RIDME spectroscopy on high-spin Mn2+ centers.

Pulsed EPR dipolar spectroscopy is a powerful tool for determining the structure and conformational dynamics of biological macromolecules, as it allows precise measurements of distances in the range of 1.5-10 nm. Utilization of high-spin Mn2+ species as spin probes for distance measurements is of significant interest, because they are biologically compatible and endogenous in numerous biological systems. However, to date dipolar spectroscopy experiments with this kind of species have been underexplored. Here we present pulsed electron electron double resonance (PELDOR also called DEER) and relaxation-induced dipolar modulation enhancement (RIDME) experiments, which have been performed at W-band (94 GHz) and J-band frequencies (263 GHz) on a bis-MnDOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) model system. The distances obtained from these experiments are in good agreement with predictions. RIDME experiments reveal a significantly higher modulation depth compared to PELDOR, which is an important consideration for biological samples. These experiments also feature higher harmonics of the dipolar coupling frequency due to effective multiple-quantum relaxation of high-spin Mn2+ as well as the multiple-component background function. Harmonics of the dipolar coupling frequency were taken into account by including additional terms in the kernel function of Tikhonov regularization analysis.

The effect of gamma-ray irradiation on the Mn(II) speciation in Deinococcus radiodurans and the potential role of Mn(II)-orthophosphates.

D. radiodurans accumulates large quantities of Mn(II), which is believed to form low molecular weight complexes with phosphate and metabolites that protect D. radiodurans from radiation damage. The concentration of Mn(II) species in D. radiodurans during the exponential and stationary phase was determined using high-field EPR and biochemical techniques. In the exponential growth phase cells a large fraction of the manganese was in the form of Mn(II)-orthophosphate complexes. By contrast, the intracellular concentration of these compounds in stationary phase cells was less than 16 µM, while that of Mn superoxide dismutase was 320 µM and that of another, yet unidentified, Mn(II) protein was 250 µM. Stationary cells were found to be equally resistant to irradiation as the exponential cells in spite of having significant lower Mn(II)-orthophosphate concentrations. Gamma irradiation induced no changes in the Mn(II) speciation. During stationary growth phase D. radiodurans favours the production of the two Mn-proteins over low molecular weight complexes suggesting that the latter were not essential for radio-resistance at this stage of growth.

Variations in Mn(II) speciation among organisms: what makes D. radiodurans different.

The manganese(II) speciation in intact cells of D. radiodurans, E. coli, S. cerevisiae and Arabidopsis thaliana seeds was measured using high-field electron paramagnetic resonance techniques. The majority of the Mn(II) ions in these organisms were six-coordinate, bound predominately by water, phosphates and nitrogen-based molecules. The relative distribution of the different phosphates in bacteria and S. cerevisiae was the same and dominated by monophosphate monoesters. Mn(II) was also found bound to the phosphate backbone of nucleic acids in these organisms. Phosphate ligation in Arabidopsis seeds was dominated by phytate. The extent of nitrogen ligation in the four organisms was also determined. On average, the Mn(II) in D. radiodurans had the most nitrogen ligands followed by E. coli. This was attributed to higher concentrations of Mn(II) bound to proteins in these species. Although constitutively expressed in all four organisms, MnSOD was only detected in D. radiodurans. As previously reported, D. radiodurans also accumulates a second abundant Mn containing protein species. The high concentration of proteinaceous Mn(II) is a unique feature of D. radiodurans.

Interstitial tonicity controls TonEBP expression in the renal medulla.

Cells in the hyperosmotic kidney medulla, express a transcriptional activator termed tonicity responsive enhancer binding protein (TonEBP). Genes targeted by TonEBP protect kidney cells from the deleterious effects of hyperosmolality by inducing the expression of organic osmolytes and molecular chaperones, and other genes that mediate urine concentration such as aquaporin-2 and urea transporters. We tested here the effect of hypertonicity and hyperosmotic salt in the renal medullary interstitium on the expression TonEBP. When massive water diuresis was induced in rats the medullary sodium concentrations did not change, neither did TonEBP expression. In these animals the medullary tonicity was unchanged despite the production of dilute urine. On the other hand, treatment with the loop diurectic furosemide resulted in a dose-dependent decrease in the medullary sodium concentration causing a reduction in interstitial tonicity. Here, TonEBP expression was blunted in the outer and inner medulla which was due, in part, to decreased mRNA abundance. As expected, the expression of TonEBP target genes in the renal medulla also decreased in response to furosemide. Hence TonEBP expression in the renal medulla is stimulated by interstitial hypertonicity.

Open anatomical coracoclavicular ligament reconstruction using a tendon graft with an Endobutton loop.

We describe a technique of open anatomical coracoclavicular ligament reconstruction restoring both parts of the native ligament, aiming at achieving maximum stability of the acromioclavicular joint without disturbing the normal anatomy. Using the same anatomical principle of ligament reconstruction as in other joints, transosseous tunnels are created at the native footprints of the conoid and trapezoid ligaments. An autologous graft is fixed using an Endobutton continuous loop and a PEEK screw; adequate healing of the ligament is ensured with an appropriate working length. Although an open procedure, this technique offers several advantages. It can be easily reproduced using basic anatomical principles and simple cost-effective instrumentation. The implant does not have to be removed, important anatomical structures are respected, normal acromioclavicular joint kinematics are restored, the scar is cosmetically acceptable and post-operative morbidity is very low.

Herbicide-induced changes in charge recombination and redox potential of Q(A) in the T4 mutant of Blastochloris viridis.

To gain new insights into the function of photosystem II (PSII) herbicides DCMU (a urea herbicide) and bromoxynil (a phenolic herbicide), we have studied their effects in a better understood system, the bacterial photosynthetic reaction center of the terbutryn-resistant mutant T4 of Blastochloris (Bl.) viridis. This mutant is uniquely sensitive to these herbicides. We have used redox potentiometry and time-resolved absorption spectroscopy in the nanosecond and microsecond time scale. At room temperature the P(+)(*)Q(A)(-)(*) charge recombination in the presence of bromoxynil was faster than in the presence of DCMU. Two phases of P(+)(*)Q(A)(-)(*) recombination were observed. In accordance with the literature, the two phases were attributed to two different populations of reaction centers. Although the herbicides did induce small differences in the activation barriers of the charge recombination reactions, these did not explain the large herbicide-induced differences in the kinetics at ambient temperature. Instead, these were attributed to a change in the relative amplitude of the phases, with the fast:slow ratio being approximately 3:1 with bromoxynil and approximately 1:2 with DCMU at 300 K. Redox titrations of Q(A) were performed with and without herbicides at pH 6.5. The E(m) was shifted by approximately -75 mV by bromoxynil and by approximately +55 mV by DCMU. As the titrations were done over a time range that is assumed to be much longer than that for the transition between the two different populations, the potentials measured are considered to be a weighted average of two potentials for Q(A). The influence of the herbicides can thus be considered to be on the equilibrium of the two reaction center forms. This may also be the case in photosystem II.

Prevalence of essential tremor: door-to-door neurologic exams in Mersin Province, Turkey.

Estimates of the prevalence of essential tremor (ET) are probably low because screening questionnaires have been used. The authors estimated the prevalence of ET in Mersin Province, Turkey, in 2,253 individuals aged >or=40 years, all of whom were examined by study neurologists. There were 89 ET cases (prevalence = 4.0%, 95% CI = 3.2 to 4.8%). The prevalence of ET may be higher than previously estimated. This is important when defining the extent of the health care problem.

[Circadian rhythm of silent myocardial ischemia. Why morning is so risky for hypertensive patients]. / Zirkadiane Rhythmik der stummen Myokardischämie. Warum der Morgen für Hypertoniker so gefährlich ist.

The circadian pattern of numerous cardiovascular events (myocardial infarction, sudden cardiac death, stroke) reveals a peak in the early hours of the morning. A circadian rhythm peaking in the morning is also found for so-called silent myocardial ischaemia, which occurs in more than 20% of patients with arterial hypertension, and can be regularly detected in combined 24-h-ABPM/EKG examinations. Comparative studies have shown that hypertensives with SMI suffer more cardiac events than those with no SMI. It has further been demonstrated that an elevated blood pressure amplitude, with is considered an independent risk factor for cardiac events, is associated with an increased incidence of SMI in patients with micro- or macro-angiopathy. Accordingly, consideration should be given to SMI when deciding on treatment, also in hypertensives with no angina pectoris symptoms.

Sensitivity of tyrosyl radical g-values to changes in protein structure: a high-field EPR study of mutants of ribonucleotide reductase.

The local electrostatic environment plays a critical role in determining the physicochemical properties of reactive radicals in proteins. High-field electron paramagnetic resonance (HF-EPR) spectroscopy has been used to determine the sensitivity of the tyrosyl radical g-values to local electrostatic environment. Site-specific mutants of ribonucleotide reductase from Escherichia coli were used to study the effect of introducing a charge group on the HF-EPR spectrum of the stable tyrosyl (Y122) radical. The changes affected by the mutations were small, but measurable. Mutation of isoleucine-74 to an arginine (I74R) or lysine (I74K) induced disorder in the hyperfine interactions. Similar effects were observed for the mutation of valine-136 to an arginine (V136R) or asparagine (V136N). For five or six mutants studied, the g(x)() component of the g-tensor was distributed. For the isoleucine-74 to lysine (I74K) and leucine-77 to phenylalanine (L77F) mutants, a shift of 1 x 10(-)(4) in g(x)() value was also detected. For the I74K mutant, it is shown that the shift is consistent with the introduction of a charged residue, but cannot be distinguished from changes in the electrostatic effect of the nearby diiron center. For the L77F mutant, the shift is induced by the diiron center. Using existing tyrosyl radical g-tensor measurements, we have developed a simple effective charge model that allows us to rationalize the effect of the local electrostatic environments in a number of proteins.

Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.

Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate that was generated by hydrogen peroxide addition to the W191G CcP mutant. The g- values of this radical (g(x)= 2.00660, g(y) = 2.00425, and g(z)= 2.00208), as well as the wild-type transient tyrosyl radical, are essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discussed.

High field EPR study of the pheophytin anion radical in wild type and D1-E130 mutants of photosystem II in Chlamydomonas reinhardtii.

The intermediate electron acceptor in photosystem II is a pheophytin molecule. The radical anion of this molecule was studied using high field electron paramagnetic resonance in a series of Chlamydomonas reinhardtii mutants. Glutamic acid 130 of the D1 polypeptide is thought to hydrogen bond the ring V carbonyl group of this radical. Mutations at this site, designed to weaken or remove this hydrogen bond, strongly affected the g tensor of the radical. The upward shift of the g(x) component followed the decreasing hydrogen bonding capacity of the amino acid introduced. This behavior is similar to that of tyrosyl and semiquinone radicals. It is also consistent with the optical spectra of the pheophytin in similar mutants. Density functional calculations were used to calculate the g tensors and rationalize the observed trend in the variation of the g(x) value for pheophytin and bacteriopheophytin radical. The theoretical results support the experimental observations and demonstrate the sensitivity of g values to the electrostatic protein environment for these types of radicals.

[Your patient self-monitors his blood pressure. Other limit values are applicable]. / Ihr Patient misst seinen Blutdruck selbst. Da gelten andere Grenzwerte.

For self-measurement of blood pressure, two points are of great importance: proper patient instruction, and the accuracy of the measuring device. The trend towards the use of wrist-worn devices is associated with increasing numbers of measuring errors. The reason for this is usually incorrect positioning of the pickup point. Since statistics show that measurements made by the patient are clearly lower than those made in the doctor's office, the First International Consensus Conference on Blood Pressure Measurement held in 1999 established the threshold for differentiating between normo- and hypertension to be 135/85 mmHg. Thus, the use of the WHO definition of normotension in the doctor's office (< 140/90) thus represents a methodological error. Indications for self-measurement are, in particular, long-term monitoring of hypertension, monitoring of blood pressure during dose titration, identification of a "white-coat hypertension", and as a means of improving compliance. For diagnostic purposes, a measurement rate of at least 12 per week is mandatory.

Orientation of the tyrosyl D, pheophytin anion, and semiquinone Q(A)(*)(-) radicals in photosystem II determined by high-field electron paramagnetic resonance.

The radical forms of two cofactors and an amino acid in the photosystem II (PS II) reaction center were studied by using high-field EPR both in frozen solution and in oriented multilayers. Their orientation with respect to the membrane was determined by using one-dimensionally oriented samples. The ring plane of the stable tyrosyl radical, Y(D)(*), makes an angle of 64 degrees +/- 5 degrees with the membrane plane, and the C-O direction is tilted by 72 degrees +/- 5 degrees in the plane of the radical compared to the membrane plane. The semiquinone, Q(A)(*)(-), generated by chemical reduction in samples lacking the non-heme iron, has its ring plane at an angle of 72 degrees +/- 5 degrees to the membrane plane, and the O-O axis is tilted by 21 degrees +/- 5 degrees in the plane of the quinone compared to the membrane plane. This orientation is similar to that of Q(A)(*)(-) in purple bacteria reaction centers except for the tilt angle which is slightly bigger. The pheophytin anion was generated by photoaccumulation under reducing conditions. Its ring plane is almost perpendicular to the membrane with an angle of 70 degrees +/- 5 degrees with respect to the membrane plane. This is very similar to the orientation of the pheophytin in purple bacteria reaction centers. The position of the g tensor with respect to the molecule is tentatively assigned for the anion radical on the basis of this comparison. In this work, the treatment of orientation data from EPR spectroscopy applied to one-dimensionally oriented multilayers is examined in detail, and improvements over previous approaches are given.

[Silent myocardial ischemia in hypertensive patients]. / Stumme Myokardischämie bei Hypertonikern.

Silent myocardial ischemia occurs in hypertensive individuals with a prevalence of approximately 35%. ST-alterations are triggered by a) hypertensive peaks and b) heart rate increase. Like in patients with coronary heart disease most ischemic events occur without angina. They are clinically silent. In daily practice silent myocardial ischemia may be detected by ECG under physical load or with 24 h Holter ECG-monitoring. The latter can detect ischemic events missed by ECG-monitored exercise tolerance. In hypertensive patients the simultaneous, ST-triggered recording of ECG and blood pressure data is more meaningful. Patients with silent ischemia are at higher risk than individuals without. Angor is not as strong a determinant of risk as silent ischemia. Hypertensive patients without coronary artery disease (CAD) who have silent ischemia may even have a worse prognosis than those with known CAD. It is therefore important to substantiate the objective extent of silent ischemia by ST-analysis. If detected it has to be included into therapeutic considerations with the goal to prevent such episodes by antihypertensive treatment.

Effect of near-infrared light on the S2-state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn cluster of Photosystem II (PSII) from Synechococcus elongatus was studied using EPR. A signal with features between g = 5 and g = 9 is reported from the S2-state. The signal is attributed to the manganese cluster in a state with a spin 5/2 state. Spectral simulations of the signal indicate zero field splitting parameters where the |E/D| was 0.13. The new signal is formed by irradiating PSII samples which contain the spin = 1/2 S2-state using 813 nm light below 200 K. This effect is attributed to a spin-state change in the manganese cluster due to absorption of the IR light by the Mn-cluster itself. The signal is similar to that reported recently in PSII of plants [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. In plant PSII the comparable signal is formed at a lower temperature (optimally below 77 K), and gradual warming of the sample in the dark leads to the formation of the state responsible for the well-known g = 4.1 signal prior to formation of the spin 1/2 multiline signal. In the present work using cyanobacterial PSII, warming of the sample in the dark leads to the formation of the spin 1/2 multiline signal without formation of the g = 4 type signal as an intermediate. These observations provide a partial explanation for the long-standing "mystery of the missing g = 4 state" in cyanobacterial PSII. The observations are rationalized in terms of three possible states which can exist for S2: (i) the spin 1/2 multiline signal, (ii) the state responsible for the g = 4.1 signal, and (iii) the new spin 5/2 state. The relative stability of these states differs between plants and cyanobacteria.

High-spin states (S >/= 5/2) of the photosystem II manganese complex.

The Mn4 complex which is involved in water oxidation in photosystem II (PSII) is known to exhibit two types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: either a multiline signal (S = 1/2) or a signal at g = 4.1 (S = 3/2 or S= 5/2). The S = 1/2 state can be converted to that responsible for the g = 4.1 signal upon the absorption of near-infrared (IR) light [Boussac, A., Girerd, J.-J., and Rutherford, A.W. (1996) Biochemistry 35, 6984-6989]. It is shown here that a third state gives rise to signals at g = 10 and 6. This state is formed by IR illumination of the S = 1/2 state at 65 K, a temperature where IR illumination leads to the loss of the S = 1/2 signal but to no formation of the g = 4.1 state. On the basis of the corresponding decrease of the S = 1/2 state, the new state can be trapped in approximately 40% of the PSII centers. Warming of the sample above 65 K, in the dark, leads to the loss of the g = 10 and 6 resonances with the corresponding appearance of the g = 4.1 signal. It is suggested that the IR-induced conversion of the S = 1/2 state into the g = 4.1 state at 150 K involves the transient formation of the new state. The new state is attributed to a S = 5/2 state of the Mn4 complex (although a S value > 5/2 is also a possibility). Spectral simulations indicate an E/D ratio of -0.05 with D