Biomedical subjects

M Saraste

Publications and source records attributed to M Saraste.

At least 37 records · Page 2Linked to original sources

PubMed2000

States and transitions during forced unfolding of a single spectrin repeat.

Spectrin is a vital and abundant protein of the cytoskeleton. It has an elongated structure that is made by a chain of so-called spectrin repeats. Each repeat contains three antiparallel alpha-helices that form a coiled-coil structure. Spectrin forms an oligomeric structure that is able to cross-link actin filaments. In red cells, the spectrin/actin meshwork underlying cell membrane is thought to be responsible for special elastic properties of the cell. In order to determine mechanical unfolding properties of the spectrin repeat, we have used single molecule force spectroscopy to study the states of unfolding of an engineered polymeric protein consisting of identical spectrin domains. We demonstrate that the unfolding of spectrin domains can occur in a stepwise fashion during stretching. The force-extension patterns exhibit features that are compatible with the existence of at least one intermediate between the folded and the completely unfolded conformation. Only those polypeptides that still contain multiple intact repeats display intermediates, indicating a stabilisation effect. Precise force spectroscopy measurements on single molecules using engineered protein constructs reveal states and transitions during the mechanical unfolding of spectrin. Single molecule force spectroscopy appears to open a new window for the analysis of transition probabilities between different conformational states.

Animals↗

PubMed2000

Efficiency of left ventricular diastolic function increases in healthy full-term infants during the first months of life. A prospective follow-up study.

Postnatal changes in left ventricular diastolic filling and systolic cardiac performance were studied monthly by serial echocardiographic measurements from days 3-5 up to six months in 20 healthy full-term infants. The fractional shortening area (FSA = (left ventricular end-diastolic area - end-systolic area)/end-diastolic area) was assessed for systolic performance, and transmitral pulsed-wave Doppler flow velocity patterns were analysed for diastolic function. FSA remained stationary during the observation. After birth, left ventricular peak early (E) and atrial (A) velocities and the respective integrals were lower than at one month of age (47+/-5 vs. 63+/-6 cm/s and 44+/-6 vs. 57+/-5 cm/s and 3.33+/-0.40 vs. 4.05+/-0.53 cm and 2.74+/-0.40 vs. 3.18+/-0.53 cm; P < 0.05). During the next five months, the early parameters (E velocity and E integral) increased but the atrial indices (A velocity and A integral) did not change. During the whole observation the E/A velocity ratio, the E/A integral ratio and the early filling fraction (EFF) increased. The early filling deceleration time was longer during the first month than later (87+/-10 vs. 72+/-13 ms; P < 0.05). In conclusion, age-related changes were observed in the diastolic but not in the systolic performance in healthy full-term infants during the first six months. The most intensive changes took place in the early and atrial transmitral parameters during the first month of life, suggesting an improvement in both left ventricular relaxation and compliance. During the following five months, the early mitral parameters increased but the atrial diastolic values remained stable. These changes may mainly be determined by the improvement in left ventricular relaxation.

Aging↗

PubMed2000

Heme centers of Rhodothermus marinus respiratory chain. Characterization of its cbb3 oxidase.

Rhodothermus (R.) marinus, a thermohalophilic gram-negative, and strict aerobic bacterium, has a rather distinct respiratory chain, containing a caa3 terminal oxidase, a novel cytochrome bc complex and a HiPIP, which is an electron carrier between this complex and a terminal oxidase (Pereira et al (1999a, c). To further elucidate this unusual respiratory system, its membrane-bound heme centers were characterized by visible and EPR spectroscopies as well as by redox potentiometry. Rhodothermus marinus contains mostly B- and C-type hemes; a small amount of A-type heme is also detected. The heme centers have relatively low reduction potentials, ranging from ca. +250 to -60 mV, at pH 7. A Rieske-type center was not detected, suggesting the absence of a canonical complex III. The major terminal oxidase expressed by R. marinus is a cbb3-type oxidase. Its presence is in agreement with molecular biology studies, which reveal the existence of a gene encoding for a FixN-type oxidase. The oxidase was partially purified and appears to have five subunits, with apparent molecular masses of 64, 57, 36, 26 (C-type heme subunit), and 13 kDa. It contains two low-spin heme C centers, one high-, and one low-spin heme B centers. A full description of the equilibrium redox behavior of the heme centers was obtained for a cbb3 oxidase for the first time. The optical spectrum for each heme center and the corresponding reduction potentials were determined at pH 7: + 195 (heme C), +120 (heme B), -50 (heme C), and -50 mV (heme B3).

Amino Acid Sequence↗

PubMed2000

Technical achievement: transthoracic Doppler echocardiography can be used to detect LAD restenosis after coronary angioplasty.

We investigated the capability of transthoracic Doppler echocardiography (TTE) to detect and quantify the severity of restenosis in the left anterior descending coronary artery (LAD) after percutaneous transluminal coronary angioplasty (PTCA). We studied 10 consecutive patients assigned for quantitative coronary angiography (qCA) due to a recurrent angina pectoris after PTCA of the LAD. The LAD was visualized by TTE, and the presence of local turbulence and an increase in the blood flow velocity was regarded to indicate coronary stenosis. To assess the severity of the stenosis, the increase of blood flow velocity was measured. Angiography showed stenoses of various degrees (27-100%) in all patients. All stenoses were detectable using TTE. Moreover, the ratio of maximal blood flow velocity at the site of stenosis to the pre-stenotic blood flow velocity (M/P-ratio) correlated significantly with the reduction of the luminal diameter of LAD (r = 0.85, P < 0.003). A M/P-ratio higher than 3.0 predicted a diameter reduction of 50% or higher with sensitivity and specificity of 100% in patients with a subtotal stenosis (n = 9). Our results indicate that stenoses in the LAD could be found and the severity of the stenoses could be quantified reliably with TTE. This approach is totally non-invasive and less expensive than coronary angiography and can be used clinically in clarifying restenosis after coronary angioplasty.

Aged↗

PubMed2000

Myocardial efficiency during levosimendan infusion in congestive heart failure.

AIMS: Levosimendan, a novel calcium-dependent calcium sensitizer of the myocardial contractile proteins, also enhances diastolic relaxation and induces peripheral vasodilation by opening potassium channels. To assess the combined energetical effects of levosimendan infusion in vivo, we performed positron emission tomography in patients with decompensated chronic heart failure. METHODS AND RESULTS: Eight hospitalized patients with New York Heart Association functional class III or IV heart failure received levosimendan or placebo intravenously in a randomized double-blind cross-over study. During steady-state, dynamic positron emission tomography with [11C]acetate was used to assess myocardial oxygen consumption and [15O]H2O to measure myocardial blood flow. Cardiac performance and dimensions were assessed by pulmonary artery catheterization and echocardiography. Compared with healthy volunteers, myocardial oxygen consumption during placebo was elevated in the right ventricle but comparable in the left ventricle. During administration of levosimendan, cardiac output increased by 32% (P = .002) mainly because of higher stroke volume. Coronary, pulmonary, and systemic vascular resistance values were significantly reduced. Mean myocardial blood flow increased from 0.76 to 1.02 mL/min/g (P = .033). Levosimendan was neutral on myocardial oxygen consumption and left ventricular efficiency, but it improved right ventricular mechanical efficiency by 24% (P = .012). CONCLUSIONS: Levosimendan has an energetically favorable short-term profile in the treatment of congestive heart failure. It enhances cardiac output without oxygen wasting, particularly by improving efficiency in the right ventricle.

Cardiotonic Agents↗

PubMed2000

Assessment of coronary flow reserve using fast velocity-encoded cine MR imaging: validation study using positron emission tomography.

OBJECTIVE: Previous studies using intravascular Doppler sonography and positron emission tomography (PET) have shown that the hemodynamic significance of coronary artery stenosis can be evaluated by measuring coronary flow reserve. The purpose of this study was to assess whether MR imaging measurements of coronary flow reserve in the left anterior descending artery are comparable with those obtained with PET in the corresponding territory. SUBJECTS AND METHODS: MR imaging and PET flow measurements were obtained in 10 healthy volunteers. Blood flow velocity in the left anterior descending artery was measured with breath-hold velocity-encoded cine MR imaging before and after IV administration of dipyridamole. The coronary flow velocity reserve measured by MR imaging was compared with the myocardial perfusion reserve in the anterior myocardium quantified on using PET and (15)O-labeled water. RESULTS: The average flow velocity reserve in the left anterior descending artery measured on MR imaging was 2.44+/-1.14 in healthy volunteers, which was comparable with the myocardial perfusion reserve measured by PET (2.52+/-0.84). MR imaging and PET measurements of the coronary flow reserve showed a significant correlation (r = 0.79, p<0.01). CONCLUSION: MR imaging measurement of the flow velocity reserve in the proximal left anterior descending artery correlates well with the myocardial perfusion reserve obtained with PET and (15)O-labeled water.

Adult↗

PubMed1999

Cardiac positron emission tomography imaging with [11C]hydroxyephedrine, a specific tracer for sympathetic nerve endings, and its functional correlates in congestive heart failure.

The integrative mechanisms of autonomic dysfunction in congestive heart failure (CHF) remain poorly understood. We sought to study cardiac retention of [11C]hydroxyephedrine (HED), a specific tracer for sympathetic presynaptic innervation, and its functional correlates in CHF. Thirty patients with mild to moderate heart failure underwent resting cardiac HED positron emission tomography imaging, spectrum analysis testing of systolic pressure and heart rate variability in the resting supine and 70 degrees head-up tilt positions, and testing of baroreflex sensitivity. Compared with control subjects, global myocardial HED retention index was reduced by 30% (p <0.01) in patients with CHF. The HED retention index did not correlate significantly with heart rate variability. However, it correlated with baroreflex sensitivity at rest (r = 0.43, p = 0.05) and with systolic pressure low-frequency (0.03 to 0.15 Hz) variability at head-up tilt (r = 0.76, p <0.01), as well as with low-frequency systolic pressure variability response from baseline to tilt (r = 0.75, p <0.01). We conclude that cardiac HED retention is reduced in patients with CHF. This correlates with blunted vascular sympathetic effector responses during posture-induced reflex activation and baroreflex control of heart rate, suggesting an interdependence between cardiac presynaptic innervation abnormalities and neural mechanisms important to blood pressure maintenance in CHF.

Blood Pressure↗

PubMed1999

The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283). In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised. It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type. This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase. The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3). Subunit I of R. marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels). However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R. marinus oxidase. Homology modelling of the R. marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre. Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV-. These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.

Amino Acid Sequence↗

PubMed1999

Structure of the alpha-actinin rod: molecular basis for cross-linking of actin filaments.

We have determined the crystal structure of the two central repeats in the alpha-actinin rod at 2.5 A resolution. The repeats are connected by a helical linker and form a symmetric, antiparallel dimer in which the repeats are aligned rather than staggered. Using this structure, which reveals the structural principle that governs the architecture of alpha-actinin, we have devised a plausible model of the entire alpha-actinin rod. The electrostatic properties explain how the two alpha-actinin subunits assemble in an antiparallel fashion, placing the actin-binding sites at both ends of the rod. This molecular architecture results in a protein that is able to form cross-links between actin filaments.

Actin Cytoskeleton↗

PubMed1999

Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate.

BACKGROUND: The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. RESULTS: We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P4). The inositol Ins (1,3,4,5)P4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-delta. A second Ins (1,3,4,5)P4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P4 to the wild type (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. CONCLUSIONS: Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1999

Oxidative phosphorylation at the fin de siècle.

Mitochondria produce most of the energy in animal cells by a process called oxidative phosphorylation. Electrons are passed along a series of respiratory enzyme complexes located in the inner mitochondrial membrane, and the energy released by this electron transfer is used to pump protons across the membrane. The resultant electrochemical gradient enables another complex, adenosine 5'-triphosphate (ATP) synthase, to synthesize the energy carrier ATP. Important new mechanistic insights into oxidative phosphorylation have emerged from recent three-dimensional structural analyses of ATP synthase and two of the respiratory enzyme complexes, cytochrome bc1 and cytochrome c oxidase. This work, and new enzymological studies of ATP synthase's unusual catalytic mechanism, are reviewed here.

Adenosine Triphosphate↗

PubMed1999

Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles.

Spectrin repeats fold into triple helical coiled-coils comprising approximately 106 amino acid residues. Using an AFM-related technique we measured the force required to mechanically unfold these repeats to be 25 to 35 pN. Under tension, individual spectrin repeats unfold independently and in an all-or-none process. The dependence of the unfolding forces on the pulling speed reveals that the corresponding unfolding potential is shallow with an estimated width of 1.5 nm. When the unfolded polypeptide strand is relaxed, several domains refold within less than a second. The unfolding forces of the alpha-helical spectrin domains are five to ten times lower than those found in domains with beta-fold, like immunoglobulin or fibronectin Ill domains, where the tertiary structure is stabilized by hydrogen bonds between adjacent strands. This shows that the forces stabilizing the coiled-coil lead to a mechanically much weaker structure than multiple hydrogen-bonded beta-sheets.

Animals↗

PubMed1999

Paramagnetic NMR studies of blue and purple copper proteins.

1H- and 13C-NMR spectroscopy is applied to investigate the CU(A) and type 1 active sites of copper proteins in solution. The analysis of hyperfine shifted 1H resonances allows the comparison of the electron spin density delocalization in the CU(A) site of the wild-type soluble domains of various cytochrome c oxidases (Thermus thermophilus, Paracoccus denitrificans, and Paracoccus versutus) and genetically engineered constructs (soluble domain of quinol oxidase from Escherichia coli and Thiobacillus versutus amicyanin). Comparable spin densities are found on the two terminal His ligands for the wild-type constructs as opposed to the engineered proteins where the spin is more unevenly distributed on the two His residues. A reevaluation of the Cys H(beta) chemical shifts that is in agreement with the data published for both the P. denitrificans and the P. versutus Cu(A) soluble domains confirms the thermal accessibility of the 2B(3u) electronic excited state and indicates the existence of slightly different spin densities on the two bridging Cys ligands. The 13C-NMR spectrum of isotopically enriched oxidized azurin from Pseudomonas aeruginosa reveals six fast relaxing signals, which can be partially identified by 1- and 2-dimensional (1-D, 2-D) direct detection techniques combined with 3-D triple resonance experiments. The observed contact shifts suggest the presence of direct spin density transfer and spin polarization mechanisms for the delocalization of the unpaired electron.

Azurin↗

PubMed1999

The PH superfold: a structural scaffold for multiple functions.

Pleckstrin homology (PH) domains form a structurally conserved family that is associated with many regulatory pathways within the cell. Domains with a nearly identical fold are found in other families that share no sequence similarity, suggesting the existence of a stable PH superfold. The PH domains generally function as regulated membrane-binding modules that bind to inositol lipids and respond to upstream signals by targeting the host proteins to the correct cellular sites. The other domains with a similar fold, such as the phosphotyrosine binding domains, recognize protein ligands.

Amino Acid Motifs↗

PubMed1999

Is routine echocardiography useful in patients hospitalized for chest pain? Evidence of areal myocardial dysfunction detected only by echocardiography.

To assess the diagnostic value of routine two-dimensional echocardiography in the coronary care unit setting, we studied 81 unselected patients admitted for acute chest pain. Using electrocardiography (ECG), clinical history and serum markers of myocardial injury, the patients were retrospectively diagnosed as having had definite acute myocardial infarction (AMI) with (n=13) or without (n=31) previous infarction, possible AMI with (n=14) or without (n=15) previous infarction, and non-coronary cardiac or other causes of chest pain (n=8). Abnormal wall motion was observed in 75/77 patients with a cardiac origin of symptoms (sensitivity 97%), and there were no false-positive wall motion findings. In the 73 patients who were finally diagnosed with coronary artery disease (CAD), echocardiography showed wall motion abnormality in at least one additional coronary territory area in which there were no diagnostic ECG changes for 56% of patients with CAD (41/73) (P<0. 001). These areas were considered to be indicative of the presence of myocardium at risk for future cardiac events. We conclude that in addition to being a sensitive and accurate tool for detection of ischaemic wall motion abnormalities, two-dimensional echocardiography can give valuable information about the area of myocardium at risk. Therefore, therapeutic decisions can be affected by the findings of the routine echocardiographic examination, which is recommended even in unselected coronary care unit patients.

Adult↗

PubMed1999

Crystallization and preliminary X-ray diffraction studies of perchloric acid soluble protein (PSP) from rat liver.

Perchloric acid soluble protein purified from the cytosol fraction of rat liver has been crystallized in a form suitable for high-resolution X-ray diffraction studies. Octahedral crystals reaching 0.5 mm in cross-sectional diameter were produced by the hanging-drop method using polyethylene glycol (Mr = 8 kDa) as precipitant. These crystals diffract to 2.44 A on an in-house X-ray source and to 1.8 A using a bending-magnet beamline at ESRF Grenoble. The crystals belong to the cubic space group P213 with a = 89.90 A and two molecules per asymmetric unit, as indicated from a Vm value of 2.12 A3 Da-1 and self-rotation function computation. Screening for heavy-atom derivatives identified a platinum compound and xenon that bind to the protein.

Animals↗

PubMed1998

Structural comparisons of calponin homology domains: implications for actin binding.

BACKGROUND: The actin-binding site of several cytoskeletal proteins is comprised of two calponin homology (CH) domains in a tandem arrangement. As a single copy, the CH domain is also found in regulatory proteins in muscle and in signal-transduction proteins. The three-dimensional structures of three CH domains are known, but they have not yet clarified the molecular details of the interaction between actin filaments and proteins harbouring CH domains. RESULTS: We have compared the crystal structure of a CH domain from beta-spectrin, which has been refined to 1.1 A resolution, with the two CH domains that constitute the actin-binding region of fimbrin. This analysis has allowed the construction of a structure-based sequence alignment of CH domains that can be used in further comparisons of members of the CH domain family. The study has also improved our understanding of the factors that determine domain architecture, and has led to discussion on the functional differences that seem to exist between subfamilies of CH domains, as regards binding to F-actin. CONCLUSIONS: Our analysis supports biochemical data that implicate a surface centered at the last helix of the N-terminal CH domain as the most probable actin-binding site in cytoskeletal proteins. It is not clear whether the C-terminal domains of the tandem arrangement or the single CH domains have this function alone. This may imply that although the CH domains are homologous and have a conserved structure, they may have evolved to perform different functions.

Actins↗

PubMed1998

The active site of the bacterial nitric oxide reductase is a dinuclear iron center.

A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.

Binding Sites↗