Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholate

Summary Recombinant human growth hormone was administered orally to carp and serum levels of absorbed bioactive hormone were investigated using a highly sensitive Nb2 rat lymphoma cell bioassay and radioimmumoassay. Serum levels of bioactive hGH reached maximum values 30 min after oral intubation and then gradually decreased. Co-administration of the hormone with deoxycholate to fasted carp resulted in up to a 1000-fold increase in absorption compared to aqueous solutions of the hormone, but had no effect on the kinetics of the absorption process. Absorption of the hormone in starved fish was significantly greater than in fed fish. A linear dose-response relationship was observed for hGH in starved fish and the level of absorption in fed fish was influenced by the time interval from the last meal. The ratio of bioactive to immunoactive hGH in fasted fish indicated little loss of bioactivity and also that deoxycholate may be protective against hGH degradation. The present study demonstrates for the first time that biologically active hGH is absorbed in the common carp after oral intubation. Furthermore, the use of a biological detergent dramatically increased the extent of hGH absorption. Additional studies are required to establish the approapriate conditions (diet composition, feeding level, and frequency, etc.) in which polypeptide hormones could be introduced orally to fish.Abbreviations hGH human growth hormone - HRP horseradish peroxidase - RIA radioimmunoassay     

Developmental changes in responsiveness to vitamin D metabolites

We have demonstrated that epiphyseal chondroblasts contain specific receptors for 24R,25-dihydroxy vitamin D3(24,25(OH)2D3) while diaphyseal osteoblasts contain specific receptors for 1 alpha 25-dihydroxy vitamin D3(1,25(OH)2D3). Both metabolites induce DNA synthesis and creatine kinase (CKBB) activity. We have also found that the responsiveness of rat kidney to these metabolites changes during development. In embryonic and early postnatal stages, the kidney responds to 24,25(OH)2D3, later to both 24,25(OH)2D3 and 1,25(OH)2D3, and the mature kidney only to 1,25(OH)2D3. These responses correlate with changes in the specific receptors present in the kidney. Furthermore, we have compared developmental changes in skeletal (epiphysis, diaphysis and mandibular condyle) and non-skeletal (kidney, cerebellum, cerebrum, liver and pituitary) tissue in both rat (a postnatal developer) and rabbit (a perinatal developer). Epiphyseal or diaphyseal chondroblasts at any stage of development were predominantly responsive to 24,25(OH)2D3, whereas osteoblasts were responsive to 1,25(OH)2D3. In contrast, condylar chondroblasts, kidney, cerebellum and pituitary responded to 24,25(OH)2D3 during early development and subsequently developed responsiveness to 1,25(OH)2D3. Using primary cell cultures from kidneys at different stages of maturation, we showed the same developmental pattern as in vivo. Chronic treatment of the cells with 24,25(OH)2D3, but not 1,25(OH)2D3, caused precocious development of responsiveness to 1,25(OH)2D3 in culture. We suggest that 24,25(OH)2D3 acts as a maturation factor, during early development in kidney, and probably in other tissues, possibly by induction of receptor to 1,25(OH)2D3, accompanied by down-regulation of its own receptor.     

Maternal-perinatal interrelationships of vitamins D metabolism in rats

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite that ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1.Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet.Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 5/25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected.Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol.Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

Cyt2Ba of Bacillus thuringiensis israelensis: activation by putative endogenous protease

The gene cyt2Ba of Bacillus thuringiensis subsp. israelensis was cloned for expression, together with p20, in an acrystalliferous strain. The large hexagonal crystals formed were composed of Cyt2Ba, which facilitated its purification. Crystal solubilization in the presence of endogenous proteases (with spores and cell debris) enabled quick and simple procedure to obtain rather pure and active toxin species by cleavage between amino acid residues 34 and 35, most likely by a camelysin-like protease that was discovered in association with activated Cyt2Ba. The product of this cleavage displayed haemolytic activity comparable to that of exogenously activated Cyt2Ba. The sequence of this putative protease shares high homology with the cell envelope-bound metalloprotease (camelysin) of the closely related species Bacillus cereus.     

Absolute binding free energy calculations: On the accuracy of computational scoring of protein–ligand interactions

Calculating the absolute binding free energies is a challenging task. Reliable estimates of binding free energies should provide a guide for rational drug design. It should also provide us with deeper understanding of the correlation between protein structure and its function. Further applications may include identifying novel molecular scaffolds and optimizing lead compounds in computer‐aided drug design. Available options to evaluate the absolute binding free energies range from the rigorous but expensive free energy perturbation to the microscopic linear response approximation (LRA/β version) and related approaches including the linear interaction energy (LIE) to the more approximated and considerably faster scaled protein dipoles Langevin dipoles (PDLD/S‐LRA version) as well as the less rigorous molecular mechanics Poisson–Boltzmann/surface area (MM/PBSA) and generalized born/surface area (MM/GBSA) to the less accurate scoring functions. There is a need for an assessment of the performance of different approaches in terms of computer time and reliability. We present a comparative study of the LRA/β, the LIE, the PDLD/S‐LRA/β, and the more widely used MM/PBSA and assess their abilities to estimate the absolute binding energies. The LRA and LIE methods perform reasonably well but require specialized parameterization for the nonelectrostatic term. The PDLD/S‐LRA/β performs effectively without the need of reparameterization. Our assessment of the MM/PBSA is less optimistic. This approach appears to provide erroneous estimates of the absolute binding energies because of its incorrect entropies and the problematic treatment of electrostatic energies. Overall, the PDLD/S‐LRA/β appears to offer an appealing option for the final stages of massive screening approaches. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.     

Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists

Expression plasmids encoding mouse and rat leptins and their L39A/D40A/F41A muteins were prepared. The proteins were expressed in Escherichia coli, refolded and purified to homogeneity, yielding electrophoretically pure, over 98% monomeric protein. Circular dichroism (CD) analysis revealed that the mutations hardly affect the leptins' secondary structure, and they were similar to previously reported CD spectra for human leptin. Both mouse and rat leptins were biologically active in promoting proliferation in BAF/3 cells stably transfected with the long form of human leptin receptor. The mutations did not change the binding properties to BAF/3 cells as compared, respectively, to non-mutated mouse, rat or human leptins, or their ability to form 1:1 complexes with the leptin-binding domain of chicken leptin receptor. In contrast, their biological activity, tested in a BAF/3 proliferation assay, was abolished and both became potent antagonists. As the LDF (amino acids 39-41) sequence is preserved in all known leptins, the present results substantiate the hypothesis that this sequence plays a pivotal role in leptins' site III and that interaction of leptin with its receptors resembles the corresponding interactions of interleukin-6 and granulocyte colony-stimulating factor their receptors.