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Biomedical subjects

L Valente

Publications and source records attributed to L Valente.

At least 19 recordsLinked to original sources
PubMed2005

Tumor necrosis factor alpha (TNFalpha) and its soluble receptor p75 (sTNF-R p75) in familial combined hyperlipidemia (FCHL).

BACKGROUND AND AIM: Familial combined hyperlipidemia (FCHL) is a genetic disorder of lipid metabolism associated with insulin resistance and abnormalities in fatty acid metabolism whose underlying mechanisms are largely unknown. Perturbations in the TNFalpha/TNF-R pathway may play a role in these abnormalities. METHODS AND RESULTS: We determined plasma levels of TNFalpha and sTNF-R p75 in 85 FCHL patients (TC 245+/-45 mg/dl; TG 260+/-148 mg/dl; apoB 148+/-37 mg/dl) and in 29 age- and sex-matched normolipemic relatives (NL) (TC 187+/-22.8 mg/dl; TG 115+/-37 mg/dl; apoB 106+/-16 mg/dl). Thirty-four normolipemic subjects (TC 180+/-34 mg/dl; TG 107+/-42 mg/dl; apoB 95+/-22 mg/dl) were also included as unrelated controls (NC). Plasma free fatty acids (NEFA) were also measured and insulin sensitivity was evaluated by HOMA. Levels of sTNF-R p75 were significantly reduced in FCHL compared to NL (2.30+/-0.55 ng/ml vs. 2.64+/-0.88 ng/ml, p<0.05) but not compared to NC (2.35+/-0.68 ng/ml). HOMA values were comparable in all groups and did not show any relation with plasma levels of sTNF-R p75. Logistic analysis demonstrated that a low concentration of sTNF-R p75 was an independent predictor of the affected status within FCHL families, but this role was no longer evident when FCHL patients were compared to NC. In FCHL, age (p<0.001) was positively, and TG (p=0.029) and HDL-C (p=0.025) were negatively correlated with plasma concentrations of sTNF-R p75. In the other groups, age (in NL) and non-HDL-C (in NC) were significantly correlated with sTNF-R p75. CONCLUSIONS: Although our data do not support a causative role of TNFalpha/TNF-R alterations in FCHL, they confirm that variation in TNF-R shedding may influence lipid phenotypic expression in FCHL families.

Adult↗
PubMed2005

Low levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in patients with newly diagnosed type 1 diabetes.

BACKGROUND AND AIMS: An epidemiological retrospective study and a recent prospective study from Finland have both concluded that vitamin D3 supplementation at birth protects individuals from type 1 diabetes later in life. Moreover, it is thought that vitamin D3 supplementation, in particular its activated form, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], may act as an immunomodulator, facilitating the shift from a Th1 to a Th2 immune response. The aim of this surveillance study was to measure levels of both 25-hydroxyvitamin D3 (25OHD3) and 1,25-dihydroxyvitamin D3 in patients with newly diagnosed type 1 diabetes as compared to normal subjects. METHODS: We measured plasma levels of 25-hydroxyvitamin D3 [25OHD3] and 1,25-dihydroxyvitamin D3 by radioimmunoassay in 88 consecutive patients with newly diagnosed type 1 diabetes (mean age 14.6 years; diagnosis within the last week), and in 57 healthy age and sex-matched subjects (mean age 16.5 years) born and residing in the Lazio region of continental Italy. RESULTS: Mean levels of both 25OHD3 and 1,25-(OH)2D3 were significantly lower in patients compared to controls (p < 0.01 and p < 0.03, respectively). There was no correlation between 1,25-(OH)2D3 plasma level and metabolic control status at disease diagnosis, age, gender, or most importantly, seasonality of disease diagnosis. This new observation endorses the findings of the Finnish study, even though Italy is a geographic area with more hours of sunlight than Finland. CONCLUSIONS: These findings suggest that vitamin D3 may be an important pathogenic factor in type 1 diabetes independent of geographical latitude, and that its supplementation should be considered not only at birth, but also at diagnosis of type 1 diabetes with the aim of favouring a Th2 immune response and protecting residual beta cells from further destruction.

Adolescent↗
PubMed2003

Mutation of a conserved CDK site converts a metazoan Elongation Factor 1Bbeta subunit into a replacement for yeast eEF1Balpha.

Elongation factor subunit eEF1Bbeta (formerly EF-1beta in plants and EF-1delta in animals) was identified and cloned in a screen for proteins from pea that interact with a cyclin-dependent kinase (CDK). CDKs are enzymes that regulate progression through meiotic and mitotic cell cycles in eukaryotes. eEF1Bbeta and the related protein eEF1Balpha (formerly EF-1beta' in plants and EF-1beta in animals and fungi) can catalyze GTP/GDP exchange on the G-protein eEF1A (formerly EF-1alpha in plants, animals and fungi) during the elongation phase of protein synthesis in eukaryotes. Recombinant Cdc2 and its native homologues from pea extracts associated both in vitro and in vivo with eEF1Bbeta. A Cdc2-cyclin B complex phosphorylated recombinant plant eEF1Bbetas, but not eEF1Balpha. These interactions between CDK and eEF1Bbeta prompted investigations into the in vivo consequences of this relationship. Expression of cDNAs encoding rice or pea eEF1Bbeta subunits failed to complement a Saccharomyces cerevisiae mutant deleted for the eEF1Balpha gene, as was previously observed for the human eEF1Bbeta. However, replacement of Thr91, the sole consensus CDK phosphorylation site in pea eEF1Bbeta, with alanine allowed the pea protein to substitute for eEF1Balpha function in vivo. In addition, this rescued strain was severely cold sensitive, and more sensitive to translational inhibitors than wild-type yeast. Taken together, these results suggest a physiological connection between the cyclin-dependent class of kinases and a translational elongation factor in mitotic cells, and provide the first in vivo evidence that an altered form of eEF1Bbeta can serve as the guanine nucleotide exchange factor for eEF1A.

Amino Acid Sequence↗
PubMed2003

Yeast as a sensor of factors affecting the accuracy of protein synthesis.

The cell monitors and maintains the fidelity of translation during the three stages of protein synthesis: initiation, elongation and termination. Errors can arise by multiple mechanisms, such as altered start site selection, reading frame shifts, misincorporation or nonsense codon suppression. All of these events produce incorrect protein products. Translational accuracy is affected by both cis- and trans-acting elements that insure the proper peptide is synthesized by the protein synthetic machinery. Many cellular components are involved in the accuracy of translation, including RNAs (transfer RNAs, messenger RNAs and ribosomal RNAs) and proteins (ribosomal proteins and translation factors). The yeast Saccharomyces cerevisiae has proven an ideal system to study translational fidelity by integrating genetic approaches with biochemical analysis. This review focuses on the ways studies in yeast have contributed to our understanding of the roles translation factors and the ribosome play in assuring the accuracy of protein synthesis.

Peptide Elongation Factors↗
PubMed2001

Crystal structures of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex.

In the elongation cycle of protein biosynthesis, the nucleotide exchange factor eEF1Balpha catalyzes the exchange of GDP bound to the G-protein, eEF1A, for GTP. To obtain more information about the recently solved eEF1A-eEF1Balpha structure, we determined the structures of the eEF1A-eEF1Balpha-GDP-Mg2+, eEF1A-eEF1Balpha-GDP and eEF1A-eEF1Balpha-GDPNP complexes at 3.0, 2.4 and 2.05 A resolution, respectively. Minor changes, specifically around the nucleotide binding site, in eEF1A and eEF1Balpha are consistent with in vivo data. The base, sugar and alpha-phosphate bind as in other known nucleotide G-protein complexes, whereas the beta- and gamma-phosphates are disordered. A mutation of Lys 205 in eEF1Balpha that inserts into the Mg2+ binding site of eEF1A is lethal. This together with the structures emphasizes the essential role of Mg2+ in nucleotide exchange in the eEF1A-eEF1Balpha complex.

Amino Acid Substitution↗
PubMed2000

Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha.

The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.

Binding Sites↗
PubMed1999

Mutations in elongation factor 1beta, a guanine nucleotide exchange factor, enhance translational fidelity.

Translation elongation factor 1beta (EF-1beta) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes. EF-1beta is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1alpha, a required step to ensure continued protein synthesis. In this work, we demonstrate that the highly conserved C-terminal region of Saccharomyces cerevisiae EF-1beta is sufficient for normal cell growth. This region of yeast and metazoan EF-1beta and the metazoan EF-1beta-like protein EF-1delta is highly conserved. Human EF-1beta, but not human EF-1delta, is functional in place of yeast EF-1beta, even though both EF-1beta and EF-1delta have previously been shown to have guanine nucleotide exchange activity in vitro. Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1beta protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors. These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis. These results indicate the critical function of EF-1beta in regulating EF-1alpha activity, cell growth, translation rates, and translational fidelity.

Alleles↗
PubMed1999

T cell reactivity to human insulinoma cell line (CM) antigens in patients with type 1 diabetes.

Autoimmune (type 1) diabetes mellitus results from a progressive destruction of insulin secreting beta cells operated by T lymphocytes in pancreatic islets. Circulating autoreactive T cells to specific beta cell antigens are detected in patients with type 1 diabetes. To date, several beta cell autoantigens have been identified in this disease (GAD, IA-2, 38kD secretory protein, insulin, ICA69 etc.), however, it is possible that also other unidentified self molecules contribute to trigger beta cell autoimmunity. In this study we used the human insulinoma cell line CM as source of beta cell antigens to detect reactive T lymphocytes in patients with type 1 diabetes mellitus. This cell line has been previously shown to express a number of recognized beta cell antigens. Since the expression of several beta cell antigens is affected by glucose stimulation we tested two preparations of CM cells cultured under different conditions containing low (0.8 mM) and high glucose concentration (11 mM). T cell proliferation was measured using cells from 32 patients with type 1 diabetes (19 of recent onset and 13 at 3 to 22 months from diagnosis) and 27 age-matched control subjects. A significant increase in T cell proliferation to CM cells grown in high glucose conditions (11 mM) (p < 0.05) was found in type 1 diabetic patients compared to controls. No significant differences were observed when using CM cells cultured at the low glucose concentration. Furthermore, the response to both extracts of CM cells was independent of disease duration (p = 0.6 for both CM cells cultured at 0.8 and 11 mM glucose). These data indicate that T cell reactivity to homogenates of CM cells is detectable in patients with type 1 diabetes and suggest that this human insulinoma cell line is an interesting potential source of beta cell material for immunological studies of autoimmune diabetes.

Adolescent↗
PubMed1997

Preclinical prediction of factors influencing the elimination of 5,6-dimethylxanthenone-4-acetic acid, a new anticancer drug.

The glucuronidation of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a newly developed anticancer drug, was investigated in vitro to determine factors likely to affect the elimination of this compound in patients. Human liver microsomal DMXAA glucuronidation followed Michaelis-Menten kinetics, with a mean apparent Km of approximately 100 microM. Two cDNA-expressed UGT isoforms, UGT1*02 and UGT2B7, had the capacity to glucuronidate DMXAA, although comparative kinetic and inhibitor studies were more consistent with a greater contribution of UGT2B7 to the human hepatic reaction. Microsomal DMXAA glucuronide formation was screened for inhibition by drugs known to be eliminated by glucuronidation. Of the drugs screened, significant inhibition was observed with diclofenac, epirubicin, indomethacin, R,S-ketoprofen, lorazepam, S-naproxen, oxazepam, and temazepam; apparent Ki values ranged from 9.5-318 microM. These values are substantially above unbound concentrations of the individual drugs achieved in vivo. DMXAA glucuronide was found to be unstable at physiological pH values, and the rate of degradation was marginally increased in the presence of albumin. Taken together, these data indicate that the kinetics of DMXAA glucuronidation in vivo are likely to be linear and unaffected by the coadministration of most glucuronidated drugs, but plasma DMXAA clearance may be decreased in patients with renal dysfunction. This study illustrates the utility of in vitro techniques for the prediction of potential drug interactions and other dispositional characteristics of newly developed anticancer drugs before their administration to patients.

Antineoplastic Agents↗
PubMed1997

Glutathione protects a human insulinoma cell line from tumor necrosis factor-alpha-mediated cytotoxicity.

It is postulated that glutathione acting as a free oxygen radical scavenger may protect beta-cells from cytokine-mediated cytotoxicity in insulin-dependent diabetes. In this study the effect of glutathione in preventing the cytotoxic damage mediated by tumor necrosis factor-a in vitro towards a human beta-cell line (CM insulinoma) was investigated. CM cells were exposed in vitro to tumor necrosis factor-alpha, tumor necrosis factor-alpha plus glutathione or glutathione alone at different concentrations. The resulting cytotoxicity was measured using a colorimetric assay. Glutathione significantly reduced the cytotoxicity mediated by tumor necrosis factor-alpha in a dose-dependent fashion (P < 0.001). These results suggest a protective effect of glutathione on beta-cell cytotoxicity induced by tumor necrosis factor-alpha and encourage the use of glutathione in trials aimed at reducing the beta-cell damage occurring in insulin-dependent diabetes.

Cell Survival↗
PubMed1997

1-Methylxanthine derived from caffeine as a pharmacodynamic probe of oxypurinol effect.

AIMS: In the present study we have investigated the use of caffeine, administered in the form of instant coffee, as a prodrug for 1MX to validate the use of the 1MU:1MX ratio following caffeine administration as a pharmacodynamic measure of oxypurinol effect on xanthine oxidase. METHODS: Five healthy volunteers took caffeine 75 mg 8 hourly administered as instant coffee over a 7 day period. They were given allopurinol 600 mg on day 4. Urine was collected in 8 h aliquots from day 1-day 7. The ratio of 1-methyluric acid (1MU) to 1-methylxanthuric (1MX) was determined. RESULTS: The relationship between the plasma oxypurinol (the active metabolite of allopurinol) concentration at the midpoint of each caffeine dosage interval and the decrement in the urinary 1MX to 1MU ratio fitted well by a sigmoid Emax model. Mean (+/-s.d.) values of the oxypurinol EC50(3.9 +/- 1.4 mg l-1), EC90(8.7 +/- 1.8 mgl-1) and the exponent, n (3.0 +/- 1.2) were similar to those obtained previously following either the direct administration of 1MX or the use of theophylline as a prodrug for 1MX. CONCLUSIONS: These data indicate that the use of caffeine as a source of 1MX could provide a simple and ethically acceptable method for monitoring oxypurinol effect in patients taking allopurinol for the treatment of gout.

Adult↗
PubMed1997

[Scleredema, acanthosis nigricans and IgA/Kappa multiple myeloma].

BACKGROUND: Scleredema is an uncommon disease of unknown origin. Characteristic thick skin with symmetrical diffuse induration develops. The infiltration begins on the face and neck then extends to the root of the upper limbs and trunk. There are three clinical types of scleredema. The first is preceded by an upper airway infection and progresses rapidly before regressing spontaneously within a few months. The second type is associated with chronic diabetes. The third type is associated with monoclonal gammapathy, rarely of myelomatous type, and develops insidiously. Acanthosis nigricans can be a paraneoplastic syndrome, often associated with a gastrointestinal cancer. Few cases associating scleredema and acanathosis nigricans have been reported. CASE REPORT: A 56-year old woman had developed scleredema over the last 6 years when acanthosis nigricans appeared together with IgA kappa multiple myeloma. Treatment with melphalan and prednisolone was effective against the myeloma as well as the scleredema and acanthosis nigricans. DISCUSSION: Only five cases of associated scleredema and multiple myeloma have been reported, four with kappa IgG myeloma and one with IgA myeloma. An association between acanthosis nigricans and sclerederma could be coincidental although the fact that the different manifestations regressed together after the myeloma treatment would suggest some relationship between these three diseases.

Acanthosis Nigricans↗
PubMed1996

Beta-cell markers and autoantigen expression by a human insulinoma cell line: similarities to native beta cells.

In the present study we have evaluated the expression of different beta-cell markers, islet molecules and auto-antigens relevant in diabetes autoimmunity by a human insulinoma cell line (CM) in order to define its similarities with native beta cells and to discover whether it could be considered as a model for studies on immunological aspects of Type 1 diabetes. First, the positivity of the CM cell line for known markers of neuroendocrine derivation was determined by means of immunocytochemical analysis using different anti-islet monoclonal antibodies including A2B5 and 3G5 reacting with islet gangliosides, and HISL19 binding to an islet glycoprotein. Secondly, the expression and characteristics of glutamic acid decarboxylase (GAD) and of GM2-1 ganglioside, both known to be islet autoantigens in diabetes autoimmunity and expressed by human native beta cells, were investigated in the CM cell line. The pattern of ganglioside expression in comparison to that of native beta cells was also evaluated. Thirdly, the binding of diabetic sera to CM cells reacting with islet cytoplasmic antigens (ICA) was studied by immunohistochemistry. The results of this study showed that beta cell markers identified by anti-islet monoclonal antibodies A2B5, 3G5 and HISL-19 are expressed by CM cells; similarly, islet molecules such as GAD and GM2-1 ganglioside are present and possess similar characteristics to those found in native beta cells; the pattern of expression of other gangliosides by CM cells is also identical to human pancreatic islets; beta cell autoantigen(s) reacting with antibodies present in islet cell antibodies (ICA) positive diabetic sera identified by ICA binding are also detectable in this insulinoma cell line. We conclude that CM cells show close similarities to native beta cells with respect to the expression of neuro-endocrine markers, relevant beta cell autoantigens in Type 1 diabetes (GAD, GM2-1, ICA antigen), and other gangliosides. Therefore, this insulinoma cell line may be considered as an ideal model for studies aimed at investigating autoimmune phenomena occurring in Type 1 diabetes.

Autoantigens↗
PubMed1995

Adjuvant therapy in recent onset type 1 diabetes at diagnosis and insulin requirement after 2 years.

Partial recovery of beta-cell function in type 1 diabetes is common after diagnosis by intensive insulin therapy. Residual beta-cell function can be improved by other therapies. Cyclosporin (CyA) and nicotinamide (NA), alone or in combination, can preserve this function, as indicated by the parameters of metabolic control (insulin dose, HbA1C). After suspension of CyA, insulin requirement returns to control values, suggesting loss of residual beta-cell function. The effects induced by withdrawal of NA after 1 year are not known. For the first time, we studied 27 type 1 diabetes patients treated with NA for 12 months and then followed up for 1 year after discontinuance of NA. Another 25 patients treated with NA + CyA and 28 control patients were followed up similarly. Insulin requirement doubled 12 months after discontinuance of NA or NA + CyA, becoming identical to that of controls. As patients showed HbA1C values similar to control subjects, it is likely that beta-cell function deteriorated after discontinuance of therapy. As NA is safer than other agents and its effects are beneficial, longer studies are warranted to investigate NA in prolonged treatments since this compound is also being considered for prevention of type 1 diabetes.

Adjuvants, Pharmaceutic↗
PubMed1995

Human hepatic cytochrome P450 2C9 catalyzes the rate-limiting pathway of torsemide metabolism.

Tolyl methylhydroxylation is the rate-limiting step in the elimination of torsemide, a newly developed diuretic, in humans in vivo. Kinetic and inhibitor studies with human liver microsomes and complementary DNA-expressed enzyme were performed to identify the cytochrome P450 (CYP) isoform responsible for torsemide tolyl methylhydroxylation to predict factors that might alter clearance in patients receiving torsemide. As in vivo, tolyl methylhydroxylation was the major biotransformation pathway in human liver microsomes. Microsomal tolyl methyl-hydroxylation kinetics followed Michaelis-Menten kinetics, with the mean apparent Km for the reaction being 11.2 +/- 1.3 microM. The microsomal reaction was almost completely abolished by the specific CYP2C9 inhibitor sulfaphenazole and was inhibited competitively by the alternative CYP2C9 substrate tolbutamide. Torsemide tolyl methylhydroxylase activity in microsomes from 16 human livers correlated significantly (rs = .81-.88) with tolbutamide and phenytoin hydroxylation, both CYP2C9-mediated reactions. Complementary DNA-expressed CYP2C9 catalyzed torsemide tolyl methylhydroxylation with an apparent Km (23 microM) similar to that observed for human liver microsomes and the IC50 values for sulfaphenazole inhibition of the reaction were essentially identical for the two enzyme sources. Taken together, these data demonstrate that human hepatic torsemide tolyl methylhydroxylation is catalyzed predominantly, if not solely, by CYP2C9. The implications of this finding for the regulation of torsemide metabolism in vivo are discussed.

Aryl Hydrocarbon Hydroxylases↗
PubMed1993

A reversal theory approach to the causes and treatment of stress in professional life.

An interactive model, which takes into account both situation and individual, is likely to be most useful in understanding the occurrence of stress in professional life. Reversal theory, when applied in the way detailed in the present paper, yields just such a model. It also, in the way discussed, provides a number of practical strategies for helping professionals identify and develop ways of coping with stress, particularly when employed within the context of the stress management workshop.

Adaptation, Psychological↗
PubMed1990

Retinol binding protein: a short half life determinant of protein non enzymatic glycation in diabetes.

The non enzymatic glycation of circulating and structural proteins is the main biochemical consequence of the chronic hyperglycaemia of diabetes mellitus. Retinol binding protein (RPB) is a 21K plasma globulin with an half life of 12 hr; its non enzymatic glycation may reflect the variation of short term metabolic control (1-4 days). In this study two blood samples were withdrawn at four days interval from 24 non insulin dependent diabetic patients. Glycated RBP was measured by a two-site immunoradiometric assay and its variations correlated with the correspondent changes in the blood glucose level. A significant correlation (r = 0.471; p less than 0.02) was found between the time 4/time 0 ratios of glycated RBP and the time 4/time 0 ratios of blood glucose. These data suggest that measurement of non-enzymatically glycated RBP may be a useful tool to evaluate the short term state of non enzymatic glycation in diabetes.

Biomarkers↗