Biomedical subjects

S Grolli

Publications and source records attributed to S Grolli.

8 recordsLinked to original sources

PubMed2000

The insect attractant 1-octen-3-ol is the natural ligand of bovine odorant-binding protein.

Bovine odorant-binding protein (bOBP) is a dimeric lipocalin present in large amounts in the respiratory and olfactory nasal mucosa. The structure of bOBP refined at 2.0-A resolution revealed an elongated volume of electron density inside each buried cavity, indicating the presence of one (or several) naturally occurring copurified ligand(s) (Tegoni et al. (1996) Nat. Struct. Biol. 3, 863-867; Bianchet et al. (1996) Nat. Struct. Biol. 3, 934-939). In the present work, by combining mass spectrometry, x-ray crystallography (1.8-A resolution), and fluorescence, it has been unambiguously established that natural bOBP contains the racemic form of 1-octen-3-ol. This volatile substance is a typical component of bovine breath and in general of odorous body emanations of humans and animals. The compound 1-octen-3-ol is also an extremely potent olfactory attractant for many insect species, including some parasite vectors like Anopheles (Plasmodium) or Glossina (Trypanosoma). For the first time, a function can be assigned to an OBP, with a possible role of bOBP in the ecological relationships between bovine and insect species.

Animals↗

PubMed2000

Mammalian odorant binding proteins.

Odorant binding proteins (OBPs) pertain to one of the most abundant classes of proteins found in the olfactory apparatus. OBPs are a sub-class of lipocalins, defined by their property of reversibly binding volatile chemicals, that we call 'odorants'. Numerous sequences of OBPs are now available, derived from protein sequencing from nasal mucus material, or from DNA sequences. The structural knowledge of OBPs has been improved too in recent years, with the availability of two X-ray structures. The physiological role of OBPs remains, however, essentially hypothetical, and most probably, not linked to a function of odor transport. The present knowledge on OBP biochemistry, sequence and structure will be examined here in relation to the different functional hypotheses proposed for OBPs.

Animals↗

PubMed2000

Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes.

Porcine odorant binding protein (pOBP) is a monomer of 157 amino acid residues, purified in abundance from pig nasal mucosa. In contrast to the observation on lipocalins as retinol binding protein (RBP), major urinary protein (MUP) or bovine odorant binding protein (bOBP), no naturally occurring ligand was found in the beta-barrel cavity of pOBP. Porcine OBP was therefore chosen as a simple model for structure/function studies with odorant molecules. In competition experiments with tritiated pyrazine, the affinity of pOBP towards several odorant molecules belonging to different chemical classes has been found to be of the micromolar order, with a 1:1 stoichiometry. The X-ray structures of pOBP complexed to these molecules were determined at resolution between 2.15 and 1.4 A. As expected, the electron density of the odorant molecules was observed into the hydrophobic beta-barrel of the lipocalin. Inside this cavity, very few specific interactions were established between the odorant molecule and the amino acid side-chains, which did not undergo significant conformational change. The high B-factors observed for the odorant molecules as well as the existence of alternative conformations reveal a non-specific mode of binding of the odorant molecules in the cavity.

Animals↗

PubMed2000

Matrix attachment region regulates basal beta-lactoglobulin transgene expression.

Nuclear matrix attachment regions (MAR) have been implicated in the regulation of gene expression. We have identified a region within the proximal 3'-flanking sequences of the ovine beta-lactoglobulin (betalg) gene that interacts with the nuclear matrix in vitro. No equivalent region was detected in the 5' flanking region. We have investigated the role of this element in regulating betalg expression in vitro and in vivo. Removal of the MAR did not affect the frequency of betalg transgene expression at the mRNA level, but betalg transgenes that lacked the MAR were expressed at a lower level than wild-type betalg transgenes. In neither in-vitro HC11 transfection experiments nor transgenic mice was hormonal induction of betalg expression significantly affected by MAR removal. Nuclear run-on analysis demonstrated that the impaired basal expression of betalg transgene loci lacking the MAR was due to a reduced transcription rate. Thus, the single MAR enhances the basal transcriptional potential of the betalg gene.

Animals↗

PubMed2000

Role of androgens in proliferation and differentiation of mouse mammary epithelial cell line HC11.

Androgens have been found in mammary epithelium and in milk throughout the cycle of the mammary gland in vivo. The aim of this study was to investigate the possible role of these substances in mammary epithelial growth and differentiation in the mouse HC11 cell line. Cells were stimulated with testosterone, dihydrotestosterone, androstenedione and 5alpha-androstane-3alpha,17beta-diol at concentrations ranging between 0.3 nM and 30 nM. Cyproterone acetate or flutamide, androgen receptor antagonists, (3 microM) were used to block specific androgen effects. Proliferative effects were measured by an MTT (tetrazolium blue) conversion test and [(3)H]thymidine uptake. HC11 cells were transfected with pbetacCAT, a chimeric rat beta-casein gene promoter-chloramphenicol acetyl transferase (CAT) gene construct and CAT ELISA was used to determine gene expression. RT-PCR was performed to detect androgen receptor expression. After 24, 48 and 72 h androgens significantly (P<0.05) increased proliferation. Androgen antagonists significantly (P<0.05) reduced the proliferative effects. Furthermore androgens potentiated the lactogenic effect of prolactin, insulin and dexamethasone (P<0.05). Finally, the androgen receptor gene was expressed in both proliferating and differentiated HC11 cells. These observations lead us to hypothesize an activity of this class of steroids in mammary physiology. In particular, androgens stimulate cell proliferation and beta-casein gene expression; this influence appears to be mediated by androgen receptors.

Androgens↗

PubMed1998

The structure of the monomeric porcine odorant binding protein sheds light on the domain swapping mechanism.

The X-ray structure of the porcine odorant binding protein (OBPp) was determined at 2.25 A resolution. This lipocalin is a monomer and is devoid of naturally occurring bound ligand, contrary to what was observed in the case of bovine OBP [Tegoni, M., et al. (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, M. A., et al. (1996) Nat. Struct. Biol. 3, 934-939]. In this latter protein, a dimer without any disulfide bridges, domain swapping was found to occur between the beta- and alpha-domains. A single Gly (121) insertion was found in OBPp when it was compared to OBPb, which may prevent domain swapping from taking place. The presence of a disulfide bridge between the OBPp beta- and alpha-domains (cysteines 63 and 155) may lock the resulting fold in a nonswapped monomeric conformation. Comparisons with other OBPs indicate that the two cysteines involved in the OBPp disulfide bridge are conserved in the sequence, suggesting that OBPp may be considered a prototypic OBP fold, and not OBPb.

Amino Acid Sequence↗

PubMed1998

Single turnover mechanism of a trypsin-reactor with high enzyme concentration.

A small column containing 2 mM CH-Sepharose 4B-immobilized trypsin was connected to a flow injection device equipped for potentiometric measurements (0.01-2 mM protons) and for post-column analysis by spectrophotometry and capillary electrophoresis (CE). The device was engaged with N alpha-benzoyl-L-arginine pNO2-anilide (BAPNA), beta-lactoglobulin (beta-Lac) and peptides of V8-protease predigested beta-Lac. At a given flow rate, the reaction with BAPNA or beta-Lac (below 2 mM) produced about 1 proton per substrate molecule in each sample (linear relation to substrate amount); with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalents per substrate molecule (hyperbolic dependence). Final experiments demonstrated that the reactor gave a correct estimate of available lysine in peptides of beta-Lac modified with 5-nitrosalicylaldehyde. The data could be predicted by a kinetic model describing the reactor performance in 'single turnover' conditions. The interplay between resident time and the non-catalytic amount of trypsin prevented each enzyme molecule from recycling as well as each substrate molecule (containing one or more cleavage sites) from encountering the enzyme more than once. In conclusion, both from the experimental and the theoretical point of view, this work permitted the analysis of trypsin behaviour in some extreme working conditions and indicates how to modulate the performance of an endoprotease-based reactor. A brief discussion on potential applications in protein mapping and tagging and in the quantitative analysis of protein bioavailability by means of a biosensorial strategy is also described.

Amino Acid Sequence↗

PubMed1997

Expression of c-myc is down-regulated as mouse mammary epithelial cells become confluent.

We have investigated the expression profile of c-myc in the mammary gland. During pregnancy when the gland is actively growing c-myc mRNA was present, while in the differentiated lactating gland no c-myc mRNA was detected. This correspondence between the differentiation state and c-myc mRNA levels in the mouse mammary gland in vivo was paralleled by HC11 mouse mammary epithelial cells in vitro. Firstly, the endogenous c-myc gene was suppressed in confluent compared to growing HC11 cells. In addition, treating the cells with lactogenic hormones did not induce c-myc expression. Secondly, a stably transfected c-myc-CAT reporter construct was similarly down-regulated. Furthermore, using this transfection model, we demonstrate that the mechanism(s) involved in regulating c-myc expression must act through the P1 and P2 core promoter and exon 1. Finally, we demonstrate that suppression of c-myc expression occurs when HC11 cells growth-arrest as they become confluent.

Animals↗