Aged ; Alzheimer Disease ; drug therapy ; physiopathology ; Brain ; physiopathology ; Chitosan ; pharmacology ; therapeutic use ; Electroencephalography ; Female ; Humans ; Male ; Phospholipids ; pharmacology ; therapeutic use

Objective To analyze the karyotypes of 11 cases of Turner syndrome with marker chromosome,and study the phenotypic effects resulting from the abnormal karyotype.Methods Eleven Turner syndrome patients had a mosaic karyotype and carried a marker chromosome,and 6 marker chromosomes were ring chromosomes.Their karyotypes were showed as mos.45,X/46,X,+mar or mos. 45,X/46,X,+r.Fluorescence in situ hybridization(FISH)technique with X/Y centromere probes was performed to determine the origin of the marker chromosome.Reverse chromosome painting technique was used to identify the breakpoints of two largest markers.Phenotype effects with different chromosome breakpoints were compared.Results All the 11 marker chromosomes were ring X chromosomes.The breakpoints of the r(X)were involved in Xp22,Xq22,Xq24 and Xq26,etc.Conclusions The marker chromosomes in Turner syndrome mainly originate from X chromosome and form ring chromosome X.Each r (X)in our patients was mosaic,indicating it was originated from mitosis error during early embryo development.To analyze the origin of the marker chromosome and the breakpoint of r(X)will provide guidance for the therapy and prognosis of the Turner syndrome patient.

Adult ; Aged ; Aged, 80 and over ; Female ; Head and Neck Neoplasms ; surgery ; Humans ; Intubation, Intratracheal ; methods ; Laryngoscopes ; Male ; Middle Aged

Objective To study the different expressions of receptor activator of nuclear factor-kappa B ligang(RANKL) mRNA in spleens of rats fed with diet of low calcium and high fluoride. Methods A 2× 2×2 factorial design was used and the factors were calcium, fluoride and action time. In the design, 40 Wistar rats [average body mass(118.9±13.5)g] were divided into four groups randomly by weight: control with normal diet (0.790%, calcium), low calcium group with low calcium intake(0.063%, calcium), high fluoride group with normal diet and high fluoride intake(100 mg/L, fluoride) and low calcium and high fluoride group with low calcium and high fluoride intake. After 4 and 8 months, 5 rats of each group were sacrificed and total RNA was extracted from spleen. And the expression levels of RANKL mRNA were determined by reverse transcription polymerase chain reaction (RT-PCR). Results At time of 4 months, the expression level of RANKL mRNA was 0.13± 0.05,0.13± 0.03,0.17±0.02,0.27± 0.05 and at time of 8 months, it was 0.11 ± 0.01,0.16 ± 0.02,0.16± 0.03,0.36 ± 0.07 in control group, low calcium group, high fluoride group, low calcium with high fluoride group, repectively. The factorial design AVONA showed that low calcium and high fluoride had significant effects on RANKL mRNA expression(F = 40.224,56.679, all P < 0.05) while action time had not(F = 2.850, P > 0.05 ). The interactions of low calcium with high fluoride or high fluoride with action time were signifieant(F = 7.247, 18.789, all P < 0.05) while the interaction of high fluoride with action time was not(F = 1.751, P > 0.05). Conclusions Low calcium alone or high fluoride alone or low calcium with high fluoride or low calcium with action time can increase the the RANKL mRNA expression level. High fluoride does not affect the RANKL mRNA level as the action time is prolonged.

OBJECTIVE: To investigate the differences of gene expression before and after the compaction to search for the control mechanism of compaction. METHODS: 8-cell embryos (626 mice) and 8-cell compacted embryos (437 mice) were collected from Kunmingbai mice, respectively. The cDNA of early 8-cell embryos was used as a tester, and the cDNA of 8-cell compacted embryos as a driver. Differences of gene expression were investigated between early 8-cell embryos and 8-cell compacted embryos using combining SMART (switching mechanism at 5o end of the RNA transcript) -PCR and suppression subtractive hybridization (SSH). RESULTS: Four hundreds and seventy eight positive clones were obtained, of which 384 clones with a range of 200~1000 bp and low redundancy were selected for further analysis. Eighty-three ESTs (expressed sequence tags) of the genes expressed differently were gained between early 8-cell embryos and 8-cell compacted embryos. Bioinformatic analysis showed that among the screened 83 putative positive ESTs, 51 ESTs matched 36 known genes, 27 ESTs matched 7 hypothetical genes, and 5 ESTs were new. Genes, which were up-regulated during the compaction, included cytoskeleton(4.8%); enzymes (9.6%); transcriptional factor/regulatory factor (12%); binding protein, protein-protein interactions and transport(13.3%); structural constituent of ribosome (14.5%); pluripotency associated gene(27.7%), and molecular function unknown(18%). Novel ESTs and 17 ESTs which matched the hypothetical genes were put into GenBank with accession numbers. CONCLUSION Combining SMART-PCR and suppression subtractive hybridization is an efficient method to investigate the gene expression difference in a few samples. During the compaction, the genes maintaining cell pluripotentiality express actively, which may be related to maintaining the embryonic cell totipotence in the differentation environment. All the 17 ESTs might be novel genes related to the compaction in the compacted embryos.
Animals ; Embryo, Mammalian ; cytology ; metabolism ; Embryonic Development ; genetics ; Expressed Sequence Tags ; Female ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Developmental ; Gene Library ; Mice ; Nucleic Acid Hybridization ; methods ; Pregnancy

OBJECTIVETo establish a method for isolating and culturing human amniotic epithelial cells (hAECs) in a serum-free medium and investigate their transdifferentiation ability into islet-like cells.

METHODSThe culture condition of hAECs was optimized using DMEM with different supplements. The genetic stability of the tenth-passage cells was assessed by chromosome analysis and G-banding method. The stem cell characteristics of the cells were identified by examination of the surface markers using immunofluorescence methods. The endocrine-related genes and hormones of the cells were tested after induced differentiation into islet-like cells.

RESULTSThe hAECs allow stable passaging in the presence of 10 ng epidermal growth factor (EGF) in the culture medium. After 10 passages, the cells maintained a normal karyotype and G-banding profile. The hAECs expressed many multi-potent stem cell markers, including SSEA4, TRA-1-60, and TRA-1-81. After induced differentiation, the endocrine-related genes were expressed in the islet-like cells, including PDX1, ngn3, insulin and glucagon. Insulin secretion increased in the differentiated islet-like cells in response to high glucose exposure.

CONCLUSIONWe established a method for isolating and expanding the hAECs in a serum-free medium. hAECs possess stem cell characteristics and can be induced to differentiate into islet-like cells in vitro.

Amnion ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; secretion ; Humans ; Insulin ; secretion ; Islets of Langerhans ; cytology ; Stem Cells ; cytology ; secretion

BACKGROUNDChromosomal aberrations are the major cause of pre- and post-implantation embryo wastage and some studies suggest that half of all human conceptions have a chromosomal abnormality. A chromosomal aberration in human sperms is also one of the causes of failure of in vitro fertilization. This study was designed to ascertain whether chromosomal aneuploidy in spermatozoa is a risk factor for male infertility.

METHODSTwelve infertile men were divided into two groups: 10 with oligoasthenoteratozoospermia (OAT, Group A) and two with a normal semen analysis (Group B). Two normal healthy sperm donors acted as controls (Group C). We used fluorescence in situ hybridization (FISH) and probes for chromosomes X, Y and 18 to determine the frequency of aneuploidy.

RESULTSThe frequencies of spermatozoa disomy for chromosomes X, Y and 18 were 0.30% and 0.30%, respectively, in Group B. The percentages were not significantly different from those of Group C (0.15% and 0.16%). The frequencies of nullisomy for chromosomes X, Y and 18 were 0.15% and 0 for Group B, and 0 and 0.15% for Group C (P > 0.05). In Group A, the incidences of disomy were 1.13% and 0.96% and the frequencies of nullisomy were 1.13% and 1.60%. In these three groups, the incidences of diploidy were 0.60%, 1.00%, and 0.30%, respectively. Both the frequencies of disomic and nullisomic spermatozoa for chromosomes X, Y, and 18 and of diploid spermatozoa were significantly higher in Group A than in Groups B and C. The estimated total aneuploidy rates in the sperm from the three groups were 42.44%, 6.05%, and 2.59%, respectively.

CONCLUSIONThese results indicate that chromosomal aneuploidy in spermatozoa may be a risk factor for infertility.

Adult ; Aneuploidy ; Chromosomes, Human, Pair 18 ; Chromosomes, Human, X ; Chromosomes, Human, Y ; Humans ; In Situ Hybridization, Fluorescence ; Infertility, Male ; etiology ; genetics ; Male ; Risk Factors

Now severe oligoasthenoteratozoospermic (OAT) patients could have offsprings because of the development of technique of intracytoplasmic sperm injection. But some researchers found these patients have increasing frequency of the aneuploid on the chromosome in their sperm. If the spermatozoa with chromosomal aneuploid were fertilized, it would be resulted in a higher rate of recurrent abortion, fetal abnormal and dead birth, so the analysis of the number of sperm chromosome will play an important role in detection on infertile men. There are many new development in the chromosomal analysis of human sperm using multi-FISH, now we have a review on them.
Aneuploidy ; Chromosomes ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Infertility, Male ; genetics ; Male ; Spermatozoa ; physiology

BACKGROUNDEvidence for the importance of genetic factors in male infertility is accumulating. This study was designed to identify a novel testis-specific gene related to spermatogenesis by a new strategy of digital differential display (DDD).

METHODSBased on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene. Multi-tissue RT-PCR was performed to analyse its tissue-specific expression. The full-length cDNA of the new gene was obtained using the BLAST program. Sequencing was performed and the result was analysed. Semi-quantitative RT-PCR and Northern blot analyses of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene. The sequence of the opening reading frame was integrated into the pQE-30 vector expressed in Escherichia coil strain M15 (pREP4). With IPTG induction, the target protein was detected.

RESULTSA full-length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified. SPATA12 was 2430 bp in length, located in chromosome 3p21.1-3p21.2. The sequence of the opening reading frame was 676-1248 bp, as was confirmed by RT-PCR and sequencing. The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20417.8 and isoelectric point of 5.23. The sequence has no significant homology with any known protein in databases. Semi-quantitative RT-PCR and Northern-blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis. The expression recombinant of SPATA12 was constructed and a high level of the histidine-tagged fusion protein was obtained.

CONCLUSIONSDDD can be confirmed by SPATA12 as a novel computational biology-based approach for identification of the testis-specific expression genes. SPATA12 may function as a testicular germ cell associated gene that plays some roles in spermatogenesis. Moreover, a great amount of SPATA12 protein could be obtained by the gene recombination technique, thus providing a reliable foundation for investigating the biological function of this new protein.

Amino Acid Sequence ; Base Sequence ; Escherichia coli ; genetics ; Expressed Sequence Tags ; Gene Library ; Genes ; Humans ; Male ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatogenesis ; genetics ; Testis ; metabolism

OBJECTIVETo construct pGEX-KG/mTSARG3 recombinant vector and prepare the fusion protein to obtain rabbit polyclonal antibody against mTSARG3.

METHODSThe open reading frame (ORF) of mTSARG3 gene was amplified from mouse testis cDNA library by PCR. The products were cloned into pGEM-T Easy vectors and sequenced. The recombinant plasmids were digested by EcoRI and SalI and subcloned into PGEX-KG vector. After identification by DNA sequence analysis, the recombinant plasmids were transformed into component E.coli BL21 cells, and the GST/mTSARG3 fusion protein was expressed with IPTG induction. The anti-mTSARG3 polyclonal antibody was produced by immunization of rabbits with the fusion protein. The resultant antibody was purified by antigen column and used for Western blotting for detecting mTSARG3 expression.

RESULTSThe recombinant vector pGEX-KG/mTSARG3 was successfully constructed. GST/mTSARG3 fusion protein was expressed abundantly at 4 h after IPTG induction and polyclonal antibodies were obtained. Western blot analysis demonstrated that the antibodies specifically detected mTSARG3 expression in E.coli BL21.

CONCLUSIONWe have successfully constructed pGEX-KG/mTSARG3 recombinant vector and obtained rabbit polyclonal antibody, which may facilitate further investigation of the role of mTSARG3 in spermatogenesis.

Animals ; Antibodies ; immunology ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Heat-Shock Proteins ; biosynthesis ; genetics ; immunology ; Male ; Mice ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology