Objective: To construct a phage-displayed random combinatorial library of IgA affibodies and to analyze its directed in vitro evolution, so as to study the relationship of IgA affibody structure with its function. Methods: The coding sequences of two affibodies, ZA1 and ZA2, were generated by overlapping PCR. The affibodies with a random linking peptide coding sequence in the 3′ terminal were randomly ligated and cloned into the K pn I site of the phagemid pCANTAB5S to construct a combinatorial phage library. Totally four rounds of in vitro human IgA directed evolution were conducted, and selected phage clones were prepared individually to test the IgA binding activity by ELISA technology. Results: The combinatorial phage library was successfully constructed; it contained about 3.4 × 107 clones with a titer of 1.6 × 1012 TU/L, and the positive clones accounted for more than 79%. Sequence analysis showed that the two single affibodies were randomly linked by random linking peptides. The composition of the phage clones displaying three or four affibodies in tandem increased remarkably along with the rounds of selection, which indicated the successful IgA directed evolution. Three new arrangements of two, three or four affibodies in tandem were obtained. ELISA results demonstrated a significantly enhanced IgA binding activity of three or four affibodies in tandem. Conclusion: A series of new IgA binding recombinants have been obtained by directed in vitro evolution of combinatorial phage library displaying randomly linked IgA affibodies. The three or four affibodies in tandem have much higher IgA binding activity than others, indicating that the in vitro molecular evolution is an effective way to produce IgA binding proteins with high activity.

This study was purposed to investigate the role of cytokines in pathogenesis of lymphoma-associated anemia. The levels of IFN-γ, IL-1β, IL-6, TNF-α and EPO in serum from 45 lymphoma patients and 12 normal controls were detected by using ELISA, the EPOR level on bone marrow cells were detected by flow cytometry, the CFU-E of bone marrow cultured in vitro was counted under inverted microscope. The results showed that 25 (55.6%) out of 45 newly diagnosed lymphoma patients had anemia before diagnosis, 13 (28.9%) had anemia during therapy, 7 (15.5%)never had anemia. The IFN-γ and TNF-α levels in serum of patients with moderate and severe anemia were significantly higher than those in patients with mild anemia and without anemia as well as normal controls. The EPO, IL-6 and IFN-γ levels correlated negatively with Hb concentration in patients, the EPOR level in patients without anemia significantly higher than that in patients with anemia and normal controls. The bone marrow CFU-E amount in patients showed positive correlation with Hb and EPOR levels. It is concluded that the increased IFN-γ, TNF-α and IL-6 may contribute to the anemia in lymphoma, and yet the EPO and EPOR levels are elevated to balance negative regulatory effects on hematopoiesis and maintain normal hematopoiesis.
Adult ; Aged ; Anemia ; blood ; etiology ; pathology ; Case-Control Studies ; Cytokines ; blood ; Erythropoietin ; blood ; Female ; Humans ; Interferon-gamma ; blood ; Interleukin-1 ; blood ; Interleukin-6 ; blood ; Lymphoma ; blood ; complications ; pathology ; Male ; Middle Aged ; Receptors, Erythropoietin ; blood ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVESTo investigate targeted blockage of BCR/ABL oncoprotein mediated cell transformation by STAT5 decoy oligodeoxynucleotide (ODN), its effect on the growth and proliferation inhibition of K562 cells and the related molecular mechanisms.

METHODSSTAT5 decoy ODN, designed and synthesized in vitro, was transfected into K562 cells by cationic lipid. The cell growth curve and colony formation assay were used to reflect the growth and proliferation capacity of K562 cells, RT-PCR to detect the expression of three genes downstream STAT5.

RESULTSConfocal microscopy demonstrated that STAT5 decoy ODN was successfully transfected into K562 cells (95.2% positive cells). STAT5 decoy ODN inhibited the growth of K562 cells (inhibition rate 77.7%) and their colony formation capacity (Decoy ODN treated group 8.3% vs control group 35.7%, P < 0.05) after the treatment with STAT5 decoy ODN, the expressions of c-myc, bcl-X(L), cyclin D1 mRNA were down-regulated by 15.4%, 30.8%, 29.1%, respectively in the K562 cells.

CONCLUSIONSSTAT5 decoy ODN inhibits the growth and proliferation of K562 cells. The mechanisms may be that decoy ODN blocks the transcriptional activation potent of STAT5 and down-regulates the expression of these tumor related genes downstream STAT5.

Cell Proliferation ; Cyclin D1 ; genetics ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Expression ; Humans ; K562 Cells ; Liposomes ; Microscopy, Confocal ; Oligodeoxyribonucleotides, Antisense ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; STAT5 Transcription Factor ; genetics ; physiology ; Transfection ; bcl-X Protein ; genetics

According to published nucleotide sequences, ORF4 gene of barley yellow dwarf virus GAV (BYDV-GAV) was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). The BYDV-GAV ORF4 gene was expressed in baculovirus -insect cell expression system efficiently, and western bolt analysis confirmed its expression product. Confocal laser scanning microscopy showed that GFP: ORF4 fusion protein was associated with the nuclear envelope of insect cells. By expressing the N- and C-terminal regions of ORF4-encoding product (P4) in insect cells combined with structure prediction, it was found that the N-terminal region of P4 containing four a-helices is required for targeting P4 to the nuclear envelope. These results provide a base for biological function of ORF4 gene during systemic infection of BYDV-GAV in host plants further.
Amino Acid Sequence ; Animals ; Baculoviridae ; genetics ; metabolism ; Genes, Plant ; genetics ; Green Fluorescent Proteins ; genetics ; Insecta ; genetics ; metabolism ; Luteovirus ; genetics ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism

OBJECTIVETo figure out the function of C/EBPalpha in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657.

METHODSThe differentiation of HL60 cells was induced by NSC67657, and the cell surface antigen CD14 expression was detected by flow cytometry. The gene and protein expressions of CCAAT enhancer binding protein alpha (C/EBPalpha) before and after the induction of cell differentiation were determined by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cells, and its expression was verified. The effect of C/EBPalpha overexpression in HL60 cells was assessed by MTT assay, Wright's staining and flow cytometry before and after NSC67657 transfection.

RESULTSHL60 cells could be induced into monocytes by 10 micromol/L ATRA within 5 days, and the coverage of CD14 positive cells reached 93.9% after 5 days of drug treatment. The eukaryotic expressing vector was successfully constructed, and over 90% positive clones were obtained after screening by G418 and electrotransfection. The results of proliferative analysis, chemical staining, ultrastructural observation, and CD11b detection confirmed that HL60 cells could be induced into granulocytic differentiation by overexpression of C/EBPalpha protein. Moreover, in the drug treatment group, transfected cells could not be induced into monocytic differentiation, and their granulocytic differentiation was also inhibited.

CONCLUSIONThe monocytic differentiation of HL60 cells induced by NSC67657 may not be via the regulation by C/EBPalpha protein-mediated signal transduction. However, the overexpression of CEBPalpha may inhibit the process of NSC67657-induced monocytic differentiation in HL60 cells.

CCAAT-Enhancer-Binding Protein-alpha ; genetics ; metabolism ; CD11b Antigen ; metabolism ; Cell Differentiation ; drug effects ; Genetic Vectors ; Granulocytes ; cytology ; HL-60 Cells ; Humans ; Lipopolysaccharide Receptors ; metabolism ; Mesylates ; pharmacology ; Monocytes ; cytology ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; Steroids ; pharmacology ; Transfection

OBJECTIVETo compare the different therapeutic effect between acupuncture at Shiqizhui (EX-B 8) only and multi acupoints on dysmenorrhea.

METHODSThirty eight cases were randomly divided into a single acupoint group and a multi-acupoints group, 19 cases in each group. The single acupoint group was treated by acupuncture at Shiqizhui (EX-B 8) only, and the multi-acupoints group by acupuncture at Shiqizhui (EX-B 8), Sanyinjiao (SP 6), Diji (SP 8), Ciliao (BL 32). They were all treated from the first day when sudden intense pain occurs, one time each day, for 3 days in each menstrual cycle, the treatment of three menstrual cycles. The therapeutic effect and Visual Analogue Scale (VAS) were compared and the score of general frequency and severity of dysmenorrhea by using Cox Menstrual Symptom Scale (CMSS) were evaluated.

RESULTSThe cured rate was 68.4% (13/19) and the effective rate was 31.6% (6/19) in the single acupoint group, being similar to 78.9% (15/19) and 21.1% (4/19) in the multi-acupoints group (P > 0.05). VAS and the scores of general frequency and severity of dysmenorrhea were all significantly decreased after treatment in both groups (all P < 0.001), with no significant difference between the two groups (all P > 0.05).

CONCLUSIONAcupuncture at Shiqizhui (EX-B 8) only can be as effective as selecting multi-acupoints to cure essential dysmenorrhea.

Acupuncture Points ; Acupuncture Therapy ; methods ; Adolescent ; Adult ; Analgesia ; Dysmenorrhea ; therapy ; Female ; Humans ; Medicine, Chinese Traditional ; Young Adult

OBJECTIVETo find a good way to diagnose VD, value the effect of Yishen Yangnao capsule on VD and try to find some rules of changes in Chinese medicine syndromes.

METHODPatients were randomly divided into treating group and western medicine comparison group. It's the phase III clinical research of Rishen Yangnao capsule curing VD, judging the validity and security of it, using dukexi slice as comparison drug. Some of the patients did the examination of P300.

RESULTThe total validity of Yishen Yangnao capsule is 56.3% (contract team is 60.0%). The improve rate of ADL is 0.1069% (contract team is 0.1134%). The scores of Chinese medicine syndrome descend.

CONCLUSIONYishen Yangnao capsule has the same effect as dukexi slice in curing VD at the side of intelligence situation and life ability.

Activities of Daily Living ; Aged ; Almitrine ; therapeutic use ; Capsules ; Dementia, Vascular ; drug therapy ; physiopathology ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; therapeutic use ; Event-Related Potentials, P300 ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; methods ; Middle Aged ; Neuroprotective Agents ; isolation & purification ; therapeutic use ; Phytotherapy ; Treatment Outcome ; Yohimbine ; therapeutic use

OBJECTIVETo investigate whether ulinastatin can alleviate the side effect in patients with gastrointestinal cancer undergoing adjuvant chemotherapy,and to explore the probable mechanism of its protective efficacy.

METHODSForty consecutive patients with gastrointestinal cancer who underwent surgical operations from May 2004 to October 2004 were recruited. The patients were randomly divided into therapeutic group and control group, receiving ulinastatin 150,000 U per day or 250 ml hydrochloric sodium before chemotherapy for 5 continuous days respectively. The prevalence of side effects and the levels IL-6 and TNF-alpha were compared between the two groups.

RESULTSThere were no differences in the clinicopathological characteristics between the two groups. The prevalences of white blood cell decline (41.2% versus 13.1%), pigmentation (23.5% versus 4.3%), baldness (17.6% versus 4.3%) were higher in the control group than those in therapeutic group (all P< 0.05). In therapeutic group, IL-6 level was significantly decreased after ulinastatin treatment, but not in the control group while the levels of TNF-alpha were not changed in the both groups.

CONCLUSIONUlinastatin can reduce the common side effects of chemotherapy, and the mechanism may be associated with the decrease of IL-6.

Aged ; Chemotherapy, Adjuvant ; methods ; Female ; Gastrointestinal Neoplasms ; drug therapy ; Glycoproteins ; therapeutic use ; Humans ; Interleukin-6 ; blood ; Male ; Middle Aged ; Postoperative Period ; Tumor Necrosis Factor-alpha ; blood

Objective To explore the difference in efficacy of metoprolol versus ivabradine in the treatment of postural orthostatic tachycardia syndrome(POTS)in the elderly after COVID-19 infection.Methods A total of 110 patients diagnosed with POTS at our department from Decem-ber 1,2022 to January 31,2023 were included.According to their drug regimen,they were divided into metoprolol group(62 patients)and ivabradine group(48 patients).On the 28th day of out-patient follow-up,the resting heart rate,heart rate of 10 min of standing,symptom disappearance rate,hospitalization rate,and mortality rate were compared between the two groups.Results On the 28th day of treatment,the resting heart rate and postural heart rate for 10 min were decreased in both groups when compared with the levels at initial diagnosis(P＜0.01).And there were no significant differences in the two types of heart rate between the two groups on the 28th day(71.0±7.0 vs 72.1±7.0,P=0.401;76.5±7.2 vs 77.4±7.6,P=0.573).No obvious differences were observed between the two groups in symptom disappearance rate,hospitalization rate,or mortality rate(88.7％vs 89.6％,3.2％vs2.1％,0％vs 0％,P＞0.05).Conclusion Metoprolol and ivabradine can effectively treat POTS in the elderly patients after COVID-19 infection.

Objective To investigate the clinical manifestations, MRI features and therapeutic methods of patients with central nervous system damage secondary to primary Sjogren syndrome.Methods The clinical features, MR1 manifestations and therapeutic method of 8 patients with central nervous system damage secondary to primary Sjogren syndrome, confirmed in our hospital, were retrospectively reviewed and analyzed. Results Wallenberg syndrome, limb weakness and mentality alteration appeared in these patients, some manifested as having multiple sclerosis and neuromyelitis optica, and optic nerve was frequently involved. MRI results indicated that 5 patients had lesion in the spinal cord, mainly located in the cervical and thoracic cords: 2 in the cervical cord, 2 in the thoracic cord, and 1 both in the cervical and thoracic cords; the lesions manifested as sheet lamellar, scattered plaques with mild/obvious enhancement or without enhancement; 5 patients had brain damage: lesious could be detected in the dorsolateral medulla oblongata, thalamus, hippocampus, aqueduct of midbrain,and sides of fourth ventricle and lateral ventricles, and these lesions could manifest as multiple minor ischemic focus white matter in the subcortex and cerebral ventricles, which might be similar to Wernicke encephalopathy; 1 patient appeared cervical artery dissection, and 7 patients got improvement after the treatment of hormone. Conclusion The clinical features and MRI manifestations of patients with central nervous system damage secondary to primary Sjogren syndrome are diverse.