Serine palmitoyltransferase (SPT, EC 2.3.1 S O ) is the key enzyme in sphingolipid biosynthesis. It catalyzes the pyridoxal-5'-phosphate-dependent condensation of L-serine and palmitoyl-CoA to 3-0x0-sphinganine. Human expressed-sequence-tag (EST) clones are similar to the two yeast genes for synthesis of long-chain bases, LCBl and LCB2, which are believed to encode two subunits of SPT [Buede, R., Pinto, W. J., Lester Human embryonic kidney cells (HEK 293) transfected with murine sequences of LCBl (mLCB1) and LCB2 (mLCB2) independently and in coexpression showed an overexpression of the transcripts on the mRNA and protein level. The enzymatic activity of cells expressing mLCB2 alone or coexpressed with mLCBl was three times higher than the activity of untransfected HEK cells. mLCBl expression was not required for the synthesis of 3-0x0-sphinganine in mammalian cells. Transcription/translation in vitro yielded mLCBl (53 kDa) and mLCB2 (63 kDa). The two proteins do not contain a signal peptide nor are they glycosylated. The endogenous and overexpressed SPT activity were both sensitive to common SPT inhibitors. Labeling studies with [1-14C]palmitic acid indicated that cell lines transfected with mLCB2 preferentially use the excess sphingoid bases for glucocerebroside and galactocerebroside synthesis. Our results provide conclusive genetic and biochemical evidence that the human and murine LCB2 genes described here encode serine palmitoyltransferase. Further studies will be required to unravel the function of the LCBl gene in mammalian cells.Keywords; serine palmitoyltransferase ; sphingolipid biosynthesis ; cloning; characterization ; overexpression.Sphingolipids are integral constituents of plasma and lysosoma1 membranes and are transiently present in the membranes of the endoplasmic reticulum. Their function as structural elements of plasma membranes remains to be elucidated. The long- Abbreviations. LCB, long-chain base; LCBl and LCB2, yeast genes for LCB synthesis; SPT, serine palmitoyltransferase; hLCB1, mLCB 1 : human, murine LCBl; hLCB2, mLCB2: human, murine LCBZ; HEK, human embryonic kidney; EST, expressed sequence tag; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle medium; RACE, rapid analysis of cDNA ends; CMV, cytomegalovirus ; GST, glutathione S-transferase.Enzymes. Serine palmitoyltransferse (EC 2.3.1.50); 5-aminolevulinate synthetase (EC 2.3.1.37), 2-amino-3-oxobutyrate:CoA-ligase (EC 2.3.1.29); 8-amino-7-oxononoate synthase (EC 2.3.1.47); mannosyl-glycoprotein endo-P-N-acetylglucosaminidase (EC 3.2. 1 .96).Nomenclature. Sphinganine, (2S,3R)-2-amino-l,3-dihydroxy-octadecane (also termed dihydrosphingosine); sphingenine, (2S,3R,4E)-2-amino-l,3-dihydroxy-octadecene (also termed sphingosine) ; phytosphinganine, (2S,3S,4R)-2-amino-l,3,4-trihydroxy-octadecane.Nore. The novel nucleotide sequence data reported in this article has been submitted to the EMBL DNA database and is available under accession numbers X95641 for mLCB1, X95642 for mLCB2, YO8685 for hLCBl and YO8686 for hLCB2.chain bases (LC...

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Angiogenesis is a complex process that can be regarded as a series of sequential events comprising a variety of tissue cells. The major problem when studying angiogenesis in vitro is the lack of a model system mimicking the various aspects of the process in vivo. In this study we have used two in vitro models, each representing different and distinct aspects of angiogenesis. Differentially expressed genes in the two culture forms were identified using the suppression subtractive hybridization technique to prepare subtracted cDNA libraries. This was followed by a differential hybridization screen to pick up overexpressed clones. Using comparative multiplex RT-PCR we confirmed the differential expression and showed differences up to 14-fold. We identified a broad range of genes already known to play an important role during angiogenesis like Flt1 or TIE2. Furthermore several known genes are put into the context of endothelial cell differentiation, which up to now have not been described as being relevant to angiogenesis, like NrCAM, Claudin14, BMP-6, PEA-15 and PINCH. With ADAMTS4 and hADAMTS1/METH-1 we further extended the set of matrix metalloproteases expressed and regulated by endothelial cells.Keywords: differential gene expression; MVEC; angiogenesis; suppression subtractive hybridization.Angiogenesis, the formation of new capillaries from preexisting blood vessels, plays a crucial role in a wide range of normal and pathological processes, and is necessary for the continous growth of solid tumors [1,2]. Angiogenesis takes not place in a single step, but is a complex sequential process that relies on a controled cross-talk between endothelial cells and the surrounding avascular environment [2,3]. Upon activation by growth factors or cytokines, endothelial cells start to degrade the surrounding extracellular matrix and invade the avascular tissue. The tight endothelial cell-cell adhesion is disrupted, the cells start to proliferate and migrate into the avascular environment. Finally they stop proliferating and differentiate to tubular structures (reviewed in [3]). Most recent studies have focused on the effect of specific growth factors and cytokines secreted by non endothelial cells on angiogenesis, but little is known about the sequential events taking place in the activated endothelial cells during the formation of new blood vessels.In this work we chose a model system where human microvascular endothelial cells (MVEC) are cultured on a gel composed of extracted basement membrane derived from mouse Engelbreth±Holm±Swarm sarcoma (matrigel) [4]. When seeded at a certain density the cells stop proliferating and virtually all cells are induced rapidly to form capillary-like, lumen containing structures [4,5]. These cells were compared against nondifferentiating, proliferating MVEC again representing an important step during the formation of new blood vessels [2,3]. The advantage of these culture systems is that they can be performed with one and the same cell type resulting in two different homogenous populatio...

Phenotypes are an important subject of biomedical research for which many repositories have already been created. Most of these databases are either dedicated to a single species or to a single disease of interest. With the advent of technologies to generate phenotypes in a high-throughput manner, not only is the volume of phenotype data growing fast but also the need to organize these data in more useful ways. We have created PhenomicDB (freely available at ), a multi-species genotype/phenotype database, which shows phenotypes associated with their corresponding genes and grouped by gene orthologies across a variety of species. We have enhanced PhenomicDB recently by additionally incorporating quantitative and descriptive RNA interference (RNAi) screening data, by enabling the usage of phenotype ontology terms and by providing information on assays and cell lines. We envision that integration of classical phenotypes with high-throughput data will bring new momentum and insights to our understanding. Modern analysis tools under development may help exploiting this wealth of information to transform it into knowledge and, eventually, into novel therapeutic approaches.

paired genes emerged early in evolution and code for homeobox transcription factors, having fundamental roles in various biological processes. We identified a novel human member of the paired-like class, which we named OTEX. A phylogenetic analysis revealed that OTEX belonged to the recently defined PEPP subfamily of paired-like homeobox genes. It was organized into three introns and, like the other PEPP genes, it was mapped to chromosome X. Its transcripts were detected mainly in the ovary, testis and epididymis, but also in the prostate and mammary gland. In the PC-3/ARwt prostate cell line, OTEX expression was stimulated dramatically following androgen treatment. Immunofluorescence studies revealed an exclusively nuclear localization of the OTEX protein. Mutation of the RARCRRHQRE amino acid sequence present at the C-terminus of the OTEX homeodomain resulted in a mainly cytoplasmic localization, indicating that this motif harboured the nuclear localization signal. No inherent transactivation function was seen for OTEX using the one-hybrid assay, and no homodimer formation was observed in the two-hybrid assay, suggesting that additional partners were needed for this activity. Taken together, the data show that OTEX represents a novel, androgen-regulated, paired-like homeobox protein, with possibly an important role in human reproduction.