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1.
Cogn Process ; 20(3): 291-298, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30569268

ABSTRACT

The concept of "presence" describes the quality of subjective experience in immersive virtual reality (IVR). Presence refers to a specific state of consciousness: we behave and feel as if we actually were in the virtual world even though we know there is nothing there. In their handbook of Virtual Reality, Burdea and Coiffet (Virtual reality technology, Wiley, New York, 2003) suggested that the experience of presence in IVR would emerge from the combination of three Is: Immersion or capacity to isolate from the external world, Interaction or capacity to naturally exploring the virtual environment, and Imagination or individual aptitudes with mental imagery. So far, several studies have investigated the technological and psychological factors affecting the degree of immersion and interaction. However, no study has explored the relationship between perceived presence and mental imagery. Here we aim at filling this gap through a correlational study comparing self-reports about sense of presence and mental imagery abilities. After experiencing two IVR scenarios (an art gallery and a living room), 142 male and female users were administered with questionnaires assessing the degree of presence (Igroup Presence Questionnaire), the degree of vividness (Vividness of Visual Imagery Questionnaire) and control (Test of Visual Imagery Control) of subjective mental images. Results showed a clear positive correlation between presence and vividness: the higher the vividness of mental images the stronger the reported sense of presence felt in IVR scenarios. Instead, the capacity to control mental imagery showed a weaker association with presence. We may conclude that individual differences in the degree of perceived presence and mental imagery ability are associated.


Subject(s)
Imagery, Psychotherapy , Individuality , Virtual Reality , Adult , Female , Humans , Imagination , Male , Surveys and Questionnaires
2.
G Ital Med Lav Ergon ; 34(3 Suppl): 526-8, 2012.
Article in Italian | MEDLINE | ID: mdl-23405707

ABSTRACT

The aim of the present article was to verify the Chronic Obstructive Pulmonary Disease (COPD) prevalence in a cohort of quarry workers who belong to the Apricena Marble District. We studied 70 workers. They received a questionnaire about the disease and confounding factors. The spirometry showed that the FEV1 was normal in 95% of workers, instead 5% showed values lower than former (Average: 73%). TNF alpha and IL-1 Beta in Exhaled breath condensate (EBC) were lower than the method limit in all workers. Our cohort is limited, but we could retain that the lung disease is not present in workers taken into consideration. Our results are in according to Rushton who demonstrated that only a prolonged occupation, higher than thirty years, is able to induce lung disease.


Subject(s)
Extraction and Processing Industry , Occupational Health , Pulmonary Disease, Chronic Obstructive , Calcium Carbonate , Forced Expiratory Volume , Humans , Italy , Pilot Projects , Pulmonary Disease, Chronic Obstructive/epidemiology , Pulmonary Disease, Chronic Obstructive/physiopathology
3.
Protein Pept Lett ; 16(9): 999-1005, 2009.
Article in English | MEDLINE | ID: mdl-19799549

ABSTRACT

Polyribonucleotide phosphorilase from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (PhPNPase) has been purified. This enzyme catalyzes both the RNA polymerisation and degradation reaction, showing the highest activity at temperatures below 40 degrees C. PhPNPase is quite sensitive to heat treatment and it is endowed with remarkable halotolerance.


Subject(s)
Polyribonucleotide Nucleotidyltransferase/chemistry , Amino Acid Sequence , Enzyme Stability , Molecular Sequence Data , Molecular Weight , Polyribonucleotide Nucleotidyltransferase/metabolism , Pseudoalteromonas/enzymology , Sequence Alignment , Temperature
4.
Br J Cancer ; 96(7): 1118-26, 2007 Apr 10.
Article in English | MEDLINE | ID: mdl-17375048

ABSTRACT

Fascin, an actin-bundling protein involved in cell motility, has been shown to be upregulated in several types of carcinomas. In this study, we investigated the expression of fascin in 228 advanced colonic adenocarcinoma patients with a long follow-up. Fascin expression was compared with several clinicopathologic parameters and survival. Overall, fascin immunoreactivity was detected in 162 (71%) tumours with a prevalence for right-sided tumours (P<0.001). Fascin correlated significantly with sex, tumour grade and stage, mucinous differentiation, number of metastatic lymph nodes, extranodal tumour extension, and the occurrence of distant metastases. Patients with fascin-expressing tumours experienced a shorter disease-free and overall survival in comparison with those with negative tumours, and fascin immunoreactivity emerged as an independent prognostic factor in the multivariate analysis. Moreover, patients with the same tumour stages could be stratified in different risk categories for relapse and progression according to fascin expression. Our findings suggest that fascin is a useful prognostic marker for colonic adenocarcinomas.


Subject(s)
Adenocarcinoma/metabolism , Carrier Proteins/metabolism , Colonic Neoplasms/metabolism , Microfilament Proteins/metabolism , Adenocarcinoma/pathology , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/metabolism , Colonic Neoplasms/pathology , Female , Humans , Immunoenzyme Techniques , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , Prognosis , Survival Rate
5.
Q J Nucl Med Mol Imaging ; 50(4): 272-87, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17043625

ABSTRACT

Neuroendocrine tumors of the lung are carcinomas characterized by different impact on the patients' prognosis, ranging from relatively indolent, low- to intermediate-grade neoplasms with longer life expectation (i.e., typical and atypical carcinoids) to very aggressive and poorly differentiated neoplasms with dismal prognosis (i.e., large cell neuroendocrine carcinoma and small cell lung cancer). The standard treatment of typical or atypical carcinoids is the complete surgical resection, whereas the role of radio-chemotherapy in a multimodality treatment or for palliation remains controversial. Conversely, high-grade neuroendocrine carcinomas are in primis treated by aggressive combination chemotherapy, deserving surgical resection for uncommon low-stage tumors. Since evidence has been accumulated that neuroendocrine tumors of the lung are supplied with a wide array of peptide receptors detectable on cell membranes by immunohistochemical methods, innovative strategies for diagnosis and radiometabolic therapy have been devised to target these molecules for the correct clinical management of the patients. In this paper, the structural and functional aspects and the clinical applications of the detection of several peptide receptors in pulmonary neuroendocrine tumors will be reviewed, including somatostatin receptors, vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide family receptors, cholecystokinin /gastrin receptors, bombesin/gastrin releasing peptide receptors, neurotensin receptors, substance P receptors, neuroepeptide Y receptors, calcitonin/calcitonin gene-related peptide receptors, atrial natriuretic peptide receptors, glucagon-like-peptide-1 receptors, oxytocin receptors and endothelin receptors. Only a detailed knowledge of the peptide receptor distribution in these tumor types, especially in uncommon neoplasms such as atypical carcinoids and large cell neuroendocrine carcinomas, is pivotal for planning the most adequate interventions for the patients' diagnosis and therapy.


Subject(s)
Carcinoma, Neuroendocrine , Lung Neoplasms , Radioisotopes , Receptors, Peptide/metabolism , Carcinoma, Neuroendocrine/diagnostic imaging , Carcinoma, Neuroendocrine/metabolism , Carcinoma, Neuroendocrine/radiotherapy , Drug Delivery Systems/methods , Humans , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/metabolism , Lung Neoplasms/radiotherapy , Radioisotopes/pharmacokinetics , Radioisotopes/therapeutic use , Radionuclide Imaging , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/therapeutic use
6.
G Ital Med Lav Ergon ; 28(2): 210-2, 2006.
Article in Italian | MEDLINE | ID: mdl-16805471

ABSTRACT

During 2005, the Unit of Occupational Health of the Local Health Centre FG/1 started to record accident at work. The results confirme "Industry" as the sector with the highest risk of accidents at work. Many accidents at work seem to be in relation with the insufficient use of D.P.I. or with the wrong use of D.P.I. The results point out the necessity of a better model to record data and in particular the data that describe what/how has the accident happened and the sector/subsector of workers. In order to continue this activity during the next years, it will be useful to control the trend.


Subject(s)
Accidents, Occupational/statistics & numerical data , Adult , Female , Humans , Italy , Male , Middle Aged , Occupational Health
7.
Biochimie ; 88(10): 1377-89, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16713057

ABSTRACT

A psychrophilic superoxide dismutase (SOD) has been characterized from the Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph). PhSOD is a homodimeric iron-containing enzyme and displays a high specific activity, even at low temperature. The enzyme is inhibited by sodium azide and inactivated by hydrogen peroxide; it is also very sensitive to peroxynitrite, a physiological inactivator of the human mitochondrial Mn-SOD. Even though PhSOD is isolated from a cold-adapted micro-organism, its heat stability is well above the maximum growth temperature of P. haloplanktis, a feature common to other Fe- and Mn-SODs. The primary structure of PhSOD was determined by a combination of mass spectrometry and automated Edman degradation. The polypeptide chain is made of 192 amino acid residues, corresponding to a molecular mass of 21251 Da. The alignment with other Fe- and Mn-SODs showed a high amino acid identity with Fe-SOD from Vibrio cholerae (79%) and Escherichia coli (70%). A significant similarity is also shared with human mitochondrial Mn-SOD. PhSOD has the unique and highly reactive Cys57 residue, located in a variable region of the protein. The three-dimensional model of the PhSOD monomer indicates that Cys57 is included in a region, whose structural organization apparently discriminates between dimeric and tetrameric SODs. This residue forms a disulfide adduct with beta-mercaptoethanol, when this reducing agent is added in the purification procedure. The reactivity of Cys57 leads also to the formation of a disulfide bridge between two PhSOD subunits in specific denaturing conditions. The possible modification of Cys57 by physiological thiols, eventually regulating the PhSOD functioning, is discussed.


Subject(s)
Cysteine/chemistry , Pseudoalteromonas/enzymology , Superoxide Dismutase/chemistry , Amino Acid Sequence , Cysteine/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Molecular Weight , Pseudoalteromonas/metabolism , Sequence Alignment , Superoxide Dismutase/isolation & purification , Temperature
8.
Int J Biol Macromol ; 39(1-3): 122-6, 2006 Aug 15.
Article in English | MEDLINE | ID: mdl-16580720

ABSTRACT

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN1.


Subject(s)
Antigens, Neoplasm/isolation & purification , Glycoproteins/isolation & purification , Membrane Proteins/isolation & purification , Sialoglycoproteins/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/metabolism , Breast Neoplasms/chemistry , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Fetus/chemistry , Fetus/metabolism , Glycoproteins/chemistry , Glycoproteins/metabolism , Humans , Leukosialin , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Sialoglycoproteins/chemistry , Sialoglycoproteins/metabolism , Thymus Gland/chemistry , Thymus Gland/metabolism
9.
Eur J Surg Oncol ; 31(3): 232-6, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15780556

ABSTRACT

AIM OF THE STUDY: Assessment of biological features and treatment of patients with breast cancer presenting during pregnancy or lactation. PATIENTS AND METHODS: Immunohistochemical analysis of estrogen receptor (ER) and progesterone receptor (PgR), Ki-67, HER2/neu, prognostic markers, treatment and follow-up of 21 patients with breast cancer during pregnancy (BCdP) and 17 with breast cancer during lactation (BCdL) are presented. RESULTS: Median age was 36 and 33 years, median tumour size was 2.4 and 2.5 cm, axillary lymph nodes were positive in 10 of 21 pregnant patients and 11 of 17 lactating patients, respectively. Both ER and PgR were not expressed in six of 21 pregnant women and nine of 17 lactating patients. All the six women who had concurrent diagnosis of breast cancer and pregnancy (first trimester) preferred termination of pregnancy although an alternative option was discussed. Five patients received anthracycline containing chemotherapy during the second and third trimester with no complications for patient and child. Conservative surgery was performed in 15 of 21 patients during pregnancy with no local reappearance after a median follow-up of 24 months. Three pregnant women underwent lymphoscintigraphy and sentinel lymph node biopsy. CONCLUSIONS: Patients who had concurrent diagnosis of breast cancer and pregnancy (early first trimester) preferred termination of pregnancy to allow easier completion of treatment. Conservative surgery was safe also in women with BCdP. Sentinel node biopsy might be considered for pregnant patients with a clinically negative axilla.


Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/diagnosis , Breast Neoplasms/therapy , Lactation , Pregnancy Complications, Neoplastic/diagnosis , Pregnancy Complications, Neoplastic/therapy , Abortion, Legal , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Axilla , Breast Neoplasms/chemistry , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Chemotherapy, Adjuvant , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Ki-67 Antigen/analysis , Lymph Node Excision , Lymphatic Metastasis , Mastectomy, Modified Radical , Mastectomy, Segmental , Predictive Value of Tests , Pregnancy , Pregnancy Complications, Neoplastic/drug therapy , Pregnancy Complications, Neoplastic/pathology , Pregnancy Complications, Neoplastic/surgery , Prognosis , Receptor, ErbB-2/analysis , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Sentinel Lymph Node Biopsy , Treatment Outcome
10.
Biochimie ; 86(12): 883-92, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15667938

ABSTRACT

A thioredoxin reductase (TrxR) has been identified in the hyperthermophilic archaeon Sulfolobus solfataricus (Ss). This enzyme is a homodimeric flavoprotein that was previously identified as NADH oxidase in the same micro-organism ('Biotechnol. Appl. Biochem. 23 (1996) 47'). The primary structure of SsTrxR is made of 323 amino acid residues and contains two putative betaalphabeta regions for the binding of FAD, and a NADP(H) binding consensus sequence in the proximity of a CXXC motif. These findings indicate that SsTrxR is structurally related to the class II of the pyridine nucleotide-disulphide oxidoreductases family. Moreover, the enzyme exhibits a NADP(H) dependent thioredoxin reductase activity requiring the presence of FAD. Surprisingly, the reductase activity of SsTrxR is reduced in the presence of a specific inhibitor of mammalian TrxR. This finding demonstrates that the archaeal enzyme, although structurally related to eubacterial TrxR, is functionally closer to eukaryal enzymes. Experimental evidences indicate that a disulphide bridge is required for the reductase but also for the NADH oxidase activity of the enzyme. These results are further supported by the significantly reduced activities exerted by the C147A mutant. The integrity of the CXXC motif is also involved in the stability of the enzyme.


Subject(s)
Archaea/enzymology , Archaea/metabolism , NADH, NADPH Oxidoreductases/isolation & purification , Sulfolobus solfataricus/enzymology , Thioredoxin-Disulfide Reductase/chemistry , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Binding Sites , Cloning, Molecular , Consensus Sequence , Cysteine/metabolism , Disulfides/chemistry , Enzyme Stability , Flavin-Adenine Dinucleotide/analysis , Kinetics , Molecular Sequence Data , Molecular Weight , Mutation , NADH, NADPH Oxidoreductases/analysis , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidases , Sequence Homology, Amino Acid , Sulfolobus solfataricus/chemistry , Sulfolobus solfataricus/genetics , Temperature , Thioredoxin-Disulfide Reductase/analysis , Thioredoxin-Disulfide Reductase/genetics , Thioredoxin-Disulfide Reductase/isolation & purification , Thioredoxin-Disulfide Reductase/metabolism
11.
Ann Ig ; 15(5): 457-67, 2003.
Article in Italian | MEDLINE | ID: mdl-14969298

ABSTRACT

From the birth of doctrines of Risk Management to today a lot of time is passed. From the initial application in the field of the insurances and the management of the enterprises, theories inspired to the identification, evaluation and correction of connected risks to the activity and the industrial trial has been figurative to the health, field in which the application of these principles results to be how much never profit and productive of benefits above all for the patients that suffer consequences of errors but also for the physicians and the personnel that, perfectly inserted in an organization aware of the trials to put into effect, can bring their contribution to underline the weak points of the relief trial. The economic cost and consequences of errors can decrease if a new culture is established inspired to the learning and the communication of the adverse events, to minimize the possibility that they again occurs.


Subject(s)
Risk Management , Risk Management/classification , Risk Management/methods
12.
Biochemistry ; 40(44): 13143-8, 2001 Nov 06.
Article in English | MEDLINE | ID: mdl-11683622

ABSTRACT

Elongation factor 1alpha from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1alpha) carries the aminoacyl tRNA to the ribosome; it binds GDP or GTP, and it is also endowed with an intrinsic GTPase activity that is triggered in vitro by NaCl at molar concentrations [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. The structural properties of SsEF-1alpha were investigated by Fourier transform infrared spectroscopy. The estimation of the secondary structure of the SsEF-1alpha*GDP complex, made by curve fitting of the amide I' band or by factor analysis of the amide I band, indicated a content of 34-36% alpha-helix, 35-40% beta-sheet, 14-19% turn, and 7% unordered structure. The substitution of the GDP bound with the slowly hydrolyzable GTP analogue Gpp(NH)p induced a slight increase in the alpha-helix and beta-sheet content. On the other hand, the alpha-helix content of the SsEF-1alpha*GDP complex increased upon addition of salts, and the highest effect was produced by 5 M NaCl. The thermal stability of the SsEF-1alpha*GDP complex was significantly reduced when the GDP was replaced with Gpp(NH)p or in the presence of NaBr or NH4Cl, whereas a lower destabilizing effect was provoked by NaCl and KCl. Therefore, the extent of the destabilizing effect of salts depended on the nature of both the cation and the anion. The data suggested that the sodium ion was responsible for the induction of the GTPase activity, whereas the anion modulated the enzymatic activity through destabilization of particular regions of SsEF-1alpha. Finally, the infrared data suggested that, in particular region(s) of the polypeptide chain, the SsEF-1alpha*Gpp(NH)p complex possesses structural conformations which are different from those present in the SsEF-1alpha*GDP complex.


Subject(s)
Guanosine Diphosphate/chemistry , Peptide Elongation Factor 1/chemistry , Sodium Chloride/pharmacology , Sulfolobus/chemistry , Sulfolobus/metabolism , Anions/chemistry , Binding Sites , Cations/chemistry , Crystallography , Guanine Nucleotides/metabolism , Peptide Elongation Factor 1/isolation & purification , Protein Denaturation , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared
13.
EMBO J ; 20(19): 5305-11, 2001 Oct 01.
Article in English | MEDLINE | ID: mdl-11574461

ABSTRACT

The crystal structure of elongation factor 1alpha from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF-1alpha.GDP) at 1.8 A resolution is reported. As already known for the eubacterial elongation factor Tu, the SsEF-1alpha.GDP structure consists of three different structural domains. Surprisingly, the analysis of the GDP-binding site reveals that the nucleotide- protein interactions are not mediated by Mg(2+). Furthermore, the residues that usually co-ordinate Mg(2+) through water molecules in the GTP-binding proteins, though conserved in SsEF-1alpha, are located quite far from the binding site. [(3)H]GDP binding experiments confirm that Mg(2+) has only a marginal effect on the nucleotide exchange reaction of SsEF-1alpha, although essential to GTPase activity elicited by SsEF-1alpha. Finally, structural comparisons of SsEF- 1alpha.GDP with yeast EF-1alpha in complex with the nucleotide exchange factor EF-1beta shows that a dramatic rearrangement of the overall structure of EF-1alpha occurs during the nucleotide exchange.


Subject(s)
Archaeal Proteins/chemistry , Guanosine Diphosphate/chemistry , Peptide Elongation Factor 1/chemistry , Sulfolobus/chemistry , Binding Sites , Crystallography , Guanine Nucleotides/metabolism , Magnesium , Protein Structure, Tertiary
14.
Ital Heart J ; 2(6): 462-7, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11453584

ABSTRACT

BACKGROUND: The aim of this study was to evaluate exercise performance in patients affected by anorexia nervosa. METHODS: We studied 19 patients (all females, mean age 23.1 +/- 5.2 years) affected by anorexia nervosa (mean weight 37.3 kg, body mass index 14.04 +/- 1.4 kg/m2) and 20 constitutionally thin women, matched for age, height and physical activity, with a body mass index < 19 kg/m2. All these women underwent clinical examination, standard ECG and a cardiopulmonary stress test. RESULTS: Patients affected by anorexia nervosa showed a lower heart rate and systolic blood pressure at peak exercise (148.8 +/- 13.8 vs 171 +/- 9.2 b/min, p < 0.001, and 130 +/- 9.5 vs 152 +/- 11.2 mmHg, p < 0.001), work load (85.5 +/- 15.1 vs 117.2 +/- 20.3 W, p < 0.001), rate-pressure product (19 371 +/- 2391 vs 25,986 +/- 2218 b/min/mmHg, p < 0.001), oxygen uptake (VO2) at rest and maximum VO2 (5.4 +/- 1.7 vs 7.1 +/- 1.1 ml/kg/min, p < 0.001, and 28.08 +/- 6.3 vs 40.2 +/- 7.1 ml/kg/min, p < 0.001), anaerobic threshold (15.7 +/- 1.9 vs 20.4 +/- 2.1 ml/kg/min, p < 0.001), VO2 during exercise (9.5 +/- 1.2 vs 12.8 +/- 1.3 ml/min/W, p < 0.001), maximum minute ventilation (34.5 +/- 9.9 vs 48.4 +/- 10.3 /min, p < 0.001), and oxygen pulse (7.2 +/- 2 vs 10.9 +/- 2.4 ml/b, p < 0.001). CONCLUSIONS: These data show an abnormal working capacity and cardiovascular responses to exercise in patients affected by anorexia nervosa. The low VO2, both at rest and during exercise, allows them to maintain a relatively high level of physical activity, which contributes to increase the energy expenditure needed for weight loss.


Subject(s)
Anorexia Nervosa/physiopathology , Exercise Test , Adolescent , Adult , Blood Pressure/physiology , Body Mass Index , Body Weight , Child , Electrocardiography , Female , Heart Rate/physiology , Humans , Oxygen/blood , Thyroid Function Tests , Thyrotropin/blood , Thyroxine/blood , Triiodothyronine/blood , Women's Health , Work Capacity Evaluation
15.
Eur J Biochem ; 268(6): 1794-801, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11248699

ABSTRACT

The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol. 286, 189--205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH(2)SO(2)F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH(2)SO(2)F. Refinement of the crystal model of SsSOD confirmed that a phenylmethanesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH(2)SO(2)F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD.


Subject(s)
Enzyme Inhibitors/pharmacology , Phenylmethylsulfonyl Fluoride/pharmacology , Sulfolobus/enzymology , Superoxide Dismutase/antagonists & inhibitors , Amino Acid Sequence , Base Sequence , Chromatography, High Pressure Liquid , DNA, Recombinant , Hot Temperature , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/isolation & purification , Superoxide Dismutase/metabolism
16.
Biochemistry ; 39(50): 15531-9, 2000 Dec 19.
Article in English | MEDLINE | ID: mdl-11112539

ABSTRACT

The elongation factor Tu was isolated from a psychrophilic eubacterial Antarctic Moraxella strain (MoEF-Tu) and its molecular and functional properties were determined. It catalyzed the synthesis of poly(Phe) and bound specifically guanine nucleotides with an affinity for GDP about 12-fold higher than that for GTP. The affinity toward guanine nucleotides was lower than that of other eubacterial EF-Tu. The intrinsic GTPase activity of MoEF-Tu was hardly detectable but was accelerated by 2 orders of magnitude in the presence of the antibiotic kirromycin (GTPase(k)). Such a property resembled Escherichia coli EF-Tu (EcEF-Tu) even though the affinity of MoEF-Tu for the antibiotic was lower. MoEF-Tu showed a thermophilicity higher than that of EcEF-Tu; its temperature for half-denaturation was 44 degrees C. The MoEF-Tu encoding gene corresponding to E. coli tufA was cloned and sequenced. The translated protein had a calculated molecular weight of 43 288 and contained the GTP-binding sequence motifs. Concerning its primary structure, MoEF-Tu showed sequence identity with E. coli and Thermus thermophilus EF-Tu equal to 84% and 74%, respectively, while the identity with EF-1 alpha from the archaeon Sulfolobus solfataricus was equal to 32%.


Subject(s)
Moraxella/chemistry , Moraxella/genetics , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , Genes, Bacterial , Molecular Sequence Data , Sequence Alignment
17.
Eur J Biochem ; 267(19): 6012-8, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10998062

ABSTRACT

The archaeal Sulfolobus solfataricus elongation factor 1alpha (SsEF-1alpha) bound to GTP or to its analogue guanyl-5'-yl imido diphosphate [Gpp(NH)p] formed a ternary complex with either Escherichia coli Val-tRNAVal or Saccharomyces cerevisiae Phe-tRNAPhe as demonstrated by gel-shift and gel-filtration experiments. Evidence of such an interaction also came from the observation that SsEF-1alphaz.rad;Gpp(NH)p was able to display a protective effect against either the spontaneous deacylation or the digestion of aminoacyl-tRNA by RNase A. Protection against the deacylation of aminoacyl-tRNA allowed evaluatation of the affinity of SsEF-1alphaz. rad;Gpp(NH)p for both aminoacyl-tRNAs used. The K'd values of the ternary complex containing S. cerevisiae Phe-tRNAPhe or E. coli Val-tRNAVal were 0.3 microM and 4.4 microM, respectively. In both cases, the affinity of SsEF-1alphaz.rad;Gpp(NH)p for aminoacyl-tRNA was three orders of magnitude lower than that of the homologous eubacterial ternary complexes, but comparable with the affinity shown by the ternary complex involving eukaryal EF-1alpha [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. 60, 47-77]. As already observed with eukaryal EF-1alpha, SsEF-1alpha in its GDP-bound form was also able to protect the ester bond of aminoacyl-tRNA, even though with a 10-fold lower efficiency compared with SsEF-1alphaz.rad;Gpp(NH)p. The overall results indicated that the archaeal elongation factor 1alpha shares several properties with eukaryal EF-1alpha but not with eubacterial EF-Tu.


Subject(s)
Archaeal Proteins/metabolism , Guanosine Triphosphate/metabolism , Peptide Elongation Factor 1/metabolism , RNA, Archaeal/metabolism , RNA, Bacterial/metabolism , RNA, Fungal/metabolism , RNA, Transfer, Amino Acyl/metabolism , Escherichia coli/metabolism , Evolution, Molecular , Guanosine Diphosphate/metabolism , Macromolecular Substances , Ribonuclease, Pancreatic/metabolism , Saccharomyces cerevisiae/metabolism , Species Specificity , Sulfolobus/metabolism
18.
J Biol Chem ; 275(2): 895-900, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10625624

ABSTRACT

A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus. The enzyme is a homodimer with M(r) 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 degrees C in the absence or presence, respectively, of 8 M urea. The enzyme did not show any reductase activity. The SsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative betaalphabeta-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47-54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase.


Subject(s)
Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/metabolism , Sulfolobus/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Enzyme Stability , Flavin-Adenine Dinucleotide/analysis , Hot Temperature , Kinetics , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , NADH, NADPH Oxidoreductases/genetics , NADPH Oxidases , Protein Denaturation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Sulfolobus/genetics , Thermodynamics , Thioredoxin-Disulfide Reductase/chemistry , Urea
19.
Biochemistry ; 38(38): 12288-95, 1999 Sep 21.
Article in English | MEDLINE | ID: mdl-10493796

ABSTRACT

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.


Subject(s)
Guanine Nucleotides/metabolism , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/metabolism , Peptide Elongation Factor Tu/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Recombinant Fusion Proteins/metabolism , Binding Sites/genetics , Computer Simulation , Escherichia coli/chemistry , Escherichia coli/genetics , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Hot Temperature , Hydrolysis , Macromolecular Substances , Models, Molecular , Peptide Biosynthesis/genetics , Peptide Elongation Factor 1/chemistry , Peptide Elongation Factor Tu/chemistry , Peptide Fragments/chemistry , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Sulfolobus/chemistry , Sulfolobus/genetics , Temperature , Tritium
20.
FEBS Lett ; 451(2): 109-12, 1999 May 21.
Article in English | MEDLINE | ID: mdl-10371148

ABSTRACT

In Sulfolobus solfataricus the binding of the exchange factor 1beta (SsEF-1beta) to SsEF-1alpha-GDP displaces the nucleotide and the SsEF-1alpha-SsEF-1beta complex is formed. The complex itself is stable, but it dissociates upon the addition of GDP or Gpp(NH)p but not ATP. Since the rate of the formation of the SsEF-1alpha-SsEF-1beta complex is significatively slower than the rate of the nucleotide exchange catalyzed by SsEF-1beta it can be inferred that in vivo the GDP/GTP exchange reaction proceeds via an SsEF-1alpha-SsEF-1beta interaction without involving the formation of a stable binary complex as an intermediate.


Subject(s)
Proteins/metabolism , Sulfolobus/chemistry , Sulfolobus/metabolism , Guanine Nucleotide Exchange Factors , Kinetics , Protein Binding , Proteins/isolation & purification , Temperature , Time Factors
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