[The clinical significance of serum C1q tumor necrosis factor-related protein-9 (CTRP9) in patients with cerebral infarction].

To explore the clinical significance of C1q tumor necrosis factor-related protein-9 (CTRP9) in patients with cerebral infarction. Our data showed that the serum CTRP9 was significantly lower than that of control group, especially in patients with large artery atherosclerotic cerebral infarction. CTRP9 was first decreased and even lower from day 4 to day 10, then gradually elevated. Logistic regression analysis suggested that high CTRP9 level was a protective factor for cerebral infarction. Thus, CTRP9 could be a factor for further classification of cerebral infarction and provides a potential option for disease prevention and treatment.

[Polymorphisms of 19 STR Loci in Guizhou Han Population and Their Forensic Application].

OBJECTIVES: To investigate the allelic distribution of 19 autosomal STR loci in Guizhou Han population, and to estimate the forensic application value. METHODS: The 19 autosomal STR loci in 520 unrelated healthy individuals from Guizhou Han population were studied using Goldeneye™ 20A kit. The 310 genetic analyzer was used for capillary electrophoresis, and the GeneMapper®ID v3.1 for genotyping. RESULTS: The heterozygosis, the discrimination power, the probability of exclusion, the polymorphism information content, the cumulative discrimination power and the cumulative probability of exclusion of the 19 STR loci were 0.603 8-0.916 4, 0.790 0-0.985 6, 0.295 5-0.826 9, 0.553 5-0.908 9, 1-1.230 0×10⁻²² and 0.999 999 99, respectively. Compared with other five Han populations in pairwise allelic frequencies, Guizhou Han only had significant differences with Shandong Han, Liaoning Han and Shanxi Han. CONCLUSIONS: The 19 autosomal STR loci such as D19S433 have a highly genetic polymorphic in Guizhou Han population, which have application values in the researches of population genetics and forensic genetics.

Agrobacterium-mediated transformation of the ß-subunit gene in 7S globulin protein in soybean using RNAi technology.

The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin ß-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the ß-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein ß-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean ß-subunit gene. The level of 7S protein ß-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

Prediction of antibiotic resistance proteins from sequence-derived properties irrespective of sequence similarity.

Increasing antibiotic resistance has become a worldwide challenge to the clinical treatment of infectious diseases. The identification of antibiotic resistance proteins (ARPs) would be helpful in the discovery of new therapeutic targets and the design of novel drugs to control the potential spread of antibiotic resistance. In this work, a support vector machine (SVM)-based ARP prediction system was developed using 1308 ARPs and 15587 non-ARPs. Its performance was evaluated using 313 ARPs and 7156 non-ARPs. The computed prediction accuracy was 88.5% for ARPs and 99.2% for non-ARPs. A potential application of this method is the identification of ARPs non-homologous to proteins of known function. Further genome screening found that ca. 3.5% and 3.2% of proteins in Escherichia coli and Staphylococcus aureus, respectively, are potential ARPs. These results suggest the usefulness of SVMs for facilitating the identification of ARPs. The software can be accessed at SARPI (Server for Antibiotic Resistance Protein Identification).

Hypermethylation leads to silencing of the SYK gene in human breast cancer.

A number of cancer-associated genes have been shown to be inactivated by hypermethylation of CpG islands during breast tumorigenesis. SYK, a candidate tumor suppressor, has been found not expressed in a subset of breast cancer cell lines, but the mechanism by which SYK is silenced is unclear. In this study, we examined the 5' CpG island methylation status of the SYK gene in breast cancer cell lines and primary breast cancer tissues. We found SYK 5' CpG hypermethylation in 30% (6/20) of breast cancer cell lines, and the aberrant methylation status was strongly associated with loss of SYK gene expression. Treatment of cells with a methylation inhibitor, 5-aza-2'-deoxycytidine, led to a reactivation of SYK expression in SYK-negative cells, as detected by reverse transcription-PCR. Using methylation-specific PCR, we demonstrated that SYK is hypermethylated in 32% (12/37) of unselected breast tumors, whereas all of the matched neighboring normal breast tissues exhibited unmethylated DNA status. We concluded that SYK is frequently inactivated through an epigenetic pathway in breast cancer. Because SYK has been shown to function as a tumor suppressor, and its loss of expression in breast cancer has been correlated with tumor invasiveness, the aberrant SYK methylation is responsible for the loss of expression and may consequently play a permissive role for tumor aggressiveness.

Dpc4 transcriptional activation and dysfunction in cancer cells.

Dpc4 (Smad4) is implicated in mediation of signals from transforming growth factor (TGF) beta and related ligands, and wild-type Dpc4 mediates TGF-beta-stimulated gene transcription at specific DNA sequences bound by Dpc4 [Smad binding element (SBE)]. We characterized panels of DPC4 tumor mutations and cancer cell lines. Amino acid substitutions within the NH2-terminal third of Dpc4 weakened or ablated SBE-mediated gene regulation by a disruption of DNA binding. An interaction of the COOH-terminal end with the DNA-binding domain of Dpc4 was evident but was not required to explain the functional impairment produced by NH2-terminal DPC4 mutations. Both substitution and truncation mutations of the COOH-terminal half of DPC4 lacked the ability to regulate transcription while retaining the sequence-specific DNA-binding function, but through differing mechanisms. A modular domain to redistribute Dpc4 to the nuclear compartment allowed SBE-mediated transcriptional activation in a cell line having a TGF-1 receptor defect and was sufficient to restore SBE-mediated transactivation ability to COOH-terminal DPC4 missense mutants. Cells harboring DPC4 alterations had a universal impairment of the TGF-beta-stimulated SBE transcriptional response. These studies identify the loss of SBE-mediated gene regulation as a uniform outcome of the selection for DPC4 alterations during tumorigenesis. They raise the possibility of restoration of some Dpc4-associated transcriptional events in cancer cells through the targeted redistribution of wild-type and some missense mutant forms of Dpc4 to the nucleus.

Two androgen response elements in the androgen receptor coding region are required for cell-specific up-regulation of receptor messenger RNA.

In most cells and tissues containing androgen receptors (ARs), androgen regulates the levels of AR messenger RNA (mRNA). As the AR concentration is correlated with androgen responsiveness, this autoregulation of AR mRNA may affect cellular sensitivity to androgens. Androgens decrease levels of AR mRNA in many cell lines and tissues; however, in some tissues and possibly also at certain developmental stages, AR mRNA is up-regulated by androgens. Sequences within the 5'-flanking region and AR promoter do not appear to be sufficient for androgen regulation of AR mRNA. We have previously shown that both down- and up-regulation of AR mRNA by androgen can be reproduced in cell lines expressing a transfected human AR complementary DNA (cDNA). Sequences within the AR cDNA confer this autoregulation in transfected cells, suggesting that sequences within the transcribed region of the AR gene are sufficient for autoregulation. In this study we have determined the mechanism of androgenic up-regulation of AR mRNA encoded by the human AR cDNA in the prostate cancer cell line, PC3, and have identified the cis-acting sequences of the AR cDNA that are required. The observations that actinomycin D blocked androgenic up-regulation of AR mRNA but cycloheximide had no effect are consistent with a model in which AR is directly involved in transcriptional up-regulation of AR cDNA expression. Nuclear run-on assays showed that androgen treatment resulted in increased transcription of the AR cDNA. Furthermore, a 350-bp AR cDNA fragment inserted 5' of a thymidine kinase promoter-chloramphenicol acetyltransferase gene conferred androgen induction of chloramphenicol acetyltransferase activity in PC3 cells. This 350-bp fragment, which is located in the AR coding region, contains two putative androgen response elements (AREs) separated by 182 bp. The 5'-most ARE (ARE-1, 5'-TGTCCT-3') resembles a half-site of the palindromic consensus hormone response element, recognized by several steroid receptors, including AR, and the 3'-sequence (ARE-2, 5'-AGTACTCC-3') is identical to a portion of an androgen-responsive region found in the rat probasin gene promoter. Analysis of either ARE-1 or ARE-2 mutants revealed that these elements function synergistically. AR protein binds to the 350-bp fragment, as demonstrated by electrophoretic mobility shift assays using a glutathione-S-transferase-AR fusion protein containing the DNA- and steroid-binding domains of AR. These results indicate that the AR coding region contains an androgen-responsive region that is involved in cell line-specific up-regulation of AR mRNA.

Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA.

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

In vitro anthelmintic activity of Zanthoxylum simulans essential oil against Haemonchus contortus.

The need for new anthelmintic agents with low impact on the environment is becoming urgent. Phytotherapy is an alternative method to control gastrointestinal nematodes in small ruminants. This study aims to determine the composition of Zanthoxylum simulans essential oil (ZSEO) and evaluate the in vitro ovicidal and larvicidal effects of ZSEO on Haemonchus contortus using egg hatch assay, larval development assay (LDA), and larval migration inhibition assay (LMIA). The chemical composition of ZSEO was determined through gas chromatography and mass spectrometry and 94 compounds were identified from the ZSEO. The major constituents of ZSEO were borneol (18.61%), ß-elemene (10.87%). ZSEO and borneol both at 40 mg/mL inhibited larval hatching by 100%, with LC50 values of 3.98 and 1.50mg/mL, respectively. The LC50 value of ß-elemene was not determined because of its insufficient activity. The results of LDA showed that ZSEO, borneol, and ß-elemene all at 40 mg/mL inhibited larval development by 99.8%, 100%, and 55.4%, respectively, and exhibited dose-dependent responses with LC50 values of 4.02, 1.99, and 32.17 mg/mL, respectively. The results of LMIA showed that ZSEO, borneol, and ß-elemene all at 40 mg/mL inhibited larval migration by 74.3%, 97.0%, and 53.2%, respectively. ZSEO presented ovicidal and larvicidal activities in vitro. Therefore, Zanthoxylum may be an alternative source of anthelmintic agents to control gastrointestinal nematodes in sheep.

Androgen and glucocorticoid regulation of androgen receptor cDNA expression.

Androgen receptor (AR) levels are regulated by androgens, other steroids and non-steroidal hormones via complex, tissue-specific processes. Since alterations in receptor levels may influence cellular sensitivity to androgens, understanding AR regulation is of fundamental and potentially therapeutic significance. In most target tissues and AR-containing cell lines, AR mRNA is down-regulated in response to androgens. We have reconstituted this androgen-mediated down-regulation of AR mRNA in COS 1 cells transfected with a human AR cDNA under the control of the cytomegalovirus (CMV) promoter. The sequences mediating receptor mRNA down-regulation are represented within the AR cDNA and not within the CMV promoter. Androgenic down-regulation of AR cDNA expression was time- and dose-dependent, resembling native AR mRNA down-regulation. In addition, androgenic regulation of the receptor cDNA was not dependent on protein synthesis suggesting that AR and/or another pre-existing protein(s) is involved in this process. In COS 1 cells co-transfected with androgen and glucocorticoid receptor cDNAs, dexamethasone mimicked the action of androgen in down-regulating AR mRNA. This response depended on glucocorticoid receptors. Androgen had little effect on steady-state levels of AR protein consistent with reports that androgen down-regulates AR mRNA but increases AR protein half-life (Kemppainen et al. (1992) J. Biol. Chem. 267, 968-974; Zhou et al. (1995) Mol. Endocrinol. 9, 208-218). However, glucocorticoids decreased AR protein levels in cells that co-expressed androgen and glucocorticoid receptors. These results indicate that sequences represented in the AR cDNA mediate AR mRNA down-regulation by both androgens and glucocorticoids. Inhibition of AR mRNA and protein by glucocorticoids suggests that these steroids may modulate androgen action in tissues, such as mammary gland and prostate, which express both androgen and glucocorticoid receptors.

Androgenic up-regulation of androgen receptor cDNA expression in androgen-independent prostate cancer cells.

The expression of the androgen receptor (AR) gene is regulated by androgens. Although androgens down-regulate AR mRNA in most cell lines and tissues, including the prostate, up-regulation occurs in some tissues. Androgen-mediated reduction in AR mRNA is reproduced in COS1 cells and in the androgen-sensitive human prostate cancer cell line LNCaP when each expresses the AR cDNA. We have previously established that the AR cDNA contains the requisite sequences for this down-regulation. Here we shown that androgen promoted up-regulation of AR mRNA in two androgen-independent human prostate cancer cell lines, PC3 and DU145, when each was transfected with a human AR cDNA. This effect was due to the AR cDNA and not to the heterologous promoter driving AR expression. In addition to up-regulation of AR mRNA, androgen induced comparable increases in AR protein levels in PC3 cells stably expressing an AR cDNA (PC3/AR). Up-regulation of AR in PC3/AR cells was accompanied by failure of these cells to undergo desensitization or inactivation of AR following prolonged (96 h) androgen administration, whereas the same conditions resulted in desensitization of AR transactivation in LNCaP cells and in CVl cells that stably express the AR cDNA. Androgen treatment of PC3/AR cells resulted in induction of an androgen-regulated reporter gene (MMTV-CAT) as well as the native prostate-specific antigen gene, which is silent in untransfected PC3 but is androgen up-regulated in LNCaP and in the prostate. These results suggest that ectopic expression of AR in androgen-independent prostate cancer cell lines establishes both typical and atypical androgenic responses in a target gene-specific manner. Androgenic up-regulation of AR cDNA expression may be due to distinct signaling mechanisms that influence androgen action in androgen-independent prostate cancer cells.

Electroacupuncture reversed the inhibition of intestinal peristalsis induced by intrathecal injection of morphine in rabbits.

Morphine elicits a series of adverse effects including the inhibition of intestinal motility in addition to the therapeutic benefit of alleviating postoperative pain. To ascertain the role of electroacupuncture (EA) in diminishing those detrimental effects on recovery, we imitated the clinical procedures in rabbits. Morphine was given via a preimplanted cannula within spinal subarachnoid space, while the duodenal motility, respiration rate and arterial pressure were simultaneously recorded. It was found that morphine (6 mg/rabbit, IT) markedly suppressed duodenal peristalsis, decreased respiration rate throughout 90 min observation. When EA was administered together with morphine, peristalsis of the duodenum was much less inhibited (P < 0.05, vs morphine alone group), but no significant improvement of respiratory depression was noticed (P > 0.05), nor obvious change of arterial pressure in both groups. The results strongly recommend extensive application of EA in postoperative care, so as to decrease both the required dosage of morphine and the subsequent occurrence of postoperative ileus, while attaining sufficient analgesia.

The attenuation effect of chlorpromazine on electro-acupuncture analgesia: involvement of dopamine system.

The effect of chlorpromazine (CPZ) (0.1 or 0.5 mg/kg, iv) on electroacupuncture analgesia (EAA) was examined by using potassium dolorimetry in rabbits. CPZ itself induced hyperanalgesia, whereas it attenuated EAA in terms of maximal increase of pain threshold as well as EAA after effect. Therefore, CPZ is not a good candidate for enhancing EAA in clinics. Monoamines and their metabolites in cerebrospinal fluid (CSF) of the rabbits were detected by high pressure liquid chromatography coupled to electrochemical detector (HPLC-ECD) method. It was revealed that CPZ enhanced DOPAC and HVA contents in CSF in both the presence and absence of electroacupuncture. CPZ attenuated EAA with elevations of DOPAC and HVA concentration in CSF. There was a positive correlation between the increases of DOPAC and HVA contents in CSF and attenuation effect of CPZ on EAA. These results suggested that activation of dopamine system be unfavorable for EAA.

C-fos expression during electroacupuncture analgesia in rats--an immunohistochemical study.

Although the central mechanisms of electroacupuncture analgesia (EAA) have been investigated, a systematic study for the involvement of neuronal populations of central nervous system (CNS) in EAA has not been well undertaken, largely due to the difficulty in tracing the neuronal pathways by traditional techniques. Recently developed c-fos expression examination by immunohistochemical method with Ab-1 antisera might be used for this purpose as a useful marker for neuronal activity in CNS. In this study, tail flick latency (TFL) was tested as an index of pain threshold in conscious rats. After unilateral electroacupuncture was applied at 'Zuan-san-li' and 'Huan-tiao', the TFL was significantly prolonged. To explore the possible involvement of certain neuronal groups of central nervous system in EAA, we examined the EAA accompanied c-fos expression throughout the neuraxis, and a lot of specific c-fos protein labelled neurons were found in lumbar spinal cord (laminae I and II), nucleus raphe magnus, nucleus raphe dorsalis, substantia grisea centralis, nucleus habenulae lateralis, nucleus habenulae medialis, nucleus medialis thalami, nucleus lateralis hypothalami, nucleus supramamillaris, nucleus supraopticus, nucleus arcuatus, nucleus preopticus medialis, nucleus amygdala, nucleus tractus diagonalis, etc. No obvious c-fos expression was shown in these areas on control rats. These results strongly suggested that the functional activation of above-mentioned nuclei by electroacupuncture was underlied in EAA action.

In vitro ovicidal and larvicidal activity of the essential oil of Artemisia lancea against Haemonchus contortus (Strongylida).

Prolonged use of chemical anthelmintics has been found to result in anthelmintic resistance and environmental issues, thereby limiting the application of these drugs in domestic animals and prompting interest in the study of plant extracts as alternative sources thereof. The aim of this study was to evaluate the in vitro effect of the essential oil (EO) of Artemisia lancea against the parasitic nematode Haemonchus contortus using egg hatch assay, larval development assay, and larval migration inhibition assay. The EO yield of extraction was 0.63% (w/w), and the major constituents were 1,8-cineole (34.56%) and camphor (16.65%). In the egg hatch assay, an inhibition greater than 99% was observed with the EO at 10 mg mL(-1) and the LC50 was 1.82 mg mL(-1). 1,8-Cineole demonstrated moderate ovicidal activity with a LC50 of 4.64 mg mL(-1), whereas camphor did not show enough activity to have its LC50 determined. In the larval development assay, the EO, 1,8-cineole, and camphor inhibited 93.6%, 65.2%, and 57% of larval development at 10 mg mL(-1) and exhibited dose-dependent responses with LC50 values of 1.66, 5.07, and 7.80 mg mL(-1), respectively. In the migration inhibition assay, the EO and 1,8-cineole at best inhibited 77% and 60.3% of larval migration at 10 mg mL(-1), respectively. Camphor showed low inhibition capacity, and its efficacy was not dose dependent. The results indicate that the in vitro anthelmintic activity of the EO of A. lancea may be associated with the additive action of the two major constituents, as well as other more minor terpenoid components.

RANKL inhibition is an effective adjuvant for docetaxel in a prostate cancer bone metastases model.

BACKGROUND: Docetaxel induces an anti-tumor response in men with advanced prostate cancer (PCa); however, the side effects associated with docetaxel treatment can be severe, resulting in discontinuation of therapy. Thus, identification of an effective adjuvant therapy to allow lower doses of docetaxel is needed. Advanced PCa is typically accompanied by skeletal metastasis. Receptor activator of NFkB ligand (RANKL) is a key pro-osteoclastic factor. Targeting RANKL decreases establishment and progression of PCa growth in bone in murine models. METHODS: The efficacy of inhibiting RANKL, using a recombinant soluble RANK extracellular domain fused with the immunoglobulin Fc domain (RANK-Fc), was tested as an adjuvant therapy with docetaxel for PCa bone metastasis in a murine intra-tibial model. RESULT: The combination of RANK-Fc and docetaxel reduced tumor burden in bone greater than either treatment alone. CONCLUSION: The combination of docetaxel with a RANKL-inhibiting agent merits further investigation for treatment of advance PCa.

Effects of phencyclidine on contractile forces of isolated rabbit papillary muscles.

Phencyclidine (PCP) (0.01-50 mumol.L-1) and its analogue, TCP (0.01-50 mumol.L-1) exhibited positive inotropic effects on electrically stimulated rabbit papillary muscle preparations. Dextrorphan (5 or 10 mumol.L-1) antagonized the actions of PCP in non-competitive manner (pD'2 = 5.25). This demonstrated the involvement of PCP receptors in the positive inotropic effects of PCP. By using high performance liquid chromatography with electrochemical detector (HPLC-ECD), an increase of DOPAC content was found in bath medium after PCP addition. Each of the dopamine receptor antagonists SCH23390, haloperidol and sulpiride (1 mumol.L-1) attenuated the maximal inotropic effects of PCP. These results suggest that PCP induces positive inotropic effects by increasing the release and/or blocking the uptake of dopamine.

Effect of tiapride on electroacupuncture analgesia.

Tiapride icv 400 micrograms/rabbit exhibited analgesic and synergistic effects on electroacupuncture analgesia (EAA) in rabbits. Both electroacupuncture (EA) and tiapride (icv 400 micrograms/rabbit) enhanced the beta-endorphin-like immunoreactive substance (beta-EPIS) level in cerebrospinal fluid (CSF) measured by radioimmunoassay (RIA). When EA and tiapride were used in combination, a further increase of beta-EPIS content was found. The results suggested that promotion of beta-EPIS release by tiapride may be one of the mechanisms of synergistic effect of tiapride on EAA.

Chlorpromazine attenuated electroacupuncture analgesia in conscious rabbits.

By measuring the defense behavior in response to the noxious stimulation induced by potassium iontophoresis on ear-lobe skin of conscious rabbit, chlorpromazine (CPZ) (0.5 mg.kg-1, i.v.) induced hyperalgesia, whereas it significantly attenuated electroacupuncture analgesia (EAA) efficacy. Monoamines and their metabolites in cerebrospinal fluid (CSF) were measured by high pressure liquid chromatography with electrochemical detector (HPLC-ECD) while the attenuation effect of CPZ on EAA was observed. CPZ markedly enhanced 3,4-dihydroxyphenylacetic acid (DOPAC) (P < 0.05) and homovanillic acid (HVA) (P < 0.01) contents in CSF both in the presence and absence of electroacupuncture. CPZ attenuated EAA with elevations of either DOPAC or HVA concentrations in CSF. There was a positive correlation between the increase of DOPAC or HVA content in CSF and the attenuation effect of CPZ on EAA (P < 0.05). These results suggested that the activation of dopamine system was unfavorable for EAA.

G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation.

The tumor suppressor Smad4/Dpc4 is a transcription activator that binds specific DNA sequences and whose nuclear localization is induced after exposure to type beta transforming growth factor-like cytokines. We explored an inducible system in which Smad4 protein is activated by translocation to the nucleus when cell lines that stably express wild-type or mutant Smad4 proteins fused to a murine estrogen receptor domain are treated with 4-hydroxytamoxifen. This induced Smad4-mediated transcriptional activation and a decrease in growth rate, attributable to a cell cycle arrest at the G1 phase and an induction of apoptosis. A tumor-derived mutation (Arg-100 --> Thr) affecting a residue critical for DNA-binding demonstrated an "oncogenic" phenotype, having decreases in both the G1 fraction and apoptosis and, consequently, an augmentation of population growth. This model should be useful in the exploration and control of components that lie further downstream in the Smad4 tumor-suppressor pathway.