Contact activation during incubation of five different polyurethanes or glass in plasma

During blood-material interaction, the enzymes factor XII fragment (factor XIIf) and kallikrein are generated (contact activation). In this study, the enzymatic activities of factor XIIf and kallikrein were examined with an assay based on the conversion of tripeptide-p-nitroanilide substrate. With the use of aprotinin to inhibit kallikrein, the proteolytic activities of factor XIIf and kallikrein could be separately determined. In this in vitro study, two commercially available polyurethanes, Pellethane and Biomer; three custom synthesized polyurethanes; a biomerlike 2000 MW polytetramethyleneoxide containing polyurethane (PU-2000); an octadecyl extended (ODCE) biomer-like 2000 MW polytetramethyleneoxide containing polyurethane (PU-2000-ODCE); a hard-segment polyurethane (HS-PU); and glass (reference material) were incubated in 25% diluted plasma. In both series of experiments, glass caused the highest amidolytic activities by factor XIIf and kallikrein compared with any of the polyurethanes. In contrast, within the polyurethane group of materials, lower amidolytic activities by factor XIIf and kallikrein were measured on the custom-made polyurethanes than on the commercially available polyurethanes, although the differences among the polyurethanes were small. In addition, the influence of different ratios of material surface to the plasma incubation volume was studied. An increased ratio of surface area over plasma volume resulted in reduced contact activation, suggesting that plasma components are the limiting factor.     

Identification of ligands for olfactory receptors by functional expression of a receptor library

The recognition of odorants by olfactory receptors represents the first stage in odor discrimination. Here, we report the generation of an expression library containing a large and diverse repertoire of mouse olfactory receptor sequences in the transmembrane II-VII region. From this library, 80 chimeric receptors were tested against 26 odorants after transfection into HEK-293 cells. Three receptors were identified to respond to micromolecular concentrations of carvone, (-) citronellal, and limonene, respectively. We also found that the mouse I7 receptor, unlike the rat I7 receptor, prefers heptanal instead of octanal, as a result of a single valine-to-isoleucine substitution. This finding represents the beginning of a molecular understanding of odorant recognition. The identification, on a large scale, of cognate receptor-odorant interactions should provide insight into olfactory coding mechanisms.     

An ester bond linking a fragment of a serine proteinase to its serpin inhibitor

Most known members of the serpin superfamily are serine proteinase inhibitors. Serpins are therefore important regulators of blood coagulation, complement activation, fibrinolysis, and turnover of extracellular matrix. Serpins form SDS-resistant complexes of 1:1 stoichiometry with their target proteinases by reaction of their P1-P1' peptide bond with the active site of the proteinases. The nature of the interactions responsible for the high stability of the complexes is a controversial issue. We subjected the complex between the serine proteinase urokinase-type plasminogen activator (uPA) and the serpin plasminogen activator inhibitor-1 (PAI-1) to proteolytic digestion under nondenaturing conditions. The complex could be degraded to a fragment containing two disulfide-linked peptides from uPA, one of which included the active site Ser, while PAI-1 was left undegraded. By further proteolytic digestion after denaturation and reduction, it was also possible to degrade the PAI-1 moiety, and we isolated a fragment containing 10 amino acids from uPA, encompassing the active site Ser, and 6 amino acids from PAI-1, including the P1 Arg. Characterization of the fragment gave results fully in agreement with the hypothesis that it contained an ester bond between the hydroxyl group of the active site Ser and the carboxyl group of the P1 Arg. These data for the first time provide direct evidence that serine proteinases are entrapped at an acyl intermediate stage in serine proteinase-serpin complexes.     

Expression of rat homeobox gene, rHOX, in developing and adult tissues in mice and regulation of its mRNA expression in osteoblasts by bone morphogenetic protein 2 and parathyroid hormone-related protein

The rat homeobox gene, rHox, was cloned from a rat osteosarcoma cDNA library. Southwestern and gel mobility shift analyses showed that rHox binds to the promoter regions of collagen (alpha1)I and osteocalcin genes while transient transfection with rHox resulted in repression of their respective promoter activities. In situ hybridization studies showed that rHox mRNA was widely expressed in osteoblasts, chondrocytes, skeletal muscle, skin epidermis, and bronchial and intestinal epithelial cells, as well as cardiac muscle in embryonic and newborn mice. However in 3-month-old mice, rHox mRNA expression was restricted to osteoblasts, megakaryocytes, and myocardium. Bone morphogenetic protein 2, a growth factor that commits mesenchymal progenitor cells to differentiate into osteoblasts, down-regulated rHox mRNA expression by 40-50% in UMR 201, a rat preosteoblast cell line, in a time- and dose-dependent manner. In contrast, PTH-related protein (PTHrP), recently shown to be a negative regulator of chondrocyte differentiation, significantly enhanced rHox mRNA expression in UMR 106-06 osteoblastic cells by 3-fold at 24 h while at the same time down-regulating expression of pro-alpha1(I) collagen mRNA by 60%. Expression of rHox mRNA in calvarial osteoblasts derived from PTHrP -/- mice was approximately 15% of that observed in similar cells obtained from normal mice. In conclusion, current evidence suggests that rHox acts as a negative regulator of osteoblast differentiation. Furthermore, down-regulation of rHox mRNA by bone morphogenetic protein 2 and its up-regulation by PTHrP support a role of the homeodomain protein, rHox, in osteoblast differentiation.     

Dogs infected with a human granulocytotropic Ehrlichia spp. (Rickettsiales: Ehrlichieae)

In contrast to the anticipation that in sepsis granulocyte colony-stimulating factor (G-CSF) would overactivate the nonspecific immune system by recruiting and priming leukocytes with consequent aggravation of inflammatory tissue lesions, recombinant (r) G-CSF pretreatment was protective in various experimental non-neutropenic models of inflammation. The mechanisms of protection, however, are not fully understood. Using intravital fluorescence microscopy, we show that rG-CSF enhances leukocyte endothelial cell interaction within the microvasculature of normal rat livers, whereas rG-CSF pretreatment of animals exposed to lipopolysaccharide (LPS) attenuates the LPS-induced leukocytic response, including stasis in sinusoids as well as rolling and adherence in postsinusoidal venules with subsequent tissue infiltration. Moreover, rG-CSF, which did not affect Kupffer cell activity in normal rat livers, reduced the immediate activation of Kupffer cells on LPS exposure, as indicated in vivo by the delayed adherence/phagocytosis of intra-arterially administered latex particles associated with attenuation of proinflammatory cytokine release (tumor necrosis factor alpha and interleukin-6). Finally, rG-CSF reduced LPS-induced nutritive perfusion failure and hepatocellular excretory dysfunction. This study provides evidence for a distinct, possibly tumor necrosis factor alpha-dependent modulation of LPS-induced cellular response within the liver by rG-CSF, thereby achieving protection against microcirculatory perfusion failure and hepatic dysfunction.     

CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat

Signal detectability was measured in three temporal conditions as a function of the bandwidth and configuration of simultaneous maskers that either did or did not spectrally overlap the signal. The 20-ms signal was 250 Hz wide and was centered at 2500 Hz (fs). Although there were marked individual differences, performance was typically poorer when signal onset came 1 ms rather than 250 ms after the onset of a 420-ms masker, and poorest when signal onset came 1 ms after the onset of a 23-ms masker. The results support the idea that two separate across-channel processes contribute to temporal changes in signal detectability. One process contributes to the improvement observed as signal onset is delayed from masker onset, and its influence is reduced by the presence of masking components at fs only when the masker extends exclusively below fs. The other process is associated with the improvement observed as masker offset is delayed from signal offset, and its influence is reduced by the presence of masking components at fs when the masker extends exclusively above, or both below and above fs. Both of these processes are primarily activated by frequencies ranging from 0.6 to 0.8fs and 1.2 to 1.4fs. The data also demonstrate that the measured critical bandwidth narrows as signal onset is delayed from masker onset.     

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.     

Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy

An arginine to glutamine missense mutation at position 403 of the beta-cardiac myosin heavy chain causes familial hypertrophic cardiomyopathy. Here we study mice which have this same missense mutation (alphaMHC403/+) using an isolated, isovolumic heart preparation where cardiac performance is measured simultaneously with cardiac energetics using 31P nuclear magnetic resonance spectroscopy. We observed three major alterations in the physiology and bioenergetics of the alphaMHC403/+ mouse hearts. First, while there was no evidence of systolic dysfunction, diastolic function was impaired during inotropic stimulation. Diastolic dysfunction was manifest as both a decreased rate of left ventricular relaxation and an increase in end-diastolic pressure. Second, under baseline conditions alphaMHC403/+ hearts had lower phosphocreatine and increased inorganic phosphate contents resulting in a decrease in the calculated value for the free energy released from ATP hydrolysis. Third, hearts from alphaMHC403/+ hearts that were studied unpaced responded to increased perfusate calcium by decreasing heart rate approximately twice as much as wild types. We conclude that hearts from alphaMHC403/+ mice demonstrate work load-dependent diastolic dysfunction resembling the human form of familial hypertrophic cardiomyopathy. Changes in high-energy phosphate content suggest that an energy-requiring process may contribute to the observed diastolic dysfunction.