L-arginine-glycine amidinotransferase, betaine-homocysteine s-methyltransferase, and neuropolypeptide h3 are diminished in renal clear cell carcinoma of humans

To identify renal clear cell carcinoma-associated marker proteins. Twelve patients with renal cell carcinoma [RCC] were collected and processed in the Department of Urology, Renmin Hospital, Wuhan University, China, between January 2008 and September 2009. Two-dimensional polyacrylamide gel electrophoresis and matrix assisted laser desorption ionisation time-of-flight mass spectrometry [MALDI-TOF-MS] were employed to investigate differentially expressed protein spots between RCC tissues and adjacent normal tissues, then reverse transcription polymerase chain reaction and western blot were employed to confirm the proteomic results. One protein spot was up regulated, 13 were downregulated, and 22 were absent in RCC tissues. Four of the absent proteins were L-arginine-glycine amidinotransferase [AGAT], Betaine-homocysteine S-methyltransferase [BHMT], Ketohexokinase [KHK], and Neuropolypeptide h3 [NPh3]. The reverse transcriptase-polymerase chain reaction analysis demonstrated mRNA expression of AGAT, BHMT, and Nph3 was significantly decreased in 12 RCC tissues. In addition, Western blot analysis showed AGAT protein was absent in 11/12, BHMT in 9/12, and Nph3 in 5/12 RCC tissues. Absence of AGAT, BHMT, and Nph3 is common events in clear cell RCC; hence, it may be involved in the development of RCC; therefore, they have the potential to be tumor markers for diagnosis, treatment, and prognosis of RCC patients

Long-term culture and identification of spermatogonial stem cells from BALB/c mice in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation.</p><p><b>METHODS</b>Testis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis.</p><p><b>RESULTS</b>After 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia.</p><p><b>CONCLUSION</b>SSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.</p>