Taxonomic study of Scytosiphon (Phaeophyceae) from temperate coasts of Argentina.

Scytosiphon is a common intertidal genus widely distributed on temperate coasts worldwide. Recently, eight species have been delimited with molecular tools. Although S. lomentaria is the only species that predominates in the macroalgal literature of the Southwestern Atlantic Ocean (SwAO), unpublished molecular data obtained for a population study of S. lomentaria revealed hidden species diversity of Scytosiphon among the individuals collected from four localities at the SwAO. The aim of this study was to revise the identity and phylogenetic relationships of Scytosiphon from temperate coasts of the SwAO using DNA data. Thalli were collected from the Argentinean coast between 39° S and 43° S, from which cox1 and rbcL gene sequences were obtained. Phylogenies and haplotype networks were inferred and morphology of gametophytes was studied. Four species were recognized, S. lomentaria, S. promiscuus, S. shibazakiorum, and one species that belongs to a complex of species known as "Scytosiphon Atlantic complex." This complex was known to occur only in the North Atlantic, however, the results found in this study revealed that it has an extended distribution range that includes the southern hemisphere, where its populations have high genetic diversity and unique haplotypes. The morphological differences among the four species were subtle; denoting that previous Scytosiphon records from the SwAO attributed to the renowned S. lomentaria could represent different species. In addition, sex ratio and genome-wide single nucleotide polymorphisms (SNPs) analyses were done for populations of S. promiscuus presumably introduced to the SwAO, and the results indicated that they included female-dominant parthenogenetic populations, which were probably introduced from Japan.

An Introduction to the Relationship Between Lewis x and Malignancy Mainly Related to Breast Cancer and Head Neck Squamous Cell Carcinoma (HNSCC).

Lewis x functions as an adhesion molecule in glycolipids and glycoproteins since it mediates homophilic and heterophilic attachment of normal and tumoral cells. During malignancy, altered glycosylation is a frequent event; accumulating data support the expression of Lewis x in tumors although controversial results have been described including its relationship with patient survival. This report has been developed as an introduction to the relationship between Lewis x expression and breast cancer and head and neck squamous cell carcinoma (HNSCC). Results obtained in our laboratory are presented in the context of the literature.

Geographical parthenogenesis in the brown alga Scytosiphon lomentaria (Scytosiphonaceae): Sexuals in warm waters and parthenogens in cold waters.

Geographical parthenogenesis, a phenomenon where parthenogens and their close sexual relatives inhabit distinct geographical areas, has been considered an interesting topic in evolutionary biology. Reports of geographical parthenogenesis from land and freshwater are numerous, but this occurrence has been rarely reported from the sea. Brown algae are mostly marine and are thought to include numerous obligate parthenogens; still, little is known about the distribution, origin and evolution of parthenogens in this group. Here we report a novel pattern of geographical parthenogenesis in the isogamous brown alga Scytosiphon lomentaria. Sex ratio investigation demonstrated that, in Japan, sexual populations grew in the coast along warm ocean currents, whereas female-dominant parthenogenetic populations grew mainly in the coast along a cold ocean current. In the two localities where sexual and parthenogenetic populations were parapatric, parthenogens grew in more wave-exposed areas than sexuals. Population genetic and phylogenetic analyses, including those based on genome-wide single nucleotide polymorphism data, indicated that parthenogens have initially evolved at least twice and subsequent hybridizations between the parthenogens and sexuals have generated multiple new parthenogenetic lineages. The origin of the initial parthenogens is not clear, except that it would not be interspecies hybridization. Interestingly, we found that the production of sex pheromones, which attract male gametes, has been independently lost in the initial two parthenogenetic lineages. This parallel loss of the sexual trait may represent the direct origin of parthenogens, or the regressive evolution of a useless trait under asexuality.

Meridionella gen. nov., a New Genus of Cystocloniaceae (Gigartinales, Rhodophyta) from the Southern Hemisphere, Including M. obtusangula comb. nov. and M. antarctica sp. nov.

The classification of Cystoclonium obtusangulum has been questioned since the species was first described by Hooker and Harvey as Gracilaria? obtusangula. The objective of this study was to provide the first comprehensive taxonomic analysis of Cystoclonium obtusangulum, based on DNA sequences coupled with morphological observations made on syntype specimens and new collections. Sequence divergences of rbcL, UPA, and COI-5P, and maximum-likelihood phylogenies for rbcL and 18S demonstrated that specimens identified as Cystoclonium obtusangulum represent a clade of two distinct species that are distantly related to the generitype Cystoclonium purpureum. A new genus, Meridionella gen. nov., is proposed for this clade. The two species placed in this new genus were morphologically indistinguishable cryptic species, but have disjunct distributions, with Meridionella obtusangula comb. nov. found from temperate to cold coasts of South America and the Falkland Islands and Meridionella antarctica sp. nov., occurring in Antarctic waters. Vegetative and reproductive characters of Meridionella gen. nov. are described, and implications of our results for the biogeography of the family Cystocloniaceae are discussed.

Unravelling genomic diversity of Zygosaccharomyces rouxii complex with a link to its life cycle.

Zygosaccharomyces rouxii and the related species Zygosaccharomyces sapae (hereafter referred to as Z. rouxii complex) are protoploid hemiascomycete yeasts relevant in the elaboration and spoilage of foodstuff. Divergence of Z. rouxii complex before whole genome duplication, leading to the genus Saccharomyces, makes these yeasts very attractive for genome evolution study. Relatively little is known, however, about the diversity in this branch at the genetic and physiological levels. In this work, we investigated Z. rouxii complex, encompassing strains that in other works have been studied separately and comparing them in a comprehensive way. We showed that the majority of strains are unusually heterogeneous in their ribosomal DNA, a signal of relaxation of concerted evolution. Further analysis showed that they have hypervariable karyotypes, different levels of ploidy, and that housekeeping markers vary both in copy number and sequence. Overall, the results provide compelling evidence that the strains considered in this study are a complex of haploid, aneuploid and diploid mosaic lineages. The reproductive mode and life cycle of Zygosaccharomyces could lead to this unsuspected diversity.

Expression of conserved mucin domains by epithelial tissues in various mammalian species.

UNLABELLED: Mucins are related to infectious and non-infectious diseases in Veterinary and Human Medicine. MUC1 mucin is a transmembrane glycoprotein expressed on the apical surface of human epithelia while MUC5AC is the predominant secreted mucin expressed in human gastric epithelium and goblet cells of lung and eyes. MUC5AC C-terminus cysteine rich regions and the cytoplasmic tail of MUC1 domains are conserved among several mammalian species. OBJECTIVE: to compare the expression of MUC1 and MUC5AC mucins in mammalian epithelia. CT33 anti-MUC1 cytoplasmic tail (MUC1CT) polyclonal antibody and 45M1 anti-MUC5AC monoclonal antibody were employed. By immunohistochemistry, MUC1CT was expressed in most tissues while MUC5AC was restricted to gastric surface epithelium and goblet cells from trachea and lung. By western blot, MUC1CT showed a band at approximately 35 kDa in most tissues; MUC5AC revealed bands at >180 kDa in stomach and lung secretions from rat, cat, pig and cow. When rat MUC5AC was immunoprecipitated, a band at about 180 kDa was obtained.

Genome size and ploidy level: new insights for elucidating relationships in Zygosaccharomyces species.

Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7+/-0.2 Mb for strain CBS 732T, 21.9+/-0.2 Mb for ATCC 42981, 28.1+/-1.3 Mb for ABT 301, and 39.00+/-0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes.

Lewis x Antigen is Associated to Head and Neck Squamous Cell Carcinoma Survival.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with poor prognosis without appropriate prognostic markers. Previous research shows that Lewis antigens have been involved in carcinoma dissemination and patients´ survival. Fucosyl and sialyltransferases are the enzymes implicated in the Lewis antigens synthesis. The purpose of this study was to evaluate the prognostic utility of Lewis antigens in HNSCC. We conducted a prospective research including histological samples from 79 patients with primary HNSCC. Lewis x and sialyl Lewis x expression were detected by immunohistochemistry; patient's data, progression free, and overall survival were documented. A statistical correlation study of antigenic expression and patients´ histopathological variables was performed. Cox regression models with internal validation procedures were employed to analyze survival data. By immunohistochemistry, Lewis x was detected in 34/79 (43%) tumor samples, while sialyl Lewis x only in 11/79 (14%). Lewis x expression showed a positive correlation with tumor differentiation and a better overall survival for Lewis x + patients was detected. Moreover, multivariate Cox's regression analysis showed that Lewis x is an independent predictor of better overall survival. The in silico analysis supported the presence of deregulated fucosyl (FUT4) and sialyltransferase (ST3GAL4) in the Lewis synthetic pathway related to patient survival. These results suggest that Lewis x expression is associated with a better outcome in patients with HNSCC.

Alternative splicing variant of RHBDD2 is associated with cell stress response and breast cancer progression.

RHBDD2 is an intramembrane pseudoprotease member of the Rhomboid superfamily. Our previous studies in breast and colorectal cancer indicate an association between RHBDD2 overexpression and advanced tumor stages. Two alternative transcriptional variants have been described for RHBDD2, which would be encoding for different RHBDD2 protein isoforms. The expression of these RHBDD2 variants/isoforms and its association with breast cancer was the focus of this study. First, expression of RHBDD2 splicing variants was evaluated in normal and breast tumor samples. RHBDD2 variant 2 overexpression was detected in tumors in respect to normal breast tissues at the mRNA and protein levels (P<0.05). Moreover, RHBDD2 variant 2 expression was associated with poor prognostic factors such as basal­like intrinsic subtype (P<0.05), high proliferation (P<0.01) and long­term risk­of­recurrence (P<0.01) scores. Second, the expression of both variants was evaluated under nutritional­deprived conditions in breast cancer cell lines. Results demonstrated that RHBDD2 splicing was switched from mRNA variant 1 to variant 2 in association with a significant increment of protein isoform B in response to glucose starvation treatment. Therefore, we propose that the switch from the RHBDD2 variant 1, expressed in normal epithelial cells, to variant 2 occurs as an adaptive phenotype to bypass the stressful tumor microenvironment and promote tumor progression. Finally, the RHBDD2 subcellular localization was corroborated at the Golgi apparatus and their associated v­SNARE transport vesicles, suggesting a putative new role for RHBDD2 in the protein trafficking of human breast cancer cells.

Lewis x is highly expressed in normal tissues: a comparative immunohistochemical study and literature revision.

An immunohistochemical analysis was employed to determine the expression of carbohydrate antigens associated to mucins in normal epithelia. Tissue samples were obtained as biopsies from normal breast (18), colon (35) and oral cavity mucosa (8). The following carbohydrate epitopes were studied: sialyl-Lewis x, Lewis x, Lewis y, Tn hapten, sialyl-Tn and Thomsen-Friedenreich antigen. Mucins were also studied employing antibodies against MUC1, MUC2, MUC4, MUC5AC, MUC6 and also normal colonic glycolipid. Statistical analysis was performed and Kendall correlations were obtained. Lewis x showed an apical pattern mainly at plasma membrane, although cytoplasmic staining was also found in most samples. TF, Tn and sTn haptens were detected in few specimens, while sLewis x was found in oral mucosa and breast tissue. Also, normal breast expressed MUC1 at a high percentage, whereas MUC4 was observed in a small number of samples. Colon specimens mainly expressed MUC2 and MUC1, while most oral mucosa samples expressed MUC4 and MUC1. A positive correlation between MUC1VNTR and TF epitope (r=0.396) was found in breast samples, while in colon specimens MUC2 and colonic glycolipid versus Lewis x were statistically significantly correlated (r=0.28 and r=0.29, respectively). As a conclusion, a defined carbohydrate epitope expression is not exclusive of normal tissue or a determined localization, and it is possible to assume that different glycoproteins and glycolipids may be carriers of carbohydrate antigens depending on the tissue localization considered.

Surface engineering of Solid Lipid Nanoparticle assemblies by methyl α-d-mannopyranoside for the active targeting to macrophages in anti-tuberculosis inhalation therapy.

This study describes the development of new mannosylated Solid Lipid Nanoparticle assemblies (SLNas) delivering rifampicin for an inhaled treatment of tuberculosis. SLNas were surface engineered with mannose residues to recognize mannose receptors located on infected alveolar macrophages and facilitate cell internalization. Two sets of SLNas were produced by the melt emulsifying technique using biocompatible lipid components, i.e. cholesteryl myristate combined with palmitic acid (PA set) or tripalmitin (TP set), in the presence of the targeting moiety, methyl α-d-mannopyranoside. Mannosylated SLNas were examined for their physical properties, drug payloads and release, as well as respirability in terms of emitted dose and respirable fraction determined by Next Generation Impactor. The most appropriate formulations were assessed for mannosylation using FTIR, XPS, SEM coupled with EDX analysis, and wettability assay, in comparison with the respective non-functionalized SLNas. Besides, cytotoxicity and cell internalization ability were established on J774 murine macrophage cell line. Mannosylated SLNas exhibited physical properties suitable for alveolar macrophage passive targeting, adequate rifampicin payloads (10-15%), and feasible drug maintenance within SLNas along the respiratory tract before macrophage internalization. Despite respirability impaired by powder cohesiveness, surface mannosylation provided quicker macrophage phagocytosis, giving evidence of an active targeting promotion.

GATA3 protein as a MUC1 transcriptional regulator in breast cancer cells.

INTRODUCTION: Recent studies have demonstrated that members of the GATA-binding protein (GATA) family (GATA4 and GATA5) might have pivotal roles in the transcriptional upregulation of mucin genes (MUC2, MUC3 and MUC4) in gastrointestinal epithelium. The zinc-finger GATA3 transcription factor has been reported to be involved in the growth control and differentiation of breast epithelial cells. In SAGE (serial analysis of gene expression) studies we observed an intriguing significant correlation between GATA3 and MUC1 mRNA expression in breast carcinomas. We therefore designed the present study to elucidate whether MUC1 expression is regulated by GATA3 in breast cancer cells. METHODS: Promoter sequence analysis of the MUC1 gene identified six GATA cis consensus elements in the 5' flanking region (GATA1, GATA3 and four GATA-like sequences). Chromatin immunoprecipitation and electrophoretic mobility-shift assays were employed to study the presence of a functional GATA3-binding site. GATA3 and MUC1 expression was analyzed in vitro with a GATA3 knockdown assay. Furthermore, expression of GATA3 and MUC1 genes was analyzed by real-time RT-PCR and immunohistochemistry on breast cancer-specific tissue microarrays. RESULTS: We confirmed the presence of a functional GATA3-binding site on the MUC1 promoter region in the MCF7 cell line. We determined that GATA3 knockdown assays led to a decrease in MUC1 protein expression in MCF7 and T47D cells. In addition, we detected a statistically significant correlation in expression between GATA3 and MUC1 genes at the mRNA and protein levels both in normal breast epithelium and in breast carcinomas (p = 0.01). GATA3 expression was also highly associated with estrogen receptor and progesterone receptor status (p = 0.0001) and tumor grade (p = 0.004) in breast carcinomas. CONCLUSION: Our study provides evidence indicating that GATA3 is probably a mediator for the transcriptional upregulation of MUC1 expression in some breast cancers.

MUC1 expression and anti-MUC1 serum immune response in head and neck squamous cell carcinoma (HNSCC): a multivariate analysis.

BACKGROUND: HNSCC progression to adjacent tissue and nodes may be mediated by altered glycoproteins and glycolipids such as MUC1 mucin. This report constitutes a detailed statistical study about MUC1 expression and anti-MUC1 immune responses in relation to different clinical and pathological parameters which may be useful to develop new anti HNSCC therapeutic strategies. PATIENTS AND METHODS: Fifty three pre treatment HNSCC patients were included: 26 (49.1%) bearing oral cavity tumors, 17 (32.1%) localized in the larynx and 10 (18.8%) in the pharynx. Three patients (5.7%) were at stage I, 5 (9.4%) stage II, 15 (28.3%) stage III and 30 (56.6%) at stage IV. MUC1 tumor expression was studied by immunohistochemistry employing two anti-MUC1 antibodies: CT33, anti cytoplasmic tail MUC1 polyclonal antibody (Ab) and C595 anti-peptidic core MUC1 monoclonal antibody. Serum levels of MUC1 and free anti-MUC1 antibodies were detected by ELISA and circulating immune complexes (CIC) by precipitation in polyethylene glycol (PEG) 3.5%; MUC1 isolation from circulating immune complexes was performed by protein A-sepharose CL-4B affinity chromatography followed by SDS-PAGE and Western blot. Statistical analysis consisted in Multivariate Principal Component Analysis (PCA); ANOVA test (Tukey's test) was employed to find differences among groups; nonparametrical correlations (Kendall's Tau) were applied when necessary. Statistical significance was set to p < 0.05 in all cases. RESULTS: MUC1 cytoplasmic tail was detected in 40/50 (80%) and MUC1 protein core in 9/50 (18%) samples while serum MUC1 levels were elevated in 8/53 (15%) patients. A significant statistical correlation was found between MUC1 serum levels and anti-MUC1 IgG free antibodies, while a negative correlation between MUC1 serum levels and anti-MUC1 IgM free antibodies was found. Circulating immune complexes were elevated in 16/53 (30%) samples and were also statistically associated with advanced tumor stage. MUC1 was identified as an antigenic component of IgG circulating immune complexes. Moreover, poorly differentiated tumors were inversely correlated with tumor and serum MUC1 detection and positively correlated with node involvement and tumor mass. CONCLUSION: Possibly, tumor cells produce MUC1 mucin which is liberated to the circulation and captured by IgG antibodies forming MUC1-IgG-CIC. Another interesting conclusion is that poorly differentiated tumors are inversely correlated with tumor and serum MUC1 detection.

From Molecular Biology to Clinical Trials: Toward Personalized Colorectal Cancer Therapy.

During the past years, molecular studies through high-throughput technologies have led to the confirmation of critical alterations in colorectal cancer (CRC) and the discovery of some new ones, including mutations, DNA methylations, and structural chromosomal changes. These genomic alterations might act in concert to dysregulate specific signaling pathways that normally exert their functions on critical cell phenotypes, including the regulation of cellular metabolism, proliferation, differentiation, and survival. Targeted therapy against key components of altered signaling pathways has allowed an improvement in CRC treatment. However, a significant percentage of patients with CRC and metastatic CRC will not benefit from these targeted therapies and will be restricted to systemic chemotherapy. Mechanisms of resistance have been associated with specific gene alterations. To fully understand the nature and significance of the genetic and epigenetic defects in CRC that might favor a tumor evading a given therapy, much work remains. Therefore, a dynamic link between basic molecular research and preclinical studies, which ultimately constitute the prelude to standardized therapies, is very important to provide better and more effective treatments against CRC. We present an updated revision of the main molecular features of CRC and their associated therapies currently under study in clinical trials. Moreover, we performed an unsupervised classification of CRC clinical trials with the aim of obtaining an overview of the future perspectives of preclinical studies.

Usefulness of triphasic CT aortic angiography in acute and surveillance: Our experience in the assessment of acute aortic dissection and endoleak.

INTRODUCTION: Computed tomography angiography (CTA) has been widely used in the diagnostic evaluation of many aortic diseases, but no standardized techniques actually exist for aortic CTA. The aim of this study was to describe the usefulness of triphasic CTA in aortic assessment in both non-traumatic emergency and surveillance conditions. METHODS: We performed non ECG-gated CTA examinations with a 64-slice CT scanner using a triphasic protocol consisting of an unenhanced acquisition, and two (early and delayed) contrastographic phases with a delay of 25-30 s and 100-120 s respectively after the injection of contrast medium. Were retrospectively selected adult patients with imaging findings of acute aortic dissection (AAD) or endoleak (EL) from November 2012 to November 2014. RESULTS: AAD was detected in 36 (67%) patients: 23 type A-AADs, and 13 type B-AADs. The presence of EL was observed in 18 (33%) patients: 1 type Ia, 5 types IIa, 2 types IIb, 1 type IIIa and 9 types IIIb. DISCUSSION: Triphasic CTA is useful to provide correct and prompt diagnosis of AAD in emergency, allowing the evaluation of type and atypical forms of AAD, and the identification of possible branch-vessel involvement and complications. During surveillance, triphasic CTA assures accurate and complete assessment of all known and unknown ELs and it is essential for first follow-up examination. CONCLUSION: Triphasic CTA represents a reliable imaging tool for aortic assessment in both non-traumatic emergency and surveillance after endovascular aneurysm repair. Modified protocol could be employed in selected patients and tailored in their known disease.

Influence of sialic acid removal on MUC1 antigenic reactivity in head and neck carcinoma.

To investigate the influence of sialic acid removal on MUC1 peptidic and carbohydrate epitope reactivity in head and neck squamous cell carcinoma (HNSCC), tumor samples belonging to 24 HNSCC patients were studied by standard immunohistochemistry (IHC) with and without desialylation with 0.1 U/ml neuraminidase. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and Western blotting (WB). Three monoclonal antibodies (MAbs) were used: C595 MAb directed to MUC1 protein core, an anti-Tn hapten MAb, and an anti-sTn hapten MAb; a comparative analysis between desialylated and sialylated samples was performed. By IHC without neuraminidase treatment, 19 of 24 samples reacted with anti-MUC1 peptidic epitope, while Tn hapten was not detected and sTn was found in 1 of 24 cases. Desialylation increased either the number of reacting cells or the intensity of the reaction with C595 and anti-Tn MAbs, and some negative samples became positive. On the other hand, sTn expression decreased with desialylation. By WB, several bands from >200 to 25 kDa were found; desialylation increased high-molecular-weight bands, diminishing the detection of low-molecular-weight ones. The use of desialylation is a suitable treatment that contributes to the exposure of MUC1-associated epitopes, which may be related to the spreading of HNSCC.

Nuclear localization of MUC1 extracellular domain in breast, head and neck, and colon cancer.

BACKGROUND: The glycoprotein MUC1 is overexpressed and underglycosylated in cancer cells. MUC1 is translated as a single polypeptide that undergoes autocleavage into 2 subunits (the extracellular domain and the cytoplasmic tail), and forms a stable heterodimer at the apical membrane of normal epithelial cells. The MUC1 cytoplasmic tail localizes to the cytoplasm of transformed cells and is targeted to the nucleus. AIMS: To study the expression of the MUC1 extracellular subunit in cell nuclei of neoplastic breast, head and neck, and colon samples. MATERIALS AND METHODS: 330 primary tumor samples were analyzed: 166 invasive breast carcinomas, 127 head and neck tumors, and 47 colon tumors; 10 benign breast disease (BBD) and 40 normal specimens were also included. A standard immunohistochemical method with antigen retrieval was performed. Nuclear fractions from tissue homogenates and breast cancer cell lines (ZR-75, MDA-MB-231, MCF7, and T47D) were obtained and analyzed by Western blotting (WB). The anti-MUC1 extracellular subunit monoclonal antibody HMFG1 was used for immunohistochemistry. RESULTS: 37/166 breast cancer specimens, 5/127 head and neck cancer specimens, 2/47 colon cancer samples, and 3/10 BBD samples showed immunohistochemical staining at the nuclear level. No nuclear reaction was detected in normal samples. By WB, breast and colon cancer purified nuclear fractions showed reactivity at 200 kDa in 3/30 breast and 3/20 colon cancer samples as well as purified nuclear fractions obtained from breast cancer cell lines. CONCLUSIONS: This study shows that the MUC1 extracellular domain might be translocated to the cell nucleus in breast, head and neck, and colon cancer as well as BBD.

Functional analysis of the promoter of human sterol 27-hydroxylase gene in HepG2 cells.

Human sterol 27-hydroxylase catalyses the first step in the alternative pathway of bile acids biosynthesis in hepatocytes. However the gene encoding this enzyme (CYP27 gene) is expressed in every tissue and some evidence suggests that this enzyme plays a role in cholesterol homeostasis. Although modulation of CYP27 expression has been reported, the mechanisms underlying the regulation of this gene in human tissues is still poorly understood. To elucidate the mechanism governing CYP27 expression we cloned a 4.3 kb fragment of the 5' flanking region of the human CYP27 gene and constructed deletion mutants which were transfected into HepG2 cells. Functional assays showed that the -217/-10 nucleotide region from the translation start site (minimal promoter), devoid of TATA and CAAT boxes, contains all the elements for basal transcription. Foot-printing analysis of minimal promoter showed four protected regions (A-D). Regions A, B and D each contain one Sp1 binding site, and region C contains a HNF4 site. Electrophoretic mobility shift assays demonstrated that Sp1, Sp3 and HNF4 transcription factors bind these sites. Mutagenesis of any of these sites resulted in the loss of promoter activity. Co-transfection of the minimal promoter with Sp1 and Sp3 expression vectors transactivated CYP27 gene promoter in Drosophila SL2 cells, which lack endogenous Sp proteins. Transactivation of the minimal promoter was also observed in HeLa cells co-transfected with HNF4 expression vector. Therefore, Sp1, Sp3 and HNF4 co-operate in the expression of the human CYP27 gene in HepG2 cells.