In Vitro Pharmacological Characterization of RXFP3 Allosterism: An Example of Probe Dependency

Recent findings suggest that the relaxin-3 neural network may represent a new ascending arousal pathway able to modulate a range of neural circuits including those affecting circadian rhythm and sleep/wake states, spatial and emotional memory, motivation and reward, the response to stress, and feeding and metabolism. Therefore, the relaxin-3 receptor (RXFP3) is a potential therapeutic target for the treatment of various CNS diseases. Here we describe a novel selective RXFP3 receptor positive allosteric modulator (PAM), 3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea (135PAM1). Calcium mobilization and cAMP accumulation assays in cell lines expressing the cloned human RXFP3 receptor show the compound does not directly activate RXFP3 receptor but increases functional responses to amidated relaxin-3 or R3/I5, a chimera of the INSL5 A chain and the Relaxin-3 B chain. 135PAM1 increases calcium mobilization in the presence of relaxin-3NH2 and R3/I5NH2 with pEC50 values of 6.54 (6.46 to 6.64) and 6.07 (5.94 to 6.20), respectively. In the cAMP accumulation assay, 135PAM1 inhibits the CRE response to forskolin with a pIC50 of 6.12 (5.98 to 6.27) in the presence of a probe (10 nM) concentration of relaxin-3NH2. 135PAM1 does not compete for binding with the orthosteric radioligand, [125I] R3I5 (amide), in membranes prepared from cells expressing the cloned human RXFP3 receptor. 135PAM1 is selective for RXFP3 over RXFP4, which also responds to relaxin-3. However, when using the free acid (native) form of relaxin-3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Thus one can exchange the entire A-chain of the probe peptide while retaining PAM activity, but the state of the probe's c-terminus is crucial to allosteric activity of the PAM. These data demonstrate the existence of an allosteric site for modulation of this GPCR as well as the subtlety of changes in probe molecules that can affect allosteric modulation of RXFP3

Rev-erb beta regulates the expression of genes involved in lipid absorption in skeletal muscle cells - Evidence for cross-talk between orphan nuclear receptors and myokines

Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Rev-erbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for > 30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (> 15-fold) in mRNA expression of interleukin-6, an exercise-induced myokine that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (> 20- fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth

The reference frame for encoding and retention of motion depends on stimulus set size

YesThe goal of this study was to investigate the reference frames used in perceptual encoding and storage of visual motion information. In our experiments, observers viewed multiple moving objects and reported the direction of motion of a randomly selected item. Using a vector-decomposition technique, we computed performance during smooth pursuit with respect to a spatiotopic (nonretinotopic) and to a retinotopic component and compared them with performance during fixation, which served as the baseline. For the stimulus encoding stage, which precedes memory, we found that the reference frame depends on the stimulus set size. For a single moving target, the spatiotopic reference frame had the most significant contribution with some additional contribution from the retinotopic reference frame. When the number of items increased (Set Sizes 3 to 7), the spatiotopic reference frame was able to account for the performance. Finally, when the number of items became larger than 7, the distinction between reference frames vanished. We interpret this finding as a switch to a more abstract nonmetric encoding of motion direction. We found that the retinotopic reference frame was not used in memory. Taken together with other studies, our results suggest that, whereas a retinotopic reference frame may be employed for controlling eye movements, perception and memory use primarily nonretinotopic reference frames. Furthermore, the use of nonretinotopic reference frames appears to be capacity limited. In the case of complex stimuli, the visual system may use perceptual grouping in order to simplify the complexity of stimuli or resort to a nonmetric abstract coding of motion information

Vampires in the village Žrnovo on the island of Korčula: following an archival document from the 18th century

Središnja tema rada usmjerena je na raščlambu spisa pohranjenog u Državnom arhivu u Mlecima (fond: Capi del Consiglio de’ Dieci: Lettere di Rettori e di altre cariche) koji se odnosi na događaj iz 1748. godine u korčulanskom selu Žrnovo, kada su mještani – vjerujući da su se pojavili vampiri – oskvrnuli nekoliko mjesnih grobova. U radu se podrobno iznose osnovni podaci iz spisa te rečeni događaj analizira u širem društvenom kontekstu i prate se lokalna vjerovanja.The main interest of this essay is the analysis of the document from the State Archive in Venice (file: Capi del Consiglio de’ Dieci: Lettere di Rettori e di altre cariche) which is connected with the episode from 1748 when the inhabitants of the village Žrnove on the island of Korčula in Croatia opened tombs on the local cemetery in the fear of the vampires treating. This essay try to show some social circumstances connected with this event as well as a local vernacular tradition concerning superstitions

A First Implementation of Modular Smalltalk

A "first" implementation of the Modular Smalltalk object-oriented programming language is presented. The implementation includes an object-oriented parser, object-oriented representation for code fragments and an object-oriented C-code generator, all implemented in Smalltalk-80. This implementation validates two of the five design principles of the Modular Smalltalk language and provides a vehicle for validating the other three design principles. The macro-based C-code generator is easily adaptable to generating production code in other languages like assembler. In addition, the generation technique applies to source languages other than Modular Smalltalk. The implementation includes an efficient method dispatch based on new extensions to incremental cache table coloring. KEY WORDS: Modular Smalltalk, object-oriented, programming language, compiler, interpreter, method dispatch, code generation. - 2 - Modular Smalltalk Technical Report 93-07 1. Introduction Modular Smalltalk (MS) i..

Inheritance Management and Method Dispatch in Reflexive Object-Oriented Languages

A collection of algorithms and data structures are presented which represent a generalized framework for inheritance management and method dispatch in reflexive, dynamically typed, single-receiver languages with type/implementation-paired multiple inheritance. By storing a small amount of information, the algorithms can incrementally maintain the entire dispatch environment during the four fundamental environment modification requests -- adding/removing selectors to/from classes and adding/removing class hierarchy links. By merging inheritance management, inheritance conflict detection and method dispatch calculation, the algorithms are computationally efficient, and can be used to maintain dispatch information even in a reflexive environment. The algorithms are applicable to all table-based method dispatch techniques and require the use of only a few dispatch-specific functions. A group of object-oriented classes are used to implement both the technique-independent and technique-depen..

Incremental Table-Based Method Dispatch for Reflective Object-Oriented Languages

A collection of algorithms and data structures are presented which provide incremental dispatch table modification. Incrementally modified dispatch tables allow table-based dispatch techniques to be used in reflective, dynamically typed, single-receiver languages with type/implementation-paired multiple inheritance. By storing a small amount of information, the algorithms can incrementally maintain the entire dispatch environment during the four fundamental environment modification requests: adding/removing selectors to/from classes and adding/removing class hierarchy links. The algorithms combine method dispatch calculation, inheritance management, and inheritance conflict detection into a single process, making the algorithms highly efficient. The algorithms also incrementally compute hierarchy information to establish which method addresses can be uniquely identified during compilation