Biomolecular Characterization Research Articles

Biomolecular characterization of human glioblastoma cells in primary cultures: Differentiating and antiangiogenic effects of natural and synthetic PPARγ agonists

Gliomas are the most commonly diagnosed malignant brain primary tumors. Prognosis of patients with high-grade gliomas is poor and scarcely affected by radiotherapy and chemotherapy. Several studies have reported antiproliferative and/or differentiating activities of some lipophylic molecules on glioblastoma cells. Some of these activities in cell signaling are mediated by a class of transcriptional factors referred to as peroxisome proliferator-activated receptors (PPARs). PPARgamma has been identified in transformed neural cells of human origin and it has been demonstrated that PPARgamma agonists decrease cell proliferation, stimulate apoptosis and induce morphological changes and expression of markers typical of a more differentiated phenotype in glioblastoma and astrocytoma cell lines. These findings arise from studies mainly performed on long-term cultured transformed cell lines. Such experimental models do not exactly reproduce the in vivo environment since long-term culture often results in the accumulation of further molecular alterations in the cells. To be as close as possible to the in vivo condition, in the present work we investigated the effects of PPARgamma natural and synthetic ligands on the biomolecular features of primary cultures of human glioblastoma cells derived from surgical specimens. We provide evidence that PPARgamma agonists may interfere with glioblastoma growth and malignancy and might be taken in account as novel antitumoral drugs.

531 POSTER Questionable usefulness of Braf-V600E mutation to distinguish between sporadic and hereditary colorectal tumors with microsatellite instability in young patients

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal-dominant disorder characterized by predisposition to colorectal cancer and extracolonic malignancies, frequent multiple primary tumors in the same patient, and early age of cancer onset. A main clinical variant of Lynch syndrome, Muir–Torre syndrome (MTS) is characterized by the association between one or more visceral malignancies, with at least one sebaceous skin tumor or keratoacanthoma. In our study, we have screened a cohort of 538 HNPCC patients, related to 57 HNPCC families, to detect sebaceous skin tumors and keratoacanthomas and the role of mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, in their pathogenesis. Among the 57 HNPCC families, we have identified four MTS families and one suspected MTS family, in which sebaceous carcinoma was found in one HNPCC mutation carrier subject who did not show visceral malignancy. In four of these families, linked to two MLH1 mutations and to two MSH2 mutations, biomolecular characterization showed concordance among immunohistochemistry analysis and gene mutations. The evidences of our investigations show that MLH1 and MSH2 gene mutations have an equivalent etiopathological role both for Lynch syndrome and for MTS; hence, we propose a broadened clinical criteria for definition of Lynch syndrome that will include sebaceous adenoma, carcinoma, and keratoacanthoma.

Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction, the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.

Isolation and attempts of biomolecular characterization of fungal strains associated to foxing on a 19th century book

Abstract The brownish spots known as foxing, commonly found on old paper artefacts contain sometimes structures, which look like filamentous fungi. Attempts to grow these structures in vitro have been always unsuccessful so far. In order to study the role of these biological elements in paper decay, it is essential to identify them. This study is aimed at the identification of isolates from these brownish areas by culture-independent approaches using molecular biology techniques. The two Internal Transcribed Spacers and the 5.8 S gene (ITS1-5.8S-ITS2) from the nuclear ribosomal DNA were amplified, cloned and sequenced. Following a preliminary treatment with cellulase from Trichoderma reesei , DNA extractions were successfully achieved directly from paper samples. From 22 selected stained spots from a book dating from the 19th century, 8 extracts of genomic DNA were entirely analysed, which yielded 145 sequenced clones. No DNA could be sampled in unstained areas. Multiple alignment of the ITS sequences and comparison with reference sequences published in the NCBI database allowed to identify 14 groups of fungi belonging mainly to the following genera: Aspergillus , Bjerkandera , Chaetomium , Gloeotinia , Penicillium , Polyporus , Saccharicola , Trichoderma and Ulocladium . Some of these fungi are cellulolytic species but are not commonly found as indoor contaminant in storage rooms of graphic documents. The majority of the foxing spots exhibited sequences identified as Penicillium minioluteum . Gloeotinia temulenta occurred also frequently. Few isolates such as Aspergillus japonicus , Aspergillus oryzae , Chaetomium globosum , Penicillium citrinum , Trichoderma citrinoviride , Ulocladium chartarum and Ulocladium cucurbitae were present in only 1 or 2 clones. One of the foxing spots produced only one type of sequence similar to both Cordyceps sinensis and Fusarium lateritium , which have identical sequences in this rDNA region. This molecular approach, simple and rapid, could provide additional data for further discussion on the origin of the phenomenon of biological foxing.

Biomolecular characterisation of leucocytes by infrared spectroscopy

Over the last 15 years, infrared (IR) spectroscopy has developed into a novel and powerful biomedical tool that has multiple applications in the field of haematology. By revealing subtle alterations in both the conformation and concentration of key macromolecules, such as DNA, protein and lipids, IR spectroscopy has been employed to investigate multiple aspects of leucocyte physiology. IR spectroscopy has been used, for example, to diagnose and prognose leukaemia; to characterise differentiation and apoptotic processes; to predict drug sensitivity and resistance in leukaemic patients undergoing chemotherapy; to monitor the response of leucocytes to chemotherapy and to perform human leucocyte antigen matching for bone marrow transplant patients. Such studies have provided insight into pathogenic mechanisms underlying specific leucocyte disorders, especially leukaemia. While it is likely to be some considerable time before IR spectroscopy is sufficiently developed to displace the established technologies, IR spectroscopy has the potential to become a valuable analytic tool in basic and clinical haematology.

Value of MLH1 and MSH2 Mutations in the Appearance of Muir–Torre Syndrome Phenotype in HNPCC Patients Presenting Sebaceous Gland Tumors or Keratoacanthomas

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal-dominant disorder characterized by predisposition to colorectal cancer and extracolonic malignancies, frequent multiple primary tumors in the same patient, and early age of cancer onset. A main clinical variant of Lynch syndrome, Muir-Torre syndrome (MTS) is characterized by the association between one or more visceral malignancies, with at least one sebaceous skin tumor or keratoacanthoma. In our study, we have screened a cohort of 538 HNPCC patients, related to 57 HNPCC families, to detect sebaceous skin tumors and keratoacanthomas and the role of mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, in their pathogenesis. Among the 57 HNPCC families, we have identified four MTS families and one suspected MTS family, in which sebaceous carcinoma was found in one HNPCC mutation carrier subject who did not show visceral malignancy. In four of these families, linked to two MLH1 mutations and to two MSH2 mutations, biomolecular characterization showed concordance among immunohistochemistry analysis and gene mutations. The evidences of our investigations show that MLH1 and MSH2 gene mutations have an equivalent etiopathological role both for Lynch syndrome and for MTS; hence, we propose a broadened clinical criteria for definition of Lynch syndrome that will include sebaceous adenoma, carcinoma, and keratoacanthoma.

Does Biomolecular Characterization of Stage II/III Colorectal Cancer Have Any Prognostic Value?

As new improvements in the treatment of colorectal cancer have become available, it has become important to understand the benefits of new therapies or the deleterious effects stemming from the increased risk of toxicity. In particular, a more rational approach to adjuvant chemotherapy for patients with stage II/III disease should be defined by understanding which patients have a higher recurrence risk. Many studies have investigated several molecular markers, but none has been definitively associated with patient outcome. We present a review of studies that have evaluated the immunohistochemical correlation between expression of some biomarkers, such as thymidylate synthase, p53, Ki-67, Bcl-2, and microsatellite instability status expressed by Mut-L homologue 1 and Mut-S homologue 2 proteins, and the prognosis of patients with stage II/III colorectal cancer. We have evaluated studies in which > or = 100 patients were involved in an effort to ensure a representative study group. The only biomarker likely to have a prognostic value is microsatellite instability status, which correlated with a better prognosis.

Different phenotypes in Muir-Torre syndrome: clinical and biomolecular characterization in two Italian families

The Muir-Torre syndrome (MTS) is an autosomal dominant genodermatosis characterized by the presence of sebaceous gland tumours, with or without keratoacanthomas, associated with visceral malignancies. We describe and characterize two families in which the ample phenotypic variability of MTS was evident. After clinical evaluation, the skin and visceral tumours of one member of a family with 'classic' MTS and one member of a family with a 'peculiar' MTS phenotype without sebaceous lesions, but with only multiple keratoacanthomas, were analysed for microsatellite instability (MSI) and by immunohistochemistry. Tumours of both individuals showed MSI, with a concomitant lack of MSH2 immunostaining in all evaluated skin and visceral lesions; moreover, in the proband of family 2 a constitutional mutation (C-->T substitution leading to a stop codon) in the MSH2 gene was identified. We conclude that the diagnosis of MTS, which is mainly clinical, should take into account an ample phenotypic variability, which includes both cases with typical cancer aggregation in families and cases characterized by the association of visceral malignancies with multiple keratoacanthomas (without sebaceous lesions), without an apparent family history of cancer.

Occurrence of Stem-Pitting Strains of Citrus tristeza virus in Croatia.

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30'N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field-infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33°C. Eight months later, the bark of terminal twigs was peeled off, and the wood was examined for the occurrence of pits. Typical tristeza stem-pitting (SP) was observed in four isolates originating from cvs. Fukumoto navel, Washington navel, and Ichimaru Satsuma and C. wilsonii. The bark from the infected sources was analyzed using immunocapture reverse transcription-polymerase chain reaction (RT-PCR) with primers CTV1 and CTV10 (1), targeting the whole coat protein (CP) gene. The PCR products of the expected size (669 nucleotides) were obtained and TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells. Thirty-two clones harboring the CTV CP gene were sequenced. Two of the SP isolates contained four genomic variants that differed an average of 2.0% from the severe SwO SP strains SY568 and Nuaga (4) from California and Japan, respectively. The other two SP isolates contained four variants that differed as little as 1.6% from the severe SwO SP from India, CTV-Puna, and CTV-Bangalore (2). The net average distance between these two clusters of sequences is 5.2%. One sequence from each of the four SP isolates was deposited in GenBank (Accession Nos. AY791841 to AY791844). These findings were confirmed by direct observation of SP symptoms in a Satsuma orchard in the Neretva Valley during the spring of 2004. No other conspicuous symptoms that could be attributed to CTV were observed in the field. Most Satsumas were introduced to the Neretva Region from Japan between 1964 and 1984. Together with the fact that the related Nuaga strain was also isolated from Satsumas in Japan (4), our results suggest that SwO SP strains were introduced into Croatia at the same time and have been spreading for several decades. It has been generally believed that this kind of CTV strains either do not exist in the Mediterranean basin or, when found (e.g., Spain), are immediately eradicated. The findings reported here suggest that the epidemiological scenario for the Mediterranean Basin requires revision.

Biospecificity of glass surfaces: streptavidin attachment to silica

An investigation of biomolecular attachment to silicate glass surfaces as a function of forming and composition has been carried out. This paper concentrates on the modes of forming for one composition: silica, and on one biomolecular species: streptavidin. The relative sensitivity measurements associated with biomolecular characterization of the glass surface are discussed. The results show that streptavidin is sensitive to the forming conditions of silica and is sensitive to variation in surface treatment. The binding data suggest that the molecular attachment occurs through the OH species bound to the silica surface and it is hypothesized that this attachment occurs by hydrogen bonding.

Biomolecular characterization of glass surfaces

This paper introduces the concept of biomolecular characterization of inorganicsurfaces. The choice of biomolecule is discussed followed by techniques that can beused to analyse the quantity of bound species, strength of binding, the nature ofbinding sites, conformational changes and the layer morphology. The prospects ofmodelling this data using a combination of molecular dynamics simulation andprotein structural modelling and the correlation to measured data are outlined.The studies described in this paper are directed toward assessing thefeasibility of biomolecular characterization, however, the data collected inthe process are designed to also help elucidate our understanding of theinteraction between biomolecular species and inorganic materials interfaces.

Olive biophenols: biomolecular characterization, distribution and phytoalexin histochemical localization in the drupes

Olive biophenols (BP) comprise a major group of secondary metabolites that display a wealth of structural variety and a great diversity of important biological activities. Recently, an explosion of interest in the olive biophenol pathway has been observed which has been closely matched by related investigations into olivegrowing practices, for the production of extra virgin olive oil (EVOO) and table olives (TO). Major advances have been stimulated by multidisciplinary research on olive biophenol composition, histological distribution, and histochemical enzymatic localization of BPs to determine their biomolecular function, activated by native β-glucosidase on secoiridoid (seco) conjugates, such as oleuropein, which releases phytoalexins against pathogens.