A tailored multi-frequency EPR approach to accurately determine the magnetic resonance parameters of dynamic nuclear polarization agents: application to AMUPol.

To understand the dynamic nuclear polarization (DNP) enhancements of biradical polarizing agents, the magnetic resonance parameters need to be known. We describe a tailored EPR approach to accurately determine electron spin-spin coupling parameters using a combination of standard (9 GHz), high (95 GHz) and ultra-high (275 GHz) frequency EPR. Comparing liquid- and frozen-solution continuous-wave EPR spectra provides accurate anisotropic dipolar interaction D and isotropic exchange interaction J parameters of the DNP biradical AMUPol. We found that D was larger by as much as 30% compared to earlier estimates, and that J is 43 MHz, whereas before it was considered to be negligible. With the refined data, quantum mechanical calculations confirm that an increase in dipolar electron-electron couplings leads to higher cross-effect DNP efficiencies. Moreover, the DNP calculations qualitatively reproduce the difference of TOTAPOL and AMUPol DNP efficiencies found experimentally and suggest that AMUPol is particularly effective in improving the DNP efficiency at magnetic fields higher than 500 MHz. The multi-frequency EPR approach will aid in predicting the optimal structures for future DNP agents.

Overhauser effects in insulating solids.

We report magic angle spinning, dynamic nuclear polarization (DNP) experiments at magnetic fields of 9.4 T, 14.1 T, and 18.8 T using the narrow line polarizing agents 1,3-bisdiphenylene-2-phenylallyl (BDPA) dispersed in polystyrene, and sulfonated-BDPA (SA-BDPA) and trityl OX063 in glassy glycerol/water matrices. The (1)H DNP enhancement field profiles of the BDPA radicals exhibit a significant DNP Overhauser effect (OE) as well as a solid effect (SE) despite the fact that these samples are insulating solids. In contrast, trityl exhibits only a SE enhancement. Data suggest that the appearance of the OE is due to rather strong electron-nuclear hyperfine couplings present in BDPA and SA-BDPA, which are absent in trityl and perdeuterated BDPA (d21-BDPA). In addition, and in contrast to other DNP mechanisms such as the solid effect or cross effect, the experimental data suggest that the OE in non-conducting solids scales favorably with magnetic field, increasing in magnitude in going from 5 T, to 9.4 T, to 14.1 T, and to 18.8 T. Simulations using a model two spin system consisting of an electron hyperfine coupled to a (1)H reproduce the essential features of the field profiles and indicate that the OE in these samples originates from the zero and double quantum cross relaxation induced by fluctuating hyperfine interactions between the intramolecular delocalized unpaired electrons and their neighboring nuclei, and that the size of these hyperfine couplings is crucial to the magnitude of the enhancements. Microwave power dependent studies show that the OE saturates at considerably lower power levels than the solid effect in the same samples. Our results provide new insights into the mechanism of the Overhauser effect, and also provide a new approach to perform DNP experiments in chemical, biophysical, and physical systems at high magnetic fields.

Ballistic movements of jumping legs implemented as variable components of cricket behaviour.

Ballistic accelerations of a limb or the whole body require special joint mechanisms in many animals. Specialized joints can be moved by stereotypic or variable motor control during motor patterns with and without ballistic components. As a model of variable motor control, the specialized femur-tibia (knee) joints of cricket (Acheta domesticus) hindlegs were studied during ballistic kicking, jumping and swimming and in non-ballistic walking. In this joint the tendons of the antagonistic flexor and the extensor muscles attach at different distances from the pivot and the opposed lever arms form an angle of 120 deg. A 10:1 ratio of their effective lever arms at full knee flexion helps to prepare for most ballistic extensions: the tension of the extensor can reach its peak while it is restrained by flexor co-contraction. In kicks, preparatory flexion is rapid and the co-contraction terminates just before knee extensions. Therefore, mainly the stored tension of the extensor muscle accelerates the small mass of the tibia. Jumps are prepared with slower extensor-flexor co-contractions that flex both knees simultaneously and then halt to rotate both legs outward to a near horizontal level. From there, catapult extension of both knees accelerates the body, supported by continued high frequency motor activity to their tibia extensor muscles during the ongoing push-off from the substrate. Premature extension of one knee instantly takes load from the lagging leg that extends and catches up, which finally results in a straight jump. In swimming, synchronous ballistic power strokes of both hindlegs drive the tibiae on a ventral-to-posterior trajectory through the water, well coordinated with the swimming patterns of all legs. In walking, running and climbing the steps of the hindlegs range between 45 deg flexion and 125 deg extension and use non-ballistic, alternating activity of knee flexor and extensor muscles. Steep climbing requires longer bursts from the extensor tibiae muscles when they support the extended hindlegs against gravity forces when the body hangs over. All ballistic movements of cricket knees are elicited by a basic but variable motor pattern: knee flexions by co-contraction of the antagonists prepare catapult extensions with speeds and forces as required in the different behaviours.

Investigations of dynamic amyloid-like structures of the Wnt signalling pathway by solid-state NMR.

We report solid-state Nuclear Magnetic Resonance (ssNMR) studies on amyloid-like protein complexes formed by DIX domains that mediate key protein interactions in the Wnt signalling pathway. Our results provide insight into the 3D fold of the self-associated Axin-DIX domain and identify a potential lipid cofactor.

Biomolecular NMR: recent advances in liquids, solids and screening.

The past year has witnessed significant advances in NMR analysis of bio-macromolecules from a broad array of disciplines. First, great progress in the development of methods for measuring residual dipolar couplings in nematic media promises to increase both the size of systems to be studied and the accuracy with which structures can be determined. Second, the ability of solid-state NMR to provide structural information on biological systems is undergoing rapid expansion as a result of recent developments. The structural details that can be derived for biomolecules in the liquid and solid states can be used for the rational design of high-affinity ligands. Such studies are now complemented by NMR screening of synthetic and natural molecular libraries. Several NMR screening protocols have been designed that employ both rational and random elements.

Multiple-quantum cross polarization in quadrupolar spin systems during magic-angle spinning.

We describe the concept of multiple-quantum cross polarization (CP) between an I = 32 and an I = 12 spin during magic-angle spinning. Experimental and theoretical results for (23)Na-(1)H pairs are presented that elucidate the transfer mechanism and the beneficial effect of adiabatic amplitude modulations of the CP field. The multiple-quantum CP approach is shown to be beneficial for improving the sensitivity of CP-MQMAS experiments and for detecting dipolar correlations.

Transferrin receptor assay and zinc protoporphyrin as markers of iron-deficient erythropoiesis in end-stage renal disease patients.

To evaluate storage iron deficiency and iron-deficient erythropoiesis we determined, in a cross-sectional study of 95 patients mainly including end-stage renal disease patients (ESRD) with (32) and without rh-EPO therapy (55), the following parameters: hemoglobin, mean corpuscular red cell volume, ferritin, transferrin saturation (TS), zinc protoporphyrin (ZPP) and soluble transferrin receptor (TfR). In the dialysis group the percentage of positive samples with each marker of tissue iron supply defined as TS < 20%, ZPP > 40 mumol/mol Heme and TfR > 3.05 microgram/ml was as follows: TS 43.7% and 32.2% at a diagnostic threshold level of < 16%, ZPP 33.3% and TfR 17.2%. Manifest storage iron deficiency defined as ferritin < 30 ng/ml was observed in 5.7% of the samples while the mean ferritin concentration of the rh-Epo treated dialysis patients was 509.3 ng/ml compared to 262.5 ng/ml in the group without rh-EPO therapy. These data reflect a generous iron substitution in our series taking a TS < 20% as an intervention criterion. Looking at the different results of the three markers the best correspondence was found between ZPP and TfR resulting in a weak positive correlation (+0.64). In conclusion, we found quite different results with different assays when evaluating endogenous iron availability in our series of mainly ESRD patients in a cross-sectional study. Because a gold-standard is not defined further firm conclusions cannot be drawn from this type of study. The adequacy of the different parameters of iron metabolism including threshold levels and, consequently, the decision and route of iron substitution deserve an evaluation in a longitudinal study to characterize the best marker or marker combination in this setting.

[From diagnosis to decision--decision processes of women in the context of prenatal diagnosis]. / Von der Diagnose zur Entscheidung--Entscheidungsprozesse von Frauen im Kontext pränataler Diagnostik.

Prenatal diagnosis is a growth industry. The constant introduction of new prenatal tests poses great challenges to prospective parents. In Germany, guidelines for prenatal care include an early nuchal-translucency-sonogram as a routine screening for down syndrome. Developer of this screening predict a 90% discovery rate. This rate can be achieved through the combination of early maternal serum examinations, computer assisted risk calculation and the nuchal-translucency measurement. The extensive use of diverse new technologies is driven by two forces; first, the parents' fear of giving birth to a child with a disability, and second, the offensive marketing strategies by the test-making industry. The information that these tests can yield is vast, yet parents' range of choices in response to these test results remain very limited. After a battery of diagnostic tests, parents confronted with the diagnosis of down syndrome can choose only between continuing or terminating the pregnancy. In the future, more and more women and their partners will be confronted with such a difficult decision. Adequate professional counseling is needed to help parents cope with the critical life event of being told a positive test result. Solutions have to be developed on an individual basis and need to be grounded on the parents' needs. Informing parents of a positive diagnosis can be a challenging moment in professional life. The professional needs to act with sensitivity and competence. The informations he or she provides have to been well balanced. It is necessary to develop quality assurance standards for counseling, diagnosis and crisis intervention.

Magic-angle-spinning NMR spectroscopy applied to small molecules and peptides in lipid bilayers.

ssNMR (solid-state NMR) spectroscopy provides increasing possibilities to study the structural and dynamic aspects of biological membranes. Here, we review recent ssNMR experiments that are based on MAS (magic angle spinning) and that provide insight into the structure and dynamics of membrane systems at the atomic level. Such methods can be used to study membrane architecture, domain formation or molecular complexation in a way that is highly complementary to other biophysical methods such as imaging or calorimetry.

[Visceral leishmaniasis (kala-azar). A rare differential diagnosis of splenomegaly and pancytopenia]. / Viszerale Leishmaniose (Kala-Azar). Eine seltene Differentialdiagnose bei Splenomegalie und Panzytopenie.

A 53-year-old man developed a septic fever up to 40 degrees C, pancytopenia and hepatosplenomegaly after a holiday in Spain. Administration of piperacillin and amikacin was ineffective, but the fever subsided and partial haematological remission occurred when 1 mg/kg methylprednisolone daily was added. After six months his general condition worsened and pancytopenia with typical inclusion bodies in bone-marrow macrophages was noted, leading to the diagnosis of visceral leishmaniasis (Kala-Azar). The diagnosis was confirmed by serological tests. The causative organism was eliminated and the abnormal findings regressed during treatment with sodium stibogluconate, at first 600 mg/d for two weeks, then 850 mg/d over 16 days, interrupted for 14 days because of side effects.

Serum level changes of endogenous and postheparin diamine oxidase (histaminase) in clinical and experimental hepatitis.

In patients suffering from acute viral hepatitis (n = 12) an about 50 per cent decrease of serum diamine oxidase (DAO, histaminase, E.C.N. 1.4.3.6) was detected as compared to a healthy control group (n = 24). Normally, the intravenous injection of heparin is promptly followed by a marked rise of plasma DAO. In viral hepatitis, however, after application of heparin (200 IU/kg b.w., i.v.) the enzyme release from the visceral organs into the plasma was markedly decreased. There was an inverse correlation between the serum glutamic pyruvic transaminase (SGPT) and the postheparin enzyme. Normalization of SGPT occurred before the normalization of post-heparin diamine oxidase (PHD). In galactosamine "hepatitis" of rats (800 mg Gal-N/kg b.w., i.p.), in contrary to human viral hepatitis plasma DAO increased about 5-fold after heparin application. This increased PHD in plasma of Gal-N rats was correlated to enhanced animal's endogenous plasma DAO activity (r=0.685, p less then 0.0005, n = 54). The cause of these enzyme activity changes and its possible pathophysiological meaning are still unknown. It is concluded from these experiments that in Gal-N "hepatitis" of rats plasmatic DAO level changes are mediated by endogenous heparin, released from disrupted mast cells. Increased basal DAO levels correspond to the enhanced release after heparin application, both possibly induced by less stable binding of the enzyme to the cells of the small intestine in inflammation. Both, decreased endogenous and post-heparin DAO levels in human hepatitis would correspond to a depletion of the enzyme containing organs.

Broadband dipolar recoupling in rotating solids: a numerical comparison of some pulse schemes.

A numerical comparison of the dipolar recoupling performance of several previously published homonuclear recoupling schemes under magic angle-spinning conditions is presented. Emphasis is put on the recoupled polarization transfer in a two-spin system where the efficiency is studied as a function of resonance offsets in the presence and absence of chemical-shielding anisotropies. In addition, the effect of the rf field strength is investigated. Powder pattern line shapes are shown in the on-resonance case that reveal the distribution of dipolar couplings for each recoupling scheme. These results are compared to data computed with a purposely misset rf field strength to estimate the pulse scheme sensitivity to rf-inhomogeneity and experimental missettings.

A case of pure red cell aplasia: follow-up on different immunosuppressive regimens.

A 66-year-old patient was admitted to our hospital in January 1992 for further evaluation of severe normocytic anemia. Hemoglobin (Hb) was 3.5 g/dl, reticulocyte count 1%. Bone marrow showed a nearly complete lack of red cell precursors, thus favoring the diagnosis of acquired pure red cell aplasia (PRCA). Immunosuppressive therapy with prednisolone was started but had to be supplemented with azathioprine because of a further rapid decrease in Hb to 3.7 g/dl after an initial transfusion of 6 U red blood cells. However, with this regimen a renewed decrease in Hb to 6.6 g/dl was noted, and further transfusions were required. Therefore therapy was switched to cyclosporine A (CyA) while tapering off prednisolone. Four months after the initial diagnosis a positive parvovirus B19 IgM antibody was found. After the failure of hematological remission with three immunosuppressive regimens a course of high-dose intravenous immunoglobulins (IVIG) was administered in July 1992. Six weeks after IVIG therapy a peak hemoglobin concentration of 12.3 g/dl was noted, and further transfusion was not required. CyA was tapered off in October 1992. One month later CyA was reinstituted because of a relapse of PRCA but was unsuccessful until January 1993. At this time immunosuppressive CyA therapy was discontinued because of a periodontal abscess. In February 1993 a second IVIG infusion was given, and a second remission of PRCA was noted, showing an increase in hemoglobin up to 14.5 g/dl by November 1993. At the last follow-up visit in February 1994 our patient was still in complete hematological remission.

Detection of primary HIV infection by a second-generation HIV(p24) antigen test.

We studied the course of a primary HIV infection in a 54-year-old woman. Probably the source of infection was sexual intercourse, since other risks, such as i.v. drug use, acupuncture and transfusion were excluded. On admission she presented with fever, a maculopapular rash, and moderate enlargement of nuchal lymph nodes. At that time the anti-HIV 1,2 enzyme immunoassay was negative. However, the HIV-p24 Ag test, which was performed in every HIV screening in our laboratory, was positive. The suspicion of an acute HIV infection was supported by a positive HIV-cDNA-PCR and confirmed by Western blot after seroconversion. As additional finding, the blood smear showed abnormal white cell differential count, indicating viral infection. Aminotransferases were slightly increased, and antibodies to hepatitis B surface and core antigens demonstrated former hepatitis B infection. It is concluded that in this case the HIV-p24 Ag test proved its suitability for early diagnosis of an acute HIV infection. In case of testing blood donors, none of the compulsory serological screening methods would have detected the HIV infection.

[Morphological changes alpha-actinin in skeletal striated muscle in the rabbit in experimental ischemia]. / Alterazioni morfologiche dell' alpha-actinina nel muscolo striato scheletrico di coniglio in condizioni di ischemia sperimentale.

In this work we studied the alteration of Z-line alpha-actinin in striated skeletal muscle, under conditions of experimental ischemia, by immunohistochemical techniques. alpha-Actinin was extracted and purified from rabbit striated skeletal muscle and used, as an antigen, for the production of antibodies, in sheep. Electrophoresis was performed on PAA sla-gel and the Ag-Ab reaction test on agarose slab-gel (immunodiffusion). Antibodies were used for the indirect immunoperoxidase technique on semithin sections of rabbit striated skeletal muscle, included in in Araldehyde, and observed with phase-contrast microscopy. alpha-Actinin from ischemic muscle because of stenosis of the iliac artery, undergoes a progressive alteration, from the third hour of ischemia, going out of its usual sites. From the fifth to the eighth hour it gradually disappears until it leaves rare and residual points of positive reaction only in correspondence to the Z-lines.