Inhibition of L-type Ca<Superscript>2+</Superscript> current in guinea pig ventricular myocytes by cisapride

The effect of cisapride on L-type Ca²⁺ current (I _Ca,L) was studied in guinea pig ventricular myocytes using a whole-cell voltage-clamp technique and a conventional action potential recording method. Myocytes were held at –40 mV, and internally dialyzed and externally perfused with Na⁺- and K⁺-free solutions; cisapride elicited a concentration-dependent block of peakI _Ca,L, with a half-maximum inhibition concentration (IC₅₀) of 46.9 µM. There was no shift in the reversal potential, nor any change in the shape of the current-voltage relationship ofI _Ca,L in the presence of cisapride. Inhibition of cisapride was not associated with its binding to serotonin or to -adrenergic receptors because ketanserin, SB203186, and prazosin had no effect on the inhibitory action of cisapride onI _Ca,L. Cisapride elicited a tonic block and a use-dependent block ofI _Ca,L. These blocking effects were voltage dependent as the degree of inhibition at –40 mV was greater than that at –70 mV. Cisapride shifted the steady-state inactivation curve ofI _Ca,L in the negative direction, but had no effect on the steady-state activation curve. Cisapride also delayed the kinetics of recovery ofI _Ca,L from inactivation. At a slow stimulation frequency (0.1 Hz), the action potential duration in guinea pig papillary muscles showed biphasic effects; it was prolonged by lower concentrations of cisapride, but shortened by higher concentrations. These findings suggest that cisapride preferentially binds to the inactivated state of L-type Ca²⁺ channels. The inhibitory effect of cisapride onI _Ca,L might play an important role in its cardiotoxicity under pathophysiological conditions, such as myocardial ischemia.     

radish encodes a phospholipase-A2 and defines a neural circuit involved in anesthesia-resistant memory

BACKGROUND: In both vertebrate and invertebrate animals, anesthetic agents cause retrograde amnesia for recently experienced events. In contrast, older memories are resistant to the same treatments. In Drosophila, anesthesia-resistant memory (ARM) and long-term memory (LTM) are genetically distinct forms of long-lasting memory that exist in parallel for at least a day after training. ARM is disrupted in radish mutants but is normal in transgenic flies overexpressing a CREB repressor transgene. In contrast, LTM is normal in radish mutants but is disrupted in CREB repressor transgenic flies. To date, nothing is known about the molecular, genetic, or cell biological pathways underlying ARM. RESULTS: Here, we report the molecular identification of radish as a phospholipase-A2, providing the first clue about signaling pathways underlying ARM in any animal. An enhancer-trap allele of radish (C133) reveals expression in a novel anatomical pathway. Transgenic expression of PLA2 under control of C133 restores normal levels of ARM to radish mutants, whereas transient disruption of neural activity in C133 neurons inhibits memory retention. Notably, expression of C133 is not in mushroom bodies, the primary anatomical focus of olfactory memory research in Drosophila. CONCLUSIONS: Identification of radish as a phospholipase-A2 and the neural expression pattern of an enhancer-trap allele significantly broaden our understanding of the biochemistry and anatomy underlying olfactory memory in Drosophila.     

Protein phosphatase 2A dephosphorylation of phosphoserine 112 plays the gatekeeper role for BAD-mediated apoptosis

BAD, a proapoptotic molecule of the BCL2 family, is regulated by reversible phosphorylation. During survival, BAD is sequestered by 14-3-3 through serine 136 phosphorylation and is dissociated from BCL-X(L) through serine 155 phosphorylation. We report that phosphoserine 112 (pSer112) dephosphorylation functions as a gatekeeper for BAD-mediated apoptosis. During apoptosis, dephosphorylation of pSer112 preceded pSer136 dephosphorylation. Dephosphorylation of pSer112 accelerated dephosphorylation of pSer136, and inhibition of pSer112 dephosphorylation prevented pSer136 dephosphorylation, indicating that dephosphorylation of pSer112 is required for dephosphorylation of pSer136. Protein phosphatase 2A (PP2A) is the major pSer112 phosphatase. PP2A competed with 14-3-3 for BAD binding, and survival factor withdrawal enhanced PP2A association with BAD. Dephosphorylation of the critical residue, pSer136, could only be blocked by inhibition of all known subfamilies of serine/threonine phosphatases, suggesting that multiple phosphatases are involved in pSer136 dephosphorylation. Inhibition of PP2A rescued FL5.12 cells from apoptosis, demonstrating a physiologic role for PP2A-mediated pSer112 dephosphorylation. Thus, PP2A dephosphorylation of pSer112 is the key initiating event regulating the activation of BAD during interleukin-3 withdrawal-induced apoptosis.     

A sense of danger from radiation

Tissue damage caused by exposure to pathogens, chemicals and physical agents such as ionizing radiation triggers production of generic "danger" signals that mobilize the innate and acquired immune system to deal with the intrusion and effect tissue repair with the goal of maintaining the integrity of the tissue and the body. Ionizing radiation appears to do the same, but less is known about the role of "danger" signals in tissue responses to this agent. This review deals with the nature of putative "danger" signals that may be generated by exposure to ionizing radiation and their significance. There are a number of potential consequences of "danger" signaling in response to radiation exposure. "Danger" signals could mediate the pathogenesis of, or recovery from, radiation damage. They could alter intrinsic cellular radiosensitivity or initiate radioadaptive responses to subsequent exposure. They may spread outside the locally damaged site and mediate bystander or "out-of-field" radiation effects. Finally, an important aspect of classical "danger" signals is that they link initial nonspecific immune responses in a pathological site to the development of specific adaptive immunity. Interestingly, in the case of radiation, there is little evidence that "danger" signals efficiently translate radiation-induced tumor cell death into the generation of tumor-specific immunity or normal tissue damage into autoimmunity. The suggestion is that radiation-induced "danger" signals may be inadequate in this respect or that radiation interferes with the generation of specific immunity. There are many issues that need to be resolved regarding "danger" signaling after exposure to ionizing radiation. Evidence of their importance is, in some areas, scant, but the issues are worthy of consideration, if for no other reason than that manipulation of these pathways has the potential to improve the therapeutic benefit of radiation therapy. This article focuses on how normal tissues and tumors sense and respond to danger from ionizing radiation, on the nature of the signals that are sent, and on the impact on the eventual consequences of exposure.     

Ectopical expression of human MUC18 increases metastasis of human prostate cancer cells

MUC18, a cell adhesion molecule (CAM), has been reported to be a diagnostic marker for the early detection of the metastatic potential of prostate cancers as well as implicated to be an important determinant for mediating the tumorigenesis and metastasis of prostate cancer. To test the hypothesis, we further investigated the possible role of MUC18 in the malignant progression of human prostate cancer. The human MUC18-minus, non-metastatic human prostate cancer LNCaP cells were transfected with the human cytomegalovirus immediate-early gene (HCMV-IE) promoter-driven human MUC18 (huMUC18) cDNA. The G418-resistant (G418R)-LNCaP clones that expressed a high level of huMUC18 were selected and used for testing the effect of huMUC18 expression on the in vitro growth, motility, and invasiveness as well as on the in vivo metastasis (via orthotopical injection) in a xenograft nude mouse model. HuMUC18 expression increased by four- to fivefold of in vitro motility and invasiveness of LNCaP cells. Anti-huMUC18 antibody significantly inhibited the in vitro motility and invasiveness of huMUC18-expressing LNCaP clones, but not the control clones. We suggest that huMUC18 expression is responsible for increasing these behaviors of LNCaP cells. HuMUC18 expression also directly increased the in vivo metastatic abilities of the LNCaP cells from the prostate gland to multiple distant organs. Western blot and immunohistochemistry analyses showed that the prostatic tumors as well as metastatic lesions expressed high levels of MUC18, indicating that they originated from the injected huMUC18-expressing LNCaP cells. We therefore conclude that HuMUC18 is an important determinant in increasing metastasis of human prostate cancer LNCaP cells to distant organs in a nude mouse model.     

Mechanisms and functional properties of two peptide transporters, AtPTR2 and fPTR2

The Arabidopsis AtPTR2 and fungal fPTR2 genes, which encode H+/dipeptide cotransporters, belong to two different subgroups of the peptide transporter (PTR) (NRT1) family. In this study, the kinetics, substrate specificity, stoichiometry, and voltage dependence of these two transporters expressed in Xenopus oocytes were investigated using the two-microelectrode voltage-clamp method. The results showed that: 1) although AtPTR2 belongs to the same PTR family subgroup as certain H+/nitrate cotransporters, neither AtPTR2 nor fPTR2 exhibited any nitrate transporting activity; 2) AtPTR2 and fPTR2 transported a wide spectrum of dipeptides with apparent affinity constants in the range of 30 microM to 3 mM, the affinity being dependent on the side chain structure of both the N- and C-terminal amino acids; 3) larger maximal currents (Imax) were evoked by positively charged dipeptides in AtPTR2- or fPTR2-injected oocytes; 4) a major difference between AtPTR2 and fPTR2 was that, whereas fPTR2 exhibited low Ala-Asp- transporting activity, AtPTR2 transported Ala-Asp- as efficiently as some of the positively charged dipeptides; 5) kinetic analysis suggested that both fPTR2 and AtPTR2 transported by a random binding, simultaneous transport mechanism. The results also showed that AtPTR2 and fPTR2 were quite distinct from PepT1 and PepT2, two well characterized animal PTR transporters in terms of order of binding of substrate and proton(s), pH sensitivity, and voltage dependence.     

Executive Function Changes before Memory in Preclinical Alzheimer’s Pathology: A Prospective,Cross-Sectional,Case Control Study

Background

Early treatment of Alzheimer’s disease may reduce its devastating effects. By focusing research on asymptomatic individuals with Alzheimer’s disease pathology (the preclinical stage), earlier indicators of disease may be discovered. Decreasing cerebrospinal fluid beta-amyloid₄₂ is the first indicator of preclinical disorder, but it is not known which pathology causes the first clinical effects. Our hypothesis is that neuropsychological changes within the normal range will help to predict preclinical disease and locate early pathology.

Methods and Findings

We recruited adults with probable Alzheimer’s disease or asymptomatic cognitively healthy adults, classified after medical and neuropsychological examination. By logistic regression, we derived a cutoff for the cerebrospinal fluid beta amyloid₄₂/tau ratios that correctly classified 85% of those with Alzheimer’s disease. We separated the asymptomatic group into those with (n = 34; preclinical Alzheimer’s disease) and without (n = 36; controls) abnormal beta amyloid₄₂/tau ratios; these subgroups had similar distributions of age, gender, education, medications, apolipoprotein-ε genotype, vascular risk factors, and magnetic resonance imaging features of small vessel disease. Multivariable analysis of neuropsychological data revealed that only Stroop Interference (response inhibition) independently predicted preclinical pathology (OR = 0.13, 95% CI = 0.04–0.42). Lack of longitudinal and post-mortem data, older age, and small population size are limitations of this study.

Conclusions

Our data suggest that clinical effects from early amyloid pathophysiology precede those from hippocampal intraneuronal neurofibrillary pathology. Altered cerebrospinal fluid beta amyloid₄₂ with decreased executive performance before memory impairment matches the deposits of extracellular amyloid that appear in the basal isocortex first, and only later involve the hippocampus. We propose that Stroop Interference may be an additional important screen for early pathology and useful to monitor treatment of preclinical Alzheimer’s disease; measures of executive and memory functions in a longitudinal design will be necessary to more fully evaluate this approach.     

Tryptase inhibition blocks airway inflammation in a mouse asthma model

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a beta-strand template (K(i) = 45 nM) that does not inhibit trypsin (K(i) = 1,061 nM), thrombin (K(i) = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18-21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.