Regulation of sterol biosynthesis in sunflower by 24(R,S),25-epiminolanosterol, a novel C-24 methyl transferase inhibitor

Whereas sitosterol and 24(28)-methylene cycloartanol were competitive inhibitors (with Ki = 26 microM and 14 microM, respectively), 24(R,S)-25-epiminolanosterol was found to be a potent non-competitive inhibitor (Ki = 3.0 nM) of the S-adenosyl-L-methionine-C-24 methyl transferase from sunflower embryos. Because the ground state analog, 24(R,S)-oxidolanosterol, failed to inhibit the catalysis and 25-azalanosterol inhibited the catalysis with a Ki of 30 nM we conclude that the aziridine functions in a manner similar to the azasteriod (Rahier, A., et al., J. Biol. Chem. (1984) 259, 15215) as a transition state analog mimicking the carbonium intermediate found in the normal transmethylation reaction. Additionally, we observed that the aziridine inhibited cycloartenol metabolism (the preferred substrate for transmethylation) in cultured sunflower cells and cell growth.     

Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase

Two major transmembranal polypeptides of bovine olfactory epithelium were identified by SDS electrophoretic analysis of Triton X-114 solubilized membranes. Both polypeptides were present in large amounts in membranes of the olfactory epithelium but were barely detectable in membranes of the nasal respiratory epithelium. Both polypeptides are enriched in the deciliated epithelium as compared with isolated cilia. One of them is a glycoprotein with an apparent molecular mass of 56 kDa (gp56); the other is an unglycosylated protein with an apparent molecular mass of 52 kDa (p52). Sequence analysis of peptides obtained by CNBr cleavage of purified gp56 indicates that it is highly homologous to UDP-glucuronosyl transferase (UDPGT). Parallel analysis shows that p52 is highly homologous to cytochrome P-450 sequences of the IIA subfamily. This protein is assigned the name P-450olf2. Polyclonal antibodies were raised against synthetic peptides corresponding to gp56 and p52 peptide sequences. Immunoblots with these antibodies reveal the following properties of gp56 and p52: (1) they are enriched in the microsomal fraction of the bovine olfactory epithelium; (2) they are possibly specific to the olfactory epithelium, as we could not detect reactivity in microsomes derived from respiratory epithelium or lung, and only a very small amount of basal reactivity was seen with liver microsomes; (3) cross-reacting proteins exist in microsomes derived from the rat olfactory epithelium. These results are consistent with a mechanism whereby the microsomal enzymes are involved in odorant modification and clearance from the nasal tissue.     

Effect of divalent metal ions on the digestibility of concanavalin A by endopeptidases.

Demetallized concanavalin A is degraded rapidly at pH 7.0 and 8.2 by alpha-chymotrypsin, thermolysin or trypsin, yielding peptide fragments devoid of ability to bind to Sephadex G-75. Addition of Ni2+ and of Ca2+ confers on concanavalin A high resistance towards proteolytic attack so that even after long periods of exposure to the enzymes, almost all of the saccharide-binding capacity is preserved. Ni2+ alone protects strongly at pH 7.0 but not at pH 8.2. Apparently, both the transition metal ion and Ca2+ play an important role in stabilizing the native conformation of the protein molecule. Digestion of demetallized concanavalin A with alpha-chymotrypsin or thermolysin readily yields small peptide fragments (Mr less than 10 000), while trypsin yields as the major product(s) larger peptide(s) (Mr approximately 20 000) of appreciable resistance to further fragmentation.     

The cleavage of transfer RNA by a single strang specific endonuclease from Neurospora crassa.

Endonuclease from Neurospora crassa (NcNase), an enzyme with specificity for polynucleotides lacking an ordered structure, was shown to cleave su+3 tRNATyr from E. coli preferentially in the anticodon region. The enzyme cleaved unfractionated tRNA primarily to 30 - 50 nucleotide size fragments, implying that most or all tRNA species are also cleaved in the anticodon region. The 3' terminal sequence C-A was cleaved as well. The results are discussed with respect to the three dimensional structure of tRNA.     

Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double‐knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor.     

Incorporating physiology into species distribution models moderates the projected impact of warming on selected Mediterranean marine species

Species distribution models (SDMs) correlate species occurrences with environmental predictors, and can be used to forecast distributions under future climates. SDMs have been criticized for not explicitly including the physiological processes underlying the species response to the environment. Recently, new methods have been suggested to combine SDMs with physiological estimates of performance (physiology-SDMs). In this study, we compare SDM and physiology-SDM predictions for select marine species in the Mediterranean Sea, a region subjected to exceptionally rapid climate change. We focused on six species and created physiology-SDMs that incorporate physiological thermal performance curves from experimental data with species occurrence records. We then contrasted projections of SDMs and physiology-SDMs under future climate (year 2100) for the entire Mediterranean Sea, and particularly the ‘warm’ trailing edge in the Levant region. Across the Mediterranean, we found cross-validation model performance to be similar for regular SDMs and physiology-SDMs. However, we also show that for around half the species the physiology-SDMs substantially outperform regular SDM in the warm Levant. Moreover, for all species the uncertainty associated with the coefficients estimated from the physiology-SDMs were much lower than in the regular SDMs. Under future climate, we find that both SDMs and physiology-SDMs showed similar patterns, with species predicted to shift their distribution north-west in accordance with warming sea temperatures. However, for the physiology-SDMs predicted distributional changes are more moderate than those predicted by regular SDMs. We conclude, that while physiology-SDM predictions generally agree with the regular SDMs, incorporation of the physiological data led to less extreme range shift forecasts. The results suggest that climate-induced range shifts may be less drastic than previously predicted, and thus most species are unlikely to completely disappear with warming climate. Taken together, the findings emphasize that physiological experimental data can provide valuable supplemental information to predict range shifts of marine species.     

Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins

Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.     

Mutant p53 Attenuates the Anti-Tumorigenic Activity of Fibroblasts-Secreted Interferon Beta

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.     

Kinetic Properties of a Novel Fusarium solani (phospho)lipase: A Monolayer Study

Using the monomolecular film technique, we studied interfacial properties of Fusarium solani lipase (FSL). This lipolytic enzyme was found to be unique among the fungal lipases possessing not only a lipase activity but also a high phospholipase one.The FSL was able to hydrolyze dicaprin films at various surface pressures. The surface pressure dependency, the stereospecificity, and the regioselectivity of FSL were performed using optically pure stereoisomers of diglyceride (1,2‐sn‐ dicaprin and 2,3‐sn‐dicaprin) and a prochiral isomer (1,3‐sn‐dicaprin) spread as monomolecular films at the air–water interface. The FSL prefers adjacent ester groups of the diglyceride isomers (1,2‐sn‐dicaprin and 2,3‐sn‐dicaprin) at low and high surface pressures. Furthermore, FSL was found to be markedly stereospecific for the sn‐1 position of the 1,2‐sn‐enantiomer of dicaprin at both low and high surface pressures.Moreover, FSL shows high activities on phospholipids monolayers. However, this enzyme displays high preference to zwitterionic phospholipids compared to the negatively charged ones. Chirality, 25:35‐38, 2012. © 2012 Wiley Periodicals, Inc.