Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.     

Co-expressed Cyclin D variants cooperate to regulate proliferation of germline nuclei in a syncytium

The role of the G1-phase Cyclin D-CDK 4/6 regulatory module in linking germline stem cell (GSC) proliferation to nutrition is evolutionarily variable. In invertebrate Drosophila and C. elegans GSC models, G1 is nearly absent and Cyclin E is expressed throughout the cell cycle, whereas vertebrate spermatogonial stem cells have a distinct G1 and Cyclin D1 plays an important role in GSC renewal. In the invertebrate, chordate, Oikopleura, where germline nuclei proliferate asynchronously in a syncytium, we show a distinct G1-phase in which 2 Cyclin D variants are co-expressed. Cyclin Dd, present in both somatic endocycling cells and the germline, localized to germline nuclei during G1 before declining at G1/S. Cyclin Db, restricted to the germline, remained cytoplasmic, co-localizing in foci with the Cyclin-dependent Kinase Inhibitor, CKIa. These foci showed a preferential spatial distribution adjacent to syncytial germline nuclei at G1/S. During nutrient-restricted growth arrest, upregulated CKIa accumulated in arrested somatic endoreduplicative nuclei but did not do so in germline nuclei. In the latter context, Cyclin Dd levels gradually decreased. In contrast, the Cyclin Dbβ splice variant, lacking the Rb-interaction domain and phosphodegron, was specifically upregulated and the number of cytoplasmic foci containing this variant increased. This upregulation was dependent on stress response MAPK p38 signaling. We conclude that under favorable conditions, Cyclin Dbβ-CDK6 sequesters CKIa in the cytoplasm to cooperate with Cyclin Dd-CDK6 in promoting germline nuclear proliferation. Under nutrient-restriction, this sequestration function is enhanced to permit continued, though reduced, cycling of the germline during somatic growth arrest.     

Genistein对大鼠垂体前叶细胞增殖的抑制作用

应用细胞培养、^3H-TdR掺入、流式细胞和电镜技术,观察酪氨酸蛋白激酶（PTK）抑制剂genistein对正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20增殖的影响,并探讨其可能的机制。结果显示：genistein作用48h后可明显抑制正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20增殖。流式细胞仪检测发现,50和100μmol/L genistein可将AtT-20细胞阻断于G0／G1期及G2／M期,并出现凋亡峰,凋亡率分别灰19.9％和36.4％。电镜照片显示有凋亡细胞。结果表明,PTK抑制剂可以明显抑制正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20的殖,并诱导细胞凋亡,说明PTK活性对细胞增殖和分化有重要作用。     

Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents

Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55 nM and 60 nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells.     

交联琼脂糖珠固定化蛋白酶在纯化牛肺Kunitz抑制剂中的应用

本文介绍了珠状交联琼脂糖及以此作为载体,经氯代环氧丙烷活化后与蛋白酶(胰蛋白酶或糜蛋白酶)结合,制成固定化蛋白酶亲和吸附剂,进而用以亲和层析牛肺提取液中的Kunitz抑制剂的方法。纯化出的抑制剂在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带,与参照物Trasytol(商品Kunitz抑制剂)具有相对应的电泳迁移率,其分子量也相符。纯化产品每毫克蛋白的抑制活力相当于16 000胰蛋白酶BAEE单位。纯化效果为90倍,收率约85％。     

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of ∼7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 μg porcine aprotinin inhibited 6 μg trypsin whereas 1 μg commercial soybean inhibitor inhibited only 1 μg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.     

Turnover of Brain Monoamine Oxidase Measured In Vivo by Positron Emission Tomography Using l-[11C]Deprenyl

The distribution of carbon-11-labeled L-deprenyl, an irreversible inhibitor of monoamine oxidase type B (MAO-B), was determined in the baboon brain by positron emission tomography. The irreversible blood-to-brain transfer constant (influx constant, K_i) was measured using a complete metabolite-corrected arterial plasma concentration curve. This influx constant was used as a measure of functional enzyme activity for sequential determinations of MAO-B recovery following a single high dose of unlabeled l -deprenyl. The half-life for turnover of MAO-B was thus determined to be 30 days. Using appropriate irreversible inhibitors, this procedure should be generally useful for determining enzyme turnover rates in any organ in vivo and can be applied to some human studies as well.     

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.     

Comparative effects of a vasopeptidase inhibitor vs. an angiotensin convertin enzyme inhibitor on cardiomyocyte apoptosis in rats with heart failure

Apoptosis is involved in ventricular remodeling after myocardial infarction (MI). We investigated the effects of the vasopeptidase inhibitor (VPI) omapatrilat on cardiomyocyte apoptosis and compared it to the angiotensin converting enzyme inhibitor (ACEI) captopril in the rat post-MI model and in cultured neonatal rat cardiomyocytes. Wistar males rats surviving 4 h post-MI were assigned to omapatrilat (40 or 80 mg/kg/day), captopril (160 mg/kg/day) or no treatment. After 56 days, hemodynamic measurements were performed (n = 96) and rats were sacrificed. One group had assessment of cardiac remodeling and detection of DNA fragments by in situ end labelling method (ISEL), while the other had morphologic measurements and DNA laddering assessed. In addition, cultured neonatal rat cardiomyocytes (n = 6) were treated for 72 h with vehicle, captopril or omapatrilat in the presence or absence of the apoptosis inducing agent H₂O₂. Omapatrilat and captopril resulted in similar improvements of hemodynamic measurements, ventricular weight and dilatation, cardiac fibrosis and myocardial cell cross-section in large MI rats. Omapatrilat increased scar thickness more than did captopril. All sham-operated groups had little evidence of apoptosis. In the large MI group, there was a significant increase in ISEL-positive cells in the control (0.095 ± 0.016%) and captopril (0.124 ± 0.024%) groups in comparison with control sham-operated (0.006 ± 0.006%), but this increase was limited to the peri-MI area. Omapatrilat (0.012 ± 0.012% for both doses) prevented the increase in apoptosis in the peri-MI area. Also, omapatrilat but not captopril reduced DNA laddering in large MI. Moreover, in cultured neonatal rat cardiomyocytes, omapatrilat but not captopril reduced apoptosis as assessed by DNA laddering. The VPI omapatrilat, with its combination of NEP and ACE inhibition, suppresses cardiomyocyte apoptosis post-MI and in neonatal cultured rat cardiomyocytes more than the ACEI captopril, but this does not result in significant hemodynamic or morphologic differences between omapatrilat and captopril.