Psychrophilic superoxide dismutase from Pseudoalteromonas haloplanktis: biochemical characterization and identification of a highly reactive cysteine residue.

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.

Iodine deficiency in Calabria: characterization of endemic goiter and analysis of different indicators of iodine status region-wide.

The distribution of goiter prevalence in schoolchildren (no.=13,984, age 6-14 yr), the neonatal TSH results obtained from the congenital hypothyroidism screening program and the urinary iodine excretion values (no.=284) were employed for the assessment of iodine deficiency in Calabria, a Southern Italy region. Data were collected during the years 1990-1996. In the inland territory, goiter prevalence ranged from 19 to 64%. At sea level, there was a great variability of goiter prevalence, with values varying from 5.3 to 25.7%. The analysis of the neonatal hypothyroidism screening program data (no.=21,078) showed a 14.8% frequency of TSH levels >5 microU/ml whole blood in newborns from the inland territory and a 14.1% frequency at sea level. Urinary iodine excretion resulted (mean+/-SD) 53.8+/-43.4 microg/l (range: <20 to 189 microg/l) in the inland territory and 89.6+/-59.8 microg/l (range: 26 to 333 microg/l) at sea level. Median urinary iodine excretion values in 13 villages or small towns of the inland territory ranged from 31 to 57 microg/l. In 2 major towns located at sea level, the median iodine excretion values were 72 microg/l in Crotone main city and 94 microg/l in Reggio Calabria main city. The data indicated that moderate, with pockets of severe iodine deficiency is present in the inland region while iodine supply varies from sufficient to marginally low in the coastal areas. Mild iodine deficiency was found in a major town located at sea level.

[Extraction and characterization of the lipopolysaccharide of Bartonella quintana] / Estrazione e caratterizzazione del lipopolisaccaride di Bartonella quintana.

Bartonella quintana has been reported as the cause of trench fever, persistent endocarditis, bacteriaemia and has been isolated with an increasing incidence in clinical specimens from AIDS patients. One of the main pathogenic factors of gram-negative bacteria, including B. quintana, is the lipopolysaccharide (LPS). However, very little information is available on the features of Bartonella LPS. The aim of the present study was to extract, purify and characterise B. quintana LPS. The effect of the LPS under scrutiny was also evaluated on TNFa release by means of the "in vitro" human whole blood model of sepsis. The Oklahoma strain of B. quintana was grown on sheep blood agar, at 37 C, in a moist atmosphere containing 5% carbon dioxide. Cells were harvested and washed in sterile and apyrogenic saline solution and LPS extracted following the procedure of Westphal e Jann (1965), modified by Minnick (1994). The LPS of B. quintana showed the migration pattern of a deep rough chemotype, and the chromogenic limulus amoebocyte lysate test (LAL test) revealed strong reactivity at low concentrations (6.2 pg/ml). Samples of human whole blood stimulated by 1000 ng/ml of B. quintana LPS released 1707 378 pg/ml of TNFa.

[Areas in Calabria with an elevated incidence of congenital hypothyroidism and iodine content of drinking water]. / Aree della Calabria con elevata incidenza di ipotiroidismo congenito e contenuto di iodio nelle acque potabili.

The screening of 128,442 newborns, carried out in our laboratory from 1/1/1991 to 31/12/1996, detected 51 cases of congenital hypothyroidism (CH). Due to a 98% coverage, the accurate regional map of CH distribution has been obtained. Two rural and/or mountainous areas showed high incidence, 1/1145 (area A) and 1/1193 (area B), respectively. The incidence of the remaining region is 1/4621, whereas the mean regional incidence is 1/2518. In order to verify a relationship between CH incidence and iodine deficiency, we analyzed the iodine concentration in drinkable water from 148 localities. The high incidence of CH in the A and B areas corresponds with a high percentage of water samples with iodine concentration < 2 micrograms/l (92% and 85% respectively). In 15 coastal localities with low CH incidence, iodine concentration was between 7 and 80 micrograms/l. For populations of the A and B areas showing dietary iodine deficiency due to local product diet, we suggest the iodine prophylaxis as soon as possible.

The statistical analysis of neonatal TSH results from congenital hypothyroidism screening programs provides a useful tool for the characterization of moderate iodine deficiency regions.

TSH data from the congenital hypothyroidism screening program were analyzed in a mild to moderate iodine deficiency region. Neonatal TSH levels were measured at day 4-5 of life in 22,384 infants (99% coverage; 51.1% males, 48.9% females). The cut off TSH value for recall was established at 20 microUl/ml whole blood. TSH values > 20 microUl/ml were excluded from further analysis of the data. The frequency distribution analysis showed that the median neonatal TSH level was 2 microUl/ml and the mode (28% of newborns) corresponded to neonatal TSH values < 1 microUl/ml. TSH levels above 5 microUl/ml were observed in 14.4% children and the 97% cut off was 11 microUl/ml. When examined in relation to the areas of newborn origin, the individual 97% cut off values varied from 8 to 14 microUl/ml. Accordingly, the frequency of TSH levels above the 97% cut off value calculated for the entire newborn series (> 11 microUl/ml) ranged from 2.1% to 4.6%. A significant correlation was found between the frequency of neonatal TSH levels > 11 microUl/ml and both goiter prevalence (r2 = 0.88; p = 0.0019) and median urinary iodine excretion (r2 = 0.86; p = 0.0077) observed in those areas for which epidemiological data were available (n = 7). The results indicate that neonatal TSH data from the congenital hypothyroidism screening programs can be used for monitoring mild to moderate iodine deficiency regions.

[Incidentally discovered masses: diagnostic and therapeutic features]. / Gli incidentalomi: aspetti diagnostici e terapeutici.

The term "incidentaloma", which was first used to indicate a mass discovered by chance at an adrenal level, is currently used to indicate any lesion identified accidentally, namely in the absence of specific symptomatology, during echographic or tomographic examination. The authors report their experience of just over one year and, through a review of the literature, draw attention to the fact that these incidentalomas raise unresolved problems both from a diagnostic and therapeutic point of view. They hope that routine abdominal echography will be extended at least to a selected hospital population given that these lesions, diagnosed by chance, may be malignant or even if benign may have a potential evolution. Obviously surgery in these cases would lead to a marked prognostic improvement.

Hormonal responses in strenuous jumping effort.

In order to test the possibility for rapid responses of blood hormone levels in short-term supramaximal exercises, serum concentrations of corticotropin (ACTH), cortisol (C), total testosterone (tT), free testosterone (fT), growth hormone (GH), thyrotropin (TSH), free thyroxine (fT4), free triiodothyronine (fT3), prolactin (PRL), insulin-like growth factor (IGF-I), and sex hormone-binding globulin (SHBG) were determined by RIA procedures in blood samples obtained before and immediately after a 60-s period of consecutive vertical jumps (Bosco test). The study subjects were 16 Italian professional soccer players. Immediately after exercise, significant increases (p < 0.05) were found in the concentrations of ACTH (by 39%), C (by 14%), TSH (by 20%), fT3 (by 28%), fT4 (by 30%), tT (by 12%), fT (by 13%), and SHBG (by 21%). Significant changes were not detected in the blood levels of GH, IGF-I and PRL. Most pronounced testosterone responses were typical for persons of high jumping performance (the increase of serum tT correlated with average power output, r = 0.61 and jumping height, r = 0.66). The larger the drop in power output during 60-s jumping, the higher was the thyroid response: the difference in jumping height between the first and last 15-s period correlated with increases in TSH (r = 0.52) and in fT4, (r = 0.55). In conclusion, the obtained results indicate that in intense exercise, causing the rapid development of fatigue, rapid increases in serum levels of hormones of the pituitary-adrenocortical, pituitary-gonadal and pituitary-thyroid systems occur.

The subacute levodopa test for evaluating long-duration response in Parkinson's disease.

The clinical relevance of a long-duration response (LDR) to levodopa therapy in Parkinson's disease (PD) has not been widely recognized. In 25 patients with moderate PD, we measured LDR on motor function after short periods of treatment with levodopa (subacute tests). Each subacute test lasted 15 days and consisted of the oral administration of levodopa at various interdose intervals (IDIs) of 48, 24, 12, 8, 6, and 5 hours. The goal for a subacute test was to achieve a satisfactory antiparkinsonian effect before the last levodopa dose (day 15), i.e., an LDR greater than 50% of the maximal response following an acute levodopa test (LDR-endpoint). Twenty-one patients (84%) reached the LDR-endpoint. The IDI at which levodopa was administered clearly differentiated patients who were otherwise clinically indistinguishable when evaluated at baseline off medication or after an acute levodopa test. The IDI schedule that produced a satisfactory LDR was specific for each patient, since longer DIs failed to produce the required LDR, and a shorter IDI schedule (resulting in larger cumulative dosage of levodopa) did not significantly enhance the response. Also, the LDR decay rate after discontinuation of treatment was individual for each patient and independent of the cumulative amount of levodopa administered. Both the IDI schedule and the LDR decay rate may reflect the ability of nigrostriatal neurons to store and to release dopamine formed from the exogenous precursor. The assessment of the LDR to levodopa by subacute tests is useful for establishing the appropriate dose of the drug, as well as for developing levodopa sparing strategies in PD patients.

Elongation factor Tu D138N, a mutant with modified substrate specificity, as a tool to study energy consumption in protein biosynthesis.

Substitution Asp138-->Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP [Hwang & Miller (1987) J. Biol. Chem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle [Weijland & Parmeggiani (1993) Science 259, 1311-1313]. Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process. In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N.XTP was 30% higher than with EF-Tu wild type (wt).GTP. However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems. The K'd values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5-40 degrees C). With rate-limiting amounts of XTP the K'm of its XTPase activity corresponded to the K'm for XTP of poly(phenylalanine) synthesis (0.3-0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

[Diverticulitis of the right colon. Description of our experience]. / Diverticolite del colon destro. Descrizione delle nostre esperienze.

Two cases of diverticultis of the right colon are described which received surgical treatment. These lesions often simulate acute appendicitis and thus represent a intraoperative surprise Differential diagnosis using a neoblastic mass is often impossible. The paper stresses the importance of surgical treatment which in general terms is the same as the treatment of the diverticular mass of the left colon. In cases where it is difficult to decide between a phlogostic and neoblastic pathology, it is advised that a right hemicolectomy should be performed in line with criteria for oncological requirements.

[Treatment of acute cholecystitis and its complications]. / Terapia della colecistite acuta e delle sue complicazioni.

Forty cases of acute cholecystitis and its complications have been reviewed. Examination of the literature and a study of the personal series leads to a discussion on the need or otherwise for early surgery, particularly in those patients in whom a more unfavourable course for the clinical picture can be objectively presumed.

Effect of propan-2-ol on enzymic and structural properties of elongation factor G.

Elongation factor G (EF-G) can support a GTPase activity in vitro even in the absence of ribosomes when propan-2-ol is present [GTPasep; De Vendittis, Masullo & Bocchini (1986) J. Biol. Chem. 261, 4445-4450]. In the present work the GTPasep activity of EF-G was further studied by investigating (i) the effect of ionic environment on GTPasep and (ii) the influence of propan-2-ol on the molecular structure of EF-G as determined by fluorescence and c.d. measurements. In the presence of 1-300 mM univalent cations (M+) alone, no detectable GTPasep activity was measured; however, in the presence of 1 mM-Mg2+ a considerable stimulation was observed at 40 mM-Li+ or 75 mM-NH4+. Among bivalent cations (M2+), 1 mM-Sr2+, 2-5 mM-Ca2+ and 1 mM-Ba2+ were the most effective, but, in the presence of 75 mM-NH4+, Mg2+ and Mn2+ became the most efficient, whereas the stimulation by other M2+ species was considerably decreased. C.d. measurements showed that the alcohol increased the mean molar residue ellipticity of EF-G at 285 nm, but not at 220 nm. As estimated from fluorescence measurements, in the presence of 20% (v/v) propan-2-ol the value of the dissociation constant of the complex formed between EF-G and 8-anilino-1-naphthalene-sulphonate decreased from 8 to 5 microM; similarly, the number of binding sites on EF-G for the fluorescent probe decreased from 13 to 6. Finally, the alcohol enhanced the quenching of the intrinsic fluorescence of EF-G caused by either acrylamide or KI. The data support the hypothesis that propan-2-ol induces moderate conformational changes of EF-G that make the catalytic centre accessible to the substrate even in the absence of ribosomes. Kinetics of GTPasep studied at different temperatures did not reveal additional structural changes of EF-G occurring with time or temperature.

[Neoplasm on cholecysto-jejunal anastomosis: an endoscopic diagnosis]. / Neoplasia su anastomosi colecisto-digiunale: una diagnosi endoscopica.

Bilio-digestive anastomoses represent a tried and tested surgical procedure in lithiasic or neoplastic type obstructions of the main biliary way. Among long-term postoperative complications the case of neoplasia in the stomy is rare. A clinical case of neoplasia on cholecystojejunal anastomosis diagnosed endoscopically is reported. The physiopathological aspects are discussed.

Superoxide dismutase activity in the skin of rats irradiated by He-Ne laser.

The activity of the enzyme superoxide dismutase has been measured in the skin of rats irradiated by a low-power He-Ne laser. The irradiation was performed at the doses of 4, 8 and 20 J/cm2 in a single or repeated treatment. The increase in activity of superoxide dismutase in the skin of irradiated animals was not statistically significant.

Interaction between the different domains of aminoacyl-tRNA and the elongation-factor-Tu x kirromycin complex.

In this work, we have studied the effect of aa-tRNA and derived 3' aminoacylated fragments on the EF-Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes. The aa-tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C-A-Val and Val-tRNA Val1 3' half molecule, as well as a purification method for short 3' aminoacylated fragments based on the amino group charge, were newly developed for this work. Aminoacyl-adenosine was found to be able to stimulate the EF-Tu x kirromycin GTPase, but only to a very small extent. Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A-Val much less than C-A-Val less than C-C-A-Val less than 3' valyladenosine dodecanucleotide much less than Val-tRNA Val1 3' half molecule less than Val-tRNA Val1. The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF-Tu x kirromycin and the stimulation by aa-tRNA, its 3' half molecule and even more by its 3' short fragments. The effect of aa-tRNA and derived 3' fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5-30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa-tRNA and, to a lesser extent, also the effect of the C-C-A-aa as well as the basic activity of the EF-Tu x kirromycin GTPase. The extent of the stimulation by aa-tRNA, and even more by C-C-A-aa, depends on the nature of the amino acid. Among the aminoacyl side chains tested (Arg-, Phe-, Val-, Met-, Leu-, Lys-) arginine was found to be the most active and leucine the least. Our results show that (a) the 3' aminoacylated extremity is of prime importance for the stimulation of the EF-Tu GTPase, (b) in the 3' extremity there are critical sequences for the interaction with EF-Tu and (c) other domains of the aa-tRNA molecule are capable of influencing this reaction: one of the most important is the region including the T psi C loop and stem.

Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu.

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule. The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val. The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys). In the absence of ribosomes and at low [Mg2+], the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs. Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu. The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.