Bactericidal/permeability-increasing protein originates in both the testis and the epididymis and localizes in mouse spermatozoa.

Bactericidal/permeability-increasing protein (BPI) is an endogenous antibiotic protein with activity against gram-negative bacteria. In the present study, we examined the expression of BPI in postnatal mouse testes and epididymides as well as the subcellular localization within epididymal spermatozoa. Our results showed that, BPI mRNA was expressed in testis and epididymis independently. Throughout the epididymis, the BPI protein level gradually decreased in the epididymal epithelium in a spatial manner, specialized within the cytoplasm of clear cells in the cauda part. We detected BPI proteins in intact acrosome, implying its testicular origin; on the other hand, after the acrosome reaction, BPI proteins were observed dispersed across the entire sperm head, especially enriched at the equatorial segment. Our findings suggested a dual origin of the BPI that generated both in the testis and epididymis, and associated with mouse spermatozoa. BPI protein might be involved in the dynamics modification of the sperm plasma membrane and also the fertilization process.

Study on early postoperative nutritional support in elderly patients with gastric cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To explore the optimal postoperative nutritional support in elderly patients with gastric cancer.</p><p><b>METHODS</b>One hundred and twenty elderly patients with gastric cancer undergoing radical gastrectomy were prospectively enrolled from January 2010 to March 2013 and randomly divided into total parenteral nutrition group(TPN, n=40), early total enteral nutrition group (TEN, n=40) and enteral plus parenteral nutrition group(EN+PN, n=40). Clinical charasteristics including treatment tolerance, nutritional indexes, immune indexes, time to first flatus, incidence of postoperative infection and anastomotic leakage, were analyzed and compared.</p><p><b>RESULTS</b>Treatment tolerance in EN+PN group(97.5%, 39/40) was significantly higher than that in TPN group(82.5%, 33/40) and TEN group(80.0%, 32/40)(both P<0.05). The nutritional indices, including prealbumin, albumin, transferrin, body mass index, and the incidence of anastomotic leakage were similar in the 3 groups(P>0.05). The immune indices, including CD3, CD4, CD4/CD8, were significantly reduced after operation in each group. However, they were significantly higher in EN+PN group and TEN group than those in TPN group(both P<0.05). Furthermore, compared to the TPN group, the incidence of postoperative infection(surgical site infection, pulmonary infection, abdominal infection) was significantly lower and time to first flatus was significantly shorter in EN+PN group and TEN group.</p><p><b>CONCLUSIONS</b>Early enteral nutrition after gastric cancer surgery is safe, simple and feasible. EN plus PN is the best way to administer postoperative nutritional support in elderly patients with gastric cancer.</p>

[Estrogens affect Sertoli cells and the blood-testis barrier in pubertal rats].

OBJECTIVE: To investigate the correlation of exogenous estrogens with the expression of FasL in Sertoli cells and the blood-testis barrier during the differentiation and maturation period of Sertoli cells, and to discuss the related factors that influence the blood-testis barrier of pubertal rats. METHODS: Super-physiological doses of exogenous estrogenic compounds (diethylstilbestrol and estradiol) were administered to pubertal Sprague-Dawley rats in vitro and in vivo, the FasL expression in the Sertoli cells of the rats detected by immunohistochemistry and Western blot, and the changes in the blood-testis barrier observed with the electron microscope. RESULTS: After the exposure to exogenous estrogens, the FasL expression was markedly up-regulated in the immature Sertoli cells (P < 0.05) as well as in the Sertoli cell membrane and the blood-testis barrier of the epithelium. The tracer lanthanum passed through the blood-testis barrier and reached the whole layer of the epithelium at 18 days. CONCLUSION: Super-physiological dose of exogenous estrogens can change the expression and distribution of FasL in immature Sertoli cells and affect the structure of the blood-testis barrier.

Expression of the retinoic acid-metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development.

AIM: To study the expression pattern of the retinoic acid metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development at both mRNA and protein levels. METHODS: Real-time polymerase chain reaction and Western blot analysis were performed to determine the relative quantity of RALDH2 and CYP26b1 at both mRNA and protein levels at postnatal day 1, 5, 10, 20, and in adult mice (70 days testes). Testicular localization of RALDH2 and CYP26b1 during mouse postnatal development was examined using immunohistochemistry assay. RESULTS: Aldh1a2 transcripts and its protein RALDH2 began to increase at postnatal day 10, and remained at a high level through postnatal day 20 to adulthood. Cyp26b1 transcripts and CYP26b1 protein did not change significantly during mouse postnatal testis development. RALDH2 was undetectable in the postnatal 1, 5 and 10 day testes using immunohistochemistry assay. At postnatal day 20 it was detected in pachytene spermatocytes. Robust expression of RALDH2 was restricted in round spermatids in the adult mouse testis. In the developing and adult testis, CYP26b1 protein was confined to the peritubular myoepithelial cells. CONCLUSION: Our results indicate that following birth, the level of retinoic acid in the seminiferous tubules might begin to increase at postnatal day 10, and maintain a high level through postnatal day 20 to adulthood.

Specific killing effects of combination of recombinant adenovirus containing double suicide gene driven by KDR promoter and survivin antisense oligonucleotide on colorectal cancer cells and vascular endothelial cells / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To evaluate the specific killing effects of combination of recombinant adenovirus mediated double suicide gene driven by KDR promoter and survivin antisense oligonucleotide(ASODN) on colorectal cancer cells and vascular endothelial cells.</p><p><b>METHODS</b>The 293 packaging cells were transfected with the plasmids of pAdEasy-CDglyTK and the recombinant adenovirus were generated. The KDR expressive cells of SW620, ECV304 were infected with adenovirus, meanwhile survivinASODN was transferred into the same cells. The infection rate of adenovirus and transfection efficiency of survivinASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on SW620, ECV304 were examined through MTT method.</p><p><b>RESULTS</b>The cells which were infected with the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection efficiency. The high expression of CDglyTK gene was found in SW620, ECV304 cells infected with recombinant adenovirus and survivin ASODN decreased the survivin protein level. The survival rate of gene therapy group was significantly lower than that of negative group. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate of SW620 and ECV304 cells as compared with the AdKDR-CDglyTK or survivin ASODN used alone (P<0.05). The survival rate was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that AdKDR-CDglyTK used alone (P>0.05). The combined therapy of AdKDR-CDglyTK and survivin ASODN showed synergistic killing efficacy and more significant bystander effects.</p><p><b>CONCLUSION</b>The combined gene therapy of AdKDR-CDglyTK system and survivin ASODN has stronger specific killing effects on colorectal cancer cells and vein endothelial cells.</p>

Age-dependent expression of the cystatin-related epididymal spermatogenic (Cres) gene in mouse testis and epididymis.

AIM: To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic (Cres) gene in mouse testis and epididymis during postnatal development. METHODS: The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. RESULTS: (1) In both the testis and epididymis, Cres mRNA was first detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. (2) In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. (3) The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. CONCLUSION: The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.

Targeted killing of colorectal tumor cells by lentiviral constructs containing CD/TK suicide genes and KDR promoter / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the selective killing of colorectal tumor cells by lentivirus-mediated double suicide gene under the regulation of KDR promoter.</p><p><b>METHODS</b>293T packaging cells were transfected with the plasmid FGW-KDRP-CD/TK to obtain the infectious viruses. KDR-expressing LoVo cells and LS174T cells that did not produce KDR were transfected with the recombinant virus, and the transfection efficiency was evaluated by the fluorecence microscope. RT-PCR was employed to examine the expression of CDglyTK. After treatment of the cells with 5-FC and GCV, the killing effects on the two cell lines were evaluated.</p><p><b>RESULTS</b>The recombinant construct showed similar infection rate of the two cell lines. RT-PCR demonstrated that CDglyTK gene was expressed only in LoVo cells infected with FGW-KDRP-CD/TK but not in LS147T cells, and the sensitivity of the two cell lines to the prodrugs was significantly different (P<0.001). The killing effect of the double suicide gene was much stronger than that of single suicide gene administered (P<0.001).</p><p><b>CONCLUSION</b>The double suicide gene driven by KDR promoter has specific killing effect on the KDR-expressing colorectal tumor cells.</p>

Effect of KDR recombinant adenovirus containing double suicide gene on proliferation of human stomach adneocarcinoma SCG7901 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of adenovirus (Ad)-mediated fusion gene system driven by KDR promoter on the proliferation of human stomach adneocarcinoma SCG7901.</p><p><b>METHODS</b>The KDR-expressing SCG7901 cells and HepG2 cells that did not express KDR were both transfected with AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-FC and/or GCV at different concentrations. The killing effects of the transfection on the cells were evaluated.</p><p><b>RESULTS</b>The expression of green fluorescent protein (GFP) was observed in 95% of the infected SCG7901 and HepG2 cells with the multiple of infection (MOI) of the Ads of 100. Transfection of SCG7901 and HepG2 cells did not produce significant changes in the cell growth, and the infected cells exhibited different sensitivities to the two prodrug: SCG7901 cells infected with rAd were highly sensitive to the prodrugs, but the infected HepG2 cells were not (P<0.01). The killing effect of CDglyTK fusion gene on the target cells was much stronger than that of either the single suicide gene (P<0.01).</p><p><b>CONCLUSION</b>CDglyTK fusion gene system driven by KDR promoter selectively kills the KDR-CDglyTK SCG7901 cells and inhibits their proliferation.</p>

Specific killing effects of combination of double suicide gene and survivin antisense oligonucleotide on breast tumor cells and vein endothelial cells / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the selectively killing effects of combination of adenovirus mediated double suicide gene driven by kinase-domain insert containing receptor (KDR) promoter and survivin antisense oligonucleotide on breast tumor cells and vein endothelial cells.</p><p><b>METHODS</b>Human embryonal kidney cells 293 were transfected with the plasmids of pAdEasy-KDR-CDglyTK and the adenovirus was generated. The KDR expressive cells of MCF-7, ECV304 were infected by adenovirus and survivin ASODN was transferred into the same cells meanwhile. The infection rates of adenovirus and transfection efficiency of survivin ASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on cells were assessed by MTT assay.</p><p><b>RESULTS</b>The cells infected by the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection rate. The high expression of CDglyTK gene was found in the two kinds of cells and survivin ASODN decreased the survivin protein level. When survivin ASODN was transferred into MCF-7, ECV304 cells, the survival rates were 56.4% +/- 3.8% and 55.9% +/- 3.6% respectively. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate comparing with using each treatment alone (P < 0.05) and the survival rate decreased gradually with the increasing of the concentration of GCV and 5-FC. But the survival rate for combined gene therapy group was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that of AdKDR-CDglyTK group (P > 0.05). The combination of survivin ASODN and AdKDR-CDglyTK therapy showed synergistic killing efficacy and more significant bystander effects.</p><p><b>CONCLUSION</b>The combined therapy with AdKDR-CDglyTK system and survivin ASODN shows obvious killing effects on breast tumor cells and vein endothelial cells.</p>

In vitro killing effect of adenovirus-mediated fusion gene system driven by KDR promoter on gastric cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the killing effect of adenovirus(Ad)-mediated double suicide gene driven by kinase domain-containing receptor (KDR) promoter on gastric cancer MGC-803 cells.</p><p><b>METHODS</b>The 293 packaging cells were transfected by the plasmids pAdEasy-KDR-CDglyTK to generate infectious viruses. The gastric cancer MGC-803 cells were infected by the Ad followed by treatment with 5-FC and/or ganciclovir at different concentrations. The cell-killing effects were evaluated and the bystander effects analyzed after coculture of the cells without AdKDR-CDglyTK infection with the infected cells at different ratios. The cell cycle distribution was detected by flow cytometry and the pathological changes of the cells were observed by electron microscopy.</p><p><b>RESULTS</b>The infection rate of the resultant recombinant Ad in the cells increased gradually with increment of the multiplicity of infection (MOI) of the Ads. The killing effect of CD/TK fusion gene on the MGC-803 cells was much stronger than that of either of the single suicide gene (P<0.001), and considerable bystander effect was observed. The Ad infection caused MGC-803 cell growth arrest at G(1) phase with onset of apoptotic and necrotic morphologies of the cells as seen under electron microscope.</p><p><b>CONCLUSION</b>The CD/TK fusion gene system driven by the KDR promoter possesses effective killing effect on the KDR-expressing gastric cancer MGC-803 cells.</p>

Adenovirus mediated double suicide gene under controlling by KDR promoter selectively kills vascular endothelial cells and colorectal tumor cells / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the selectively killing effect of adenovirus (Ad) mediated double suicide gene under the regulation of KDR promoter on vascular endothelial cells and colorectal tumor cells.</p><p><b>METHODS</b>293 packaging cells were transfected with the plasmids of pAdEasy- KDR- CDglyTK and pAdEasy- CMV- CDglyTK and the infectious viruses were generated. The KDR expressive cells of ECV304,SW620 and the KDR inexpressive cells of LS174T were infected by two Ads. The infection rate was observed and the expression of CDglyTK was detected by RT- PCR. After treatment with different concentrations of 5- FC and GCV,the killing effect and bystander effect on ECV304,SW620 and LS174T were examined.</p><p><b>RESULTS</b>The titers of these two purified Ads were 2.0 x 10(12 ) pfu/ml. There was no significant difference in infection rate between two recombinant Ads infecting various cells,and the infection rate increased in accordance with the enhancing titers of Ads. RT- PCR demonstrated that there existed the product of CDglyTK gene in all the cells infected by Ad- CMV- CDglyTK and the cells infected by Ad- KDR- CDglyTK except in the SL174T. The curative effect in this system on various cells was shown as follows: (1) All cells infected with Ad- CMV- CDglyTK and some cells of ECV304 and SW620 infected with Ad- KDR- CDglyTK were highly sensitive to the prodrugs,but there was no significant differences among them (P > 0.05); compared with ECV304 and SW620 cells,LS174T cells were not sensitive to the two prodrugs (P< 0.001). (2) The efficacy of double suicide gene was better than that of single suicide gene (P< 0.001). (3) The system had considerable bystander effect.</p><p><b>CONCLUSION</b>The double suicide gene under the regulation of KDR promoter has specific killing effect on the KDR- expressing endothelial cells and colorectal tumor cells.</p>

Anthraquinones sensitize tumor cells to arsenic cytotoxicity in vitro and in vivo via reactive oxygen species-mediated dual regulation of apoptosis.

Cellular oxidation/reduction state affects the cytotoxicity of a number of chemotherapeutic agents, including arsenic trioxide. Reactive oxygen species (ROS), the major intracellular oxidants, may be a determinant of cellular susceptibility to arsenic. Our previous studies showed that a naphthoquinone and an anthraquinone (emodin) displayed the capability of producing ROS and facilitating arsenic cytotoxicity in both leukemia and solid tumor cell lines. We therefore attempted to test emodin and several other kinds of anthraquinone derivatives on EC/CUHK1, a cell line derived from esophageal carcinoma, and on a nude mouse model, with regard to their effects and mechanisms. Results showed that anthraquinones could produce ROS and sensitize tumor cells to arsenic both in vivo and in vitro. The combination of emodin and arsenic promoted the major apoptotic signaling events, i.e., the collapse of the mitochondrial transmembrane potential, the release of cytochrome c, and the activation of caspases 9 and 3. Meanwhile a combination of emodin and arsenic suppressed the activation of transcription factor NF-kappaB and downregulated the expression of a NF-kappaB-specific antiapoptotic protein, survivin. These two aspects could be antagonized by the antioxidant N-acetyl-L-cysteine. Therefore anthraquinones exert their effects via a ROS-mediated dual regulation, i.e., the enhancement of proapoptosis and the simultaneous inhibition of antiapoptosis. In vivo study showed that emodin made the EC/CUHK1 cell-derived tumors more sensitive to arsenic trioxide with no additional systemic toxicity and side effects. Taken together, these results suggest an innovative and safe chemotherapeutic strategy that uses natural anthraquinone derivatives as ROS generators to increase the susceptibility of tumor cells to cytotoxic therapeutic agents.

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period.

AIM: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. METHODS: The indirect immunofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. RESULTS: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary spermatocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. CONCLUSION: GCNF may play important roles in spermatogenesis, capacitation and fertilization.

Spatial and temporal expression of germ cell nuclear factor in murine epididymis.

AIM: To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period. METHODS: The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope. RESULTS: GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis. The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis. In adults, GCNF exhibited a region-specific expression pattern, i.e., it was expressed predominantly in the initial segment, caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis. GCNF could be found in the nuclei of the principal, apical, narrow, clear and halo cells. CONCLUSION: GCNF may play an important role in epididymal differentiation and development and in sperm maturation.

Mechanisms of glucocorticoid-induced Leydig cell apoptosis.

The high levels of corticosterone (CORT) that are typically achieved during stress induce apoptotic death of Leydig cells. The intracellular mechanisms by which CORT acts on Leydig cells to induce apoptosis are unknown, and the present study tested for mediation by Fas ligand (FasL), a member of the tumor necrosis factor ligand family, in association with caspase activation. In addition, another apoptotic pathway involving in the participation of mitochondria was studied by evaluation of mitochondrial membrane potential (DeltaPsi) loss and generation of reactive oxygen species (ROS), which are early apoptotic events in many cell types. Rat Leydig cells were isolated from adrenalectomized rats on day 90 postpartum at 3, 6, 12, 24 and 48 h after the start of CORT administration (at a dose of 5 mg total/100 g body weight per day intraperitoneally in two daily injections starting 3 days after surgery). Both FasL and Fas receptor protein levels, analyzed by Western blot and fluorescent immunohistochemistry, increased at 6 h after the start of CORT administration, peaking at 24 h and declining thereafter. Leydig cell caspase-3 activity was analyzed in vitro. Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nM CORT in vitro, and the abundance of the fragments was more pronounced at 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Western blotting analysis revealed that procaspase-3 was present only at low levels in untreated control Leydig cells, and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced, and the cleaved, active caspase-3 forms appeared and increased through 24 h. These results indicated that FasL/Fas and caspase were implicated in CORT-mediated Leydig cell apoptosis. Decreased DeltaPsi and increased ROS generation were also measurable in Leydig cells for up to 2 days following CORT administration in vitro. These data indicate that activation of the Fas system, cleavage of procaspase-3, loss of DeltaPsi and increased ROS generation are all implicated in the process of CORT-induced Leydig cell death.