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The feasibility to obtain highly porous collagen-based scaffolds (SCs) integrated with uniformly dispersed poly(lactide-co-glycolide) (PLGA) microparticles (MPs) was evaluated. PLGA-MPs were prepared by double emulsion technique and SCs with different amounts of MPs (SC/PLGA-MPs) were produced. Results showed that SC/PLGA-MPs could be successfully manufactured, the PLGA-MPs being physically retained in the SCs, without affecting the porous structure. PLGA-MPs also increased the SC hydrophilicity, acted as a mechanical reinforcement and retarded the degradation rate. In addition, the interactions between MPs and Caco-2 cells did not interfere with the correct morphological, adhesion and proliferation pathways. [ABSTRACT FROM AUTHOR]

We studied the expression and the hormonal regulation of the PDS gene product, pendrin, which is, in thyrocytes, responsible for the iodide transport out of the cell. We show that PC Cl3 cells, a fully differentiated thyroid cell line, grown without TSH and insulin, express very low level of PDS mRNA; such expression is greatly increased after stimulation with insulin or TSH. 125I pre-loaded cells showed an 125I efflux accelerated in chloride-containing buffer with respect to chloride-free buffer, suggesting that this efflux is chloride dependent. By immunoblotting, pendrin was found in agonists-stimulated cells, whereas it was barely detectable in un-stimulated cells. An increase in both PDS mRNA and protein was also obtained using phorbol ester PMA, or using 8-Br-cAMP and forskolin. Stimulation with insulin (1 µg/ml; 0–40 min) provoked the cytosol-to-membrane translocation of pendrin and a decrease of intracellular I- content in 125I pre-loaded cells. Insulin- or PMA-treated cells also showed a cytosol-to-membrane translocation of PKC-δ and -ℇ. Inhibition of both PKC-δ and -ℇ activities by GF109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC-δ by rottlerin did not affect the insulin-provoked translocation of pendrin whilst it was inhibited by a PKC-ℇ translocation inhibitor peptide and also by PKC-ℇ downregulation using the small interfering RNA, thus indicating that such translocation was due to PKC-ℇ activity. In conclusion, our study demonstrates that, in PC Cl3 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC-ℇ-dependent intracellular pathway. J. Cell. Physiol. 217: 103–112, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Membrane potential depolarization due to electrogenic peptide transport activity was examined in eel ( Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) by monitoring the fluorescence quenching of the voltage-sensitive dye 3,3′-diethylthiadicarbocyanine iodide. Our experimental approach consisted of generating an internal negative membrane potential mimicking in vivo conditions and measuring membrane potential depolarization due to different extravesicular dipeptides. Peptide-dependent membrane potential depolarization was observed in both the presence and absence of extravesicular Na+ and was inhibited by diethylpyrocarbonate, which is consistent with the involvement of electrogenic, Na+-independent, H+-dependent peptide transport activity. Kinetic analysis indicated that peptide-dependent membrane potential depolarization is a saturable process ( Km,app ∼ 1.5 mmol L−1) and that within the 0.1–10 mmol L−1 peptide range a single carrier system is involved in the transport process. Our results suggest that a peptide transport activity, kinetically resembling the PepT1(Slc15A1)-type-mediated H+/peptide cotransport action, can be monitored in eel intestinal BBMV using an easy and inexpensive fluorescence assay. [ABSTRACT FROM AUTHOR]

The pharmacokinetic properties of cephalexin were studied in sea bream (mean weight 77 g), Sparus aurata (L.) after a single intraperitoneal injection (200 mg kg-1). Pharmacokinetic analysis of the serum concentrations vs time points obtained was performed using non-compartmental analysis and a compartmental pharmacokinetic model approach. In the latter case, a two-compartment open model with a lag time gave the best fitting. The maximum peak serum concentration was 5.018 mg ml-1 kg-1 1 h post-treatment. The area under the curve (AUC) cephalexin was 17.394 mg·h kg-1 and the elimination half-life of 1.83 h. [ABSTRACT FROM AUTHOR]

d-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated. We have studied Na+-dependent d-glucose transport (Na+/d-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions. As assessed by brush-border membrane vesicle studies, Na+/d-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential. Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic. When assayed in purified R and B cell suspensions, Na+/d-glucose cotransport activity was restricted to B cells only. Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na+/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions. Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA. The molecular nature of this Na+/d-glucose cotransport system is still to be established. [ABSTRACT FROM AUTHOR]

The goal of the present study was to evaluate the changes in the cell type composition and ATPase activities (total ATPase, ouabain-sensitive Na+/K+-ATPase, furosemide-sensitive Na+-ATPase) that occur during the different stages of the moulting cycle in the hepatopancreas of the Marsupenaeus japonicus. The results clearly suggest that the number of resorptive and fibrillar cell types changes significantly during the different stages. An inverse correlation between resorptive and fibrillar cells is observed during moulting (both in normally fed and fasted animals). Fasting, but not the moulting cycle, affects the number of blister-like cells. In the resorptive cells the enzymatic activities (total ATPases and ouabain-sensitive Na+/K+-ATPase) also change during the moulting in a cyclical manner. All these results are in agreement with and confirm the different functions carried out by the two cell types within the hepatopancreas. [ABSTRACT FROM AUTHOR]

H[sup +]/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H[sup +]/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (K[sub m,app]) of 0.806±0.161 mmol l[sup -1]. Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H[sup +]/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)[sup +] RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated DPhe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H[sup +]/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguiila, the maximal activity rate occurring at approximately 0°C for the former and 25°C for the latter. Temperature dependence of icefish and eel intestinal mRNAstimulated uptake in the heterologous system (oocytes) was comparable. [ABSTRACT FROM AUTHOR]