Intermolecular relaxation in glycerol as revealed by field cycling 1H NMR relaxometry dilution experiments.

(1)H spin-lattice relaxation rates R(1) = 1/T(1) have been measured for partly deuterated glycerol-h(5) diluted in fully deuterated glycerol-h(0) for progressively lower concentrations of glycerol-h(5). By means of the field cycling (FC) technique relaxation dispersion data, R(1)(ω), have been collected for several temperatures in the frequency range of 10 kHz-20 MHz. In order to disclose the spectral shape of the intra- and intermolecular relaxation, extrapolation of the relaxation data to the zero concentration limit has been performed. The paper confirms that the low frequency excess contribution to the total relaxation rate R(1)(ω) previously reported for several liquids is of intermolecular origin and reflects translational motion, whereas the high-frequency part is attributed to molecular rotation. Thus, intra- and intermolecular relaxation contributions are spectrally separated. The intermolecular relaxation itself contains also a contribution from rotational motion, which is due to non-central positions of the interacting nuclei in the molecule. This eccentricity effect is quantitatively reproduced by treating the intermolecular spectral density as a sum of translational-like (described by the free diffusion model) and rotational-like contributions (described by a Cole-Davidson function). Applying frequency-temperature superposition master curves as well as individual relaxation dispersion data, R(1)(ω), are analyzed. It is demonstrated that, in spite of the rotational influence, the translational diffusion coefficients, D(T), can be extracted from the (1)H relaxation dispersion which gives (1)H NMR relaxometry the potential to become a routine technique determining the diffusion coefficient in liquids.

2H nuclear magnetic resonance study on the molecular motion in cyanoadamantane. II. Orientationally ordered and glassy crystalline phase.

The orientationally ordered crystalline and glassy plastically crystalline phase of cyanoadamantane were investigated using (2)H NMR. Solid-echo line shape, two-dimensional spectrum, and spin-lattice relaxation were analyzed. In both phases, the molecules display solely a rotation around the molecular C(3) symmetry axis. For the orientationally ordered phase, a single correlation time characterizes the motion, and the time constant shows an Arrhenius temperature dependence. In contrast, a broad distribution G[ln(tau)] of correlation times is observed for the glassy plastically crystalline phase that leads to characteristically different NMR features such as "two-phase" spectra and pronounced nonexponential relaxation. The distribution G[ln(tau)] can be derived from a temperature independent distribution of activation energies g(E(a)), with its mean value lying significantly below the activation energy corresponding to the ordered phase. Thus, the molecular uniaxial rotation proves to be a sensitive probe for the energy landscape of the orientationally disordered glassy crystalline phase of cyanoadamantane.

2H nuclear magnetic resonance study of the molecular motion in cyanoadamantane. I. Supercooled plastically crystalline phase.

The supercooled plastically crystalline phase (glassy crystal) of cyanoadamantane was investigated by multidimensional 2H NMR (T>Tg). Although the orientationally disordered crystalline phase always coexisted with the orientationally ordered crystalline phase, we were able to single out the signal from the glassy crystal by selective excitation and it was possible to carry out line shape measurements and two-dimensional (2D) experiments (in frequency and time domain). The latter directly reveal sixfold jumps with an reorientation of the molecular C3 axis via 90 degrees angles, thus reflecting the symmetry of the lattice. The motion around the C3 axis is found to be always fast. We can reproduce the line shape by random walk simulations properly taking into account these molecular motions. Both methods (line shape and 2D experiments) yield time constants which agree with those reported by other techniques. Refining the analysis a narrow distribution of correlation times is introduced to account for a weak stretching of the correlation function. We did not find any indication of a small angle process usually found in structural glasses. Thus, the motional process in the glassy crystal appears to be simple and quite different from that in structural glasses.

Combined k-space q-space pulsed ESR imaging: mapping of restricted diffusion in (FA)(2)PF(6).

A (FA)(2)PF(6) crystal from the family of the quasi-one-dimensional organic conductors was selectively damaged by a beam of Helium ions with a slitted mask placed in the beam's trajectory. Pulsed ESR density weighted imaging of the damaged crystal revealed the appearance of regions where the ESR signal was absent. The one-dimensional motion of the charge carriers was thus restricted to the undamaged sections. The local charge carrier spin dynamics in these restricted areas was probed by combined k-space q-space pulsed ESR imaging. The local expected appearance of the restricted pulsed gradient spin echo (PGSE) "diffusive diffraction" effect is shown. The position of the diffraction minima is compatible with the density imaging results.

A clinical advancement program: creating an environment for professional growth.

Clinical ladders help maintain expert, motivated, and effective nurses in direct patient care roles. While many institutions were phasing out clinical ladders for registered nurses, Miami Valley Hospital was proactively evaluating the original version. The authors describe their clinical advancement program, based on Benner's model, with program evaluation results. The program participation rate has increased overall. Participants perceive their work and environment more positively than nonparticipants.

Critical care nurse perceptions of family needs.

Family needs research has for the most part focused on the families' perceptions when a significant other is admitted to the intensive care unit. We examined critical care nurse perceptions of family needs. The questionnaire "Needs of Families of Critically Ill Patients" was given to 126 intensive care unit nurses. The tool was an adaptation of Molter's questionnaire "Needs of Relatives of Critically Ill Patients." The revised tool examined nurse perception of family needs, perception of time available to meet the needs in daily practice, and the best professional to meet the family need if the need was identified as best met by someone other than the nurse. The majority of the nurses perceived family needs as important or very important, and 85% of the nurses indicated that they were able to meet family needs and had the time to do so. Cognitive family were ranked higher than psychologic or personal and physical needs. Nurses from the four intensive care units ranked family needs significantly differently, a result that may be influenced by differing patient acuity and patient length of stay on individual units. Nurses' perceptions of family needs were influenced by units worked, length of time practicing in critical care, educational preparation, and length of time in nursing.

Influence of beta-lactam antibiotics and ciprofloxacin on cell envelope of Escherichia coli.

The effects of subinhibitory concentrations of different beta-lactam antibiotics and one quinolone on the quantitative composition of the outer membrane (OM) of two strains of Escherichia coli, on lipid translocation into the OM, and on the production of capsular K1 polysaccharide were studied. The phospholipid/amino acid ratio was reduced in almost all OM preparations from antibiotic-treated bacteria. In one strain, antibiotic treatment increased the lipopolysaccharide/amino acid ratio. The amount of peptidoglycan fragments bound to the OM was increased by all the antibiotics. In pulse-chase experiments with a radioactive lipid precursor, ciprofloxacin, imipenem, and aztreonam inhibited phospholipid translocation into the OM. Furthermore, imipenem, cephaloridine, and ciprofloxacin induced a pronounced reduction of the production of capsular K1 polysaccharide. Thus, antibiotics seem to induce marked changes of the quantitative composition of the cell envelope of E. coli. Possible connections of these data with findings on the influence of antibiotics on functional parameters of the host-parasite relationship such as OM immunogenicity and serum resistance are discussed.

Murein biosynthesis in synchronized cells of Proteus mirabilis. Quantitative analysis of O-acetylated murein subunits and of chain terminators incorporated into the sacculus during the cell cycle.

Cells of Proteus mirabilis, synchronized by sucrose density gradient centrifugation, were grown in complex medium containing radioactive N-acetylglucosamine. At various times, labelled murein sacculi were isolated and digested with endo-N,O-acetylmuramidase from Chalaropsis. The murein fragments thus obtained were separated into disaccharide peptides as the monomeric subunits and into peptide-cross-linked subunits by gel filtration. The subunits were further differentiated into O-acetylated and non-O-acetylated species, and into subunits containing anhydro-N-acetylmuramic acid which were glycan chain terminators in the native sacculi. Quantification of the subunit species gave the following results. At specific times during the cell cycle, murein subunits were lost from the polymer and a transient decrease in cross-linkage was observed. The overall degree of cross-linkage in mature murein, i.e. the ratio of peptide-cross-linked subunits versus uncross-linked subunits, was 1.15 as determined by regression analysis. Anhydro-N-acetylmuramic-acid-containing murein subunits representing glycan chain terminators were found either peptide-cross-linked or uncross-linked as monomers. Since these two subunit species were recovered in a defined ratio of 1.6, mature murein consisted of at least two different types of glycan chains. On average, each chain contained 15.4 murein subunits. About 60% of the murein subunits in mature murein were O-acetylated and showed a higher degree of cross-linkage than the non-O-acetylated portion. Finally, following the composition of the sacculus during the cell cycle revealed a complex precursor-product relationship between non-O-acetylated and O-acetylated subunits during murein maturation. The data allowed us to deduce several features of the assembly process of murein sacculi.

Cell cycle parameters of Proteus mirabilis: interdependence of the biosynthetic cell cycle and the interdivision cycle.

We investigated the time periods of DNA replication, lateral cell wall extension, and septum formation within the cell cycle of Proteus mirabilis. Cells were cultivated under three different conditions, yielding interdivision times of approximately 55, 57, and 160 min, respectively. Synchrony was achieved by sucrose density gradient centrifugation. The time periods were estimated by division inhibition studies with cephalexin, mecillinam, and nalidixic acid. In addition, DNA replication was measured by thymidine incorporation, and murein biosynthesis was measured by incorporation of N-acetylglucosamine into sodium dodecyl sulfate-insoluble murein sacculi. At interdivision times of 55 to 57 min murein biosynthesis for reproduction of a unit cell lasted longer than the interdivision time itself, whereas DNA replication finished within 40 min. Surprisingly, inhibition of DNA replication by nalidixic acid did not inhibit the subsequent cell division but rather the one after that. Because P. mirabilis fails to express several reactions of the recA-dependent SOS functions known from Escherichia coli, the drug allowed us to determine which DNA replication period actually governed which cell division. Taken together, the results indicate that at an interdivision time of 55 to 57 min, the biosynthetic cell cycle of P. mirabilis lasts approximately 120 min. To achieve the observed interdivision time, it is necessary that two subsequent biosynthetic cell cycles be tightly interlocked. The implications of these findings for the regulation of the cell cycle are discussed.

Alteration of the immunoglobulin G subclass responses in mice to lipopolysaccharide: effects of nonbacterial proteins and bacterial membrane phospholipids or outer membrane proteins of Proteus mirabilis.

The immunoglobulin M (IgM) and the IgG1, IgG2ab, and IgG3 subclasses of plaque-forming cells (PFC) specific for lipopolysaccharide (LPS) were measured after immunization of mice with LPS alone and compared with the responses to LPS in combination with nonbacterial proteins and with bacterial membrane phospholipid vesicles or two major outer membrane proteins from Proteus mirabilis. The relative numbers of IgG PFC belonging to the IgG1, IgG2, or IgG3 subclasses induced by immunization with LPS alone depended upon the type of LPS administered. Phospholipids and the proteins effected characteristic alterations in not only the strength but also the subclass of the IgG responses to LPS. The results suggest that the hydrophobic-hydrophilic nature or state of aggregation of the preparations plays a role in the induction of IgG1 and IgG2 subclasses of PFC specific for LPS. Complex formation with LPS and adjuvant was apparently necessary to obtain these effects.

Murein and lipopolysaccharide biosynthesis in synchronized cells of Escherichia coli K 12 and the effect of penicillin G, mecillinam and nalidixic acid.

The incorporation of radioactive N-acetyl-glucosamine into murein and lipopolysaccharide of synchronized cells of Escherichia coli K 12 was followed over 100 min in the presence of antibiotics. At 20 min intervals cell walls were prepared. Lipopolysaccharide and murein sacculi were isolated and the radioactivity was quantified in both polymers. Labelled, newly synthesized murein was characterized according to murein subunits linked to lipoprotein, and the degree of crosslinkage. Furthermore, murein subunits containing anhydromuramic acid were determined, permitting the calculation of the average glycan chain length. The results indicated that penicillin G at 30 micrograms/ml stimulated the incorporation of new murein subunits into sacculi followed by a sudden increase in lipopolysaccharide incorporation into the outer membrane. The degree of crosslinkage in murein synthesized in the presence of 30 micrograms/ml penicillin G was higher than in the control, and almost twice as high as in murein synthesized in the presence of 20 micrograms/ml nalidixic acid. Both antibiotics inhibited cell division at the concentrations indicated. Murein synthesized in the presence of 2 micrograms/ml mecillinam also showed higher crosslinkage. However, about twice as much anhydromuramic acid-containing subunits were observed as in the control. At the same time lipopolysaccharide incorporation into the outer membrane was stimulated two- to three-fold.

Anterior and posterior cruciate ligament reconstruction in rhesus monkeys.

Although numerous procedures have been described for the reconstruction of the anterior and posterior cruciate ligaments, there has been little evaluation of the viability and strength of these substitutes. Using microangiographic, histological, and biomechanical techniques, we studied the vascularity and tensile strength of the medial one-third of the patellar tendon at intervals after it had been inserted as a substitute for either the anterior or the posterior cruciate ligament in twenty-nine young adult Rhesus monkeys. For the anterior cruciate reconstruction (nineteen knees), we used and medial one-third of the patellar tendon elongated by a portion of the patella. For the posterior cruciate reconstruction (ten knees), we used the medial one-third of the patellar tendon lengthened by attached portions of the patella and tibia as a free graft. Both the anterior and the posterior cruciate ligament substitutes were revascularized at eight weeks, and at one year they had approximately 80 per cent of the tensile strength that they had prior to transfer.

Evidence from a carbohydrate incorporation assay for direct activation of bone marrow myelopoietic precursor cells by bacterial cell wall constitutents.

The stimulation of incorporation of [3H]galactose into membrane glycoconjugates, measured in a precipitation test, was used as a criterion for activation of bone marrow cells. In this assay, purified bacterial lipopolysaccharide, lipoprotein, and murein monomer and dimer fragments all activated rat bone marrow cells in vitro. The response was dose dependent, followed a defined time course, and was not serum dependent. O-Acetylated murein dimer fragments from Proteus mirabilis were much less active than their unsubstituted counterparts, indicating a structural specificity for murein activation. Removal of adherent and phagocytizing cells from the marrow suspensions did not alter these results. The labeled, activated cells constituted a distinct population of buoyant density 1.064 to 1.069 g/cm3 when centrifuged on a continuous gradient of Percoll. Enrichment of the target cell population was achieved by a combination of adherent cell removal and discontinuous density gradient centrifugation to remove granulocytes and erythropoietic cells. It was concluded that a population of myelopoietic precursors could be activated by direct contact with bacterial cell wall constituents. The stimulation of galactose incorporation was not coupled to active deoxyribonucleic acid synthesis in the marrow cells. Thus, the activation was interpreted as an induction of differentiation rather than a mitotic event.

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis.

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.

Membranes of the protoplast L-form of Proteus mirabilis.

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysacchartide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. Considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of D-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypeptidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10). which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.

Molecular composition of the outer membrane of Escherichia coli and the importance of protein-lipopolysaccharide interactions.

Whole cells of Escherichia coli strains 0111, K12 and B as well as the ampicilln-resistant mutant K12 D21 and several lipopolysaccharide (LPS) mutants derived from this strain were analyzed for their molar LPS content per mg dry weight. An increase of the LPS concentration in some LPS mutants was substantiated by analyzing isolated cell walls and relating the molar LPS content to the murein subunit as measure of cell surface area. The increase of LPS was paralleled by increasing amounts of phospholipid while the overall protein content in the outer membrane decreased. According to the pattern of major outer membrane proteins in the various strains and the respective LPS structures, protein-LPS interactions are discussed as important requirements for outer membrane assembly and stability.

Identification of peptide-cross-linked trisdisaccharide peptide trimers in murein of Escherichia coli.

Purified murein from Escherichia coli K-12 was degraded into disaccharide peptide fragments by endo-N-acetylmuramidase from Chalaropsis. About 5% of the total murein fragments were recovered as peptide-cross-linked trisdisaccharide peptide trimers.