Psychological status and behavior changes of the public during the COVID-19 epidemic in China.

BACKGROUND: A cluster of pneumonia cases were reported by Wuhan Municipal Health Commission, China in December 2019. A novel coronavirus was eventually identified, and became the COVID-19 epidemic that affected public health and life. We investigated the psychological status and behavior changes of the general public in China from January 30 to February 3, 2020. METHODS: Respondents were recruited via social media (WeChat) and completed an online questionnaire. We used the State-Trait Anxiety Inventory, Self-rating Depression Scale, and Symptom Checklist-90 to evaluate psychological status. We also investigated respondents' behavior changes. Quantitative data were analyzed by t-tests or analysis of variance, and classified data were analyzed with chi-square tests. RESULTS: In total, 608 valid questionnaires were obtained. More respondents had state anxiety than trait anxiety (15.8% vs 4.0%). Depression was found among 27.1% of respondents and 7.7% had psychological abnormalities. About 10.1% of respondents suffered from phobia. Our analysis of the relationship between subgroup characteristics and psychological status showed that age, gender, knowledge about COVID-19, degree of worry about epidemiological infection, and confidence about overcoming the outbreak significantly influenced psychological status. Around 93.3% of respondents avoided going to public places and almost all respondents reduced Spring Festival-related activities. At least 70.9% of respondents chose to take three or more preventive measures to avoid infection. The three most commonly used prevention measures were making fewer trips outside and avoiding contact (98.0%), wearing a mask (83.7%), and hand hygiene (82.4%). CONCLUSIONS: We need to pay more attention to public psychological stress, especially among young people, as they are likely to experience anxiety, depression, and psychological abnormalities. Different psychological interventions could be formulated according to the psychological characteristics of different gender and age groups. The majority of respondents followed specific behaviors required by the authorities, but it will take time to observe the effects of these behaviors on the epidemic.

MicroRNA-4461 derived from bone marrow mesenchymal stem cell exosomes inhibits tumorigenesis by downregulating COPB2 expression in colorectal cancer.

Colorectal cancer (CRC) is one of the main cause of cancer-related deaths. It's reported that bone marrow mesenchymal stem cells (BMSCs) affects tumor development through secreting exosomes. This study aims to investigate the function of BMSCs-derived exosome miR-4461 in CRC. The results of qRT-PCR showed that miR-4461 expression in DLD1, HCT116 and SW480 CRC cells and CRC tissues was lower than that in FHC cells and normal tissues, respectively. And COPB2 mRNA expression was negatively correlated with miR-4461. Western blot was used to detect COPB2 protein expression. Dual-luciferase reporter assay results revealed that miR-4461 targeted COPB2. Transwell assay and CCK-8 assay demonstrated that COPB2 knockdown inhibited HCT116 and SW480 cells proliferation, migration and invasion abilities. Furthermore, BMSCs-derived exosome miR-4461 downregulated COPB2 expression and inhibited HCT116 and SW480 cells migration and invasion. The findings demonstrated that miR-4461 could be a potential target for the diagnosis and treatment of colorectal cancer.

[Effect of Metformin on Proliferation Capacity, Apoptosis and Glycolysis in K562 Cells].

OBJECTIVE: To investigate the effect of metformin on the proliferation, apoptosis and energy metabolism of acute myeloid leukemia (AML) K562 cells and the possible mechanism. METHODS: Different doses (0, 5, 10, 20 and 30 mmol/L) of metformin was added into the K562 cells, which were cultivated for 24 h, 48 h and 72 h. The inverted optical microscope was used to observe the cell growth, CCK 8 was used to detect the cell vitality. The appropriate metformin doses (0, 10, 20 and 30 mmol/L) and the best time (48 h) were selected for subsequent experiments. The flow cytometer with Annexin V-FITC /PI doulde staining was used to detect apoptosis; the glucose detection kit and lactate detection kit were used to detect glucose consumption and lactate production; fluorescence quantitative PCR was used to detect glycolysis-related gene expression, and Western blot was used to detect protein expression. RESULTS: Metformin inhibited the proliferation of K562 cells in a dose-dependent manner (r=0.92), and the relative survival in the 30 mmol/L group was as low as 19.84% at 72 h. When treated with metformin for 48 h, the apoptosis rates of 0, 10, 20 and 30 mmol/L groups were 5.14%, 12.19%, 26.29% and 35.5%, respectively. Compared with the control group, the glucose consumption and lactate secretion of K562 cells treated with metformin were significantly reduced (P<0.05), and showed a dose-dependent effect(r=0.94,r=0.93,respectively). Metformin inhibited the expression of GLUT1, LDHA, ALDOA, PDK1, and PGK1 genes of K562 cells (P<0.05) showing a dose-dependent manner(r=0.83，r=0.80，r=0.72，r=0.76，r=0.73,respectively). Metformin inhibited the expression of P-Akt, P-S6, GLUT1, LDHA proteins of K562 cells(P<0.05), showing a dose-dependent relationship(r=0.80，r=0.92，r=0.83，r=0.92,respectively). CONCLUSION: Metformin can inhibit the growth and proliferation of K562 cells and promote the apoptosis of K562 cells by inhibiting glycolysis energy metabolism. PI3K/Akt/mTOR signaling pathway may be one of the molecular mechanisms of metformin on k562 cells.

Acoustic Radiation Force Impulse Technology in the Differential Diagnosis of Solid Breast Masses with Different Sizes: Which Features Are Most Efficient?

PURPOSE: To evaluate diagnostic performance of acoustic radiation force impulse (ARFI) technology for solid breast masses with different sizes and determine which features are most efficient. MATERIALS AND METHODS: 271 solid breast masses in 242 women were examined with ARFI, and their shear wave velocities (SWVs), Virtual Touch tissue imaging (VTI) patterns, and area ratios (ARs) were measured and compared with their histopathological outcomes. Receiver operating characteristic curves (ROC) were calculated to assess diagnostic performance of ARFI for small masses (6-14 mm) and big masses (15-30 mm). RESULTS: SWV of mass was shown to be positively associated with mass size (P < 0.001). For small masses, area under ROC (Az) of AR was larger than that of SWV (P < 0.001) and VTI pattern (P < 0.001); no significant difference was found between Az of SWV and that of VTI pattern (P = 0.906). For big masses, Az of VTI pattern was less than that of SWV (P = 0.008) and AR (P = 0.002); no significant difference was identified between Az of SWV and that of AR (P = 0.584). CONCLUSIONS: For big masses, SWV and AR are both efficient measures; nevertheless, for small masses, AR seems to be the best feature.

Involvement of CaSR in hyperglycemia-induced macroangiopathy and related mechanism.

In order to clarify the potential role of calcium sensing receptor (CaSR), a typical G protein coupled receptor (GPCR), in hyperglacemia-induced macroangiopathy, experimental hyperglycemia models in vivo and in vitro were prepared. Firstly, SD rats were divided into control group (n=10) and diabetes group (n=10), and diabetic model was induced via high-fat diet feeding and streptozotocin (STZ, 30 mg/kg) injection. Hydroxyproline level, determined via Choramnie T oxidation method, in vessel wall in diabetic rats was 30% more than that in control group. The gene transcription and expression levels were detected by real-time PCR and Western blotting, respectively. Both of collagen I and III mRNA levels in diabetic aorta were nearly twice those in normal aorta. The cleaved caspase-3 and -9 were elevated 1.5 and 2.5 times respectively in diabetic vascular cells. As compared with controls, mRNA and protein levels of CaSR in aorta were increased by 3 and 1.5 times in diabetes group. The expression levels of Bax as well as pro-apoptotic kinases (phospho-p38 and phosphor-JNK) were also increased 2, 0.5 and 0.5 times respectively in diabetic rats. To further validate the involvement of CaSR in cell apoptosis and explore the potential mechanism, the endothelial cell line (human umbilical vascular endothelial cells, HUVECs) was stimulated with high concentration of glucose (33 mmol/L) to mimic hyperglycemia in vitro. Cell-based assays also showed that the CaSR level and key apoptotic proteins (cleaved caspase-3 and -9, Bax, phospho-p38 and phosphor-JNK) were elevated in response to stimulation, and inhibition of CaSR by using specific inhibitor (NPS-2143, 10 µmol/L) could protect cells against apoptosis. Our results demonstrated that CaSR might take important part in the development of diabetic macroangiopathy through promoting cell apoptosis induced by hyperglycemia.

B3GNT2, a polylactosamine synthase, regulates glycosylation of EGFR in H7721 human hepatocellular carcinoma cells.

The epidermal growth factor receptor (EGFR) is an important surface receptor with N-glycans in its extracellular domain, whose glycosylation is essential for its function, especially in tumor cells. Here, we demonstrated that polylactosamine is markedly increased in H7721 hepatocellular carcinoma cells after treatment with EGF, while it apparently declined after exposure to all-trans retinoic acid (ATRA). In the study of the enzymatic mechanism of this phenomenon, we explored changes in the expression of poly-N-acetyllactosamine (PLN) branching glycosyltransferases using RT-PCR. Among the four glycosyltransferases with altered expression, GnT-V was most elevated by EGF, while GnT-V and B3GNT2 were most declined by ATRA. Next, we conducted co-immunoprecipitation experiments to test whether B3GNT2 and EGFR associate with each other. We observed that EGFR is a B3GNT2-targeting protein in H7721 cells. Taken together, these findings indicated that the altered expression of B3GNT2 will remodel the PLN stucture of EGFR in H7721 cells, which may modify downstream signal transduction.

[Multilocus sequence typing of Candida albicans bloodstream isolates in an intensive care unit].

OBJECTIVE: Investigate the molecular epidemiological characteristics of bloodstream infections (BSI) due to Candida albicans (C. albicans) in the intensive care unit (ICU) of West China Hospital, Sichuan University. METHODS: C. albicans isolates recovered from blood cultures in West China Hospital, Sichuan University between 2009 and 2011 were collected. Multilocus sequence typing (MLST) was performed to assess the genetic relationships among BSI isolates of C. albicans collected from the ICU. RESULTS: 135 BSI isolates of Candida species were obtained from 2009 to 2011. C. albicans was the leading pathogen (51 isolates, 37.8%). 17 C. albicans BSI isolates from 15 patients of ICU were analyzed by MLST. Among the 17 isolates, 15 were recovered from peripheral blood and 2 from central venous catheters (CVC) (Peripheral blood and CVC were sent for culture and both had positive results for 2 patients). The 17 isolates yielded 15 unique sequence types (STs) by MLST. While 14 STs were each derived from a single isolate, 1 STs were shared by 3 isolates. 5 (29.4%) isolates were clustered within Group 46, 2 (11.8%) isolates were clustered within Group 47, and 10 isolates (58.8%) typed as singletons. The strains (Calb-36 and Calb-40) recovered from one blood sample and one CVC from one patient were indistinguishable by MLST, while two distinct strains were found in one blood sample and one CVC from another patient. CONCLUSION: C. albicans was the most frequently isolated species of candidemia in West China Hospital. Predominant strains of C. albicans caused BSI in the ICU belonged to Group 46 and Group 47. There was not yet an outbreak of BSI caused by C. albicans, but catheter-related candidemia was confirmed by our research.

Expression of ß1,3-N-acetylglucosaminyltransferases during differentiation of human acute myeloid leukemia cells.

The expressions of ß1,3-N-acetylglucosamonyltransferase-2 and -8 (ß3GnT-2, ß3GnT-8),-the two main glycosyltransferases responsible for the synthesis of poly-N-acetyllactosamine (polyLacNAc) in glycans, and ß3GnT-5 participating in the syntheses of sphingoglycolipids were studied in leukemia cell lines during differentiation using RT-PCR method. ß3GnT-2 and ß3GnT-8 distribute widely in six myeloid and monocytoid leukemia cell lines with different abundances, while ß3GnT-4 was only present in NB4 cells. ATRA (all-trans retinoic acid) and dimethylsulfoxide (DMSO), which induce the differentiation of HL-60 and NB4 (two human acute myeloid leukemia cell lines) to myelocytic lineage, up-regulated these two enzymes with various degrees at 2 and 72 h of treatment. In HL-60 cells treated with ATRA, the increase of ß3GnT-8 was more than ß3GnT-2, while in NB4 cells treated with DMSO, the increase of ß3GnT-2 was more than ß3GnT-8. However, when HL-60 and NB4 were differentiated to monocytic lineage induced by phorbol 12-myristate 13-acetate the expressions of ß3GnT-2 and ß3GnT-8 showed no alterations or the increase of expressions was far less than those in myelocytic differentiation. By means of FITC-labeled tomato lectin affinity staining and flow-cytometry, it was found that the product of ß3GnT-2 and -8, polyLacNAc was also increased on the cell surface of HL-60 and NB4 treated with ATRA or DMSO, but unchanged when treated with PMA. These results were in accordance with the up-regulation of the mRNAs of ß3GnT-2 and -8. The expression of ß3GnT-5, however, was not changed both in myelocytic and monocytic differentiations. The difference in the up-regulation of ß3GnT-2 and -8, especially their products may become a useful index to discriminate the myelocytic and monocytic differentiation of leukemia cells.

Bis{2-[1-(8-hydroxy-2-quinolylmethyl)-1H-benzimidazol-2-yl]quinolin-8-ol} toluene solvate.

Crystals of the title compound, 2C(26)H(18)N(4)O(2)·C(7)H(8), were obtained from the reaction of 8-hydroxy-quinoline with 1,2-phenyl-enediamine in methanol and recrystallized from toluene. The compound contains three essentially planar ring systems: the benzimidazole ring (r.m.s. deviation = 0.039 Å) and two 8-hydroxy-quinoline rings (r.m.s. deviations of 0.0056 Šin both rings). The benzimidazole ring and one 8-hydroxy-quinoline ring are almost co-planar, forming a dihdral angle of 3.1 (2)°. The other 8-hydroxy-quinoline ring is almost perpendicular to the benzimidazole plane with a dihedral angle of 86.2 (2)°. Intra-molecular O-H⋯N contacts are present. The crystal structure is stabilized by inter-molecular O-H⋯N inter-actions. The complete toluene molecule is generated by crystallographic inversion symmetry; therefore its methyl group is disordered over two sites of equal occupancy.

[Etiology of infections in the wounded victims of Wenchuan Earthquake].

OBJECTIVE: To analyze the etiology of infections in the wounded victims of Wenchuan Earthquake. METHODS: 2135 smears of secretion were made from 1823 hospitalized wounded victims of Wenchuan Earthquake to detect the pathogens. Specimens were delivered to be cultured. The bacteria thus obtained were identified. Drug sensitivity test was conducted. RESULTS: 2002 specimens, 1243 specimens of secretion (62.1%), 600 blood specimens (30.0%), 102 specimens of pus or secretion of respiratory tract (5.1%), 45 specimens from catheter (2.2%), and 12 urine specimens (0.5%). Pathogens were found in 725 cases. The top five pathogenic bacteria isolated within 1 month after the quake were Acinetobacter baumannii (16.7%), Staphylococcus haemolyticus (16.7%), Escherichia coli (12.5%), Klebsiella pneumoniae (12.5%), and Candida tropicalis (8.3%), quite different from the pathogen spectrum of the common in-patients within one month before the quake: Escherichia coli (18.2%), Staphylococcus aureus (11.4%), Candida glabrata (11.4%), Klebsiella pneumoniae (11.4%) and Staphylococcus epidermidis (9.1%). The isolation rate of methicillin resistant Staphylococcus aureus after the earthquake was significantly lower, and the isolation rates of extended spectrum beta-lactamase-producing Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca were all significantly higher than those from the common surgical patients before the quake (all P < 0.05). There were not significant differences in the isolation rates of multi-drug resistant Pseudomonas alcaligenes and Acinetobacter baumannii before and after the quake. CONCLUSION: Infection is frequent after natural disasters. It is necessary to summarize the changes of spectrum of pathogens and drug-resistant spectrum.

[DNA homology between methicillin-resistant Staphylococcus aureus strains isolated from the wounds caused by earthquake and those from patients from other areas during the same period].

OBJECTIVE: To investigate the DNA homology between the methicillin-resistant Staphylococcus aureus (MRSA) isolated from the wounds caused by earthquake and those from the patients from non-earthquake-stricken areas. METHODS: Five MRSA isolates were obtained from the wounds caused by earthquake of 5 in-patients admitted into the West China Hospital, and 6 MRSA isolates were obtained from the inpatient of the same hospital during the same period. DNA fingerprint alas was obtained by repetitive extragenic palindromic sequence-based PCR (Rep-PCR) and homolog analysis was conducted with the help of Rep-based DiversiLab Microbial Typing system. RESULTS: Five different patterns (A-E) were found among the 11 MRSA strains. The 5 strains isolated from the wounds caused by earthquake included subtype A (n = 1), subtype B (n = 2), subtype C (n = 1), and subtype E (n = 1). And the 6 stains isolated from the patient that come from non-earthquake-stricken areas included A (n = 3), subtype B (n = 1), subtype C (n = 1), and subtype D (n = 1). CONCLUSION: The MRSA stains isolated from the wounds caused by earthquake are highly homologous with those isolated from the patient from non-disastrous areas during the same period.

[Association of genotype of OXA-carbapenemases of Acinetobacter baumannii and antibiotic resistance].

OBJECTIVE: To determine the association of the antibiotic resistance of clinical isolates of Acinetobacter baumannii and the genotype of OXA-carbapenemases. METHODS: One hundred and twenty Acinetobacter baumannii strains were isolated from clinical specimens in the West China Hospital from January 2005 to June 2006. The MICs of 12 common antimicrobial agents were determined by 2-fold agar dilution method followed by NCCLS recommendations. The bla(OXA-23) and bla(OXA-24) of those with resistance to IMP were amplified by PCR. RESULTS: The resistant rates of the 120 isolates to 11 common antimicrobial agents exceeded 50% except for IMP (44.4%). One hundred and ten strains were resistant to more than three common antimicrobial agents, with a resistant rate of 91.67%. Of the 50 strains resistant to IMP, 13 stains carried bla(OXA-23). No bla(OXA-24) was found in the resistant isolates. CONCLUSION: Multiple antibiotic resistances are common in Acinetobacters baumannii isolated in the West China Hospital. OXA-23-type carbapenemase is the major carbapenemase that contributes to the nosocomial infection of Acinetobacters baumannii.

[Genetic types of three community-acquired methicillin-resistant Staphylococcus aureus isolates].

OBJECTIVE: To investigate the genetic features of the community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) prevalent in West China. METHODS: Three CA-MRSA isolates obtained in Chengdu, Sichuan, underwent SCCmec (Staphylococcal Cassette Chromosome mec) multi-PCR, Staphylococcal protein A (spa) typing and multi-locus sequence typing (MLST) method, and their Panton-Valentine leucocidin (PVL) gene was also detected. RESULTS: All 3 CA-MRSA isolates were positive of mecA gene (147 bp), and the other PCR product of 750 bp was confirmed to be type IVa SCCmec. Spa typing showed that the MRSA strains s29635 and s35301 were typed as t437, and the strain s19165 was typed as t008. MLST showed that the MRSA strains s29635 and s35301 were typed as ST59, and the strain s19165 was typed as ST8. The strains s19165 and s35301 were all positive for PVL gene, and the strain s29635 was negative for PVL gene. CONCLUSION: CA-MRSA clones ST8-t008 and ST59-t437 have been isolated in West China.

alpha1,3 fucosyltransferase-VII up-regulates the mRNA of alpha5 integrin and its biological function.

After transfection of alpha1,3fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the expression of alpha5, but not beta1 integrin was significantly up-regulated. This was evidenced by the increase of alpha5 integrin on cell surface as well as the increase of alpha5 mRNA and protein in the cells. However, the expressions of sialyl Lewis X (SLe(x), the product of alpha1,3FucT-VII) on both alpha5 and beta1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involve in the signal transduction of integrin alpha5beta1) were up-regulated, while the Tyr-527 phosphorylated Src was down-regulated. The above-mentioned alterations were correlated to the expressions of alpha1,3FucT-VII in different alpha1,3FucT-VII transfected H7721 cell lines. In addition, after alpha1,3FucT-VII transfection, cell adhesion to fibronectin (Fn) and chemotaxic cell migration were obviously promoted. The cell adhesion could be blocked by alpha5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both alpha5 integrin and SLe(x). These findings suggest that the increased surface alpha5 integrin caused by the up-regulation of alpha5 mRNA promotes the cell adhesion to Fn, cell migratiom, and Fn-induced signaling of alpha5beta1 integrin. The up-regulation of surface SLe(x) originated from the over expression of alpha1,3FucT-VII also led to the stimulation of cell migration. This is the first time to report that alpha1,3FucT-VII can regulate the mRNA expression of integrin.

Alpha1,3 Fucosyltransferase-VII modifies the susceptibility of apoptosis induced by ultraviolet and retinoic acid in human hepatocarcinoma cells.

The role of alpha1,3fucosyltransferase-VII (alpha1,3 FucT-VII) in cell apoptosis was studied in human hepatocellular carcinoma H7,721 cells. After the cells were transfected with alpha1,3 FucT-VII cDNA, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB and phospho-Bad were increased as compared with mock (vector) transfected cells, indicating that alpha1,3FucT-VII is a potential anti-apoptotic factor in H7,721 cells. After "alpha1,3FucT-VII" cells were irradiated by UV to induce apoptosis, the anti-apoptotic potential of alpha1,3FucT-VII became more apparent, as evidenced by the less apoptotic cell % and active cleaved caspase-3, more phospho-p38 MAPK and JNK (two anti-apoptotic signaling molecules in H7,721 cells responsible to UV stress) when compared with the "Mock" cells. In contrast, "alpha1,3FucT-VII" cells facilitated the apoptosis induced by all-trans retinoic acid (ATRA), which was verified by the greater sub-G1 (apoptotic cells) peak in flow cytometry analysis, more expressions of active caspase-3 and pro-apoptotic protein Bax, as well as less expressions of anti-apoptotic proteins, Bcl-2 and Bcl-X(L). The up regulation of alpha1,3FucT-VII mRNA and cell surface SLe(x) (alpha1,3FucT-VII product) by UV and down regulation of them by ATRA was speculated to be one of the mechanisms that alpha1,3FucT-VII decreased and increased the susceptibility of apoptosis induced by UV and ATRA respectively.

Alpha 1,3-fucosyltransferase-VII regulates the signaling molecules of the insulin receptor pathway.

Two H7721 human hepatocarcinoma cell lines showing moderate and high expression of alpha1,3-fucosyltransferase (FucT)-VII cDNA were established and designated FucTVII-M and FucTVII-H, respectively. In alpha1,3-FucT-VII-transfected cells, expression of insulin receptor (InR) alpha- and beta subunits and epidermal growth factor receptor (EGFR) on the cell surface and in cells, as well as the sialyl Lewis X (SLe(x), the product of alpha1,3-FucT-VII) content of the EGFR were unchanged. However the level of SLe(x) on the InR alpha subunit (InR-alpha) was increased dramatically. Tyrosine autophosphorylation of InR-beta , but not EGFR, was elevated. Concomitantly, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Ser/Thr phosphorylation of protein kinase B (PKB; Akt), p42/44 mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), and the protein of some other signaling molecules, such as phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase (PKN), c-Raf-1 and beta-catenin were also upregulated. The activities of PKB and transcription factor TCF were concomitantly stimulated. Upregulation of InR signaling molecules and their phosphorylation was correlated with the level of SLe(x) on InR-alpha and alpha1,3-FucT-VII expression in cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different levels of alpha1,3-FucT-VII expression were attenuated significantly by the inhibitor of InR tyrosine kinase and by the mAb to SLe(x). Furthermore, insulin-induced signaling was facilitated in alpha1,3-FucT-VII-transfected cells, particularly FucTVII-H. These findings provide strong evidence that alpha1,3-FucT-VII may affect insulin signaling by upregulating the phosphorylation and expression of some signaling molecules involved in the InR-signaling pathway. These effects are likely mediated by its product, SLe(x), on the glycans of the InR. This is the first study to report that changes in the terminal structure of glycans on a surface receptor can modify cell signaling.

Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D3] on two leukemia cell lines, K562 and SHI-1, and its relation to the expression of different subtypes of polypeptide: N-acetylgalactosaminyltransferase (pp-GalNAc-T) was studied. With morphological and cell flow-cytometric method, it was found that 1,25(OH)(2)D3 induced the differentiation of both leukemia cell lines toward monocytic lineage, but not affected the cell growth and apoptosis. The expressions of different subtypes of pp-GalNAc-T, the initial glycosyltransferase in O-glycan synthesis, were studied with RT-PCR before and after the treatment of different concentrations of 1,25(OH)(2)D3. Among fourteen subtypes of pp-GalNAc-T (T1 approximately T14), K562 cells obviously expressed pp-GalNAc-T2, T4, T5, T7 (T2 was the highest) and SHI-1 cells apparently expressed pp-GalNAcT1, T2, T3 and T4 (T4 was the highest) only. After K562 cells were treated 1, 25(OH)(2)D3 for 72 h, pp-GalNAc-T2, T4, T5, T7 were increased in a dose dependent manner. In contrast, pp-GalNAc-T1 and T2, especially T1, were up-regulated in SHI-1 cells by 1,25(OH)(2)D3, but T3 was unchanged and T4 was down-regulated. The different alterations of pp-GalNAc-Ts in these two cell lines were probably related to the different structural changes of O-glycans during 1,25(OH)(2)D3 induced differentiation.

Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7,721 cells.

N-acetylglucosaminyltransferase V (GnT-V) is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line (GnT-V-AS/7,721) was constructed from 7,721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7,721 and parental 7,721 cells. The data indicated that GnT-V-AS/7,721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7,721 by the analysis on key molecules in both two unfolded protein response (UPR) pathways. For ATF6 and Ire1/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form of XBP-1. As for PERK/eIF2alpha pathway, the activation of ER eIF2alpha kinase PERK was observed. To confirm the results from GnT-V-AS/7,721 cells, the key molecules in the UPR were examined again in 7,721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7,721 cells. Rate of (3)H-Man incorporation corrected with rate of (3)H-Leu incorporation in GnT-V-AS/7,721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7,721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.

Down regulation of N-acetylglucosaminyltransferase V facilitates all-transretinoic acid to induce apoptosis of human hepatocarcinoma cells.

After N-acetylglucosaminyltransferase V (GnT-V) activity was down-regulated by the transfection of its antisense cDNA(GnTV-AS), apoptosis of H7721 cells was appeared and the apoptosis induced by 80 microM all-transretinoic acid (ATRA) was facilitated, while ATRA itself could not induce apparent apoptosis in mock cells transfected with the vector. In the study of the molecular mechanism of this phenomenon, it was found that GnTV-AS reduced the expressions of anti-apoptotic proteins, such as phosphorylated protein kinase B and phosphorylated Bad as well as Bcl-2 and Bcl-X (L), and elevated those of pro-apoptotic proteins, including Bax, full length caspase-3 and its activated fragments as well as anti-oncoprotein p53. In the contrast, ATRA up regulated the expressions of Bax and activated caspase-3 fragments only. After the GnTV-AS transfected cells were treated with ATRA, phosphorylated PKB and Bad were further decreased, while Bax and activated caspase-3 fragment were further increased, leading to the enhanced apoptosis in flow-cytometry analysis when compared with GnTV-AS cells not treated with ATRA. It was speculated that the decreased phospho-Bad resulted from the reduced phospho-PKB and the up regulation of p53 caused the elevated activity of Bax. The increased active caspase-3 was the consequence of the elevated Bax/ Bcl-2(Bcl-X(L)) activity ratio in the cells.