AP106P Sigma-AldrichRabbit Anti-Goat IgG Antibody, HRP conjugate

Na(+) transport in the renal distal convoluted tubule (DCT) by the thiazide-sensitive NaCl cotransporter (NCC) is a major determinant of total body Na(+) and BP. NCC-mediated transport is stimulated by aldosterone, the dominant regulator of chronic Na(+) homeostasis, but the mechanism is controversial. Transport may also be affected by epithelial remodeling, which occurs in the DCT in response to chronic perturbations in electrolyte homeostasis. Hsd11b2(-/-) mice, which lack the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) and thus exhibit the syndrome of apparent mineralocorticoid excess, provided an ideal model in which to investigate the potential for DCT hypertrophy to contribute to Na(+) retention in a hypertensive condition. The DCTs of Hsd11b2(-/-) mice exhibited hypertrophy and hyperplasia and the kidneys expressed higher levels of total and phosphorylated NCC compared with those of wild-type mice. However, the striking structural and molecular phenotypes were not associated with an increase in the natriuretic effect of thiazide. In wild-type mice, Hsd11b2 mRNA was detected in some tubule segments expressing Slc12a3, but 11βHSD2 and NCC did not colocalize at the protein level. Thus, the phosphorylation status of NCC may not necessarily equate to its activity in vivo, and the structural remodeling of the DCT in the knockout mouse may not be a direct consequence of aberrant corticosteroid signaling in DCT cells. These observations suggest that the conventional concept of mineralocorticoid signaling in the DCT should be revised to recognize the complexity of NCC regulation by corticosteroids.

Diabetic neuropathic pain (DNP) is one of the most common clinical manifestations of diabetes mellitus (DM), which is characterized by prominent mechanical allodynia (DMA). However, the molecular mechanism underlying it has not fully been elucidated. In this study, we examined the spatio-temporal expression of a major nociceptive channel protein transient receptor potential vanilloid 1 (TRPV1) and analyzed its functional involvement by intrathecal (i.t.) application of TRPV1 antagonists in streptozocin (STZ)-induced DMA rat models. Western blot and immunofluorescent staining results showed that TRPV1 protein level was significantly increased in the soma of the dorsal root ganglion (DRG) neurons on 14 days after STZ treatment (DMA 14 d), whereas those in spinal cord and skin (mainly from the central and peripheral processes of DRG neurons) had already been enhanced on DMA 7 d to peak on DMA 14 d. qRT-PCR experiments confirmed that TRPV1 mRNA level was significantly up-regulated in the DRG on DMA 7 d, indicating a preceding translation of TRPV1 protein in the soma but preferential distribution of this protein to the processes under the DMA conditions. Cell counting assay based on double immunostaining suggested that increased TRPV1-immunoreactive neurons were likely to be small-sized and CGRP-ergic. Finally, single or multiple intrathecal applications of non-specific or specific TRPV1 antagonists, ruthenium red and capsazepine, at varying doses, effectively alleviated DMA, although the effect of the former was more prominent and long-lasting. These results collectively indicate that TRPV1 expression dynamically changes during the development of DMA and this protein may play important roles in mechanical nociception in DRG neurons, presumably through facilitating the release of CGRP.

The airway is covered by multicilia that beat in a metachronous manner toward the mouth to eliminate debris and infectious particles. Coordinated one-directional beating is an essential feature of multicilia in the airway to guarantee proper mucociliary clearance. Defects in ciliary motility lead to primary ciliary dyskinesia (PCD), with major symptoms including bronchitis and other chronic respiratory diseases. Recent work suggested that ciliary motility and planar polarity are required in the process of ciliary alignment that produces coordinated beating. However, the extent to which cilia motility is involved in this process in mammals has not yet been fully clarified. Here, to address the role of ciliary motility in the process of coordinated ciliary alignment, we analyzed Kintoun mice mutants (Ktu(-/-)). Ktu(-/-) exhibited typical phenotypes of PCD with complete loss of ciliary motility in trachea and another ciliated tissue, the brain ependyma. Immunohistochemistry using antibodies against axonemal dynein confirmed the loss of multiple axonemal dynein components in mutant cilia. Observation of cilia orientation based on basal foot directions revealed that ciliary motility was not required in the alignment of airway cilia, whereas a strong requirement was observed in brain ependymal cells. Thus we conclude that the involvement of ciliary motility in the establishment of coordinated ciliary alignment varies among tissues.

Complement plays a vital role in both the innate and adaptive immune systems. It recognizes a target, opsonizes it, generates anaphylatoxins, and directly kills cells through the membrane attack complex (MAC). This final function, which assembles C5b-9(n) on viable cell surfaces, can kill host cells through bystander lysis. Here we identify for the first time compounds that can inhibit bystander lysis while not interfering with the other essential functions of complement. We show that aurin tricarboxylic acid (ATA), aurin quadracarboxylic acid (AQA), and aurin hexacarboxylic acid (AHA), block the addition of C9 to C5b-8 so that the MAC cannot form. These molecules inhibit hemolysis of human, rat, and mouse red cells with a half maximal inhibitory concentration (IC(50)) in the nanomolar range. When given orally to Alzheimer disease type B6SJL-Tg mice, they inhibit MAC formation in serum and improve memory retention. On autopsy, they show no evidence of harm to any organ. Aurin tricarboxylic acid, aurin quadracarboxylic acid, and aurin hexacarboxylic acid may be effective therapeutic agents in Alzheimer disease and other degenerative disorders where self damage from the MAC occurs.

BACKGROUND The role of the plasminogen-plasmin (PLG-PLA) system in fertilization is unknown, although its dysfunction has been associated with subfertility in humans. We have recently detected and quantified plasminogen in the oviductal fluid of two mammals and showed a reduction in sperm penetration during IVF when plasminogen is present. The objective of this study was to describe the mechanism by which PLG-PLA system regulates sperm entry into the oocyte. METHODS AND RESULTS By combining biochemical, functional, electron microscopic, immunocytochemical and live cell imaging methods, we show here that (i) plasminogen is activated into the protease plasmin, by gamete interaction; (ii) urokinase-type and tissue-type plasminogen activators are present in oocytes, but they are not of cortical granule origin; (iii) sperm binding to oocytes triggers the releasing of plasminogen activators and (iv) the generated plasmin causes sperm detachment from the zona pellucida. CONCLUSIONS Our results describe a novel mechanism for the success or failure of fertilization in mammals, by which molecules present in the oviductal environment are activated by molecules originating within the gametes. We anticipate that therapeutic up- or down-regulation of this physiological mechanism may be used to help in conception or as a contraceptive tool. Since components of the PLG-PLA system are already available as drugs for heart attacks or cancer therapies, basic research on this novel function would be rapidly transferable for clinical application.

The diagnosis of Parkinson's disease (PD) is currently based on the clinical evaluation of extrapyramidal signs with a considerable error rate. The identification of specific markers might allow PD diagnosis before the onset of classical motor symptoms. By two-dimensional electrophoresis we identified proteome alterations in T-lymphocytes of 17 control subjects and 15 PD patients. The observed changes were used to build predictive models that were verified by the leave-one-out cross-validation. We further built two functions able to stage the subjects. We chose to verify by Western blotting the identity of spots corresponding to β-fibrinogen and transaldolase, two recurrent proteins in six out of 20 spots. β-Fibrinogen levels are lowered in PD patients, whereas a heavy transaldolase set of isoforms was more abundant. Eventually, we identified a list of seven proteins showing different levels in early-onset with respect to late-onset PD patients.

Exposure to stress can result in an increased risk for psychiatric disorders, especially among genetically predisposed individuals. Neuregulin 1 (NRG1) is a susceptibility gene for schizophrenia and is also associated with psychotic bipolar disorder. In the rat, the neurons of the hypothalamic paraventricular nucleus show strong expression of Nrg1 mRNA. In patients with schizophrenia, a single nucleotide polymorphism in the 5' region of NRG1 interacts with psychosocial stress to affect reactivity to expressed emotion. However, there is virtually no information on the role of NRG1 in hypothalamic-pituitary-adrenal axis function, and whether the protein is expressed in the paraventricular nucleus is unknown. The present studies utilize a unique line of Nrg1 hypomorphic rats (Nrg1(Tn)) generated by gene trapping with the Sleeping Beauty transposon. We first established that the Nrg1(Tn) rats displayed reduced expression of both the mRNA and protein corresponding to the Type II NRG1 isoform. After confirming, using wild type animals, that Type II NRG1 is expressed in the neurocircuitry involved in regulating hypothalamic-pituitary-adrenal axis responses to environmental stimuli, the Nrg1(Tn) rats were then used to test the hypothesis that altered expression of Type II NRG1 disrupts stress regulation and reactivity. In support of this hypothesis, Nrg1(Tn) rats have disrupted basal and acute stress recovery corticosterone secretion, differential changes in expression of glucocorticoid receptors in the pituitary, paraventricular nucleus and hippocampus, and a failure to habituate to an open field. Together, these findings point to NRG1 as a potential novel regulator of neuroendocrine responses to stress as well as behavioral reactivity.

The trout heart is 10-fold more sensitive to Ca(2+) than the mammalian heart. This difference is due, in part, to cardiac troponin C (cTnC) from trout having a greater Ca(2+) affinity than human cTnC. To determine what other proteins are involved, we cloned cardiac troponin I (cTnI) from the trout heart and determined how it alters the Ca(2+) affinity of a cTn complex containing all mammalian components (mammalian cTn). Ca(2+) activation of the complex was characterized using a human cTnC mutant that contains anilinonapthalenesulfote iodoacetamide attached to Cys53. When the cTn complex containing labeled human cTnC was titrated with Ca(2+), its fluorescence changed, reaching an asymptote upon saturation. Our results reveal that trout cTnI lacks the N-terminal extension found in cTnI from all other vertebrate groups. This protein domain contains two targets (Ser23 and Ser24) for protein kinase A (PKA) and protein kinase C. When these are phosphorylated, the rate of cardiomyocyte relaxation increases. When rat cTnI in the mammalian cTn complex was replaced with trout cTnI, the Ca(2+) affinity was increased ∼1.8-fold. This suggests that trout cTnI contributes to the high Ca(2+) sensitivity of the trout heart. Treatment of the two cTn complexes with PKA decreased the Ca(2+) affinity of both complexes. However, the change for the complex containing rat cTnI was 2.2-fold that of the complex containing trout cTnI. This suggests that the phosphorylation of trout cTnI does not play as significant a role in regulating cTn function in trout.

β-Adrenergic stimulation of the mammalian heart increases heart rate, the strength of contraction as well as the kinetics of force generation and relaxation. These effects are due to the phosphorylation of select membrane and thin filament proteins by cAMP-activated protein kinase (PKA). At the level of the sarcomere, it is typically the phosphorylation of cardiac myosin binding protein C (cMyBP-C) and cardiac troponin I (cTnI) that is responsible for the change in the kinetics of contraction and relaxation. Trout cTnI (ScTnI) lacks two critical PKA targets within the N-terminus of the protein that, when phosphorylated in mammalian cTnI, cause a reduction in myofilament Ca(2+) affinity. To determine what role the contractile element plays in the response of the trout heart to β-adrenergic stimulation, we characterized the influence of PKA treatment on the Ca(2+) activation of skinned preparations dissected from ventricular trabeculae. In these experiments, isometric force generation and the rate of force development were measured over a range of Ca(2+) concentrations. The results demonstrate that PKA treatment does not influence the Ca(2+) sensitivity of force generation but it decreases maximum force generation by 25% and the rate of force re-development at maximal activation by 46%. Analysis of the trabeculae preparations for phosphoproteins revealed that PKA treatment phosphorylated myosin light chain 2 but not cTnI or cMyBP-C. These results indicate that the function of the trout cardiac contractile element is altered by PKA phosphorylation but in a manner different from that in mammalian heart.

In this study, we investigated whether the anti-inflammatory drug PP56 (alpha-trinositol) may improve cancer-induced metabolic disorders. We implanted human MiaPaCa2 pancreatic cancer cells in the pancreas of 14 athymic mice for 12 weeks, using six intact littermates as normal controls. During the 12 weeks, seven tumor-cell recipients were treated with PP56 by daily injection (PPT mice). The tumor-cell recipients that were otherwise untreated were used as tumor controls (TC mice). Impaired glucose tolerance and decreased body weight gain were seen in TC but not PPT mice. When an enzyme for fatty acid beta-oxidation namely medium-chain acyl-CoA dehydrogenase (MCAD) was determined in tumor grafts; tumors from PPT mice showed more MCAD than those from TC mice. This suggests that PP56 stimulated fatty acid beta-oxidation in MiaPaCa2 cells in vivo. In keeping with this notion, PPT mice had decreased plasma free fatty acids. In vitro, we demonstrated that MiaPaCa2 cells consumed more fatty acids in the presence of PP56. In another experiment, we infused PP56 or vehicle in normal mice and found that PP56 decreased circulating glucose in the animals. We also showed that PP56 increased glucose transport in L6 skeletal muscle cells in vitro. In conclusion, PP56 increases the turnover of circulating nutrients such as glucose and helps maintain energy homeostasis in mice with pancreatic cancer.