Crystallization and preliminary X-ray study of two liver basic fatty acid-binding proteins.

The fatty acid-binding proteins (FABPs) are a very well known protein family which includes the liver basic FABPs (Lb-FABPs), a subgroup so far characterized in several vertebrates but not in mammals. The most important difference recognized between the proteins in this subgroup and the better known mammalian liver FABPs (L-FABPs) is the stoichiometry of ligand binding: two fatty acid molecules in L-FABPs compared with one in Lb-FABPs. The only Lb-FABP with a known three-dimensional structure is that of chicken Lb-FABP, but the details of ligand binding are still unresolved as the crystals of the protein are grown at an acidic pH and the protein has been shown to lose its ligand under these conditions. The two proteins whose crystallizations are reported here are the second and third members of this subfamily to be crystallized. The crystals of axolotl Lb-FABP belong to either space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 65.38, c = 60.90 A, and diffract to a resolution of 2.0 A on a conventional source at room temperature. The crystals of toad Lb-FABP belong to either space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 48.14, c = 135.23 A, and diffract to 2.5 A resolution under the same conditions. It is expected that the solution of these two structures will help to clarify the structural differences between Lb-FABPs and L-FABPs and will possibly explain the different binding stoichiometries observed in these otherwise so similar protein subfamilies.

Interaction of chicken liver basic fatty acid-binding protein with fatty acids: a 13C NMR and fluorescence study.

Two different groups of liver fatty acid-binding proteins (L-FABPs) are known: the mammalian type and the basic type. Very few members of this second group of L-FABPs have been characterized and studied, whereas most of the past studies were concerned with the mammalian type. The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with 1-(13)C-enriched palmitic acid (PA) and oleic acid (OA) were investigated by (13)C NMR spectroscopy. Samples containing fatty acids (FA) and Lb-FABP at different molar ratios exhibited only a single carboxylate resonance corresponding to bound FA, and showed a binding stoichiometry of 1:1 both for PA and for OA. Fluorescence spectroscopy measurements yielded the same binding stoichiometry for the interaction with cis-parinaric acid [K(d) = 0.38(4) microM]. Competition studies between cis-parinaric acid and the natural ligands indicated a decreasing affinity of chicken Lb-FABP for PA, OA, and retinoic acid (RA). (13)C NMR proved that pH and ionic strength affect complex stability. The carboxyl signal intensity reversibly decreased upon lowering the pH up to 5. The pH dependence of the bound carboxyl chemical shift yielded an apparent pK(a) of 4.8. A decrease of the integrated intensity of the bound carboxylic signal in the NMR spectra was observed while increasing the chloride ion concentration up to 200 mM. This body of evidence indicates that the bound FA is completely ionized at pH 7.4, that its polar head is positioned in a solvent-accessible region, that a FA-protein strong ionic bond is not present, and that high ionic strength causes the release of the bound FA. The reported results show that, insofar as the number of bound ligands and its relative affinity for different FAs are concerned, chicken Lb-FABP is remarkably different from the mammalian liver FABPs, and, within its subfamily, that it is more similar to catfish Lb-FABP while it behaves quite differently from shark or axolotl Lb-FABPs.

Behaviour of inorganic and organic cations in the Debye-Hückel layer of DNA.

Inorganic, monovalent cations (Li, Na, K, Rb, Cs), when present in the Debye-Hückel layer of DNA, are found to bind to the negatively charged groups of the helix solely on the basis of their charge/mass ratio. Thus, when an electric field is applied, the free mobility of the DNA is seen to increase from Li- to Cs-equilibrated DNAs, since the latter cation, having a weaker surface charge distribution and a larger physical size (in the non-hydrated state), is more loosely bound to the DNA helix, thus providing less screening of its negative charges. On the contrary, organic amines (Tris and a number of Good's buffers) are found to bind not only via electrostatic interactions, but by additional bonds, notably H-bonds. In particular, Tris can form two H-bonds, with a purine and pyrimidine, respectively, and a third H-bond shared between the -OH groups of two adjacent Tris. Hence, these buffer components may be unwitting participants in reactions carried out in in vitro systems.

Properties of a stationary phase based on immobilised chicken liver basic fatty acid-binding protein.

The fatty acid-binding proteins (FABPs) are a class of low-molecular-mass proteins that bind fatty acids and are thought to be involved in their intracellular transport. FABPs have been isolated and studied from several tissues, but their precise function and mechanism of action are still not clear. Chicken liver (basic) fatty acid-binding protein (bFABP) was immobilised on aminopropyl silica and the developed stationary phase was used to examine the enantioselective properties of this protein and to study the binding of drugs to bFABP. The retention and enantioselectivity of the new column for a large number of chiral drugs was investigated. The enantiomers of basic and neutral compounds were poorly retained and not resolved by the bFABP column. On the contrary the resolution of the enantiomers of some acidic compounds was obtained. Therefore the influence of the mobile phase pH and organic modifier on the chromatographic performance of acidic compounds was studied. In order to clarify the retention mechanism, competitive displacement studies were also carried out by adding short-chain fatty acids to the mobile phase as displacing agents and preliminary quantitative structure-retention relationship correlations were developed to describe the nature of the interactions between the chemical structures of the analytes and the observed chromatographic results.

Crystal structure of a truncated form of porcine odorant-binding protein.

The odorant-binding proteins (OBPs) are a family of structurally related molecules that are found in high concentrations in the nasal mucus of vertebrates and bind with moderate affinity a large family of hydrophobic odorants. On the basis of their quaternary structure, the OBPs have been classified as monomers, homodimers, and heterodimers. Porcine OBP was believed for a long time to be a monomer under physiological conditions but there are recent data that support the existence of a monomer-dimer equilibrium. We have determined the crystal structure of a monoclinic form of porcine OBP and found that the truncated molecules, which lack the first 8 amino acids, pack in the cell as dimers that appear to have physiological relevance. The presence in the maps of electron density for an endogenous ligand has also let us identify the side chain of the amino acids that are at the ligand-binding site. In addition, an alternative way of access to the central cavity that binds the ligands is suggested by the particular packing of the molecules in this unit cell. Proteins 2001;42:201-209.

Crystallization of chicken liver (basic) fatty acid binding protein after purification in multicompartment electrolyzers with isoelectric membranes.

A preparation of chicken liver (basic) fatty acid binding protein was purified to homogeneity in multicompartment electrolyzers with isoelectric membranes. Large amounts of the isoelectric point (pI) 9.7 protein were collected into a compartment delimited by pI 8.8 and 11.0 membranes. The protein thus purified produced crystals which diffract to higher resolution than those obtained by purification via preparative isoelectric focusing (IEF) in soluble carrier ampholytes. In addition, a novel orthorhombic form with a different molecular packing was obtained. It is hypothesized that, when using conventional IEF, traces of carrier ampholytes could adhere to the protein, particularly in the hydrophobic ligand-binding pocket, rendering the interpretation of the electron density maps difficult. Multicompartment electrolyzers do not present this drawback, since they are based on insoluble buffering species.

The carbohydrates of the isoforms of three avian riboflavin-binding proteins.

The carbohydrate chains of nine isoforms of chicken egg-white riboflavin-binding protein (RfBP) and six isoforms each of quail egg-white and yolk RfBP have been structurally characterized. The two N-glycosylation sites, Asn36 and Asn147, of the most abundant isoform of each of the three proteins were analyzed in further detail leading to the identification of different glycosylation patterns. In both chicken and quail egg-white RfBP the carbohydrates attached to position 36 had a lower degree of branching and, in the case of the quail protein, this site was only partially glycosylated. A very heterogeneous mixture of complex structures was characteristic of the other glycosylation site. Analysis of the two sites in quail yolk RfBP confirmed this result which agrees with what has been established for hen yolk RfBP. The presence in the three proteins of a highly heterogeneous mixture of differently branched glycans suggests that the differences in isoelectric points, which is a peculiarity of the different isoforms, are probably indeed due to differences in carbohydrate structure.

Capillary zone electrophoresis of ds-DNA in isoelectric buffers: effect of adding of competing, nonamphoteric ions.

When separating ds-DNA in isoelectric His buffer (pH=pI=7.6), in the 50-250 mM concentration range, some unique phenomena were observed: improved resolution for smaller DNA fragments, up to ca. 150 bp, and a rapid deterioration of resolution above this critical length (which corresponds to the persistence length). Such phenomenon depended also on voltage and concentration of sieving liquid polymer. Direct binding of His to the DNA helix was hypothesized, with resultant stiffening and an increment of diameter of the DNA fragments, thus inducing an early onset of reptation at the applied voltage in the 100-300 V/cm range. In order to prove this hypothesis, "competing ions" (notably NaCl and KBr) were added to the His background electrolyte: a partial reversal of the His effect was already apparent at low concentrations of such ions (10 mM) and was complete at higher concentrations (30 and 50 mM). By molecular modeling, it was found that His could be docking on the negatively charged oxygen (bound to the phosphate) by offering both charged (primary and tertiary amino) groups to simultaneous binding, thus forming a salt and neutralizing the negative charge borne by the oxygen. The following characteristic bond distances were found: 0.34 nm between the N (imidazolic) and O; 0.32 nm between the primary N and O; 0.36 nm between the two nitrogens engaged in salt formation with the oxygen. In addition, for complexation to occur, the distance between the noncharged nitrogen in the imidazole ring and the nearest phosphate oxygen (engaged in the phosphodiester bridge) should be 0.44 nm. Under these conditions, the two rings present (a six-membered, ideal one, salt-linked with the oxygen and rather highly elongated, and the imidazole) will not be precisely coplanar, since the primary and tertiary nitrogens will be one slightly above and one slightly below the plane of the drawing. Upon extensive binding, occupying every available phosphate site, pi-pi interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.

Fungaemia in hospitalized patients.

At various clinics of IRCCS S. Matteo Hospital, Pavia, Italy, 269 blood cultures recovered from immunocompromised patients over 4 years have been examined mycologically. Of the 269 cultures, 101 were from HIV-infected patients and five were from cardiac transplant recipients. Of the total examined 96 blood cultures were positive (36%). The most frequent genus was Candida: C. albicans (48%), C. tropicalis and C. parapsilosis (8% each), C. glabrata and C. guillermondii (3% each). Cryptococcus neoformans was detected in 21 patients (22%).

Clinical efficacy of azithromycin in lower respiratory tract infections.

A total of 51 patients with acute exacerbation of chronic bronchitis and pneumonia were enrolled: 27 treated with azithromycin (500 mg once a day for 3 days), and 24 with roxithromycin (150 mg every 12 hours for 7 days). The two regimens were equally effective, with clinical cure in 80% and 72% of patients respectively. Bacteriological eradication on day 19-23 was obtained in 7/11 cases (64%) and in 6/13 cases (46%) in the two groups, respectively. No side effects occurred in patients treated with azithromycin, while they occurred in the roxithromycin group (2 vomiting and 1 gastritis). Clinical and bacteriological efficacy, excellent tolerability, simplified dosage (single daily dose) and short-course (3 days) therapeutic regimen make azithromycin, in our experience, efficacious for the treatment of acute exacerbation of chronic bronchitis and community-acquired pneumonia.

Fluorescence polarization immunoassay to determine aminoglycoside modifying enzymes activity.

The possible use of polarized-immunofluorescence to evaluate aminoglycoside-modifying enzyme activities has been investigated. The TDX instrument was used to determine the residual quantity of aminoglycoside following inactivation during the assay procedure. Through the spectrum of the modified aminoglycoside antibiotics the enzymes encoded on eleven R-factors from clinical isolates of Enterobacteriaceae have been identified.

Third-generation cephalosporins resistance in clinical isolates of Enterobacteriaceae.

Seven clinical isolates of Enterobacteriaceae resistant to third-generation cephalosporins were investigated in order to assess the role played in resistance by permeability barrier and by beta-lactamase production. The addition of subinhibitory concentrations of EDTA increased susceptibility to ceftriaxone in five strains showing that the permeability barrier is involved. All strains produced different amounts of beta-lactamases that were always increased by cefoxitin induction. Hydrolytic activity of investigated enzymes varied in different cephalosporins; ceftriaxone and ceftazidime are the most stable compounds.

Coronavirus inhibitor in human sera: age distribution and prevalence.

We studied the distribution, in human sera, of non antibody serum inhibitor active towards human OC43 and bovine NCDCV coronaviruses. Antibodies to coronaviruses, present with high prevalence in human sera, were filtered by Protein A Sepharose CL 4B column. In the present work we tested sera of newborns, infants, young children and adults. Inhibitory activity was revealed in all sera except these from newborns. This non antibody inhibitor was tested by a plaque-reduction assay.

Serum inhibitor of coronaviruses OC43 and NCDCV: a study in vivo.

It is well known that some strains of coronaviruses, such as human OC43 and bovine NCDCV, are highly inhibited by non antibody factors present in sera of different mammalian species and sensitive to phospholipase-C treatment. So far this inhibitor has only been revealed in vitro experimental procedures with coronavirus strains adapted to growth in human lung fibroblast cell cultures. The purpose of this work was to ascertain whether this inhibiting activity was also effective "in vivo". In order to detect the "in vivo" activity, we used suckling mice which have been shown to be the animals most sensitive to infection with these viruses. Our results demonstrated that the same non antibody inhibitor is able to neutralize viral infection both "in vivo" and "in vitro".

Recent advances of knowledge on the mechanism of action of drugs modifying the bronchial secretions.

In order to explain the mechanism of action of drugs modifying the bronchial secretions, their subdivision into agents with a direct or an indirect action seems justified: the former primarily modify already formed secretions, whilst the latter act on the bronchial structures originating mucus. In hypersecretive states with increased viscoelasticity of mucus, a widespread therapy is represented by the use of direct reducing drugs, which act by breaking the mucofibrillar network. Among them the most important are those with free sulphydryl groups (-SH), which can break the disulphide bonds of the bronchial secretions. The mechanism of action of substances such as acetylcysteine and tiopronin, that are included in those with a direct reducing action, was already verified by researches with the modified thrombo-elastograph (MTE) and has been recently confirmed by the present authors by employing a simple biochemical method, by which the action of such drugs on the structure of serum IgM can be studied in vitro. The same method permits the classification of carbocisteine and letosteine as amongst the drugs with indirect action. From the results obtained by such in vitro experiences, the necessity to examine more closely the consequences of secondary effects which direct mucolytic agents may cause by modifying the rheological, biochemical and immunological properties of bronchial secretions has been demonstrated.

Phosphatidyl-serine inhibition of OC43 and NCDCV coronavirus infectivity.

Human OC43 and bovine neonatal calf diarrhoea coronavirus (NCDCV) are known to be inhibited by non-antibody factors present in sera of different mammalian species, including man and cattle. Such an inhibitor is also present in fetal calf serum generally used for growing and maintaining cell cultures, thus influencing the viral infectivity titer. Previous reports refer to the effectiveness of phospholipase C treatment in eliminating the inhibiting activity. In order to detect the compound possibly involved in coronavirus growth inhibition (among the phospholipids naturally present in mammalian sera), we tested several phospholipids with hemagglutino-inhibition test and plaque reduction assay. Only phosphatidyl-serine seemed to be effective on Coronavirus OC43 and NCDCV, as detected by plaque-reduction assay, but not by hemagglutino-inhibition test.

Herpes simplex virus infection in patients affected by dental caries.

Herpes simplex virus (HSV) excretion was studied in patients with dental diseases. Two groups of patients were examined: the first with dental caries and the second without dental decay. Results showed a significantly higher elimination rate of HSV in dental caries patients in comparison with control patients.

R factor-mediated adhesiveness to mammalian cells in E. coli K12.

We checked the possible effects of the standard plasmids of known compatibility groups on the ability of three strains of E. coli K12, J62, J53 and C600, to agglutinate human and guinea pig erythrocytes and to adhere to cultures of human epithelial cells. The results obtained showed that under defined experimental conditions one plasmid, R478, from a clinical isolate of Serratia marcescens is able to modulate the adhesive properties of the strain J62 which lacks adhesiveness. On the contrary, the same factor did not alter the ability of strains J53 and C600 to agglutinate erythrocytes and to adhere to human epithelial cells in culture, nor did it induce adhesive properties in wild-type strains of E. coli from clinical isolates that lacked them. This would suggest a possible plasmidic control of chromosomally encoded surface structures that mediated adhesiveness of E. coli to mammalian cells.

[Prospective use of immunofluorescence polarization in the study of enzymes modifying aminoglycosides]. / Prospettive di utilizzazione della immunofluorescenza polarizzata per lo studio degli enzimi modificanti gli aminoglicosidi.

A number of assays have been described for aminoglycoside-modifying enzymes; the aim of our investigation was to check the validity of polarized immunofluorescence assay for aminoglycoside enzymes. We have studied three enzymes encoded by R-factors from clinical isolates of Enterobacteriaceae conferring various patterns of aminoglycoside-resistance. The assay resulted valid: this is a simple, rapid and sensitive method.

[The role of beta-lactamases and the permeability barrier in resistance to beta-lactam antibiotics]. / Ruolo delle beta-lattamasi e della barriera di permeabilità nella resistenza agli antibiotici beta-lattamici.

The role played by beta-lactamases and by permeability barrier in resistance to beta-lactam antibiotics was investigated in 250 clinical isolates of Enterobacteriaceae. The data obtained showed that in 73 strains (52.9%) was a significant correlation between the specific enzyme activity and resistance, however in 38 strains (27.5%) an essential role was played by permeability barrier because the addition of subinhibitory levels of EDTA produced markedly reduction in the MIC.