OBJECTIVE: Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system. METHODS: Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were also fused to the GFP expression vector. The recombinant vectors were transfected into HEK-293T cells plated onto coated glass coverslips. The transfected cells were stained with 4',6-diamidino-2-phenylindol and photographed using a fluorescence microscope. RESULTS: Mutants containing the HD region as well as full-length Obox4 were clearly localized to the nucleus. In contrast, the other mutants of either the N-terminal or C-terminal region without HD had impaired nuclear localization. We also found that the N-terminal and C-terminal of the Obox HD contributed to nuclear localization and the entire HD was necessary for nuclear localization of Obox4. CONCLUSION: Based on the results of the present study, we demonstrated that the intact HD region of Obox4 is responsible for the nuclear localization of Obox4 protein in cells.
Fluorescence ; Genes, Homeobox ; Glass ; Meiosis ; Oocytes

OBJECTIVETo investigate the differently expressed Homeobox genes between adenoid cystic carcinoma of salivary gland and normal gland tissue, and find out the effect of homeobox genes on oncogenesis and differentiation of adenoid cystic carcinoma of salivary gland.

METHODSSix strictly paired specimens including adenoid cystic carcinoma and its surrounding normal gland tissue and two pairs of specimens including cell strain of adenoid cystic carcinoma and its surrounding normal gland tissue were established. Customized Oligo microarray which contains probes of 232 human homeobox genes was used to analyze and conclude two groups of different genes data. RT-PCR technique was used to examine the mRNA expressing level of highly suspected relevant genes of adenoid cystic carcinoma in different specimens. Obvious differently expressed Homeobox genes were found through statistical analyses.

RESULTSIn tissue specimens homeobox genes were found 67 up-regulated and 54 down-regulated, and in cell specimens homeobox genes were found 12 up-regulated and 15 down-regulated. One up-regulated gene and 7 down-regulated genes were found both in tissue and cell specimens, among which EVX1 and PITX1 were the most frequent. RT-PCI showed that there was statistical expressing difference between TGIF, EVX1 and normal gland tissue in ACC-M.

CONCLUSIONAs the key gene to cellular proliferation and differentiation, homeobox genes are closely relevant to the oncogenesis of adenoid cystic carcinoma of salivary gland.

Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population.
Animals ; Dependovirus ; Fluorescence ; Genes, Homeobox ; Genes, Reporter ; Immunohistochemistry ; Mice ; Mice, Transgenic ; Neurons* ; RNA, Small Interfering

OBJECTIVE: To compare the clinical effect of the combined treatment of acupuncture, moxibustion, Chinese herbal medicine and western medication and simple western medication on polycystic ovary syndrome (PCOS) of kidney deficiency and blood stagnation pattern and explore the effect on endometrial receptivity and the expression of serum homeobox gene A10 (HOXA10). METHODS: A total of 60 patients with PCOS of kidney deficiency and blood stagnation pattern were randomized into a combined treatment group and a western medication group, 30 cases in each one. In the western medication group, on the fifth day of menstruation, clomiphene citrate tablets were taken orally, 50 mg each time, once daily, consecutively for 5 days. On the day when the follicle diameter was ≥ 18 mm, chorionic gonadotrophin for muscular injection, a dose of 10 000 U was given. Before sleep, the aspirin enteric-coated tablets were taken orally, 50 mg (except during menstruation). In the combined treatment group, on the base of the treatment as the western medication group, acupuncture and moxibustion were adopted and the Chinese herbal for tonifying the kidney and activating blood circulation was taken orally. The acupoints were Guanyuan (CV 4), Qihai (CV 6), Zusanli (ST 36), Sanyinjiao (SP 6), Zigong (EX-CA 1), etc. Acupuncture was remained for 30 min each time, once every two days and discontinued during menstruation. Chinese herbal was given from the 3rd day of menstruation till the onset of the next menstruation, one dose each day. After consecutive treatment for 3 menstrual cycles in the two groups, the real-time polymerase chain reaction (RT-PCR) method was adopted to determine the expression of serum HOXA10 before and after treatment in the patients of the two groups. The endometrial thickness at ovulatory phase, uterine arterial flow 7 days after ovulation [including uterine arterial pulsatility index (PI), resistance index (RI), peak systolic velocity (PSV)/end diastolic velocity (EDV), meaning S/D], pregnancy rate and the score of Chinese medicine symptoms before and after treatment were compared in the patients between the two groups. RESULTS: ① After treatment, the expression of serum HOXA10 was higher than that before treatment in the patients of the two groups ( CONCLUSION The combined treatment with acupuncture, moxibustion and medication effectively improves endometrial receptivity and uterine arterial flow in the patients with PCOS of kidney deficiency and blood stagnation pattern and increases pregnancy rate. The therapeutic effect is better than the simple western medication and its mechanism is probably related to the regulation of serum HOXA10 expression.
Acupuncture Points ; Acupuncture Therapy ; Female ; Genes, Homeobox ; Homeobox A10 Proteins ; Humans ; Kidney ; Moxibustion ; Polycystic Ovary Syndrome/genetics* ; Pregnancy

HOX gene encodes a group of homeodomain transcription factors which are highly conserved. The caudal-type homeobox (CDX) , ten-eleven translocation (TET) genes and polycomb group (PcG) , trithorax group (TrxG) proteins act as upstream regulators of HOX genes that manipulate the targeted gene expression through genetic and epigenetic mechanisms. The abnormal expression of HOX genes and their fusions contribute to myelodysplastic syndromes (MDS) pathogenesis. Aberrant DNA methylation and NUP98-HOX translocation serve as molecular mediators of dysfunction in MDS which can be used for the evaluation of biology and therapy. This article provides an overview of recent advances of studies on HOX gene and its abnormal molecular mechanisms, as well as potential correlation with MDS.
DNA-Binding Proteins ; Drosophila Proteins ; Epigenesis, Genetic ; Genes, Homeobox ; Humans ; Myelodysplastic Syndromes ; Translocation, Genetic

In order to investigate the special role of HOXB4 in expansion and self renewal of hematopoietic stem cells, the cDNA of HOXB4 was extracted and cloned from umbilical cord blood mononuclear cells by using RT-PCR. Then the eukaryotic expression bicistronic plasmid vector pIRES2-EGFP/HOXB4 was designed and constructed after cutting HOXB4 and pIRES2-EGFP respectively by restriction enzyme EcoRI and BamHI. The recombinant plasmid was delivered into competent cells of Escherichia coli. The successful construction of plasmid was confirmed by the identification of endonuclease cutting and sequencing. The results showed that the HOXB4 cDNA was cloned successfully from umbilical cord blood mononuclear cells and the recombinant eukaryotic expression bicistronic plasmid vector was constructed, and then introduced it into 293T cells successfully. It is concluded that a pIRES2-EGFP/HoxB1 eukaryotic expression bicistronic plasmid vector has been constructed successfully, which results provide a useful material basis for exploration of HoxB4 function in the proliferation and differentiation of hematopoietic cells.
Cell Line ; Cloning, Molecular ; Gene Expression ; Genes, Homeobox ; Genetic Vectors ; Humans ; Plasmids ; Transfection

OBJECTIVEMuscle segment homeobox gene (MSX)1 has been proposed as a gene in which mutations may contribute to nonsyndromic cleft lip with or without cleft palate (NSCL/P). To study MSX1 polymorphisms in NSCL/ P by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), and investigate the association of MSX1 exons 1 polymorphisms with NSCL/P.

METHODSDNA were extracted from blood samples from NSCL/P and unrelated normal subjects. Genome DNA from peripheral leukocyte with these blood samples were extracted, which was used as template to amplify desired gene fragment of MSX1 exons 1 by means of polymerase chain reaction (PCR). The PCR products were examined by single-strand conformation polymorphism (SSCP). The MSX1 exons 1 polymorphisms were examined by sequencing if mutations were found.

RESULTSMSX1 genes of exon 1 mutation was not been found in the NSCL/P and unrelated normal subjects by SSCP.

CONCLUSIONNo correlation between MSX1 exon 1 and NSCL/P was found. MSX1 exon 1 may not be a key gene (susceptibility gene) in NSCL/P.

In medicolegal investigations, correct identification of the necrophagous fly species collected around and on the corpse is an essential step for estimating the postmortem interval (PMI). Therefore, forensic pathologists and entomologists investigating deaths due to violent crimes need a rapid, easy-to-use protocol to identify fly species found on corpses. A rapid and robust DNA-based tool that can distinguish between various immature and mature species from the Calliphoridae, Muscidae, and Sarcophagidae families would be ideal for such investigations. To date, the DNA barcode initiative is the best approach for identifying species-specific nucleotide sequences. We have developed 3 sequence-characterized amplified region (SCAR)-based identification systems derived from the Abdominal-B homeobox sequences of 17 fly species belonging to the Muscidae and Sarcophagidae. The flies used in this study were collected in Korea. These assay systems can classify 17 forensically important fly species into the dipteran family group and reliably distinguish them from inter- and intraspecific fly species through a 2-step multiplex PCR. This novel approach may also be used as an alternative to conventional DNA-based identification methods.
Base Sequence ; Cadaver ; Crime ; Diptera ; DNA ; Genes, Homeobox ; Humans ; Korea ; Multiplex Polymerase Chain Reaction ; Muscidae ; Sarcophagidae

This study was purposed to construct the recombinant retroviral vector containing human homeobox gene hoxA10 and to establish the packaging cell lines which stably produce viruses. The whole coding region of hoxA10 gene was amplified by PCR and inserted into the retroviral vector MSCVneo. The recombinant vector was identified by DNA sequencing. The recombinant and control retroviral vectors were transfected into the packaging cell line PT67 by liposome Lipofectamine(TM) 2000. These cell lines stably producing retrovirus were isolated following G418 selection. The viral suspension was harvested and the viral titer was determined by NIH3T3 cells. The results showed that the recombinant retroviral vector was proved to encode hoxA10 genes by sequencing. The cell lines efficiently producing virus were screened by G418 and designated as PT67/MSCVneo and PT67/MSCVneo-hoxA10. The titers of them were 5 x 10(5) CFU/ml and 4 x 10(4) CFU/ml respectively. It is concluded that the recombinant retroviral vector containing homeobox gene hoxA10 and the stably packaging cell lines which efficiently and correctly produce viruses are successfully constructed, which provides a basis for further exploration of the hoxA10 gene function in the regulation of hematopoiesis.
Cell Line ; Genes, Homeobox ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Retroviridae ; genetics ; Transfection

Thymic carcinomas are uncommon malignant tumors, and thymic adenocarcinomas are extremely rare. Here, we describe a case of primary thymic adenocarcinoma in a 59-year-old woman. Histological examination of the tumor revealed tubular morphology with expression of cytokeratin 20 and caudal-type homeobox 2 according to immunohistochemistry, suggesting enteric features. Extensive clinical and radiological studies excluded the possibility of an extrathymic primary tumor. A review of the literature revealed only two global cases of primary tubular adenocarcinomas of the thymus with enteric immunophenotype.
Adenocarcinoma* ; Female ; Genes, Homeobox ; Humans ; Immunohistochemistry ; Keratin-20 ; Middle Aged ; Thymoma ; Thymus Gland*