RESUMEN

The equilibrium and kinetic folding properties of a small oncogene product, P13(MTCP1), of novel topology have been investigated using perturbation by guanidine hydrochloride and observation by fluorescence, circular dichroism and two-dimensional heteronuclear NMR spectroscopy. The structure of P13(MTCP1) is comprised of a canonical filled beta-barrel, although the topology of the structure is absolutely unique, rendering the folding properties of this protein of great interest. Equilibrium measurements of the intrinsic fluorescence emission spectrum, the fluorescence decay, the circular dichroism spectrum and the (15)N-(1)H heteronuclear single quantum coherence (HSQC) correlation spectrum as a function of increasing concentrations of denaturant showed no evidence for the population of any equilibrium intermediates, although negative amplitudes on the blue edge of the tryptophan emission and loss of intensity of the native HSQC correlation peaks were indicative of increased conformational dynamics at low denaturant concentrations. The free energy and cooperativity of unfolding as observed by fluorescence and circular dichroism were in relatively good agreement, also consistent with a two-state transition. Kinetics measurements of the fluorescence emission as a function of denaturant concentration revealed that P13(MTCP1) is the slowest folding beta-structure protein reported to date. Comparison of the activation cooperativity values (m(f) and m(u)) indicates that the structure of the transition state is quite close to the folded state in terms of exposed surface area. The calculated contact order of P13(MTCP1) is relatively low and does not appear to explain its slow rate of folding. We suggest that the complex topology of this protein, which would require the ordering of the beta-barrel through a long loop joining the two L-shaped components of the barrel, could provide an explanation for this slow folding.

Asunto(s)

RESUMEN

This study deals with the exploitation of the three classical 15N relaxation parameters (the longitudinal relaxation rate, R1, the transverse relaxation rate, R2, and the 1H-15N cross-relaxation rate, sigmaNH) measured at several magnetic fields in uniformly 15N-labeled proteins. Spectral densities involved in R1, R2 and sigmaNH are analyzed according to the functional form A + B/(1 + omega(2) taus(2)), where taus is the correlation time associated with slow motions sensed by the NH vector at the level of the residue to which it belongs. The coefficient B provides a realistic view of the backbone dynamics, whereas A is associated with fast local motions. According to the "model free approach", B can be identified with 2tausS(2) where S is the generalized order parameter. The correlation time taus is determined from the field dependency of the relaxation parameters while A and B are determined through linear equations. This simple data processing is needed for obtaining realistic error bars based on a statistical approach. This proved to be the key point for validating an extended analysis aiming at the determination of nitrogen chemical shift anisotropy. The protein C12A-p8(MTCP1) has been chosen as a model for this study. It will be shown that all data (obtained at five magnetic field strengths corresponding to proton resonance of 400, 500, 600, 700, and 800 MHz) are very consistently fitted provided that a specific effective correlation time associated with slow motions is defined for each residue. This is assessed by small deviations between experimental and recalculated values, which, in all cases, remain within experimental uncertainty. This strategy makes needless elaborate approaches based on the combination of several slow motions or their possible anisotropy. Within the core of the protein taus fluctuates in a relatively narrow range (with a mean value of 6.15 ns and a root-mean-square deviation of 0.36 ns) while it is considerably reduced at the protein extremities (down to approximately 3 ns). To a certain extent, these fluctuations are correlated with the protein structure. A is not obtained with sufficient accuracy to be valuably discussed. Conversely, order parameters derived from B exhibit a significant correlation with the protein structure. Finally, the multi-field analysis of the evolution of longitudinal and transverse relaxation rates has been refined by allowing the 15N chemical shift anisotropy (csa) to vary residue by residue. Within uncertainties (derived here on a statistical basis) an almost constant value is obtained. This strongly indicates an absence of correlation between the experimental value of this parameter obtained for a given residue in the protein, the nature of this residue, and the possible involvement of this residue in a structured area of the protein.

Asunto(s)

RESUMEN

Helical coiled-coils and bundles are some of the most common structural motifs found in proteins. Design and synthesis of alpha-helical motifs may provide interesting scaffolds that can be useful as host structures to display functional sites, thus allowing the engineering of novel functional miniproteins. We have synthesized a 38-amino acid peptide, alpha2p8, encompassing the alpha-helical hairpin present in the structure of p8MTCP1, as an alpha-helical scaffold particularly promising for its stability and permissiveness of sequence mutations. The three-dimensional structure of this peptide has been solved using homonuclear two-dimensional NMR techniques at 600 MHz. After sequence specific assignment, a total of 285 distance and 29 dihedral restraints were collected. The solution structure of alpha2p8 is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics, using simulated annealing protocol with the AMBER force field. The RMSD values for the backbone and all heavy atoms are 0.65+/-0.25 and 1.51+/-0.21 A, respectively. Excised from its protein context, the alpha-hairpin keeps its native structure: an alpha-helical coiled-coil, similar to that found in superhelical structures, with two helices spanning residues 4-16 and 25-36, and linked by a short loop. This motif is stabilized by two interhelical disulfide bridges and several hydrophobic interactions at the helix interface, leaving most of its solvent-exposed surface available for mutation. This alpha-helical hairpin, easily amenable to synthetic chemistry and biological expression system, may represent a stable and versatile scaffold to display new functional sites and peptide libraries.

Asunto(s)

RESUMEN

The ubiquitous form of the sodium-hydrogen exchanger, NHE1, is devoted to the regulation of intracellular pH and cell volume. In addition, NHE1 activity is stimulated by growth factors and increased NHE rates are found in both circulating and immortalized cells during diabetes or diabetic nephropathy. In this context, we searched for polymorphisms of the 5'-flanking regulatory region of NHE1 gene in subjects with type-I diabetes. We identified a C/T transition 696 bases upstream the translation initiation start site which disrupts a repeated palindromic GC sequence. The TT genotype was significantly more frequent in type-1 diabetics and may have functional importance. Genetic linkage between NHE1 and diabetes has been previously described in NOD mice strains with consequences on NHE rates. Hence, the polymorphism described hereby may act as a predisposition factor to type-I diabetes or to diabetic complications, and may be useful to investigate the genetic involvement of NHE1 in human pathophysiology.

Asunto(s)

RESUMEN

Thyroid carcinomas usually have a good prognosis, particularly well-differentiated tumors confined to the gland. An analysis was made of the treatment and prognosis of thyroid neoplasms with invasion of the upper respiratory and digestive tract. Of 211 patients treated at our institution from 1975 to 1994, only 18 had invasion of the upper respiratory and digestive system. All patients were treated surgically. The procedures included total thyroidectomy, laryngectomy, partial esophagectomy, and tracheal resection. The neoplasm recurred locally after complete resection in 33%. Cervical lymph node metastases occurred in 17% and distant metastases in 33%. The 5-year survival rate was 46% for well-differentiated carcinomas. Invasion of the respiratory and digestive tract had negative prognostic significance in the statistical analysis. Locally advanced thyroid neoplasms had a poorer prognosis than those limited to the thyroid gland. However, complete surgical resection of the malignancy was achieved in most patients without functionally mutilating procedures and with satisfactory survival results.

Asunto(s)

RESUMEN

The heat stable inhibitor of cAMP-dependent protein kinase (PKIalpha) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIalpha to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CaH chemical shifts revealed two regions of upfield chemical shifts characteristic of alpha-helices. When PKIalpha was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIalpha (1-25), contains an alpha-helix from residues 1-13. This helix extends by one turn the helix observed in the crystal structure of a PKIalpha (5-24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35-47, overlaps with the nuclear export signal in the PKIalpha (26-75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

Asunto(s)

RESUMEN

MTCP1 (for Mature-T-Cell Proliferation) was the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8MTCP1 protein encoded by the MTCP1 oncogene has been previously determined by homonuclear proton two-dimensional NMR methods at 600 MHz: it consists of an original scaffold comprising three alpha-helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an alpha-hairpin which resembles an antiparallel coiled-coil. The third helix is orientated roughly parallel to the plane defined by the alpha-antiparallel motif and appears less well defined. In order to gain more insight into the details of this new scaffold, we uniformly labeled with nitrogen-15 a mutant of this protein (C12A-p8MTCP1) in which the unbound cysteine at position 12 has been replaced by an alanine residue, thus allowing reproducibly high yields of recombinant protein. The refined structure benefits from 211 additional NOEs, extracted from 15N-edited 3D experiments, and from a nearly complete set of phi angular restraints allowing the estimation of the helical content of the structured part of the protein. Moreover, measurements of 15N spin relaxation times and heteronuclear 15N¿1H¿NOEs provided additional insights into the dynamics of the protein backbone. The analysis of the linear correlation between J(0) and J(omega) was used to interpret relaxation parameters. It appears that the apparent relative disorder seen in helix III is not simply due to a lack of experimental constraints, but associated with substantial contributions of sub-nanosecond motions in this segment.

Asunto(s)

RESUMEN

MTCP1 (for Mature-T-Cell Proliferation) is the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8(MTCP1) protein encoded by the MTCP1 oncogene was determined by homonuclear proton two-dimensional NMR methods at 600 MHz. After sequence specific assignments, a total of 931 distance restraints and 57 dihedral restraints were collected. The location of the three previously unassigned disulfide bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of p8(MTCP1) is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics using a simulated annealing protocol with the AMBER force field. The r.m.s.d. values with respect to the mean structure for the backbone and all heavy atoms for a family of 30 structures are 0.73(+/-0.28) and 1.17(+/-0.23) A, when the structured core of the protein (residues 5 to 63) is considered. The solution structure of p8(MTCP1) reveals an original scaffold consisting of three alpha helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an alpha-hairpin which resembles an antiparallel coiled-coil. The third helix is oriented roughly parallel to the plane defined by the alpha-antiparallel motif and its axis forms an angle of approximately 60 degrees with respect to the main axis of this motif.

Asunto(s)

RESUMEN

The effectiveness of two-stage surgery was studied in several skin-flap models in rabbits. The first group (n = 8) of 7 cm x 2 cm skin flaps perpendicular to major regional blood vessels became necrotic after the first stage of surgery. In the second group (n = 16) of 4 cm x 2 cm skin flaps, survival was no better in the two-stage group (n = 8) than in controls (n = 8). In the third group (n = 32) of 7 cm x 2 cm skin flaps parallel to major blood vessels, survival was better (p < 0.001) in the two-stage subgroup (n = 16). We conclude that effective two-stage skin-flap surgery requires the use of narrow skin flaps that are parallel to major blood vessels.

Asunto(s)

Asunto(s)

RESUMEN

The aim of this study was to determine which BP measurement is the best related to target organ damage. There were 49 young adults with a history of childhood hypertension (school check-up) and to increase the prevalence of hypertension in the study group, 25 males 24 females, aged 20.1 +/- 2.2 years old were included in the study. All patients had a diurnal ambulatory BP monitoring and office BP measurements taken with a sphygmomanometer and with an oscillometric device during a 15 minute period monitoring (Dinamap BP). Echocardiography and blood samples (cholesterol and triglycerides) were performed in all patients. In contrast with office BP, a mean ambulatory SBP and mean Dinamap SBP significantly correlated to left ventricular mass index (LVMI) (P < 0.01). There was also a correlation between triglycerides and mean Dinamap SBP (P < 0.01) which could suggest an early insulin resistance. In this selected population, a short period BP monitoring, as well as ambulatory BP, is more closely related to LVMI than office BP.

Asunto(s)

RESUMEN

Thirty-four cases of sino-nasal adenocarcinoma treated between 1976 and 1992 are presented. The purpose of this work is the epidemiological analysis of this disease and to put forward a classification of the lesions according to their degree of differentiation, based on the histopathological examination of 29 tumors. The risk increase of having a sino-nasal neoplasia was significant in the population exposed to wood dust in their working environment--896 times greater (540 in the male population) for adenocarcinoma and 20 times greater for squamous-cell adenocarcinoma. The proposed histopathological classification divides these lesions according to their degree of differentiation. In this series 23 well-differentiated cases were found, three moderately differentiated and three colloid-type. The five-year determinate survival of this series was 30%. The tumor stage (especially intracranial involvement) was the only significant clinico-pathological parameter that impaired the survival.

Asunto(s)

RESUMEN

Thirty four patients with allergic rhinitis were included in two randomized groups and treated with Cetirizine and Astemizole respectively, 10 mg/day in oral administration during 15 days. This study was designed in order to compare the efficacy and tolerance of Cetirizine versus Astemizole, with special reference on the collateral effects on the central nervous system. Although both drugs showed good results. Cetirizine had a statistically significant better outcome in the evolution of symptoms, above all at the second day of treatment. Also it showed superior results, based on a subjective approach, related to the rapidity, power and efficacy of its action.

Asunto(s)

RESUMEN

In order to assess the potential role of the plasma membrane sodium-proton (Na+/H+) exchanger in the pathogenesis of diabetic nephropathy, we investigated 32 insulin dependent (type 1) diabetic patients and 21 control subjects. We tested the Na+/H+ exchange as the rate of amiloride sensitive and sodium dependent volume gain of platelets suspended in sodium propionate. Patients with diabetic nephropathy had significantly increased rates of Na+/H+ exchange (0.31 +/- 0.06 s-1 x 10(-2)) when compared to those without nephropathy (0.24 +/- 0.07, p less than 0.05) or to a control group (0.23 +/- 05, p less than 0.05). Nine patients who were classified as hypertensive had a highly significant increase in the Na+/H+ exchange rates when compared to 23 non-hypertensive diabetic patients: 0.33 +/- 0.04 versus 0.24 +/- 0.06 (p less than 0.001). There was no significant correlation between the Na+/H+ exchange rates and age, diabetes duration, glycated hemoglobin or fructosamine levels on the day of the test. In summary, the data presented here demonstrate an increase in the Na+/H+ exchange rate in insulin-dependent diabetic patients with nephropathy and hypertension.

Asunto(s)

RESUMEN

Therapeutic apheresis in children is limited by technical considerations, such as circuit blood volume, venous access and hemodynamic instability. Here we report our experience with 310 plasma-filtration sessions in 44 children (aged 9 months-17 years). We used a miniaturized circuit with a unipuncture technique and a "balancing system" which assured a permanent fluid equilibrium. This procedure is well adapted to children and always made pediatric plasma exchange therapy easier and safer. The more common indications remain Guillain-Barré syndrome and acute severe glomerular diseases.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

The tendency of subjects to maintain their relative position within the distribution of blood pressure (BP) has been defined as "tracking". Regarding this phenomenon, the purpose of the study was to evaluate the interest of ambulatory BP monitoring (ABPM) in the assessment of arterial hypertension in young adults (YA) with childhood hypertension history (CHH). 52 subjects, 20.1 +/- 2.4 years old, 26 men, 26 women issued from a cohort of 150 children with high BP levels (greater than 97.5 th percentile) during their infancy (school check-up), were included in the study. An ABPM was performed with space-labs system 90202 from 8 a.m. to 6 p.m., measurements every 15 minutes (37.6 +/- 7.4 readings). Left ventricular mass index (LVMI) was determined with echocardiography, (Penn convention). Office BP, measured with mercury apparatus in lying and standing position, was respectively, 131.0 +/- 14.6/81.9 +/- 9.7 and 130.1 +/- 14/86.6 +/- 9.9. According to JNC 1988, this casual BP identified 40 normotensives (NT), 9 borderlines (BL) and 5 hypertensives (HT); 10 of them had a "high normal" diastolic BP (85-90 mmHg) ABP recordings of the study group were compared to day-time reference values of NT. Three subgroups are individualized: G1 NT, G2 HT, G3 BL. [table; see text] *p: less than 0.001; p: less than 0.01. Wall thickness (WTh) and LVMI were significantly higher in hypertensives (G2 + G3) than in normotensives (G1): [table; see text] There was a significant correlation between LVMI and mean systolic ABP (p less than 0.01: r = 0.44), but not with office SBP.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

In order to assess the relationships between increased cellular sodium-proton (Na+/H+) exchange and cardiovascular abnormalities in essential hypertension (EH), 21 young subjects as part of an ongoing longitudinal study were tested for the platelets Na+/H+ exchange using the amiloride sensitive sodium dependent component of platelet volume change under cytoplasmic acidification induced by a sodium propionate medium; cell volumes were determined by electronic cell sizing (Livne et al., Lancet 1987; i: 533-6). 24 normal subjects with normotension and without familial history of hypertension were taken as controls. Data of ambulatory blood pressure recording (ABPR) defined 2 groups according to the presence of normotension (group I, n = 10), or of hypertension (group II, n = 11): established (n = 2) or borderline (n = 9) hypertension. Hypertensive subjects (group II) had increased values of Na+/H+ exchange (k coefficient, mean (SEM): 0.287 (0.07) vs 0.228 (0.05) in control group (p less than 0.01). Na+/H+ rates were significantly related to ABPR data (r = 0.46, p less than 0.02 with diastolic charge during ABPR), but not to left ventricular mass index in g/m2 by echocardiography. Increased rates of platelets Na+/H+ exchange which were related to diastolic blood pressure levels by ABPR, and perhaps to the level of peripheric vascular resistances, may play a significant role in the development of EH in the early stages.

Asunto(s)

RESUMEN

Abscisic acid (ABA) uptake by Amaranthus tricolor cell suspensions was found to include both a nonsaturable component and a saturable part with K(m) of 3.74 +/- 0.43 micromolar and an apparent V(max) of 1.5 +/- 0.12 nanomoles per gram per minute. These kinetic parameters as well as the uptake by intact cells at 0 degrees C or by frozen and thawed cells, are consistent with operation of a saturable carrier. This carrier-mediated ABA uptake was partially energized by DeltapH: it increased as the external pH was lowered to pH 4.0; it decreased after the lowering of the DeltapH by the proton ionophore carbonylcyanide-m-chlorophenylhydrazone or after the altering of metabolically maintained pH gradient by metabolic inhibitors (KCN, oligomycin). The carrier is specific for ABA among the plant growth regulators tested, is unaffected by (RS)-trans-ABA and was inhibited by (S)-ABA, (R)-ABA, and also by the ABA analog LAB 173711.