Evolutionary analysis of genes of two pathways involved in placental malaria infection

Placental malaria is a special form of malaria that causes up to 200,000 maternal and infant deaths every year. Previous studies show that two receptor molecules, hyaluronic acid and chondroitin sulphate A, are mediating the adhesion of parasite-infected erythrocytes in the placenta of patients, which is believed to be a key step in the pathogenesis of the disease. In this study, we aimed at identifying sites of malaria-induced adaptation by scanning for signatures of natural selection in 24 genes in the complete biosynthesis pathway of these two receptor molecules. We analyzed a total of 24 Mb of publicly available polymorphism data from the International HapMap project for three human populations with European, Asian and African ancestry, with the African population from a region of presently and historically high malaria prevalence. Using the methods based on allele frequency distributions, genetic differentiation between populations, and on long-range haplotype structure, we found only limited evidence for malaria-induced genetic adaptation in this set of genes in the African population; however, we identified one candidate gene with clear evidence of selection in the Asian population. Although historical exposure to malaria in this population cannot be ruled out, we speculate that it might be caused by other pathogens, as there is growing evidence that these molecules are important receptors in a variety of host-pathogen interactions. We propose to use the present methods in a systematic way to help identify candidate regions under positive selection as a consequence of malaria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of peptide nucleic acid-peptide chimeras carrying the c-myc tag-sequence

The preparation of peptide nucleic acids (PNA)carrying a c-myc tag-peptide sequence isdescribed. These PNA-peptide chimeras have higheraffinity to complementary DNA than unmodifiedoligonucleotides. Moreover, they can be used asnonradioactive probes with sensitivity similar toother nonradioactive methods.     

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.     

Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence

Isolation and characterization of transposon-generated Mycoplasma genitalium gliding-deficient mutants has implicated mg200 and mg386 genes in gliding motility. The proposed role of these genes was confirmed by restoration of the gliding phenotype in deficient mutants through gene complementation with their respective mg386 or mg200 wild-type copies. mg200 and mg386 are the first reported gliding-associated mycoplasma genes not directly involved in cytadherence. Orthologues of MG200 and MG386 proteins are also found in the slow gliding mycoplasmas, Mycoplasma pneumoniae and Mycoplasma gallisepticum, suggesting the existence of a unique set of proteins involved in slow gliding motility. MG200 and MG386 proteins share common features, such as the presence of enriched in aromatic and glycine residues boxes and an acidic and proline-rich domain, suggesting that these motifs could play a significant role in gliding motility.     

Analysis of galanthamine-type alkaloids by capillary gas chromatography-mass spectrometry in plants

Galanthamine, an acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease, and galanthamine-type alkaloids are synthesised in different plants of the family Amaryllidaceae. A capillary gas chromatographic-mass spectroscopic (CGC-MS) method for the separation of 7 galanthamine type alkaloids, including galanthamine and epigalanthamine, is described in the present paper. A simple method for the routine quantification of galanthamine in plants was developed using pre-packed columns with diatomaceous earth (Isolute HM-N), allowing simultaneous preparation of a large number of samples. Galanthamine showed excellent linearity in the range from 50 to 1000 microg/mL and the limit of quantification was 5 microg/mL in total ion current mode and 1.6 ng/mL in selected ion monitoring mode. The recovery of galanthamine was more than 90%. Interday reproducibility (RSD) of the extraction was 2.74%. A method to find and to microextract Amaryllidaceae alkaloids in low-mass plant samples is also described.     

Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells

Abstract. Objectives: More than 50 years ago, we learned that breast cancer cells (and those of many other types of tumour) endogenously synthesize 95% of fatty acids (FAs) de novo, despite having adequate nutritional lipid supply. Today, we know that breast cancer cells benefit from this phenomenon in terms of enhanced cell proliferation, survival, chemoresistance and metastasis. However, the exact role of the major lipogenic enzyme fatty acid synthase (FASN) as cause, correlate or facilitator of breast cancer remains unidentified. Materials and methods: To evaluate a causal effect of FASN‐catalysed endogenous FA biosynthesis in the natural history of breast cancer disease, HBL100 cells (an SV40‐transformed in vitro model for near‐normal gene expression in the breast epithelium), and MCF10A cells (a non‐transformed, near diploid, spontaneously immortalized human mammary epithelial cell line) were acutely forced to overexpress the human FASN gene. Results: Following transient transfection with plasmid pCMV6‐XL4 carrying full‐length human FASN cDNA (gi: NM 004104), HBL100 cells enhanced their endogenous lipid synthesis while acquiring canonical oncogenic properties such as increased size and number of colonies in semisolid (i.e. soft‐agar) anchorage‐independent cultures. Anchorage‐dependent cell proliferation assays in low serum (0.1% foetal bovine serum), MTT‐based assessment of cell metabolic status and cell death ELISA‐based detection of apoptosis‐induced DNA‐histone fragmentation, together revealed that sole activation of endogenous FA biosynthesis was sufficient to significantly enhance breast epithelial cell proliferation and survival. When analysing molecular mechanisms by which acute activation of de novo FA biosynthesis triggered a transformed phenotype, HBL100 cells, transiently transfected with pCMV6‐XL4/FASN, were found to exhibit a dramatic increase in the number of phosphor‐tyrosine (Tyr)‐containing proteins, as detected by 4G10 antiphosphor‐Tyr monoclonal antibody. Phosphor‐Tyr‐specific antibodies recognizing the phosphorylation status of either the 1173 Tyr residue of epidermal growth factor receptor (HER1) or the 1248 Tyr residue of HER2, further revealed that FASN‐induced Tyr‐phosphorylation at ～180 kDa region mainly represented that of these key members of the HER (erbB) network, which remained switched‐off in mock‐transfected HBL100 cells. ELISA and immunoblotting procedures demonstrated that FASN overactivation significantly increased (> 200%) expression levels of epidermal growth factor receptor and HER2 proteins in HBL100 cells. Proteome Profiler? antibody arrays capable of simultaneously detecting relative levels of phosphorylation of 42 phospho‐receptor Tyr‐kinases (RTKs) confirmed that acute activation of endogenous FA biosynthesis specifically promoted hyper‐Tyr‐phosphorylation of HER1 and HER2 in MCF10A cells. This FASN‐triggered HER1/HER2‐breast cancer‐like phenotype was specifically inhibitable either by FASN inhibitor C75 or by Tyr‐kinase inhibitors (TKIs) gefitinib (Iressa?) and lapatinib (Tykerb?) but not by chemotherapeutic agents such as cisplatin. Transient overexpression of FASN dramatically increased HBL100 breast epithelial cells’ sensitivity to cytotoxic effects of C75, gefitinib and lapatinib (～8, 10 and > 15 times, respectively), while significantly decreasing (～3 times) cisplatin efficacy. Conclusions: Although we cannot definitely establish FASN as a novel oncogene in breast cancer, this study reveals for the first time that exacerbated endogenous FA biosynthesis in non‐cancerous epithelial cells is sufficient to induce a cancer‐like phenotype functionally dependent on the HER1/HER2 duo. These findings may perhaps radically amend our current perspective of endogenously synthesized fat, as on its own, it appears to actively increase signal‐to‐noise ratio in the HER1/HER2‐driven progression of human breast epithelial cells towards malignancy.