Supplementary Figure 4 from Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Catastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage
Supplementary Figure 1 from Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Catastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage
HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear.Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation.In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence.In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner.Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤4 µg/ml) and stimulates CSR at high concentrations (≥8 µg/ml).On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription.Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1.Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs.We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.
Objective To study the clinical and genetic features of a family with hereditary spastic paraplegia(HSP).Methods The patients were from a large Linyi family.Five members were clinically diagnosed with HSP according to Harding's criteria Blood samples were collected from family members.Genomic DNA was extracted from total blood samples using a standard phenol-chloroform extraction.The genetic linkage analysis was performed using microsatellite markers.Two-point linkage analysis was performed using the LINKAGE program.Five members underwent detailed neurological examinations and 4 members underwent electrophysiological analysis,cervical and thoracic MRI and serum enzymes.Results Linkage analysis mapped the AD-HSP locus to chromosome 2p12(SPG31)in this family.Positive LOD scores were obtained for SPG31 markers on chromosome 2 with a maximum multipoint LOD score of Z=1.8.Analysis of the REEP1 gene revealed a heterozygous G-to-A mutation at nucleotide position c417+1 donor site in exon 5.resulting in splice-site mutation.The symptoms of the patients manifested as stiffness,instability or weakness of the legs.MRI of the thoracic revealed atrophies of the spinal cord in the proband's son.Conclusions SPC31 patients have the clinical features of the typical HSP characteristics.REEP1 gene is the pathogenic gene.with REEP1 c417+1G＞A heterozygous mutation.
Key words:
Spastic paraplegia,hereditary; Pedigree; Membrane transport proteins; Heterozygote; Mutation
Abstract Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor β chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1β induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1β to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1βR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation.
The infection with high-risk human papillomavirus is linked to cervical cancer, nevertheless, the role of miRNAs regulated by HPV oncogenes in cancer progression remain largely unknown. Here, we knocked down endogenous E6/E7 in HPV16-positive CaSki cell lines, screened differences in miRNA expression profile with control using miRNA array. 38 miRNAs were down-regulated and 6 miRNAs were up-regulated in the E6/E7 silenced CaSki cells (>2-fold changes with P <0.05). The levels of miR-27b, miR-20a, miR-24, miR-93, and miR-106b were verified by qPCR in E6/E7 silenced CaSki and SiHa cells. MiR-27b, up-regulated by E7, promoted CaSki and SiHa cell proliferation and invasion, inhibit paclitaxel-induced apoptosis. Dual-luciferase experiment confirmed miR-27b down-regulated its target gene PLK2 through the "seed regions". The tumor suppressor PLK2 inhibited SiHa cell proliferation, reduced cell viability, and promoted paclitaxel/cisplatin -induced apoptosis. Furthermore, DGCR8 was found to mediate the up-regulation of miR-27b by HPV16 E7. Our study demonstrated that HPV16 E7 could increase DGCR8 to promote the generation of miR-27b, which accelerated cell proliferation and inhibited paclitaxel-induced cell apoptosis through down-regulating PLK2. These findings provide an insight into the interaction network of viral oncogene, miR-27b and PLK2, and support the potential strategies using antisense nucleic acid of miR-27b for therapy of cervical cancer in the future.
Objective
To study the clinical and electrophysiological features of the patients with hereditary neuropathy with liability to pressure palsy (HNPP) diagnosed by gene analysis.
Methods
Seven patients from two HNPP families were assessed on medical history, physical examination, electrophysiology findings and gene analysis.
Results
A clinical manifestation of acute, painless, recurrent peripheral nerve palsies was typical for HNPP. Median, ulnar and peroneal nerves were usually affected. Electrophysiology study revealed that prolonged distal motor latency and slowing nerve conduction velocity were prominent. Gene studies exhibited a deletion of the peripheral myelination protein 22 gene in all the seven patients.
Conclusions
HNPP usually affects areas where nerves are subject to entrapment, and many episodes are preceded by minor compression on the affected nerve. As a reliable screening tool in detecting HNPP, the electrophysiological study shows that segmental demyelination is most commonly seen at common nerve entrapment sites.
Key words:
Hereditary sensory and motor neuropathy; Myelin proteins; Gene deletion; Electrophysiology; Polymerase chain reaction
Mucosal-associated invariant T (MAIT) cells participate in both protective immunity and pathogenesis of diseases. Most murine MAIT cells express an invariant TCRVα19-Jα33 (iVα19) TCR, which triggers signals crucial for their development. However, signal pathways downstream of the iVα19TCR and their regulation in MAIT cells are unknown. Diacylglycerol (DAG) is a critical second messenger that relays the TCR signal to multiple downstream signaling cascades. DAG is terminated by DAG kinase (DGK)-mediated phosphorylation and conversion to phosphatidic acid. We have demonstrated here that downregulation of DAG caused by enhanced DGK activity impairs late-stage MAIT cell maturation in both thymus and spleen. Moreover, deficiency of DGKζ but not DGKα by itself causes modest decreases in MAIT cells, and deficiency of both DGKα and ζ results in severe reductions of MAIT cells in an autonomous manner. Our studies have revealed that DAG signaling is not only critical but also must be tightly regulated by DGKs for MAIT cell development and that both DGKα and, more prominently, DGKζ contribute to the overall DGK activity for MAIT cell development.
Altered abundance or activity of the dual-function transient receptor potential melastatin-like 7 (TRPM7) protein is implicated in neurodegenerative disorders, including Alzheimer’s disease (AD). Toxic aggregation of amyloid-β (Aβ) in neurons is implicated in AD pathology. Here, we found that the kinase activity of TRPM7 is important to stimulate the degradation of Aβ. TRPM7 expression was decreased in hippocampal tissue samples from patients with AD and two mouse models of AD ( APP/PS1 and 5XFAD ). In cultures of hippocampal neurons from mice, overexpression of full-length TRPM7 or of its functional kinase domain M7CK prevented synapse loss induced by exogenous Aβ. In contrast, this neuroprotection was not afforded by overexpression of either the functional ion channel portion alone or a TRPM7 mutant lacking kinase activity. M7CK overexpression in the hippocampus of young and old 5XFAD mice prevented and reversed, respectively, memory deficits, synapse loss, and Aβ plaque accumulation. In both neurons and mice, M7CK interacted with and activated the metalloprotease MMP14 to promote Aβ degradation. Thus, TRPM7 loss in patients with AD may contribute to the associated Aβ pathology.
Background/Aims: Interleukin-17 (IL-17) is a major pro-inflammatory cytokine that initiates and maintains inflammation. However, the molecular mechanisms as to how IL-17 influences endothelial cells to promote neutrophil recruitment are not fully understood. Methods: Human endothelial cells (HMECs) were stimulated with IL-17, and investigated for proliferation, migration, and tubule formation activities. Transwell chemotaxis and adhesion assays were performed to assess neutrophil recruitment. Cytokine production was measured by Cytokine Array Chip and ELISA. Western blotting and immunofluorescent analysis were used to detect the phosphorylation and translocation of STAT3. Specific inhibitors, small interfering RNA, and phosphorylation mutants were used to confirm that IL-17 induced STAT3 activation via IL-17RA signaling. Results: Activation of HMECs with IL-17 induced STAT3 phosphorylation and nuclear translocation, which were associated with induction of GRO-α, GM-CSF and IL-8, and neutrophil recruitment. Phosphorylation of STAT3 was identified mainly at the tyrosine in position 705 (Y705), and the Y705F mutants attenuated IL-17-mediated STAT3 activation. Moreover, specific inhibitors, FLLL31, or siRNA silencing of STAT3 attenuated HMECs activation, resulting in inhibition of GRO-α, GM-CSF, IL-8 production, and neutrophil recruitment. Furthermore, phosphorylation of STAT3 was identified as downstream of IL-17RA signaling. Conclusions: IL-17 induced STAT3 activation as a necessary step in endothelial cell activation and neutrophil recruitment.
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