Conformational ensemble of human α-synuclein physiological form predicted by molecular simulations.

We perform here enhanced sampling simulations of N-terminally acetylated human α-synuclein, an intrinsically disordered protein involved in Parkinson's disease. The calculations, consistent with experiments, suggest that the post-translational modification leads to the formation of a transient amphipathic α-helix. The latter, absent in the non-physiological form, alters protein dynamics at the N-terminal and intramolecular interactions.

Disruption of zinc and copper interactions with Aß(1-40) by a non-toxic, isoniazid-derived, hydrazone: a novel biometal homeostasis restoring agent in Alzheimer's disease therapy?

Disruptions of biometal-Aß(1-40) interactions by an isoniazid-derived hydrazone, INHHQ, were demonstrated via in vitro NMR titrations. The compound has adequate theoretical BBB absorption properties, assessed by in silico studies. In vivo acute toxicity assays indicate that INHHQ is innocuous up to 300 mg kg(-1), showing potential as an anti-Alzheimer's drug.

Axial ligand modulation of the electronic structures of binuclear copper sites: analysis of paramagnetic 1H NMR spectra of Met160Gln Cu(A).

Cu(A) is an electron-transfer copper center present in heme-copper oxidases and N2O reductases. The center is a binuclear unit, with two cysteine ligands bridging the metal ions and two terminal histidine residues. A Met residue and a peptide carbonyl group are located on opposite sides of the Cu2S2 plane; these weaker ligands are fully conserved in all known Cu(A) sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba3 oxidase has been studied by NMR spectroscopy. In its oxidized form, the binuclear copper is a fully delocalized mixed-valence pair, as are all natural Cu(A) centers. The faster nuclear relaxation in this mutant suggests that a low-lying excited state has shifted to higher energies compared to that of the wild-type protein. The introduction of the Gln residue alters the coordination mode of His114 but does not affect His157, thereby confirming the proposal that the axial ligand-to-copper distances influence the copper-His interactions (Robinson, H.; Ang, M. C.; Gao, Y. G.; Hay, M. T.; Lu, Y.; Wang, A. H. Biochemistry 1999, 38, 5677). Changes in the hyperfine coupling constants of the Cys beta-CH2 groups are attributed to minor geometrical changes that affect the Cu-S-C(beta)-H(beta) dihedral angles. These changes, in addition, shift the thermally accessible excited states, thus influencing the spectral position of the Cys beta-CH2 resonances. The Cu-Cys bonds are not substantially altered by the Cu-Gln160 interaction, in contrast to the situation found in the evolutionarily related blue copper proteins. It is possible that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible Cu(A) excited states by tuning axial ligand interactions.

Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state.

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.

Utility of Doppler velocimetry in predicting outcome in twin reversed-arterial perfusion sequence.

OBJECTIVE: The aim of this study was to describe Doppler velocimetric findings in pregnancies complicated by the twin reversed-arterial perfusion sequence and to determine the association of these findings with pregnancy outcome. STUDY DESIGN: Six twin pregnancies complicated by twin reversed-arterial perfusion sequence had ultrasonographic and Doppler studies performed between 1990 and 1997. Pulsatile vessels in the umbilical cords of the pump and acardiac twins were insonated, and reversal of flow was confirmed in all cases. Resistive index values were calculated, and the difference in resistive index between the pump and acardiac twin in each pair was evaluated as a marker of pregnancy outcome. RESULTS: Five of 6 pump twins survived the immediate neonatal period. Although 5 of the acardiac twins had abnormally elevated Doppler index values, no ratio of systolic to diastolic velocity or resistive index value of the acardiac twin alone was associated with either a good or poor prognosis for the pump twin. Among the 3 pump twins with good outcomes, all had a resistive index difference >0.20. Among the 3 pump twins with poor outcomes, all had small resistive index differences (<0.05). CONCLUSION: We found larger differences in resistive index to be associated with improved outcome of the pump twin in pregnancies complicated by twin reversed-arterial perfusion sequence. Smaller resistive index differences were associated with poor outcome, including cardiac failure and central nervous system hypoperfusion.

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.

Changing trends in patient decisions concerning genetic amniocentesis.

OBJECTIVE: This study was undertaken to determine whether there was a change in patient decisions concerning genetic amniocentesis during the period 1995-1998. STUDY DESIGN: All patients referred for genetic counseling because of advanced maternal age, abnormal serum triple-screen results, or ultrasonographic abnormalities between January and March 1995 and between January and March 1998 were evaluated through a retrospective chart review. Patient characteristics included age, race, and gestational age. Group 1 consisted of patients from 1995. Group 2 consisted of patients from 1998. Data on patient decisions concerning amniocentesis before and after genetic counseling and ultrasonographic examination were compared in each group. Groups 1 and 2 were then compared with respect to decisions before and after genetic counseling and ultrasonographic evaluation. RESULTS: A total of 112 patients were studied. Group 1 consisted of 53 patients and group 2 consisted of 59 patients. When the groups were compared, no differences in age, race, or gestational age were noted. In group 1, before counseling, 18 of 53 patients desired genetic testing, compared with 44 of 53 after counseling (P =.02). In group 2, before counseling, 4 of 59 patients desired genetic testing, compared with 15 of 59 after counseling (P =.01). A significantly greater number of patients in group 1 than in group 2 desired genetic testing both before counseling (n = 18/53 vs n = 4/59; P =.01) and after counseling (n = 44/53 vs n = 15/59; P =.01). CONCLUSION: Fewer patients at risk for Down syndrome in 1998 than in 1995 desired amniocentesis both before and after genetic counseling and ultrasonographic examination.

Probing the interaction between N(1),N(4)-dibenzylputrescine and tRNA through (15)N NMR: biological implications.

NMR spectroscopy was used to characterize the binding properties of polyamines to Escherichia coli tRNA. The (15)N NMR spectra of three (15)N-enriched N-substituted putrescine derivatives (DMP, DEP and DBP) were recorded in the presence of tRNA, and the spin relaxation times of the nitrogen nuclei were measured. From these data, the activation parameters for the rotational correlation times of the (15)N nuclei were determined. The present data indicate that the nature of the amino substituents does play a relevant role in controlling the polyamine-tRNA interaction. This study also provides a rationale for the in vivo antiproliferative effect of DBP against tumoral cells.

[Varicella pneumonia in the previously healthy adult]. / Neumonía varicelosa en el adulto previamente sano.

Varicella (chickenpox) is a contagious, self-limited, usually benign disease common in childhood but uncommon in adulthood. Pneumonia is the most frequent complication of the disease in adults. We retrospectively review 7 cases of varicella pneumonia in previously healthy adults diagnosed in our hospital between 1992 and 1996. All of them were treated with intravenous acyclovir with good therapeutic response save for a patient who developed respiratory insufficiency and died 8 days after his admission. Smoking was the only risk factor detected. Clinical features of our patients are described and the need of an early diagnosis and treatment of varicella pneumonia is emphasized.

Neumonía varicelosa en el adulto previamente sano / Varicella penumonia in the previously healthy adult

La varicela es una enfermedad contagiosa autolimitada y generalmente benigna propia de la infancia, y poco común en el adulto. La neumonía es la complicación mas frecuente de la enfermedad en el adulto. Revisamos retrospectivamente 7 casos de neumonía varicelosa en adultos previamente sanos diagnosticados en nuestro hospital entre 1992 y 1996. Todos ellos fueron tratados con Aciclovir endovenoso, presentando buena evolución salvo un paciente que desarrolló insuficiencia respiratoria y falleció 8 días tras su ingreso. El tabaco fue el único factor de riesgo detectado. Presentamos las particularidades clínicas de nuestros pacientes y destacamos la necesidad de un diagnóstico y tratamiento precoz de la neumonía por varicela (AU)

The four-chamber view and its sensitivity in detecting congenital heart defects.

We sought to determine the sensitivity of detecting congenital heart defects with routine antenatal ultrasound including the four-chamber view in a population at low risk for cardiac anomalies. Neonatal outcome and anomaly databases were reviewed to identify cases of morphologic cardiac defects from 1988-1992. Of 176 cases identified, 62 (35%) had routine antenatal ultrasound including the four-chamber view. Thirteen of these 62 (21%) were diagnosed by ultrasound and 18 of the 116 (15%) total defects present were seen. Of these, only 16 of 25 (64%) defects reasonably expected to be seen by the four-chamber cardiac view alone were detected. The routine ultrasound four-chamber cardiac evaluation is limited and detection depends on the type of cardiac defect present.

A randomized placebo-controlled evaluation of terbutaline for external cephalic version.

OBJECTIVE: To evaluate the efficacy of subcutaneous terbutaline therapy on the success rate of external cephalic version in term gestation. METHODS: Women with singleton noncephalic gestations were assigned randomly to receive either terbutaline (0.25 mg) or placebo. Physicians were blinded to the assignment. Fifteen to 30 minutes after the study drug was administered, external cephalic version was attempted. It was discontinued after three attempts, for patient discomfort, for fetal heart rate decelerations, or when successful. Patients were discharged home after the procedure and allowed to enter spontaneous labor. Primary outcomes evaluated included initial success of version, presentation in labor, and route of delivery. RESULTS: One hundred three women were enrolled in the study between January 1994 and June 1995, of whom 52 were assigned to terbutaline and 51 to placebo. External cephalic version was successful in 27 of 52 (52%) women receiving terbutaline compared with 14 of 51 (27%) of those receiving placebo (P = .019). This comparison yielded a relative risk (RR) of 1.9 (95% confidence interval [CI] 1.3, 6.5). Four of the 27 (15%) successful versions in the terbutaline group and three of the 14 (21%) successful versions in the placebo group spontaneously reverted to breech presentation. Ultimately, in labor there were 24 (46%) cephalic presentations in the terbutaline group and 13 (25%) in the placebo group (P = .048, RR 1.84, 95% CI 1.1, 5.8). Cesarean delivery rates were 11 of 41 (27%) for women with successful versions and 58 of 62 (94%) among those with failed versions (P < .001). CONCLUSION: Terbutaline (0.25 mg) administered subcutaneously before an attempted version in women at term with noncephalic presentations significantly increased the initial success rate of version and the rate of cephalic presentations in labor while decreasing the rate of cesarean delivery.

Alkaline transition of Rhus vernicifera stellacyanin, an unusual blue copper protein.

Stellacyanin from Rhus vernificera is a blue copper protein in which the metal is coordinated to a Cys, two His, and a Gln residue. It displays a low redox potential, a fast electron exchange rate, and a reversible alkaline transition. We have studied this transition in Cu(II)- and Co(II)-stellacyanin by means of electronic and NMR spectroscopy. The data indicate that a conformational rearrangement of the metal site occurs at high pH. A drastic alteration in the Gln coordination mode, as initially proposed, is discarded. These results show that the metal site in stellacyanin is more flexible than the sites of other blue copper proteins. The present study demonstrates that the paramagnetic shifts of the bound Cys in the Co(II) derivative are sensitive indicators of the electron delocalization and conformational changes experienced by this residue.

MURCS association: ultrasonographic findings and pathologic correlation.

MURCS association is a rare, lethal and unusual constellation of nonrandom findings that includes mullerian duct aplasia, renal aplasia, and cervicothoracic somite dysplasia.1-3 It has been described in 30 patients by Duncan and coworkers2 in 1979, in which report the authors proposed an embryologic cause for these defects.3 Antenatal ultrasonographic findings included a massive, cystic umbilical cord related to a patent urachus, enlarged bladder, single small kidney, and suspicion of urethral obstruction in a fetus of female phenotype. These findings are rare in a case of MURCS and were all confirmed on pathologic examination.

Blindness associated with preeclampsia and eclampsia.

OBJECTIVE: Over a 14-year period at Parkland Hospital, the clinical courses of 15 women with severe preeclampsia or eclampsia were further complicated by blindness. Our purpose is to describe their management and outcome, as well as to offer insight to the pathophysiologic characteristics of blindness complicating pregnancy-induced hypertension. STUDY DESIGN: Prospective ascertainment of women with blindness and pregnancy-induced hypertension was done. These cases were managed according to the standardized preeclampsia-eclampsia regimen used at our hospital since 1955. Briefly, this regimen includes magnesium sulfate given intramuscularly to prevent or control seizures, hydralazine to lower dangerously elevated blood pressure, intravenous fluid restriction, and delivery. RESULTS: There were 15 women with blindness that persisted from 4 hours to 8 days; it subsequently resolved completely in all. Of the 13 women who underwent computed tomography, 8 had low-density areas localized predominantly in the occipital lobes. Five of these 13 subsequently underwent magnetic resonance imaging and 2 showed corresponding hyperintense lesions in the occipital areas. CONCLUSIONS: On the basis of previously published experiences with computed tomography in women with eclampsia, as well as the experiences described here, we conclude that cortical blindness associated with preeclampsia-eclampsia results from petechial hemorrhages and focal edema in the occipital cortex. These lesions are likely stimulated by disparity in cerebral regional blood flow that is characterized by vasospasm and diminished flow primarily affecting the posterior circulation.

Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis.

N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities. 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA. When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM-4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced. From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA. These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N-bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding). The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2. The decrease of the V1/2 values of the -NH2(+)-groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA. A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities.

Interactions between natural polyamines and tRNA: an 15N NMR analysis.

15N NMR spectroscopy was used to explore the interactions between natural polyamines and Escherichia coli tRNA. It was found that when tRNA is added to solutions of 15N-labeled spermine or spermidine, there is a considerable decrease in the relative heights of the -NH(2+)--resonances with respect to the signals arising from the -NH3+ groups. The presence of tRNA was also found to reduce the longitudinal relaxation times T1 of the nitrogens, mainly those of the -NH(2+)- groups. The longitudinal relaxation times of the nitrogens were used to characterize the temperature dependence of the binding, and they allowed us to calculate the activation energies that determine the correlation times of amino groups in the presence of tRNA. Both the thermodynamic and the relaxation results indicate that (i) spermine binds more strongly to tRNA than spermidine does and (ii) within each of these molecules the -NH(2+)- groups bind more strongly to tRNA than the more electropositive -NH3+ moieties. This specificity suggests that the interaction between polyamines and tRNA cannot be described exclusively in terms of electrostatic forces and that other interactions (most likely, hydrogen bonding) are very important for establishing the polyamine-tRNA link. Some of the factors that may conspire against the binding of -NH3+ groups to tRNA are briefly discussed.