Antimicrobial nano-assemblies of tryptocidine C, a tryptophan-rich cyclic decapeptide, from ethanolic solutions.

Tryptocidine C (TpcC), a Trp-rich cyclodecapeptide is a minor constituent in the antibiotic tyrothricin complex from Brevibacillus parabrevis. TpcC possesses a high tendency to oligomerise in aqueous solutions and dried TpcC forms distinct self-assembled nanoparticles. High-resolution scanning electron microscopy revealed the influence of different ethanol:water solvent systems on TpcC self-assembly, with the TpcC, dried from a high concentration in 15% ethanol, primarily assembling into small nanospheres with 24.3 nm diameter and 0.05 polydispersity. TpcC at 16 µM, near its CMC, formed a variety of structures such as small nanospheres, large dense nanospheroids and facetted 3-D-crystals, as well as sheets and coarse carpet-like structures which depended on ethanol concentration. Drying 16 µM TpcC from 75% ethanol resulted in highly facetted 3-D crystals, as well as small nanospheres, while those in 10% ethanol preparation had less defined facets. Drying from 20 to 50% ethanol led to polymorphic architectures with a few defined nanospheroids and various small nanoparticles, imbedded in carpet- and sheet-like structures. These polymorphic surface morphologies correlated with maintenance of fluorescence properties and the surface-derived antibacterial activity against Staphylococcus aureus over time, while there was a significant change in fluorescence and loss in activity in the 10% and 75% preparations where 3-D crystals were observed. This indicated that TpcC oligomerisation in solutions with 20-50% ethanol leads to metastable structures with a high propensity for release of antimicrobial moieties, while those leading to crystallisation limit active moieties release. TpcC nano-assemblies can find application in antimicrobial coatings, surface disinfectants, food packaging and wound healing materials.

Structures of the 2-nitrophenol alkali complexes in solution and the solid state.

The materials studied in this investigation were aqueous solutions (0.02-25.0 mM) of the salts of alkali metal ion (Me(+)) and 2-nitrophenol (2-NP). In the investigation, small-angle X-ray scattering, wide-angle X-ray scattering, and membrane-pressure osmometry were used to study the 2-NP-Me(+) molecular salt structures and the onset of crystallization as a function of concentration and temperature. The experimental methods used to examine the 2-NP-Me(+) molecular salt complexes provided corroborative evidence for the existence of spherical clusters with hydrodynamic diameters between ∼12 Å (Li) and 14 Å (Cs). Guinier plots of the zero-angle scattering peak were characteristic of the scattering from lamellae-like shapes with thicknesses of ∼290 Å. Tetramer and pentamer 2-NP-Me(+) molecular clusters for Me(+) = Li, Na, K, and Rb were assembled from four or five 2-NP molecules bound to a central alkali metal ion. The coordination symmetry around the six coordinated Li(+), Na(+), and K(+) ions was that of a trigonal prism (D3h), with an octahedral arrangement (D2h). The Rb(+) also revealed six-coordinate geometry and the central Rb(+) ion adopted an octahedral arrangement (D2h). The eight-coordinated Cs(+) ions with six 2-NP ligands were characteristic of a square antiprism (D4d). The square antiprism was the outcome of leaving two o-nitro groups and two phenolic oxygens being left intermolecularly uncoordinated to the Cs(+) ion. The 2-NP residues were strictly planar and contained short non-bonded intramolecular distances. van der Waals forces were present between the adjacently stacked phenyl rings. No water molecules were involved as ligands for any of the 2-nitrophenol-Me(+) complexes.

Two dimensional crystallization of three solid lipid A-diphosphate phases.

Surface-tension-induced liquid-crystal growth of monomeric lipid A-diphosphate in aqueous dispersions is reported as a function of concentration, (c), and temperature, (T), and at low ionic strength (10(-3) M). As the temperature was varied, a solid-liquid transition was revealed in the surface layer at a fixed lipid A-diphosphate bulk concentration. Here, the development of different two-dimensional (2-d) faceted crystal morphologies was observed and, as growth proceeded, these faceted 2-d crystals became unstable. Selected area electron microscopy diffraction (SAED) and X-ray diffraction (XRD) measurements of the faceted 2-d crystalline lipid A-diphosphate layers exhibited a pseudohexagonal molecular arrangement. The crystalline layer was a smectic F, S(F), phase below the critical temperature, T(C), and a smectic I, S(I), phase above T(C) (15 °C). Both phases could be described in terms of the same C-centered monoclinic unit cell. The in-plane order extended for a limited distance although the layers were coupled. The analysis of the SAED patterns revealed short-range order in the S(F) phase (5-15 °C), but long-range order in the S(I) phase, for the temperature range 15-30 °C. The observed 2-d solid hexatic phase and the 2-d liquid hexatic phase had correlation lengths of 220 Å. This, the hexatic phase, displayed short-range in-plane positional order and quasi long-range, sixfold bond-orientational order. The S(I) phase showed long-range order characteristics of a hexatic 2-d crystal. The two-, four-, or six-layer crystalline lipid A-diphosphate films exhibited 2-d hexatic order and 6n-fold bond-orientational order. These films did not evolve into the S(F) phase, demonstrating that the two phases were thermodynamically distinct. A finite tilt angle of φ = 15° was calculated for the lipid A-diphosphate molecule; the tilt was toward the small side of the rectangular 2-d lattice. The constraint of six close-packed acyl chains in two distinct phases with the same symmetry suggests that the S(F) → S(I) transition was first-order.

Phase transformations in lipid A-diphosphate initiated by sodium hydroxide.

The nature of the fluid phase transitions of charged-stabilized spherical lipid A-diphosphate clusters in aqueous dispersions was explored using a combination of small-angle X-ray scattering (SAXS) and electron microscopy. In contrast to previous studies, rather than removing NaCl, NaOH was added to the dispersions to promote crystallization. The fluid phase experiments were carried out employing titrations with mM·L(-1) NaOH (c(S)), along with variations in the particle-number density, n. When c(S) was increased, a new fluid disordered phase of self-assembled lipid A-diphosphate was encountered, followed by a crystalline bcc phase and then a new fluid phase containing 70 nm lipid A-diphosphate particles. The bcc crystal structure found in this regime had a lattice constant of 35.6 nm. By varying c(S) (mM L(-1)), it was possible to determine the effective charge, z(eff), for various n values and the screening parameter, k, for the excess electrolyte. For sufficiently large values of n, lipid A-diphosphate crystallized because of an increase in z(eff) at a constant c(S). When the c(S) was increased, the crystals melted with little change in z(eff). The existence of a bcc-fluid phase transition for different values of c(S) was supported by applying the Debye correlation function to the obtained data. An increase in c(S) enhanced interparticle interaction and attraction. The effective charge and k accounted for counterion condensation and many-body effects. If the effective charge determined from scattering measurements was used in the simulations, the equilibrium phase boundaries were consistent with predicted universal melting-line simulations. At any particle-number densities, n, when the melting line was reached, 70 nm clusters were formed.

Studies on structures of lipid A-monophosphate clusters.

Single crystalline clusters of lipid A-monophosphate were grown from organic dispersions containing 5-15% (v/v) water at various volume fractions, φ, and temperatures. The morphology of the single lipid A-monophosphate crystals was either rhombohedral or hexagonal. The hexagonal crystals were needlelike or cylindrical in shape, with the long dimension parallel to the c axis of the unit cell. The crystalline clusters were studied using electron microscopy and x-ray powder diffraction. Employing molecular location methods following a Rietveld refinement and whole-pattern refinement revealed two monoclinic crystal structures in the space groups P2(1) and C2, both converged with R(F) = 0.179. The two monoclinic crystal structures were packing (hydrocarbon chains) and conformational (sugar) polymorphs. Neither of these two structures had been encountered previously. Only intramolecular hydrogen bonding was observed for the polymorphs, which were located between the amide and the carboxyl groups. Another crystalline structure was found in the volume-fraction range 2.00 × 10(-3) ≤ φ ≤ 2.50 × 10(-3), which displayed hexagonal symmetry. The hexagonal symmetry of the self-assembled lipid A-monophosphate crystalline phase might be reconciled with the monoclinic symmetry found at low-volume-fractions. Therefore, lowering the symmetry from cubic, i.e., Ia 3d, to rhombohedral R 3 m, and finally to the monoclinic space group C2 was acceptable if the lipid A-monophosphate anion was completely orientationally ordered.

Diabetogenic glucose and insulin concentrations modulate transcriptome and protein levels involved in tumour cell migration, adhesion and proliferation.

BACKGROUND: During the last decade, epidemiological studies uncovered the tremendous impact of metabolic syndrome/diabetes mellitus type 2 (DM T2) as risk factors of the progression of cancer. Therefore, we studied the impact of diabetogenic glucose and insulin concentrations on the activities of tumour cells, because little is known about how high glucose and insulin levels are influencing gene activities causing changes in the signal cascade activities with respect to kinases involved in the proliferation and migration of cancer cells. METHODS: To address this question we analysed the activity of more than 400 gene signatures related to (i) cell cycle, (ii) cell movement as well as (iii) signal transduction. We examined transcriptomes of kinases (PKCα, PI3K), cadherins (E-, N- VE-), integrins and cyclins by comparing physiological (5.5 mM) vs diabetogenic (11 mM) glucose concentrations (without and with insulin). RESULTS: Proliferation assays revealed that high levels of glucose (11 mM) and insulin (100 ng ml(-1)) did promote the proliferation of the tumour cell lines HT29, SW480, MCF-7, MDA MB468, PC3 and T24. Using a 3D-migration assay, we have shown that high glucose concentrations caused increased motility rates of the tumour cells. The increase in migratory activity at high glucose and insulin concentrations was mediated by an activation of PI3K, PKCα and MLCK, as figured out by the pharmacological inhibitors wortmannin, Go6976 and ML-7. CONCLUSION: We present molecular and functional data, which could help to understand how hyperglycaemia and hyperinsulinemia might trigger tumour cell proliferation and motility in patients, too.

14N and 81Br quadrupolar nuclei as sensitive NMR Probes of n-alkyltrimethylammonium bromide crystal structures. An experimental and theoretical study.

This is the first time a comprehensive study has been carried out on n-alkyltrimethylammonium bromide salts using (14)N and (81)Br solid state NMR, X-ray diffraction, and theoretical calculations. The investigation represents a necessary step toward further (14)N and (81)Br NMR characterization of the environment of cationic and anionic groups in materials, accounting for the amphiphilic properties of cationic surfactants. The NMR spectra of five C(x)H(2x+1)(CH(3))(3)N(+)Br(-) polycrystalline samples with different n-alkyl chain lengths (x = 1, 12, 14, 16, 18) were recorded and modeled. The (14)N and (81)Br quadrupolar coupling interaction parameters (C(Q), eta(Q)) were also estimated from spectrum modeling and from computer simulation. The obtained results were discussed in depth making use of the experimental and reoptimized crystal structures. In the study, both (14)N and (81)Br nuclei were found to be sensitive probes for small structural variations. The parameters which influence the NMR properties the most are mobility, deviation of C-N-C bond angles from T(d) angles, and variations in r(N-Br) distances.

Two new colloidal crystal phases of lipid A-monophosphate: order-to-order transition in colloidal crystals.

A study of the structure of stable regular-shaped nanocrystals of hexa-acylated (C(14)) lipid A-monophosphate from Escherichia coli was carried out using dilute electrostatically stabilized aqueous dispersions at low ionic strength (I=1.0x10(-5)M NaCl). An order-to-order transition of colloidal clusters of lipid A-monophosphate was found at two volume fractions: phi=5.9x10(-4) and phi=11.5x10(-4). The clusters belonged to the cubic space groups Pm3n and Ia3d with unit-cell dimensions of a=4.55 nm and a=6.35 nm, respectively, as revealed by small-angle x-ray diffraction and electron-diffraction results of thin nanocrystals of lipid A-monophosphate. When viewed in the scanning electron microscope these fragile clusters displayed a number of shapes: cubic, cylindrical, and sometimes-rounded hexagons, which were extremely sensitive when exposed to an electron beam. The smallest and most numerous of the clusters appeared as approximately 7 nm cubes. Crystalline cluster formation occurred over a wide volume-fraction range, between 1.5x10(-4) and 40.0x10(-4), and at temperatures of 20 and 35 degrees C. The crystalline networks of the lipid A-monophosphate clusters may be represented by space-filling models of two pentagonal dodecahedra with six tetrakaidecahedra arrangements of lipid A-"micelles" in the cubic space group Pm3n. The simulated electron density profiles are in accord with spherical clusters of lipid A-monophosphate at the corners and at the body centers of the cubic Pm3n unit cell. The profiles are rounded tetrahedrally at distances of 1/4 and 3/4 along one of the bisectors of each face of the cubic unit cell. These nanocrystalline systems provide examples of "cellular" crystalline networks, which rearrange themselves spontaneously into three-dimensional polyhedral structures. It appears that a closely related analogy exists between the tetrahedrally close-packed networks as revealed for the lipid A-mono- and diphosphates [C. A. Faunce, H. Reichelt, H. H. Paradies, et al., J. Chem. Phys. 122, 214727 (2005); C. A. Faunce, H. Reichelt, P. Quitschau, et al., J. Chem. Phys. 127, 115103 (2007)]. However, the cubic Ia3d phase consists of two three-dimensional networks of rods, mutually intertwined but not connected. For this cubic Ia3d phase each junction involves three coplanar rods at an angle of 120 degrees, showing an interwoven labyrinth of lipid A-monophosphate rods which are connected three by three. The rod diameter is approximately 2.2 nm, which is similar in diameter to the disk-shaped aliphatic chiral core of lipid A-monophosphate (2.14 nm) with an ellipticity of 0.62 seen for the "c" position of the tetrakaidecahedra in the Pm3n cubic unit cell. An epitaxial relationship appears to exist between the {211} planes of the cubic Ia3d phase and the (001) planes of the lamellar phase as well as with the {10} planes of the hexagonal phase. The transformation of the cubic into the hexagonal phase can be reconciled by the growth of a cylinderlike assembly of lipid A-monophosphate molecules of the hexagonal phase parallel to the 111 directions of the cubic Ia3d phase. Upon cooling from 35 to 20 degrees C the cubic Ia3d lipid A-monophosphate phase unexpectedly transforms and gives rise to an intermediate R3m structure (a=3.90+/-0.12 nm, c=7.82+/-0.05 nm, and gamma=120 degrees). Both cubic Ia3d and hexagonal R3m phases originate from similar rodlike units of lipid A-monophosphate clusters. However, the overall shapes of the assemblies are different because of their spatial distribution. Both assemblies morphologically bridge the lipid A-monophosphate hexagonal and and lamellar phases. The structural path followed during the phase transitions is governed by topological similarities between the phase which forms and the one from which it originates. Although the two phases, Ia3d and R3m, have similar curvature energies on cooling, the topology is more than likely to be the initial factor determining the overall phase transition path.

Nucleation of calcium carbonate as polymorphic crystals in the presence of lipid A-diphosphate.

The well-defined structure of lipid A-diphosphate in aqueous solutions provides a way of observing the formation of calcium carbonate crystals. The crystals are either tetrahedral or rhombohedral calcite at a volume fraction of phi = 5.4 x 10 (-4) at pH 5.8 or the vaterite polymorph of CaCO(3) at a volume fraction of phi = 7.8 x 10 (-4) at pH 5.8. In both cases, nucleation, adsorption pH, and the shape-dependent template of lipid A-diphosphate control the formation of the calcite and vaterite.

The phase diagram of charged colloidal lipid A-diphosphate dispersions.

Small-angle X-ray-scattering, light-scattering, and electron microscope experiments were used to determine the phase transitions of colloidal lipid A-diphosphate aqueous dispersions. The phases detected were a correlated liquid phase, a face-centered cubic (Fd3m) and a body-centered cubic (Im3m) colloidal crystal phase and a new glass phase. These experimentally determined phases were shown to be in accord with theoretically predicted equilibrium phases.

Observations of liquidlike order of charged rodlike lipid A diphosphate assemblies at pH 8.5.

A new structural form of charged lipid A diphosphate, with a molecular weight of 5.9x10(6) Da and a rodlike shape (L=800 nm), was found in aqueous solutions at pH 8.5. The experimental techniques used in the investigation were light scattering, small-angle x-ray scattering (SAXS), and electron microscopy. Measurements of the static-structure factor S(Q) as a function of the ionic strength are presented over the concentration regimes C>C(*) and C or =C(*) show that strong interparticle correlations exist even at concentrations as high as 15C(*), in contrast with results for hard-rod systems. The magnitude of the correlations depends on both the lipid A diphosphate concentration at pH 8.5 and the Debye screening length k(-1). For a constant lipid A diphosphate concentration at pH 8.5, as the amount of salt was increased a decrease in structure was observed. There was also a shift in the peak of the first maximum position Q(max) to larger scattering wave vectors. The observed phase behavior (C=15C(*)) exhibited an isotropic I-Sm transition and an I-N-Sm transition, which were recorded on electron microscope images.

Ordering of lipid A-monophosphate clusters in aqueous solutions.

In this investigation, a study of the self-assembly of electrostatically stabilized aqueous dispersions of nanometric lipid A-monophosphate clusters from Escherichia coli was carried out in three different volume-fraction regimes. The experimental techniques used in the investigation were osmotic pressure, static and quasielastic light scattering, scanning electron microscopy and transmission electron microscopy, and small-angle x-ray scattering. Experiments were carried out at low ionic strength (I=0.1-5.0 mM NaCl) at 25 degrees C. At volume fractions between 1.5x10(-4)

Elusive forms and structures of N-hydroxyphthalimide: the colorless and yellow crystal forms of N-hydroxyphthalimide.

A comprehensive structural characterization of the colorless and yellow forms of N-hydroxyphthalimide (NHP), the deuterated form (NDP), and the ethoxylated form (ethoxy-NHP) has been carried out using single-crystal X-ray diffraction, FTIR and Raman spectroscopies, and scanning electron microscopy. Both NHP and NDP forms crystallize in the monoclinic space group (P21/c, No. 14). The various forms of NHP differ in the way in which the molecules adjoin one another through their N-hydroxyl groups and how the carbonyls of the isoindole-1,3-dione ring differ through intermolecular hydrogen bonding. Although the hydrogen bonding about the b axis is virtually the same, the isoindole-1,3-dione ring experiences different twists for the two NHP forms. Both the colorless and yellow forms of NHP exhibit strong intermolecular hydrogen bonding between O(3) and H(1). In the yellow form, the N-hydroxyl group is significantly out of the plane (approximately 1.19 degrees ), but the N-hydroxyl group in the colorless form is only approximately 0.06 degrees out of the plane. Both forms of NHP reveal an infinite chain of intermolecular hydrogen-bonded molecules in the direction of the b axis; however, the molecules are ordered differently within the unit cells. The hydrogen-bond geometry for the yellow form of NHP is O(2)-H(1)...O(3), with an angle of 185 degrees , intermolecular distances of O(2)...O(3) = 2.68 A and H(1)...O(3) = 1.70 A, and an intramolecular hydrogen bond of O(1)...H(1) = 1.17 A. The colorless form of NHP shows an intermolecular hydrogen-bond geometry between O(3) and H(1) with a distance of 1.78 A; the O(2)-O(3) distance is 2.71 A. The O(2)-H(1)...O(3) angle is 159 degrees, and the intramolecular distance is O(1)...H(1) = 0.97 A. The N-ethoxy derivative of NHP crystallizes in an orthorhombic space group (Pnma, No. 62) and exhibits no hydrogen bonding, displaying a strong head-to-tail stacking of the planar rings along the needle axis direction.

Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur-oxidizing system in Paracoccus pantotrophus GB17.

The novel genes soxFGH were identified, completing the sox gene cluster of Paracoccus pantotrophus coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction. soxF coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein FccB of flavocytochrome c sulfide dehydrogenase of Allochromatium vinosum. The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol. soxG coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif. The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer. soxH coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide. The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer. SoxXA, SoxYZ, SoxB, and SoxCD (C. G. Friedrich, A. Quentmeier, F. Bardischewsky, D. Rother, R. Kraft, S. Kostka, and H. Prinz, J. Bacteriol. 182:4476-4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome c reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in soxF and complementation analysis, it was evident that the soxFGH gene products were not required for lithotrophic growth with thiosulfate.

[Restoration of blood circulation in reperfusion of ischemic tissues]. / Vidnovlennia krovobihu pry reperfusiï ishemizovanykh tkanyn.

In experiment there were studied changes of the tissue blood circulation and microcirculation during postischemic reperfusion of skeletal muscles with various density of capillaries. It was established that reactive hyperemia, caused mainly by enhancement of extracapillary tissue blood flow. While persistence of durable arterial hypertension the capillaries quantity and potential volume of microcirculatory bed are reducing, negatively influencing the blood circulation restoration process during postischemic reperfusion. In chronic ischemia the potential volume of microcirculation is increasing, securing complete restoration of blood circulation in postischemic reperfusion.

["Reperfusion collapse" phenomenon during post-ischemic reperfusion in experiment]. / Fenomen "reperfuziinoho kolapsu" pid chas postishemichnoï reperfuziï v eksperymenti.

Postischemic changes of central and regional hemodynamic while various reperfusion regimens have been studied on animal experimental model. We had revealed that despite vasodilatation, peripheral vascular resistance in the postischemic extremity rises, as a result of reperfusion by the lowered perfusion volume. Such phenomenon has been named as "reperfusion-collapse". In spite of the microcirculation disorders progression there have been no significant rising of the peripheral vascular resistance as well as worsening of the regional hemodynamic indexes in the postischemic limb during reperfusion by either normal or increased perfusion volume.

SPIO-MR imaging versus double-phase spiral CT in detecting malignant lesions of the liver.

PURPOSE: To assess the diagnostic performance of superparamagnetic iron oxide MR (SPIO-MR) imaging compared with double-phase spiral CT in detecting liver metastases and hepatocellular carcinoma. MATERIAL AND METHODS: Thirty-eight patients with a total of 144 malignant lesions of the liver were examined by CT and SPIO-MR. After definition of a gold standard by a panel of experts, the patient images were randomized and presented to a blinded jury of 5 independent observers whose task was to identify as many lesions as possible. The results were tested for statistical significance using multifactorial analysis of variance (alpha=5%). RESULTS: SPIO-MR produced the highest detection rate and was significantly superior (p<0.05) to unenhanced MR imaging and double spiral-phase contrast-enhanced CT (DPS-CECT). Maximum performance in DPS-CECT was obtained during the portal venous contrast phase but was significantly inferior to SPIO-MR imaging. The scores for unenhanced CT and unenhanced MR were significantly lower than for the corresponding enhanced procedures. SPIO-MR imaging produced a higher incidence of false-positive findings (n=39). CONCLUSION: SPIO-MR produced a significantly better detection rate for malignant focal liver lesions compared with double-phase spiral DPS-CECT but was associated with a higher rate of false-positive findings.

[Erythrocytes after cryopreservation with HES: molecular, structural and functional characteristics]. / Erythrozyten nach Kryokonservierung mit HES. Molekulare, strukturelle und funktionelle Eigenschaften.

The use of hydroxyethylstarch (HES) as an alternative to glycerol for cryopreservation of erythrocytes is presented. Immediately after thawing erythrocytes showed alterations in the membrane skeleton and a lowered rigidity of their membrane. However, a few minutes after resuspension in a physiological salt solution the initial poicilocytosis changed back to normocytotic forms. Intravital microscopy of the dog's mesentery revealed a homogeneous distribution of labeled previously cryopreserved erythrocytes in the capillary bed. The lowering of the ATP-level in the erythrocyte by about 20 to 40% was seen to be harmless, because the ATP-turn over rate was unchanged and the glucose support by the GluT 1 carrier was seen to be guaranteed. There was no decrease in the 2,3-DPG level on one hand, but a shift to the right in the O2-association as well as dissociation functions; the saturation capacity of hemoglobin with O2 on the other hand was constant. Free radical oxygen species, generated as a consequence of the freezing/thawing process, were inactivated by the erythrocyte's own antioxidation system. The 24-hour post-transfusion survival in dog amounted to about 95% and the in vivo-life time was normal. The hemolysis of human erythrocytes after thawing was about 5%. Most methodological and scientific problems concerning erythrocyte cryopreservation are considered to be solved. After official permission for use in humans, major benefits to many fields in surgery are expected: no more short-cuts in blood supply, practically complete exclusion of infectious risks, even unlimited use of autologous blood.

Action of hydroxyethyl starch (HES) on the activity of plasmatic clotting factors.

Studies were carried out on the effects of different doses of hydroxyethyl starch 200/0.5 (HES) on plasma clotting factors in dogs, as an animal model for the human clotting system. In 8 German shepherd dogs 15% of the total blood was isovolemically substituted either by Ringer's solution with lactate alone (controls) or with 0.6, 1.3, 1.9, 2.5 g HES/kg b.w. Immediately after the infusion, the HES concentration in the recipients' plasma amounted to 8 mg/ml up to 38 mg/ml. In the following 6 h, the HES decreased to 25% in each case. It was found that the higher the plasma HES content was, the lower the haematocrit. Neither the thrombin-nor the batroxobin-time showed any significant change, irrespective of the plasma HES concentration. The prothrombin-time was decreased directly after the infusion in parallel to the haematocrit. The single clotting factors FI, FII, FV, FVII, FVIII, FX, and FXII behaved approximately in the same way: their activities directly after infusion, but also 6 h later, were lowered in proportion to the amount of HES infused. The loss of factor activity correlated with the volume-expanding effect of HES shortly after the infusion, but not 6 h later. It is concluded that there are two different modes of HES action on clotting factors: the dilution by plasma volume expansion and a non-dilutional action. Cautious handling might be required in patients with clotting disturbances as well as in long-term treatment.