Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin.

The retinol carrier retinol-binding protein (RBP) forms a complex with the thyroid hormone binding protein transthyretin in the plasma of a number of vertebrate species. The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with transthyretin have been investigated by means of fluorescence anisotropy studies. The presence of two independent and equivalent RBP binding sites per transthyretin molecule has been established for proteins purified from species distant in evolution. Although the natural ligand retinol participates in the interaction between retinol-RBP and transthyretin, its binding to RBP is not a prerequisite for protein-protein interaction. The dissociation constants of human transthyretin binding liganded and unliganded forms of human RBP were determined to be: all-trans retinol-RBP, Kd approximately 0.2 microM; apoRBP, Kd approximately 1.2 microM; all-trans retinoic acid-RBP, Kd approximately 0.8 microM; all-trans retinyl methyl ether-RBP, Kd approximately 6 microM. The complex of RBP with the synthetic retinoid fenretinide, which bears the bulky hydroxyphenyl end group, exhibits negligible affinity for transthyretin. The replacement of RBP-bound retinol with synthetic retinoids affects RBP-transthyretin recognition to an extent that appears to be well correlated with the nature and steric hindrance of the groups substituting the retinol hydroxyl group, consistent with their location at the interface between the contact areas of RBP and transthyretin.

Specific interaction of lipoate at the active site of rhodanese.

Dihydrolipoate is an acceptor of the rhodanese-bound sulfane sulfur atom, as shown by analysis of the elementary steps of the reaction catalyzed by rhodanese. The crystal structure of sulfur-substituted rhodanese complexed with the non-reactive oxidized form of lipoate has revealed that the compound is bound at the enzyme active site, with the dithiolane ring buried in the interior of the cavity and the carboxylic end pointing towards the solvent. One of the sulfur atoms of the ligand in the unproductive complex is relatively close to the sulfane sulfur bound to Cys-247, the sulfur that is transferred during the catalytic reaction. This mode of binding of lipoate is likely to mimic that of dihydrolipoate. The results presented here support the possible role of dihydrolipoate as sulfur-acceptor substrate of rhodanese in an enzymatic reaction that might serve to provide iron-sulfur proteins with inorganic sulfide.

Structure of chicken plasma retinol-binding protein.

The crystal structure of the specific carrier of retinol (retinol-binding protein, RBP) purified from chicken plasma has been determined (space group P2(1)2(1)2(1), with a=46.06(5) A, b=53.56(6) A, c=73.41(8) A, and one protein molecule in the asymmetric unit). Despite being obtained from a species phylogenetically distant from mammals, chicken holoRBP has an overall structure that closely resembles the previously determined structures of mammalian holoRBPs. The lack in chicken RBP of eight carboxy-terminal amino acid residues characteristic of mammalian RBPs does not significantly affect the protein structure. A distinctive feature of the avian protein is a better definition of the loop 63-67, close to the opening of the beta-barrel cavity accommodating the retinol molecule, which is rather disordered in the structures of mammalian RBPs.

Structure at 1.44 A resolution of an N-terminally truncated form of the rat serum complement C3d fragment.

Complement component C3 plays a key role in the complement-mediated immune defence, and occupies a central position within the complement cascade system. One of its degradation products, C3dg, was purified from rat serum and crystallised in two different crystal forms as N-terminally truncated fragment. Despite the truncation and the lack of a significant portion of the N-terminus as compared to C3d, the structure of the fragment is highly similar to that of recombinant human C3d (Nagar et al., Science 280 (1998) 1277-1281). Structural details of the reactive site have been obtained, suggesting a possible mode of thioester bond formation between Cys-1010 and Gln-1013 and thioester bond cleavage in the transacylation reaction involving His-1126. The truncation at the N-terminus of C3d leads to the exposure of a surface of the molecule that favours dimerisation, so that in both crystal forms, the fragment is present as a dimer, with monomers related by a two-fold axis.

Crystallization of human plasma apo-retinol-binding protein.

Crystals of three forms of human plasma apo-retinol-binding protein have been obtained using the procedure described for the holoprotein. The apoprotein was prepared by a novel method, which uses hydrophobic interaction and immobilized dye chromatography. The three forms were separated by fast protein liquid chromatography. All of the crystals are isomorphous and diffract to 2.5 A resolution. These crystals will be useful for studies of the mechanism of binding of retinol to its carrier using X-ray diffraction techniques.

Crystal structure of the trigonal form of human plasma retinol-binding protein at 2.5 A resolution.

The three-dimensional structures of the liganded and unliganded forms of human plasma retinol binding protein (RBP) in the trigonal crystal form have been solved at 2.5 A resolution. The final model of RBP complexed with retinol (holoRBP, space group R3, a = b = 104.0 A, c = 74.4 A) has a crystallographic R factor of 0.176 for 9652 reflections. The unliganded form, obtained through a purification procedure which included steps based on hydrophobic interaction chromatography, crystallized isomorphously with holoRBP and its structure has been refined to an R factor of 0.190 for 9614 reflections. The structure of the trigonal holo protein is quite similar to that of the orthorhombic form: the root-mean-square deviation of all the equivalent alpha-carbons in the two chains is 0.53 A. The structural comparison between the liganded and unliganded forms of RBP in the crystal did not reveal gross conformational changes. The most significant difference between the two forms of the protein is a conformational change involving residues from 34 to 37. In this region, the movements of side-chains of Leu35 and Phe36 are most noticeable. In particular, in the unliganded form the side-chain ring of the latter residue is in the place previously occupied by the alcoholic moiety of retinol. Our data are consistent with a model in which a region comprising these residues and at least part of the opening of the beta-barrel is involved in the recognition between RBP and transthyretin. In the case of the unliganded form, the central cavity, that is occupied by the vitamin in the two human crystalline holoRBPs, is filled by electron density that, at the present resolution, we interpret as solvent.

Inflammatory mediators and eosinophilia in atopic and non-atopic patients with nasal polyposis.

Nasal polyps are characterized by eosinophilic infiltration and presence of inflammatory mediators, such as total IgE, eosinophil cationic protein (ECP) and cytokines. The role of atopy in nasal polyp pathogenesis is still unclear. Therefore, we evaluated serum IgE levels, nasal mucus concentrations of ECP and cytokines and the number of infiltrating eosinophils in nasal tissue of polyps from atopic and non-atopic patients. Samples were obtained from a randomized population of 31 patients with nasal polyposis having endonasal sinus surgery and of 13 control subjects undergone corrective surgery of the nasal septum. On the basis of medical history of allergy, positive skin-prick tests and total IgE levels, patients with polyposis were divided in atopic (n = 13) and non-atopic (n = 18) patients. We determined levels of IgE in blood, ECP and cytokines (IL-4, IL-6, IL-8, IFN-gamma and IL-2) in nasal mucus, and number of infiltrating eosinophils in nasal tissue. The concentrations of total IgE, ECP, IL-4 and IL-8 and eosinophilia were significantly higher in all patients with nasal polyps compared with controls. Inside, all patients with nasal polyposis showed lower levels of IL-6, IFN-gamma and IL-2 compared with controls. The atopic patients showed significant differences when compared with non-atopic patients for the higher concentrations of total IgE (698.80+/-322.24 vs. 279.63+/-234.11; P < 0.0001) and IL-8 (1437.2 pg/ml+/-1250.7 vs. 605.5 pg/ml+/-481.1; P < 0.015). These findings suggest that inflammation still remains the major factor in the etiology of nasal polyposis and show different levels of inflammatory mediators into atopic and non-atopic patients.

Interaction of rhodanese with intermediates of oxygen reduction.

Cyanide-promoted inactivation of the enzyme rhodanese [thiosulfate sulfurtransferase (EC 2.8.1.1)] in the presence of ketoaldehydes is caused by reduced forms of molecular oxygen generated during autoxidation of the reaction products. The requirement of both catalase and superoxide dismutase to prevent rhodanese inactivation indicates that hydroxyl radical could be the most efficient inactivating agent. Rhodanese, also in the less stable sulfur-free form, shows a different sensitivity towards oxygen activated species. While the enzyme is unaffected by superoxide radical, it is rapidly inactivated by hydrogen peroxide. The extent of inactivation depends on the molar ratio between sulfur-free enzyme and oxidizing agent. Fully inactive enzyme is reactivated by reduction with its substrate thiosulfate.

In vitro interaction of fenretinide with plasma retinol-binding protein and its functional consequences.

The synthetic retinoid fenretinide (4-HPR; N-[4-hydroxyphenyl] all-trans-retinamide) interacts with plasma apo-retinol-binding protein (RBP) to form a tight complex (K'd approximately 0.2 microM) which does not exhibit binding affinity to transthyretin (TTR). Therefore, a substantial modification of the retinol hydroxyl group does not appear to affect the interaction with RBP but does drastically interfere with the protein-protein recognition. The remarkable early reduction in plasma retinol level induced by fenretinide administration may be associated with the high binding affinity of this retinoid to RBP and to its interference with the RBP-TTR complex formation.

The chemical composition of standard cotton dust.

Cotton dust samples from Cotton Incorporated were investigated by X-ray fluorescence and proximate analysis methods. These dust samples are known as "standard cotton dust" and have been used by many researchers investigating the causative agent(s) and physiological mechanisms of byssinosis. Silicon, calcium, potassium, and aluminum were present in relatively high concentrations (1-4%) in the dust fractions. The ash content of the dust fractions increased as the fraction particle size decreased. Proximate analyses demonstrate that "noncellulosic organics" are the major class of constituents (35-45%) in cotton dust. Cellulose comprises only 10-15% of the dust, while water-extractable materials comprise approximately 20% of the dust. Capillary gas chromatography performed on silylated, freeze-dried, aqueous extracts of the less than or equal to 38 micron dust fraction revealed the presence of phosphate, malate, arabitol, citrate, fructose, glucose, and mannitol.

Social stress, myocardial damage and arrhythmias in rats with cardiac hypertrophy.

In rat models of cardiac hypertrophy (moderate aortic coarctation: ACm, n=18; severe aortic coarctation: ACs, n=27; aging: OLD, n=25; spontaneous chronic hypertension: SHR, n=18) and properly matched control animals (C(ACm), n=17; C(ACs), n=19; C(OLD), n=24; C(SHR), n=22), we investigated the relative contribution of intense autonomic activity and cardiac structural damage to ventricular arrhythmogenesis. We used an "in vivo" to tissue level approach, by correlating in the same animal: (i) social stress-induced ventricular arrhythmias, telemetrically recorded, and (ii) left ventricular weights (LVW) and amount and geometrical properties of myocardial fibrosis (MF). Arterial blood pressure was significantly higher in ACm (+11%), ACs (+28%) and SHR (+34%) than in controls. LVW were approximately 20% greater in ACm, ACs and OLD and 50% greater in SHR. MF was about twice as great and characterized by more frequent occurrence of microscopic scarring in ACm and ACs, and eight times greater and associated with both a higher number and a larger size of fibrotic foci in OLD and SHR compared to controls. Social stress increased ventricular arrhythmia vulnerability in all models of cardiac hypertrophy, as well as in controls. The arrhythmogenic action of stress was facilitated in ACs, OLD and SHR. A correlation between structural cardiac remodeling and ventricular arrhythmias was found only in SHR and OLD, which exhibited the greatest increase in LVW and/or MF. Social stress proved to be a valuable tool for analyzing the combined effects of autonomic stimulation and altered myocardial substrate on the genesis of potentially life-threatening arrhythmias in social animals.

[An increase in bile acids in the serum of patients with chronic kidney failure]. / Incremento degli acidi biliari nel siero dei pazienti affetti da insufficienza renale cronica.

Measurement of serum bile acids was performed in 86 uremic patients without any liver or bile tract diseases. Thirty-six patients (23 males and 13 females, aged 21-60 years) were on conservative dietary treatment, whereas 50 uremics (31 males and 19 females, aged 23-82 years) were on maintenance hemodialysis. The assays were made by means of enzymatic procedure and confirmed by RIA method too. Elevated serum concentrations of bile acids (> 6 mumol/L) were found in 24 out of the 86 uremics (27.9%), and the prevalence was similar in patients on conservative (10/36, 27.7%) and on dialysis treatment (14/50, 28%). Then, abnormally elevated concentrations of circulating bile acids are present in a quite high percentage of uremics both on hemodialysis and on conservative dietary therapy. The existence of a subclinical liver disease or an abnormal entero-hepatic cycle of bile acids might explain these findings.

IgE modification of the specific antidermatophagoides during the first year of specific immunotherapy (SIT).

Six subjects (4 female, 2 male), aged from 16 to 25 years, presented with allergic rhinitis to Dermatophagoides mites and received SIT by the sub-cutaneous route with delayed-release alpha fraction Bayropharm at the standard doses. Diagnosis was based on clinical history, skin tests and measurement of specific IgE at 0, 3, 9, and 12 months, by the fluoro-enzymatic technique (FAST). For comparison, in a reference group (n = 20) the IgE varied between 0.32 and 0.11 IU/ml for D1 and 0.31 to 0.09 IU/ml for D2. The eight patients had specific IgE titres of D1 = 0.96, D2 = 0.99. For these authors, the FAST technique used for the measurement of specific IgE, although less sensitive than the RIA technique of RAST, gives a good evaluation of SIT.

Extremely preterm births: end-of-life decisions in European countries.

OBJECTIVE: To explore the influence of end-of-life decisions (EoL-D) on survival and mortality data in the light of differences reported among European countries. DESIGN: We collected the published data of several epidemiological studies: Epicure, Epipage, Epibel and the Norwegian study performed in the UK, France, Belgium and Norway, respectively. The data concerning the Epibel and Norwegian studies are considered for a preliminary analysis, while the data relating to the Epicure and Epipage studies are compared based on the total published data. The statistical analysis was performed through the class of generalised linear models, and more specifically, through log-linear models. The data considered were the number of babies who died in neonatal intensive care units after active withdrawal classified according to the country and gestational age. RESULTS The probability of death after active withdrawal was significantly higher at 22 and 24 weeks' gestational age compared with week 25, when considering both countries (OR, 2.35 and 1.29, respectively). For the week 23(0)-->(6), the probability of death after active withdrawal was not significant; however, it is relevant when considering the OR (1.31). When considering the country, there was a higher probability of death after active withdrawal in France than in the UK, or alternatively, with the assumed baseline French parameter, in the UK, there was a lower probability of death after active withdrawal, with a significant OR=0.69. CONCLUSIONS The attitude towards EoL-D could in part explain the differences in survival data of extremely preterm infants and should be taken in mind when comparing international survival rates.