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Biomedical subjects

M Marra

Publications and source records attributed to M Marra.

At least 37 records · Page 2Linked to original sources
PubMed2003

The eukaryotic initiation factor 5A is involved in the regulation of proliferation and apoptosis induced by interferon-alpha and EGF in human cancer cells.

Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.

Antineoplastic Agents↗
PubMed2003

Cryptococcus neoformans gene expression during experimental cryptococcal meningitis.

Cryptococcus neoformans, an encapsulated basidiomycete fungus of medical importance, is capable of crossing the blood-brain barrier and causing meningitis in both immunocompetent and immunocompromised individuals. To gain insight into the adaptation of the fungus to the host central nervous system (CNS), serial analysis of gene expression (SAGE) was used to characterize the gene expression profile of C. neoformans cells recovered from the CNS of infected rabbits. A SAGE library was constructed, and 49,048 tags were sequenced; 16,207 of these tags were found to represent unique sequences or tag families. Of the 304 most-abundant tags, 164 were assigned to a putative gene for subsequent functional grouping. The results (as determined according to the number of tags that identified genes encoding proteins required for these functions) indicated that the C. neoformans cells were actively engaged in protein synthesis, protein degradation, stress response, small-molecule transport, and signaling. In addition, a high level of energy requirement of the fungal cells was suggested by a large number of tags that matched putative genes for energy production. Taken together, these findings provide the first insight into the transcriptional adaptation of C. neoformans to the host environment and identify the set of fungal genes most highly expressed during cerebrospinal fluid infection.

Animals↗
PubMed2003

Agreement among neurologists on the clinical diagnosis of dystonia at different body sites.

OBJECTIVE: To study the reliability of the diagnosis of blepharospasm, oromandibular dystonia, cervical dystonia, and writer's cramp among neurologists. METHODS: 12 patients with adult onset focal segmental dystonia were videotaped in a standardised way. The tape was sent to six neurologists who are involved in clinical practice without a specific interest in movement disorders (general neurologists), and to four neurologists expert in movement disorders. The observers had to recognise whether the patients were affected by dystonia and to distinguish among blepharospasm, oromandibular dystonia, cervical dystonia, and writer's cramp. Interobserver reliability was assessed by kappa statistics, and the degree of agreement was classified according to the Landis classification. RESULTS: The 10 neurologists reached slight to moderate agreement on the diagnosis of these four disorders. When the observers were subdivided according with their professional experience in the field, a moderate to perfect agreement on the diagnosis was achieved by specialists in movement disorders, and a fair to moderate agreement by the general neurologists. CONCLUSIONS: Neurologists may have different ability to recognise adult onset focal dystonia, depending on their experience and on the type of dystonia.

Adult↗
PubMed2003

A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence.

We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel. The resulting map contains 70 loci spanning the length of bovine chromosome 19. Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones. Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10. Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map.

Animals↗
PubMed2003

Voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, enhances the cytotoxic effect of doxorubicin on multidrug-resistant tumor cells.

Multidrug-resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the chemotherapy. In order to overcome MDR, substances, such as verapamil and cyclosporin A (CsA), were employed. As these P-gp modulating agents did not seem promising in clinical practice, new compounds with a low degree of undesirable side effects, were introduced. In this study, bisindolic alkaloid voacamine was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on drug resistant cells. Two different pairs of tumor cell lines were analyzed: the parental lymphoblastoid cell line CEM-WT and its MDR derivative CEM-R, the parental osteosarcoma cell line U-2 OS-WT and its resistant counterpart U-2 OS-R. These cell lines were characterized for their morphological features by scanning electron microscopy (SEM) and for the expression of the main drug transporters by flow cytometric analysis. The effects of voacamine on the cell survival and on both accumulation and efflux of DOX were then investigated. The intracellular distribution of DOX, given alone or in association with CsA or voacamine, was observed by laser scanning confocal microscopy. A differential effect of voacamine between sensitive and resistant cells on the intracellular DOX concentration and distribution was shown. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. The results of cell survival experiments revealed an enhancement of the cytotoxic effect of DOX induced by voacamine, confirmed by evident morphological changes observed by SEM. These findings suggest promising applications of this natural substance against MDR tumors.

Alkaloids↗
PubMed2002

Are the general equations to predict BMR applicable to patients with anorexia nervosa?

AIM: To determine whether the general equations to predict basal metabolic rate (BMR) can be reliably applied to female anorectics. INDIVIDUALS AND METHODS: Two hundred and thirty-seven female patients with anorexia nervosa (AN) were divided into an adolescent group [n=43, 13-17 yrs, 39.3+/-5.0 kg, body mass index (BMI) (weight/height) 15.5+/-1.8 kg/m2] and a young-adult group (n=194, 18-40 yrs, 40.5+/-6.1 kg, BMI 15.6+/-1.9 kg/m2). BMR values determined by indirect calorimetry were compared with those predicted according to either the WHO/FAO/UNU or the Harris-Benedict general equations, or using the Schebendach correction formula (proposed for adjusting the Harris-Benedict estimates in anorectics). RESULTS: Measured BMR was 3,658+/-665 kJ/day in the adolescent and 3,907+/-760 kJ/day in the young-adult patients. In the adolescent group, the differences between predicted and measured values were (mean+/-SD) 1,466 529 kJ/day (+44+/-21%) for WHO/FAO/UNU, 1,587+/-552 kJ/day (+47+/-23%) for the Harris-Benedict and -20+/-510 kJ/day for the Schebendach (+1+/-13%), while in the young-adult group the corresponding values were 696+/-570 kJ/day (+24+/-24%), 1,252+/-644 kJ/day (+37+/-27%) and -430+/-640 kJ/day (-9+/-16%). The bias was negatively associated with weight and BMI in both groups when using the WHO/FAO/UNU and Harris-Benedict equations, and with age in the young-adult group for the Harris-Benedict and Schebendach equations. CONCLUSIONS: The WHO/FAO/UNU and Harris-Benedict equations greatly overestimate BMR in AN. Accurate estimation is to some extent dependent on individual characteristics such as age, weight or BMI. The Schebendach correction formula accurately predicts BMR in female adolescents, but not in young adult women with AN.

Adolescent↗
PubMed2002

Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells.

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Body composition changes in patients with anorexia nervosa after complete weight recovery.

OBJECTIVE: To assess body composition changes occurring in female anorectic patients after complete weight recovery. DESIGN: Longitudinal study. SUBJECTS: : Ten female patients with anorexia nervosa (age at baseline: 19.7+/-5.8 y) were studied both when undernourished (body mass index, BMI 14.8+/-1.3 kg/ m(2)) and after the achievement of a BMI >18.5 kg/m(2). The control group comprised 18 well-nourished women (age 20.1+/-4.4 y; BMI 19.0-23.0 kg/ m(2)). INTERVENTIONS: Fat mass and fat-free mass were determined by dual-energy X-ray absorptiometry. Skinfold thicknesses and circumferences were also measured. Arm muscle area and arm fat area were calculated by standard formulas. RESULTS: The undernourished patients had lower fat-free mass, fat mass, skinfold thicknesses and circumferences. After refeeding, fat mass represented 25-71% (mean 56%) of the mass regained, this percentage being directly related to the extent of weight gain. The increases in skinfolds and circumferences depended upon the site considered and were correlated to a various extent with those in weight or BMI. Skinfolds at biceps and abdominal sites and the waist-to-hip ratio remained significantly higher, whereas arm muscle circumference was significantly lower, in the refed group than in the control one. CONCLUSION: The percentage of fat in the weight regained by refed female anorectic patients was directly related to the extent of body mass increase. Refed anorectic patients appear to preferentially regain fat in the abdominal and triceps regions. Abnormalities in skinfolds (at biceps and abdominal sites), arm muscle area and waist-to-hip ratio still persist in refed anorectic patients in comparison to control healthy controls.

Adolescent↗
PubMed2002

Percutaneous ethanol injection efficacy in the treatment of large symptomatic thyroid cystic nodules: ten-year follow-up of a large series.

We present a prospective study on the long-term efficacy of percutaneous ethanol injection (PEI) treatment of a large series of symptomatic thyroid cystic nodules (STCN). Ninety-eight patients (72 females and 26 males) were treated. The mean basal volume of the STCN was 35.3 mL. In 92 of 98 patients PEI treatment induced a greater than 50% nodule shrinkage, only 6 of 92 responder patients relapsed at a follow-up of 9 years. Moreover, all the patients had a significant clinical benefit because a significant reduction of the cyst-associated symptoms was recorded. Furthermore, a limited number of sessions was required for the treatment of cysts larger than 40 mL (mean +/- standard deviation [SD]: 2.7 +/- 0.75) demonstrating the feasibility of the procedure also in the treatment of large cysts. In conclusion, PEI is an effective and inexpensive procedure with a high patient compliance and long-lasting effects in the treatment of cysts larger than 40 mL.

Administration, Cutaneous↗
PubMed2002

What is the relationship between P-glycoprotein and adhesion molecule expression in melanoma cells?

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Programmed cell death takes flight: genetic and genomic approaches to gene discovery in Drosophila.

Programmed cell death (PCD) is an essential and wide-spread physiological process that results in the elimination of cells. Genes required to carry out this process have been identified, and many of these remain the subjects of intense investigation. Here, we describe PCD, its functions, and some of the consequences when it goes awry. We review PCD in the model system, the fruit fly, Drosophila melanogaster, with a particular emphasis on cell death gene discovery resulting from both genetics and genomics-based approaches.

Animals↗
PubMed2002

Resuspension of DNA sequencing reaction products in agarose increases sequence quality on an automated sequencer.

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.

Cell Fractionation↗
PubMed2002

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Adenosine 5'-monophosphate inhibits the association of 14-3-3 proteins with the plant plasma membrane H(+)-ATPase.

Although a well ascertained evidence proves that the activity of the plant plasma membrane H(+)-ATPase is regulated by 14-3-3 proteins, information about physiological factors modulating the phosphorylation-dependent association between 14-3-3 proteins and the proton pump is largely incomplete. In this paper we show that the 5'-AMP-mimetic, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), inhibits the fusicoccin-promoted proton extrusion in maize roots. We also demonstrate that 5'-AMP inhibits the association of 14-3-3 proteins with the C-terminal domain of the H(+)-ATPase in an overlay assay as well as the 14-3-3-dependent stimulation of the Arabidopsis thaliana H(+)-ATPase AHA1 isoform expressed in yeast membranes. Finally, by means of affinity chromatography with immobilized 5'-AMP and trinitrophenyl-AMP fluorescence analysis, we demonstrate that the 14-3-3 isoform GF14-6 from maize is able to bind 5'-AMP. The possible role of 5'-AMP as a general regulator of 14-3-3 functions in the plant cell is discussed.

14-3-3 Proteins↗
PubMed2001

Dissection of the promoter region of the inositol 1,4,5-trisphosphate receptor gene, itr-1, in C. elegans: a molecular basis for cell-specific expression of IP3R isoforms.

Inositol 1,4,5-trisphosphate receptors in Caenorhabditis elegans are encoded by a single gene, itr-1. This provides a powerful system in which to dissect the mechanisms that control the tissue-specific expression of molecules that determine the specificity of calcium signalling. We first identified the Caenorhabditis briggsae orthologue of itr-1, Cbitr-1. Comparison of the two itr-1 genes revealed that the chromosomal organisation, gene structure and predicted cDNA and protein sequences were all conserved. The conserved gene structure supports the hypothesis that the itr-1 gene has three promoters, each of which gives rise to an alternative mRNA and hence unique protein. To test this and to identify the roles of the three putative promoters (pA, pB and pC) in regulating itr-1 expression we fused each promoter to the green fluorescent protein gene and identified their expression patterns. Introduction of these transgenes into C. elegans identified unique and defined patterns of green fluorescent protein expression directed by each promoter: pA directs expression in the pharyngeal terminal bulb, the rectal epithelial cells and vulva; pB directs expression in the motor neurone PDA, the amphid socket cells and the spermatheca; pC directs expression in the spermathecal valve, uterine sheath cells, pharyngeal isthmus and intestine. Thus tissue-specific expression of itr-1 variants is directed by three promoters and this results in adjacent cells in the same tissue containing different inositol trisphosphate receptor isoforms. Within pA, four short regions (pA-A to pA-D) of sequence conservation between C. elegans and C. briggsae were identified. Deletion analysis demonstrated that the region containing pA-C is required for expression in the terminal bulb and rectal epithelial cells and the region containing pA-D is required for expression in the vulva. pA-C includes sequences similar to the binding sites for transcription factors that have been demonstrated to be important in pharyngeal development and gene expression.

Amino Acid Sequence↗
PubMed2001

A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

Chromosomes, Artificial, Bacterial↗
PubMed2001

A gender--dependent genetic predisposition to produce high levels of IL-6 is detrimental for longevity.

Current literature indicates that elevated IL-6 serum levels are associated with diseases, disability and mortality in the elderly. In this paper, we studied the IL-6 promoter genetic variability at -174 C/G locus and its effect on IL-6 serum levels in a total of 700 people from 60 to 110 years of age, including 323 centenarians. We found that the proportion of homozygotes for the G allele at -174 locus decreases in centenarian males, but not in centenarian females. Moreover, we found that, only among males, homozygotes for the G allele at -174 locus have higher IL-6 serum levels in comparison with carriers of the C allele. On the whole, our data suggest that those individuals who are genetically predisposed to produce high levels of IL-6 during aging, i.e. -174 locus GG homozygous men, are disadvantaged for longevity.

Aged↗