A form of apolipoprotein a-I is found specifically in relapses of focal segmental glomerulosclerosis following transplantation.

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) following kidney transplantation occurs in a large percentage of patients. Accurate prediction of recurrence and elucidation of its pathogenesis are major therapeutic goals. To detect differential proteins related to FSGS recurrence, proteomic analysis was performed on plasma and urine samples from 35 transplanted idiopathic FSGS patients, divided into relapsing and nonrelapsing. Several proteins were detected increased in urine of relapsing FSGS patients, including a high molecular weight form of apolipoprotein A-I, named ApoA-Ib, found exclusively in relapsing patients. This finding was verified by Western blot individually in the 35 patients and validated in an independent group of 40 patients with relapsing or nonrelapsing FSGS, plus two additional groups: FSGS-unrelated patients showing different proteinuria levels (n = 30), and familial FSGS transplanted patients (n = 14). In the total of 119 patients studied, the ApoA-Ib form was detected in 13 of the 14 relapsing FSGS patients, and in one of the 61 nonrelapsing patients. Only one of the 30 patients with FSGS-unrelated proteinuria tested positive for ApoA-Ib, and was not detected in familial patients. Urinary ApoA-Ib is associated with relapses in idiopathic FSGS and warrants additional investigation to determine its usefulness as biomarker of relapse following transplantation.

Photocatalytic degradation of formaldehyde containing wastewater from veterinarian laboratories.

The photocatalytic destruction of methanol, formaline (mixture of formaldehyde, methanol and water) and formaline wastes from the preservation of vertinarian physiologic samples has been attempted by two different processes, at high concentrations of reagents and by dossification of reagents, varying pH in both. Experiment evolution has been monitored by measuring the organic matter such as TOC and formaldehyde concentrations [H2CO]. Also, methanol and methanol-formaldehyde interactions with the TiO2 surface have been analysed by FTIR spectroscopy. Results indicate that at high concentrations the catalyst surfacial alterations given by methoxy, formates or carbonates, according to the pH of the sample can profoundly affect catalyst behaviour. It has been established that reagent dossification is advantageous for enhancing photonic efficiency as it minimizes the adsorbate presence that hampers the photocatalytic process.

Adaptations of the beta-adrenoceptor-adenylyl cyclase system in rat skeletal muscle to endurance physical training.

beta-Adrenergic mechanisms may be important in the adaptation of skeletal muscle to endurance training. beta-Adrenergic signal transduction was examined in the gastrocnemius muscle of rats submitted to a progressive, 12-week treadmill running program and compared with sedentary controls. beta-Adrenoceptor density was significantly lower in exercised rats than in controls. The affinity constant for [125I]-(-) iodocyanopindolol binding was not different among the various groups. Adenosine cyclic monophosphate formation was significantly decreased in trained animals when isoproterenol plus guanosine triphosphate or forskolin plus Mn2+ were used to stimulate adenylyl cyclase. Immunoblot analyses revealed that the amount of the alpha-subunit of stimulatory guanine nucleotide-binding protein (Gs,alpha), both the small and the large isoforms, also decreased with physical exercise. Thus, the present report shows that endurance training results in alterations in beta-adrenergic receptor density, adenylyl cyclase activity and Gs protein level in rat gastrocnemius muscle.

Clusters of Bell's palsy.

The idiopathic facial paralysis or Bell's palsy installs abruptly or within a few hours, without any apparent cause. It corresponds to approximately 75% of all peripheral facial palsies. Three theories try to explain its pathogenecity: vascular-ischemic, viral and auto-immune. We reviewed the records of the EMG Sector, Hospital do Servidor Público Estadual (São Paulo, Brazil), from 1985 to 1995 and found 239 cases of Bell's palsy. Data were analysed according to age, gender, seasonal distribution of cases. There was a predominance of cases in the 31-60 age bracket (40.59%). The female gender was responsible to 70.71% of cases. There was a predominance of cases in winter (31.38%) and autumn (30.13%), which was statistically significant. These findings let us to suppose that Bell's palsy predominates in females, in 41-60 years age bracket, and occurs predominantly in cold months. There are groups of clusters throughout temporal distribution of cases and cases are dependent on one each other or on factors affecting them all, which reinforces the infectious hypothesis (there is a rise in the incidence of viral upper respiratory tract infection during cold months).

Three-dimensional structure of acidic fibroblast growth factor in solution: effects of binding to a heparin functional analog.

Acidic and basic fibroblast growth factors (aFGF and bFGF; FGFs) are paradigms of a group of nine closely related proteins known as the fibroblast growth factor family. FGFs induce mitosis in most mesoderm- and neuroectoderm-derived cells, and appear to be involved in diseases caused by anomalous cell proliferation. In vitro assays show that binding to heparin-like glycosaminoglycans is required to elicit the mitogenic activity of these proteins. It has been shown that myo-inositol hexasulfate (MIHS) emulates heparin in the mitogenesis assays of aFGF, and a low-resolution three-dimensional structure in solution of this protein bound to MIHS has been reported. Here we describe the 1H-NMR three-dimensional structure in solution of the free aFGF. Comparison of this structure with that of the protein bound to MIHS, upgraded to a level of refinement equivalent to that of the free protein, shows that MIHS binding causes some slight conformational changes with an increase in the definition of the structure. In addition, amide exchange H/2H rates of the most protected protons, and exchange data of the intermediate and fast-exchanging ones show that the free protein is less stable (< or = 2 kcal/mol) and more flexible in terms of local unfolding equilibria, respectively, than the MIHS-bound one. Thus, MIHS binding to aFGF causes a decrease of its flexibility, which translates into an enhancement of the definition of its three-dimensional structure. The increase of aFGF rigidity affects regions that include those involved in recognizing the cell membrane receptor. Thus, our data suggest that enhancement of structural definition may play a key role in the modulation of the affinity of aFGF by its receptor, and, consequently, of its specific mitogenic activity.

Effect of endurance physical training on rat liver adenylyl cyclase system.

The adaptive response to endurance exercise of the catecholamine- and glucagon-sensitive adenylyl cyclase system was studied in rat liver plasma membranes. Endurance exercise enhanced adenylyl cyclase system activation by cellular agonists (glucagon, isoproterenol), by stimulators of the enzyme catalytic subunit (forskolin, Mn2+), and by Gs-protein activators (GppNHp, fluoride). In addition, endurance exercise increased the levels of G50, Gi alpha, and G beta subunits. These results show that the adenylyl cyclase system becomes sensitized in response to physical training.

Adenylyl cyclase system is affected differently by endurance physical training in heart and adipose tissue.

Adaptive changes in the beta-adrenergic adenylyl cyclase (AC) system in response to endurance training were studied in heart and adipose tissue. Training was performed by making male Wistar rats run on a motor-driven treadmill. The changes following exercise training were opposite in the two tissue studied. The density of beta-adrenergic receptors in left ventricular membranes of trained rats showed a marked decrease. Comparison of AC activities in cardiac membranes prepared from trained and sedentary rats revealed a depressing effect of endurance training on: 1. the beta-adrenergic stimulatory pathway and the inhibition of AC via receptor; 2. the Gs component and the Gs-adenylyl cyclase coupling, as shown by the response of adenylyl cyclase to GppNHp and NaF; and 3. the enzyme catalytic activity in the presence of Mn2+ or forskolin. The levels of Gsalpha subunits in the left ventricle, as measured in terms of ADP-ribosylated and immunologically reactive proteins, were decreased by endurance exercise, whereas immunodetectable levels of Gialpha2 increased in the membranes of trained myocardium. In contrast to the diminished sensitivity that characterizes the behavior of the cardiac beta-adrenergic-AC system, endurance physical training increased sensitivity of this signal transduction system in adipose tissue. Thus, the density of beta-ARs as well as AC activity and the beta-adrenergic stimulatory pathway were increased in adipose membranes of trained rats compared with the corresponding sedentary controls. In addition, the levels of Gsalpha subunits were higher in the adipose plasma membranes of trained rats. However, immunodetectable levels of Gi1alpha and Gi3alpha increased with training, whereas the amount of Gi2alpha decreased in membranes of trained rats. In conclusion, the present study shows that chronic exercise is associated with a tissue-specific adaptation of the beta-adrenergic AC system.

Conformational investigation of designed short linear peptides able to fold into beta-hairpin structures in aqueous solution.

BACKGROUND: Formation of secondary structure plays an important role in the early stages of protein folding. The conformational analysis of designed peptides has proved to be very useful for identifying the interactions responsible for the formation and stability of alpha-helices. However, very little is known about the factors leading to the formation of beta-hairpins. In order to get a good beta-hairpin-forming model peptide, two peptides were designed on the basis of beta-sheet propensities and individual statistical probabilities in the turn sites, together with solubility criteria. The conformational properties of the two peptides were analyzed by two-dimensional NMR methods. RESULTS: Long-range cross-correlations observed in NOE and ROE spectra, together with other NMR evidence, show that peptide IYSNPDGTWT forms a highly populated beta-hairpin in aqueous solution with a type I beta-turn plus a G1 beta-bulge conformation in the chain-bend region. The analogous peptide with a Pro5 substituted by Ser forms, in addition to the previous conformation, a second beta-hairpin with a standard type I beta-turn conformation, and the two forms are in fast dynamic equilibrium with one another. The effect of pH demonstrates the existence of a stabilizing interaction between the Asn and Asp sidechains. The populations of beta-hairpin conformations increase in the presence of trifluoroethanol (a structure-enhancing solvent). On the other hand, some residual structure persists at a high denaturant concentration (8 M urea). CONCLUSIONS: This work highlights the importance of the beta-turn residue composition in determining the particular type of beta-hairpin adopted by a peptide, though a role of interstrand sidechain interactions in the stabilization of the formed beta-hairpin is not discarded. The fact that trifluoroethanol can stabilize alpha-helices or beta-hairpins depending on the intrinsic properties of the peptide sequence is again shown. An additional example of the presence of residual structure under denaturing conditions is also presented.

Interactions responsible for the pH dependence of the beta-hairpin conformational population formed by a designed linear peptide.

In a previous work [Blanco, F.J., Jiménez, M.A., Herranz, J., Rico, M., Santoro, J. & Nieto, J. L. (1993) J. Am. Chem. Soc. 115, 5887-5888] we showed that a short, designed linear peptide, YQNPDGSQA (peptide 1), can form a monomeric beta hairpin in aqueous solution. The pH dependence of the beta-hairpin conformation formed by the designed peptide and a series of related peptides has been examined in this work using 1H-NMR methods. Three pH-dependent interactions have been identified: a local interaction, unimportant structurally, between the C-terminal carboxylate group and the side-chain amide group of Q8; an electrostatic interaction between the main-chain N-terminus and C-terminus; and a hydrogen bond involving the side-chain amide protons of N3 and the side-chain carboxylate group of D5. The latter two interactions are particularly relevant as they increase the population of the beta-hairpin conformation. We also observe in the mutant peptide A9H that the interaction between Y1 and H9 (of the type proposed to exist in proteins) does not contribute to beta-hairpin stabilisation in our peptide system. Peptide 1 is, therefore, a very suitable model to examine the different interactions that contribute to beta-hairpin stability.

1H-NMR assignment and solution structure of human acidic fibroblast growth factor activated by inositol hexasulfate.

A major fragment of human acidic fibroblast growth factor of 132 amino acid residues is shown to be as active and stable as the 139 residue molecule initially described, and commonly used in physiological studies. It is shown that inositol hexasulfate is a good substitute for heparin in both activating and protecting acidic fibroblast growth factor. The complex between the shortened form of the protein and inositol hexasulfate was used to determine the structure of activated acidic fibroblast growth factor in solution. The 1H-NMR spectrum of the complex was totally assigned, and a low-resolution, three-dimensional structure of the protein computed. The global fold of the activated acidic fibroblast growth factor is similar to that proposed for a crystallized variant of the protein obtained by genetic engineering whose activity is not dependent on heparin. The inositol hexasulfate binds to the protein through the positively charged groups of Lys126, Lys127, Arg133 and Lys142 side-chains. The computed three-dimensional structure suggests that inositol hexasulfate may stabilize and activate the protein by conferring rigidity to the hairpin involving beta-strands 10 and 11.

Helix formation by the phospholipase A2 38-59 fragment: influence of chain shortening and dimerization monitored by nmr chemical shifts.

The solution structure of a peptide fragment corresponding to the 38-59 region of porcine phospholipase A2 has been investigated using CD, nmr chemical shifts, and nuclear overhauser effects (NOEs). This isolated fragment of phospholipase forms an alpha-helix spanning residues 38-55, very similar to the one found in the native protein, except for residues 56-58, which were helical in the crystal but found random in solution. Addition of triflouroethanol (TFE) merely increased helix population but it did not redefine helix limits. To investigate how the folding information, in particular that concerning eventual helix start and stop signals, was coded in this particular amino acid sequence, the helices formed by synthetic peptides reproducing sections of this phospholipase 38-59 fragment, namely 40-59, 42-59, 38-50, and 45-57, were characterized using NOEs and helix populations quantitatively evaluated on different peptide chain segments using nmr chemical shifts in two solvents (H2O and 30% TFE/H2O). A set of nmr spectra was also recorded and assigned under denaturing conditions (6M urea) to obtain reliable values for the chemical shifts of each peptide in the random state. Based on chemical shift data, it was concluded that the helix formed by the phospholipase 38-59 fragment was not abruptly, but progressively, destabilized all along its length by successive elimination of residues at the N end, while the removal of residues at the C end affected helix stability more locally and to a lesser extent. These results are consistent with the idea that there are not single residues responsible for helix initiation or helix stability, and they also evidence an asymmetry for contributions to helix stability by residues located at the two chain ends. The restriction of molecular mobility caused by linking with a disulphide bridge at Cys 51 two identical 38-59 peptide chains did not increase helix stability. The helix formed by the covalently formed homodimer was very similar in length and population to that formed by the monomer.

NMR solution structure of the isolated N-terminal fragment of protein-G B1 domain. Evidence of trifluoroethanol induced native-like beta-hairpin formation.

The solution structure of the isolated N-terminal fragment of streptococcal protein-G B1 domain has been investigated in H2O and TFE/H2O solution by CD and NMR to gain insight into the possible role that native beta-hairpin secondary structure elements may have in early protein folding steps. The fragment also has been studied under denaturing conditions (6 M urea), and the resulting NMR chemical shifts were used as a reference for the disordered state. On the basis of CD and NMR data, it is concluded that in aqueous solution the fragment is basically flexible, with two local low populated chain bends involving residues 8-9 and 14-15, respectively, in close agreement with secondary structure predictions, a structure that is different from the final folded state of that segment of the protein. The changes in the CD spectrum, the presence of several medium-range NOEs plus two long-range NOEs, and the sign of the H alpha conformational shifts reveal that the addition of TFE facilitates the formation of a set of transient beta-hairpins involving essentially the same residues that form the native beta-hairpin found in the final three-dimensional structure of the B1 domain. The stabilization of native-like structures by TFE is known to occur for helices, but, to our knowledge, this is the first time the stabilization of a native-like beta-hairpin structure by TFE is reported. Since long-range tertiary interactions are absent in the isolated fragment, our results support the idea that, in addition to helices, beta-hairpins may play an active role in directing the protein folding process.

Cardiac beta-adrenoceptors, G-proteins and adenylate cyclase regulation during myocardial hypertrophy.

The role of the beta-adrenoceptor-G-protein-adenylate cyclase system in the pathogenesis of cardiac hypertrophy was studied. We have used a minipig model of pressure-overload cardiac hypertrophy secondary to aortic banding. Four groups of five animals were used: minipigs made hypertrophic were evaluated 2 months (CH2 group) and 9 months (CH9 group) later and compared to controls (C2 and C9 groups, respectively). A decrease in beta-adrenergic receptor density and an increase in antagonist affinity were shown in left ventricular membranes of hypertrophied animals compared with controls. In both groups, CH2 and CH9, an increase in EC50 for isoproterenol-stimulated adenylate cyclase activity, an increase in forskolin-stimulated adenylate cyclase activity and a diminished inhibition by carbachol of isoproterenol-stimulated adenylate cyclase were observed. In contrast, fluoride-stimulated adenylate cyclase activity was markedly increased only in the end stage of hypertrophy. alpha s-cholera toxin-catalysed ADP-ribosylation is increased in early hypertrophy and then decreases with late hypertrophy and a similar pattern is observed with alpha o pertussis toxin-catalysed ADP-ribosylation, whereas alpha i-ADP-ribosylation remains unchanged. Tissue content of Gs-, Gi- and Go-proteins, as assessed by specific antibodies, was found unchanged in CH9 and CH2 groups when compared with that in C9 and C2 control groups, respectively. Modifications in Gs functional activity in later hypertrophic stages, expressed as alterations in cholera toxin ADP-ribosylation and adenylate cyclase fluoride responsiveness, may be important in the pathogenesis of decompensation from compensated hypertrophy to cardiac failure.

High-resolution three-dimensional structure of ribonuclease A in solution by nuclear magnetic resonance spectroscopy.

High-resolution three-dimensional structures of bovine pancreatic ribonuclease A in aqueous solution have been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. The structures are based on: (1) 464 interproton distance constraints with accurate upper and lower limits, determined from build-up rates of nuclear Overhauser effects (NOE) by using the complete relaxation matrix; (2) 999 more approximate upper limits for interproton distances; and (3) 42 dihedral angle constraints (37 for phi and 5 for chi 1). A total of 16 structures were calculated, which show a root-mean-square (r.m.s.) deviation of 0.66 A for the backbone atoms and 1.68 A for all heavy-atoms. The converged structures are highly similar to those found in the crystal state. r.m.s. deviation of backbone atom positions in the crystal as compared to those in the average solution structure is 0.92 A. Observed differences are concentrated in loop regions and in the neighborhood of His119 and His48 side-chains. Dynamic aspects, such as H/D amide proton exchange and side-chain mobility have been examined.

CD and 1H-NMR studies on the conformational properties of peptide fragments from the C-terminal domain of thermolysin.

The propensity of the peptide fragments 233-248, 245-260, 258-276, 279-298 and 299-316 from the thermolysin C-terminal domain to form non-random structures has been examined by CD and two-dimensional NMR spectroscopy. The conformational properties of these fragments have been studied in aqueous solution and in the mixed solvent trifluoroethanol/H2O (3:7 by vol.). Small but detectable populations of helical structures (up to 10-20%) in aqueous solution have been found for the fragments 233-248, 279-298 and 299-316. These populations are remarkably enhanced (50-70%) in the more hydrophobic mixed solvent, where the fragment 258-276 also forms a comparable helical population. These four fragments are helical in the native crystal structure and the spanning of the corresponding helices in the isolated peptides in solution matches very closely the ones in the native structure. In contrast, the fragment 245-260, an omega-loop in the crystal, remains unstructured in both solvents. Medium-range NOE between protons in sidechains indicate the adoption of preferred sidechain conformations accompanying helix formation. Results are in agreement with the framework model of folding, in which native elements of secondary structure are formed first and folding follows from the collapse of these structural elements.

Characterization of a synthetic calmodulin-binding peptide derived from Bacillus anthracis adenylate cyclase.

A 34-amino acid peptide corresponding to residues 532-565 of Bacillus anthracis adenylate cyclase (P532-565), a calmodulin (CaM)-activated enzyme, was synthesized by solid phase method. Although not homologous to any known CaM binding sequence, P532-565 exhibits molecular features characteristic of this class of peptides: a higher proportion of basic and hydrophobic residues, segregated onto the two faces of the alpha-helical structure. Fluorescence measurements and gel retardation analysis showed that P532-565 binds CaM in a Ca(2+)-dependent manner, with a binding energy that represents 80% of the binding energy of the adenylate cyclase-CaM complex. Circular dichroism analysis showed that P532-565 exists in solution as a mixture of random-coil and alpha-helical structures and that trifluoroethanol increases the relative proportion of alpha-helical population. Analysis of proton NMR spectrum in H2O allowed identification of the different amino acid spin systems and complete spectral assignment. The pattern of nuclear Overhauser effect connectivities, intense NN(i,i + 1) and medium range alpha N(i,i + 3) and alpha beta (i,i + 3) indicate the presence of an alpha-helix in the carboxylterminal end (between residues 551 and 563) in fast exchange with extended structures. These data, together with CaM-binding properties of Bordetella pertussis adenylate cyclase, show that despite rather divergent primary structures, the two bacterial enzymes possess similar structural organization of their binding sites for activator protein.

Periodic properties of proton conformational shifts in isolated protein helices. An experimental study.

In this work, the helix-forming residues in fragments of several proteins (ribonuclease, thermolysin, tendamistat and angiogenin) were identified by NOE and the helix proton shifts were measured as delta changes associated with helix-population increments driven by trifluoroethanol addition. When estimated in this way, a regular pattern of helix conformational shifts was clearly seen in the delta delta versus sequence profiles of all the peptides studied. The helix periodicity of the H alpha and H beta resonances was especially clear, an observation that earlier statistical studies of protein delta values failed to predict. Amide protons showed the largest helix shifts, but with a less-sharply defined periodic character. Aromatic residues considerably distorted the periodicity of the helix amide shifts in some peptides, as evidenced by the delta shifts of a RNase A fragment 1-15 analog in which the two aromatic residues were replaced by Ala. The relationship between helix periodicity and peptide amphiphatic character is discussed.

The homologous angiogenin and ribonuclease N-terminal fragments fold into very similar helices when isolated.

The solution structure of the N-terminal hexadecapeptide of human angiogenin, a protein of unknown tertiary structure, has been precisely delineated by the combined use of CD, NOE and secondary shift data. A helix that starts just after Ser 3 and ends at Asp 14 was stabilized in 30% trifluoroethanol. This helix is strikingly similar in origin and length to the one formed by its homologous, the S-peptide of Ribonuclease (conformationally reexamined here), despite their quite different sequences (only four conserved residues). These results support the idea that individual start and stop signals indeed govern the location and size of natural isolated helices.

Tendamistat (12-26) fragment. NMR characterization of isolated beta-turn folding intermediates.

In order to determine whether regions of a protein that are turns in the native structure are able to maintain such a structure when isolated, we have studied the conformational properties of various peptide fragments corresponding to the 12-26-peptide region of the alpha-amylase inhibitor tendamistat, by NMR. Amide solvent accessibility, NOE spectroscopy (NOESY) and rotating-frame NOE spectroscopy (ROESY) data strongly support the conclusion that the 12-26 and 15-23 peptides adopt in aqueous solution, a set of turn-like structures located around the central region of their corresponding polypeptidic chains, the same region where a beta turn exists in the native protein. Such a set of structures are destabilized when one residue located within the native beta turn of the 15-23 peptide is modified Trp18----Ser. Our results indicate that the tendency to bend in a predetermined region of a protein chain seems to exist from the very beginning of the folding process and therefore it could drive the folding instead of being a consequence of the tertiary assembly of the protein.

3D structure of bovine pancreatic ribonuclease A in aqueous solution: an approach to tertiary structure determination from a small basis of 1H NMR NOE correlations.

A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of 1H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the phi and psi backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His119 side chain in the solution and crystal structures has been detected.