OBJECTIVETo explore the effect of 17-allylamide-17-demethoxygeldanamycin (17AAG), a heat shock protein 90 (HSP90) inhibitor, on the growth, differentiation and apoptosis of leukemic Kasumi-1 cells.

METHODSKasumi-1 cells were treated with 17AAG at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry. KIT protein was analysed by Western blot and c-kit mRNA by RT-PCR.

RESULTS17AAG treatment caused a dose-dependent inhibition of the cell proliferation with the IC(50) of 0.62 micromol/L. A dose-dependent increase in early apoptosis occurred at 24 hours treatment and in late apoptosis at 48 hours treatment. 17AAG induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD11b and CD15, a progressive decline in S-phase cell fraction and an increase in G(0)/G(1) cells. When Kasumi-1 cells were incubated with 1 micromol/L of 17AAG, KIT protein began to decrease at 2 hours and KIT protein could hardly be detected at 20 hours, but c-kit mRNA was not decreased.

CONCLUSION17AAG treatment of Kasumi-1 cells could lower KIT protein expression, inhibit cell proliferation, induce cell partial differentiation, apoptosis and accumulation in G(0)/G(1) phase.

Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; RNA, Messenger ; genetics

This study was aimed to investigate the expression of c-MPL in acute myeloid leukemia (AML) and the correlation of the c-MPL expression with CD34 and CD38, so as to define the expression of c-MPL in leukemic stem cells. The expression levels of CD34, CD38 and c-MPL were detected by flow cytometry in bone marrow cells from 29 newly diagnosed AML patients. The relationship of c-MPL positive cell ratio with clinical parameters and correlation of c-MPL with CD34 and CD38 expression in AML patients were analyzed. The results showed that expression level of c-MPL in AML patients was significantly higher than that of normal controls (P < 0.05), and the expression level of c-MPL did not correlate with age, sex, white blood cell count, AML1-ETO fusion gene and remission after chemotherapy, but the expression of c-MPL in M2 and M5 patients was higher than that of normal control (P < 0.05). Expression of c-MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P < 0.01). c-MPL was significantly higher expressed in CD34(+) cells than that in CD34(-) cells (P < 0.001), while c-MPL expression was not significantly different between CD34(+)CD38(-) and CD34(+)CD38(-) cell groups. Positive correlation between c-MPL and CD34 expression was observed (r = 0.380, P = 0.042). It is concluded that expression of c-MPL is higher in AML patients, and positively correlates with the expression level of CD34. The c-MPL expresses in leukemic stem cells.
Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; Female ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Male ; Middle Aged ; Neoplastic Stem Cells ; metabolism ; Receptors, Thrombopoietin ; metabolism ; Young Adult

OBJECTIVEThe activity of deer serum albumin on proliferation of rat osteogenic-like cells UMR-106 and the IGF-I secretion were investigated in order to elucidate pilose antler's bone-strengthening mechanism.

METHODDeer serum albumin was isolated from freeze-dry pilose antler powder extract. The methods were Sephacryl S-200HR gel filtration, POROS 20QE ion-exchange and TSK G3000SW chromatographies. The effect of deer serum albumin on proliferatio of UMR-106 cells was assaied by MTT, and the secretion of IGF-I of UMR-106 cells was assaied by RIA.

RESULTDeer serum albumin, with the molecular weight of 56.3 kDa, significantly increased the proliferation rate of the osteoblast-like UMR-106 cell and IGF-I secretion. When concentration of deer serum albumin reached 0.149 microg x mL(-1), UMR-106 cell proliferation rate was 241.03% and IGF-I secretion was 66.89 ng x mL(-1).

CONCLUSIONThe concentration of deer serum albumin, from 14.9 ng x mL(-1) to 14.90 microg x mL(-1), significantly increased the proliferation rate of the osteoblast-like UMR-106 cell and IGF- I secretion.

Animals ; Antlers ; chemistry ; Bone Neoplasms ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deer ; Insulin-Like Growth Factor I ; secretion ; Materia Medica ; isolation & purification ; pharmacology ; Osteoblasts ; metabolism ; pathology ; Osteosarcoma ; pathology ; Rats ; Serum Albumin ; isolation & purification ; pharmacology

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Catalytic Domain ; Enzyme Inhibitors ; chemistry ; pharmacology ; Flavonoids ; chemistry ; pharmacology ; Hydro-Lyases ; antagonists & inhibitors ; chemistry ; Molecular Sequence Data ; Mycobacterium tuberculosis ; drug effects ; enzymology ; Protein Binding ; drug effects ; Protein Multimerization ; drug effects ; Protein Structure, Secondary ; Sequence Alignment

Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.
Amino Acid Sequence ; Catalytic Domain ; Crystallography, X-Ray ; Humans ; Malonates ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Nitrophenols ; metabolism ; Organophosphorus Compounds ; metabolism ; Phosphoric Monoester Hydrolases ; chemistry ; classification ; metabolism ; Tartrates ; metabolism ; Water ; metabolism

Objective To explore the selection rules of acupuncture and moxibustion for treating ovarian dysfunction-related diseases by applying data mining technology.Methods Computer searches were conducted to find clinical research literature on acupuncture and moxibustion for ovarian reserve dysfunction-related diseases(including diminished ovarian reserve,premature ovarian insufficiency,premature ovarian failure)in major databases,such as China National Knowledge Infrustructure(CNKI),Wanfang Data Knowledge Service Platform(Wanfang),and China Science and Technology Journal Database(VIP),Excel 2021 was used to establish a prescription database of acupoints selection,and SPSS Modeler 18.0 and SPSS Stastics 26.0 software were used to analyse the frequency,meridian tropism,site,special acupoints,analysis of association rule,and cluster analysis of acupoints to study the rules of acupuncture and moxibustion for treating ovarian reserve dysfunction-related diseases.Results A total of 215 articles were screened to meet the requirements,in which,96 acupoints were used,with a total frequency of 2 110 times.The high-frequency used acupoints were Guanyuan(RN4),Sanyinjiao(SP6),and Shenshu(BL23),etc,.The commonly used meridians were the conception vessel(CV)and bladder meridian of foot-taiyang,and the acupoints were mostly located in the abdomen and lower limbs,with the majority being the five-shu point and the yuan front-mu points.The core acupoints and four effective clusters were analysed.Conclusion The acupoint selection of acupuncture and moxibustion for ovarian reserve dysfunction-related diseases focuses on tonifying qi and blood,cultivating the vital essence and tonifying the kidneys.Most of the meridians related to reproduction were selected in this method,which aiming at regulating menstruation and tonifying blood,and pre-cultivating its loss.This research focuses on the matching of neighboring point and distant point selection,and pays attention to the use of specific acupoints,and treates multiple organs simultaneously.

OBJECTIVETo study the effect of hypoxia-inducible factor-1α (HIF-1α) on human gastric cancer cells apoptosis in simulated CO2 pneumoperitoneum environment.

METHODSApplied closed box to simulated CO2 pneumoperitoneum environment under the pressure of 0, 5, 10 and 15 mm Hg (1 mm Hg = 0.133 kPa). Compared HIF-1α mRNA and protein expression of MKN-45 cells before and after silencing HIF-1α by RT-PCR and Western blot. Study the changes of bcl-2/bax expression in MKN-45 cells before and after silencing HIF-1α by immunohistochemistry. The apoptosis ratio of MKN-45 was measured by using Annexin V-FITC/PI double labelled staining.

RESULTSIn 15 mm Hg group, HIF-1α mRNA and protein expression of MKN-45 cells (1.48 ± 0.22, 1.34 ± 0.09) and HIF-1α protein expression in 10 mm Hg group (1.25 ± 0.10) were significantly higher than those in control group (0.55 ± 0.17, 0.83 ± 0.04) (P < 0.05). But there was no significant differences among 0, 5, 10 mm Hg group and control group in HIF-1α mRNA (P > 0.05); and no obvious difference was found among 0, 5 mm Hg group and the control group in HIF-1α protein expression (P > 0.05). In 15 mm Hg CO2 pressure, bcl-2/bax expression (0.78 ± 0.05) was obviously lower than that in the control group (1.43 ± 0.15) (P < 0.05) and the apoptosis ratio (11.70 ± 0.12) was significantly higher than the control group (0.22 ± 0.07) (P < 0.01) before silencing HIF-1α. But once HIF-1α was silenced, HIF-1α mRNA (0.52 ± 0.11), HIF-1α protein expression (0.92 ± 0.02), bcl-2/bax ratio (1.57 ± 0.04) and apoptosis ratio (0.45 ± 0.11) in MKN-45 were not significantly different between 15 mm Hg group and the control group (P > 0.05).

CONCLUSIONSThe apoptosis ratios of MKN-45 under 0, 5, 10 mm Hg CO2 pneumoperitoneum are comparable with that in the control group before the silencing of HIF-1α. The apoptosis ratio of MKN-45 is increased under 15 mm Hg CO2 pneumoperitoneum environment and HIF-1α may be one of the important factor to improve the apoptosis of human gastric cancer cell.

Apoptosis ; Carbon Dioxide ; physiology ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Pneumoperitoneum, Artificial ; RNA Interference ; Stomach Neoplasms ; metabolism ; pathology

This study is to explore the mechanism and effect of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation and apoptosis of A549 cells in vitro and on A549 xenograft tumor in nude mice. With different concentrations of ZGDHu-1 at different times were used to treat A549 cells in vitro. The proliferation was determined by living cell count, SRB assay and Brdu-ELISA. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin V/PI and Hoechst 33258 labeling method. The nude mice model of A549 xenograft tumor was established by subcutaneous inoculation. The suppression activity of ZGDHu-1 by intraperitoneal injection on xenograft mice model was detected. The expressions of bcl-2, bax and p53 gene and protein were analyzed by RT-PCR and flow cytometry. ZGDHu-1 can inhibit A549 cell proliferation viability within a certain range of treating time and does, and a majority of A549 cells were arrested in G2-M phase. The A549 cells apoptosis was confirmed by typical cell morphology, DNA fragment, Sub G1 phase, Hoechst 33258 and Annexin V/PI labeling method with a time and dose related manner. When the xenograft tumor mice model were treated with 10, 20 and 40 mg x kg(-1) ZGDHu-1 for 14 days, the tumor growth inhibition rate were 43.7%, 56.9% and 60.0%, respectively. The expression of bax, bax/bcl-2 and p53 gene and protein increased significantly and bcl-2 decreased slightly by the treatment of ZGDHu-1. ZGDHu-1 can significantly suppress the growth of A549 xenograft tumor in vivo and inhibited proliferation by inducing tumor cell apoptosis in vitro. The mechanism may associate with its up-regulation of bax and p53 during the apoptosis process.
Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Female ; Flow Cytometry ; Heterocyclic Compounds, 1-Ring ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; prevention & control ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; biosynthesis ; genetics

Objective To study the incidence of lactase deficiency and the risk factors affecting intestinal lactase secretion in newborns with lactase deficiency.Method From February to December 2016,newborns admitted to the neonatal ward of the Affiliated Hospital of Hangzhou Normal University were enrolled in this prospective study.Urine samples were taken within one to two hours after feeding for galactose qualitative tests,and the related clinical data were recorded.The newborns were assigned into lactase deficient group and non-lactase deficient group according to the test results.Then the risk factors of lactase deficiency were analyzed comparing the clinical data between the two groups.Result A total of 1 022 newborns were hospitalized during the research period,of whom 213 were enrolled in this study according to the inclusion criteria.154 cases had positive results in the urine galactose qualitative tests,yielding the incidence of lactase deficiency of 72.3 ％.42 cases had lactose intolerance symptoms,and the incidence of lactose intolerance was 27.3 ％ (42/154).Age and positive family history in lactase deficient group were higher than non-lactase deficient group (10.3 ±6.4 d vs.8.1 ±5.8 d and 23.4％ vs.10.2％),while the gestational age of lactase deficient group was lower than non-lactase deficient group (37.8 ±2.9 weeks vs.39.0 ± 1.7 weeks),and the differences between the two groups were statistically significant (P ＜ 0.05).No significant differences existed in gender,birth weight,antibiotics use and feeding volumes between the two groups (P ＞ 0.05).Multivariate Logistic regression analysis showed that age (OR =1.065,95％CI 1.007 ～ 1.127) and positive family history (OR =2.912,95％ CI 1.053 ～ 8.056) were the risk factors of lactase deficiency.Gestational age (OR =0.747,95％ CI 0.617 ～ 0.904) was the protective factor of lactase deficiency in newborns.Conclusion The incidence of lactase deficiency in newborns is high,but not all the newborns manifest lactose intolerance symptoms.Age and positive family history were the risk factors while gestational age was the protective factor for lactase deficiency in newborns.

Ebola virus (EBOV) is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA (-ssRNA) viruses, EBOV possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain (NP(core)) possesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an α-helix of EBOV NP(core) itself, which is highly conserved among filoviridae family. Combined with other biochemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.
Crystallography, X-Ray ; Ebolavirus ; physiology ; Humans ; Nucleoproteins ; chemistry ; genetics ; metabolism ; Protein Structure, Tertiary ; Structure-Activity Relationship ; Virus Assembly ; physiology