Day-night changes in c-fos expression in the fetal sheep suprachiasmatic nucleus at late gestation

The suprachiasmatic nucleus (SCN) is a circadian oscillator in mammals and shows day-night changes in metabolic activity. To investigate whether the fetal sheep SCN behaves as a circadian oscillator, day-night changes in c-fos expression, a marker of neuronal activity, were measured. Eight fetal sheep were sacrificed at 135 days gestation--four at day-time (1200 hours) and four at night-time (2400 hours). Fetal brains were fixed, removed and cut in 40-microns serial coronal sections. Alternate sections were incubated with anti-Fos antibody (1:500) and Fos expression was revealed with extra-avidin-peroxidase and 3,3'-diaminobenzidine or stained with cresyl violet. The number of Fos-immunoreactive (Fos-ir) neurons per mm2 in the rostral, intermediate and caudal regions of the fetal sheep SCN was counted. Fetuses sacrificed in the day-time showed a higher number of Fos-ir neurons per mm2 (mean +/- s.e.; 516.7 +/- 60.1) in the three regions of the SCN than fetuses sacrificed at night-time (140.5 +/- 21.8). In addition, at night-time Fos-ir neurons were mainly localized to the ventrolateral area of the SCN. These findings demonstrate day-night changes in molecular activity consistent with the presence of a circadian oscillator in the fetal sheep SCN.     

Differential contact of disparate class I/peptide complexes as the basis for epitope cross-recognition by a single T cell receptor

In an effort to better understand functional recognition of structurally dissimilar ligands by a single TCR, a model system for studying cross-recognition of disparate peptide/class I complexes was developed using the murine (H-2b) CTL clone AHIII12.2, which is reactive to a human self-peptide (p1049) bound to an HLA-A2.1 molecule. We identified a second complex comprised of a synthetic peptide, designated p1058, bound to H-2Db that is recognized by clone AHIII12.2. In cytolysis assays, dose-response profiles for peptides p1049 and p1058 pulsed onto the appropriate target cells were comparable, suggesting that p1049/A2.1 and p1058/Db form functionally equivalent epitopes. To probe the interaction between each complex and the TCR of AHIII12.2, singly substituted analogues of each peptide were tested for their activity in lysis assays. Differences were observed between the two epitopes with respect to permissible residue substitutions at each peptide position from P3 to P8; marked differences were evident at P3 and at P8. The results obtained suggest that this TCR forms critical contacts with atoms at peptide positions P3 and P5 of p1049/A2.1 and at P5 and P8 of p1058/Db, and that TCR cross-recognition of these ligands is a function of both shared and complex-specific contacts made with each epitope. These findings further highlight the versatile reactivity that may be shown by a single TCR and suggest a basis for the recognition of peptide ligands sharing only a limited set of structural features.     

Cidofovir transport in the pigmented rabbit conjunctiva

PURPOSE: To characterize cidofovir (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine) transport in the pigmented rabbit conjunctiva and to evaluate the formulation influence on its transport. METHODS: The excised pigmented rabbit conjunctiva was mounted in the modified Ussing chamber. Cidofovir transport was initiated by applying 3H-cidofovir to the donor compartment and assayed by measuring the radioactivity accumulated in the receiver fluid over 180 min. RESULTS: Cidofovir flux in the mucosal-to-serosal direction increased proportionally with drug concentration over the 0.01 to 1 mM range. Cidofovir transport (0.01 mM) at 37 degrees C in the mucosal-to-serosal direction was not significantly different from that in the opposite direction or from that at 4 degrees C. Hypotonicity (80 mOsm/kg), 0.5% EDTA, and 0.0125% benzalkonium chloride increased the apparent permeability coefficient of cidofovir 3, 21, and 49 times, respectively. This was accompanied by a corresponding 43%, 86%, and 96% reduction in the transconjunctival electrical resistance over 180 min. The reduction in transepithelial electrical resistance elicited by hypotonicity was reversible. There was a good correlation between apparent permeability coefficient and the transconjunctival conductance, suggesting that cidofovir may undergo paracellular passive diffusion in the conjunctiva. CONCLUSION: Cidofovir transport in the rabbit conjunctiva may be via paracellular passive diffusion. Formulation changes may improve cidofovir absorption from the conjunctival route.     

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O2) yet PIGF expression decreased 73 +/- 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of function flt-1 on normal trophoblast suggests that VEGF/PIGF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.     

Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase AT submicromolar iron concentrations: evidence for metal-catalyzed oxidation

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe2+ + H2O2 --> HO. + HO- + Fe3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca2+-ATPase by oxygen reactive species at Fe2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe2+, H2O2, and ascorbate. Measuring the rate constants of inactivation (k inact) at different Fe2+ concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 microM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 microM Fe2+. Inhibition was not affected by addition of 200 microM Ca2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu2+ was used instead of Fe2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.     

Results of cemented metal-backed acetabular components: a 10-year-average follow-up study

We have developed a computer program for the rapid assessment of the primary structure differences between a protein of unknown sequence and a homologous known protein. Both proteins are reduced, alkylated, and digested with the same hydrolytic agent. The unfractionated peptide mixtures are submitted to automatic sequence analysis. Based on the knowledge of the reference sequence, the program utilizes the analysis data to identify all the potential peptides present in the two mixtures, determining their primary structure, homology degree, and molecular weight calculated both as integer MH+ and average mass variables. These fingerprints allow the user to easily identify the structural differences between the two proteins and clarify possible doubts by a mass spectrometric analysis of the two mixtures. In order to verify the utility of the program, we provide an application example using the already reported data of two homologous proteins.