A novel cell tool for α2δ-1-NMDAR target-based analgesic drug discovery / 生物工程学报

The α2δ-1 protein coded by Cacna2d1 is dramatically up-regulated in dorsal root ganglion (DRG) neurons and spinal dorsal horn following sensory nerve injury in various animal models of neuropathic pain. Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. The α2δ-1-NMDAR interaction promotes surface trafficking and synaptic targeting of NMDARs in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury, as well as in other pathological conditions such as in the paraventricular nucleus (PVN) with neurogenic hypertension and in the brain with ischemic stroke. The lentiviral transfection method was used to construct a human embryonic kidney HEK293T cell line that could stably express α2δ-1-NMDAR complex. A stably transfected cell line was observed by florescence microscope, and identified by RT-qPCR and Western blotting. The results showed that the HEK293T cell line was successfully transfected and the genes could be stably expressed. Subsequently, the transfected cell line was successfully developed into a target drug screening system using patch clamp techniques. It provides a promising cell model for further research on the interaction mechanism of α2δ-1-NMDAR complex and drug screening for chronic pain and related diseases with low side effects.

mTORC1 signaling specifically promotes proliferation of hair follicle stem cells during the telogen-to-anagen transition / 中华皮肤科杂志

Objective@#To identify the time window during which the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway plays a key role in telogen-to-anagen transition of hair follicles, and to explore whether the pathway specifically promotes the proliferation of hair follicle stem cells (HFSCs) .@*Methods@#Totally, 36 newborn ICR mice were randomly and equally divided into 3 groups: RAPA-P19 group intraperitoneally injected with 5 mg·kg-1·d-1 sirolimus on days 19-24 after birth, RAPA-P21 group intraperitoneally injected with 5 mg·kg-1·d-1 sirolimus on days 21-24 after birth, and control group intraperitoneally injected with the same volume of solvent on days 19-24 after birth. Four mice were sacrificed in each group on days 22, 23 and 24 separately. Skin tissues were resected from the back, and hematoxylin-eosin staining of the skin tissues were performed followed by observation of hair follicle morphology to evaluate whether murine hair follicles progressed into the anagen phase on day 24. Immunofluorescence costaining was conducted to determine the expression and localization of mTORC1 downstream molecular marker pS6 and cell proliferation marker Ki67 on days 22 and 23.@*Results@#On day 24, hematoxylin-eosin staining showed anagen hair follicles in the control group and RAPA-P21 group, but telogen hair follicles in the RAPA-P19 group. On days 22 and 23, immunofluorescence costaining revealed positive staining for both pS6 and Ki67 in HFSCs in the control group, negative staining for both pS6 and Ki67 in the RAPA-P19 group, negative staining for pS6 and positive staining for Ki67 in the RAPA-P21 group. On day 23, epidermal cells and sebaceous gland cells in the upper hair follicle bulge were stained positively for Ki67 in all the 3 groups.@*Conclusion@#mTORC1 signaling specifically promotes the proliferation of HFSCs during telogen-to-anagen transition, but not affects proliferation of other cells in hair follicles.

Role of DNA methyltransferase 1 in mouse skin aging / 中南大学学报(医学版)

Objective:To explore the role ofDNA methyltransferase 1 (DNMT1) in mouse skin aging.Methods:Epidermal conditional K14 Cre-mediated DNA methyltransferase 1 (DNMT1) knockout mice (Mut group,n=4) and the littermate normal mice with the same age (WT group) n=4) were used in this study.HE staining was used to detect the pathological changes of skin;the changes of number in the dermal elastic fibers were detected by Gomori aldehyde fuchsin staining,the number of 5-bromo-2-deoxyuridine (BrdU)-labeled transit amplifying cells (TAC) in epidermis were detected by immunohistochemical staining;the number of chlorodeoxyuridine (CldU)-labelretaining cells (LRC) in epidermis were detected by immunofluorescent staining.Results:Compared with the WT group,the skin showed premature aging symptoms in the Mut group concomitant with the decreased epidermal thickness as well as the number of dermal collagen fibers,while the increased dermal elastic fiber fracture.Compared with the WT group,the number of TAC in the epidermis was significantly increased (P＜0.05),and the number of LRC was significantly decreased (P＜0.05) in the Mut group.Conclusion:The phenotype of skin premature aging in epidermal stem cell conditional DNMT1-knockout mice suggests an important role of DNMT 1 in skin aging.

Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI / 生物工程学报

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.