Nuclear Envelope Protein MAN1 Regulates the Drosophila Circadian Clock via Period / 神经科学通报·英文版

Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes. These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, we have shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. Here, we further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced mRNA and protein levels of the core clock gene period (per), likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.

Physiological Cyclic Stretch Inhibits Vascular Smooth Muscle Cell Apoptosis via Inducing Expression of Nuclear Envelope Protein Emerin / 医用生物力学

Objective To investigate the mechanical response of Emerin, a nuclear envelope protein, and its role in apoptosis of vascular smooth muscle cells (VSMCs) during cyclic stretch, and the potential effect of transcriptional factors in this process. Methods Physiological cyclic stretch with the magnitude of 5% and frequency of 1.25 Hz was subjected to VSMCs in vitro by using FX-5000T cyclic stretch loading system. VSMCs cultured under the same conditions but without applying mechanical stretch were used as the static control. The apoptosis of VSMCs was detected by using Cleaved-caspase3 ELISA kit, and the expression of Emerin was revealed by using Western blotting. The effects of Emerin on activities of 345 kinds of transcriptional factors in VSMCs were demonstrated with Protein/DNA array after Emerin specific RNA interference (RNAi) under static condition, and the potential transcriptional factors involved in VSMC apoptosis were analyzed with Ingenurity Pathway Analysis (IPA) software. Furthermore, the binding abilities of Emerin to the motif of 2 kinds of apoptosis-related transcriptional factors were detected with chromatin immunoprecipitation (CHIP) and qPCR. ResultsCompared with the static control, the apoptosis of VSMCs was significantly decreased by 5% cyclic stretch, which suggested a protective effect of physiological cyclic stretch. The expressions of Emerin in VSMCs was remarkably increased with 5% cyclic stretch applied for 6 h, 12 h and 24 h. Specific RNAi under static condition decreased the expressions of Emerin but increased the apoptosis of VSMCs. Emerin siRNA transfection remarkably increased (more than 2 times) the activities of 10 transcriptional factors that participated in cellular apoptosis, i.e. CREB-BP1, p300, p55, MAX, NRF-1, STAT1, STAT3, TEF1, TR and BZP. CHIP-qPCR result revealed that the binding ability of Emerin to specific mofit of STAT1 or STAT3 was significantly repressed with Emerin RNAi. Conclusions Physiological cyclic stretch could increase the expression of Emerin which might modulate the binding of Emerin to motifs of apoptosis-related transcriptional factors such as STAT1 and STAT3, regulate the activities of these factors, and then subsequently repress the VSMC apoptosis. The investigation on mechanobiological mechanisms of VSMC apoptosis induced by cyclic stretch may contribute to further understanding the physiological and pathological mechanisms of vascular homeostasis and vascular remodeling.

Clinical value of combined detection of anti-nuclear envelope protein antibody, anti-soluble acid resistant nucleoprotein and anti-mitochondrial antibody M2 subtype in diagnosis sjogren syndrome and primary biliary cirrhosis / 中国综合临床

Objective To investigate the clinical significance of anti-nuclear envelope protein antibody (gp210),anti-soluble acid resistant nucleoprotein (sp100) and anti-mitochondrial antibody M2 subtype (AMA-M2) in sjogren syndrome (SS) and primary biliary cirrhosis (PBC).Methods A total of 241 hospitalized patients diagnosed with connective tissue disease (CTD) were recruited.Anti-gp210,anti-sp100 and AMA-M2 were detected by indirect immunofluorescence.Results (1) Positive rate of AMA-M2,anti-sp100 and antigp210 in 241 cases CTD patient were 10.4％ (25/241),3.3％ (8/241) and 2.9％ (7/241) respectively.(2) There were 16 cases with SS,5 cases with SS-PBC overlap syndrome and 17 cases with PBC in 241 patients with CTD.Distinction among groups of PBC,SS,SS overlapping PBC of positive incidence of AMA-M2 antibody (x2 =6.584,P =0.03) and anti-gp210 (x2 =8.735,P ＜ 0.01) were significantly different,while there was no apparent difference about positive rate of anti-sp100 among the three groups (x2 =3.343,P =0.18).(3) Positive expression of either antibody of anti-gp210 or anti-sp100 in the three groups of SS,SS overlapping PBC,PBC were 3 cases,4 cases,4 cases respectively.The positive rates of any of three autoantibodies in three groups of were 8 cases,5 cases,13 cases respectively.(4) There were significant difference in terms of serum ALB(t =3.858,P＜0.000 1),TSB(t =5.473,P＜0.000 1),ALT(t =2.235,P=0.026),AKP(t =3.141,P =0.002) and γ-GT (t =2.317,P =0.021) in liver damaged patients of all CTD between AMA-M2 positive and negative patients (P ＜ 0.05).However,serum TSB in anti-sp100 positive and negative patients were differed (t =7.892,P ＜ 0.000 1).Serum AKP was different between anti-gp210 positive and negative patients (t =2.451,P =0.015).Conclusion Positive rate of anti-gp210,anti-sp100 and AMA-M2 are the highest in patients with SS overlap of the PBC among CTD patients.Combined detection can improve the sensitivity of diagnosis.Antisp100 and anti-gp210 are valuable for the diagnosis of SS-PBC overlaps syndrome with negative AMA-M2.

Lymphohematopoietic licence: Sterol C-14 reductase activity of lamin B receptor (Lbr) is essential for neutrophil differentiation.

Bertie Wooster, PG Wodehouse’s fictional character, proudly fancied himself a writer for having once contributed an article (‘What the Well-Dressed Man Is Wearing’) to Milady’s Boudoir, his Aunt Dahlia’s weekly magazine for women. My conceit of expertise in vertebrate lamin B receptor (Lbr) research is slightly less dubious. I co-authored two papers (Papavinasasundaram and Kasbekar 1994 and Prakash et al. 1999) that established that the C-terminal two-thirds of Lbr has sterol Δ14,15 reductase activity. An interloping article (Silve et al. 1998) reached pretty much the same conclusion (and to my chagrin, garnered the lion’s share of the citations). So the recent demonstration by Peter Gaines and coworkers that the Lbr sterol reductase regulates differentiation of neutrophils (Subramanian et al. 2012) filled me with proprietary pride, especially since neutrophils are the most abundant white blood cells in circulation and present the critical first line of defence against infectious microbes. Lbr is an integral protein of the vertebrate nuclear envelope inner membrane. Its N-terminal ~200 residues are hydrophilic, bind to B-type lamins, DNA and HP1-type chromatin proteins, and provide a substrate for p34cdc2, a key mitotic protein kinase. The ~420 residue hydrophobic, membrane-spanning, C-terminal domain (CTD) with sterol reductase activity anchors the nucleoplasmic domain to the inner nuclear membrane. Mutations in human LBR cause Pelger-Huët anomaly (PHA), a benign dominant disorder characterized by hyposegmentation of the neutrophil nucleus (Hoffmann et al. 2002). A spontaneously aborted fetus with Greenberg/HEM dysplasia was homozygous for LBR mutations, and peripheral blood neutrophils from the fetus’ mother displayed PHA (Waterham et al. 2003). Greenberg/HEM dysplasia and PHA reflect the pleiotropism of LBR mutations. In mouse, the Lbr gene is defined by the ichthyosis (ic) mutations (Shultz et al. 2003). Neutrophils from ic/ic mice display bilobed or ovoid nuclei typical of PHA. Additionally, ic/ic homozyogotes exhibit sparse hair, decreased body size and occasionally hydrocephalus and syndactyly. It was of interest to understand the functional significance of the Lbr protein’s sterol reductase activity, especially since KO mice for another locus, Tm7sf2, that encodes SR-1, a 418 residue protein with 58% identity with the Lbr CTD and possessing sterol C−14 reductase activity, do not display an observable phenotype (Bennati et al. 2006, 2008). First, a quick flashback to another 1994 paper: Tsai et al. (1994) had shown that transduction of normal mouse bone marrow cells with a retroviral vector harbouring a dominant-negative retinoic acid receptor (RARα403) could reproducibly immortalize lymphohematopoietic progenitors as stem-cell-factor-dependent clonal lines, designated as EML cells for their ability to subsequently undergo erythroid, myeloid and lymphoid differentiation in vitro. A 3-day treatment of EML cells with stem cell factor, IL-3, and high concentrations of all-trans retinoic acid, and then washing and switching them into GM-CSF, induced their differentiation into promyelocytes, designated as EPRO cells (EML-derived promyelocytes), that can be maintained in GM-CSF. Treatment of EPRO cells with high concentrations of retinoic acid in the presence of GM-CSF induced them to terminally differentiate into mature neutrophils with characteristic nuclear lobulation and respiratory burst response phenotypes. Several years later, Gaines et al. (2008) generated EML- and EPRO-like cells frombonemarrow of a C57BL/6J-Lbric-J/Lbric-J (ic/ic) mouse and a normal (+/ic) littermate, and found that neutrophils derived from EPRO-ic/ic cells exhibited nuclear hypolobulation identical to that seen in ichthyosis mice and displayed a deficient respiratory burst, whereas those from.

Lamins, laminopathies and disease mechanisms: Possible role for proteasomal degradation of key regulatory proteins.

Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.

Association of progerin-interactive partner proteins with lamina proteins:Mel18 is associated with emerin in HGPS / 北京大学学报(医学版)

Objective :The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is a childhood disorder with features of premature aging and is caused by mutations in the lamin A gene resulting in the production of an abnormal protein, termed progerin. To investigate the underlying pathogenic mecha-nism, we studied the nuclear co-localization and association of progerin interactive partner proteins (PIPPs) with lamina proteins. Methods:Both wild-type (WT) and progeria fibroblasts were studied by various methods including eonfocal microscopy, immunopreeipitation and Western blot. Results:All PIPPs discovered so-far co-localized with lamin A/C. In addition, the PIPPs were selectively associated with lamina proteins. An increased immunofluorescent staining signal was found for Mel18 in HGPS as com-pared to WT cells. An association of Me118 with emerin was observed in HGPS, but not in WT cells.Conclusion: Based on these findings, we propose that PIPPs, along with associated lamina proteins may form a pathogenic progerin-containing protein complex.

Towards understanding lamin gene regulation.

The lamins are components of the nuclear lamina, which forms a fibrous meshwork lining the inner nuclear membrane. Lamina-membrane interactions play a crucial role during nuclear disassembly and reassembly at mitosis, whereas lamina-chromatin association has been proposed to be essential for chromatin organization. The composition of the lamina changes considerably during embryonic development and cell differentiation. Recent studies have provided insights into the regulation of the lamin genes.

ULTRASTRUCTURAL STUDY ON THE PROCESS OF NUCLEAR RECONSTITUTION IN A CELL-FREE SYSTEM FROM XENOPUS EGGS / 解剖学报

Nuclear reconstitution around Lambda DNA in a cell-free system from Xenopus eggs involves distinct steps at ultrastructural level. First, Lambda DNA polymers were induced to form chromatin-like structures with the proteins in egg extracts. Then, along with membrane vesicles and nuclear pore components attached to them to assemble double nuclear membranes, these chromatin-like structures underwent variations from condensation to decondensation, simultaneously. It is different from the nuclear reconstitution induced by chromatin in that, while membrane vesicles were attaching to the chromatin-like structures to fuse each other, the assembly of nuclear pore complexes occurred practically.