatives. For the two-step process different energy-saving options were considered, such as a higher concentration of water-insoluble solids in the filter cake before the second step, and the possibility of excluding the pressure reduction between the steps. The most optimistic configuration, with 50% water-insoluble solids in the filter cake in the feed to the second pretreatment step, no pressure reduction between the pretreatment steps, and 77% overall ethanol yield (0.25 kg EtOH/kg dry wood), resulted in a production cost of 3.90 SEK/L (52.0 cent /L). This shows the potential for the two-step pretreatment process, which, however, remains to be verified in pilot trials.     

Oncostatin M-stimulated apical plasma membrane biogenesis requires p27(Kip1)-regulated cell cycle dynamics

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.     

Polarized membrane traffic and cell polarity development is dependent on dihydroceramide synthase-regulated sphinganine turnover

Sphingoid bases have been implicated in various cellular processes including cell growth, apoptosis and cell differentiation. Here, we show that the regulated turnover of sphingoid bases is crucial for cell polarity development, i.e., the biogenesis of apical plasma membrane domains, in well-differentiated hepatic cells. Thus, inhibition of dihydroceramide synthase or sphinganine kinase activity with fumonisin B1 or N,N-dimethylsphingosine, respectively, dramatically perturbs cell polarity development, which is due to increased levels of sphinganine. Consistently, reduction of free sphinganine levels stimulates cell polarity development. Moreover, dihydroceramide synthase, the predominant enzyme responsible for sphinganine turnover, is a target for cell polarity stimulating cAMP/protein kinase A (PKA) signaling cascades. Indeed, electrospray ionization tandem mass spectrometry analyses revealed a significant reduction in sphinganine levels in cAMP/PKA-stimulated cells. These data suggest that sphinganine turnover is critical for and is actively regulated during HepG2 cell polarity development. Previously, we have identified an apical plasma membrane-directed trafficking pathway from the subapical compartment. This transport pathway, which is part of the basolateral-to-apical transcytotic itinerary, plays a crucial role in apical plasma membrane biogenesis. Here, we show that, as a part of the underlying mechanism, the inhibition of dihydroceramide synthase activity and ensuing increased sphinganine levels specifically perturb the activation of this particular pathway in the de novo apical membrane biogenesis.     

Association of a promoter variant in the inducible cyclooxygenase-2 gene (PTGS2) with type 2 diabetes mellitus in Pima Indians

Recent studies have suggested that prostaglandin-endoperoxide synthase-2 (PTGS2), also known as cyclo-oxygenase 2, plays an etiological role in the development of type 2 diabetes mellitus (T2DM). PTGS2 generates prostaglandins, which negatively modulate glucose-stimulated insulin secretion, and functions as a mediator of the inflammatory response, which is associated with decreased insulin sensitivity. Moreover, the gene encoding this enzyme, PTGS2, is located on 1q25.2, a region that has been linked with early onset T2DM in Pima Indians. To determine the possible role played by PTGS2 in modulating susceptibility to T2DM, we screened approximately 7.0 kb of the gene, corresponding to the promoter, coding sequence, and flanking exon-intron boundaries, and identified five variants, including three single nucleotide polymorphisms (SNPs) in the promoter, one intronic SNP, and one in the 3' untranslated region. With the exception of one rare promoter SNP (minor allele frequency <0.03), all SNPs were typed in ~1000 Pima Indians. The range of frequencies for the more common alleles was 0.65–0.88, and we found substantial linkage disequilibrium between all PTGS2 SNP pairs (D0.95). Variant alleles at two markers, rs20417 and rs2066826, which are located in the promoter and intron 6, respectively, were in strong linkage disequilibrium with each other (D=0.97) and were associated with a higher prevalence of T2DM. For marker rs20417, individuals with the variant CC genotype had a 30% higher T2DM prevalence compared with subjects with the GG genotype (odds ratio=1.6 per copy of C allele; P=0.01). The variant C allele of rs20417 has been associated with decreased PTGS2 promoter activity, thereby suggesting a possible biological consequence attributable to this polymorphism. These findings indicate that genetic variants in PTGS2 may play a role in mediating susceptibility to T2DM in Pima Indians and are consistent with the hypothesis that chronic inflammation may contribute to the development of T2DM in some individuals.     

More is not necessarily better: prozone-like effects in passive immunization with IgG

Despite a century of study, the relationship between Ag-specific Ig concentration and protection remains poorly understood for the majority of pathogens. In certain conditions, administration of high Ab doses before challenge with an infectious agent can be less effective than smaller Ab doses, a phenomenon which is consistent with a prozone-like effect. In this study, the relationship between IgG1, IgG2a, IgG2b, and IgG3 dose, infective inocula, and protection was investigated in a mouse model of Cryptococcus neoformans infection. The activity of each IgG subclass ranged from protective to disease-enhancing depending on both the Ab dose and infective inocula used. Enhanced dissemination to the brain was observed in mice given a high IgG2a dose and a relatively low inoculum. Ab administration had immunomodulatory effects, with cytokine expression in lung, brain, and spleen varying as a function of the infective inoculum Ab dose and IgG subclass. In vitro studies did not predict or explain the mechanism of in vivo prozone-like effects, because all isotypes were opsonic and elicited NO release from macrophages. IgG2a was most efficient in inducing a macrophage oxidative burst. These results reveal that an individual Ab can be protective, nonprotective, or disease-enhancing depending on its concentration relative to a challenge inoculum. Our findings have implications for the potential contribution of Ab responses to defense against microbial diseases because Ab-mediated immunity may be protective, nonprotective, or even deleterious to the host.     

Post-translational import of the prion protein into the endoplasmic reticulum interferes with cell viability: a critical role for the putative transmembrane domain

Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability.