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

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 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

This study was purposed to investigate the molecular basis for RhD negative phenotype in Yiwu population in Zhejiang Province of China. The RhD negative samples were screened by saline agglutination test in blood donors. Some real RhD negative and RhDel phenotypes were identified using anti-human globulin test and absorbtion elution test. Ten exons of RHD gene in these samples were amplified by PCR-SSP, and positive exons were DNA sequenced. The results indicated that 30 real RhD negative and 8 RhDel phenotypes were identified in 38 initial RhD negative samples. Ten exons were complete negative in 28 real RhD negative samples and only exon 1, 2 and 10 were positive in 2 real RhD negative samples amplified by PCR. All 10 exons in 8 RbDel samples were positive and a DNA variant (1227G > A) was found in 8 RhDel samples. It is concluded that all exons are absence in most real RhD negative phenotypes, and the partial exons absence is also found in some real negative phenotypes among Yiwu population in Zhejiang province of China. The G to A mutation at position 1227 is found in all RhDel phenotypes.
Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Exons ; Genotype ; Humans ; Molecular Sequence Data ; Phenotype ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; genetics ; immunology

OBJECTIVETo explore the molecular basis of an individual featuring an ABx variant of ABO blood group system.

METHODSSerological assays were used to characterize the erythrocyte phenotypes and salivary ABH secretors. All of the seven exons and flanking introns of ABO glycosyltransferase gene were amplified with polymerase chain reaction (PCR). And the products were sequenced bidirectionally following enzyme digestion. Exons 6 and 7 were also subcloned and analyzed for haplotypes of the ABO gene.

RESULTSErythrocytes of the proband have expressed a strong A antigen and a weak B antigen, which was identified as a rare ABx variant in addition with other serological features. Nine heterozygous sites in exon 6 (297A/G) and exon 7 (467C/T, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 808T/A, 930G/A) of the coding region of the ABO gene were identified. Based on haplotype analysis, one allele was determined as common A102, whilst another was consistent with B101 except for an 808T>A mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B.

CONCLUSIONThe 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.

ABO Blood-Group System ; genetics ; Adult ; Exons ; Female ; Glycosyltransferases ; genetics ; Haplotypes ; Humans ; Mutation

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

BACKGROUNDThis study was designed to investigate the hot spots of microsatellite loss of heterozygosity (LOH) on 9p13-23 in laryngeal squamous cell carcinoma and to find out the correlation between the incidence of microsatellite LOH and the clinicopathological parameters.

METHODSTumor tissues were obtained from paraffin embedded sections with microdissection. Genomic DNA was extracted from tumor tissues and peripheral blood lymphocytes with the phenol-chloroform. Polymerase chain reaction (PCR) amplification and denaturing gel electrophoresis were carried out in a set of 42 squamous cell carcinoma (SCC) of larynx and corresponding peripheral blood lymphocytes using 13 highly polymorphic microsatellite markers on 9p13-23. The correlation was analyzed between microsatellite LOH at the high frequency on 9p13-23 and clinicopathological parameters in the patients with squamous cell carcinoma of larynx.

RESULTSOf the 42 laryngeal cancers, 41 (97.6%) showed LOH in at least one of the microsatellite markers tested on 9p13-23. The most frequently deleted marker was D9S162 in 17 of the 19 (89.5%) informative samples. The marker D9S171, which is located on 9p21, had LOH detected in 12 of the 15 informative cases (80.0%). LOH at the D9S1748 marker (closest to the p16 gene locus) was detected in 18 of the 36 informative cases (50.0%). Allelic deletion mapping revealed two minimal regions of LOH encompassing markers D9S161-D9S171 on 9p21 and IFNA-D9S162 on 9p22-23. Multiple LOH (> or = 4) on 9p21-23 was found more frequently in the patients under 60 years, with supraglottic SCC or cervical lymph node metastasis than those over 60 years, with glottic SCC or without cervical lymph node metastasis (P < 0.01 or 0.01, 0.05, respectively). On the contrary, there was no correlation between T stages or pathologic classification and the frequency of LOH on 9p21-23 in 42 SCC of Larynx.

CONCLUSIONSThese findings imply the presence of at least two putative tumor suppressor genes on 9p13-23 in laryngeal SCC. Multiple genetic alterations are probably implicated in supraglottic SCC with cervical lymph node metastasis in younger patients.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; genetics ; pathology ; Chromosomes, Human, Pair 9 ; Female ; Humans ; Laryngeal Neoplasms ; genetics ; pathology ; Loss of Heterozygosity ; Lymphatic Metastasis ; Male ; Microsatellite Repeats ; Middle Aged