Near-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S'.

Near-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-,1epsilon,7epsilon-diguanidono-[Asn14]-, [Phe(F)8, Orn10]- and 1epsilon, 7epsilon-diguanidino-S-peptide, with S-protein. Environmental alterations of Phe-8 in the S-peptide and Tyr-25 in the S-protein, derived from the association process, lead to strong optical signals whose location and magnitude were clearly defined by means of a comparative analysis of the above spectra. Additionally, the spectroscopic effects resulting from insertion of a tyrosyl residue into an hydrophobic environment in the presence or absence of hydrogen-bonding partners were identified and compared with similar findings obtained from the model compound p-cresol.

Far-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S'.

Far-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-, 1epsilon, 7epsilon-diguanidino-[Asn14]-, [Phe(F)8, Orn10]-, [Cha8, Orn10]- and 1epsilon, 7epsilon-diguanidono-S-peptide, with S-protein. The aromatic chromophores contributions to the absorption spectra in the 220-250 nm wavelengths interval strongly exceed the hyperchromism due to the random coil to right handled alpha-helix transition of the S-peptide, accompanying the association process. Contributions resulting from peptide transitions constitute a large portion of the total dichroism, nevertheless substitution of the non aromatic cycloexylalanine residue in position 8 of the S-peptide with phenylalanine, p-fluorophenylalanine and tyrosine leads to CD negative maxima located at 215, 215 and 212,5 nm, respectively. The strongest ellipticity increment (28%), relative to that of the Cha-derivative, was observed at 212,5 nm for the [Tyr8]-S-peptide analog, while in the narrow 222 nm range minimal differences were found upon insertion into the position 8 of the S-peptide of the above aromatic residues. Information derived from above data and from a comparison of RNAase A, S and S-protein CD spectra enabled us to assign the positive CD band at 240 nm in RNAase A spectrum to transitions of phenylalanines and inaccessible tyrosines.

Protamines. II. Circular dichroism study of the three main components of clupeine.

The three main components YI, YII, and Z of clupeine, a protamine from herring, have been purified and characterized. The conformational preferences of clupeines have been examined as a funciton of pH, temperature, added salts, and presence of structure-disrupting agents and helix-supporting solvents using circular dichroism. It was found that these small basic proteins assume predominantly an unordered conformation in aqueous solution. Addition of counter ions, in particular perchlorate, and 2-chloroethanol induces in various amounts the onset of the right-handed alpha-helical conformation. Urea favors the statistical coil state. It was also demonstrated that in the 0.1--4.0 . 10(-1) M range, in contrast to clupeines YI and Z, the circular dichroic properties of the YII component do not seem to be sensitive to the addition of mono- and diphosphate.

Deflavination of flavo-oxidases by nucleophilic reagents.

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.

A novel thermostable inhibitor of trypsin and subtilisin from the seeds of Brassica nigra: amino acid sequence, inhibitory and spectroscopic properties and thermostability.

A novel thermostable protein inhibitor of trypsin and subtilisin, called BN, was isolated from the seeds of Brassica nigra. The purified protein gave a single band on SDS-PAGE, corresponding to a molecular mass of 15 500 +/- 1000 Da. The inhibitor is composed of two disulfide-linked polypeptide chains, consisting of 39 and 90 residues, respectively. The amino acid sequence of the two chains was determined by Edman degradation of peptides, isolated from enzyme hydrolysates with TPCK-trypsin, EndoLysC proteinase and a Glu-specific proteinase of reduced and vinylpyridinated protein samples. A segment of the 'heavy' chain, between residues 65 and 81, showed homology with the reactive site loop region of the 6-kDa trypsin inhibitors from Nicotiana alata. The basic residue in position 39 (N. alata) or 70 (napins) is conserved as arginine or lysine in all inhibitors from N. alata and in all napins hitherto sequenced. Probably, the two families of trypsin inhibitors have structurally similar reactive sites. BN exhibits an extremely high thermostability: CD measurements showed that during heating to 97 degrees C it preserves a considerable part of the polypeptide backbone folding. Studies on the fluorescence properties of the inhibitor BN in the absence and presence of neutral or ionic quenchers demonstrated that the intrinsic emission of this protein is dominated by a tryptophyl residue, buried in the interior of the protein matrix. 20% of the light absorbed by Tyr 63 of the 'heavy' chain is transferred to Trp 26 of the 'light' chain.

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein.

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Activation of acetylcholinesterase by aluminium(III): the relevance of the metal species.

The present paper reports how aluminium [Al(III)] at a concentration of 3.7 microM can activate the bovine erythrocytic enzyme acetylcholinesterase (AChE) by about 38% in vitro. This same activating effect was observed on AChE form human as well as from rat erythrocyte ghosts and murine neuroblastoma cells. The interaction between Al3+ and gamma-peripheral sites of the enzyme produces AChE structural modifications as evidenced by circular dichroism measurements. This may provide a molecular explanation of the raised enzymatic activity.

Effects of beta 2-agonists during cardiopulmonary exercise test in COPD patients.

The aim of this study was to evaluate endocrine-metabolic, respiratory and cardiovascular effects of two beta 2-sympathomimetic selective agents, such as broxaterol and salbutamol, before and during cardiopulmonary exercise test (CPX). Twelve in-patients with chronic obstructive pulmonary disease (COPD) (with partially reversible airways obstruction) were included. Broxaterol (400 micrograms) and salbutamol (400 micrograms) were administered i.v., according to a double-blind, cross-over study. Before treatment and within 60 min after the administration of each agent, the patients underwent incremental CPX by bicycle ergometer to the maximum tolerable threshold. At these times the following variables were assessed: minute ventilation (VE), oxygen consumption (VO2), carbon dioxide production (VCO2), VE/VO2 ratio and O2 pulse, glycaemia, insulinaemia, plasma norepinephrine (NE) and epinephrine (E), arterial oxygen and carbon dioxide tension (PaO2 and PaCO2), plasma lactates, heart rate (HR), systolic (SBP) and diastolic (DBP) blood pressure. Spirometry was performed before and after the administration of each beta 2-adrenoceptor agonist. CPX brought about a significant increase in VE, VO2, VCO2, and O2 pulse. Broxaterol or salbutamol administration did not significantly modify the increases caused by CPX. At rest, 60 min after treatment, both bronchodilators caused a significant rise in glycaemia. A significant reduction of PaO2 (after broxaterol) and PaCO2 (after salbutamol) was observed at rest. In contrast, both agents caused no modification to potassium and insulin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

[Changes in the heart rhythm and slow-release beta-2 agonist therapy (peroral) in asthmatic patients. A Holter study]. / Alterazioni del ritmo cardiaco e terapia beta-2-agonista a lento rilascio (per via orale) in pazienti asmatici. Studio Holter.

Slow-release formulations of salbutamol, used in asthmatic patients, may have few effects on cardiac rhythm. This study was designed to compare incidence and type of cardiac arrhythmias in a group of ten patients with stable bronchial asthma before and after 6 weeks treatment with a slow-release formulation of salbutamol. After a baseline spirometric and electrocardiographic evaluation, patients received placebo for a two-weeks period. At the end of this period patients repeated spirometry and carried out a 24-hour-ambulatory-E-Holter-monitoring. Subsequently, patients received for 6 weeks a treatment with a slow-release formulation of oral salbutamol. At the end of this period patients repeated spirometry, a 24-hour-ambulatory-EG-Holter-monitoring and potassium serum levels were controlled. We concluded from our study that, in patients with moderate bronchial asthma the treatment with an oral formulation of slow-release salbutamol may carry out a significant and durable bronchodilator effect, without causing important cardiac arrhythmias.

[Etiopathogenesis of hand syndactyly in the light of several experimental findings]. / Etiopatogenesi delle sindattilie della mano alla luce di alcune osservazioni sperimentali.

The limb developmental program is comprised of processes of cell multiplication and of cell determination, differentiation and morphogenesis. Many of these processes depend on cell-cell communications within tissues and between different tissue types. The Authors present a review of the more significant data about the etiology of the syndactylies of the hand, both in their physiopathogenic aspects and in their clinical typology. The peculiarity of these malformations is outlined, supported by literature data and personal experimental experiences.

Aluminum-triggered structural modifications and aggregation of beta-amyloids.

We investigated the structural effects induced by Al3+ on different beta-amyloid (Abeta) fragments at pH 7.4 and T=25 degrees C, with particular attention given to the sequences 1-40 and 1-42. Al3+ caused peptide enrichment in beta sheet structure and formation of solvent-exposed hydrophobic clusters. These intermediates evolved to polymeric aggregates which organized in fibrillar forms in the case of the Al3+-Abeta(1-42) complex. Comparative studies showed that Zn2+ and Cu2+ were much less efficient than Al3+ in stimulating the spontaneous aggregation/fibrillogenesis of Abetas. Studies with liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of Al3+-Abeta complexes, suggesting a major role of Al3+ in Abeta-induced cell dysfunction. Al3+ effects were abolished by desferrioxamine mesylate (DFO) only in solution. We concluded that, in vivo, DFO may act as a protective agent by preventing or reverting Abeta aggregation in the extracellular spaces.