DNA was isolated from 0 5-1 g of cell paste using MasterPure Gram

DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer, with modification st/DL for cell lysis as described in Wu et al. [40]. DNA is available through the DNA Bank Network [42]. Genome sequencing and assembly The draft genome was generated at the DOE inhibitor licensed Joint Genome Institute (JGI) using a combination of Illumina and 454 technologies (Roche). For this genome, we constructed and sequenced an Illumina GAii shotgun library which generated 26,937,600 reads totaling 969.8 Mb, a 454 Titanium standard library which generated 238,617 reads and a paired end 454 library with an average insert size of 14.3 kb which generated 112,671 reads totaling 141.8 Mb of 454 data.

All general aspects of library construction and sequencing performed at the JGI can be found at [43]. The initial draft assembly contained 28 contigs in one scaffold. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 0.7.63 [44], and the consensus sequences were computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS – 4.24 (High Performance Software, LLC).

The software Consed [45] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI [46]. Possible mis-assemblies were corrected using gapResolution [43], Dupfinisher [47], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 388 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 508.6 �� coverage of the genome.

Genome annotation Genes were identified using Prodigal [48] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [49]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction Brefeldin_A analysis and functional annotation was performed within the Integrated Microbial Genomes – Expert Review (IMG-ER) platform [50].

9998 �� 0 0102 The LOD and LOQ were 20 33 and 60 72 ng/band, res

9998 �� 0.0102. The LOD and LOQ were 20.33 and 60.72 ng/band, respectively. The proposed HPTLC methods were validated for sellckchem intra and interday variations. The values of percent relative standard deviations (RSDs) were found to be 0.76 and 0.94, respectively which indicate that the method was precise. The method was also evaluated by the assay of commercially available tablets containing mycophenolate mofetil. Six replicate analyses were performed on accurately weighed amount of tablets. The percent assay was found to be 99.29 �� 0.77 for mycophenolate mofetil. To study the accuracy of the method, recovery studies were performed. For mycophenolate mofetil, the recovery ranged from 99.26 to 100.5%, with values of percent RSD ranging from 0.29 to 0.72, indicating that the proposed HPTLC method was highly accurate [Table 1].

When the specificity of the method was checked, it was found that the Rf and UV spectrum of the drug standard were the same as those from the sample. The study of robustness of the method revealed that the peak areas were unaffected (RSD < 2%) by small changes in the operating conditions, and can be inferred to be more robust. The method validation parameters are presented in Table 2. Figure 2 Typical HPTLC densitogram of mycophenolate mofetil standard (Rf: 0.55 �� 0.02) Figure 3 Typical HPTLC densitogram of mycophenolate mofetil extracted from tablet formulation (Rf: 0.55 �� 0.02) Table 1 Results from recovery studies of mycophenolate mofetil Table 2 Method validation parameters of mycophenolate mofetil CONCLUSION The developed HPTLC technique is precise, specific, and accurate.

Statistical analysis proves that the method is suitable for the analysis of mycophenolate mofetil as bulk drug and as formulation without interference from its excipients. It may be extended for the quantitative estimation of the said drug in plasma and other biological fluids. ACKNOWLEDGMENT The authors wish to thank Intas Pharmaceuticals Ltd., Ahmedabad, for providing the gift sample of mycophenolate mofetil. They are also grateful to the management of the Hindu College of Pharmacy for providing Drug_discovery the necessary facilities to carry out this project. Footnotes Source of Support: Nil Conflict of Interest: None declared.
Cefpodoxime proxetil is an orally-absorbed prodrug of cefpodoxime, an extended-spectrum, semi-synthetic cephalosporin developed by Sankyo Co. Ltd Japan. Cefpodoxime proxetil, chemically a relatively new broad-spectrum third-generation cephalosporin, has very good in vitro activity against Enterobacteriaceae, Hemophilus spp., and Moraxella spp., including fl-lactamase producers and many strains resistant to other oral agents. It also has activity against gram-positive bacteria, especially against streptococci.


Formulation Calcitriol supplier analysis The percentage Assay of LORN and PCM in two different tablet samples Lornasafe-plus and Lorsaid-P were calculated and recorded in Table 4. Table 4 Summary of validation Specificity The specificity of the method was ascertained by the absence of any other peak of placebo [Figure 2].The validation summary is given in Table 4. Figure 2 Chromatogram of constituted tablet placebo CONCLUSION The developed HPTLC method was found to be simple, specific, precise, accurate, and reproducible and can be used for the routine estimation of LORN and PCM in the combined tablets dosage form available in market. The developed method is found to be less sensitive but more accurate and specific than the previously published method. ACKNOWLEDGMENT Authors are thankful to Cirex pharmaceutical Ltd (Gundla, Mandal district, A.

P., India) and Cadila Pharmaceutical Ltd (Dholka, Ahmedabad, Gujarat, India) for providing the gift sample of LORN and PCM standard. Footnotes Source of Support: Nil Conflict of Interest: None declared.
Capsaicin (CAP) is the main alkaloid responsible for pungency in chillies and is used as a counterirritant balm for external application and it is also used in creamy to provide external pain relief for arthritis patients.[1] To date, research has shown that capsaicinoids, and CAP in particular, have a wide variety of biological and physiological activities which provide them functions, such as antioxidants, anticarcinogenics, promotion of energy metabolism and suppression of fat accumulation, and inflammations.

However, the potential applications of these molecules are limited by the irritation caused by their pungency.[2] Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a crystalline, lipophilic, colorless, and odorless alkaloid Cilengitide with the molecular formula C18H27NO3 [Figure 1]. Its molecular weight is 305.40 g/mol, and it is fat-, alcohol- and oil-soluble. CAP displays cis/trans isomerism because the double bond prevents internal rotation. CAP is always found as the trans isomer because in the cis form, the �CCH(CH3)2 and the longer chain on the other side of the double bond will be close together, causing them to repel each other slightly. This additional strain imposed causes the cis isomer to be a less stable arrangement than the trans isomer.[2] Figure 1 Chemical structure of capsaicin CAP is known to be effectively absorbed topically from the skin. In a study of 12 subjects topically administered, a 3% CAP solution in three different vehicles (70% isopropyl alcohol; mineral oil; and propylene glycol in 20% alcohol), CAP was shown to be rapidly absorbed and to quickly reach maximum concentration when CAP is applied topically. The half-life of CAP was approximately 24 h.[3].

Exclusion criteria for minimally invasive approach were the same

Exclusion criteria for minimally invasive approach were the same of traditional laparoscopic surgery. Clinical or radiological signs of complicated appendix or gallbladder disease (masses and abscesses) and of voluminous neoplasms, the presence of liver cirrhosis, peritonitis, previous upper abdominal Imatinib chemical structure surgery, or severe obesity were exclusion criteria for SILS. 2.2. Single-Port Access Technique: Surgical Glove Port Construction An access device was made by a standard wound protector (a small size or extra small size ALEXIS wound retractor; Applied Medical, CA, USA) (Figure 1) and size 6, nonlatex sterile glove. The wound retractor was introduced through the small umbilical incision. The surgical glove was fixed to the outer ring of the wound retractor (Figure 2).

A little access was made on the tip of one finger, and the CO2 pipe was connected to induce pneumoperitoneum (Figure 3). Other accesses were made on the others fingers to create a working channel for the laparoscopic instruments (Figure 4). Five- or three-millimeter traditional or curved laparoscopic instruments were used. Figure 1 Placement of wound protector. Figure 2 Placement of surgical glove. Figure 3 Induction of pneumoperitoneum. Figure 4 Placement of instruments. 3. Results SILS was successfully completed in 34 patients: 20 appendectomy was performed in female patients (median age 15, range 9�C32 years), cholecystectomy in 12 patients (11 female and 1 male, median age 35, range 17�C83 years), and right hemicolectomy in 2 female patients (55 and 64 years old).

In no patient conversion to standard laparoscopy or to open surgery was needed. The median operative time for appendectomy, cholecistectomy and right hemicolectomy was 35, 45, and 67.5 minutes, respectively. Blood loss was minimal in all cases. No wound complication occurred; a picture of the scare at the end of a procedure is showed in the Figure 5. Figure 5 The umbilical scare at the end of a procedure. The postoperative course was uneventful in all patients. The median postoperative in-hospital stay was 2 days for appendectomy and cholecistectomy and 6 days for right hemicolectomy. The characteristics of patients and the perioperative results are resumed in Table 1. Table 1 Patients and perioperative results. An analytical analysis of postoperative pain was not performed; however, no patient needed any opiates drugs and no discharged was conditioned by sorrow.

In right hemicolectomy, the resection margins were oncologically correct and the number of regional limphonodes was adequate: in the surgical specimen of the first patient, 17 limphonodes were found with 2 micrometastases; in the second patient, Brefeldin_A 14 limphonodes were found without any sign of disease. An adequate preoperative staging was performed: thoracic and abdominal CT with contrast enhancement and colonoscopy excluded, respectively distant metastases and other cancer colonic localization. An analysis of costs of this technique was made too.

Clostridium dakarense strain FF1T (= CSUR P243 = DSM 27086), is t

Clostridium dakarense strain FF1T (= CSUR P243 = DSM 27086), is the type strain of Clostridium dakarense sp. nov. This bacterium is a Gram-positive, anaerobic, spore-forming, indole negative bacillus that was isolated from the stool of a 4-month-old Senegalese child suffering from gastroenteritis as part of a ��culturomics�� www.selleckchem.com/products/lapatinib.html study aiming at cultivating individually all species within human feces. The elevated cost and lack of intra- and inter-laboratory reproducibility of the ��gold standard�� of taxonomic tools. i.e. DNA-DNA hybridization and G+C content determination [1], put bacterial taxonomic classification in a precarious state. In addition, the internationally-validated cutoff values of 16S rRNA sequence comparison [2] do not apply to all validly published genera and species.

Recently, high throughput genome sequencing and mass spectrometric analyses of bacteria have allowed unprecedented access to a wealth of genetic and proteomic information [3]. As a consequence, we proposed to use a polyphasic approach [4] to describe new bacterial taxa, including genome sequence, MALDI-TOF spectrum and main phenotypic characteristics [5-11]. The genus Clostridium (Prazmowski, 1880), classified among the Firmicutes, was created in 1880 [12] and consists of obligate anaerobic rod-shaped bacilli capable of producing endospores [12]. More than 180 Clostridium species have been described to date [13]. Members of the genus Clostridium are mostly environmental bacteria or associated with the commensal digestive flora of mammals, but several are major human pathogens, including C.

botulinum, C. difficile, C. tetani and C. perfringens [14,15]. A few species, such as C. butyricum and C. pasteurianum, fix nitrogen and have gained importance in agricultural and industrial applications [16,17]. Here we present a summary classification and a set of features for C. dakarense sp. nov. strain FF1T (= CSUR P243 = DSM 27086) together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species C. dakarense sp. nov. Classification and features A stool specimen was collected from a 4-month-old Senegalese child suffering from gastroenteritis. Informed consent was obtained from the child��s parents and approval from the ethics committee from the Institut Federatif de Recherche 48 (Facult�� de M��decine, Marseille, France).

The fecal specimen was preserved at -20��C after collection and sent to Marseille. Strain FF1T (Table 1) was isolated in July 2011 by anaerobic cultivation on 5% sheep blood-enriched Columbia agar (BioMerieux, Marcy GSK-3 l��Etoile, France). This strain exhibited a 96.90% 16S rRNA nucleotide sequence similarity with C. lituseburense, the phylogenetically closest validated Clostridium species (Figure 1). Although sequence similarity of the 16S rRNA is not uniform across taxa, this value was lower than the 98.

, Chicago, IL, USA) Statistically significant differences were e

, Chicago, IL, USA). Statistically significant differences were evaluated using the chi-square test, with significance set at P<.05. RESULTS The numbers and percentages of mandibular premolars directly and molars evaluated in this study population are shown in Table 2. Table 2 Number and percentage of mandibular premolars and molars in study population. Mandibular first premolars and second premolars In total, 790 teeth (99.9%) were detected to have a one-rooted mandibular first premolar. Only one tooth (0.1%) had a two-rooted mandibular first premolar. The overall occurrence of the roots between female and male participants and the occurrence on the left side and the right side did not display any significant difference (P>.05; Table 3 and and44).

Table 3 Classification of permanent mandibular premolars and molars by root number and topology (right and left side). Table 4 Classification of permanent mandibular premolars and molars by gender. In addition, 784 mandibular second premolars were one-rooted (99.4%), and five second premolars (0.6%) were two-rooted teeth. There were no significant differences between females and males regarding the overall occurrence of the roots (P>.05). Likewise, the occurrence on the left side and the right side did not show any statistically significant difference (P>.05; Tab. 3 and and44). Mandibular first molars The majority of first molars (77.4%) had one mesial and one distal root (Table 3 and and4).4). One hundred sixty-two mandibular first molars (22.3%) had DL roots, and two first molars (0.3%) were one-rooted teeth.

The bilateral incidence of three-rooted mandibular first molars was similar between male (n=28) and female subjects (n=25; P>.05; Table 5). Regardless of gender, the overall occurrence on the left side and the right side showed a statistically significant difference (P=.011). The right mandibular first molar (tooth n=90, 13.5%) had a higher incidence of being a three-rooted tooth when compared with the left side (tooth n=59, 8.9%). Table 5 Analysis of gender distribution in unilateral and bilateral cases of (1) mandibular first molars with two distal roots in patients having bilateral mandibular first molars, (2) mandibular second molars with two distal roots in patients having both mandibular … Mandibular second molars Most mandibular second molars (54.5%) were two-rooted teeth with one mesial and one distal root; 2.

3% of the second molars had three roots having one DL root, and one tooth (0.1%) had three roots with two mesial roots. In total, 293 teeth (41.3%) had Entinostat C-shaped roots (Table 3 and and44). The bilateral incidence of a three-rooted mandibular second molar having one DL root was similar between females (n=2) and males (n=2; Table 5). The right mandibular second molars (tooth n=6) had a similar incidence of being a three-rooted tooth when compared with the left side (tooth n=7).

5%) and 167 boys Complete parasitological data (i e , quadruplic

5%) and 167 boys. Complete parasitological data (i.e., quadruplicate Kato-Katz thick smears, duplicate POC-CCA cassette tests, and duplicate urine filtrations) at the baseline survey before treatment were available for 242 children, 133 from Azagui�� M’Brom�� (55.0%) and 109 from Azagui�� Makougui��. There were 127 girls (52.5%) and 115 boys with a mean age of 3.2 years (range: 2 months KPT-330 buy to 5.5 years). Figure 1 Flowchart showing study participation. Three weeks after the administration of praziquantel, only 86 out of the 242 children had complete parasitological data. There were 43 girls (50.0%) and 43 boys with a mean age of 3.6 years (range: 15 months to 5 years). The two population groups (children with complete data records before and after treatment) were similar in terms of average age, sex, arithmetic mean FECs of S.

mansoni, and co-infection status (all p>0.05). S. mansoni and Soil-Transmitted Helminth Infections before Treatment Table 2 shows the baseline prevalence and intensity of S. mansoni infection, as assessed by Kato-Katz and POC-CCA. Among the 242 children with complete data records, 56 (23.1%) were found positive for S. mansoni by quadruplicate Kato-Katz thick smears. Most infections were of light intensity (n=40; 71.4%), whereas 12 children (21.4%) had a moderate (100�C399 EPG) and four children (7.1%) had a heavy infection (��400 EPG). The group arithmetic mean FEC was 23.4 EPG (95% confidence interval (CI): 13.0�C33.7 EPG). A single CCA(t?) test identified 83 children (34.3%) harboring active schistosome infections.

Table 2 Baseline prevalence of helminths according to diagnostic approach (n=242). The youngest child infected with S. mansoni, as determined by the presence of S. mansoni eggs in stool using the Kato-Katz technique, was 8 months. According to the CCA(t?) test results, the earliest infection was observed in a child aged 3 months. According to quadruplicate Kato-Katz thick smears before treatment, among the 242 preschool-aged children with complete data records, 22 (9.1%), 15 (6.2%) and nine (3.7%) were positive for T. trichiura, hookworm and A. lumbricoides, respectively (Table 2). Hookworm and T. trichiura infections were exclusively of light intensity (<2,000 EPG and <1,000 EPG, respectively), whereas a third of the A. lumbricoides infections were of moderate intensity (5,000�C49,999 EPG).

Among the 40 children who were infected with S. mansoni according to a single CCA(t?) test, but showed no S. mansoni eggs in any of the four Kato-Katz thick smears, three (7.5%), two (5.0%), and two (5.0%) children were positive for T. trichiura, S. haematobium and A. lumbricoides, Drug_discovery respectively. None of these children were infected with hookworm. S. haematobium Infections before Treatment Among 242 children at the baseline survey, 26 were infected with S.

Treatment of Hep-G2 with Doxo alone induced apoptosis in 24% of c

Treatment of Hep-G2 with Doxo alone induced apoptosis in 24% of cells, whereas Doxo and TRAIL in combination led to apoptosis rates of 43% exactly (Figure (Figure3B,3B, lower panel). Figure 3 Treatment of HCC cells with TRAIL in combination with 5-FU and doxorubicin. Values are expressed as mean �� SD. A: Huh7 and Hep-G2 cells were analyzed for cell viability after treatment with the chemotherapeutic agents 5-FU and doxorubicin alone. … Treatment of HCC cells with TRAIL in combination with specific kinase inhibitors Antiapoptotic pathways such as PI3K/Akt, epidermal growth factor receptor (EGFR), [mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) kinase] (MEK)/ERK are well known to be activated in malignant cells, thus contributing to cell cycle progression and tumor growth.

Therefore, we analyzed whether inhibition of kinases involved in these pathways could overcome resistance towards TRAIL-mediated apoptosis. Firstly, we applied the multi-kinase inhibitor Sorafenib to inhibit RAF/MEK/ERK signaling, in escalating concentrations (2.5, 5 and 10 ��mol/L). A dose-dependent decrease of cell viability in Huh7 and Hep-G2 was observed (Figure (Figure4A,4A, upper panel). In a second step, we analyzed the impact of Sorafenib on TRAIL treatment. In Huh7 cells, Sorafenib (10 ��mol/L) induced apoptosis rates of 50%. Strikingly, the combination of SkTRAIL (50 ng/mL) and Sorafenib (10 ��mol/L) induced apoptosis in 80% of the cells. In Hep-G2 cells Sorafenib caused only minor apoptosis rates (33%). However, combination of TRAIL and Sorafenib led to 98% apoptotic cells (Figure (Figure4A,4A, lower panel).

Figure 4 Treatment of HCC cells with TRAIL in combination with specific kinase inhibitors. Viability of HCC cells treated with kinase inhibitors alone (upper panels). On day one after seeding of Huh7 and Hep-G2 cells onto 96-well plates, cells were treated with … Next, we inhibited the PI3K/Akt pathway by application of the PI3K inhibitor LY294002. A slight decrease of cell viability was observed in Huh7 and Hep-G2 after 48 h treatment in concentrations lower than 50 ��mol/L (Figure (Figure4B,4B, upper panel). However, combination of LY294002 (10 ��mol/L) and SkTRAIL (50 ng/mL) doubled apoptosis rates in Hep-G2 cells to 59% compared to SkTRAIL treatment alone.

In Huh7, we observed an increased rate of apoptosis after treatment with the combination of LY294002 and SkTRAIL compared to SkTRAIL alone (23% vs 11%, respectively. Figure Figure4B,4B, lower panel). Furthermore, we used AG1478 to inhibit EGFR kinase. Interestingly, inhibition of EGFR Dacomitinib kinase increased cell viability in Hep-G2 cells in concentrations up to 5 ��mol/L. In Huh7 cells, AG1478 caused no significant changes in cell viability when applied in low concentrations (Figure (Figure4C,4C, upper panel).

11��-HSD1 primary antibodies were as described above Alexa Fluor

11��-HSD1 primary antibodies were as described above. Alexa Fluor 488 donkey anti sheep IgG and Alexa Flour 546 rabbit anti mouse IgG were used at a dilution of 1100 and slides were covered in foil for the remainder of the procedure. Slides were mounted in VectaShield www.selleckchem.com/products/Perifosine.html hard set mounting medium with DAPI (Vector Labs). Preparation of Liver Microsomes Human liver microsomes were prepared from 4 human normal livers and 5 livers with NASH livers by differential centrifugation techniques as described previously [21]. Microsomal fractions were resuspended in a buffer containing 20 mm NaCl, 1 mm MgCl2, 100 mm KCl, 20 mm Mops, pH 7.2, and were snap-frozen under liquid nitrogen. Microsomal protein concentration was determined using the Bio-Rad protein assay with bovine serum albumin as a standard as per the manufacturer’s instructions (Bio-Rad).

The integrity of the microsomal membranes was assessed by using the mannose-6-phosphatase assay [22], which showed a latency greater than 95% in all preparations. Immunoblotting SDS-PAGE was performed by the method of Laemmli [23] with 10 ��g of liver microsomal protein on 11% acrylamide minigels using a Bio-Rad Mini-PROTEAN II apparatus (Bio-Rad). Following electrophoresis, proteins were transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA). Nonspecific protein binding was blocked by incubating membranes in 20% nonfat milk, 0.1% Tween 20 in phosphate-buffered saline at 25��C for 1 h. Membranes were then incubated with an in-house raised polyclonal antibody to human 11��-HSD1 at a dilution of 11000 for 12 h at 4��C.

Following 3��10-min washes in phosphate-buffered saline, 0.1% Tween 20, membranes were incubated with secondary antibody (goat anti-sheep IgG peroxidase-conjugate) at a dilution of 125,000 for 1 h at room temperature. Bound peroxidase-conjugated IgG was visualized using ECL detection kit (Amersham Biosciences, Buckinghamshire, UK) by exposing membranes to x-ray film (Kodak, France). Membranes were reprobed with anti-beta Actin antibody [mAbcam 8226] (HRP) as a loading Control at 120,000. Statistical Analysis Data are presented as means �� SE unless otherwise stated. Area under the curve (AUC) analysis was performed using the trapezoidal method. For comparison of single variables between control, steatosis and steatohepatitis groups, one way analysis of variance (ANOVA) was used to identify variables with differences between groups and t tests were used (Mann Whitney test was used where data were not normally distributed).

Analysis was performed using SPSS Statistics 17.0 software. Results Clinical and biochemical characteristics of participants We characterised the metabolic phenotype and hepatic cortisol metabolism in patients Carfilzomib with histologically proven NAFLD compared to healthy obese control. Compared with the control group, waisthip ratios and sagittal height were significantly higher in the NASH group.

We analysed the possible involvement of AP-2 in the increased ERB

We analysed the possible involvement of AP-2 in the increased ERBB2 expression, because of the well-characterised role of http://www.selleckchem.com/products/ganetespib-sta-9090.html this transcription factor in breast cancer cells. Indeed, the 500bp proximal ERBB2 promoter contains two AP-2 binding sites, which contribute to the gene overexpression. The first site is located 215bp upstream from the transcription initiation site (Bosher et al, 1995), whereas the second lies 501bp upstream from the transcription start site (Grooteclaes et al, 1999; Vernimmen et al, 2003). Nuclear AP-2�� levels and AP-2 DNA binding activities were considerably lower in prostate, colon, ovary and pancreatic cell lines than in breast cancer cells. Since AP-2 could be cytoplasmic rather than nuclear (see below), we also analysed whole-cell extracts by Western blotting.

We did not observe any difference between nuclear and total AP-2 levels, indicating that the low levels of the nuclear factor is not the consequence of its cytoplasmic retention (data not shown). Besides the full-length 50kDa protein, we detected lower and higher molecular weight proteins as well. The lower molecular weight bands might result from the proteolysis of the transcription factor. Higher molecular weight immunoreactive forms were observed mainly in the pancreatic cells (Figure 2A) and might represent the recently described, 60kDa sumolated form of the protein. The transcriptional activity of these sumolated factors is reduced (Eloranta and Hurst, 2002). AP-2�� levels were not correlated with ERBB2 expression in non-breast cancer cell lines.

This indicates that AP-2 is not involved in ERBB2 gene overexpression. In other cancers, the role of AP-2 has been shown to vary according to the tumour type. Some authors described the loss of AP-2�� expression early in the development of prostate adenocarcinoma (Ruiz et al, 2001). Moreover, AP-2B, a dominant-negative variant of AP-2��, was detected by RT�CPCR in LNCaP cells (Buettner et al, 1993). Others did detect AP-2�� by immunohistochemistry in some prostate cancer specimens (Lipponen et al, 2000). In primary colorectal (Ropponen et al, 2001) and ovarian cancers (Anttila et al, 2000), AP-2�� was detected in the nucleus or the cytoplasm of tumour cells. However, the presence of the transcription factor in the cytoplasm precludes its function in transcription.

Interestingly, high AP-2�� mRNA levels were detected in some tumours which were negative for the protein (Karjalainen et al, 2000). To analyse the molecular mechanisms leading to ERBB2 overexpression in non-breast cancer cell lines, we transfected four GSK-3 LUCIFERASE reporter vectors containing 200bp�C6kb fragments of the ERBB2 promoter in colon and ovary cell lines. The relative transcriptional activity of each reporter was compared to the luciferase activity induced by the p255-LUC vector, considered as equal to one (Grooteclaes et al, 1994).