The aim of this study was to clone human RHD gene and to investigate its expression in transduced K562 cells. Total RNA was extracted from reticulocyte of cord blood. RHD and RHCE genes were amplified using RT-PCR method. The amplified products were cloned into pGEM-T plasmid by TA ligation and several clones were screened by direct sequencing method in order to obtain the RHD gene. RHD gene was subcloned into pcDNA3.1(-) expression vector, then the recombined plasmids were transduced into K562 cells with superfect transfection reagent kit. Finally transcription and expression of RHD gene in K562 cells were detected. The result showed that RHD gene has been cloned sucessfully, the inserted sequence and direction of RHD cDNA in its recombined pcDNA3.1(-) vector were identified using enzyme cutting and sequencing method. After transduced with recombined pcDNA3.1(-) vector, K562 cells could transcribe RHD mRNA in its cytoplasm and express RhD antigen on its membrane surface. In conclusion, RhD antigen can expressed in K562 cells with RHD cDNA transduction, and the expression system in vitro may be helpful to further investigate the molecular basis of RhD variants.
Base Sequence ; Cell Membrane ; metabolism ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Molecular Sequence Data ; RNA, Messenger ; biosynthesis ; genetics ; Rh-Hr Blood-Group System ; biosynthesis ; genetics ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo analyze the influence of benign prostatic hyperplasia (BPH) drugs on incidence and pathology grading of prostate cancer in China.

METHODSRetrospectively investigated the history of drug treatment in 1029 cases of BPH in patients from February 1998 to December 2004. According to the history of drug use, the patients were divided into 4 groups: finasteride group, alpha-receptor inhibitor group, finasteride and alpha-receptor inhibitor combination group and control group (untreated group). We gathered pathology sections of patients in all groups, and gave Gleason Score to each. The difference of incidence and pathology grading of prostate cancer were analyzed by Stata 7.0.

RESULTSThe incidence of prostate cancer in the population of our study was 13.5%; The incidence in finasteride group, alpha-receptor inhibitor group, combination group and control group was 9.8%, 16.0%, 10.3% and 18.6%, respectively. There was significant difference between the two groups with the use of finasteride and the two groups without it (P < 0.05). In our study, the ratio of middle or high level pathology grading (Gleason ≥ 7) in prostate cancer patients was 58.3%, the ratio of middle or high level pathology grading prostate cancer patients in the four groups was 71.4%, 59.6%, 67.7% and 40.0%, respectively. In the comparison of composition ratio of middle or high level prostate cancer, there was significant difference between the two groups with the use of finasteride and the two groups without it (P < 0.05).

CONCLUSIONSFinasteride can lower the risk of prostate cancer, but increase the pathology grade of the prostate cancer which has occurred in the same time. The alpha-receptor inhibitor does not have the same effect.

Adrenergic alpha-Antagonists ; therapeutic use ; Aged ; Aged, 80 and over ; Finasteride ; therapeutic use ; Humans ; Incidence ; Male ; Middle Aged ; Prostatic Hyperplasia ; drug therapy ; Prostatic Neoplasms ; epidemiology ; pathology ; Retrospective Studies

To investigate the molecular genetics basis for one para-Bombay phenotype, the red blood cell phenotype of the proband was characterized by standard serological techniques. Exon 6 and 7 of ABO gene, the entire coding region of FUT1 gene and FUT2 gene were amplified by polymerase chain reaction from genomic DNA of the proband respectively. The PCR products were purified by agarose gels and directly sequenced. The PCR-SSP and genescan were performed to confirm the mutations detected by sequencing. The results showed that the proband ABO genotype was A(102)A(102). Two heterozygous mutations of FUT1 gene, an A to G transition at position 682 and AG deletion at position 547-552 were detected in the proband. A682G could cause transition of Met-->Val at amino acid position 228, AG deletion at position 547-552 caused a reading frame shift and a premature stop codon. The FUT2 genotype was heterozygous for a functional allele Se(357) and a weakly functional allele Se(357), 385 (T/T homozygous at position 357 and A/T heterozygous at 385 position). It is concluded that the compound heterozygous mutation--a novel A682G missense mutation and a 547-552 del AG is the molecular mechanism of this para-Bombay phenotype.
ABO Blood-Group System ; genetics ; China ; DNA Mutational Analysis ; Female ; Fucosyltransferases ; genetics ; Genotype ; Humans ; Male ; Mutation ; Mutation, Missense ; Pedigree ; Phenotype ; Sequence Deletion

To analyse the reason for one case of hemolytic transfusion reaction, antibodies in a patient's serum were identified using panel cells and Le (a-b-) phenotype cells, patient phenotype was identified by using anti-Le(a) and anti-Le(b) blood grouping reagents and the entire coding region of FUT3 gene was amplified by PCR and sequenced directly. The results showed that both IgM anti-Le(a) and anti-Le(b) antibodies were detected in patient's serum. Red cells was typed as Le (a-b-) phenotype and the FUT3 genotype was homozygote for non-functional le(59, 508) alleles. In conclusion, anti-Le(b) antibody can result in hemolytic transfusion reaction, FUT3 gene is homozygous for le(59, 508) allele resulting in Le (a-b-) phenotype.
Adult ; Antibodies ; adverse effects ; immunology ; Blood Grouping and Crossmatching ; Female ; Fucosyltransferases ; genetics ; Genotype ; Hematologic Diseases ; Humans ; Lewis Blood-Group System ; immunology ; Serology ; Transfusion Reaction

The only difference of primary structure between single-chain prourokinase (pro-UK or scu-PA) and two-chain urokinase (UK or tcu-PA) is the cleavage of a single peptide bond (Lys158-Ile159) and transform scu-PA into its active two-chain form. A 13-peptide (Thr-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Cys), which spans the cleavage peptide bond, was synthesized and linked to KLH (Keyhole limpet hemocyanin). The Balb/c mice were immunized by the conjugated protein with proper adjuvant. According to the Kohler and Milstein's methods, a hybridoma cell line G7 secreting monoclonal antibody specific for scu-PA was obtained. The anti-scu-PA McAb, purified from the supernatant of porous microcarrier hybridoma cell culture, was conjugated to CNBr-activated Sepharose 4B to prepare an immuno-affinity chromatography column. The u-PA was purified only by this affinity column from the supernatant of cultivating the u-PA-producing recombinant CHO cell, the u-PA recovery ratio is 90.4%, the purification factor was about 50, with the specific activity of 1.2 x 10(5) IU/mg, the scu-PA ratio in the u-PA product was 96.3%. Compared to immuno-affinity chromatography, the 3-step process for purifying u-PA (cation-exchange column, gel filtration column and benzamidine affinity column) has a u-PA recovery ratio of about 65%, with a specific activity of 1.0 x 10(5) IU/mg, and an scu-PA ratio of about 90%. These results showed that immuno-affinity chromatography is simple to recover u-PA and effective to separate scu-PA from tcu-PA.
Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; immunology ; isolation & purification ; Urokinase-Type Plasminogen Activator ; immunology ; isolation & purification

OBJECTIVETo probe into the molecular genetics basis for para-Bombay phenotype.

METHODSRed blood cell phenotype of the proband was characterized by serological techniques. Exons 6 and 7 of ABO gene, the entire coding region of alpha(1,2) fucosyltransferase (FUT1) gene and FUT2 gene were amplified by polymerase chain reaction (PCR) from genomic DNA of the proband respectively. The PCR products were excised and purified from agarose gels and were directly sequenced.

RESULTSAG at 547-552 deletion homozygous allele was found in the proband, which caused a reading frame shift and a premature stop codon. Parents of proband were heterozygous carriers.

CONCLUSIONTwo base deletion at position 547-552 of alpha (1,2) fucosyltransferase gene may cause para-Bombay phenotype.

ABO Blood-Group System ; genetics ; Fucosyltransferases ; genetics ; Humans ; Male ; Mutation ; Phenotype

OBJECTIVETo study the molecular genetic basis for p phenotype.

METHODSRed blood cell phenotype of the proband was characterized by standard serological technique. Exon 3 of the alpha (1,4) galactosyltransferase gene was amplified by polymerase chain reaction from genomic DNA of the proband. The amplified PCR products were excised and purified from agarose gels and direct sequenced.

RESULTSHomozygous single nucleotide G deletion at position 300 or 301 was found in the proband, which caused a reading frame shift at codon 101,resulting in a premature stop at codon 113. Parents of the proband were heterozygous carriers.

CONCLUSIONA novel single guanosine deletion at position 300 or 301 of alpha (1,4) galactosyltransferase gene was determined, which may be one facet of the molecular basis for p phenotype.

Galactosyltransferases ; genetics ; Gene Deletion ; Humans ; Male ; Phenotype ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

RHD gene has different alternative transcripts. This study was aimed to construct expression vector of normal mRNA, DEL9 and DEL89 transcripts from RHD gene. Total RNA was extracted from Rh(D) positive umbilical blood cells of newborn. Intact RhD cDNA, DEL9 and DEL89 transcripts were obtained by one-step and two-step RT-PCR, respectively. The obtained products were cloned into pCR4 TOPO sequencing vector for choosing the right transcript. RHD gene was amplified again from the sequencing plasmid DNA, and then subcloned into pcDNA3.1/V5-His TOPO expression vector; DEL9 and DEL89 were cloned into the expression vector directly. Gene sequence and direction were identified by sequencing. The results showed that the sequence and direction of target genes were right, thus these 3 different expression vectors were correctly constructed. It is concluded that expression vector is constructed from different transcripts of RHD gene, which lays a foundation for further exploring the membranous protein expression of Rh(D) antigen.
Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA

OBJECTIVETo investigate the application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression.

METHODSThe results of flow cytometry of 30 lymphoma/leukemia cases with aberrant antigen expression, of which 3 cases being lymphomas, 8 B-cell leukemia, 1 T-cell leukemia, 17 acute non-lymphoid leukemia and 1 acute non-lymphoid leukemia involving lymph nodes were analyzed. Immunohistochemistry (EnVision) for CD79a, CD3 and MPO was performed on all cases.

RESULTSEleven cases of B-cell lymphoma/leukemia were cytoplasmic CD79a (cCD79a)-positive, cytoplasmic CD3 (cCD3epsilon) and cytoplasmic MPO (cMPO)-negative. Five of these cases were positive for CD5 and 2 for CD5, 1 or 2 for myeloid marker(s). The T-cell leukemia cases were cCD3epsilon-positive, cCD79a and cMPO-negative, they also co-expressed CD13 and CD33. The mantle cell lymphoma cases were positive for CD3, CD13 and CD33. Of the 8 B-cell leukemia cases, 4 were positive for CD5, 3 for CD13 and 1 for CD13 and CD33. The 18 acute non-lymphoid leukemia cases (including 1 acute non-lymphoid leukemia case involving lymph nodes) were cMPO-positive and cCD79a and cCD3epsilon-negative. Eight of the 18 expressed T-cell markers (including 1 case of acute non-lymphoid leukemia involving lymph nodes), 8 expressed B-cell markers, 2 expressed both T and B-cell markers.

CONCLUSIONSFlow cytometry can demonstrate aberrant antigen expression in lymphoma/leukemia cells and is helpful in delineating their cell origin. The technique is thus useful in the differential diagnosis of lymphoma/leukemia.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; CD13 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD5 Antigens ; metabolism ; CD79 Antigens ; metabolism ; Diagnosis, Differential ; Flow Cytometry ; Humans ; Leukemia, B-Cell ; diagnosis ; immunology ; Leukemia, T-Cell ; diagnosis ; immunology ; Lymphoma, Mantle-Cell ; diagnosis ; immunology ; Peroxidase ; metabolism ; Retrospective Studies ; Sialic Acid Binding Ig-like Lectin 3

OBJECTIVETo explore the treatment and prognosis on patients with tracheal invasion by papillary thyroid carcinoma (PTC).

METHODSForty-five patients treated for PTC with tracheal invasion between 1980 and 1995 were retrospectively analyzed. The different kinds of surgical modalities were performed according to the extent and degree of tracheal invasion by PTC. Neck dissect was performed in 39 patients. External beam radiotherapy was used postoperatively in patients with gross residual tumor or microscopic residual tumor in pathologic margins after resection. Survival was evaluated using the Kaplan-Meier method.

RESULTS(1) Twenty-eight patients with limited tracheal invasion were treated with shave excision, the 5- and 10-year survival rates were 85.0% and 62.6%, respectively. After a shave excision, the differences of 5- and 10-year survival rates between irradiated and nonirradiated patients were not statistically significant (P > 0.05). (2) Ten patients were radical excision for intraluminal involvement extending through the tracheal cartilage, including circumferential sleeve resection (4 cases), tracheal window resection (5 cases) and total laryngectomy (1 case), the survival rate was 80.0% for five years and 58.3% for ten years. After a radical excision, the differences of 5- and 10-year survival rates between irradiated and nonirradiated patients were not statistically significant (P > 0.05). (3) For 7 patients performing the palliative operation, the 5-and 10-year survival rates were 42.9% and 28.6%, respectively. For 4 patients received postoperative radiotherapy, the 5-and 10-year survival rates were 50.0% and 50.0%, respectively. Three patients didn't received postoperative radiotherapy, the 5-year survival rate was 33.3%, no patient survived for ten years. In these patients of incomplete resection, the differences of 5-and 10-year survival rates between irradiated and nonirradiated patients were not statistically significant (P > 0.05).

CONCLUSIONSPTC with limited involvement of the trachea could be treated successfully by shaving tumor off the tracheal cartilage. Intraluminal involvement extending through the tracheal cartilage could be resected radically in patients with PTC. Postoperative radiotherapy could improve the survival of the patients with PTC with tracheal invasion who have been performed incomplete resection.

Adult ; Aged ; Carcinoma, Papillary ; diagnosis ; pathology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; Retrospective Studies ; Survival Rate ; Thyroid Neoplasms ; diagnosis ; pathology ; therapy ; Trachea ; pathology ; Tracheal Neoplasms ; diagnosis ; secondary ; therapy