Sensitivity of 2H NMR spectroscopy to motional models: proteins and highly viscous liquids as examples.

In order to study to what extent mechanisms of molecular motion can be unambiguously revealed by (2)H NMR spectroscopy, (2)H spectra for proteins (chicken villin protein headpiece HP36, selectively methyl-deuterated at leucine-69, C(δ) D(3)) and binary systems of high viscosity (benzene-d(6) in tricresyl phosphate) have been carefully analyzed as illustrative examples (the spectra are taken from the literature). In the first case, a model of restricted diffusion mediated by jumps between rotameric orientations has been tested against jump- and free diffusion models which describe rotational motion combined with jump dynamics. It has been found that the set of (2)H spectra of methyl-deuterated at leucine-69 chicken villin protein headpiece HP36 can be consistently explained by different motional models as well as by a gaussian distribution of correlation times assuming isotropic rotation (simple brownian diffusion model). The last finding shows that when the possible distribution of correlation times is not very broad one might not be able to distinguish between heterogeneous and homogenous (but more complex) dynamics by analyzing (2)H lineshapes. For benzene-d(6) in tricresyl phosphate, the dynamics is heterogeneous and it has been demonstrated that a gaussian distribution of correlation times reproduces well the experimental lineshapes, while for a Cole-Davidson distribution the agreement is somewhat worse. For inquires into the sensitivity of quadrupolar NMR spectral analysis (by "quadrupolar NMR spectroscopy we understand NMR spectroscopy of nuclei possessing quadrupole moment), the recently presented theoretical approach [Kruk et al., J. Chem. Phys. 135, 224511 (2011)] has been used as it allows simulating quadrupolar spectra for arbitrary motional conditions by employing the stochastic Liouville equation.

1H NMR relaxation in glycerol solutions of nitroxide radicals: effects of translational and rotational dynamics.

(1)H spin-lattice relaxation rates in glycerol solutions of selected nitroxide radicals at temperatures between 200 K and 400 K were measured at 15 MHz and 25 MHz. The frequency and temperature conditions were chosen in such a way that the relaxation rates go through their maximum values and are affected by neither the electron spin relaxation nor the electron-nitrogen nucleus hyperfine coupling, so that the focus could be put on the mechanisms of motion. By comparison with (1)H spin-lattice relaxation results for pure glycerol, it has been demonstrated that the inter-molecular electron spin-proton spin dipole-dipole interactions are affected not only by relative translational motion of the solvent and solute molecules, but also by their rotational dynamics as the interacting spins are displaced from the molecular centers; the eccentricity effects are usually not taken into account. The (1)H relaxation data have been decomposed into translational and rotational contributions and their relative importance as a function of frequency and temperature discussed in detail. It has been demonstrated that neglecting the rotational effects on the inter-molecular interactions leads to non-realistic conclusions regarding the translational dynamics of the paramagnetic molecules.

Nuclear quadrupole resonance lineshape analysis for different motional models: stochastic Liouville approach.

A general theory of lineshapes in nuclear quadrupole resonance (NQR), based on the stochastic Liouville equation, is presented. The description is valid for arbitrary motional conditions (particularly beyond the valid range of perturbation approaches) and interaction strengths. It can be applied to the computation of NQR spectra for any spin quantum number and for any applied magnetic field. The treatment presented here is an adaptation of the "Swedish slow motion theory," [T. Nilsson and J. Kowalewski, J. Magn. Reson. 146, 345 (2000)] originally formulated for paramagnetic systems, to NQR spectral analysis. The description is formulated for simple (Brownian) diffusion, free diffusion, and jump diffusion models. The two latter models account for molecular cooperativity effects in dense systems (such as liquids of high viscosity or molecular glasses). The sensitivity of NQR slow motion spectra to the mechanism of the motional processes modulating the nuclear quadrupole interaction is discussed.

Variable Coupling Scheme for High Frequency Electron Spin Resonance Resonators Using Asymmetric Meshes.

The sensitivity of a high frequency electron spin resonance (ESR) spectrometer depends strongly on the structure used to couple the incident millimeter wave to the sample that generates the ESR signal. Subsequent coupling of the ESR signal to the detection arm of the spectrometer is also a crucial consideration for achieving high spectrometer sensitivity. In previous work, we found that a means for continuously varying the coupling was necessary for attaining high sensitivity reliably and reproducibly. We report here on a novel asymmetric mesh structure that achieves continuously variable coupling by rotating the mesh in its own plane about the millimeter wave transmission line optical axis. We quantify the performance of this device with nitroxide spin-label spectra in both a lossy aqueous solution and a low loss solid state system. These two systems have very different coupling requirements and are representative of the range of coupling achievable with this technique. Lossy systems in particular are a demanding test of the achievable sensitivity and allow us to assess the suitability of this approach for applying high frequency ESR to the study of biological systems at physiological conditions, for example. The variable coupling technique reported on here allows us to readily achieve a factor of ca. 7 improvement in signal to noise at 170 GHz and a factor of ca. 5 at 95 GHz over what has previously been reported for lossy samples.

A physician-pharmacist model for the surveillance of blood pressure in the community: a feasibility study.

Hypertension is poorly managed. Hospital-based pharmacists working with physicians have been shown to improve the rate of achievement of "target" blood pressure in selected patients. It is unknown if such schemes can operate in the community and to what extent they would attract volunteers with poorly managed blood pressure. We assessed the feasibility of pharmacists to provide community-based, open-access, blood pressure monitoring. In addition, we describe the blood pressure profile of the group in comparison to that of the 1994 Health Survey of England (HSE). Pharmacists from six pharmacies were trained to deliver the service. Adults living within the postal districts of the pharmacies were invited, through an advertising campaign, to volunteer to have their blood pressure measured. Blood pressure data and information on treatment for hypertension and/or diabetes were collected on 263 registrants. Patients were advised to have their blood pressure managed by the general practitioner immediately (category 1), re-measured within 2-3 months (category 2) or in 12 months time (category 3). The mean (s.d.) blood pressure of patients in categories 1 (n = 16), 2 (n = 117) and 3 (n = 130) was 186(16)/97(29), 151(13)/94(9) and 139(22)/86(13) mm Hg respectively; P < 0.001. Ninety-one patients (35%) were in receipt of antihypertensive therapy. Forty-five percent of the treated group had controlled blood pressure (<160/95 mm Hg) compared with 30% in the HSE dataset. A large proportion of known hypertensive patients with poor blood pressure control who had visited their general practitioner within the previous 6 months were detected by the pharmacist-led service. Pharmacists operating an open-access blood pressure monitoring service may be of value in improving the management of hypertension.

Electron spin resonance in studies of membranes and proteins.

We provide a review of current electron spin resonance (ESR) techniques for studying basic molecular mechanisms in membranes and proteins by using nitroxide spin labels. In particular, nitroxide spin label studies with high-field/high-frequency ESR and two-dimensional Fourier transform ESR enable one to accurately determine distances in biomolecules, unravel the details of the complex dynamics in proteins, characterize the dynamic structure of membrane domains, and discriminate between bulk lipids and boundary lipids that coat transmembrane peptides or proteins; these studies can also provide time resolution to studies of functional dynamics of proteins. We illustrate these capabilities with recent examples.

Lipid hydroperoxide and markers of renal disease susceptibility in African-Caribbean and Caucasian patients with Type 2 diabetes mellitus.

AIMS: The reasons for the increased incidence of diabetic nephropathy in African-Caribbean compared with Caucasian subjects are poorly understood. Increased oxidative stress is linked to the development of endothelial dysfunction and histological abnormalities associated with diabetic renal disease. Therefore, it was assessed whether a marker of oxidative stress, lipid hydroperoxide (LOOH) and endothelial damage, von Willebrand factor (vWF) varied according to ethnic origin in the presence or absence of early diabetic nephropathy. METHODS: Eighty-eight patients with Type 2 diabetes mellitus of African-Caribbean or Caucasian origin without a history of cardiovascular disease or clinical proteinuria were studied. Patients were classified as having microalbuminuria or normal albumin excretion. Fasting plasma vWF and LOOH were measured by an inhouse enzyme-linked immunoassay and ferrous oxidation with xylenol orange (FOX) assay, respectively. The relationship of LOOH to urinary albumin status, the metabolic disturbances of diabetes, blood pressure and ethnic origin were assessed using multivariate analysis. RESULTS: Compared with Caucasian patients those of African-Caribbean origin had higher systolic blood pressure and HDL-cholesterol (157.8 +/- 17.0 vs. 147.8 +/- 24.4, P = 0.041 and 1.6 +/- 0.4 vs. 1.3 +/- 0.5, P = 0.018) but lower total triglycerides (1.3 +/- 0.8 vs. 1.9 +/- 1.1, P = 0.008). LOOH was significantly higher in the African-Caribbean patients compared with Caucasians (5.98 +/- 2.49 vs. 4.49 +/- 2.19, P = 0.006). vWF tended to be higher in microalbuminuric patients but showed no variation with ethnicity. In logistic regression analysis, LOOH was the only independent predictor of a raised albumin excretion rate (P = 0.008). In multiple regression analysis, African-Caribbean ethnicity (P = 0.020) HDL-cholesterol (P = 0.036), total triglycerides (P = 0.002) and a raised albumin excretion rate (P = 0.002) were independent predictors of LOOH. CONCLUSIONS: In this group of Type 2 diabetic patients an increase in LOOH is associated with abnormal urinary albumin excretion. African-Caribbean origin was a determinant of LOOH independently of microalbuminuria. It is postulated that increased oxidative stress is of pathophysiological significance in accelerating the development of renal disease in African-Caribbean patients.

QT dispersion in microalbuminuric Type 1 diabetic patients without myocardial ischemia.

Microalbuminuria is associated with an increased risk of cardiac death. We assessed whether urinary albumin excretion is related to abnormalities of the QT interval independently of myocardial ischemia. Thirty-four patients with type 1 diabetes who were free from ischemic heart disease on the basis of normal stress electrocardiography and echocardiography were studied. Maximal QT interval and dispersion were significantly greater in the group with microalbuminuria (n=17) compared to controls (n=17) with normal urinary albumin excretion (394 [26.1] vs. 373.8 [27.8] ms; P=.044 and 62.4 [21.8] vs. 42.7[11.6] arbitrary units; P=.009). Autonomic function was similar between the groups. Urinary albumin excretion correlated positively with QT dispersion (P=.023). These data suggest that in type 1 diabetic patients, QT abnormalities can occur independently of autonomic dysfunction or myocardial ischemia and may be related to the processes which increase urinary albumin leakage.

Pattern of angiogenic cytokine release from human vascular smooth muscle cells programmed by amino acid deprivation.

Vascular smooth muscle cells (VSMC) may be programmed by nutrient deprivation. We found that after 2 and 12 h exposure to 75% reduced amino acids, the release of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF beta 1) from VSMC was significantly greater than that from cells maintained in control medium [2572.0 (546.3) vs 602.1 (241.7), P=0.001 and 16 028.0 (2192. 4) vs 13 027.3 (1233.5) pg/10(6)cells, P=0.022 respectively]. These differences were magnified after two passages of exposure for both bFGF (P=0.0001) and TGF beta 1 (P=0.0001). The stimulated release of VEGF by hypoxia and bFGF was unaffected. Amino acid deprivation of human VSMC is associated with a patterned release of angiogenic cytokines which could be relevant to the programmed changes in VSMC phenotype.

Jones matrix formalism for quasioptical EPR.

The Jones matrix formalism that has been used to analyze quasioptical millimeter-wave circuits is extended for specific application to high-frequency electron paramagnetic resonance (EPR). A survey of general expressions for Jones matrices of elements commonly used in quasioptical EPR spectrometers is given. The Jones matrix representations of quasioptical transmission and reflection cavities are derived, and their relationship to the equivalent circuit and transmission line representations used for conventional EPR cavities is demonstrated. The formalism is applied to selected quasioptical EPR spectrometer designs and experimental tests of the formalism are presented for two configurations of a quasioptical spectrometer operating at 220 GHz.

Synergistic effect of formulated plasmid and needle-free injection for genetic vaccines.

PURPOSE: A plasmid-based gene expression system was complexed with protective, interactive, and non-condensing (PINC) polymer system and administered with Medi-Jector, a needle-free injection device (NFID), to achieve high and sustained levels of antigen-specific antibodies in blood circulation. METHODS: Human growth hormone (hGH) or bacterial beta-galactosidase gene expression plasmids driven by a cytomegalovirus (CMV) promoter were formulated in saline or complexed with a PINC polymer, polyvinylpyrrolidone (PVP), and intramuscularly or subcutaneously administered into dogs and pigs using a 22-gauge needle or a NFID. The hGH-specific IgG titers in serum were measured by an ELISA. Beta-galactosidase expression was measured in injected muscles by an enzymatic assay or immunohistochemistry. The effect of NFID on DNA stability and topology was assessed by gel electrophoresis. RESULTS: Intramuscular (i.m.) or subcutaneous (s.c.) injection of a hGH expression plasmid pCMV-hGH (0.05-0.5 mg/kg) in dogs and pigs elicited antigen-specific IgG antibody titers to expressed hGH. With both routes of injection, pDNA delivery by a NFID was superior to pDNA injection by needle. The magnitude of hGH-specific IgG titers with NFID was 15-20-fold higher than needle injection when pDNA was complexed with PVP, and only 3-4-fold higher with pDNA in saline. The transfection efficiency in the injected muscle, as measured by beta-galactosidase expression, following i.m. injection of pCMV-betagalactosidase/PVP, was not significantly different between needle and NFID-injected groups. CONCLUSIONS: These data demonstrate that the combination of pDNA/ PVP complexes and a NFID act synergistically to achieve high and sustained levels of antigen-specific IgG response to expressed antigen. This gene delivery approach may offer advantage over needle injection of naked DNA for the development of genetic vaccines.

Amino acid depletion modulates vascular endothelial growth factor production during the life span of human vascular smooth muscle cells.

The role of nutrient supply in the replicative capacity and secretory phenotype of cultured human diploid cells is unclear. We examined the relationship between amino acid privation, the secretion of vascular endothelial growth factor (VEGF) and growth phenotype of vascular smooth muscle cells (VSMC), and endothelial cells. Cultures of VSMCs, but not endothelial cells, were growth inhibited by exposure to medium that was 75% deficient in leucine, methionine, arginine, and cysteine over two passages. Exposed VSMC cultures exhibited an increased vulnerability to apoptosis. The maximal cumulative population doubling of the exposed cells was reduced significantly compared with the control cells (25.7 +/- 2.0 doublings vs. 27.9 +/- 2.1 doublings; P < 0.03). Constitutive VEGF production first became evident in the later passages of the exposed and nonexposed cell cultures. However, production of VEGF was 17-fold greater in the exposed cultures at the tenth passage (P < 0.001). The replicative capacity and constitutive production of VEGF in VSMCs in culture may be programmed by transient privation of amino acids. These observations are relevant to new concepts concerning the pathogenesis of vascular disease.

Permissive role of hypertension in the development of proteinuria and progression of renal disease in insulin-dependent diabetic patients.

BACKGROUND: In insulin-dependent diabetic subjects, heritable factors related to hypertension and cardiovascular disease are associated with nephropathy. OBJECTIVE: To determine the relationship of blood pressure, glycaemic control, family history of cardiovascular disease and sodium-lithium countertransport activity to the onset of proteinuria and decline in glomerular filtration. DESIGN: A retrospective analysis of the rate of onset of proteinuria and a longitudinal study of the progression of established renal disease. SETTING: Guy's Hospital Diabetes Renal Clinic, a secondary referral centre. PATIENTS: Fifty-four insulin-dependent diabetic patients with nephropathy, persistent total protein excretion rate > 500 mg/24 h, enrolled between 1978 and 1992. MAIN OUTCOME MEASURES: Rate of decline in glomerular filtration rate. Duration of diabetes at onset of nephropathy (time to proteinuria). Blood pressure and glycosylated haemoglobin at the time of diagnosis of nephropathy (baseline). Family history of cardiovascular disease and hypertension. Erythrocyte sodium-lithium countertransport activity in a subset of patients (n = 41) not being administered renal replacement therapy in 1992. RESULTS: The estimated (95% confidence interval) time to proteinuria was shortened in relation to increments in diastolic blood pressure at baseline and to a family history of cardiovascular disease in both parents [1.9 (0.2-3.2) years/10 mmHg, P < 0.05 and 6.7 (-0.3-13.7) years, P < 0.07, respectively]. During follow-up 15% (n = 8) of the patients who did not require antihypertensive therapy had slower rates of decline in glomerular filtration and lower rates of sodium-lithium countertransport activity than did those who had been administered treatment [median (range): 2.88 (0.2 to -11.28) versus 7.8 (0.1 to -20.4) ml/min per 1.73 m2/year, P < 0.05 and 0.28 (0.14-0.54) versus 0.43 (0.18-0.88) mmol/l per erythrocyte/h, P < 0.03, respectively]. In this group there was an inverse relationship between the time to proteinuria and glycosylated haemoglobin (r = -0.79, P = 0.018). For the whole group a multivariate analysis showed hypertension and initial glomerular filtration rate to be related independently to the rate of decline in renal function; glycaemic control just failed to attain statistical significance (P < 0.06). CONCLUSION: Elevation of blood pressure accelerates the onset of nephropathy and its progression; its absence, a reduced familial predisposition to cardiovascular disease, low sodium-lithium countertransport activity and good blood glucose control favour a more benign prognosis.

Premature cell ageing and evolution of diabetic nephropathy.

The rate of development and progression of renal disease varies greatly in insulin-dependent diabetic (IDDM) patients. The cellular and molecular reasons for this difference are largely unknown but could be related to early cell differentiation, a phenomenon recently reported in IDDM patients with nephropathy. In this study we compared cell differentiation and cell volume between IDDM patients with and without nephropathy and investigated the cell ageing characteristics in relation to the rate of evolution of renal disease in the IDDM patients with diabetic nephropathy. Cell volume was larger and the percentage of post-mitotic fibrocytes was higher in skin fibroblasts derived from IDDM patients with diabetic nephropathy compared to those from IDDM patients without kidney disease (mean +/- SD in arbitrary units 817.3 +/- 25.7 vs 760 +/- 32.8; p = 0.005; and mean +/- SD % 33.6 +/- 11.8 vs 20.8 +/- 10; p = 0.02 respectively). Analysis of the interaction of the time to proteinuria (TTP) and the rate of change of glomerular filtration rate (GFR) with glycaemic control, arterial blood pressure and cell volume and the state of cell differentiation showed that glycated haemoglobin and the percentage of post-mitotic fibrocytes were negatively correlated to TTP (r = -0.68; p = 0.008; r = 0.52; p = 0.05 respectively) and positively associated with the rate of change of GFR (r = 0.76; p = 0.03; r = 0.56; p = 0.037 respectively). Cell volume was negatively correlated to TTP (r = -0.53; p = 0.05). Diastolic blood pressure was also related to the rate of GFR change (r = 0.56; p = 0.039). In a multiple linear regression analysis glycated haemoglobin maintained its significance independent relationship with TTP at the 1% level, while the strength of the association between the percentage of post-mitotic cells and cell volume was reduced to the 11 and 9% level, respectively. Cultured skin fibroblasts from IDDM patients with nephropathy show signs of early differentiation. Glycaemic control is a key factor in the rate of onset of proteinuria and different rates of cell ageing appear to contribute to the rate of development and progression of diabetic nephropathy. Their interaction may be responsible for the severity of renal involvement in susceptible IDDM patients.

Premature senescence of skin fibroblasts from insulin-dependent diabetic patients with kidney disease.

Diabetic glomerulopathy develops in a subset only of patients with insulin-dependent diabetes (IDDM) and early, in its course, is characterized by cell hypertrophy and by excessive extracellular matrix production. These observations suggest that an alteration in the control of cell growth processes may contribute to its pathogenesis and be related to the susceptibility to kidney disease. We therefore investigated whether the development of diabetic nephropathy is associated with abnormalities of cell growth and morphology. Cultured skin fibroblasts from 14 IDDM patients with nephropathy (DN) were compared with those of 10 IDDM patients without nephropathy (D) and of 14 control non-diabetic subjects (C). Cell volume (in arbitrary units) and total protein content (microgram/10, 000 cells) were increased in serially passaged skin fibroblasts of IDDM patients with nephropathy (DN = 809.5 +/- 33.1 and 1.93 +/- 0.38 vs. D = 764.4 +/- 31.5 and 1.5 +/- 0.37, P = 0.005 and P = 0.03, respectively; vs. C = 756.2 +/- 36.3 and 1.5 +/- 0.38, P = 0.0006 and P = 0.03, respectively). These hypertrophic cells had a tendency to a slower duplication rate and exhibited a dissociation of the DNA and cytoplasmic cell-cycles, resulting in a higher proportion of tetraploid cells (DN = 25 +/- 15% vs. D = 6 +/- 4%, P = 0.005; and vs. C = 10 +/- 8%, P = 0.04). The frequency of terminally differentiated post-mitotic fibrocytes, cells specialized for extracellular matrix production, was higher in patients with nephropathy compared to that of patients without nephropathy and normal controls (DN = 34 +/- 14% vs. D = 21 +/- 10%, P = 0.02; and vs. C = 19 +/- 12%, P = 0.008). That early differentiation was a specific feature of cells derived from patients with diabetic nephropathy was confirmed by the study of cell life-span which demonstrated that these cells aged prematurely (log rank test, chi 2 = 10,012; P = 0.0067). We conclude that an acceleration of cell aging is a peculiar feature of diabetic kidney disease and may contribute to its pathological tissue changes.

Microalbuminuria as a marker of silent myocardial ischaemia in IDDM patients.

Insulin-dependent diabetic (IDDM) subjects with microalbuminuria have an increased long-term risk of overt cardiovascular disease; however, the early exposure to cardiovascular risk factors may increase their predisposition to current silent myocardial ischaemia. The frequency of silent myocardial ischaemia detected by stress echocardiography and electrocardiography was significantly greater in 32 asymptomatic IDDM patients with microalbuminuria compared to 32 normoalbuminuric IDDM patients (25% [n = 8] vs 6.3% [n = 2]; p = 0.03, odds ratio [95% CI] 6.3 [1.2, 37.8]). Elective coronary artery bypass grafting was required in 1 patient with microalbuminuria and silent myocardial disease. Microalbuminuria and poorer autonomic function were independently associated with silent myocardial ischaemia in multivariate analysis (p = 0.03 and p = 0.02, respectively). Screening for silent myocardial ischaemia using these non-invasive tests may be warranted in microalbuminuric IDDM which patients could be of considerable clinical importance.

Use of seclusion with children and adolescents in public psychiatric hospitals.

A review of seclusion orders issued over a one-month period at three public psychiatric centers found that youngsters who had been secluded, notably those with high use of seclusion, differed from other children at the centers. Seclusion was frequently resorted to by direct-care staff at times of high staff-child interaction but low programming. Findings suggest the need for clinicians to use treatment goals and interventions as means of altering seclusion patterns and minimizing duration of seclusion.

250-GHz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes.

Rigid-limit 250-GHz electron spin resonance (FIR-ESR) spectra have been studied for a series of phosphatidylcholine spin labels (n-PC, where n = 5, 7, 10, 12, 16) in pure lipid dispersions of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as well as dispersions of DPPC containing the peptide gramicidin A (GA) in a 1:1 molar ratio. The enhanced g-tensor resolution of 250-GHz ESR for these spin labels permitted a careful study of the nitroxide g-tensor as a function of spin probe location and membrane composition. In particular, as the spin label is displaced from the polar head group, Azz decreases and gxx increases as they assume values typical of a nonpolar environment, appropriate for the hydrophobic alkyl chains in the case of pure lipid dispersions. The field shifts of spectral features due to changes in gxx are an order of magnitude larger than those from changes in Azz. The magnetic tensor parameters measured in the presence of GA were characteristic of a polar environment and showed only a very weak dependence of Azz and gxx on label position. These results demonstrate the significant influence of GA on the local polarity along the lipid molecule, and may reflect increased penetration of water into the alkyl chain region of the lipid in the presence of GA. The spectra from the pure lipid dispersions also exhibit a broad background signal that is most significant for 7-, 10-, and 12-PC, and is more pronounced in DPPC than in POPC. It is attributed to spin probe aggregation yielding spin exchange narrowing. The addition of GA to DPPC essentially suppressed the broad background signal observed in pure DPPC dispersions.