[Comparison of long-term clinical outcomes between transvaginal mesh and pelvic floor reconstruction with native tissue repair in the treatment of advanced pelvic organ prolapse].

Objective: To study the long-term clinical effect of transvaginal mesh (TVM) and pelvic floor reconstruction with native tissue repair (NTR) in the treatment of advanced pelvic organ prolapse (POP). Methods: Totally 207 patients with advanced POP who were treated in Hunan Provincial Maternal and Child Health Care Hospital from Jan. 2016 to Sep. 2019 were enrolled. The patient's pelvic organ prolapse quantification were all at degree Ⅲ or above, and they all complained for different degree of symptoms. They were divided into two groups according to the different surgical methods, TVM group and NTR group. In TVM group, the mesh was implanted through the vagina for pelvic floor reconstruction, while in NTR group, the traditional transvaginal hysterectomy combined with uterosacral ligament suspension and anterior and posterior wall repair, as well as perineal body repair were performed. The median follow-up time was 60 months, during the follow up time, 164 cases (79.2%, 164/207) had completed follow-up, including 76 cases in TVM group and 88 cases in NTR group. The perioperative data and complication rates of the two groups were compared, and the subjective and objective outcomes of the two groups at 1, 3 and 5 years were observed, respectively. The objective efficacy was evaluated by three composite criteria, namely: (1) the distance from the farthest end of the prolapse of the anterior and posterior wall of the vagina to the hymen is ≤0 cm, and the descending distance of the top is ≤1/2 of the total length of the vagina; (2) determine the disappearance of relevant POP symptoms according to "Do you often see or feel vaginal mass prolapse？"; (3) no further operation or pessary treatment was performed due to prolapse. If the above three criteria were met at the same time, the operation is successful; otherwise, it was recurrence. The subjective efficacy was evaluated by the pelvic floor distress inventory-short form 20 (PFDI-20) and pelvic floor impact questionnaire-short form 7 (PFIQ-7). Results: The median follow-up time of the two groups was 60 months (range: 41-82 months). Five years after the operation, the subjective and objective cure rates of TVM group were 89.5% (68/76) and 94.7% (72/76), respectively. The subjective and objective cure rates in NTR group were 80.7% (71/88) and 85.2% (75/88), respectively. There were significant differences in the subjective and objective cure rates between the two groups (χ2=9.869, P=0.002; χ2=3.969, P=0.046). The recurrence rate of TVM group was 5.3% (4/76), and that of NTR group was 14.8% (13/88). There was a significant difference between the two groups (P=0.046). The postoperative PFDI-20 and PFIQ-7 scores of the two groups were significantly lower than those before surgery, and there were significant differences of the two groups before and after surgery (all P<0.05). Postoperative mesh exposure in TVM group was 1.3% (1/76). Conclusions: The long-term outcomes between the two groups show that the subjective and objective outcomes of pelvic floor reconstruction in TVM group are significantly higher than those in NTR group, and the recurrence rate is significantly lower than that in NTR group. TVM has certain advantages in the treatment of advanced POP.

[Comparison of clinical and immunological features between clinically amyopathic dermatomyositis and typical dermatomyositis].

OBJECTIVE: To study the differences between clinically amyopathic dermatomyositis (CADM) and typical dermatomyositis (DM) on clinical and immunological features. METHODS: By collecting clinical data of 106 CADM patients and 158 DM patients from January 2010 to June 2019 in the department of Rheumatology and Immunology, Peking University People's Hospital, the clinical characteristics and immunological features in the two groups were compared, and the distribution characters and the clinical meanings of myositis autoantibodies were discussed in the two groups respectively. Myositis autoantibodies were measured by immunoblotting according to the manufacturers' instructions. RESULTS: In the aspects of clinical manifestations, CADM presented more with onset of interstial lung diseases (ILD) compared with DM (20.7% vs. 7.6%, P=0.002), and CADM-ILD was more likely to be acute ILD (58.3% vs. 26%, P < 0.001), and there were no differences between CADM and DM in cutaneous manifestations, accompanied with connective tissue disease (CTD) and malignancy. In CADM, the positive rate of rheumatoid factors and antinuclear antibodies was lower in DM. The most common myositis specific autoantibodies (MSAs) in CADM were anti-MDA5 (36%), anti-PL-7 (11.2%) and anti-TIF-1γ (10.1%). The most common MSAs in DM were anti-Jo-1 (19.2%), anti-TIF-1γ (11.5%) and anti-MDA5 (11.5%). Anti-MDA5 was correlated with acute ILD and skin ulceration both in CADM and DM; in CADM, skin ulceration was not associated with the titer of anti-MDA5; while in DM, skin ulceration was associated with high titer of anti-MDA5. In DM, anti-TIF-1γ was correlated with heliotrope eruption, V/shawl neck sign, perionychia erythma and malignancy, and higher rate of malignancy was seen in all titers of the anti-TIF-1γ positive patients. In CADM, anti-TIF1-γ showed no correlation with clinical manifestations. The most common myositis associated autoantibody was anti-Ro-52 both in CADM and DM. In CADM, anti-Ro-52 was associated with Raynaud's phenomenon and chronic ILD, while in DM, anti-Ro-52 was associated with mechanic's hands, noninfectious fever and accompanied CTD. CONCLUSION: Compared with DM, ILD is more likely to be acute in CADM. It is different between CADM and DM about the distribution of myositis autoantibodies and the clinical significance of the same myositis antibody, and the clinical significance of some myositis antibodies is related to titers.

lncRNA SNHG3 accelerates the proliferation and invasion of non-small cell lung cancer by downregulating miR-340-5p.

Small nucleolar RNA host genes (SNHGs) as a subset of long non-coding RNAs (lncRNAs) act critical roles in tumor progression. The present study aimed to elucidate the role and mechanisms of SNHG3 in non-small cell lung cancer (NSCLC). The correlation of SNHG3/miR-340-5p/HOXA10 with the clinicopathological features and outcomes in NSCLC was analyzed by TCGA cohort. In vitro and in vivo functional experiments were conducted to assess the role of SNHG3 in NSCLC cells. Bioinformatic analysis and luciferase gene reporter were used to estimate the interaction between miR-340-5p and SNHG3/HOXA10 3'UTR. The effects of SNHG3 and (or) miR-340-5p on HOXA10 expression were detected by qRT-PCR and Western blot analysis. As a consequence, the elevated expression of SNHG3 and HOXA10 or lowered expression of miR-340-5p was related to the lymph node infiltration, distant metastases and unfavorable prognosis in NSCLC. Ectopic expression of SNHG3 boosted the proliferation and invasion of NSCLC cells in vitro and in vivo, whereas downregulation of SNHG3 reversed these effects. Moreover, SNHG3 could bind with miR-340-5p and reduce its expression levels, and miR-340-5p attenuated SNHG3-induced tumor proliferation and HOXA10 expression in NSCLC cells. Our findings unveiled that SNHG3 might be an oncogenic factor in NSCLC by downregulating miR-340-5p.

Long noncoding RNA LINC00858 promotes the proliferation, migration and invasion of gastric cancer cells via the miR-363-3p/FOXP4 axis.

OBJECTIVE: Long non-coding RNA (lncRNA) LINC00858 has been found to exert oncogenic activity in several types of cancers, except gastric cancer (GC). In the present study, we aimed to explore the potential role of LINC00858 in GC and the underlying molecular mechanism. PATIENTS AND METHODS: The expression patterns of LINC00858 were determined using qRT-PCR in GC samples and cell lines. Cell proliferation was examined utilizing CCK-8 assay. Cell migration and invasion were evaluated using transwell assays. We used the bioinformatics software StarBase and TargetScan to predict lncRNA-miRNA and miRNA-mRNA interactions. RESULTS: Our findings revealed that LINC00858 expression was markedly upregulated in GC tissues and cell lines. Loss-of-function experiments demonstrated that LINC00858 silencing inhibited the proliferation, migration and invasion of GC cells. Bioinformatics analysis showed that there were several complementary binding sites between LINC00858 and microRNA (miR)-363-3p, and further Luciferase reporter assay confirmed the interaction between LINC00858 and miR-363-3p. In addition, forkhead box P4 protein (FOXP4) was found to be a target gene of miR-363-3p in GC cells. FOXP4 overexpression reversed the inhibitory effects of miR-363-3p mimics on cell proliferation, migration and invasion of BGC-823 cells. CONCLUSIONS: Collectively, LINC00858 acted as an oncogene in GC via regulating miR-363-3p/FOXP4 axis, which indicated that LINC00858 might be a novel therapeutic target for the treatment of GC.

[Intraocular lens power calculation for high myopic eyes with cataract: comparison of three formulas].

Objective: To compare the accuracy of three different formulas for intraocular lens power calculation in high myopic eyes with cataract and analyze their influencial factors. Methods: One hundred and three high myopic patients of cataract (103 eyes), with average age of 60.2±8.8 years old (39.0-77.0), including 45 male and 54 female and with axial length ≥ 26 mm were enrolled in this retrospective case-series study. All of them underwent routine ocular examination and IOLMastermeasurement preoperatively and then underwent phacoemulsification through temporal clear-corneal incision with implantation of HumanOptic posterior chamber Intraocular lens (IOL). All analyses were conducted using SPSS version 19.0. Repeated-measures analysis of variance was applied to compare the refractive results one month postoperatively with the predicted IOL powers calculated by SRK/T, Holladay 1, or Haigis formula before surgery. The differences were further compared based on different grouping of axial length (AXL), corneal curvature (K) and corneal astigmatism (CA). The accuracies of the three formulas were analyzed using Bland-Altman analyses and the possible error sources of each formula were analyzed using multiple regression model. Results: The majority of patients enrolled had hyperopic shift after cataract surgery. The mean errors (ME) of the three formulas were SRK/T: 0.70±0.89D, Holladay 1: (1.20±0.88) D and Haigis: (0.60±0.88) D; the mean absolute errors (MAE) of the three formulas were (0.81±0.79) D, (1.23±0.84) D and (0.76±0.74) D, respectively. Both ME and MAE of Holladay formula were significantly greater than the other two formulas (F=86.31, P<0.01). Besides, the proportion of patients having a prediction error within 0.50 D was lower in those using Holladay formula (20.4%, 21/103) than the other two (SRK/T: 38.8%, 40/103, χ(2)=8.41, P<0.01, Haigis: 45.6%, 47/103, χ(2)=14.84, P<0.01). Bland-Altman analyses showed that the accuracies of all the three formulas were acceptable in patients of cataract with high myopia in clinical practice. ME and MAE tended to be larger with longer axial length, larger corneal curvature and astigmatism of the patients in all three formulas. However, in eyes with axial length> 30 mm or corneal curvature ≤43.00 D, the MAE of Haigis formula was lower than that of SRK/T formula (F=63.26,63.94, both P<0.01). The prediction error of SRK/T formula was positively correlated with axial length and corneal astigmatism (F=33.97, r=0.66, ß=0.48, P<0.01 and ß=0.42, P<0.01), while for Holladay and Haigis formulas, in addition to the previous two factors, the errors were also positively correlated with mean corneal curvature (Holladay 1: F=31.26, r=0.72, AXL: ß=0.52, P<0.01, K: ß=0.20, P<0.05 and CA: ß=0.37, P<0.01; Haigis: F=30.96, r=0.72, AXL: ß=0.33, P<0.01, K: ß=0.40, P<0.01 and CA: ß=0.37, P<0.01). Conclusions: In the selection of IOL formula for high myopic patients with cataract, Haigis or SRK/T would reduce the prediction error and serve as the more accurate formulas than Holladay 1. Haigis formula may be more accurate than SRK/T formula in case of AXL>30 mm or K≤43.00 D. (Chin J Ophthalmol, 2017, 53: 260-265).

[Analysis of CT perfusion imaging in chronic cerebral circulatory insufficiency and its relationship with crossed cerebellar diaschisis].

Objective: To investigate the change of cerebral microcirculation of chronic cerebral circulation insufficiency(CCCI) patients and the relationship between CCCI and crossed cerebellar diaschisis(CCD)by using 320-detector row of low-dose volume CT perfusion imaging. Methods: A total of 158 patients (103 males, 55 females, from 45 to 82 years old, the mean age was 62.9) with symptoms of CCCI were admitted to the First Affiliated Hospital of Wenzhou Medical University from June 2013 to January 2016. Low-dose CTP imaging of whole brain was performed to them using 320-detector row volume CT scanner. The perfusion parameters such as cerebral blood flow(CBF), cerebral blood volume(CBV), mean transit time(MTT), time to peak(TTP) and DLY in both cerebral blood supply areas and cerebellum were got, so were the 4-dimensional CTA images, and rCBF, rCBV, rMTT and rTTP were calculated by ipsilateral/contralateral value. Comparative t-test and independent t-test were applied to analyzing these parameters quantitatively.Chi-square test and Logistic regression model were applied to analyzing the related clinical risk factors. Results: (1) All 108 patients in CCCI group showed asymmetric perfusion within two cerebral hemispheres in CTP images. The CBF, CBV of diseased side were lower than the contralateral mirror area (t(CBF)=-12.89, t(CBV)=-7.031, P(CBF, CBV)<0.001); the MTT of the diseased side was shorter than the contralateral mirror area (t(MTT) =13.310, P(MTT)<0.001); the TTP of the diseased side was longer than the contralateral mirror area (t(TTP)=-4.012, P(TTP)<0.001). The rCBF and rCBV of CCCI group were lower than that in non-CCCI group (t(rCBF)=3.079, t(rCBV)=2.760, P(rCBF, rCBV)<0.01), while the rTTP of CCCI group was longer than that in non-CCCI group (t(rTTP)=4.846, P(rTTP)<0.001). (2)The results of Chi-square test showed that the differences of gender (χ(2)=4.036, P=0.045), hyperlipidemia (χ(2)=7.687, P=0.006), as well as smoking (χ(2)=11.868, P=0.001) had statistical significance between CCCI group and non-CCCI group.Multi-factor Logistic regression analysis showed that hyperlipidemia (OR value=3.736, P=0.016) and smoking (OR value=4.641, P=0.01) were the risk factors of CCCI, while gender had no relationship with it.(3)The incidence of CCD was 18.5% in the CCCI group, and at the same time, the supratentorial corresponding blood supply areas were classified.A total of 10(34.5%) cases were in blood supply area of posterior cerebral artery, 6(20.7%) cases were in blood supply area of middle cerebral artery, 12(41.4%) cases were of anterior cerebral artery, while only 1(3.5%) case was of basal ganglia, in which 4 cases were in blood supply area of posterior cerebral artery, another 4 cases were middle cerebral artery, 7 cases were of anterior cerebral artery and no case of basal ganglia respectively leading CCD alone. Conclusions: CTP could display the microcirculation situation of abnormal brain tissue perfusion area intuitively and quantitatively. Additionally, it could reflect the degree of relationship between cerebral several blood supply areas and cerebellum.

EPHX1 Tyr113His and His139Arg polymorphisms in esophageal cancer risk: a meta-analysis.

Microsomal epoxide hydrolase 1 (EPHX1) is an important biological phase II metabolic enzyme that is extensively involved in the metabolism of diverse environmental carcinogens such as polycyclic aromatic hydrocarbons and heterocyclic amines. Many articles have reported the association between EPHX1 (Tyr113His and His139Arg) polymorphisms and esophageal cancer risk, but the results are controversial. This study aimed to identify the association between EPHX1 (Tyr113His and His139Arg) polymorphisms and esophageal cancer risk by meta-analysis. The odds ratio (OR) with 95% confidence interval (95%CI) was used to evaluate the strength of the associations. Heterogeneity was estimated by the chi-square-based Q-statistic test and the P value. Meanwhile, the random-effect or fixed-effect model was used according to the between-study heterogeneity. Begg's funnel plot and the Egger test were performed to assess the publication bias of articles. Finally, 8 case-control studies involving 1158 cases and 1868 controls for the Tyr113His polymorphism and 7 case-control studies involving 901 cases and 1615 controls for the His139Arg polymorphism were included in this meta-analysis. Meta-analysis showed that the Tyr113His polymorphism was a stronger power trend towards risk for esophageal cancer using a recessive model (CC versus CT+TT, OR = 1.204, 95%CI = 1.001-1.450, P = 0.049). However, no significant associated risk was found between the His139Arg polymorphism and esophageal cancer. These findings suggest that the Tyr113His polymorphism might be a stronger power trend towards risk for esophageal cancer. However, no evidence was found for the association between the EPHX1 His139Arg polymorphism and esophageal cancer risk.

Nicotine-induced over-exposure to maternal glucocorticoid and activated glucocorticoid metabolism causes hypothalamic-pituitary-adrenal axis-associated neuroendocrine metabolic alterations in fetal rats.

Fetuses with intrauterine growth retardation (IUGR) induced by prenatal nicotine exposure are susceptible to adult metabolic syndrome. Our goals for this study were to investigate the effects of prenatal nicotine exposure on the fetal hypothalamic-pituitary-adrenal (HPA) axis and glucose and lipid metabolism and to explain the susceptibility to adult metabolic syndrome for fetuses with nicotine induced-IUGR. Pregnant Wistar rats were administered 0.25, 0.5, and 1.0 mg/kg nicotine subcutaneously twice a day from gestational day 11 to 20. Nicotine exposure significantly increased the levels of fetal blood corticosterone and decreased the expression of placental 11ß-hydroxysteroid dehydrogenase-2 (11ß-HSD-2). Moreover, nicotine exposure significantly increased the expressions of fetal hippocampal 11ß-HSD-1 and glucocorticoid receptor (GR) and decreased the expressions of fetal hypothalamus corticotropin-releasing hormone, adrenal steroid acute regulatory protein, and cholesterol side-chain cleavage enzyme. Additionally, increased expressions of 11ß-HSD-1 and GR were observed in fetal liver and gastrocnemius muscle, and these tissues also expressed lower levels of insulin-like growth factor-1 (IGF-1), IGF-1 receptor, and insulin receptor, while expressing increased levels of adiponectin receptor, leptin receptors, and AMP-activated protein kinase α2. Prenatal nicotine exposure causes HPA axis-associated neuroendocrine metabolic alterations in fetal rats. The underlying mechanism may involve activated glucocorticoid metabolism in various fetal tissues.

Yin Yang 1 positively regulates BRCA1 and inhibits mammary cancer formation.

Expression of the breast cancer-associated gene 1 (BRCA1) in sporadic breast cancers is usually reduced, yet the underlying mechanisms remains elusive. To identify factors that are responsible for reduced BRCA1 expression, we screened 92 known transcription factors for their ability to regulate expression of BRCA1. Among several potential regulators, the Gli-Krueppel-related transcription factor Yin Yang 1 (YY1) showed the most dramatic transactivation of the BRCA1 promoter. YY1 binds to the promoter of BRCA1, and its overexpression resulted in increased expression of BRCA1 and a number of BRCA1 downstream genes. We further showed that overexpression of YY1 in cancer cells inhibited cell proliferation, foci formation and tumor growth in nude mice. To assess the clinical relevance between YY1 and BRCA1, we studied expression of YY1 and BRCA1 from human breast cancer samples and tissue arrays, and detected a significant positive correlation between the level of YY1 and BRCA1 expression in these cancers. Taken together, these findings suggest that YY1 is a key regulator of BRCA1 expression and may be causally linked to the molecular etiology of human breast cancer.

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment.

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.

Cloning, expression and chromosome locations of the human DNMT3 gene family.

DNA methylation plays an important role in animal development and gene regulation. In mammals, several genes encoding DNA cytosine methyltransferases have been identified. DNMT1 is constitutively expressed and is required for the maintenance of global methylation after DNA replication. In contrast, the murine Dnmt3 family genes appear to be developmentally regulated and behave like de novo DNA methyltransferases in vitro. In this study, we have cloned human DNMT3A and DNMT3B that encode full-length DNMT3A and DNMT3B proteins with 98% and 94% amino acid sequence identity to their murine homologues. The DNMT3A and DNMT3B show high homology in the carboxy terminal catalytic domain and contain a conserved cysteine-rich region, which shares homology with the X-linked ATRX gene of the SNF2/SWI family. We have mapped human DNMT3A and DNMT3B to chromosomes 2p23 and 20q11.2 respectively, and determined the DNMT3B genomic structure. We further show that DNMT3A expression is ubiquitous and can be readily detected in most adult tissues, whereas DNMT3B is expressed at very low levels in most tissues except testis, thyroid and bone marrow. Significantly, both DNMT3A and DNMT3B expression is elevated in several tumor cell lines to levels comparable to DNMT1. The cloning of the human DNMT3 genes will facilitate further biochemical and genetic studies of their functions in establishment of DNA methylation patterns, regulation of gene expression and tumorigenesis.

Reed-Sternberg cell genome expression supports a B-cell lineage.

The malignant Reed-Sternberg cell of Hodgkin's disease, first described a century ago, has resisted in-depth analysis due to its extreme rarity in lymphomatous tissue. To directly study its genome-wide gene expression, approximately 11,000,000 bases (27,518 cDNA sequences) of expressed gene sequence was determined from living single Reed-Sternberg cells, Hodgkin's tissue, and cell lines. This approach increased the number of genes known to be expressed in Hodgkin's disease by 20-fold to 2,666 named genes. The data here indicate that Reed-Sternberg cells from both nodular sclerosing and lymphocyte predominant Hodgkin's disease were derived from an unusual B-cell lineage based on a comparison of their gene expression to approximately 40,000,000 bases (10(5) sequences) of expressed gene sequence from germinal center B cells (GCB) and dendritic cells. The data set of expressed genes, reported here and on the World Wide Web, forms a basis to understand the genes responsible for Hodgkin's disease and develop novel diagnostic markers and therapies. This study of the rare Reed-Sternberg cell, concealed in its heterogenous cellular context, also provides a formidable test case to advance the limit of analysis of differential gene expression to the single disease cell.

A novel human prostate-specific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer.

We have isolated a prostate-specific gene (NKX3.1) in humans that is homologous to the Drosophila NK homeobox gene family. Northern blot analyses indicate that this gene is expressed at high levels in adult prostate and at a much lower level in testis, but is expressed little or not at all in several other tissues. In an androgen-dependent prostate carcinoma line, LNCaP, NKX3.1 mRNA is expressed at a basal level that was increased markedly upon androgen stimulation; the NKX3.1 mRNA was undetectable in several other human tumor cell lines including two androgen-independent prostate carcinoma lines. The NKX3.1 gene maps to chromosome band 8p21, a region frequently reported to undergo a loss of heterozygosity associated with tissue dedifferentiation and loss of androgen responsiveness during the progression of prostate cancer. Based on these data we propose that NKX3.1 is a candidate gene for playing a role in the opposing processes of androgen-driven differentiation of prostatic tissue and loss of that differentiation during the progression of prostate cancer.

ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B.

Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACGG pentapeptide, rather than the QACRG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.

ICE-LAP3, a novel mammalian homologue of the Caenorhabditis elegans cell death protein Ced-3 is activated during Fas- and tumor necrosis factor-induced apoptosis.

Members of the ICE/ced-3 gene family have been implicated as components of the cell death pathway. Based on similarities with the structural prototype interleukin-1 beta-converting enzyme (ICE), family members are synthesized as proenzymes that are proteolytically processed to form active heterodimeric enzymes. In this report, we describe a novel member of this growing gene family, ICE-LAP3, which is closely related to the death effector Yama/CPP32/Apopain. Pro-ICE-LAP3 is a 35-kDa protein localized to the cytoplasm and expressed in a variety of tissues and cell lines. Overexpression of a truncated version of ICE-LAP3 (missing the pro-domain) induces apoptosis in MCF7 breast carcinoma cells. Importantly, upon receipt of a death stimulus, endogenous ICE-LAP3 is processed to its subunit forms, suggesting a physiological role in cell death. This is the first report to demonstrate processing of a native ICE/ced-3 family member during execution of the death program and the first description of the subcellular localization of an ICE/ced-3 family member.

The androgen receptor in the testicular feminized (Tfm) mouse may be a product of internal translation initiation.

Androgen insensitivity in the testicular feminized (Tfm) mouse is caused by frame-shift mutation in the androgen receptor (AR) mRNA, which results in a stop codon in the amino terminus. Despite this mutation, a smaller sized protein corresponding to the DNA- and steroid-binding domain of the AR can be synthesized from the cloned Tfm AR cDNA by in vitro translation. The Tfm AR construct was demonstrated to express a protein capable of binding androgen with an affinity similar to the cloned wild-type AR. Although the Tfm AR product failed to transactivate mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter when low concentrations (100 ng) of Tfm AR vector were cotransfected, higher concentrations (5000 ng) resulted in a residual amount of transactivation, suggesting lower level transactivating capabilities. By cotransfecting the Tfm AR expression vector with the wild-type receptor, it was demonstrated that the product of the Tfm AR gene is capable of inhibiting the transactivation activity of the wild-type receptor. These data suggest that although the Tfm AR mRNA fails to produce a full-length AR because of the frame-shift mutation, a smaller protein capable of binding both steroid and DNA can be produced by translation from an internal initiation codon. This product could account for the low levels of androgen-binding activity detected previously in the Tfm mouse.

A frame-shift mutation in the androgen receptor gene causes complete androgen insensitivity in the testicular-feminized mouse.

The testicular feminized (Tfm) mouse lacks completely androgen responsiveness; and therefore, is unique for studying the role of androgenic steroids in different biological processes. In order to understand the molecular basis of this mutation, 2.8 kilobases of cDNA encoding the Tfm mouse androgen receptor (AR) were amplified with a polymerase chain reaction (PCR) technique. No large deletion in the coding region of the Tfm mouse AR was detected. However, sequence analysis revealed a single base deletion in the coding region of the Tfm AR mRNA. This mutation, which is located in the amino-terminus domain of the receptor, is predicted to cause a frame-shift in translation resulting in a premature termination of AR synthesis at amino acid 412. In vitro translation studies of the recombinant wild type and Tfm AR's demonstrated that the Tfm AR cDNA failed to produce a full-length receptor. Furthermore, the Tfm AR was demonstrated to lack transcriptional activation capability by cotransfection experiments using the Tfm AR with a reporter plasmid of mouse mammary tumor virus long terminal repeat linked to the chloramphenicol acetyltransferase gene. These studies provide evidence of the molecular defect which causes androgen insensitivity in the Tfm mouse.

In situ hybridization of prostate-specific antigen mRNA in human prostate.

Prostate-specific antigen (PSA) mRNA was detected by in situ hybridization utilizing a 428 base pair [35S]-labelled cDNA probe from the 3' noncoding region of the PSA gene. Thirty six fresh surgical specimens were collected from patients undergoing radical retropubic prostatectomy for carcinoma of the prostate. Quantitative analysis of the levels of PSA mRNA in both the benign and malignant tissues was performed using an IBAS 2000 Image Analysis System. The results of this study demonstrated that there is a significant decrease in the expression of PSA mRNA in the carcinoma tissue when compared to the benign epithelium. The average binding (number of silver grains/1 x 10(4) microns. 2) for 20 specimens of malignant epithelium was 475 +/- 161 and 586 +/- 140 for 16 specimens of benign epithelium (p less than 0.05). Eleven patients had both benign and malignant tissue from the same surgical specimen available for study. From these paired specimens, the PSA mRNA expression was also significantly reduced in the malignant epithelium when compared to the benign epithelium, 445 +/- 162 and 588 +/- 135 respectively (p less than 0.005). The PSA protein was detected using a monoclonal antibody to PSA with an immunohistochemical staining technique. The PSA protein expression paralleled the expression of the PSA mRNA in the majority of the tissue sections. Many of the tumor specimens showed a heterogeneous expression of PSA, whereas all of the benign epithelium had a uniform high level of PSA expression. In conclusion, PSA mRNA and protein are located only within the glandular epithelial tissue, the expression of PSA protein parallels that of the PSA mRNA, and both the PSA protein and PSA mRNA are significantly decreased in the malignant epithelium when compared to benign prostatic epithelium.

Molecular cloning of androgen receptors from divergent species with a polymerase chain reaction technique: complete cDNA sequence of the mouse androgen receptor and isolation of androgen receptor cDNA probes from dog, guinea pig and clawed frog.

We have cloned and sequenced 2.8 kilobases of cDNA encoding the mouse androgen receptor by RNA amplification with transcript sequencing. Sequence analysis predicts that this cDNA contains an open reading frame of 2697 nucleotides encoding a polypeptide of 899 amino acids. Androgen receptor cDNA probes of dog, guinea pig, and frog were also isolated and sequenced using consensus primers derived from human and rat androgen receptor cDNAs. Northern blot analysis with the species-specific probes revealed similarities in size between amphibian and mammalian mRNAs. These results demonstrate the utility of this technique in obtaining nucleic acid probes and sequence information of steroid receptors from different species. The sequence data and the Northern blot analysis of the receptors in different species demonstrate that the androgen receptor has been well-conserved during evolution.