Expression of a novel sodium-hydrogen exchanger in the gastrointestinal tract and kidney

Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP.     

Development of an in vivo active,dual EP1 and EP3 selective antagonist based on a novel acyl sulfonamide bioisostere

Recent preclinical studies demonstrate a role for the prostaglandin E₂ (PGE₂) subtype 1 (EP1) receptor in mediating, at least in part, the pathophysiology of hypertension and diabetes mellitus. A series of amide and N-acylsulfonamide analogs of a previously described picolinic acid-based human EP1 receptor antagonist (7) were prepared. Each analog had improved selectivity at the mouse EP1 receptor over the mouse thromboxane receptor (TP). A subset of analogs gained affinity for the mouse PGE₂ subtype 3 (EP3) receptor, another potential therapeutic target. One analog (17) possessed equal selectivity for EP1 and EP3, displayed a sufficient in vivo residence time in mice, and lacked the potential for acyl glucuronide formation common to compound 7. Treatment of mice with 17 significantly attenuated the vasopressor activity resulting from an acute infusion of EP1 and EP3 receptor agonists. Compound 17 represents a potentially novel therapeutic in the treatment of hypertension and diabetes mellitus.     

Changes in odor quality discrimination following recovery from olfactory nerve transection

Following recovery from olfactory nerve transection, animals regain their ability to discriminate between odors. Odor discrimination is restored after new neurons establish connections with the olfactory bulb. However, it is not known if the new connections alter odor quality perception. To address this question, 20 adult hamsters were first trained to discriminate between cinnamon and strawberry odors. After reaching criterion (> or = 90% correct response), half of the animals received a bilateral nerve transection (BTX) and half a surgical sham procedure. Animals were not tested again until day 40, a point in recovery when connections are re-established with the bulb. When BTX animals were tested without food reinforcement, they could not perform the odor discrimination task. Sham animals, however, could discriminate, demonstrating that the behavioral response had not been extinguished during the 40 day period. When reinforcement was resumed, BTX animals were able to discriminate between cinnamon and strawberry after four test sessions. In addition, their ability to discriminate between these two familiar odors was no different than that of BTX and sham animals tested with two novel odors, baby powder and coffee. These findings suggest that, after recovery from nerve transection, there are alterations in sensory perception and that restoration of odor quality discrimination requires that the animal must again learn to associate individual odor sensations with a behavioral response.      

The accuracy of several multiple sequence alignment programs for proteins

Background  

There have been many algorithms and software programs implemented for the inference of multiple sequence alignments of protein and DNA sequences. The "true" alignment is usually unknown due to the incomplete knowledge of the evolutionary history of the sequences, making it difficult to gauge the relative accuracy of the programs.     

Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys

Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.     

Evidence for the presence of a critical disulfide bond in the mouse EP3γ receptor

To determine the contribution of cysteines to the function of the mouse E-prostanoid subtype 3 gamma (mEP3γ), we tested a series of cysteine-to-alanine mutants. Two of these mutants, C107A and C184A, showed no agonist-dependent activation in a cell-based reporter assay for mEP3γ, whereas none of the other cysteine-to-alanine mutations disrupted mEP3γ signal transduction. Total cell membranes prepared from HEK293 cells transfected with mEP3γ C107A or C184A had no detectable radioligand binding. Other mutant mEP3γ receptors had radioligand affinities and receptor densities similar to wild-type. Cell-surface ELISA against the N-terminal HA-tag of C107A and C184A demonstrated 40% and 47% reductions respectively in receptor protein expression at the cell surface, and no radioligand binding was detected as assessed by intact cell radioligand binding experiments. These data suggest a key role for C107 and C184 in both receptor structure/stability and function and is consistent with the presence of a conserved disulfide bond between C107 and C184 in mouse EP3 that is required for normal receptor expression and function. Our results also indicate that if a second disulfide bond is present in the native receptor it is non-essential for receptor assembly or function.