Wernicke´s encephalopathy as rare complication after sleeve gastrectomy.

The authors report a case study of a 24-year-old female patient after sleeve gastrectomy with subsequent manifestation of Wernickes encephalopa-thy. After 5 weeks following the surgery, which were uneventful, the patient was repeatedly hospitalized in the surgical department for extreme vomiting; however, her health status rapidly improved with infusion therapy followed by careful re-alimentation. At the same time, her GIT was closely examined without any clear finding. Problems progressively deteriorated into neurological manifestations. The final diagnosis was established by MR imaging of the brain. In addition, the patients condition was complicated by sudden febrile status with elevated CRP values; therefore, with regard to the unclear CT finding in the abdominal cavity, a laparoscopic revision was made 8 weeks after the primary surgery. The patient survived thanks to the final correct diagnosis and administration of adequate therapy in the form of thiamine substitution. After 30 days following the last admission, the patient was discharged for home treatment in a satisfactory health condition. The aim of the case study is to describe the onset of atypical symptoms in a patient following bariatric surgery - particularly from the point of view of a surgeon - and difficult and prolonged diagnosis of this syndrome due to its relative rarity, also from the point of view of other colleagues with various specializations who participated in the establishment of the diagnostics.

Real-time G-protein-coupled receptor imaging to understand and quantify receptor dynamics.

Understanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 µm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

Urea tolerance as a molecular adaptation of elasmobranch hemoglobins.

Urea is maintained at moderately high concentrations in the blood and tissues of marine elasmobranchs. Functional properties of the hemoglobins fromn several elasmobranch species are unaffected by urea concentrations as high as 5 molar. This in. sensitivity to urea, which is not observed with human hemoglobin, is accompanied by an increased sensitivity to sodium chloride.

Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient.

The binding of oxygen to heme irons in hemoglobin promotes the binding of nitric oxide (NO) to cysteinebeta93, forming S-nitrosohemoglobin. Deoxygenation is accompanied by an allosteric transition in S-nitrosohemoglobin [from the R (oxygenated) to the T (deoxygenated) structure] that releases the NO group. S-nitrosohemoglobin contracts blood vessels and decreases cerebral perfusion in the R structure and relaxes vessels to improve blood flow in the T structure. By thus sensing the physiological oxygen gradient in tissues, hemoglobin exploits conformation-associated changes in the position of cysteinebeta93 SNO to bring local blood flow into line with oxygen requirements.

Head-Up Tilt Test in Risk Stratification of Patients with Hypertrophic Cardiomyopathy.

Conflicting results have been published considering the role of head-up tilt test (HUTT) positivity as a prognostic factor in patients with hypertrophic cardiomyopathy (HCM). The relationship between HCM patients' genotype and their HUTT results has not been previously reported. The aim of this study was to evaluate patients with HCM and their HUTT results in regard to its value for outcome prediction and to investigate the relation of patients' genotype and their HUTT results. Seventy-four (51 ± 15 years; 42% women; median follow-up 72 months) HCM patients were divided into two groups based on their HUTT results and were retrospectively analyzed. In 67 (90.5%) subjects included in the analysis, next-generation sequencing-based genomic testing was performed. A composite end point of unexplained syncope, heart failure hospitalization, and death was defined. A total of 14 patients (18.9%) had positive HUTT (HUTT+), whereas 60 (81.1%) had negative HUTT (HUTT-). Except for the New York Heart Association functional class ( p = 0.01), both groups had similar characteristics. Positive genotype was evenly distributed between the two groups. Composite end point occurred in 5 patients (35.7%) in HUTT+ group versus 14 (23.3%) patients in HUTT- group ( p = 0.33). We did not find a relationship between HUTT results and long-term outcome. We found no association between HUTT results and genotype.

Hemoglobin Rahere, a human hemoglobin variant with amino acid substitution at the 2,3-diphosphoglycerate binding site. Functional consequences of the alteration and effects of bezafibrate on the oxygen bindings.

We encountered an abnormal hemoglobin (Rahere), with a threonine residue replacing the beta 82 (EF6) lysine residue at the binding site of 2,3-diphosphoglycerate, which was responsible for overt erythrocytosis in two individuals of a Japanese family. Hemoglobin Rahere shows a lower oxygen affinity on the binding of 2,3-diphosphoglycerate or chloride ions than hemoglobin A. Although a decrease in the positive charge density at the binding sites of 2,3-diphosphoglycerate in hemoglobin Rahere apparently shifts the allosteric equilibrium toward the low affinity state, it greatly diminishes the cofactor effects by anions. The oxygen affinity of the patient's erythrocytes is substantially lowered by the presence of bezafibrate, which combines with sites different from those of 2,3-diphosphoglycerate in either hemoglobin Rahere or hemoglobin A.

Determination of L-lactate binding stoichiometry and differences in allosteric interactions of structurally distinct homohexamers from Panulirus interruptus hemocyanin.

The role of structurally distinct subunits from the hemocyanin of Panulirus interruptus was investigated by the analysis of the oxygen-binding properties of reassembled homohexamers. Homohexamers reassembled from subunits a and b exhibited cooperative oxygen binding, whereas subunit c did not. The oxygen affinity of homohexamers from subunits b and c was specifically increased by the addition of L-lactate, whereas that of subunit a was not. Both native hexamers and the homohexamers from subunit b have approximately one oxygen-linked lactate binding site per hexamer.

Functional properties of normal and sickle cell hemoglobins in polyethylene glycol 6000.

The functional properties of human hemoglobin A and S were studied in concentrated solutions of polyethylene glycol. Polyethylene glycol solutions are frequently used as media for protein crystallization. In particular, sickle cell hemoglobin, which does not make X-ray quality crystals in high salt solutions, will form high-quality crystals in polyethylene glycol. Comparison of the functional properties of normal and sickle cell hemoglobin in polyethylene glycol show that pH, anion effects and cooperativity of ligand binding are largely unaffected by polyethylene glycol. This suggests that the crystals grown in this medium are representative of the native structure.

Amphitrite ornata erythrocruorin. II. Molecular controls of function.

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1-3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3-4 are observed with 50 mM CaCl2, 50 mM MgCl2 or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl2 is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl2 and 12 degrees C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature for 12 to 20 degrees C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12 degrees C, the erythrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.

The hemoglobin system of the primitive fish, Amia calva: isolation and functional characterization of the individual hemoglobin components.

Blood from the primitive holostean fish, the bowfin, Amia calva, contains 2 mo of ATP per mol of hemoglobin. The hemolysates contain at least five tetrameric hemoglobin components which differ in their oxygen affinities and their response to cofactors such as ATP. The binding of oxygen by each chromatographically isolated component, including a cathodal component, is influenced by pH and organic phosphates; there is no significant differentiation of function or structure as seen in trout and certain other fish hemolysates. Kinetic analyses of ligand binding indicate that the Bohr and Root effects of Amia calva hemoglobins are best explained by changes in both the "on" and "off" constants. At low pH, the increase in the "off" constant is smaller than for most other Root hemoglobins. The hemoglobin system of Amina calva is functionally undifferentiated and may be representative of the ancestral condition in teleosts.

Functional consequences of ligand-linked dissociation in hemoglobin from the sea cucumber Molpadia arenicola.

It has been established that Molpadia hemoglobin tends to dissociate into subunits as oxygen is bound. The kinetics and equilibria of carbon monoxide and ethylisocyanide binding reported here show a dependence on protein concentration that supports the conclusion that the aggregated hemoglobin has a lower ligand affinity than the dissociated subunits. This is true for the isolated D-chain as well as for the unfractionated hemolysate that contains four distinct polypeptide chains (A-D). This indicates that even homopolymers of Molpadia hemoglobin have lower ligand affinity than the dissociated subunits. At high protein concentration hemolysates of Molpadia hemoglobin show slight cooperativity. The time course of ligand binding to the deoxy D-chain also suggests cooperative interactions. The low affinity of the aggregated state may have a different molecular explanation than in human hemoglobin where tetramers of identical subunits (as in Hb H) show high oxygen affinity. The absence of tyrosine and histidine at the C-terminal of the Molpadia D-chains also suggests a different stabilization of the low affinity deoxy state. An additional functional difference between Molpadia hemoglobin and human hemoglobin is that organic phosphates do not alter the ligand affinity of the sea cucumber hemoglobin.

Amphitrite ornata erythrocruorin. I. Structural properties and characterization of subunit interactions.

A high molecular weight erythrocruorin (Mr approx. 3 . 10(6)) is found in the vascular system of the marine terebellid worm Amphitrite ornata, while a low molecular weight hemoglobin is contained in the coelomic cells. Polyacrylamide gel electrophoresis indicates that Amphitrite erythrocruorin contains three different types of polypeptide chain, of molecular weight approx. 15,000, whereas the molecular weight per heme group is approx. 20,000. These data suggest that only two of three polypeptide chains may be associated with a heme group. The coelomic hemoglobin, which occurs as a monomer, has an apparent molecular weight of approx. 14,000. The circular dichroism spectra of Amphitrite erythrocruorin and of the coelomic protein reveal marked differences in the heme environment, while the alpha-helical contents are not very different (60% and 70%, respectively). Amphitrite erythrocruorin is unusual in its dissociation behavior. Divalent cations are required for maintaining the quaternary structure. In the pH range 7.75--8.5, when the Ca2+ concentration is reduced below 1 mM, the whole molecule (57 S) dissociates into a number of lower molecular weight species (25, 15, 10 and 3 S) which have been correlated with specific subunit structures by electron microscopy. Whole molecules and 25 S subunits are not in equilibrium with the lower molecular weight species and can be isolated from partially dissociated mixtures. In contrast, the lower molecular weight subunits are themselves in a state of rapid equilibrium which is sensitive to cations, protons and oxygen. Of special interest is the dimerization reaction of the 10 S subunits, which appears to be mediated by Ca2+ and conforms to the predictions of the Cann and Goad theory on ligand mediated equilibria. The dissociation of Amphitrite erythrocruorin is readily reversible when the Ca2+ concentration is increased. The subunits obtained at physiological (7.8) or slightly acid (6.5) pH completely reassemble into whole molecules. Reassembly, however, is only partial when dissociation occurs at high pH. The presence of stable intermediates, such as the 15 S species, may facilitate the reassociation process.

Chloride masks effects of opposing positive charges in Hb A and Hb Hinsdale (beta 139 Asn-->Lys) that can modulate cooperativity as well as oxygen affinity.

In the human hemoglobin variant Hb Hinsdale, lysine is substituted for asparagine at position beta 139 (H17), which lies in the water-filled cavity that runs through the center of the molecule. This substitution adds two extra cationic residues to the excess of four cationic residues normally lining this cavity. Moo-Penn and colleagues who discovered this hemoglobin, found its oxygen affinity in 0.5 M bis-Tris buffer to be lower than that of Hb A. Their finding conflicted with our prediction that additional cationic groups lining the central cavity would destabilize the T-structure by increased electrostatic repulsion and thereby increase the oxygen affinity. We have, therefore, remeasured the ligand-binding properties of Hb Hinsdale. In chloride-free Hepes buffer, Hb Hinsdale has greatly increased oxygen affinity and lower cooperativity than Hb A. A comparison of the properties of Hb A, Hb Hinsdale, Hb Deer Lodge (beta 2 His-->Arg) and Hb Abruzzo (beta 143 His-->Arg) in 0.05 M Hepes versus 0.05 M bis-Tris buffers shows that very low chloride concentrations can significantly alter cooperativity as well as oxygen affinity. The apparent conflict between the findings of Moo-Penn and colleagues and our prediction arises from the enhanced chloride effects exhibited by Hb Hinsdale. On going from 0.05 M Hepes to 0.05 M bis-Tris at pH 7.0, log P50 values for Hb A and Hb Hinsdale are increased by 0.28 and 1.12, respectively. The Bohr effect, the kinetics of oxygen dissociation, the second-order rate constant of CO binding and the rate of CO recombination after flash photolysis were also determined for Hb Hinsdale. The enhanced chloride sensitivity of Hb Hinsdale is consistent with the allosteric mechanism of chloride interactions proposed by Perutz et al. in the accompanying paper.

Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18 A resolution: clues for a mechanism for allosteric regulation.

The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine N epsilon atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N delta atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5 degrees with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 degrees rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

The site of the redox-linked proton pump in eukaryotic cytochrome c oxidases.

The electronic spectra of fully oxidized derivatives of some cytochrome c oxidase preparations are distinctly pH dependent. In general, the observed spectral shifts are greater in the case of pulsed derivatives compared to resting preparations and also, greater for preparations of the enzyme from shark skeletal muscle compared to beef heart. The low temperature near-infrared magnetic circular dichroism spectrum of the fully oxidized shark enzyme is not pH dependent in the experimental range, indicating the sensitivity of the visible region electronic spectrum to variation in pH to be due principally to changes at the heme a3-CuB chromophore. The results are discussed in relation to proposed mechanisms of proton translocation in cytochrome c oxidase.

Nitric oxide is required for tactile learning in Octopus vulgaris.

Nitric oxide, produced by nitric oxide synthase in brain tissue, is essential for several different kinds of learning in vertebrates. We present the first evidence that it is also essential for learning in an invertebrate. Intramuscular injections of an inhibitor of the enzyme completely block touch learning in Octopus vulgaris. Eight control animals learned a touch paradigm, but none of eight synthase-inhibited ones learned it.

Nitric oxide is necessary for visual learning in Octopus vulgaris.

We recently reported that inhibition of nitric oxide synthase (NOS) in Octopus vulgaris by intramuscular injections of an analog of L-arginine, N-omega-nitro-L-arginine methyl ester (L-NAME), blocked touch learning in Octopus vulgaris. The inactive enantiomorph (D-NAME), which had no effect on learning, was used for control. We now report that essentially the same procedures block visual learning in this animal. We used a visual paradigm in which the octopus was trained to respond positively to a smooth black plastic ball 2.5 cm diameter and negatively to a similar white ball, or vice versa. One set of eight animals was trained to the black ball positive, and another set of six to the white ball positive. Each set was trained at different times by two different trainers. We found that a 1 h pretreatment with the nitric oxide synthase inhibitor L-NAME blocks visual learning in Octopus vulgaris in both sets of animals.

Effects of the calcium channel blocker flunarizine on the hemodynamics and oxygenation of tumor microvasculature.

Flunarizine is a diphenylpiperazine calcium entry blocker that has been shown previously to increase tumor blood flow and sensitivity to radiotherapy via reduction in the radiobiologically significant hypoxic fraction. Two mechanisms of action have been proposed previously (vasodilation, altered blood viscosity), but no studies have been performed to examine its mechanisms of action in vivo. Such information would be invaluable in determining the role of flunarizine in multimodality approaches to reduce tumor hypoxia. Fisher-344 rats bearing R3230Ac tumors transplanted into dorsal flap window chambers were used to examine microcirculatory changes after administration of flunarizine (1.0 mg/kg, iv). The drug increased the diameters of the microvasculature and red cell velocities specifically in central tumor regions (producing an average increase in vessel flow by a factor of 1.96), which was accompanied by an increase in perivascular pO2 of 12 mm Hg, on the average. The drug did not change the diameters of tumor "feeding" vessels, nor did it change vascular length densities. Thus the improvement in central tumor blood flow and oxygenation could not be attributed to dilation of feeding vessels. The oxygen-carrying capacity of the blood was not altered either since hemoglobin saturation (measured in vitro) and the hematocrits of the microvasculature were unchanged after drug administration. Therefore, by a process of elimination, the most likely explanation for the effect of the drug is modification of blood viscosity. Additional studies are under way in this laboratory to examine whether changes in viscosity occur after flunarizine administration.

Stroma-free human hemoglobin A decreases R3230Ac rat mammary adenocarcinoma blood flow and oxygen partial pressure.

We examined the effect of a nitric oxide (NO) quencher, stroma-free human hemoglobin A (HbA0; 0.01, 0.05, 0.1, 0.2 g/kg), on the blood flow measured using the Doppler flow technique, tumor oxygen pressure (pO2) and the diameter of the arterioles using R3230Ac mammary adenocarcinoma as the tumor model. In female Fischer 344 rats with 1-cm-diameter tumors implanted in the lateral aspect of the left quadriceps, intravenous infusion of 0.1 and 0.2 g/kg HbA0 decreased both central tumor and peripheral tumor blood flow by 20-30% (P < 0.05). Tumor pO2 decreased 28% with 0.2 g/kg HbA0, from 15 mm Hg (baseline) to 11 mm Hg at 10 min (P = 0.02). Although 0.2 g/kg HbA0 increased blood flow 55% in the left quadriceps muscle proximal to the implanted tumor (P < 0.05), HbA0 had little effect on blood flow in right quadriceps muscle with no tumor implanted, and increased right quadriceps pO2, from 21 mm Hg (baseline) to 23 mm Hg at 10 min (P = 0.03). HbA0 increased mean arterial pressure 5-10% in a manner that was dependent on dose while heart rate concurrently decreased 9-19%. The diameter of the arterioles supplying the tumor was rapidly reduced 10% by 0.2 g/kg HbA0 (P = 0.037) and remained stable through 60 min of observation (P = 0.005). HbA0 selectively reduces tumor blood flow and tumor pO2 through vasoconstriction of the arterioles supplying the tumor. Vascular NO quenching provides an alternative to NO synthase inhibition as a means to achieve the goal of selective tumor hypoxia.