Investigation into the mechanism of bacterial adhesion to hydrogel-coated surfaces.

As a model for hydrogel-coated biomaterials, self-assembled monolayers of polyoxyethylene (POE) derivatives on sheets of polymeric biomaterials were prepared. The POE derivatives consisted of hydrophilic chains with different lengths and a long-chain alkyl group that served as an anchor function. The coatings obtained were analyzed with XPS and contact angle measurements showing hydrophilic chains of different lengths extending away from the surface. Bacterial adhesion was measured with a clinically relevant Klebsiella pneumoniae type strain and measurements reproduced 12 times. Bacterial adhesion decreased markedly with increasing hydrophilic chain length. Based upon these findings a new model for bacterial adhesion to hydrogel-coated surfaces is suggested: steric repulsion effects that increase with increasing chain length of grafted hydrophilic chains play an important role in bacterial adhesion to hydrogel-coated surfaces.

A new method of mechanical circulatory support with an implantable multichamber pump system (IMPS): presentation and first experimental results.

This paper presents a new cardiac support device for left ventricular failure which consists of two inflatable bellows positioned dorsally and ventrally to the left ventricle. The implantable multichamber pump system (IMPS) is driven by a pneumatic pump system and controlled by a microcomputer using ECG-trigger and pacemaker modules. It was implanted via thoracotomy in 8 pigs. The circulatory parameters were measured in the animals on beta-blockers, with cardiac failure and in ventricular fibrillation with an activated (IMPS on) and deactivated (IMPS off) system. IMPS significantly increased the left ventricular pressure (LVPsys IMPS off: 63 +/- 6 mmHg vs IMPS on: 96 +/- 8 mmHg) and the blood pressure in the common carotid artery (BPca, IMPS off: 69/38 mmHg vs IMPS on: 95/40 mmHg). The IMPS proved to be highly efficient in the therapy of animals with acute cardiac failure and in ventricular fibrillation in the experimental model. Apart from its efficiency the advantages with this system are the ease of handling and its high biocompatibility due to the lack of contact with circulating blood.

Solution NMR study of the structural basis of the Bohr effect in the monomeric hemoglobins from Chironomus thummi thummi.

The larva of the midge Chironomus thummi thummi possesses two monomeric hemoglobins. HbIII and HbIV, with extensive sequence homology, which exhibit marked but differential Bohr effects (pH influence on ligand affinity). These Hbs serve as ideal models for allosteric control of ligand affinity via tertiary-only structural changes. The cyanomet derivatives of these two Hbs have been shown to possess essentially indistinguishable heme cavity structures in solution at low pH (Zhang et al., 1996) that are also very similar to that of the low pH form of HbIII in the crystal (Steigemann & Weber, 1979). 2D 1H NMR has been utilized to elucidate the solution heme cavity structure of the alkaline form of the cyanomet derivatives of HbIII and HbIV to identify the Bohr proton binding site and characterize the nature of the structural changes that accompany the allosteric transition. Significant structural changes with pH have been identified in two regions of the heme cavity, near the axial His and at the junction of pyrroles B and C. The Bohr proton site is identified as His94, which at low pH makes a salt bridge to the terminal Met136. The rupture of this salt bridge at high pH leads to the expulsion of the Met136 side chain next to the His F8 ring where it serves as a spacer between the heme and F-helix, and leads to a cascade of side chain reorientations in the densely packed hydrophobic interior involving five Phe (65, 66, 128, 129, 133), Val132, and Ile69, all on the E- and H-helices. The terminal member of the cascade, Phe65, which acts as a spacer between the E- and F-helices at low pH, is rotated toward the heme plane. The conversion of the low pH, low-affinity "tense" to the high pH, high-affinity "relaxed" state is primarily due to the removal of the Met136 and Phe65 spacers. A central residue in transmitting the Bohr effect from His94 to Phe65 is residue 132. In HbIV, Val132 provides a cavity in the hydrophobic core to readily accommodate the initial step in rotating the Phe129 side chain. In HbIII, the Ile132 provides tight packing to all neighboring side chains and hence would inhibit the rotation of the Phe129 side chain. It is proposed that the lone internal residue difference between HbIII (Ile132) and HbIV (Val132) is the primary basis for the different amplitudes of their Bohr effect.

Solution 1H-NMR structure of the heme cavity in the low-affinity state for the allosteric monomeric cyano-met hemoglobins from Chironomus thummi thummi. Comparison to the crystal structure.

Solution 1H-NMR studies of the heme cavity were performed for the cyanomet complexes of monomeric hemoglobins III and IV from the insect Chironomus thummi thummi, each of which exhibit marked Bohr effects. The low pH 5, paramagnetic (S = 1/2) derivatives were selected for study because the large dipolar shifts provide improved resolution over diamagnetic forms and allow distinction between the two isomeric heme orientations [Peyton, D. H., La Mar, G. N. & Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82-94]. The crystal structure for the low-pH form of the hemoglobin III derivative, moreover, has been reported and showed that the functionally implicated distal His58 side chain adopts alternative orientation, either in or out of the pocket [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338]. All heme pocket residues for the low-pH forms of the two hemoglobins were located, at least in part, and positioned in the heme cavity on the basis of nuclear Overhauser effects to the heme and each other, dipolar shifts, and paramagnetic-induced relaxation. The resulting structure yielded the orientation of the major axis of the paramagnetic susceptibility tensor. The heme pocket structure of the cyanomet hemoglobins III and IV were found to be indistinguishable, with both exhibiting a distal His58 oriented solely into the heme cavity and in contact with the ligand, and with two residues, Phe100 and Phe38, exhibiting small but significant displacements in solution relative to hemoglobin III in the crystal.

NMR studies on water and polymer diffusion in dextran gels. Influence of potassium ions on microstructure formation and gelation mechanism.

At room temperature aqueous solutions of dextrans with concentrations > 25% (w/w) exhibit a sol-gel transition in the presence of > 1.0 M potassium chloride. In dextrans the gelation was unexpected due to missing anionic groups that usually provide the binding sites for cations. The quantitative investigation of the gel formation is based on changes of the diffusibility of water and dextran chains. The apparent diffusion coefficients of bulk water (in the order of 10(-6) cm2/s) and of water trapped in the junction zones as well as of polymer chains (in the order of 10(-7) to 10(-8) cm2/s) are determined by employing pulsed field gradient stimulated echo (PFGSTE) NMR. The restricted diffusion of bulk water in viscous sols and in soft and rigid gels has been quantitatively analyzed providing data for interbarrier distances (pore size), permeabilities of the diffusion barriers (density of junction zones) and interbarrier diffusion coefficients of water. Based on already published x-ray structure data and in accordance with the diffusion data presented in this paper "potassium-bonding" is assumed to be the most important interaction for the formation of a microstructure and for the stabilization of cross-links. The ionic radius of the potassium ion perfectly fits to the cage established by six oxygen atoms of glucose units of three polymer chains. Other cations, such as Li+, Na+, Rb+ and Cs+, according to their nonfitting ionic radii, do not provoke dextran gelation under these conditions. The mechanism of the transitions from sol to soft gel and further to rigid gel is discussed on the basis of restricted diffusion and x-ray structure data.

The concept of "direct mechanical ventricular assistance" in the treatment of left-ventricular failure. Part 2. Results of the new intrathoracic implantable multi-chamber pump system (IMPS) in an animal study.

An assist device was developed which is able to support the pumping function of the heart by direct application of pressure to the left ventricle. The goal of this animal study in pigs was to determine whether it is possible to maintain sufficient blood circulation with the aid of the new system when the heart is fibrillating or its capacity has been greatly reduced. Following sternotomy complete invasive monitoring was installed. The intrathoracic implantable mechanical multi-chamber pump system (IMPS) was placed around the left ventricle. By means of the beta-blocker carazolol, systolic left-ventricular pressure (LVPsys), cardiac output, heart rate, and left-ventricular dp/dtmax (LVdp/dtmax) were gradually lowered and the pump system was tested intermittently. Then the heart was fibrillated and the system was tested again. When cardiac output, LVdp/dtmax, and systolic blood pressure were reduced by approximately 50% IMPS was able to increase LVPsys by 83% (IMPS) on: 96 +/- 9 mmHg vs. IMPS off: 63 +/- 6 mmHg), and the blood pressure in the carotid artery by 86% (IMPS on: 95/40 +/-15 mmHg vs. IMPS off: 69/38 +/- 9 mmHg). The mean cardiac output was 64% (IMPS on: 4.3 L/min vs. IMPS off: 3.9L/min); in most cases a great variability could be observed depending on the preload, the heart rate, and the mode of pressure application. When the heart was fibrillating, IMPS was able to maintain adequate circulatory conditions with LVPsys = 88%, blood pressure in the carotid artery = 85%, and LVdp/dtmax = 57% of the control values measured before fibrillation and beta-blockade. The system presented here is able to support the impaired left ventricle and to replace its pumping function. The advantages of the system are its efficiency and the lack of contact of the circulating blood with foreign surfaces. Whether the system is suited for bridging and recovery support shall be clarified in further studies.

Principles of multimodal imaging.

Current medical practice deals with a variety of multimodal information (X-ray film, ultrasound, CT, MR, ECG and EEG, laboratory results, medical records, etc.) Diagnosis and treatment demand an integrated view of this information including the patient's record and history. This paper describes multimodal imaging approaches to such a system with regard to (i) user interface, (ii) data management (including access control), (iii) registration and modality matching based on reference models, and (iv) interface to the modalities.

The concept of "direct mechanical ventricular assistance" in the treatment of left-ventricular failure. Part 1: Idea, development and construction of an implantable multi-chamber pump system partially surrounding the heart.

In this paper we present a therapeutic concept for the treatment of heart failure due to muscular inability to pump properly. The basic principle of this concept triggered numerous studies, then with the aim of cardiopulmonary resuscitation, back in the 1970s. In general, it dealt with mechanical systems which led to an increase in stroke volume, systolic blood pressure, and cardiac output through the application of pressure directly to the left ventricle. After a critical appraisal of the relevant literature from technical, physical, and medical viewpoints, and our own preliminary studies on animal hearts within the framework of mock circulation experiments, we have conceived a new functional principle for direct mechanical ventricular assistance based on squeezing the left ventricle only. We have begun development of the system which has the advantages of ease of use, high biocompatibility due to lack of contact between blood and system components, the prevention of infection through complete intrathoracic implantation (long-range goal), and the fact that the patient's own heart can be supported by the system without being removed from the circulatory system (support of the residual myocardial function). Technical as well as medical prerequisites are indicated, and the materials selection and construction principles of the control, pressure generation, pressure transduction, and ventricular compression unit are described. It has proved possible to construct a prototype system to be used in animal experiments, which, through pneumatic inflation of a chamber system partially surrounding the left ventricle, should be able to augment or take over the pumping function of the left ventricle.

Multiexponential proton relaxation processes of compartmentalized water in gels.

The proton relaxation times, T1 and T2, of water in Sephadex gels, exhibiting pores of varying size (i.e., with exclusion limits of molecular weight between 10(3) and 10(5)) and water contents in the range 30 to 70% (w/w, weight of water to total weight), were measured at 20 MHz in the temperature range 5 to 50 degrees C. Multiexponential analysis of the relaxation curves revealed the existence of two relaxation components in all gel systems. A component with long T1 and T2 (T1,1 and T2,1) is associated with a large water fraction alpha 1,1 and alpha 2,1 and a component with short T1 and T2 (T1,2 and T2,2) with a small water fraction alpha 1,2 and alpha 2,2. An analysis of the temperature behavior of the relaxation components gives insight into the relaxation mechanisms. The relaxation process in water, compartmentalized in the gel matrix, is mainly controlled by dipole-dipole interactions. In addition, proton exchange processes between hydration water and hydroxyl groups of the matrix chain contribute under specific conditions and lead to a dramatic enhancement of the relaxation rate. In particular, for gels with small pores and with low water content proton exchange is observed. Compartments of water in gels could be models for compartments of water in biological tissues.

MR microimaging of articular cartilage and contrast enhancement by manganese ions.

Saline solutions of manganese ions (Mn2+) were used as articular contrast agents in magnetic resonance microscopy (9.4 T) of cartilage of chicken femoral condyles and pig temporomandibular joints. The diffusion of Mn2+ from the articular surface into the cartilage matrix led to a strong contrast enhancement in the cartilage. The combination of the high spatial resolution and the contrast enhancement allowed the visualization of fine structures (tissue types) in the cartilage, which correlate with the tissue zones in histological sections stained with cationic dyes. We assume that the electrostatic interactions between the negatively charged groups of the proteoglycans and Mn2+ are most important for the mechanism of contrast enhancement. Hence, the different signal intensities of the various zones of cartilage indicate differences in density of proteoglycans. The intraarticular injection of the cationic contrast agent could improve the possibility of an early diagnosis of cartilage dysfunction and degeneration.

T1 rho dispersion imaging and localized T1 rho dispersion relaxometry: application in vivo to mouse adenocarcinoma.

The dispersion (frequency dependence) of the spin-lattice relaxation time in the rotating frame, T1 rho, is considered for tissue characterization. Methods for the volume-selective determination of the proper T1 rho dispersion and for imaging of parameters characterizing this frequency dependence are described. On- and off-resonance versions of the techniques are demonstrated. In vitro studies of excised rat tissues and in vivo applications to mice with implanted adenocarcinoma are reported. T1 rho dispersion images show clear contrasts of the malignant tissue, whereas muscle tissue is completely suppressed. No contrast agent is required. The measuring time is only twice as long as that for conventional magnetic resonance images. The results suggest that the T1 rho dispersion is less susceptible to the biological variability than the absolute values of the relaxation times.

Proton nuclear magnetic resonance study of the solution distal histidine orientation in monomeric Chironomus thummi thummi cyanomet hemoglobins. Dynamic stability of the heme pocket as monitored by labile proton exchange.

The 1H nuclear magnetic resonance spectral characteristics of the cyano-Met form of Chironomus thummi thummi monomeric hemoglobins I, III and IV in 1H2O solvent are reported. A set of four exchangeable hyperfine-shifted resonances is found for each of the two heme-insertion isomers in the hyperfine-shifted region downfield of ten parts per million. An analysis of relaxation, exchange rates and nuclear Overhauser effects leads to assignments for all these resonances to histidine F8 and the side-chains of histidine E7 and arginine FG3. It is evident that in aqueous solution, the side-chain from histidine E7 does not occupy two orientations, as found for the solid state, rather the histidine E7 side-chain adopts a conformation similar to that of sperm whale myoglobin or hemoglobin A, oriented into the heme pocket and in contact with the bound ligand. Evidence is presented to show that the allosteric transition in the Chironomus thummi thummi hemoglobins arises from the "trans effect". An analysis of the exchange with bulk solvent of the assigned histidine E7 labile proton confirms that the group is completely buried within the heme pocket in a manner similar to that found for sperm whale cyano-Met myoglobin, and that the transient exposure to solvent is no more likely than in mammalian myoglobins with the "normal" distal histidine orientation. Finally, a comparison of solvent access to the heme pocket of the three monomeric C. thummi thummi hemoglobins, as measured from proton exchange rates of heme pocket protons, is made and correlated to binding studies with the diffusible small molecules such as O2.

[Error analysis, biological modification factors and variance of peri-ungual video-capillary microscopy]. / Fehleranalyse, biologische Einflussfaktoren und Varianz der periungualen Videokapillarmikroskopie.

Error analysis or the evaluation of the precision of microscopic measurements must distinguish between technical errors inherent in the measuring system, and biological variability. The technical error inherent in the overall system in the case of linear measurements is smaller than the lengths to be measured by 1 or 2 orders of magnitude. Thus, the erythrocyte column lengths can be correctly, reproducibly and linearly quantified over the entire measuring range. The biological influencing factors can largely be taken into account or excluded by suitable standardisation of the measuring process. Despite a considerable individual fluctuation in the erythrocyte velocity, there is, on average, no significant dependence of the measuring parameter within the daily profile or from day to day. Differences in capillary perfusion in the presence of diseases associated with microcirculatory disorders or a therapeutic influence on erythrocyte velocity can thus be reliably demonstrated.

[NMR-microscopic studies of the condylar cartilage of the temporomandibular joint]. / NMR-mikroskopische Untersuchungen am kondylären Knorpel des Kiefergelenks.

The objectifying of growth mechanisms of the TMJ is very important for orthodontic diagnosis, treatment as well as clinical research. Condylar cartilage of the TMJ from neonatal pigs was investigated on in vivo preparations by NMR-microscopy. NMR-micro-pictures and histologic slide preparations of condylar cartilage were compared and discussed in connection with its implications for treatment with functional appliances.

Formation of free radicals and nitric oxide derivative of hemoglobin in rats during shock syndrome.

Free radicals have been postulated to play an important role as mediators in the pathogenesis of shock syndrome and multiple-organ failure. We attempted to directly detect the increased formation of radicals by Electron Spin Resonance (ESR) in animal models of shock, namely the endotoxin (ETX) shock or the hemorrhagic shock of the rat. In freeze-clamped lung tissue, a small but significant increase of a free radical signal was detected after ETX application. In the blood of rats under ETX shock, a significant ESR signal with a triplet hyperfine structure was observed. The latter ESR signal evolved within several hours after the application of ETX and was localized in the red blood cells. This signal was assigned to a nitric oxide (NO) adduct of hemoglobin with the tentative structure [alpha 2+ NO)beta 3+)2. The amount of hemoglobin-NO formed, up to 0.8% of total hemoglobin, indicated that under ETX shock a considerable amount of NO was produced in the vascular system. This NO production was strongly inhibited by the arginine analog NG-monomethyl-arginine (NMMA). The ESR signal of Hb-NO was also observed after severe hemorrhagic shock. There are three questions, namely (i) the type of vascular cells and the regulation of the process forming such a large amount of NO during ETX shock, (ii) the pathophysiological implications of the formed NO, effects which have been described as cytotoxic mediator, endothelium-derived relaxing factor (EDRF) or inhibitor of platelet aggregation, and (iii) the possible use of Hb-NO for monitoring phases of shock syndrome.

Resonance Raman enhancement of the Mn-N-O bending mode in nitrosyl manganese "strapped" and "open" heme complexes.

Resonance Raman spectra of the MnII-NO moiety in synthetic nitrosyl manganese heme complexes with and without steric hindrance are reported. The "strapped" hemes having a hydrocarbon strap (variable length) across one face of the heme hinder the perpendicular bonding of a linear ligand. These complexes were employed to investigate the effects of ligand distortion (primarily tilting) on Mn-NO stretching, Mn-N-O bending, and N-O stretching modes. It is demonstrated that ligand distortion in the MnII-NO system is a valid mechanism for causing the resonance enhancement of the Mn-N-O bending mode, similar to that observed in the FeII-CO system (Yu, N.-T., E. A. Kerr, B. Ward, and C. K. Chang. 1983. Biochemistry. 22:4534-4540). More interesting is the observation of the delta(Mn-N-O) enhancement caused by the tilting of the trans Mn-N epsilon bond in the "open" heme complexes (e.g., heme-5 and proto-1X dimethylester) with 1,2-dimethylimidazole or piperidine as a base. The nu(Mn-NO) and nu(N-O) modes exhibit an increase and a decrease, respectively, as the strap length decreases (hence the steric hindrance increases). Both nu(Mn-NO) and nu(N-O) frequencies are insensitive to the strength of the trans base. The results from "strapped" and "open" model heme systems imply that the Mn-N-O geometry is essentially linear and perpendicular in the nitrosyl complexes of monomeric manganese insect hemoglobin CTT IV and sperm whale myoglobin. The unusually low nu(N-O) frequency in the manganese myoglobin complex may be caused by the distal histidine-NO interaction. The delta(Mn-N-O) enhancement in both nitrosyl manganese CTT IV and nitrosyl manganese myoglobin may be caused by a tilting of the Mn"-Nf (proximal histidine) bond.

Resonance Raman assignment and evidence for noncoupling of individual 2- and 4-vinyl vibrational modes in a monomeric cyanomethemoglobin.

We have investigated the resonance Raman spectra of monomeric insect cyanomethemoglobins (CTT III and CTT IV) reconstituted with (1) protohemes IX selectively deuterated at the 4-vinyl as well as the 2,4-divinyls, (2) monovinyl-truncated hemes such as pemptoheme (2-hydrogen, 4-vinyl) and isopemptoheme (2-vinyl, 4-hydrogen), (3) symmetric hemes such as protoheme III (with 2- and 3-vinyls) and protoheme XIII (with 1- and 4-vinyls), and (4) hemes without 2- and 4-vinyls such as mesoheme IX, deuteroheme IX, 2,4-dimethyldeuteroheme IX, and 2,4-dibromodeuteroheme IX. Evidence is presented that the highly localized vinyl C = C stretching vibrations at the 2- and 4-positions of the heme in these cyanomet CTT hemoglobins are noncoupled and inequivalent; i.e., the 1631- and 1624-cm-1 lines have been assigned to 2-vinyl and 4-vinyl, respectively. The elimination of the 2-vinyl (in pemptoheme) or the 4-vinyl (in isopemptoheme) does not affect the C = C stretching frequency of the remaining vinyl. Furthermore, two low-frequency vinyl bending modes at 412 and 591 cm-1 exhibit greatly different resonance Raman intensities between 2-vinyl and 4-vinyl. The observed intensity at 412 cm-1 is primarily derived from 4-vinyl, whereas the 591-cm-1 line results exclusively from the 2-vinyl. Again, there is no significant coupling between 2-vinyl and 4-vinyl for these two bending modes.

Effect of cadmium ions on dioxygen affinity and polyphosphate activity of human red blood cells.

The effect of cadmium ions on the dioxygen affinity, the time-dependent depletion of intracellular polyphosphates, and the elongation of human red blood cells (RBC's) was examined. The incubation of RBC's in the presence of 1 mM Cd2+ at 37 degrees C for more than one hour results in a decrease of the p50 value by 2.5-3.0 mmHg in comparison to controls. The p50 of stripped (phosphate-free) hemoglobin is not affected by the presence of 1 mM Cd2+ (p50 = 4.8 mmHg at pH 7.2 and 37 degrees C). Experiments with RBC cryolysates demonstrate an apparently competitive effect of 2.3-bisphosphoglycerate (DPG) with cadmium ions on the dioxygen affinity. From 31P NMR spectra, 31P T1 relaxation, and 31P T2 relaxation behavior a more direct evidence for DPG-Cd2+ complexation is obtained. 31P NMR spectra of RBC cryolysates also indicate DPG-Cd2+ complexation. The hydrolysis of free polyphosphates in RBC's incubated at 37 degrees C as monitored by 31P NMR spectra can be noticed after a three-hour lag phase (constant polyphosphate level). This lag phase is lengthened from three hours to four hours in the presence of Cd2+ ions. RBC elongation, as a measure of deformability, decreases slightly upon incubation with 1 mM Cd2+.