Effect of Sirtuin-1 on Synaptic Plasticity in Nucleus Accumbens in a Rat Model of Heroin Addiction.

BACKGROUND Synaptic plasticity plays an important role in the process of addiction. This study investigated the relationship between synaptic plasticity and changes in addictive behavior and examined the expression of synaptic plasticity-associated proteins and genes in the nucleus accumbens (NAc) region in different rat models. MATERIAL AND METHODS Heroin addiction, SIRT1-overexpression, and SIRT1-silenced rat models were established. Polymerase chain reaction gene chip technology, immunohistochemistry, Western blotting, and transmission electron microscopy were used to detect changes in synaptic plasticity-related gene and protein expression, and changes in the ultrastructure of synapses, in the NAc. RESULTS Naloxone withdrawal symptoms appeared in the SIRT1-overexpression group. In the SIRT1-silenced group the symptoms were reduced. Immunohistochemistry and Western blotting results showed that FOXO1 expression decreased in the heroin addiction (HA) group but increased in the SIRT1-silenced group (p<0.05). The expression of Cdk5, Nf-κB, PSD95, and Syn was enhanced in the HA group (p<0.05) and further increased in the SIRT1-overexpression group but were reduced in the SIRT1-silenced group (p<0.05). The number of synapses increased in the HA group (p<0.05) along with mitochondrial swelling in the presynaptic membrane and obscuring of the synaptic cleft. CONCLUSIONS SIRT1 and other synaptic plasticity-related genes in NAc are involved in the regulation of heroin addiction. SIRT1 overexpression can increase behavioral sensitization in the NAc of rats, and SIRT1 silencing might ease withdrawal symptoms and reduce conditioned place preferences.

Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

Humanization of Yeasts for Glycan-Type End-Products.

Yeasts are often considered microorganisms for producing human therapeutic glycosylated end-products at an industrial scale. However, the products with non-humanized glycans limited their usage. Therefore, various methods to develop humanized glycosylated end-products have been widely reported in yeasts. To make full use of these methods, it is necessary to summarize the present research to find effective approaches to producing humanized products. The present research focuses on yeast species selection, glycosyltransferase deletion, expression of endoglycosidase, and expression of proteins with galactosylated and or sialylated glycans. Nevertheless, the yeasts will have growth defects with low bioactivity when the key enzymes are deleted. It is necessary to express the corresponding repairing protein. Compared with N-glycosylation, the function of yeast protein O-glycosylation is not well-understood. Yeast proteins have a wide variety of O-glycans in different species, and it is difficult to predict glycosylation sites, which limits the humanization of O-glycosylated yeast proteins. The future challenges include the following points: there are still many important potential yeasts that have never been tried to produce glycosylated therapeutic products. Their glycosylation pathway and related mechanisms for producing humanized glycosylated proteins have rarely been reported. On the other hand, the amounts of key enzymes on glycan pathways in human beings are significantly more than those in yeasts. Therefore, there is still a challenge to produce a large body of humanized therapeutic end-products in suitable yeast species, especially the protein with complex glycans. CRISPR-Cas9 system may provide a potential approach to address the important issue.

Understanding the cardioprotective effects of flavonols: discovery of relaxant flavonols without antioxidant activity.

3',4'-Dihydroxyflavonol (DiOHF) is a cardioprotective flavonol that can reduce injury after myocardial ischemia and reperfusion and thus is a promising small molecule for the treatment of cardiovascular disease. Like all vasoactive flavonols reported to date, DiOHF is both relaxant and antioxidant, hindering investigation of the relative contribution of each activity for the prevention of reperfusion injury. This study investigates structure-activity relationships of variations at the 3' and 4' positions of the B ring of DiOHF and vasorelaxant and antioxidant activities. Relaxation of rat isolated aortic rings precontracted with KCl revealed that the most active flavonols were those with a 4'-hydroxyl group, with the opening of potassium channels as a possible contributing mechanism. For the antioxidant activity, with the exception of DiOHF, none of the flavonols investigated were able to significantly scavenge superoxide radical, and none of the three most potent vasorelaxant flavonols could prevent oxidant-induced endothelial dysfunction. The discovery of single-acting vasorelaxant flavonols without antioxidant activity, in particular 4'-hydroxy-3'-methoxyflavonol, will assist in investigating the mechanism of flavonol-induced cardioprotection.

Conformational dynamics of the essential sensor histidine kinase WalK.

Two-component systems (TCSs) are key elements in bacterial signal transduction in response to environmental stresses. TCSs generally consist of sensor histidine kinases (SKs) and their cognate response regulators (RRs). Many SKs exhibit autokinase, phosphoryltransferase and phosphatase activities, which regulate RR activity through a phosphorylation and dephosphorylation cycle. However, how SKs perform different enzymatic activities is poorly understood. Here, several crystal structures of the minimal catalytic region of WalK, an essential SK from Lactobacillus plantarum that shares 60% sequence identity with its homologue VicK from Streptococcus mutans, are presented. WalK adopts an asymmetrical closed structure in the presence of ATP or ADP, in which one of the CA domains is positioned close to the DHp domain, thus leading both the ß- and γ-phosphates of ATP/ADP to form hydrogen bonds to the ℇ- but not the δ-nitrogen of the phosphorylatable histidine in the DHp domain. In addition, the DHp domain in the ATP/ADP-bound state has a 25.7° asymmetrical helical bending coordinated with the repositioning of the CA domain; these processes are mutually exclusive and alternate in response to helicity changes that are possibly regulated by upstream signals. In the absence of ATP or ADP, however, WalK adopts a completely symmetric open structure with its DHp domain centred between two outward-reaching CA domains. In summary, these structures of WalK reveal the intrinsic dynamic properties of an SK structure as a molecular basis for multifunctionality.

Suppressing effect of Ca

As local signals, calcium puffs arise from the concerted opening of a few nearby inositol 1,4,5-trisphospate receptor channels to release Ca^{2+} ions from the endoplasmic reticulum. Although Ca^{2+} puffs have been well studied, little is known about the modulation of cytosolic basal Ca^{2+} concentration ([Ca^{2+}]_{Basal}) on puff dynamics. In this paper we consider a puff model to study how the statistical properties of puffs are modulated by [Ca^{2+}]_{Basal}. The puff frequency and lifetime trivially increase with the increasing [Ca^{2+}]_{Basal}, but an unexpected result is that the puff amplitude and the maximum open-channel number of the puff show decreasing relationship with the increasing [Ca^{2+}]_{Basal}. The underlying dynamics is related not only to the increasing puff frequency which gives a shorter recovery time, but also to the increasing frequency of blips with only one channel open. We indicate that Ca^{2+} blips cause the channels to be inhibited and prevent their recovery during interpuff intervals, resulting in the suppressing effect on puff amplitudes. With increasing [Ca^{2+}]_{Basal}, more blips occur to cause more channels to be inhibited, leaving fewer channels available for puff events. This study shows that the blips may play relevant functions in global Ca^{2+} waves through modulating puff dynamics.

Conformational Dynamics of Response Regulator RegX3 from Mycobacterium tuberculosis.

Two-component signal transduction systems (TCS) are vital for adaptive responses to various environmental stresses in bacteria, fungi and even plants. A TCS typically comprises of a sensor histidine kinase (SK) with its cognate response regulator (RR), which often has two domains-N terminal receiver domain (RD) and C terminal effector domain (ED). The histidine kinase phosphorylates the RD to activate the ED by promoting dimerization. However, despite significant progress on structural studies, how RR transmits activation signal from RD to ED remains elusive. Here we analyzed active to inactive transition process of OmpR/PhoB family using an active conformation of RegX3 from Mycobacterium tuberculosis as a model system by computational approaches. An inactive state of RegX3 generated from 150 ns molecular dynamic simulation has rotameric conformations of Thr79 and Tyr98 that are generally conserved in inactive RRs. Arg81 in loop ß4α4 acts synergistically with loop ß1α1 to change its interaction partners during active to inactive transition, potentially leading to the N-terminal movement of RegX3 helix α1. Global conformational dynamics of RegX3 is mainly dependent on α4ß5 region, in particular seven 'hot-spot' residues (Tyr98 to Ser104), adjacent to which several coevolved residues at dimeric interface, including Ile76-Asp96, Asp97-Arg111 and Glu24-Arg113 pairs, are critical for signal transduction. Taken together, our computational analyses suggest a molecular linkage between Asp phosphorylation, proximal loops and α4ß5α5 dimeric interface during RR active to inactive state transition, which is not often evidently defined from static crystal structures.

Deciphering the underlying mechanisms of Diesun Miaofang in traumatic injury from a systems pharmacology perspective.

Diesun Miaofang (DSMF) is a traditional herbal formula, which has been reported to activate blood, remove stasis, promote qi circulation and relieve pain. DSMF holds a great promise for the treatment of traumatic injury in an integrative and holistic manner. However, its underlying mechanisms remain to be elucidated. In the present study, a systems pharmacology model, which integrated cluster ligands, human intestinal absorption and aqueous solution prediction, chemical space mapping, molecular docking and network pharmacology techniques were used. The compounds from DSMF were diverse in the clusters and chemical space. The majority of the compounds exhibited drug-like properties. A total of 59 compounds were identified to interact with 16 potential targets. In the herb-compound-target network, the majority of compounds acted on only one target; however, a small number of compounds acted on a large number of targets, up to a maximum of 12. The comparison of key topological properties in compound-target networks associated with the above efficacy intuitively demonstrated that potential active compounds possessed diverse functions. These results successfully explained the polypharmacological mechanism underlying the efficiency of DSMF for the treatment of traumatic injury as well as provided insight into potential novel therapeutic strategies for traumatic injury from herbal medicine.

Understanding the polypharmacological anticancer effects of Xiao Chai Hu Tang via a computational pharmacological model.

Xiao Chai Hu Tang (XCHT), a traditional herbal formula, is widely administered as a cancer treatment. However, the underlying molecular mechanisms of its anticancer effects are not fully understood. In the present study, a computational pharmacological model that combined chemical space mapping, molecular docking and network analysis was employed to predict which chemical compounds in XCHT are potential inhibitors of cancer-associated targets, and to establish a compound-target (C-T) network and compound-compound (C-C) association network. The identified compounds from XCHT demonstrated diversity in chemical space. Furthermore, they occupied regions of chemical space that were the same, or close to, those occupied by drug or drug-like compounds that are associated with cancer, according to the Therapeutic Targets Database. The analysis of the molecular docking and the C-T network demonstrated that the potential inhibitors possessed the properties of promiscuous drugs and combination therapies. The C-C network was classified into four clusters and the different clusters contained various multi-compound combinations that acted on different targets. The study indicated that XCHT has a polypharmacological role in treating cancer and the potential inhibitory components of XCHT require further investigation as potential therapeutic strategies for cancer patients.