OBJECTIVETo clone a novel human testis-specific gene TDRG1.

METHODSA new contig of expression sequence tags (ESTs) Hs.180197 was identified from the testis libraries using digital differential display (DDD) to screen the novel human testis-specific gene. To validate the use of bioinformatics approaches in gene discovery, the ESTs Hs.180197, which was predicted to be testis specific, was chosen for further study. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on different normal tissues to identify the expression of Hs.180197 in human testis. Using bioinformatics methods and IMAGE cloning of this contig, the full-length cDNA sequence of the noval human gene was cloned.

RESULTSThis novel gene was 1197 bp in length, located in chromosome 6p21.1-p21.2. The sequence of the open reading frame was 504-806 bp, as confirmed by RT-PCR and sequencing in human testis. The cDNA encodes a novel protein of 100 amino acids with a theoretical molecular weight of 10 000 and isoelectric point of 6.81. The sequence shares no significant homology with any known protein in the databases. Semi-quantitative RT-PCR analysis of multiple tissues further showed that the novel gene was expressed specifically in adult human testis. Considering a possible relation of this novel gene with the function of human testis, we named this new gene TDRG1 (testis development related gene 1, GenBank accession number: DQ168992).

CONCLUSIONDDD combined with laboratory validation is an efficient method for identifying new human functional genes.

Adult ; Cloning, Molecular ; DNA, Complementary ; genetics ; Databases, Genetic ; Gene Expression Regulation ; Homeodomain Proteins ; genetics ; Humans ; Male ; Molecular Sequence Data ; Organ Specificity ; Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment ; Testis ; metabolism

BACKGROUNDUrotensin II (UII) is a new vasoconstrictive peptide that may activate the adventitial fibroblasts. Transforming growth factor-β1 (TGF-β1) is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-β1 is involved in UII-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.

METHODSAdventitial fibroblasts were prepared by the explant culture method. TGF-β1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression of α-smooth nuscle actin (α-SM-actin), the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR (real-time RT-PCR) and Western blotting, respectively.

RESULTSUII stimulated the secretion of TGF-β1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% (P < 0.01), than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10(-8) mol/L of UII (P < 0.01), which was increased by 59.9%, compared with in the control group (P < 0.01). The secretion of TGF-β1 induced by UII was significantly blocked by SB-710411 (10(-7) mol/L), a specific antagonist of UII receptor. In addition, both UII (10(-8) mol/L) and TGF-β1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UII was inhibited by the specific neutralizing antibody (20 µg/ml) of TGF-β1, while the α-SM-actin expression stimulated by TGF-β1 (20 ng/ml) was inhibited by SB-710411 (10(-7) mol/L), the UII receptor antagonist.

CONCLUSIONThis study suggests that UII could induce TGF-β1 secretion in adventitial fibroblasts via UT activation, and TGF-β1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UII, and TGF-β1 signaling might be one of the important pathways by which UII is involved in vascular fibrosis.

Actins ; analysis ; genetics ; Animals ; Aorta ; cytology ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; Male ; Myofibroblasts ; cytology ; Phenotype ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Signal Transduction ; Transforming Growth Factor beta1 ; physiology ; Urotensins ; antagonists & inhibitors ; pharmacology

【Objective】To provide a rabbit model for dry eye using a dry environment induced by Controlled Drying System.【Methods】Twenty-four male New Zealand rabbits were used in the experiment. They were randomly divided into control group and dry group，each one with 12 rabbits. The dry group was randomly housed in Controlled Drying System（CDS）for 14 days. The relative humidity，airflow and temperature were kept at（22±4）%，3~4 m/s and（23~25）℃， respectively. The control group were fed in a normal environment where relative humidity，airflow and temperature were kept at 60%~70%，0.2 m/s and（23~25）℃. The Schirmer test，corneal fluorescein staining，conjunctival lissamine green staining were performed during the experimental process on days 0，3，7，and 14. On the last day，the rabbits were euthanized and the eye tissues were made into paraffin-cut sections. After staining，we evaluated the corneal epithelial thickness and goblet cell number in the conjunctiva using light microscopy. MUC5AC in the conjunctival epithelium was detected by immunofluorescence. The apoptosis level changes on the ocular surface were evaluated using Caspase- 3 by immunohistochemistry. 【Results】 Decreased tear production ，increased corneal fluorescein staining and increased conjunctival lissamine green staining were found on days 3，7，and 14 in the dry group compared with the control group（P < 0.001）. Corneal epithelial thickness of control group and dry group were （58.0±7.2）μ m and（47.8±7.6）μ m ，which showed corneal epithelial thickness of dry group was decreased（P＜0.05）. Goblet cells in the conjunctiva of control group and dry group were 15 ± 4 and 10 ± 2，which showed goblet cells of dry group was decreased（P＜0.01）. The expression of MUC5AC（consistent with goblet cells deficiency） was also reduced. Caspase- 3 was highly expressed on the corneal epithelium in the dry group. IOD/field of control group and dry group were（17±2）% and（20±2）%（P＜0.01）.【Conclusions】 Dry environment can make rabbits have pathological changes of dry eye on ocular surface epithelium. This dry eye model of rabbit caused by Controlled Drying System would be an effective tool to study the pathogenesis of dry eye.

¹⁷⁷Lu- prostate specific membrane antigen (PSMA) radio-ligand therapy has been approved abroad for advanced prostate cancer and has been in several clinical trials in China. Based on domestic clinical practice and experimental data and referred to international experience and viewpoints, the expert group forms a consensus on the clinical application of ¹⁷⁷Lu-PSMA radio-ligand therapy in prostate cancer to guide clinical practice.