OBJECTIVETo investigate the expression of androgen receptor (AR) isoforms in normal human prostate.

METHODSFourteen normal prostatic specimens from donors, aged 25 on average (21-28 yr), were analyzed by high resolution isoelectric focusing (IEF). The expression of AR isoforms was demonstrated in all 14 normal human prostatic tissues.

RESULTSFour types of AR isoforms were detected with isoelectric point value at 6.5, 6.0, 5.8 and 5.3 in 14 prostatic specimens. Binding of 3H-dihydrotestosterone (DHT) to these four AR isoforms was inhibited by the addition of 100-fold excess of DHT and testosterone. No effect of progesterone, estradiol and diethylstilbestrol on tritiated hormone binding was observed.

CONCLUSIONSThere are four AR isoforms in normal human prostate. The expression of AR isoforms is different from one another.

Adult ; Humans ; Isoelectric Focusing ; Isoelectric Point ; Male ; Prostate ; metabolism ; Protein Isoforms ; biosynthesis ; Receptors, Androgen ; biosynthesis

OBJECTIVETo explore the mechanism of cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2).

METHODSThe expression and translocation of different isoforms of protein kinase C (PKC) in cells were observed with immunocytochemistry and Western blot techniques. The content of diacylglycerol (DAG) was analyzed with high performance thin layer chromatography (HPTLC) technique.

RESULTSTransfection of PEMT2 can inhibit the expression of cPKC alpha, but obviously promotes the expression and translocation from cytosol to plasma membrane of cPKC beta2. At the same time, the content of DAG was decreased in the transfected cells. Expression and translocation of other PKC isoforms were not changed by PEMT2 transfection.

CONCLUSIONEffects of overexpression of PEMT2 on the expression and translocation of different PKC isoforms might be related to the mechanism of cell proliferation inhibition and apoptosis induced by transfecting PEMT2.

Animals ; Liver Neoplasms, Experimental ; enzymology ; pathology ; Phosphatidylethanolamine N-Methyltransferase ; biosynthesis ; genetics ; Protein Isoforms ; Protein Kinase C ; biosynthesis ; genetics ; Rats ; Transfection

The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.
Anemia, Aplastic/*blood ; Antigens, CD34/*metabolism ; Cells, Cultured ; Colony-Forming Units Assay ; Cyclins/*biosynthesis ; Cyclins/genetics ; Fetal Blood/cytology ; Hematopoietic Stem Cells/*cytology ; Protein Isoforms/biosynthesis ; Protein Isoforms/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Serum

OBJECTIVETo investigate the effects of estrogen on the expression of phosphofructokinase muscle-specific isoform (PFK-M) in genioglossus of chronic intermittent hypoxia (CIH) rats.

METHODSFifty male SD rats were randomly divided into five groups: the normal control group (NC), the chronic intermittent hypoxia group (CIH), and three doses of estrogen plus hypoxia groups (LE, ME, HE). Rats in the latter four groups were used to build CIH models (8 h/d, 5 weeks). In the mean time, rats in the latter three groups were injected with three dose levels of estrogen (0.1, 0.2, 0.3 mg/kg), and rats in NC and CIH groups were injected with sterile olive oil as control. At the end of the treatment, the genioglossus was isolated and quickly removed. The mRNA levels of PFK-M were determined by real-time RT-PCR and the protein content of PFK-M was detected by Western blotting analysis.

RESULTSPFK-M mRNA and protein in CIH group (2.144 ± 0.260, 0.875 ± 0.025) were both higher than those (1.000 ± 0.259, 0.413 ± 0.013) in NC group (P < 0.05). The expression of PFK-M mRNA in LE, ME and HE groups were 1.424 ± 0.193, 1.395 ± 0.251 and 1.310 ± 0.094, respectively. The expression of protein in LE, ME and HE groups were 0.638 ± 0.015, 0.576 ± 0.017 and 0.505 ± 0.021, respectively. Compared with CIH group, the expression of PFK-M mRNA and protein in LE, ME and HE groups were all inhibited significantly (P < 0.05). Among the three treatment groups, decreased protein content of PFK-M was observed only in HE group when compared with LE group (P < 0.05), but no significant difference was detected in the expression of PFK-M mRNA.

CONCLUSIONSCIH exposure could increase the expression of PFK-M mRNA and protein in rat genioglossus, while estrogen administration could dose dependently inhibit the overexpression.

Animals ; Estrogens ; physiology ; Hypoxia ; Male ; Muscle, Skeletal ; metabolism ; Phosphofructokinases ; biosynthesis ; Protein Isoforms ; RNA, Messenger ; Rats ; Tongue ; metabolism

OBJECTIVETo investigate the feasibility of metallothionein (MT) gene expression level in human peripheral blood lymphocytes (HPBLs) as a biomarker in cadmium exposure.

METHODSThe MT gene expression level in HPBLs of workers exposed to cadmium was examined using RT-PCR technique, and the exposure assessment and effect assessment were conducted in exposed workers.

RESULTSThe basal MT-1A, IE, IF, IX and MT-2A expression level in workers exposed to cadmium were significantly higher than those in the control group (P < 0.05). The basal MT-1A, IE, IF, IX and MT-2A expression level would be significantly increased with the increase of the blood cadmium (BCd) level (P < 0.05). There was a trend of increase for the mRNA expression of the basal MT-1A, 1E, IF, IX, MT-2A, especially for the mRNA expression of MT-1A and MT-2A (P < 0.05) with the increase of the level of the urine cadmium (UCd). There was a good dose-response relationship between basal MT-1A expression and UCd. The basal MT-1A, IE, IF, IX and MT-2A expression level were significantly correlated with BCd (P < 0.05) while the basal MT-1A, IF and MT-2A expression level were significantly correlated with UCd (P < 0.05). There were dose-effect relationships of BCd to the basal MT-1E, MT-1F, MT-1X and MT-2X expression level respectively and there were also dose-effect relationships of UCd, beta(2)-MG and the urine metallothionein to the basal MT-1A expression.

CONCLUSIONThe expression of the MT gene isoforms in HPBLs can serve as the biomarker for the cadmium exposure and MT-1A can also serve as the effective biomarkers for the cadmium-induced renal toxicity.

Adult ; Biomarkers ; metabolism ; Cadmium ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Gene Expression ; Humans ; Lymphocytes ; metabolism ; Male ; Metallothionein ; biosynthesis ; genetics ; Occupational Exposure ; Protein Isoforms ; biosynthesis ; RNA, Messenger ; genetics

Animals ; Hepatic Encephalopathy ; genetics ; metabolism ; Male ; Protein Isoforms ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; biosynthesis ; genetics

Mechano-growth factor (MGF) is one of IGF-1 isoforms. MGF is mechanosensitive and has important functions in muscle hypertrophy, regeneration and nerve injury recovery. In this study, MGF cDNA (330 bp) was cloned from stretched osteoblasts by RT-PCR. In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5' end sequence was truncated by primer, then the obtained truncated MGF (des(1-3)MGF) cDNA (321 bp) was subcloned in pET32a(+) vector to construct a prokaryotic recombination expression plasmid. Trx/des(1-3)MGF fusion protein, existing in forms of solution, was expressed in transformed Escherichia coli strain BL21(DE3) by IPTG induction at 30 degres C. The supernatant of cell lysates was subjected to ion exchange chromatography and Ni2+ metal affinity chromatography, and the fusion protein was obtained with the purity over 95%. After the fusion protein was cleaved by enterokinase, Trx and des(1-3)MGF was isolated by reverse-phase HPLC. Through these procedures, des(1-3) MGF was obtained with the purity of 98%. The protein molecular mass was conformity to the theoretical value by SDS-PAGE and mass spectrometry analysis. The purified des(1-3)MGF was incubated with MC3T3-E1 for cell proliferation and migration assays. The results show that des(1-3)MGF exhibited more facilitative effects on proliferation and migration of MC3T3-E1 than that of des(1-3)IGF-1.
Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Insulin-Like Growth Factor I ; Osteoblasts ; metabolism ; Protein Isoforms ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; STAT5 Transcription Factor ; biosynthesis ; genetics ; Tumor Suppressor Proteins ; biosynthesis ; genetics

The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.
Anemia, Aplastic ; blood ; Antigens, CD34 ; metabolism ; Cells, Cultured ; Colony-Forming Units Assay ; Cyclin D3 ; Cyclins ; biosynthesis ; genetics ; Female ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Male ; Protein Isoforms ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Serum

Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. To construct recombinant adenoviral vectors expressing hSef-L and hSef-S, the coding sequences of the two isoforms were amplified and ligated into pAdTrack-CMV, forming shuttle vectors pAdTrack-CMV/hSef-L-Myc and pAdTrack-CMV/hSef-S-Myc. After sequence confirmation, these two shuttle vector plasmids were linearized by Pme I and then co-transformed respectively with the adenoviral genome vector pAdEasy-1 into E. coli BJ5183. The successful recombinants were selected by Kanamycin and confirmed by Pac I digestion. The recombinant vectors Ad-hSef-L-Myc and Ad-hSef-S-Myc were finally digested with Pac I and transfected into HEK293 cells to pack into viral particles. The virus were amplified in 293 cells and used to infect MEF cells. Western blotting analysis was used to demonstrate the expression of hSef-L-Myc and hSef-S-Myc proteins. The inhibitory effects of the adenovirus mediated Sef expression on FGF signaling was further evaluated by Elk luciferase reporter assay. Our results indicated the constructed virus could produce effectively the proteins and then inhibit FGF signaling in MEF cells.
Adenoviridae ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Defective Viruses ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Protein Isoforms ; biosynthesis ; genetics ; Receptors, Interleukin ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Virus Cultivation ; methods

Crystallography, X-Ray ; Escherichia coli ; metabolism ; Fibroblast Growth Factors ; chemistry ; genetics ; metabolism ; Heparin ; metabolism ; Humans ; Models, Molecular ; Protein Binding ; Protein Isoforms ; chemistry ; metabolism ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Sulfates ; chemistry ; metabolism