Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

AIM:To investigate the role of nuclear factor of activated T cell 2(NFAT2),the major NFAT protein in peripheral T cells,in sustained T cell activation and intractable inflammation in human ulcerative colitis(UC). METHODS:We used two-dimensional gel-electrophoresis, immunohistochemistry,double immunohistochemical staining,and confocal microscopy to inspect the expression of NFAT2 in 107,15,48 and 5 cases of UC, Crohn's disease(CD),non-specific colitis,and 5 healthy individuals,respectively. RESULTS:Up-regulation with profound nucleo- translocation/activation of NFAT2 of lamina propria mononuclear cells(LPMC)of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC,as compared to CD or NC(P<0.001,Kruskal- Wallis test).Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T,but was less prominent in CD4+T cells or CD20+B cells.It was strongly associated with the disease activity,including endoscopic stage (τ=0.2145,P=0.0281)and histologic grade(τ=0.4167, P<0.001). CONCLUSION:We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis.Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity.Since activation of NFAT2is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways,our results not only provide new insights into the mechanism for sustained intractable inflammation,but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

Proliferating cell nuclear antigen of Ulva prolifera is involved in the response to temperature stress

Ulva prolifera is the most common specie causative to green tide,and its growth is sensitive to temperature stress.However,the mechanisms of U.prolifera response to temperature stress remain elusive.In this study,high temperature(36℃)stimulus promoted the death of unformed cell wall protoplasts and delayed the division of formed cell wall protoplasts,while low-temperature(4℃)stimulus did not,suggesting that the mechanisms of the response of U.prolifera to high and low temperature stresses are different.Transcriptome results show that proliferation-related genes were differentially expressed under high and low-temperature stresses,especially the proliferating cell nuclear antigen(PCNA)and cyclins(CYCs).Subsequently,the interaction between PCNA and Cyclin A was confirmed by Co-immunoprecipitation,yeast two-hybrid,and so on.Furthermore,high-and low temperature stresses induced the expression of PCNA and Cyclin A in varying of degrees,and activated extracellular signal-regulated kinase(ERK)signal pathway.These results suggest,PCNA,Cyclin A,and ERK signal pathway played important roles in the resistance of U.prolifera to temperature stress.Interestingly,high-temperature stress induced an increase of miR-2916 in abundance,and exhibiting reverse expression of PCNA;and PCNA was target gene of miR-2916,suggesting that miR-2916 protected U.prolifera from high-temperature stress via post-transcriptionally regulation of PCNA.This study laid a foundation for understanding the function of PCNA and Cyclin A,moreover,it has a guiding significance to explore the mechanisms of the response to temperature stress from proliferation-related genes regulatory networks in U.prolifera.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

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 immun-ofluorescence 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 sperma-tocytes 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.

Influence of norcantharidin on proliferation,proliferation-related gene proteins prolifera-ting cell nuclear antigen and Ki-67 of human gallbladder carcinoma GBC-SD cells

BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and should be consi- dered for palliative treatment such as chemotherapy and ra- diotherapy. Unfortunately, reports of chemotherapy and radiotherapy for gallbladder carcinoma are disappointing. We investigated the influence of norcantharidin (NCTD) on proliferation, proliferation-related gene proteins PCNA and Ki-67 of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: GBC-SD cell lines of human gallbladder carci- noma were cultured by the cell culture technique. The ex- periment was divided into NCTD group and control group. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The streptavidin-biotin complex method was used to determine the expressions of prolifera- tion-related gene proteins PCNA and Ki-67 of human gall- bladder carcinoma GBC-SD cells. RESULTS: NCTD inhibited the growth and proliferation of GBC-SD cells from 10 mg/L or after 6 hours in a dose- and time-dependent manner, with the IC50 value of 56.18 μg/ ml at 48 hours. After treatment with NCTD, the expression of PCNA (0.932 ±0.031 vs. 0.318 ±0.023, P<0.001) and Ki-67 (0.964 ±0.092 vs. 0.297 ±0.018, P<0.001) proteins were decreased significantly. CONCLUSION: NCTD inhibits the proliferation of human gallbladder carcinoma GBC-SD cells in vitro and the expres- sion of their proliferation-related gene proteins PCNA and Ki-67.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

The Effect of the LysoPC-induced Endothelial Cell Conditioned Medium on Proliferating Cell Nuclear Antigen Expression of the Calf Thoracic Aorta Smooth Muscle Cells

In order to study the effect of and mechanism of lysophosphatidylcholine (LysoPC) on proliferation of the calf thoracic aorta smooth muscle cells (ASMCs), the ASMCs were used to observe the effects of LysoPC induced endothelial cell conditioned medium on the DNA content and proliferating cell nuclear antigen (PCNA) expression in the calf thoracic ASMCs by flow cytometry and Western Blot technique. It was found that LysoPC induced endothelial cell conditioned medium could significantly promote PCNA expression of the calf ASMCs, induce the converting of ASMCs from G 0 /G 1 phase to S phase of DNA synthesis, and increase the tyrosine phosphorylation protein expression. Tyrosine protein kinase inhibitor (TPKi) RG50864 could obviously inhibit proliferation of LysoPC induced ASMCs in a dose dependence manner. The results indicated that the effect of LysoPC promoting the proliferation of ASMCs is partly evoked by endothelial cell derived growth factors such as PDGF and so on.

Human Pro-insulin Transgenic Calf Derived from Somatic Cell Nuclear Transfer

The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.

Probiotic Bio-Three induces Th1 and anti-inflammatory effects in PBMC and dendritic cells

AIM:To investigate the immune response of peripheral blood mononuclear cells(PBMCs)and dendritic cells (DCs)that were stimulated by probiotic preparations. METHODS:PBMCs were isolated,cultured,and stimulated with Bio-Three(a mixture of Bacillus mesentericus, Clostridium butyricum and Enterococcus faecalis;105, 10 6 and 10 7 CFU/mL for 24 h).Cytokine production of (1)circulating PBMCs;(2)PBMCs stimulated by probiotic preparation;(3)monocyte-derived DCs;and(4)DC andT cell co-culture was determined by enzyme-linked immunosorbent assay.Phenotypic analysis of circulating PBMCs was also investigated by flow cytometry.Blood was obtained from individuals who consumed Bio-Three (10 9 CFU/d B.mesentericus,C.butyricum and E.faecalis) for 2 wk,or those who did not take probiotics orally. RESULTS:In culture supernatants,interferon-γ(IFN-γ) and interleukin(IL)-10 production increased,but IL-4 and tumor necrosis factor-α(TNF-α)production by PBMCs decreased after 1 and 2 wk of probiotic treatment.Flow cytometry was also performed on day 14 and detected enhanced expression of CD11b,HLA-DR, CD4,CD45RA,CD25,CD44 and CD69 in response to Bio-Three.Furthermore,IL-10 and IL-12 were upregulated in supernatants of monocyte-derived DCs,and IFN-γand IL-10 were enhanced in supernatants of CD4 + T cells co-cultured with DCs. CONCLUSION:Bio-Three appeared to stimulate the Th1 immune response,downregulate pro-inflammatory cytokines(TNF-α)and upregulate anti-inflammatory cytokine(IL-10).Probiotics could be effective in activation of PBMCs and DCs.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

Effect of pre-moxibustion on apoptosis and proliferation of gastric mucosa cells

AIM： TO observe the effects of pre-moxibustion on apoptosis and proliferation of gastric mucosal cell in rats with stress-induced ulcer, and to analyze the relationship between those effects and the expression of heat-shock protein 70 （HSP70）. METHODS： Sixty healthy Sprague Dawley rats were randomly assigned into four groups, namely group A, B, C and D. The animal model of stress ulcer was established by water immersion and restraint stress. The rats in group A, B, and D served as the restraint, model, and non-acupoint controls, respectively, while those in group C received moxibustion at Zusanli and Uangmen points. Immunohistochemical methodology was used to detect the expression of HSP70, apoptosis index （AI, × 10^-6/μm^2） and proliferation index （PCNA-LI, × 10^-6/μm^2）. The mucosal expression of transforming growth factor α （TGF-α） was detected by radioimmunoassay. RESULTS： IVloxibustion at Zusanli and Liangmen points significantly decreased the gastric injury and the apoptosis of gastric mucosal cells, while markedly increased the mucosal expression of TGF-α and HSP70 as well as the proliferation of gastric mucosal cells. Compared with group A, ulcer index （UI） （26.8 ± 9.8 vs 12.0 ± 5.9, P 〈 0.01）, AI （9.6 ± 4.2 vs 4.4 ± 2.6, P 〈 0.05） and expression of HSP70 （9.6 ± 4.2 vs 4.4 ± 2.6, P 〈 0.05） were significantly increased, but the content of TGF-α （104.7 ± 51.2 pg/mL vs 254.0 ± 86.9 pg/mL, P 〈 0.01） and PCNA-LI （6.9 ± 4.7 vs 14.9 ± 4.6, P 〈 0.05） were significantly decreased in group B. However, ulcer index values （UI） and AI were obviously lower in group C compared to groups B and D （14.1 ± 5.4 vs 26.8 ± 9.8 and 26.2 ± 7.7, P 〈 0.01, 3.0 ± 1.6 vs 9.6 ± 4.2 and 8.2 ± 5.2, P 〈0.05, respectively）, but content of TGF-α （237.0 ± 72.6 pg/mL vs 104.7 ± 51.2 pg/mL and 154.1 ± 61.3 pg/mL, P 〈0.01） and expression of HSP70 （0.13 ± 0.03 vs 0.08 ± 0.06 and 0.06 ± 0.04, P 〈 0.05） were higher in group C. Furthermore, the PCNA-LI was significantly higher in group C than in group B （21.6 ± 4.1 vs 6.9 ± 4.7, P 〈 0.01）. CONCLUSION： Moxibustion at Zusanli and Liangmen points has a protective effect on rats gastric mucosa in stress-induced gastric ulcer, which is closely related to its actions in promoting synthesis of TGF-α and proliferation of gastric mucosal cells, suppressing gastric mucosal cell apoptosis, and up-regulating HSP70 expression.

Clinical significance of expression of proliferating cell nuclear antigen and E-cadherin in gastric carcinoma

AIM to investigate the expression of proliferating cell nuclear antigen(p CNA)and E-cadherin in gastric carcinoma and to analyze their clinical significance.METHODS A total of 146 patients were selected for this study,including 38 patients with intestinal metaplasia,42with dysplasia,and 66 with primary gastric cancer.In addition,40 patients with normal gastric tissues were selected as controls.the expression of p CNA and E-cadherin was detected by immunohistochemistry.Differences in p CNA and the E-cadherin labeling indexes among normal gastric mucosa,intestinal metaplasia,dysplasia,and gastric carcinoma were compared.Subjects with normal gastric tissues were assigned to a normal group,while gastric cancer patients were assigned to a gastric cancer group.the difference in p CNA and E-cadherin expression between these two groups was compared.the relationship between expression of p CNA and E-cadherin and clinicopathological features was also explored in gastric cancer patients.furthermore,prognosis-related factors,as well as the expression of p CNA and E-cadherin,were analyzed in patients with gastric cancer to determine the 3-year survival of these patients.RESULTS the difference in p CNA and the E-cadherin labeling indexes among normal gastric mucosa,intestinal metaplasia,dysplasia,and gastric carcinoma was statistically significant(p<0.05).During the transition of normal gastric mucosa to gastric cancer,the p CNA labeling index gradually increased,while the E-cadherin labeling index gradually decreased(p<0.05).the p CNA labeling index was significantly higher and the E-cadherin labeling index was significantly lower in gastric cancer than in dysplasia(p<0.05).the expression of p CNA was significantly higher in the gastric cancer group than in the normal group,but E-cadherin was weaker(p<0.05).there was a negative correlation between the expression of p CNA and E-cadherin in gastric carcinoma(r=-0.741,p=0.000).p CNA expression differed significantly between gastric cancer patients with and without lymph node metastasis and between patients at different t stages.E-cadherin expression also differed significantly between gastric cancer patients with and without lymph node metastasis(p<0.05).High t stage and positive p CNA expression were risk factors for the prognosis of patients with gastric cancer(RR>1),while the positive expression of E-cadherin was a protective factor(RR<1).the sensitivity,specificity,and accuracy of p CNA positivity in predicting the 3-year survival of patients with gastric cancer were 93.33%,38.89%,and0.64,respectively;while these values for E-cadherin negativity were 80.0%,41.67%,and 0.59,respectively.When p CNA positivity and E-cadherin negativity were combined,the sensitivity,specificity,and accuracy were66.67%,66.67%,and 0.67,respectively.CONCLUSION Combined detection of p CNA and E-cadherin can improve the accuracy of assessing the prognosis of patients with gastric cancer.

Clinical Significance of p53, Proliferating Cell Nuclear Antige and bcl-2 Expression in Bladder Transitional Cell Carcinoma

Objective: To correlate the frequency of p53 mutations, bcl-2 expression and the proliferation status (proliferating cell nuclear antigen, PCNA) in patients with bladder cancer with cell proliferation, apoptosis and their clinico-pathologic ?ndings. Methods: Para?n-embedded sections from 39 super?cial (T1G1-G3) and 23 invasive (T2-T4a G3 N0M0) primary transitional cell carcinomas (TCC) in the bladder were investigated immunohistochemically for p53, bcl-2 and PCNA. The median follow-up was 37 months; 24 had recurrences. The proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL- positive tumor cells. Results: P53 mutation was identi?ed in 50 patients (80.6%). The mutation was most common in tumors of grade 3 (91.3%) as compared to grade 2 (78.5%) and grade 1 (72.7%, P<0.05). Stage pT2 tumors had a higher frequency of p53 mutation (95.7%) as compared to pTa-1 tumors (74.3%, P<0.01). Only 14 tumors (22.5%) expressed bcl-2; grade 3 tumors expressed bcl-2 signi?cantly more frequently (P<0.05); there was no correlation between bcl-2 and tumor stage. There was no interrelation between p53 mutation and bcl-2 expression (P>0.05). The PI ranged from 17.2% to 41.8% (median 22.4%) and the AI from 1.9% to 3.5% (median 2.9%) in bladder cancer. Statistical analyses revealed a close associations between PI, AI and tumor grade and stage of bladder cancer. Conclusion: P53 mutation correlates with invasion. P53 and PCNA overexpression may o?er valuable additional prognostic information in bladder tumors. With the progression of the tumor grade, cell proliferation may be accompanied by frequent apoptosis in bladder cancer, but the PI increased much more than the AI.

ECRG2 enhances the anti-cancer effects of cisplatin in cisplatin-resistant esophageal cancer cells via upregulation of p53 and downregulation of PNCA

AIM To explore the anti-tumor effects of esophageal cancerrelated gene 2(ECRG2) in combination with cisplatin(DDP) in DDP-resistant esophageal cancer cells(EC9706/DDP).METHODS A drug-resistant cell model was established, with EC9706/DDP cells being treated with ECRG2 and/or DDP. Cell viability was examined by MTT assay. The rate of cell apoptosis was determined by flow cytometry. The mR NA expression levels of proliferating cell nuclear antigen(PCNA), metallothionein(MT), and p53 were determined by RT-PCR and PCNA, while MT and p53 protein expression levels were determined by western blotting.RESULTS The anti-proliferative effect of ECRG2 in combination with DDP was superior when compared to ECRG2 or DDP alone. The inhibition rate for the combination reached its peak(51.33%) at 96 h. The early apoptotic rates of the control, ECRG2 alone, DDP alone, and ECRG2 plus DDP groups were 5.71% ± 0.27%, 12.68% ± 0.61%, 14.15% ± 0.87%, and 27.96% ±0.36%, respectively. Although all treatment groups were significantly different from the control group(P < 0.05), the combination treatment of ECRG2 plus DDP performed significantly better when compared to either ECRG2 or DDP alone(P < 0.05). The combination of ECRG2 and DDP significantly upregulated p53 m RNA and protein levels and downregulated PCNA m RNA and protein levels compared to ECRG2 or DDP alone(P < 0.05). However, no changes were seen in the expression of MT mR NA or protein.CONCLUSION ECRG2 in combination with DDP can inhibit viability and induce apoptosis in esophageal cancer DDP-resistant cells, possibly via upregulation of p53 expression and downregulation of PCNA expression. These findings suggest that the combination of ECRG2 and DDP may be a promising strategy for the clinical treatment of esophageal cancers that are resistant to DDP.

Evaluation of germ-cell kinetics in infertile patients with proliferating cell nuclear antigen proliferating index

Aim: To explore the usefulness of proliferating cell nuclear antigen proliferating index (PCNA PI) in the pathologicaldiagnosis and treatment of male infertility. Methods: Testicular biopsy specimen obtained from 48 cases of male in-fertility and 2 normal controls were fixed and embedded. The sections were stained with anti-PCNA monoclonal anti-bodies or haematoxylin/eosin. Proliferating index (PI), expressed as the percentage of germ-cell nuclei positivelystained with PCNA antibody, was assessed from more than 20 seminiferous tubules or 600 germ-cells. Results: Theinfertile patients were divided into 4 groups: Group 1, normal spermatogenesis (14 cases); Group 2, hypospermato-genesis (16 cases); Group 3, germinal arrest (10 cases); Group 4, Sertoli cell only syndrome (8 cases). The PCNAPI of normal control testis was 86.5 % (mean value). Group 3 had a significantly lower PCNA PI (29.8 %) than nor-mal testis; Group 1 and 2 had similar Pis (82.3% and 82.3%, respectively) as the control testis. PI of the negativecontrol (Group 4) was 0 as no germ-cells were found. Conclusion: PCNA PI is useful for assessing germ-cell ki-netics, especially for pathological diagnosis of germinal arrest which is difficult to differentiate by routine HE stainingtechnique. In germinal arrest, there is a significantly lowered PCNA PI, which is an indication of DNA synthesis dete-rioration, suggesting the use of therapies be different from those for hypospermatogenesis. (Asian J Androl 2001 Mar;3: 63-66)

The Relationship of Expression of bcl-2, p53, and Proliferating Cell Nuclear Antigen (PCNA) to Cell Proliferation and Apoptosis in Renal Cell Carcinoma

To investigate the relationship of bcl-2, p53, proliferating cell nuclear antigen (PCNA) to cell proliferation, apoptosis and pathological parameters, the patterns of cell growth and turnover in renal cell carcinoma (RCC), formalin-fixed and paraffin-embedded tissue blocks from 34 patients with RCC were examined. Cell proliferation activity was detected by PCNA immunostaining and the proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy- nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL-positive cells in the tumor cells. Expressions of bcl-2 and p53 were assessed immunohistochemically. Our results showed that the PI ranged from 6.0 % to 24.0 % (median 12.3 %) and the AI from 2.0 % to 8.0 % (median 5.4 %) in RCC. The expression of the bcl-2 protein was demonstrated in 15 cases (44.1 %); the expression of the p53 protein, however, was seen in only 3 case. bcl-2 positivity was not associated with PI or AI or any pathological parameters. There were close associations between PI and tumor grade and stage, and a significant relationship between AI and the tumor grade of RCC. Our study suggests that bcl-2 positivity was not associated with PI or AI or any pathological parameters. There are close associations between PI and AI and tumor grade and stage of RCC. Active cell proliferation may be accompanied by frequent apoptosis in RCC.

Mast cell density and the context of clinicopathological parameters and expression of p185,estrogen receptor,and proliferating cell nuclear antigen in gastric carcinoma

AIM:To investigate the relationship between the mast cell density(MCD)and the context of clinicopathological parameters and expression of p185,estrogen receptor(ER), and proliferating cell nuclear antigen(PCNA)in gastric carcinoma. METHODS:Mast cell,p185,ER,and PCNA were detected using immunohistochemical S-P labeling method.Mast cell was counted in tissue of gastric carcinoma and regional lymph nodes respectively,and involved lymph nodes(ILN)were examined as usual. RESULTS:MCD was significantly related to both age and depth of penetration(x^2=4.688,P<0.05 for age and x^2=9.350, P<0.01 for depth of penetration)between MCD>21/0.03 mm^2 and MCD≤21/0.03 mm^2 in 100 patients;MCD in 1-6 ILN group patients was significantly higher than that in 7-15 ILN or>15 ILN group patients(u=6.881,8.055,P<0.01); There were significant differences intergroup in positive expression rate of p185,ER and PCNA between MCD>21/ 0.03 mm^2 and MCD≤21/0.03 mm^2 in 100 patients. CONCLUSION:Mast cell may have effect on inhibiting invasive growth of tumor,especially in the aged patients; The number of mast cells,in certain degree,may predicate the number of involved lymph nodes,which is valuable for assessment of prognosis;MCD was related to the expression of p185,ER,and PCNA in gastric carcinoma.It suggests that mast cell accumulation may inhibit the proliferation and the dissemination of the gastric carcinoma. INTRODUCTION Recently,many studies have reported on the association of mast cell with various tumorst.In several malignancies,mast cell has been found to correlate with growth,penetration and prognosis of tumor.Therefore,our study was undertaken to investigate the relationship between the mast cell density (MCD)and the context of clinicopathological parameters and expression of p 185,estrogen receptor(ER),and proliferating cell nuclear antigen(PCNA)in gastric carcinoma.

Spatial and temporal expression of germ cell nuclear factorin 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.

The Relationship between Apoptosis and the Expression of Proliferating Cell Nuclear Antigen and the Clinical Stages in Gastric Carcinoma

The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5±3.7) % and (49.8±15.9) % respectively, and the rate of AI/PI was 0.13±0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer ( P <0.01). With the advanced TNM stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage Ⅱ to Ⅳ ( P <0.05). It was suggested that the decreased apoptotic cells and the increased proliferating cells were obviously related to the tumor genesis and tumor progression in gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

RELATIONSHIP BETWEEN PROLIFERATING CELL NUCLEARANTIGEN EXPRESSION AND ITS MALIGNANCY POTENTIAL IN COLORECTAL CARCINOMA

Objective: To study the relationship between proliferating cell nuclear antigen expression and its malignancy potential in colorectal carcinoma. Methods: Paraffin sections of 86 patients with advanced colorectal carcinoma were assessed by immunohistochemical study, using a mouse monoclonal antibody (pc-10, DAKO Co. USA) to check proliferating cell nuclear antigen (PCNA). To compare PCNA with conventional clinicopathologic factor, including p53 overexpression, tissue carcinoembnyonic antigen immunoreactivity pattern and flow cytometric DNA ploidy for assessing tumor malignancy potential. In addition, recurrence and survival of patients with advanced colorectal carcinoma after curative resection were analyzed in accordance with degree of PCNA expression. Results: PCNA-labeling index (PCNA-LI) increased significantly as the tumor stage advanced (p=0.0001). Strong correlations were observed between PCNA-LI and various pathologic parameters, including histologic differentiation (P=0.0027), lymphatic invasion (P=0.0001), vascular invasion (P=0.0001), lymph node metastasis (P=0.0001), and liver metastasis (P=0.0036). Mean PCNA-LI was also significantly higher in tumor with DNA aneuploidy (P=0.0006) and negative (P=0.01). Linear relationships were demonstrated between PCNA-LI and clinical outcomes; Recurrence rate was significantly greater in the group with higher than the mean PCNA-LI, who underwent curative resection (P<0.01), and three-year survival rates for curative cases with higher than the mean PCNA-LI were significantly poorer than those with lower than mean PCNA-LI (P<0.005). Conclusion: There were correlations between PCNA-LI and various pathologic parameters, PCNA-LI increased significantly as the tumor stage advanced in colorectal carcinoma, the rates of recurrence and death got higher as PCNA-LI increased after curative resection for colorectal carcinoma.