High-pressure NMR spectroscopy of proteins and membranes.
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Equipment Design↗
Biomedical subjects
J Jonas
Publications and source records attributed to J Jonas.
Effects of amino acid substitutions on the pressure denaturation of staphylococcal nuclease as monitored by fluorescence and nuclear magnetic resonance spectroscopy.
In the present study we have used high hydrostatic pressure coupled with either time-resolved and steady-state fluorescence or NMR spectroscopy in order to investigate the effects of amino acid substitutions on the high-pressure denaturation properties of staphylococcal nuclease. This protein has been shown previously to be structurally heterogeneous in its native state. On the NMR time scale, four distinct interconverting conformational forms arise from the population of both cis and trans Xaa-Pro peptide bonds (His46-Pro47 and Lys116-Pro117) [Evans et al. (1989) Biochemistry 28, 362; Loh et al. (1991) in Techniques in Protein Chemistry II, pp 275-282, Academic Press, New York]. Mutations in the protein sequence have been shown to change the distribution among the various forms [Alexandrescu et al. (1989) Biochemistry 28, 204; Alexandrescu et al. (1990) Biochemistry 29, 4516]. Time-resolved fluorescence on a series of mutants with altered equilibria for cis/trans isomerism about the 116-117 peptide bond did not reveal any simple relationship between the position of the cis/trans equilibrium in the folded state and the heterogeneity of the fluorescence decay. However, the specific dynamic properties of each mutant, as revealed by time-resolved fluorescence, do appear to be correlated with their partial molar volume changes of denaturation. A striking finding is that mutation of either (or both) of the prolines that exhibits structural heterogeneity to glycine greatly alters the stability of the protein to pressure. These mutations also result in decreased chain mobility as assessed by time-resolved fluorescence. It appears that packing defects, which allow for peptide bond cis/trans heterogeneity in the wild-type protein, are removed by the Pro-->Gly substitutions.
Amino Acid Sequence↗
Molten-globule conformation of Arc repressor monomers determined by high-pressure 1H NMR spectroscopy.
The conformation of the pressure-dissociated monomer of Arc repressor was characterized by 1H NMR spectroscopy. The NMR spectra of the monomer under pressure (up to 5.0 kbar; 1 bar = 100 kPa) are typical of a molten globule and they are considerably different from those of the native dimer and thermally denatured monomer. The two-dimensional nuclear Overhauser effect spectra suggest that the pressure-induced molten globule retains some secondary structure. The presence of nuclear Overhauser effects in the beta-sheet region in the dissociated state suggests that the intermonomer beta-sheet (residues 8-14) in the native dimer is replaced by an intramonomer beta-sheet. Changes in one-dimensional and two-dimensional NMR spectra prior to pressure dissociation were found and suggest the existence of a "predissociated" state.
Amino Acid Sequence↗
Utility of the color pattern-electroretinogram (PERG) in glaucoma.
Pattern-onset electroretinograms (PERGs) with red-green color contrast (CC) and green-"black" luminance contrast (LC) stripe patterns (0.3 c/deg) were recorded in a group of 80 control subjects and in a group of 42 patients having glaucomas of varying etiology and severity. The PERG data were correlated with the results of static perimetry and optic disc morphometry. In the glaucoma group the PERG was reduced significantly and by relatively similar amounts with both CC and LC stimuli. A significant correlation of the PERG reduction with visual field loss was found only with the CC, not with the LC PERG. Correlations between PERG amplitudes and neuroretinal rim areas of the optic disk were similar for the LC and for the CC stimulus. The rather poor percentage of correct classification of controls and patients based on the PERG or the optic disc morphometry alone can be improved by two-dimensional discriminant analysis using both CC PERG and papillometry data.
Color Perception↗
A technique for the tensile testing of demineralised bone.
The purpose of this study was to devise a method for the tensile testing of the organic component of demineralised bone. It was hoped that the method could be applied to determine whether an observed reduction in the strength of bone in copper-deficient rats could be attributed to a reduction in the cross-linking of collagen. Tensile testing of a fully demineralised specimen proved impossible to perform because of the difficulty in gripping the material, which tended to slide out of its holding clamps. The technique devised involved the demineralisation of only the diaphysis of the bone, leaving the bone ends intact. This enabled the bone to be gripped firmly at both ends in an Instron tensile testing machine. Elongation under loading of the demineralised diaphysis was measured by the Instron and a video recording was made of the elongation of the central portion. The strain was found to be uniform. Tensile strength was found to correlate with copper levels in the liver (r = 0.54, p < 0.05), although no significant differences were found between the strength, the stiffness, or the strain to failure for bone from the copper-deficient animals and bone from the normal controls. The test method itself should be useful in other studies in which the tensile properties of the organic phase of bone need to be measured.
Animals↗
Impaired mechanical strength of bone in experimental copper deficiency.
Copper, through its role as cofactor for lysyl oxidase, is essential for intra- and inter-molecular cross-links in collagen. Copper deficiency, in man and in animals, is associated with bone fragility ascribed to defective cross-links. To assess bone strength in copper-deficient animals, we designed a sensitive torsion-testing apparatus according to biomechanical considerations. Femora from 7 copper-deficient rats and from their pair-fed controls were tested in torsional loading until fracture. Significant decreases in the maximal sustained torque (t = 2.93, p < 0.05), in the ultimate angular deformation (t = 2.52, p < 0.05) and in the toughness (t = 2.88, p < 0.05) were demonstrated. In a complementary study, it was shown that the ash weight and the calcium content of the femora from the copper-deficient animals did not differ from those of the controls. It was likely, therefore, that the impaired mechanical strength was related to defects in the collagen component of bone.
Animals↗
[Results of surgical therapy for venous insufficiency as a cause of erectile disorders].
Since penile vein ligation is usually associated with a poor postoperative outcome, a study was undertaken to evaluate possible prognostic factors for this procedure. A total of 44 patients with erectile dysfunction underwent ligation of all dorsal penile veins and resection of the deep dorsal penile vein for venous impotence. A comprehensive study of all patients was made. All patients did not respond to pharmacotherapy and had a venous leak. With a minimum follow-up period of 6 months, patients were categorized as follows: full spontaneous erections, those patients responding to pharmacotherapy and those showing no improvement. Out of 44 patients, 13 had full spontaneous erections postoperatively, 5 now responded to pharmacotherapy and 26 showed no improvement. The maintenance flow was 89 +/- 50 ml/min in the group with spontaneous erections and 85 +/- 49 ml/min in those showing no improvement (P = 0.78). Out of the 20 patients with normal single potential analysis of cavernous electric activity (SPACE), 12 had full erections postoperatively, 4 showed no improvement and 4 responded to pharmacotherapy. Of the 24 patients with abnormal SPACE, 1 developed full erections, 1 now responds to pharmacotherapy and 22 showed no improvement. Our results indicate that SPACE seems to be an important prognostic factor for the postoperative outcome of penile vein ligations for venous impotence.
Adult↗
High-resolution NMR study of the pressure-induced unfolding of lysozyme.
The pressure-induced reversible unfolding of lysozyme was investigated by high-resolution proton magnetic resonance spectroscopy by following the proton spectra of the following residues: His-15 epsilon 1, Trp-28 epsilon 3, Leu-17 delta 2, Cys-64 alpha, and Trp-108 epsilon 3. The experiments were performed at pH 3.9 and 68.5 degrees C in the pressure range from 1 bar to 5 kbar both in the absence and presence of tri-N-acetylglucosamine (tri-NAG). From the pressure-induced changes of the equilibrium between the native and denatured forms of lysozyme, the reaction volumes (delta V) were calculated for each residue. Small but statistically significant differences in delta V were found for residues located in different regions of the protein. For example, delta V for the disulfide bonded Cys-64 alpha is smaller than the delta V's found for the other residues. In particular, the effect of tri-NAG binding to lysozyme was a change of delta V from -10.3 +/- 0.6 cm3/mol to -18.1 +/- 1.7 cm3/mol for the Trp-108 epsilon 3 residue which is located close to the active site. It is important to note that the Cys-64 alpha residue also senses the binding of the substrate analog. The ability to detect statistically significant differences for delta V of individual residues located in different regions of lysozyme represents the main result of these experiments.
Hydrogen-Ion Concentration↗
High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.
High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)
1,2-Dipalmitoylphosphatidylcholine↗
[The incidence of focal liver lesions in patients with colorectal carcinoma].
The preoperative sonographic findings in 166 patients with confirmed colorectal carcinomas were analysed to determine the frequency of focal liver lesions. In 15 patients liver metastases were found at the first examination and in a further 16 patients they were detected subsequently. Homogeneous high-intensity echoes were found in only one patient. Cysts occurred in 13 and haemangiomas in 11 patients. The latter showed solitary lesions which exceeded 25 mm in only one patient and otherwise measured between 6 mm and 15 mm. Focal abnormalities of fat distribution were found in 12 patients. Even in a tumor patient, the first differential diagnosis of a solitary, homogeneous liver lesion of less than 2 cm is a hemangioma and further investigation is mandatory.
Colorectal Neoplasms↗
Biomorphometry and histomorphometry of the optic disc with special reference to the parapapillary region.
Glaucomatous optic nerve damage is typically associated with intrapapillary changes such as neuroretinal rim loss. In this study, parapapillary chorioretinal atrophy was evaluated in 691 normal eyes, 1081 glaucomatous eyes and 31 eyes with ocular hypertension. It was significantly larger and occurred more often in the glaucomatous eyes (parapapillary atrophy area: 1.07 +/- 0.83 mm2) than in the normal eyes (0.55 +/- 0.64 mm2) or the eyes with ocular hypertension (0.55 +/- 0.37 mm2). These differences were significant also for eyes with moderate glaucomatous damage (0.86 +/- 0.62 mm2). Parapapillary chorioretinal atrophy was associated with shallow glaucomatous cupping, diffuse nerve fiber loss, markedly tessellated fundus and only moderately elevated intraocular pressure. It increased with decreasing neuroretinal rim area. It showed a spatial correlation to neuroretinal loss inside the optic disc. In unilateral glaucoma, it was larger in the affected eye than in the unaffected eye. Differentiated into zones Alpha and Beta, it correlated clinico-histologically with a complete loss of retinal pigment epithelium cells and incomplete loss of photoreceptors (Zone Beta) and pigmentary and structural irregularities of the retinal pigment epithelium (zone Alpha). Parapapillary chorioretinal atrophy is associated with glaucoma.
Adult↗
[Arterial occlusive disease after surgical treatment of fractures of the lower extremity].
Chronic arterial occlusive disease of the lower limb may complicate fracture treatment. In five cases, diagnosis of vascular disease was established when delayed fracture healing or skin gangrene occurred some weeks after operative fracture treatment. The diagnosis of vascular disease was made from the case history, examination of the patient and repeated pulse control at the feet. Arterial insufficiency will be worsened by long-term elevation of the leg and by fracture treatment with a cast or traction. Angiography is indicated, and quick vascular reconstruction is recommended. In one patient with known iliac occlusion (Leriche syndrome) we performed simultaneous vascular reconstruction and operative fracture treatment.
Aged↗
Pressure effects on dipalmitoylphosphatidylcholine bilayers measured by 2H nuclear magnetic resonance.
The effects of pressure, up to 5 kbar, on multilamellar vesicles of 1,2-dipalmitoyl-sn-phosphatidylcholine perdeuterated in the acyl chains (DPPC-d62) were examined by using high-pressure NMR techniques. A deuterium probe was built, and the quadrupole splitting was measured against pressure at various temperatures. The experiments were performed on pure lipid bilayers in the liquid-crystalline state and on bilayers in the liquid-crystalline state containing the local anesthetic tetracaine. The results show that the order parameter of all segments of the acyl chains increases with pressure in the liquid-crystalline state. The more highly ordered regions of the chains are affected slightly more than the regions near the methyl ends. The addition of tetracaine increases the disorder of the chains, and pressure reverses the effect of anesthetic on the lipid as seen by the reversal of the changes in line shape and the measured order parameter.
1,2-Dipalmitoylphosphatidylcholine↗
Comprehensive school health programs in Texas public schools.
One goal of the Texas Comprehensive School Health Initiative (TCSHI) involves assessing the status of comprehensive school health (CSH) programs in Texas public schools. This project used a case study approach to establish baseline information regarding CSH programs in selected school districts across the state. Eight researchers were trained to use a case study instrument and procedures for conducting interviews. Seven school districts participated in the survey during fall 1990. Results indicated considerable concern and support for CSH programs but little coordination of programs was evident. Six districts were not organized to address health comprehensively. Generally, school districts were overwhelmed with other responsibilities, so CSH programming was not a priority. Results will be used to revise the survey instrument and further expand the data base.
Adolescent↗
[Sensitivity of preoperative diagnosis in mesenteric vascular occlusion].
In a consecutive series the sensitivity of the preoperative diagnostic in acute mesenteric vascular occlusion was investigated prosprectively in 62 patients. 22 patients had an arterial thrombosis, 20 patients a venous thrombosis, 13 patients had an embolism, six patients a non-occlusive disease and one patient suffered from a dissection of the aorta. All but three patients (95.2%) had a leukocytosis with an average of 18,700/nl. The serum lactate was increased in 88.7% of the patients. The percutaneous ultrasonography was without any pathological findings in 45% of the patients. The x-ray of the abdomen shows signs of an ileus in more than half of the patients. The angiography had a sensitivity of 74%.
Aged↗
High-pressure small-angle neutron scattering (SANS) study of 1,2-dielaidoyl-sn-glycero-3-phosphocholine bilayers.
Small-angle neutron scattering of the trans-unsaturated DEPC has been investigated as a function of pressure at 12, 18.6 and 35 degrees C. A pressure-induced structural phase transition from a liquid-crystalline state to a gel state is observed at the temperatures studied. The critical pressure of this transition increases with increasing temperature with a delta P/delta T value of 51 bar/C degrees. The small-angle neutron scattering results indicate that the effect of the trans double bonds in DEPC is to enhance the conformational disorder in the hydrocarbon chains. In DEPC bilayers, a pressure-induced conformational ordering process is observed not only in the liquid-crystalline phase but also in the gel phase, which indicates that conformational disorder exists in the liquid-crystalline phase as well as in the gel phase.
Fatty Acids, Unsaturated↗
Optic disk size in ischemic optic neuropathy.
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Eye↗
The pattern-evoked electroretinogram (PERG): age-related alterations and changes in glaucoma.
Pattern-onset electroretinograms (PERGs) were studied in 147 normal subjects of different ages (14-79 years) and in 110 eyes of 65 patients with glaucoma or ocular hypertension. The responses showed an increase (P less than 0.001) in peak latency with increasing age and a decrease (P less than 0.001) in amplitude which approximately parallels the loss of ganglion cells estimated by other authors. Many glaucoma eyes showed a loss of the normally present spatial tuning. In the age group above 50 years 50% of the onset responses were significantly diminished and the peak latencies were not significantly different. A negative correlation (P less than 0.001) was found between the size of the PERG and the cup/disc ratio and a positive correlation (P less than 0.001) with the area of the neuroretinal rim of the optic disc. The PERG decreased (P less than 0.01) with increasing visual field losses.
Adolescent↗