Isolation of interstitial fluid from rat mammary tumors by a centrifugation method

Access to interstitial fluid is of fundamental importance to understand tumor transcapillary fluid balance, including the distribution of probes and therapeutic agents. Tumors were induced by gavage of 9,10-dimethyl-1,2-benzanthracene to rats, and fluid was isolated after anesthesia by exposing tissue to consecutive centrifugations from 27 to 6,800 g. The observed (51)Cr-EDTA (extracellular tracer) tissue fluid-to-plasma ratio obtained from whole tumor or from superficial tumor tissue by centrifugation at 27-424 g was not significantly different from 1.0 (0.92-0.99), suggesting an extracellular origin only. However, fluid collected from excised central tumor parts had a significantly lower ratio (0.66-0.77) for all imposed G forces, suggesting dilution by fluid deriving from a space unavailable for (51)Cr-EDTA. The colloid osmotic pressure in tumor fluid was generally higher than in fluid isolated from the subcutis, attributable to less selective capillaries and impaired lymphatic drainage in tumors. HPLC analysis of tumor fluid showed that low-molecular-weight macromolecules not present in arterial plasma were present in tumor fluid obtained by centrifugation and in venous blood draining the tumor, most likely representing proteins derived from tumor cells. We conclude that low-speed centrifugation may be a simple and reliable method to isolate interstitial fluid from tumors.     

Essential role of the apolipoprotein E receptor-2 in sperm development

The apolipoprotein (apo) E receptor-2 (apoER2) is a member of the low density lipoprotein receptor gene family and an important regulator of neuronal migration. It acts as a receptor for the signaling factor Reelin and provides positional cues to neurons that migrate to their proper position in the developing brain. Besides brain formation defects, apoER2-deficient mice also exhibit male infertility. The role of the receptor in male reproduction, however, remained unclear. Here we demonstrate that apoER2 is highly expressed in the initial segment of the epididymis, where it affects the functional expression of clusterin and phospholipid hydroperoxide glutathione peroxidase (PHGPx), two proteins required for sperm maturation. Reduced PHGPx expression in apoER2 knockout mice results in the inability of the sperm to regulate the cell volume and in abnormal sperm morphology and immotility. Because insufficient expression of PHGPx is a major cause of infertility in men, these findings not only highlight an important new function for apoER2 that is unrelated to neuronal migration, but they also suggest a possible role for apoER2 in human infertility.     

Differential binding of ligands to the apolipoprotein E receptor 2

Apolipoprotein E receptor 2 (apoER2) is an important participant in the Reelin signaling pathway that directs cell positioning during embryogenesis. ApoER2 is a cell surface molecule that elicits intracellular signal transduction through binding of Reelin. The structural requirements for Reelin binding to apoER2 and the receptor domains involved in this process are unclear at present. Using a series of receptor mutants, we characterized the interaction of apoER2 with Reelin and compared this interaction to that of apoER2 with the receptor-associated protein (RAP), an apoER2 ligand that does not induce signaling. By surface plasmon resonance we demonstrate that apoER2 exhibits 6-fold higher affinity for Reelin than the very low density lipoprotein receptor (VLDLR), which also functions as a Reelin receptor (K(D) 0.2 nM versus K(D) 1.2 nM). Acidic amino acid residues in complement-type repeat domains 1 and 3 of apoER2 are required for Reelin binding. The same regions of the receptor are also bound by RAP with a 25-fold lower affinity (K(D) 5 nM). Whereas RAP binds to apoER2 with a 1:1 stoichiometry, experimental evidence suggests that Reelin associates with two or more receptor molecules simultaneously to achieve high-affinity interaction. This finding indicates that aggregation of apoER2 by multivalent ligands such as Reelin may be the structural basis for signal transduction.     

Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases

Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.     

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background

Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44^mapk) and ERK2 (p42^mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca²⁺ and Ca²⁺-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F_2α.

Results

Pretreatment of the cells with the Ca²⁺ chelators BAPTA-AM or EGTA, as well as the Ca²⁺ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca²⁺/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF_2α.

Conclusion

The present data indicate that Ca²⁺ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.     

Aprotinin uptake in the proximal tubules in the rat kidney. II. Uptake site relative to glomerulus

Glomerular filtration rates in whole kidney and in outer, middle and inner cortical zones have previously been estimated by measuring the amount of iodinated Aprotinin, filtered and taken up in the first two thirds of the proximal convoluted tubules, in part positioned more superficial than the parent glomerulus. Thus, an appreciable amount of the absorbed Aprotinin may be located superficial to its filtration site and lead to an underestimate of glomerular filtration in deep cortical layers. Therefore, in this study we have measured the distance from the glomerulus to the center of proximal convoluted tubular ball and the site of Aprotinin uptake. Measurements were made on photos of Microfil-injected tubules and on camera lucida drawings of tubular transections from autoradiographs of nephrons containing both Microfil and iodinated Aprotinin. Both techniques showed that the center of the tubular ball was localized more superficial in all cortical layers. The average distance, in percent of cortical thickness, from all proximal convoluted tubular transections to the parent glomerulus was 9% in deep and 13% in middle and superficial cortex. Corresponding distances for tubular transections containing Aprotinin were 7 and 12%. Grain density in five reconstructed proximal convoluted tubules showed a continuous and exponential fall of Aprotinin along the uptake segment. The results may be used to estimate single nephron filtration rate from Aprotinin uptake and glomerular density in outer, middle, and inner cortex.     

Developmental expression of the <Emphasis Type="Italic">Xenopus laevis Tbx20</Emphasis> orthologue

We have isolated the Xenopus orthologue of the T-box gene, Tbx20, and characterized its developmental expression profile. We show that Tbx20 is one of the earliest markers of heart tissue in Xenopus, and is expressed throughout all cardiac tissue during later stages of development. In addition, we also observe expression in the cement gland, the jugular vein, the lung bud, the cloacal aperture, rhombomeres 2, 4, 6 and 8, and in a subset of motor neurons.     

Glucoamylase from Thermoanaerobacterium thermosaccharolyticum: Sequence studies and analysis of the macromolecular architecture of the enzyme

A chromosomal DNA fragment with a length of 2,025 bp, carrying the structural gene coding for glucoamylase in Thermoanaerobacterium thermosaccharolyticum, was cloned and sequenced. It coded for 695 amino acids, representing a polypeptide with a predicted molecular mass of 77.5 kDa. The deduced amino acid sequence exhibited high homologies with the glucoamylase sequence of another bacterial glucoamylase (Clostridium sp. G0005) and with fungal glucoamylases. The catalytic domain (amino acids 271 to 695) of the T. thermosaccharolyticum enzyme shared a high degree of similarity (five conserved regions) with the catalytic domain of Aspergillus awamori glucoamylase. By comparing the secondary structure of the sequence of the catalytic domain of the T. thermosaccharolyticum enzyme with that of glucoamylase from A. awamori, and on the basis of X-ray crystallographic data available for the A. awamori enzyme, it turned out that, most probably, both enzymes have a catalytic domain organized into an "(alpha/alpha)(6)-barrel" and an overall size and shape that is very similar. These findings confirm and extend our working model for the macromolecular architecture of the T. thermosaccharolyticum glucoamylase obtained, in earlier experiments, by electron microscopy of negatively stained isolated enzyme molecules. Antibodies for an enzyme-specific peptide located near the active site were successfully applied for inhibition studies of enzyme activity and for electron microscopic epitope mapping. A study comparing the site of attachment of this kind of antibody to the T. thermosaccharolyticum glucoamylase molecule with the expected attachment site as deduced from the A. awamori enzyme structure confirmed the close similarity of both glucoamylases regarding the macromolecular architecture of that part of the enzyme carrying the catalytic center, though helices H9, H10, and H11 in peripheral parts of the A. awamori enzyme are missing in the T. thermosaccharolyticum enzyme.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.