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Spinosyn A is a polyketide-derived macrolide produced by Saccharopolyspora spinosa and is an active ingredient in several commercial insecticides. It is glycosylated by a tri-O-methylated rhamnose at C-9 and a forosamine at C-17. Previous studies indicated that the rhamnose methyltransferases are encoded by the spnH, spnI and spnK genes. To verify the functions of these methyltransferases and to study how they are coordinated to achieve the desired level of methylation of rhamnose, we studied the catalytic properties of the spnH, spnI and spnK gene products and validated their roles in the permethylation process of spinosyn A. Our data reported herein firmly established that SpnH, SpnI, and SpnK are the respective rhamnose 4′-, 2′-, and 3′-Omethyltransferase. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK and SpnH in sequence is productive for the permethylation of the rhamnose moiety. Moreover, the completion of rhamnose permethylation is likely achieved by the proper control of the expression levels of the methyltransferase genes involved. These results set the stage for future exploitation of spinosyn biosynthetic pathway to produce targeted spinosyn derivatives and, perhaps, new analogues.Spinosyn A (SPA, 1) is a polyketide-derived macrolide produced by Saccharopolyspora spinosa, that is an active ingredient in several commercial insecticides. 1 The structures of SPA and its many analogues have a characteristic perhydro-as-indacene core, which is glycosylated by a rhamnose (see 2) at C-9 and a forosamine at C-17. Both the aglycone (AGL, 3) and the sugar appendages contribute to the observed activity of the spinosyns, among which SPA is most potent. 2 Alteration of the tetracyclic nucleus or removal of either deoxy sugars significantly diminishes the pesticidal activity. Even subtle structural variations, such as the methylation pattern of the rhamnose moiety in spinosyns, change the LD 50 by as much as >200- 3 the study of how the corresponding methyltransferases are coordinated to achieve the desired level of methylation of rhamnose has been a focus of this research. We report herein the function and substrate specificity of the three methyltransferases involved in the methylation reactions, the preferred reaction sequence of their catalyzed reactions, and the likely regulation of permethylation of rhamnose in 1.The spinosyn biosynthetic gene cluster had been cloned from S. spinosa. 4 Sequence analysis and gene disruption experiments revealed that the spnH, spnI and spnK genes, 4 all of which show high sequence identity to those encoding S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs), are involved in the O-methylation of rhamnose in 1. 4,5 As illustrated in Scheme 1, methylations may take place before (2 → 4 → 6, route A) or after (3 → 5 → 6, route B) the attachment of rhamnose to the aglycone (AGL, 3). It is also possible that methylations are the final tailoring steps after both sugars have been coupled to the aglycone (...
Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, but its role in neuropathic pain remains unclear. In this study, we examined the ER stress and the unfolded protein response (UPR) activation in a L5 spinal nerve ligation (SNL)-induced rat neuropathic pain model. SNL-induced neuropathic pain was assessed behaviorally using the CatWalk system, and histologically with microglial activation in the dorsal spinal horn. L5 SNL induced BIP upregulation in the neuron of superficial laminae of dorsal spinal horn. It also increased the level of ATF6 and intracellular localization into the nuclei in the neurons. Moreover, spliced XBP1 was also markedly elevated in the ipsilateral spinal dorsal horn. The PERK-elF2 pathway was activated in astrocytes of the spinal dorsal horn in the SNL model. In addition, electron microscopy revealed the presence of swollen cisternae in the dorsal spinal cord after SNL. Additionally, inhibition of the ATF6 pathway by intrathecal treatment with ATF6 siRNA reduced pain behaviors and BIP expression in the dorsal horn. The results suggest that ER stress might be involved in the induction and maintenance of neuropathic pain. Furthermore, a disturbance in UPR signaling may render the spinal neurons vulnerable to peripheral nerve injury or neuropathic pain stimuli.
Tonic extrasynaptic GABAA receptor (GABAAR) activation is under the tight control of tonic GABA release from astrocytes to maintain the brain's excitation/inhibition (E/I) balance; any slight E/I balance disturbance can cause serious pathological conditions including epileptic seizures. However, the pathophysiological role of tonic GABA release from astrocytes has not been tested in epileptic seizures. Here, we report that pharmacological or genetic intervention of the GABA‐permeable Bestrophin‐1 (Best1) channel prevented the generation of tonic GABA inhibition, disinhibiting CA1 pyramidal neuronal firing and augmenting seizure susceptibility in kainic acid (KA)‐induced epileptic mice. Astrocyte‐specific Best1 over‐expression in KA‐injected Best1 knockout mice fully restored the generation of tonic GABA inhibition and effectively suppressed seizure susceptibility. We demonstrate for the first time that tonic GABA from reactive astrocytes strongly contributes to the compensatory shift of E/I balance in epileptic hippocampi, serving as a good therapeutic target against altered E/I balance in epileptic seizures.
Neuropathic pain is a complex, chronic pain state characterized by hyperalgesia, allodynia, and spontaneous pain. Accumulating evidence has indicated that the microglial Toll-like receptor 4 (TLR4) and autophagy are implicated in neurodegenerative diseases, but their relationship and role in neuropathic pain remain unclear. In this study, we examined TLR4 and its association with autophagic activity using a chronic constriction injury (CCI)-induced neuropathic pain model in wild-type (WT) and TLR4-knockout (KO) mice. The mice were assigned into four groups: WT-Contralateral (Contra), WT-Ipsilateral (Ipsi), TLR4 KO-Contra, and TLR4 KO-Ipsi. Behavioral and mechanical allodynia tests and biochemical analysis of spinal cord tissue were conducted following CCI to the sciatic nerve. Compared with the Contra group, mechanical allodynia in both the WT- and TLR4 KO-Ipsi groups was significantly increased, and a marked decrease of allodynia was observed in the TLR4 KO-Ipsi group. Although glial cells were upregulated in the WT-Ipsi group, no significant change was observed in the TLR4 KO groups. Moreover, protein expression and immunoreactive cell regulation of autophagy (Beclin 1, p62) were significantly increased in the neurons, but not microglia, of WT-Ipsi group compared with the WT-Contra group. The level of PINK1, a marker for mitophagy was increased in the neurons of WT, but not in TLR4 KO mice. Together, these results show that TLR4-mediated p62 autophagic impairment plays an important role in the occurrence and development of neuropathic pain. And what is more, microglial TLR4-mediated microglial activation might be indirectly coupled to neuronal autophage.
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