OBJECTIVETo investigate the role of NR2B subunit C-terminus in assembling and surface expression of NMDA receptor subtype composed of NR1-1a/NR2B subunits.

METHODSEight vectors NR2BDelta1-Delta8) expressing GFP-tagged NR2B subunit mutants with various deletion in the carboxyl-terminal region were generated by conventional molecular cloning techniques. Each of these vectors was transfected alone, or co-transfected with NR1-1a into HEK293 cells. NR1-1a/GFP-NR2B receptors on membrane surface of the living transfected cells were immuno-stained using rabbit antibody against GFP followed by Cy3 conjugated secondary antibody.

RESULTSThe eight vectors NR2BDelta1-Delta8 were successfully constructed. No surface labeling of GFP-tagged NMDA receptor was found for those transfected cells with NR1-1a, GFP-NR2B, and GFP-NR2BDelta1-Delta8 alone. GFP-tagged NMDA receptors were immuno-stained by anti-GFP for those cells co-transfected by NR1-1a and GFP-NR2B or GFP-NR2BDelta1-Delta6, which were mutants with partially deleted c-terminus at different region. However, positive stained was not found for those cells co-transfected by NR1-1a and GFP-NR2BDelta 7 (lack of most C-terminus and with PDZ binding motif fused with TM4) or GFP-NR2BDelta8 (lack of whole C-terminus).

CONCLUSIONSThe formation of NR1-1a/NR2B sub-type NMDA receptor requires co-expression and assembling of NR1-1a and NR2B subunits. Shield or inhibition of ER retention motif within C1 cassette of NR1-1a subunit by NR2B subunit when assembling is not dependent on any particular region in NR2B C-terminus.

Cell Line ; Genetic Vectors ; Humans ; Mutation ; Receptors, N-Methyl-D-Aspartate ; chemistry ; genetics ; Transfection

OBJECTIVETo examine the potential function of NMDA receptor NR2A subunit C-terminus in assembling and surface expression of the receptor in HEK293 cells.

METHODSFive vectors GFP- NR2ADeltaC1- DeltaC5 were constructed for expressing N-terminally GFP-tagged NR2A with C-terminal deletion at different regions by using conventional techniques of molecular cloning. The deleted region for NR2ADeltaC1-Delta C5 was 897L-1017S, 1024D-1142P, 1149D-1347G, 1354S-1464V, and 897L-1464V. These plasmids were transfected alone or co-transfected with NR1-1a into HEK293 cells. The surface NMDA receptors were immuno-stained using rabbit antibody against GFP and Cy3 conjugated secondary antibody in living cells.

RESULTThe vectors GFP-NR2ADeltaC1-DeltaC5 were generated and all of them expressed GFP fluorescence in the transfected cells. Surface NMDA receptors were detected by immuno-labeling with anti-GFP in the cells co-transfected by NR1-1a and any one of GFP-NR2ADeltaC1-DeltaC5. However, no surface expression of NR2A proteins was found in the transfected cells with any one of these plasmids alone.

CONCLUSIONWithin the region downstream from the 897L of NR2A subunit, neither a particular domain directly interacted with ER retention domain in NR1-1a C1 cassette, nor that determining ER retention of NR2A subunit itself has been found, indicating that more complicated mechanisms might exist in which the subunit assembling and targeting to plasma membrane of NMDA receptors undergo.

Cell Line ; Gene Deletion ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Mutation ; Receptors, N-Methyl-D-Aspartate ; analysis ; genetics

OBJECTIVETo explore the clinical value of Lens culinaris agglutinin-reactive alpha-fetoprotein in the differentiation diagnosis between benign and malignant liver diseases, as well as the early warning of hepatocellular carcinoma.

METHODSAlpha-fetoprotein variants from 300 patients with liver diseases were isolated with micro-spin column equipped lens culinaris agglutinin (LCA). The AFP and AFP-L3 were detected by the electrochemical luminescence (ECL) method, and the proportions of AFP-L3 were calculated.

RESULTSThe positive rates of AFP-L3 of HCC patients and chronic liver disease patients were 95% and 64% respectively, there were significant difference in two groups (chi2 = 134.72, P < 0.01), the HCC incidence rates of AFP-L3 positive and negative chronic liver disease patients showed significant difference (chi2 = 80.158, P < 0.01). there were no correlations between the proportion of AFP-L3 and AFP consistency(r = 0.046, P > 0.05).

CONCLUSIONSThe detection of AFP-L3 by micro-spin column assay show great clinical value in the differentiation diagnosis of benign and malignant liver diseases, as well as the early warning of hepatocellular carcinoma.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; analysis ; Carcinoma, Hepatocellular ; diagnosis ; Child ; Diagnosis, Differential ; Female ; Humans ; Liver Diseases ; diagnosis ; Liver Neoplasms ; diagnosis ; Male ; Middle Aged ; Plant Lectins ; chemistry ; Young Adult ; alpha-Fetoproteins ; analysis

OBJECTIVETo study the effects of lutein on apoptosis and its mechanism.

METHODThe cells of human esophageal carcinoma EC9706 were grown in RPMI medium containing 10% bovine serum and were treated with lutein at 100 microg x mL(-1) concentration. Flow cytometry was employed to investigate the effects of lutein on cell apoptosis of EC9706 cells. Histochemistry was performed to determine apoptosis-related protein expresion.

RESULTFlow cytometry analyses revealed that lutein increased EC9706 cell apoptosis ratio when treated with lutein 100 microg x mL(-1) at 96 h. Lutein decreased the expression of Bcl-2 protein and increased the expression of Bax protein in EC9706 cells.

CONCLUSIONLutein could inhibit mitosis and stimulate apoptosis of EC9706 cells. The apoptotic effect may result from the down-regulation of expression of Bcl-2 and up-regulation expression of Bax.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Lutein ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

Objective To investigate the clinical features, laboratory findings, chest CT findings and treatment of patients with COVID-19, and to analyze their relationship with prognosis. Methods From January to February 2020, the clinical data on the 42 patients with COVID-19 admitted to the Wenzhou Sixth People′s Hospital were analyzed retrospectively. Results The clinical symptoms of the 42 cases included fever (35 cases), cough (26 cases), fatigue (14 cases), aspiration (9 cases), sore throat (4 cases), muscle ache (5 cases), headache (2 cases), nausea (4 cases), diarrhea (6 cases) and abdominal pain (1 case).The absolute number of blood lymphocyte decreased to different degrees in 22 cases.Fourteen cases had lactate dehydrogenase obviously, with no obvious change in procalcitonin.The imaging manifestations were cloud-like and ground-glass-like high density shadows scattered outside the lungs, small flaky consolidation and bronchus inflating sign were seen locally.A few images showed diffuse high density, most of the lesions showed consolidation or striate change, and local fibrosis was formed in the lower lobes of both lungs. Conclusion Fever and cough are the first symptoms of COVID-19, and a few cases are associated with shortness of breath and diarrhea, accompanied by different degrees of systemic symptoms, but most of the patients improve their conditions after active antivirus, anti-infection, systematic symptoms improvement and supportive treatment.The disease is highly infectious and its condition changes rapidly.Therefore, early detection, early diagnosis and comprehensive treatment of the whole body as soon as possible are the keys to treatment.

T-cell internal antigen-1 (TIA-1) has roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SGs have been reported in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that it facilitates the assembly of canonical SGs in both human and chicken cells. Overexpression of the chicken prion-related domain (cPRD) of cTIA-1 that bore an N-terminal green fluorescent protein (GFP) tag (pntGFP-cPRD) or Flag tag (pFlag-cPRD) induced the production of typical SGs. However, C-terminal GFP-tagged cPRD induced notably large cytoplasmic granules that were devoid of endogenous G3BP1 and remained stable when exposed to cycloheximide, indicating that these were not typical SGs, and that the pntGFP tag influences cPRD localization. Finally, endogenous cTIA-1 was recruited to SGs in chicken cells and tissues under environmental stress. Taken together, our study provide evidence that cTIA-1 has a role in canonical SG formation in chicken cells and tissues. Our results also indicate that cPRD is necessary for SG aggregation.
Chickens ; Clone Cells ; Cycloheximide ; Cytoplasmic Granules ; Humans ; Protein Biosynthesis ; RNA Precursors ; RNA-Binding Proteins ; T-Lymphocytes

A cumulative evidence indicates that consumption of tea catechin, flavan-3-ol derived from green tea leaves, lowers the risk of cardiovascular diseases. However, a precise mechanism for this cardiovascular action has not yet been fully understood. In the present study, we investigated the effects of different green tea catechins, such as epigallocatechin-3 gallate (EGCG), epigallocatechin (EGC), epicatechin-3 gallate (ECG), and epicatechin (EC), on angiotensin II (Ang II) -induced hypertrophy in primary cultured rat aortic vascular smooth muscle cell (VSMC). [3H]-leucine incorporation was used to assess VSMC hypertrophy, protein kinase assay, and western blot analysis were used to assess mitogen-activated protein kinase (MAPK) activity, and RT-PCR was used to assess c-jun or c-fos transcription. Ang II increased [3H]-leucine incorporation into VSMC. However, EGCG and ECG, but not EGC or EC, inhibited [3H]-leucine incorporation increased by Ang II. Ang II increased phosphorylation of c-Jun, extracellular-signal regulated kinase (ERK) 1/2 and p38 MAPK in VSMC, however, EGCG and ECG, but not EGC or EC, attenuated c-Jun phosphorylation increased by Ang II. ERK 1/2 and p38 MAPK phosphorylation induced by Ang II were not affected by any catechins. Ang II increased c-jun and c-fos mRNA expression in VSMC, however, EGCG inhibited c-jun but not c-fos mRNA expression induced by Ang II. ECG, EGC and EC did not affect c-jun or c-fos mRNA expression induced by Ang II. Our findings indicate that the galloyl group in the position 3 of the catechin structure of EGCG or ECG is essential for inhibiting VSMC hypertrophy induced by Ang II via the specific inhibition of JNK signaling pathway, which may explain the beneficial effects of green tea catechin on the pathogenesis of cardiovascular diseases observed in several epidemiological studies.
Angiotensin II ; Angiotensins* ; Animals ; Blotting, Western ; Cardiovascular Diseases ; Catechin* ; Electrocardiography ; Hypertrophy* ; MAP Kinase Signaling System ; Muscle, Smooth, Vascular* ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Rats ; RNA, Messenger ; Tea

OBJECTIVETo recognize the clinical features of the enterovirus 71 (EV71) infection with pulmonary edema or pulmonary hemorrhage as a fulminant and often fatal illness.

METHODSWe retrospectively reviewed the medical records of the three cases with EV71 infection for clinical manifestation, laboratory data, medications, outcome etc.

RESULTSAll the cases were infants and they all died. These infants had no skin or mucosal lesions, however, they had sudden onset of cyanosis and tachypnea 1 to 2 days after the onset of the febrile disease with vomiting. All these 3 cases were misdiagnosed and were treated for shock on admission. Pulmonary hemorrhage was not considered in any of the cases on admission. All the cases received tracheal intubation when foamy secretions were discharged from mouth and nose of the patients and notable cyanosis was noted. After intubation, all had pink foamy fluid flew out from the endotracheal tube. The patients had hyperglycemia and limb weakness, two had tachycardia, and hypertension was found in one case. Chest X-ray showed bilateral or unilateral widespread air space opacity, but the cardiac size and shape were normal. All the patients had leucocytosis. EV71 infection was confirmed by detection of specific sequences of the virus in throat swab and tracheal secretions samples and in one case in cerebrospinal fluid sample.

CONCLUSIONPulmonary edema or pulmonary hemorrhage occurred in the 3 cases with EV71-infected infants. The initial presentation was often nonspecific with fever and vomiting, and sudden appearances of cyanosis, tachypnea, tachycardia, hypertension or hypotension, limb weakness may suggest pulmonary edema or hemorrhage. Excessive fluid resuscitation may deteriorate the illness, on the contrary, fluid restriction and inotropic agents, and early intubation with positive pressure mechanical ventilation may be the proper treatment.

Enterovirus A, Human ; Enterovirus Infections ; pathology ; Female ; Hemorrhage ; etiology ; virology ; Humans ; Infant ; Male ; Pulmonary Edema ; etiology ; virology ; Retrospective Studies

OBJECTIVETo explore the heterogeneous subclones in acute myeloid leukemia (AML) with t(8;21) by quantitative multicolor- fluorescence in situ hybridization (QM-FISH), and to figure out whether there is putative ancestral relationship among different subclones.

METHODSBacterial artificial chromosomes (BAC) clones that contain the targeted genes including AML1, ETO, WT1, p27 and c-kit were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP or dCTP to targeted genes by nick translation. Bone marrow mononuclear cells from t (8;21) AML patients are dropped on to the wet surface of glass slides after hypotonic treatment and fixation. After hybridization, the fluorescence signals were captured by Zeiss fluorescence microscope. The copy number of AML1, ETO, WT1, p27, c- kit and the AML1-ETO fusion gene in AML1-ETO positive cells was counted. The cells with same signals were defined as a subclone. Various subclones were recorded and their proportions were calculated, and their evolutionary relationship was deduced. The subclones in matched primary and relapsed samples were compared, the evolution of dominant clones were figured out and the genomic abnormality that is associated with relapse and drug resistance were speculated.

RESULTSIn this study, 36 primary AML with t(8;21) cases and 1 relapsed case paired with the primary case were detected. In these 36 primary cases, 4 cases (11.1%) acquired additional AML1-ETO fusion signal, 3(8.3%) had additional AML1 signal, 4(11.1%) had additional ETO signal, 20(55.6%) had additional WT1 signal, 15(41.7%) had additional p27 signal and 14(38.9%) had additional c-kit signal. In addition, 10(27.8%) displayed AML1 signal deletion, and such an aberration represents statistic significance in male patients. It seems that male patients usually accompany AML1 signal deletion. Of 36 cases, 28(77.8 %) harbored at least 2 subclones (ranged from 2 to 10). According to the genetic signature of subclones, we can assemble a putative ancestral tree, and the genetic architecture is linear or branching. In particular, the clonal architecture of the relapsed sample exhibited significant clonal evolution compared to its paired sample at diagnosis, including proportion changes in dominant clone, subclone disappearance and appearance of new dominant clones.

CONCLUSIONGenomic abnormality is very diverse in t(8;21) AML. Subclones have linear or complex branching evolutionary histories, and clonal architecture is dynamic.

Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Male ; Translocation, Genetic

To study the efficacy and compliance analysis of pollen allergen drops in the treatment of allergic rhinitis. The method of single-center controlled was used to analyze the dates' results. From July 2021 to September 2021, 80 patients with seasonal allergic rhinitis were referred to the clinic of otorhinolaryngology in First Hospital of Shanxi Medical University.40 patients received sublingual immunotherapy (SLIT group), and the other 40 patients received symptomatic drug treatment as the control group. The total rhinoconjunctivitis symptom score (TRSS), the visual analogue scale(VAS), total medication score (TMS) and combined scores of medication and rhinoconjunctivitis symptoms (CSMRS) of the patient before the start of the treatment and after the first year of the treatment were compared to assess the efficacy of sublingual immunotherapy of Artemisia pollen. Follow the shedding during the study, the safety of the drug and the causes for compliance analysis were analyzed and recorded. The results of comparison with TRSS, VAS, TMS and CSMRS in two groups in the period of pretherapy were as follows: TRSS(12.393±3.023, 12.450±3.029, t=-0.077, P=0.939), VAS(8.357±1.026, 8.400±0.982, t=-0.173, P=0.862), TMS(3.214±0.568, 3.175±0.501, t=0.301, P=0.764), CSMRS (5.286±0.680, 5.253±0.677, t=0.199, P=0.843), there was no significant difference (P>0.05); lower observed symptom scores were got in the post-treatment pollen peak SLIT group compared to the control group, TRSS(3.964±1.551, 7.750±2.169, t=-7.918, P<0.05), VAS(2.893±0.956, 5.175±1.481, t=-8.286, P<0.05), TMS (1.821±0.863, 3.175±0.501, t=-8.163, P<0.05), CSMRS (2.489±0.921, 4.468±0.601, t=-10.723, P<0.05), and the differences between the groups were statistically significant (P<0.05); the SLIT group significantly reduced all symptom scores at the first peak compared to the starting, TRSS(12.393±3.023, 3.964±1.551, t=20.576, P<0.05), VAS (8.357±1.026, 2.893±0.956, t=30.070, P<0.05), TMS (3.214±0.568, 1.821±0.863, t=7.151, P<0.05), CSMRS(5.286±0.680, 2.489±0.921, t=14.533, P<0.05) and there was statistical difference (P<0.05). No significant adverse reactions occured during medication in the SLIT group. A total of 12 cases were shed in the SLIT group, so the compliance rate was 70%. The four reasons were that patients considered the course was long (4 cases, 33%); the drugs were expensive (3 cases, 25%); patients were busy with their work and life (3 cases, 25%); patients were affected by the outbreak (2 cases, 17%). In summary, Artemisia pollen sublingual drops may improve the symptoms of the patients who got allergic rhinitis caused by Artemisia pollen after the treatment for one year. However, due to the lack of sufficient understanding of immunotherapy or the difficulty in adhering to standardized medication, the compliance with sublingual immunotherapy is still poor, the compliance with sublingual immunotherapy needs to be further improved through patient education.
Humans ; Rhinitis, Allergic, Seasonal/therapy* ; Rhinitis, Allergic ; Pollen ; Ambulatory Care Facilities ; Allergens