[Investigation on folic acid supplementation status among Chinese women in the first trimester of pregnancy].

OBJECTIVE: To investigate folic acid (FA) supplementation status among Chinese rural reproductive women in their early pregnancy, and to provide evidence for the evaluation of FA supplementation project from national basic public health services. METHODS: The data on intake status of FA supplementation from "National Free Preconception Health Examination Project (NFPHEP)" database between January 1, 2010 and December 31, 2012 was used and analyzed.A total of 902 270 women who achieved follow-up during early pregnancy from 220 pilot counties of 31 provinces were recruited. RESULTS: From 2010 to 2012, the rate of FA intake among women recuited were 73.58% (134 131/182 289), 75.88% (329 288/433 970) and 76.53% (218 896/286 011), respectively, which increased year by year (χtrend(2)=13 371.8, P<0.001). Among 682 315 women who took FA, 350 738(51.40%) women began to take FA 3 months before amenorrhea; 130 683 (19.15%) women began to take FA 1-2 months before amenorrhea, and 200 894 (29.44%) began to take FA after amenorrhea.Among 682 315 women who took FA, 645 310 (94.58%) women took it regularly, and 342 418 (50.18%) women took FA in a standardized way. CONCLUSIONS: The status of FA intake among Chinese rural women in the first trimester of pregnancy was good, and 75.62% (682 315/902 270) of them took FA.It is also important for basic public health services to improve the rate of FA intake and increase the rate of taking FA in a standardized way in Chinese rural pregnant women.

Identification and characterization of a novel enhancer for the rat neu promoter.

We used chloramphenicol acetyltransferase (CAT) assays to identify and characterize cis-acting elements responsible for rat neu promoter function. Deletion of a region of the neu promoter (-504 to -312) resulted in a marked decrease in CAT activity, indicating that this promoter region corresponds to a positive cis-acting element. Using band shift assays and methylation interference analyses, we further identified a specific protein-binding sequence, AAGATAAAACC (-466 to -456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. Our results demonstrate that RVF is the major DNA-binding protein contributing to enhancer activity. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.

Identification of an activity in B-cell extracts that selectively impairs the formation of an immunoglobulin mu s poly(A) site processing complex.

The immunoglobulin mu heavy-chain transcription unit is differentially expressed during B-cell development, producing mRNAs that encode secreted (mu s) and membrane-bound (mu m) forms of the heavy-chain polypeptide. Whereas the mu s mRNA and the mu m mRNA are produced in approximately equal abundance in B cells, an increase in the utilization of the mu s poly(A) site contributes to the production of the mu s mRNA as the predominant form in a plasma cell. Previous experiments have demonstrated a correlation between the formation of a stable complex on a poly(A) site and the relative function of the poly(A) site. We have thus investigated the parameters determining the interaction of these factors with the immunoglobulin poly(A) sites. Assays of complex formation involving the two immunoglobulin poly(A) sites by using HeLa cell activities revealed the formation of stable complexes with no apparent difference between the mu s site and the mu m site. In contrast, the mu s-specific complex was markedly less stable when a B-cell extract was used. Fractionation of B-cell extracts has revealed an activity that specifically destabilizes the mu s polyadenylation complex, suggesting that the function of this poly(A) site may be regulated by both positive- and negative-acting factors.

HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway.

HER-2/neu has been implicated in the activation of androgen receptor (AR) and in inducing hormone-independent prostate cancer growth. Here we report that HER-2/neu activates Akt (protein kinase B) to promote prostate cancer cell survival and growth in the absence of androgen. Blocking of the Akt pathway by a dominant-negative Akt or an inhibitor LY294002 abrogates the HER-2/neu-induced AR signaling and cell survival/growth effects in the absence or presence of androgen. Akt specifically binds to AR and phosphorylates serines 213 and 791 of AR. Thus, Akt is a novel activator of AR required for HER-2/neu signaling to androgen-independent survival and growth of prostate cancer cells.

Tumor suppression and sensitization to tumor necrosis factor alpha-induced apoptosis by an interferon-inducible protein, p202, in breast cancer cells.

p202, an IFN-inducible protein, interacts with several important regulatory proteins, leading to growth arrest or differentiation. In this report, we demonstrate that, in addition to inhibiting in vitro cell growth, p202 can also suppress the tumorigenicity of breast cancer cells in vivo. Furthermore, we found that p202 expression could sensitize breast cancer cells to apoptosis induced by tumor necrosis factor alpha treatment. One possible mechanism contributing to this sensitization is the inactivation of nuclear factor-kappaB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and tumor necrosis factor alpha treatment against breast cancer.

p202, an interferon-inducible protein, mediates multiple antitumor activities in human pancreatic cancer xenograft models.

p202, an IFN-inducible protein, interacts with certain transcriptional activators leading to transcriptional repression. p202 expression has been associated with inhibition of cancer cell growth in vitro and in vivo. To examine a potential p202-mediated antitumor activity in pancreatic cancer, we used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple antitumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic markers, such as interleukin 8 and vascular endothelial growth factor, and p202-expressing pancreatic cancer cells have reduced level of matrix metalloproteinase-2 activity, a secreted protease activity important for metastasis. Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice xenograft model, suggesting a feasibility of using the p202/SN2 liposome in future preclinical gene therapy experiments. Together, our results strongly suggest that p202 expression mediates multiple antitumor activities against pancreatic cancer and may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment.

Repressed expression of the HER-2/c-erbB-2 proto-oncogene by the adenovirus E1a gene products.

The E3 region of adenovirus induces down-regulation of epidermal growth factor receptor (EGFR) through endocytosis. Here we report that an EGFR-related protein, the HER-2/c-erbB-2 gene product, p185, is also down-regulated by adenovirus, but via a different mechanism. We found that the adenovirus E1a gene is responsible for the repression of HER-2/c-erbB-2 at the RNA level.

Involvement of cdc2-mediated phosphorylation in the cell cycle-dependent regulation of p185neu.

We previously reported cell cycle-dependent negative regulation of p185neu (decreased tyrosine phosphorylation and kinase activity, with electrophoretic mobility retarded by serine/threonine phosphorylation) in M phase and the escape of mutation-activated p185neu* from this regulation. Our present results showed that retardation of electrophoretic mobility occurs independently of the cells' transformed status. We found that normal p185neu lost its ability to dimerize in the M phase. We demonstrated a physical association between cdc2 (a serine/threonine kinase, active in M phase) and p185neu. We showed that the carboxy terminal portion of p185neu is phosphorylated in vitro by cdc2. Many phosphopeptides (at least three phosphoserine residues) unique to the M phase were identified, and the in vivo and in vitro phosphopeptide patterns were superimposable. In contrast, mutation-activated p185neu* dimerized in the M phase with no changes in electrophoretic mobility, failed to associate with cdc2 and no unique phosphoserine residues could be identified in the M phase (data not shown), consistent with the escape of p185neu* from cell cycle-dependent regulation. Our results suggest that this escape is an intrinsic property of the mutation-activated p185neu* independent of its ability to transform cells. Our results also suggest the involvement of serine/threonine kinases such as cdc2 in the cell cycle-dependent negative regulation of p185neu.

Reduced growth rate and transformation phenotype of the prostate cancer cells by an interferon-inducible protein, p202.

Interferons (IFNs) can exert cytostatic and immunomodulatory effects on carcinoma cells. In particular, growth inhibition of human prostate carcinoma by IFNs has been demonstrated both in vitro and in vivo. p202 is a 52 kd nuclear phosphoprotein known to be induced by IFNs. In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. More importantly, cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype of prostate cancer cells.

Targeting HER-2/neu-overexpressing breast cancer cells by an antisense iron responsive element-directed gene expression.

Overexpression of HER-2/neu proto-oncogene is found in many human cancers including 20-30% of breast cancer and is a predictor of poor prognosis. To target breast cancer cells that overexpress HER-2/neu mRNA, we previously described a novel strategy that combines the principle of antisense (AS) and translational inhibitory activity conferred by an iron-responsive element (IRE) (AS-IRE). Here, we showed that three potential AS-IREs, i.e. AS-IRE1, 4, and 5, derived from HER-2/neu antisense sequence could bind endogenous iron regulatory protein (IRP) and, when placed in 5' untranslated region (5'UTR) of a reporter gene, the gene expression could be translationally repressed by recombinant IRP in vitro. Using AS-IRE4 as our model, we demonstrated that it is regulated by iron, and importantly, such regulation is impaired in HER-2/neu-overexpressing breast cancer cells. Furthermore, we showed that AS-IRE4 could preferentially direct the expression of a reporter gene in HER-2/neu-overexpressing breast cancer cells. Interestingly, when AS-IRE4 was placed in 5'UTR of Bax gene, a pro-apoptotic protein in the Bcl-2 protein family, we observed a preferential cell killing in breast cancer cells that overexpress HER-2/neu. Taken together, our results suggest that AS-IRE behaves as a functional IRE and it may direct therapeutic gene expression to preferentially target HER-2/neu-overexpressing breast cancer cells.

Aberrant expression of the c-erbB-2/neu protooncogene in ovarian cancer.

Overexpression of the c-erbB-2/neu protooncogene has recently been shown in ovarian tumors collected from the United States. It is known that environmental and cultural factors may contribute to certain types of cancer, therefore, we examined expression of c-erbB-2/neu in ovarian tumors collected from China by immunohistochemical staining. Out of 81 tumor specimens, 57 (70.4%) were found to be immunopositive, whereas only one out of 17 (5.9%) normal ovarian tissue samples was slightly positive. Our results indicate that overexpression of c-erbB-2/neu is a general phenomenon for ovarian cancer regardless of different population. To search for a c-erbB/neu overexpressing cell line for future study on molecular mechanism, we also analyzed 13 cancer cell lines from the female genital tract for expression of c-erbB-2/neu. The c-erbB-2/neu RNA was found to be overexpressed at least 100-fold in one of the four ovarian cancer cell lines examined. An aberrant c-erbB-2/neu RNA was also found to be overexpressed in this cell line. Southern blot analysis indicated that the c-erbB-2/neu was amplified 2-4-fold in this line, and some of these alleles have structural alteration which may account for expression of the aberrant c-erbB-2/neu RNA. Since the 2-4-fold gene amplification is not proportional to the greater than 100-fold overexpression in RNA, other mechanisms such as transcriptional or posttranscriptional control must be involved in overexpression of this gene in ovarian cancer.

E1A-Mediated Gene Therapy.

Ovarian carcinoma is the most lethal tumor of the female genital tract and continues to be a major cause of female cancer deaths, largely as a function of early abdominal seeding producing carcinomatosis. The high rate of mortality is mainly caused by the difficulties of early detection of this disease and the lack of effective treatment for advanced stages of ovarian cancers when they are detected. One of the common pathological features of many primary ovarian cancers is the amplification/overexpression of a transmembrane tyrosine kinase receptor, HER-2 (also known as neu or ErbB2) gene (1). Although there are some discrepancies in the literature (2), HER-2 overexpression in cancer cells correlates well with a greater resistance to chemotherapeutic agents in certain experimental systems (3). In addition, HER-2 overexpression has been shown to enhance metastatic potential in many different model systems (4). Therefore, the HER-2 gene has become an excellent target for developing therapeutic agents that could reverse malignant transformation of HER-2-overexpressing cancer cells (5).

An experimental study on biomechanical properties of hepatic tissue using a new measuring method.

Hepatic tissue is an important internal organ for life. In this paper, we combined the moire method (an optical method) and indentation technique to investigate biomechanical response of hepatic tissue. To examine this measuring technique, we first measured the biomechanical properties of the block of pig's hepatic tissue using this method. The experimental result indicated that the pig's hepatic tissue was obviously a viscoelastic material. We closely analyzed the change of curves for force relaxation, creep, and nonlinear deformation of the block of pig's liver. Meanwhile, we also compared the changes in the biomechanical parameters of normal and cirrhosis hepatic tissue of humans.

[Study on experimental parameters for diagnosing ganyang huafeng syndrome of cerebral infarction].

One hundred patients with cerebral infarction were observed, the results showed that Ganyang Huafeng Syndrome (GYHFS) was the major syndrome of acute cerebral infarction which was accounted for 60% and Qiyin Liangxu Syndrome (QYLXS), Qixu Xueyu Syndrome (QXXYS) were mainly observed at convalescent stage of this disease which was accounted for 37.78% and 31.11% respectively. This study chose plasma norepinephrine (NE), epinephrne (E), thromboxane B2(TXB2), 6-keto-PGF1 alpha, cortisol (F) and serum triiodothyroidoglobulin (T3) as the monitoring parameters, and the results showed that the increase of plasma NE,F,TXB2, the decrease of serum T3 could be considered as the comprehensive experimental parameters for diagnosing GYHFS of cerebral infarction.

[Relationship between three extracting factors and extracting rate and stability of chlorogenic acid in the refining process of flos Lonicerae].

Content determination of chlorogenic acid in the extract of Flos Lonicerae by reversed-phase HPLC has proved that relatively long time boiling in weak acid conditions is beneficial to increasing the extracting rate of the acid. Also, verifying experiments have shown that chlorogenic acid remains stable in the above-said conditions.

Targeting human breast cancer cells that overexpress HER-2/neu mRNA by an antisense iron responsive element.

The overexpression of HER-2/neu proto-oncogene has been found in a variety of human cancers. In particular, the amplification and overexpression of HER-2/neu gene were found in 20-30% of breast and ovarian cancer patients with a decreased survival and an increased relapse rates. To target the breast cancer cells overexpressing HER-2/neu mRNA, a novel approach is described that combines the antisense principle and the biochemical property of a translation regulator, an iron responsive element (IRE). This report shows that a HER-2/neu antisense IRE-reporter gene can be preferentially expressed in the breast cancer cells that overexpress HER-2/neu mRNA. This antisense IRE-mediated gene expression system may be applied broadly to target other cell type that uniquely expresses or overexpresses a known gene.

Differential activity of the RVF enhancer element in the decreased expression of the neu oncogene in NR-6 cells versus parental Swiss Webster 3T3 cells.

The rat neu oncogene encodes a growth factor receptor-like glycoprotein, termed p185, that shares structural similarity with epidermal growth factor receptor (EGFR), particularly in the tyrosine-kinase domain. The Swiss Webster 3T3 murine embryo fibroblast (SW3T3) variant NR-6, in contrast to the parental cell line, does not express EGFR mRNA. After transfection of an activated rat neu cosmid clone, we demonstrated in this report that whereas SW3T3 cells readily expressed the exogenous rat p185 protein, NR-6 cells did not express detectable levels of this protein product. By transfection of plasmids containing the chloramphenicol acetyltransferase (CAT) gene driven by the neu promoter and subsequent CAT assays, we also showed that neu gene promoter activity was significantly less in NR-6 cells than in SW3T3 cells and that the neu promoter sequence responsible for this decreased transcription was a previously identified RVF enhancer element (Yan D-H, Hung M-C, Mol Cell Biol 11:1875-1882, 1991). That is to say, the RVF enhancer element of the neu promoter did not function as an enhancer in NR-6 cells. To investigate the mechanism responsible for the inactivation of RVF in NR-6 cells, we used southwestern blot analyses and demonstrated that the 60-kDa RVF polypeptide was present in both NR-6 and SW3T3 cell nuclear extracts. This result indicates that the DNA-binding activity of RVF was similar in these two cell lines; therefore, loss of RVF enhancer activity in NR-6 cells is probably due to inactivation of the trans-activating function and not DNA binding activity of RVF.

Amplification of the proto-neu oncogene facilitates oncogenic activation by a single point mutation.

To determine whether the amplification of the proto-neu oncogene (also called c-erbB-2) plays a role in tumorigenicity, we previously generated an NIH 3T3 transfectant (DHFR/G-8) that carried the amplified proto-neu gene. The DHFR/G-8 cells exhibited normal morphology. Their growth curve was similar to that of NIH 3T3 cells but was different from that of the B104-1 cell, and NIH 3T3 transfectant that carries the activated neu oncogene. When injected into nude mice, B104-1 cells produced tumors within 2 weeks, whereas the DHFR/G-8 cells did not produce tumors until 3 months after injection, and the NIH 3T3 cells did not produce any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in culture. The cells derived from the tumors were of transformed morphology and highly tumorigenic. The DNAs from the tumor cells were transfected into NIH 3T3 cells. The transfection resulted in foci on the NIH 3T3 monolayer. Southern analysis indicated that the foci derived from the transfection contained the neu gene. Using oligonucleotides as probes, the neu gene in the foci was found to carry a single-point mutation identical to the one previously found in the rat neuroblastoma and glioblastoma induced by the ethylnitrosourea. We conclude that the DNA region encoding the transmembrane domain of neu is a hot spot for converting the proto-neu gene into an activated oncogene and that amplification of the proto-neu gene facilitates mutation of the hot spot.

Differential expression of the neu oncogene in mouse liver and pancreatic cell lines.

To study the tissue-specific expression of neu oncogene, we used two mouse tumor cell lines derived from liver, Hep1-a, and pancreatic, 266-6, tumors as a model system. The endogenous neu gene is expressed in Hep1-a but not in 266-6 cells. We demonstrate in this report that differential expression of the neu gene in these two cell lines is mainly regulated at transcriptional level. The neu promoter sequence responsible for the differential regulation is localized within a 90 bp region and it is possibly due to lack of a specific positive transcription factor(s) interacting with this region in 266-6 cells.

Transcriptional repression of the neu protooncogene by the adenovirus 5 E1A gene products.

Amplification/overexpression of the human neu protooncogene has been frequently found in human primary breast and ovarian cancers and is correlated with the number of axillary lymph nodes positive for metastasis in breast cancer patients. Identification of the factors controlling transcription of the neu gene is essential for understanding the mechanisms of neu gene regulation and its role in tumorigenicity. The adenovirus early region 1A (E1A) gene products are pleiotropic transcription regulators of viral and cellular genes and have been identified as a viral suppressor gene for metastasis. Here we demonstrate that transcription of neu can be strongly repressed by the E1A gene products. The 13S and 12S products of E1A gene are effective at repressing neu transcription and the transcriptional repression requires the conserved region 2 of the E1A proteins. The target for E1A repression was localized within a 139-base-pair DNA fragment in the upstream region of the neu promoter. In addition, competition experiments suggest that the sequence TGGAATG, within the 139-base-pair fragment, is an important element for the E1A-induced repression. These results indicate that E1A negatively regulates neu gene expression at the transcriptional level by means of a specific DNA element.