Knockdown of the gene encoding Drosophila tribbles homologue 3 (Trib3) improves insulin sensitivity through peroxisome proliferator-activated receptor-γ (PPAR-γ) activation in a rat model of insulin resistance.

AIMS/HYPOTHESIS: Insulin action is purportedly modulated by Drosophila tribbles homologue 3 (TRIB3), which in vitro prevents thymoma viral proto-oncogene (AKT) and peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. However, the physiological impact of TRIB3 action in vivo remains controversial. METHODS: We investigated the role of TRIB3 in rats treated with either a control or Trib3 antisense oligonucleotide (ASO). Tissue-specific insulin sensitivity was assessed in vivo using a euglycaemic-hyperinsulinaemic clamp. A separate group was treated with the PPAR-γ antagonist bisphenol-A-diglycidyl ether (BADGE) to assess the role of PPAR-γ in mediating the response to Trib3 ASO. RESULTS: Trib3 ASO treatment specifically reduced Trib3 expression by 70% to 80% in liver and white adipose tissue. Fasting plasma glucose, insulin concentrations and basal rate of endogenous glucose production were unchanged. However, Trib3 ASO increased insulin-stimulated whole-body glucose uptake by ~50% during the euglycaemic-hyperinsulinaemic clamp. This was attributable to improved skeletal muscle glucose uptake. Despite the reduction of Trib3 expression, AKT2 activity was not increased. Trib3 ASO increased white adipose tissue mass by 70% and expression of Ppar-γ and its key target genes, raising the possibility that Trib3 ASO improves insulin sensitivity primarily in a PPAR-γ-dependent manner. Co-treatment with BADGE blunted the expansion of white adipose tissue and abrogated the insulin-sensitising effects of Trib3 ASO. Finally, Trib3 ASO also increased plasma HDL-cholesterol, a change that persisted with BADGE co-treatment. CONCLUSIONS/INTERPRETATION: These data suggest that TRIB3 inhibition improves insulin sensitivity in vivo primarily in a PPAR-γ-dependent manner and without any change in AKT2 activity.

Metrics for monitoring local inequalities in access to maternity care: developing a basket of markers from routinely available data.

BACKGROUND: Equal access for all based on need is part of a conceptualisation of quality underpinning recent UK NHS policies. OBJECTIVE: To develop metrics for access to maternity care from routinely available data in order to inform inequalities monitoring and commissioning. DESIGN: Cross-sectional cohort design using case-note audit and postnatal questionnaire. SETTING: London hospital, UK, in an area of relative socio-economic deprivation. METHODS: Stage 1: Identification of potential markers. Stage 2: Testing of markers via case note audit and postnatal questionnaire. Stage 3: Selection of final basket of markers of access to maternity services. RESULTS: Of 71 possible markers identified, 32 used information obtainable from maternity case notes. After testing in the case-note audit, 21 were discarded, and 11 included in the final basket covering: timely entry to maternity care; appropriate assessment and identification of needs of individuals; referral and communication with other related health and social care services. CONCLUSION: It is possible to devise a local basket of markers covering a range of important entry and in-system access metrics. Such a tool offers an unobtrusive means to audit the effectiveness of some of the processes intended to help women move through the maternity and related health and social care systems during pregnancy, and to monitor progress on reducing social inequalities in access over time.

Factors associated with postpartum physical and mental morbidity among women with known HIV status in Lusaka, Zambia.

The objective of our study was to investigate factors associated with postpartum physical and mental morbidity among women in Lusaka, Zambia with particular reference to known HIV status. Our study was part of the Breastfeeding and Postpartum Health (BFPH) longitudinal cohort study conducted between June 2001 and July 2003. Women were recruited at 34 weeks gestation and followed up to 16 weeks postpartum. Data on maternal health were collected at 3, 7, 10, and 14 days and at 3, 4, 5, 6, 9, 12, and 16 weeks postpartum. Maternal mental health data were collected from April 2002 onwards at recruitment and at seven days and six weeks postpartum. Data on physical morbidity were collected for 429 women (218 HIV-negative, 211 HIV-positive) and data on mental morbidity were collected for 272 women (134 HIV-negative, 138 HIV-positive). Multivariate logistic regression was used to examine factors associated with postpartum physical or mental morbidity. Postpartum physical morbidity was associated with HIV status, parity > or =5 and age < 20 years. Neither antenatal nor postpartum mental morbidity, as indicated by a self-reporting questionnaire 20-item (SRQ-20) score > or =7, were associated with HIV status or with postpartum physical morbidity in this population. Larger comparative studies are required to corroborate or contest these findings.

Spinal distribution and metabolism of 2'-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats.

Intrathecal (IT) delivery of antisense oligodeoxynucleotides (ASO) has been used to study the function of specific gene products in spinal nociception. However, a lack of systematic studies on the spinal distribution and kinetics of IT ASO is a major hurdle to the utilization of this technique. In the present study, we injected rats IT with 2'-O-(2-methoxyethyl) modified phosphorothioate ASO (2'-O-MOE ASO) and examined anatomical and cellular location of the ASO in the spinal cord and dorsal root ganglia (DRG) by immunocytochemistry. At 0.5 h after a single IT injection, immunostaining for ISIS 13920 (a 2'-O-MOE ASO targeting h-ras) localized superficially in the lumbar spinal cord, while at 24 h the immunostaining was distributed throughout the spinal cord and was predominantly intracellular. Double staining with cell type specific antibodies indicated that the ASO was taken up by both glia and neurons. ASO immunoreactivity was also observed in DRG after IT ISIS 13920. Capillary gel electrophoresis analysis showed that ISIS 22703, a 2'-O-MOE ASO targeting the alpha isozyme of protein kinase C (PKC), remained intact in spinal cord tissue and cerebrospinal fluid up to 24 h after the injection and no metabolites were detected. In contrast, after IT ISIS 11300, an unmodified phosphorothioate ASO with the same sequence as ISIS 22703, no full-length compound was detectable at 24 h, and metabolites were seen as early as 0.5 h. IT treatment with ISIS 22703 at doses that effectively down-regulated PKCalpha mRNA in spinal cord did not affect the mRNA expression in DRG. In summary, 2'-O-MOE ASO displayed high stability in spinal tissue after IT delivery, efficiently distributed to spinal cord, and internalized into both neuronal and non-neuronal cells. ASO are able to reach DRG after IT delivery; however, higher doses may be required to reduce target gene in DRG as compared with spinal cord.

Barriers to accessing safe motherhood and reproductive health services: the situation of women with disabilities in Lusaka, Zambia.

PURPOSE: To ascertain how well health services in Lusaka, Zambia currently meet the safe motherhood and reproductive health care needs of women who have physical impairment leading to disability. METHODS: A qualitative study was conducted in Lusaka, Zambia. In-depth tape-recorded interviews were conducted with 24 purposively selected women with disabilities and with 25 safe motherhood/reproductive public sector health service providers. Qualitative analysis was conducted using NVivo software. RESULTS: Women with disabilities encounter various social, attitudinal and physical barriers to accessing safe motherhood and reproductive health (RH) services in this particular setting. The strong desire for children and affection can increase vulnerability to sexual exploitation. At the same time, a generalized assumption among reproductive health service providers that women with disabilities will not be sexually active, and not require RH services, leads to increased vulnerability to sexually transmitted infection including HIV. Once pregnant, traditional beliefs about transmission of disabilities can create barriers to integration in ante-natal clinics. Nurse-midwives' fear of delivery complications in women with physical impairments can also result in routine over-referral to a tertiary maternity facility which is outside the locality and harder for women with mobility limitations to get to. CONCLUSION: Greater understanding of the influences underpinning societal attitudes towards sexuality and disability in this setting, and more extensive communication between health care staff and women with disabilities would facilitate positive action towards improving safe motherhood and reproductive health services for women with disabilities.

Zambian women's experiences of urban maternity care: results from a community survey in Lusaka.

Urban African maternity care systems face problems, as rapid population growth puts them under increasing pressure. In 1983 a decentralised system with midwife-run maternity units at health centres was initiated in Lusaka. A community-based survey of 1210 women conducted in 1999 examined access, coverage and quality of care in these maternity services. Results were generally positive: 99% of respondents received some antenatal check-ups and three quarters had five or more. Institutional delivery rate was 89.5%. Home birth was associated with belonging to a "very poor" household. Sixty three per cent of births were in the decentralised units. Eighty nine per cent reported care as "good" or "very good", but 21% remembered someone who had treated them badly during labour, principally by shouting or scolding. One fifth of women reported having been left alone for "too long" in labour. Less than half of the women said they would like a lay labour companion and three quarters would prefer a companion at the delivery.

Inhibition of spinal protein kinase Calpha expression by an antisense oligonucleotide attenuates morphine infusion-induced tolerance.

Protein kinase C isoforms including the alpha isozyme have been implicated in morphine tolerance. In the present study, we examined the effect of intrathecal delivery of an antisense oligonucleotide targeting rat protein kinase Calpha mRNA on the expression of spinal protein kinase Calpha isozyme and spinal morphine tolerance. Continuous intrathecal infusion of rats with morphine produced an increase in paw withdrawal threshold to thermal stimulation on day 1, which disappeared by day 5. On day 6, a bolus intrathecal injection of morphine (a probe dose) produced significantly less analgesia in morphine-infused rats than in saline-infused rats, suggesting tolerance. Intrathecal treatment with the protein kinase Calpha antisense concurrent with spinal morphine infusion not only maintained the analgesic effect of morphine during the 5-day infusion, it also significantly increased responsiveness to the probe morphine dose on day 6. In comparison, the missense used in the same treatment paradigm had no effect. The inhibitory effect of protein kinase Calpha antisense on spinal morphine tolerance was dose-dependent, and reversible. Intrathecal treatment with the antisense, but not the missense, in rats decreased expression of spinal protein kinase Calpha mRNA and protein, as revealed by real-time quantitative reverse transcription-polymerase chain reaction and western blots. Expression of the gamma isozyme was not affected by the oligonucleotides. The antisense also attenuated protein kinase C-mediated phosphorylation in spinal cord. These results demonstrate that selective reduction in the expression of the spinal protein kinase Calpha isozyme followed by a decrease of local protein kinase C-mediated phosphorylation will reverse spinal morphine infusion-induced tolerance. This finding is consistent with the view that tolerance produced by morphine infusion is dependent upon an increase in phosphorylation by protein kinase C, and also it emphasizes that the protein kinase Calpha isozyme and its activation in spinal cord may specifically participate in the phenomenon of opiate tolerance.

Tools for monitoring the effectiveness of district maternity referral systems.

It is widely accepted that substantial reductions in maternal mortality and severe morbidity are impossible to achieve without an effective referral system for complicated cases. Early detection and referral to higher levels of care might also substantially reduce neonatal deaths due to the complications of childbirth. The general goal of such a referral system is that patients are dealt with in the right place with effective treatment provided at the minimum of cost. There are real challenges, however, in monitoring the effectiveness of such referral systems once put in place. This paper describes some of the tools used to review pregnancy-related referrals in Lusaka, Zambia. The tool-mix used provided information for monitoring five different aspects of the referral system: the distribution of births across levels of facility and population coverage; the use of essential obstetric care (EOC) level facilities by women with complications; the progress towards a reduction of maternal mortality at referral facility level; inappropriate use of EOC level facility; and perinatal outcomes at peripheral facility level. Apart from the information on coverage, the data came from routinely collected facility statistics, registers and medical notes. Findings for Lusaka are reported. Consideration is given to issues of interpretation of specific indicators, and to how such tools might be used in conjunction with others, in order to help district managers to monitor the effectiveness of district maternity referral systems.

Relation between private health insurance and high rates of caesarean section in Chile: qualitative and quantitative study.

OBJECTIVES: To explore the circumstances and factors that explain the association between private health insurance cover and a high rate of caesarean sections in Chile. DESIGN: Qualitative analysis of audiotaped in-depth interviews with obstetricians and pregnant women; quantitative analysis of data from face to face semistructured interview survey conducted postnatally (with women who had given birth in the previous 24-72 hours), and of a review of medical notes at a public hospital, a university hospital, and a private clinic. SETTING: Santiago, Chile. PARTICIPANTS: Qualitative arm: 22 obstetricians, 21 pregnant women; quantitative arm: 540 postnatal women. MAIN OUTCOME MEASURES: Rates of caesarean section in different types of institutions; consultants' views on private practice; work patterns in private practice; women's reasons for choosing private care; women's preferences on method of delivery. RESULTS: Private health insurance cover requires the primary maternity care provider to be an obstetrician. In the postnatal survey, women with private obstetricians showed consistently higher rates of caesarean section (range 57-83%) than those cared for by midwives or doctors on duty in public or university hospitals (range 27-28%). Only a minority of women receiving private care reported that they had wanted this method of delivery (range 6-32%). With the diversification in the healthcare market, most obstetricians now have demanding peripatetic work schedules. Private maternity patients are a lucrative source of income. The obstetrician is committed to attend these private births in person, and the "programming" (or scheduling) of births is a common time management strategy. The rate of elective caesarean sections was 30-68% in women with private obstetricians and 12-14% in women not attended by private obstetricians. CONCLUSIONS: Policies on healthcare financing can influence maternity care management and outcomes in unforeseen ways. The prevailing business ethos in health care encourages such pragmatism among those doctors who do not have a moral objection to non-medical caesarean section.

Identification of okadaic-acid-induced genes by mRNA differential display in glioma cells.

To identify novel genes associated with apoptosis in glioma cells, we treated T98G glioma cells with okadaic acid (OA). Differential display using 15 random primers was performed on RNA extracted from these cells. Upregulated bands were excised from polyacrylamide gels and cloned. Northern blots were used to confirm RNA expression in T98G cells. 18 RNA fragments corresponding to the untranslated region of genes were identified and sequenced. Three unknown gene fragments were used to screen a fetal brain cDNA library resulting in three complete cDNA sequences. The three sequences corresponded to a human gene homologous to the yeast translation initiation factor Sui-1, a cAMP-regulated phosphoprotein, ARPP-16/19, and a novel gene designated O48. Transcription of Sui-1 increased in response to all stress factors tested, whereas ARPP only responded to OA. 2-kb and 4-kb O48 RNA species were identified. OA and stress factors increased 2-kb expression while K252a (protein kinase inhibitor) increased 4-kb expression. Differential display is effective for identifying genes associated with apoptosis. Novel genes may be identified by further analysis of the gene fragments identified in this study. The function of O48 is unknown.

Phosphorothioate oligodeoxynucleotides distribute similarly in class A scavenger receptor knockout and wild-type mice.

It has been suggested that binding of phosphorothioate oligodeoxynucleotides (P=S ODNs) to macrophage scavenger receptors (SR-AI/II) is the primary mechanism of P=S ODN uptake into cells in vivo. To address the role of scavenger receptors in P=S ODN distribution in vivo, several pharmacokinetic and pharmacological parameters were compared in tissues from scavenger receptor knockout mice (SR-A-/-) and their wild-type counterparts after i.v. administration of 5- and 20-mg/kg doses of P=S ODN. With an antibody that recognizes P=S ODN, no differences in cellular distribution or staining intensity in livers, kidneys, lungs, or spleens taken from SR-A-/- versus wild-type mice could be detected at the histological level. There were no significant differences in P=S ODN concentrations in these organs as measured by capillary gel electrophoresis as well, although the concentration of P=S ODN in isolated Kupffer cells from livers of SR-A-/- mice was 25% lower than that in Kupffer cells from wild-type mice. Furthermore, a P=S ODN targeting murine A-raf reduced A-raf RNA levels to a similar extent in livers from SRA-/- (92.8%) and wild-type (88.3%) mice. Finally, in vitro P=S ODN uptake studies in peritoneal macrophages from SR-A-/- versus wild-type mice indicate that other high- and low-affinity uptake mechanisms predominate. Taken as a whole, our data suggest that, although there may be some contribution to P=S ODN uptake by the SR-AI/II receptor, this mechanism alone cannot account for the bulk of P=S ODN distribution into tissues and cells in vivo, including macrophages.

Training initiatives for essential obstetric care in developing countries: a 'state of the art' review.

Increased international awareness of the need to provide accessible essential or emergency obstetric and newborn care in developing countries has resulted in the recognition of new training needs and in a number of new initiatives to meet those needs. This paper reviews experience in this area so far. The first section deals with some of the different educational approaches and teaching methods that have now been employed, ranging from the traditional untheorized 'chalk and talk', to competency-based training, to theories of adult learning, problem solving and transferable skills. The second section describes a range of different types of indicators and data sources (learner assessments, user and community assessments, trainer assessments and institutional data) that have been used in the assessment of the effectiveness of such training. The final section of the paper draws together some of the lessons. It considers evaluation design issues such as the inclusion of medium and long term evaluation, the importance of methods that allow for the detection of iatrogenic effects of training, and the roles of community randomized trials and 'before, during and after' studies. Issues identified for the future include comparative work, how to keep training affordable, and where training ought to lie on the continuum between straightforward technical skills acquisition and the more complex learning processes required for demanding professional work.

K252a induces cell cycle arrest and apoptosis by inhibiting Cdc2 and Cdc25c.

The indole carbazole K252a has been shown in previous studies to inhibit the platelet-derived growth factor signal transduction pathway in gliomas. Because K252a has nonspecific effects on protein kinase function, we studied its effect on cyclin-dependent kinases (CDK) and cell cycle blockade in glioma cells. K252a blocked T98G cells at the G1/S and G2/M checkpoints. Consistent with cell cycle arrest, K252a was shown to hypophosphorylate Rb, upregulate p21, and decrease Cdc2 and Cdc25c activity. Finally, cell cycle arrest in T98G cells resulted in apoptosis as determined by cell morphology and DNA laddering. K252a is a useful tool for studying the effects of CDK inhibition and cell cycle blockade in tumor cells.

Sodium vanadate inhibits apoptosis in malignant glioma cells: a role for Akt/PKB.

The dual signal hypothesis of apoptosis holds that a common signal can activate both apoptotic and proliferative pathways. The fate of a cell is dependent on which of these two pathways predominates. In the MAPK family of kinases, ERK and JNK have been proposed to mediate apoptosis whereas the PI3K-stimulated kinase, Akt/PKB, has been shown to inhibit apoptosis. The object of this study was to determine the role of these kinases in a glioma model of apoptosis. We have previously shown that K252a induces apoptosis and inhibits kinase activity. In this study we confirm these results and show that the protein tyrosine phosphatase inhibitor sodium vanadate activates ERK, JNK and Akt/PKB, but does not stimulate proliferation. Vanadate did protect T98G cells from K252a-induced apoptosis, an effect that was abolished by addition of the PI3K inhibitor wortmannin. This suggests that PI3K and Akt/PKB may be responsible for mediating vanadate's protective effect on glioma cells. We conclude that the intracellular balance between protein phosphorylation pathways is a critical determinant of both cell proliferation and cell death.

Selection and evaluation of blood- and tribologically compatible journal bearing materials.

A critical issue in the Cleveland Clinic Foundation (CCF) Innovative Ventricular Assist System (IVAS) blood pump is the selection of materials for the blood-lubricated journal bearing. Under normal operating conditions, the journal bearing geometry creates a thick blood film that separates the rotating and stationary surfaces. However, since start-up and certain transients could cause temporary contact, the material pair selected for these surfaces must be both tribologically and blood compatible. Combinations of two biocompatible alloys were tested: a titanium-zirconium-niobium alloy (Ti-13Zr-13Nb) and a zirconium-niobium alloy (Zr-2.5Nb). A standard pin-on-disk tester was used, with the contact surfaces lubricated by glycerol/saline mixtures simulating the viscosity range of blood. One test series evaluated start-up conditions; the other modeled a high-speed rub that might occur if the fluid film broke down. Results showed that the preoxidized Zr-2.5Nb pin/Ti-13Zr-13Nb disk combination was superior at all sliding velocities; a self-mated Zr-2.5Nb pair also showed promise. The oxide film on a self-mated Ti-13Zr-13Nb pair, and a Ti-13Zr-13Nb pin and Zr-2.5Nb disk combination did not show adequate wear life. More work remains to explain distinct performance differences of certain combinations, with more data needed on mechanical properties of thin, hard coatings on softer metal substrates.

K252a inhibits proliferation of glioma cells by blocking platelet-derived growth factor signal transduction.

Growth factors are known to regulate glioma proliferation. The glioma cell lines U87 and T98G were examined for evidence of an autocrine stimulatory loop involving the neurotrophin family of growth factors. Although neurotrophin-3 and TrkC RNA were detected by reverse transcription-PCR, there was no evidence of significant interaction between neurotrophin-3 and its cognate receptor TrkC. The microbial alkaloid K252a has been described to inhibit both Trk tyrosine kinase activity and neuroblastoma cell proliferation. K252a inhibited proliferation in U87 (IC50 = 1170 nM) and T98G (IC50 = 529 nM) but induced apoptosis in U87 cells only. At concentrations of 500 nM to 1 microM, K252a blocked only platelet-derived growth factor (PDGF)-mediated receptor autophosphorylation. These results suggest that an autocrine loop involving PDGF is functional and important for maintaining tumor growth. There is no evidence to support the existence of a neurotrophin-mediated autocrine loop. K252a, through inhibition of PDGF signal transduction, may be a novel therapeutic agent in the treatment of human gliomas.

Cesarean birth trends in Chile, 1986 to 1994.

BACKGROUND: Despite indications of high cesarean section rates in various parts of Latin America, relatively few comprehensive studies of national birth intervention trends have been conducted in that continent. Recent national statistics suggest that Chile may now have the highest reported cesarean section rate in the world. This paper examines cesarean birth trends in Chile with reference to changing patterns in health care financing. METHODS: The growth in the national cesarean birth rate is analyzed, with reference to regional patterns, differences according to insurance coverage, and recent shifts in the financing pattern of health care provision, using insurance fund data and hospital reporting systems data for both public and private sector care from the mid-1980s to mid-1990s. RESULTS: Chile had a cesarean birth rate of 37.2 percent for the 301,955 births covered by either the National Health Fund or private health insurance in 1994. This was a one-third increase from the 1986 rate of 27.7 percent. The private health insurance sector revealed consistently far higher cesarean section rates than the National Health Fund sector (59% vs 28.8% in 1994); intrasectoral rates remained fairly stable over the 8-year period. CONCLUSIONS: The overall increase in Chile's cesarean section rate correlates with the growth in the proportion of all births whose care was privately insured during these years (from 7.5% to 24.8%). This change may be partly explained by the doubling (to 32%) of the percentage of women with a personal obstetrician rather than a "duty" practitioner attending the birth of their baby.

Neonatal care in developing countries.

Worldwide, about eight newborn babies die every minute, yet interventions to reduce early neonatal mortality and morbidity are still not given high priority in most developing countries. It is often assumed that neonatal care is too costly for high coverage in poor populations, but in fact many deaths could be prevented or treated with low technology and improved care. There are four principles of basic newborn care: an atraumatic and clean delivery, maintenance of body temperature, initiation of spontaneous respiration and breast feeding shortly after birth. For the majority these could be facilitated at the health centre by nurse midwives or at home by TBAs or family members. Low cost special care for many of the 10%-15% who are sick or pre-term or low birth weight could be provided at district hospitals using appropriate simple technologies.

Involvement of multiple cis elements in basal- and alpha-adrenergic agonist-inducible atrial natriuretic factor transcription. Roles for serum response elements and an SP-1-like element.

In the present study, cis elements in the 5'-flanking sequence (FS) of the rat atrial natriuretic factor (ANF) gene involved in regulating basal and alpha 1-adrenergic-inducible transcription were investigated. Truncation analyses using ANF-luciferase reporter constructs transfected into primary neonatal rat cardiac myocytes showed that an A/T-rich serum response element (SRE) at -114 bp of the ANF 5'-FS, which bound serum response factor (SRF), was required for basal and inducible transcription. In constructs composed of 134 bp of rat ANF 5'-FS driving luciferase (ANF-134Luc), mutations in the SRE at -114 bp disrupted SRF binding and ANF promoter activity. However, the same mutations in ANF-638Luc had little effect, suggesting a collaborating role for more distal sequences, such as the other SRE in ANF-638 at -406 bp. In ANF-638Luc, mutations in the SRE at -406 bp that disrupted SRF binding to that site decreased ANF reporter activity by only 25%; however, mutating both of the SREs completely blocked alpha 1-adrenergic-inducible activity. Mutation analyses showed that an ... (SP-1)-like site at -69 bp, shown previously to confer inducibility in reporters with 134 bp of ANF 5'-FS, was not required in ANF-638Luc. However, double mutants in the SP-1-like region and either SRE completely blocked alpha 1-adrenergic-inducible ANF promoter activity. These findings emphasize that no single element is responsible for alpha 1-adrenergic agonist-regulated ANF transcription but that the SREs at -114 and -406 bp and the SP-1-like sequence at -69 bp mediate the effect in collaboration.