Influence of antibody binding on oxygen binding behavior of Panulirus interruptus hemocyanin

Evidence linking paclitaxel to cardiotoxicity arose from early Phase I trials in which continuous cardiac monitoring was performed because of the high incidence of major hypersensitivity reactions. A variety of cardiac manifestations have been reported, ranging from asymptomatic sinus bradycardia to fatal myocardial infarction. The following case report describes an acute myocardial infarction occurring shortly after paclitaxel therapy.     

Electron paramagnetic resonance and oxygen binding studies of alpha-Nitrosyl hemoglobin. A novel oxygen carrier having no-assisted allosteric functions

alpha-Nitrosyl hemoglobin, alpha(Fe-NO)2beta(Fe)2, which is frequently observed upon reaction of deoxy hemoglobin with limited quantities of NO in vitro as well as in vivo, has been synthetically prepared, and its reaction with O2 has been investigation by EPR and thermodynamic equilibrium measurements. alpha-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O2 binding at the heme sites of its beta-subunits. Its O2 binding is coupled to the structural/functional transition between T- (low affinity extreme) and R- (high affinity) states. This transition is linked to the reversible cleavage of the heme Fe-proximal His bonds in the alpha(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, alpha(Fe-NO)2beta(Fe)2 is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of alpha-nitrosyl hemoglobin is comparable to that of normal hemoglobin, despite the fact that the oxygenation process involves only two ligation steps. All of these structural and functional evidences have been further confirmed by examining the reactivity of the sulfhydryl group of the Cysbeta93 toward 4, 4'-dipyridyl disulfide of several alpha-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O2-carrying capacity, alpha-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O2 carrier with improved features that could deliver O2 to tissues effectively even after NO is sequestered at the heme sites of the alpha-subunits. It is concluded that the NO bound to the heme sites of the alpha-subunits of hemoglobin acts as a negative allosteric effector of Hb and thus might play a role in O2/CO2 transport in the blood under physiological conditions.     

Fluorescence studies of human semi-beta-hemoglobin assembly

The intrinsic fluorescence properties of human alpha apohemoglobin at protein concentrations from 1 to 5 microM in 0.1 M potassium phosphate buffer, pH 7 or 8 at 5 degrees C were monitored in the absence and presence of a fixed concentration (5 microM) of a fluorescence quenching heme-containing native or Des (146-His, 145-Tyr) beta chain partner. These "reverse quenching" studies revealed that the emission intensity changes observed correlated well with protein concentration and theoretical extent of semi-beta-hemoglobin assembly. Furthermore, the relative quenching efficiencies were calculated to be 0.32, 0.25 and 0.61 for beta (pH 7), beta (pH 8) and Des beta (pH 7) chains, respectively. Thus, heme-mediated quenching was sensitive to the expected pH induced alpha apohemoglobin conformational change and to alteration in beta chain structure. Intramolecular changes induced by carboxylterminal modification (decreased "beta chain self-quenching") appeared to enhance the intermolecular rearrangements (increased "alpha chain partner quenching") seen upon subunit assembly.     

The binding of Cd(II) to the hemocyanin of the Mediterranean crab Carcinus maenas

In idiopathic dilated cardiomyopathy (IDC), the relation between the signal-averaged electrocardiogram and ventricular tachycardia (VT) remains unclear. In this study, conventional time domain and frequency domain analyses (2-dimensional, spectral temporal mapping and spectral turbulence analysis) of the signal-averaged electrocardiogram were performed in 64 patients with IDC. Eight patients had a history of symptomatic sustained VT and an additional 24 had nonsustained VT recorded during ambulatory electrocardiography. Conventional time domain analysis, using the 25 and 40 Hz filter, and spectral temporal mapping, detected late potentials within the terminal QRS in 8 (13%), 14 (22%) and 18 (28%) patients, respectively. Late potentials were seen more often in patients with than without VT, and in patients with sustained versus nonsustained VT, but these differences were not significant. The predictive accuracy of these techniques in detecting either form of VT were: sensitivity, 22, 25 and 31%; specificity, 97, 81 and 75%; and overall predictive value, 59, 53 and 50%, respectively. Two-dimensional frequency domain analysis of the signal-averaged electrocardiogram revealed a higher energy and area ratio in patients with than without VT (entire QRS), and in patients with sustained versus nonsustained VT (entire QRS and terminal QRS). Spectral turbulence analysis was abnormal in 24 patients (39%), but no differences were observed between patients with and without VT. During follow-up (mean duration 18 +/- 14 months), 5 patients had arrhythmic events (3 died suddenly, 1 had aborted sudden death and 1 developed sustained VT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic studies of oxygen behavior in tantalum-based alloys

An experimental study of equilibrium thermodynamic properties of oxygen in pure tantalum and tantalum alloyed with V, Nb or Mo was made by EMF measurements on solid electrolytic cells over the temperature range of 873–1373 K (600–1100°C). The solubility of oxygen in pure tantalum in equilibrium with Ta₂O₅ was determined to be C_s = 12.4exp[−(16 kJ/mol)/(RT)] over the experimental temperature range. It was concluded that oxygen obeys Henry's law for concentrations up to the terminal solubility in tantalum for the temperature range 873–1373 K (600–1100°C). The oxygen activity coefficient increased with Mo content in TaMo alloys and decreased with V content in TaV alloys. The equilibrium results showed virtually no change in thermodynamic behavior of the oxygen solution by adding Nb to TaO alloys. The positive deviation of the oxygen activity coefficient in TaMo alloys is evidence of a repulsive interaction between molybdenum and oxygen atoms. The interaction energy was calculated assuming that the interaction extends to third nearest neighbors. It may, in fact, extend to fourth or higher neighboring shells of atoms.     

Fluorescence resonance energy transfer in studies of inter-chromophoric distances in biomolecules

Fluorescence resonance energy transfer (FRET) is a technique widely used in studies of interchromophoric distances in biomolecules such as peptides, proteins and nucleic acids. FRET is especially useful in determination of conformational changes caused by a solvent, presence of denaturing agents, diffusion and other external factors. Precision of interchromophoric distances obtained using the FRET technique is comparable with that of low-resolution X-ray diffraction and NMR data. Comparison of FRET results with the crystal structure for several proteins is reviewed. Moreover, the effect of the orientation factor kappa2 value on FRET results and determinants of kappa2 are discussed.     

Effects of hyperchloremia on blood oxygen binding in healthy calves

Three different levels of hyperchloremia were induced in healthy Friesian calves to study the effects of chloride on blood oxygen transport. By infusion, the calves received either 5 ml/kg of 0.9% NaCl (low-level hyperchloremia; group A), 5 ml/kg of 7.5% NaCl (moderate hyperchloremia; group B), or 7.5 ml/kg of 7.5% NaCl (high-level hyperchloremia; group C). Blood was sampled from the jugular vein and the brachial artery. Chloride concentration, hemoglobin content, arterial and venous pH, PCO2, and PO2 were determined. At each time point (0, 15, 30, 60, and 120 min), the whole blood oxygen equilibrium curve (OEC) was measured under standard conditions. In groups B and C, hyperchloremia was accompanied by a sustained rightward shift of the OEC, as indicated by the significant increase in the standard PO2 at 50% hemoglobin saturation. Infusion of hypertonic saline also induced relative acidosis. The arterial and venous OEC were calculated, with body temperature, pH, and PCO2 values in arterial and venous blood taken into account. The degree of blood desaturation between the arterial and the venous compartments [O2 exchange fraction (OEF%)] and the amount of oxygen released at tissue level by 100 ml of bovine blood (OEF vol%) were calculated from the arterial and venous OEC combined with the PO2 and hemoglobin concentration. The chloride-induced rightward shift of the OEC was reinforced by the relative acidosis, but the altered PO2 values combined with the lower hemoglobin concentration explained the absence of any significant difference in OEF (% and vol%). We conclude that infusion of hypertonic saline induces hyperchloremia and acidemia, which can explain the OEC rightward shift observed in arterial and peripheral venous blood.     

Fluorescence and immunochemical studies of advanced glycation-related lens pigments

PURPOSE: To establish whether advanced glycation is the major mechanism for yellowing of lens proteins. METHODS: Synchronous fluorescence (SF) and immunochemical assays were used to study glycation in vitro and in vivo. In the in vitro study, advanced glycation end products (AGEs) were prepared and used as antigens to induce antibodies to AGEs. The in vitro AGEs and classified nuclear cataracts were analyzed by SF and immunochemical assays. RESULTS: In vitro AGEs generated from various glycating agents and carrier proteins displayed strong SF above 350 nm; the spectra were well resolved with major bands at 380 nm and 420 nm. Samples from human lenses manifested a band at 395 nm in addition to the two bands shown by in vitro AGEs. SF intensity is greater for the water-insoluble (WI) than water-soluble (WS) fraction, but both increased with increasing nuclear color. The immunoreactivity data also showed that the WI fraction contained more AGEs than the WS fraction and that the amount of AGEs increased with increasing nuclear color. CONCLUSIONS: Fluorescence and immunoassays indicated that pigmented AGEs contributed to yellowing of the crystalline lens nucleus.     

Fluorescence depolarization studies on the flexibility of myosin rod

The single photon counting method has been used to measure the decay of fluorescence polarization anisotropy of myosin rods labeled with extrinsic fluorophores. Rods labeled with 8-anilino-1-naphthalenesulfonate (ANS) or 5-dimethylaminoaphthalene-1-sulfonyl chloride (DNS-Cl) exhibit negative rotational correlation times; the anisotropy increases with time. Possible artifactual causes for the negative decay times are ruled out. It is shown that such curves are to be expected for rigid rods when the fluorophore is bound so that the absorption and emission dipoles each make a small angle with the long axis of the molecule and lie on opposite sides of the rod. At pH 4 and below, rapid decay of the anisotropy (positive correlation times) indicates the presence of a freely bending region in the rod. This is probably the proteolytically sensitive region between light meromyosin and heavy meromyosin subfragment 2. At pH 8, no such free bending is observed, even at temperatures as high as 50 degrees C. From this observation and other physical properties of the rod, we conclude that, at pH 8, the hinge region has considerable resistance to bending. It is more like a spring than a free hinge. The rotational diffusion about the rod axis is faster than would be predicted for a rigid, smooth molecule.     

Primary structure of hemocyanin from Palinurus vulgaris

The primary structure of hemocyanin from the spiny lobster Palinurus vulgaris was determined using a mixture of at least four slightly different subunits. Heterogeneities were observed in 32 (5%) of the positions. The amino acid sequence differs at about 20% of the positions from that of subunit a of Panulirus interruptus hemocyanin.     

Cytobiological studies on hemocyanin metabolism in the branchial heart complex of the common cuttlefish Sepia officinalis (Cephalopoda, Dibranchiata)

The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with 125I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.     

Fluorescence quenching studies of the rat ovarian LH/hCG receptor

Fluorescence quenching method providing information about the structure and dynamics of proteins, ligand-protein and protein-lipid interactions was used in a study of the rat ovarian LH/hCG receptor. The efficiency of two different quenchers, acrylamide and iodide, was tested. Acrylamide was significantly more effective in quenching of intrinsic fluorescence of ovarian membranes than iodide and therefore it was used in all of the following experiments. Both acrylamide and iodide were not effective in quenching of membranes labelled with fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH). In the process of desensitization of ovarian LH/hCG receptors the administration to rats of hCG modified the quenching rate of protein fluorescence and intrinsic fluorescence spectral properties of membranes. Alteration in the quenching of intrinsic fluorescence of ovarian membranes was observed after chemical modification of LH/hCG receptors by 2-hydroxy-5-nitrobenzyl bromide (HNB-Br). The accessibility of tryptophan fluorophores was increased in HNB-Br-treated membranes. Delipidation of the LH/hCG receptor modified the quenching of protein fluorescence characteristic for control proteoliposomes. These results demonstrate that fluorescence quenching technique can be successfully applied in the study of the LH/hCG receptor.     

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin

Yersinia pestis produces a set of virulence proteins (Yops and LcrV) that are expressed at high levels and secreted by a type III secretion system (Ysc) upon bacterium-host cell contact, and four of the Yops are vectorially translocated into eukaryotic cells. YopD, YopB, and YopK are required for the translocation process. In vitro, induction and secretion occur at 37 degrees C in the absence of calcium. LcrH (also called SycD), a protein required for the stability and secretion of YopD, had initially been identified as a negative regulator of Yop expression. In this study, we constructed a yopD mutation in both wild-type and secretion-defective (ysc) Y. pestis to determine if the lcrH phenotype could be attributed to the decreased stability of YopD. These mutants were constitutively induced for expression of Yops and LcrV, despite the presence of the secreted negative regulator LcrQ, demonstrating that YopD is involved in negative regulation, regardless of a functioning Ysc system. Normally, secretion of Yops and LcrV is blocked in the presence of calcium. The single yopD mutant was not completely effective in blocking secretion: LcrV was secreted equally well in the presence and absence of calcium, while there was partial secretion of Yops in the presence of calcium. YopD is probably not rate limiting for negative regulation, as increasing levels of YopD did not result in decreased Yop expression. Overexpression of LcrQ in the yopD mutant had no significant effect on Yop expression, whereas increased levels of LcrQ in the parent resulted in decreased levels of Yops. These results indicate that LcrQ requires YopD to function as a negative regulator.     

Mathematical coupling in medical research: lessons from studies of oxygen kinetics

BACKGROUND: Smooth muscle hamartoma is an uncommon lesion. Diagnosis is usually made at birth in infants presenting a plaque with minimal or no infiltration and covered with long dark hairs. Congenital forms with multiple plaques are rarely reported. CASE REPORT: A 5-day-old infant (normal pregnancy and delivery) had plaques localized on the buttocks, the left thigh, leg and shoulder and the right ankle. The plaques were minimally infiltrative and covered with long black hairs. Histology examination showed hyperplastic smooth muscle bundles with varying orientation. The diagnosis was smooth muscle hamartoma. The rest of the clinical examination was normal. CONCLUSION: This case of congenital smooth muscle hamartoma showed a particular form with partially regressive multiple plaques.