Jugular Venous Response for Risk Stratification in Heart Failure.

BACKGROUND:  The response of jugular venous pressure (JVP) to increased preload with inspiration has been recognized as a method of stratifying risk in the management of heart failure (HF). Whether the JVP response to inspiration may be more effective than other simple approaches in this setting remains unclear. METHODS:  This study enrolled 79 patients with stable HF. JVP was assessed from the right internal jugular vein in the sitting position and was considered high if visible above the right clavicle at rest. JVP responses to inspiration, the five-repetition sit-to-stand test (5-STS), and squatting were also evaluated. The primary outcome was a composite of all-cause death and hospitalization for worsening HF. RESULTS:  JVP assessment after 5-STS and during squatting was not conducted in two and 14 HF patients, respectively, due to physical limitations. During a mean follow-up of 837 days, the primary outcome was associated with a high JVP at rest (hazard ratio, 2.47; 95% confidence interval [CI], 1.09 to 5.60; P <0.05), with inspiration (hazard ratio, 2.53; 95% CI, 1.17 to 5.46; P <0.05), after 5-STS (hazard ratio, 2.61; 95% CI, 1.23 to 5.97; P <0.05), and during squatting (hazard ratio, 2.40; 95% CI, 1.03 to 6.06; P <0.05). Among patients without a high JVP at rest, the specificity of the primary outcome at one year was greater for the JVP response to inspiration (89%) and squatting (92%) than for the response to 5-STS (80%). CONCLUSIONS:  JVP response to increased preload with inspiration may be a simple and practical method for risk assessment in patients with stable HF.

Jugular Venous Pressure and Kussmaul's Sign as Predictors of Outcome in Heart Failure.

Simplifying the estimation of internal jugular venous pressure (JVP) as visible or not visible above the right clavicle in the sitting position has attracted attention for risk assessment in patients with heart failure (HF). It remains unclear whether this simple assessment, combined with its inspiration response known as Kussmaul's sign, is useful in patients with HF who vary in features such as HF with reduced ejection fraction (HFrEF) and preserved ejection fraction (HFpEF).This study consisted of 246 patients who were admitted for the management of HF. JVP was assessed before discharge and considered high if visible at rest. The inspiration response was also examined. The primary outcome was a composite of all-cause death and hospitalization for worsening HF.One year after discharge, primary outcome events occurred in 91 patients (37%). The incidence of primary outcome was higher in patients with a high JVP at rest (odds ratio, 5.06; 95% confidence interval, 2.31-11.1; P = 0.0001) or with inspiration (odds ratio, 5.93; 95% confidence interval, 2.14-16.4; P < 0.01) than in patients without high JVP conditions. These findings were similarly observed among patients with HFrEF and HFpEF (odds ratios, 3.53 and 6.76; 95% confidence intervals, 1.68-8.68 and 2.19-15.5; P = 0.01 and < 0.01, respectively) and in subgroup analysis stratified by baseline characteristics of the patients.A high JVP at rest and with inspiration as assessed by this simple, practical method may be useful for risk assessment in patients with HF, independent of baseline characteristics.

Excited state dynamics of protonated dopamine: hydration and conformation effects.

Electronic and vibrational spectroscopy in a cryogenic ion trap has been applied to protonated dopamine water clusters and assigned with the help of quantum chemistry calculations performed in the ground and electronic excited states. A dramatic hydration effect is observed when dopamine is solvated by three water molecules. The broad electronic spectra recorded for the bare and small water clusters containing protonated dopamine turn to sharp, well-resolved vibronic transitions in the 1-3 complex. This reflects the change induced by hydration in the photodynamics of protonated dopamine which is initially controlled by an excited state proton transfer (ESPT) reaction from the ammonium group toward the catechol ring. Interestingly, conformer selectivity is revealed in the 1-3 complex which shows two low lying energy conformers for which the ESPT reaction is prevented or not depending on the H-bond network formed between the dopamine and water molecules.

Jugular Venous Pressure Response to Inspiration for Risk Assessment of Heart Failure.

Simplifying jugular venous pressure (JVP), visibility of the right internal jugular vein above the right clavicle in the sitting position, has been proposed in the management of heart failure (HF) because of its convenience. However, this method may be undervalued for the detection of mildly to moderately increased JVP. Increased JVP on inspiration, known as Kussmaul sign, may be a useful physical finding in this condition. This study consisted of 138 patients who were admitted for the management of HF. Using this simple method, JVP was assessed at rest in the sitting position before discharge; its response to inspiration was also examined if no high JVP was noted at rest. The primary outcome was a composite of cardiac death and hospitalization for worsening HF. Among all the patients, 16 patients (12%) had high JVP at rest and another 16 patients (12%) had high JVP not at rest but on inspiration. During a follow-up period of 249 ± 182 days, a primary outcome event occurred in 63 patients (46%). The incidence of adverse cardiac events was higher in patients with a high JVP at rest (69%; hazard ratio 3.31, 95% confidence interval 1.64 to 6.67, p = 0.0009) and in patients with a high JVP on inspiration (56%; hazard ratio 2.18, 95% confidence interval 1.02 to 4.63, p = 0.043) than in patients without a high JVP in both conditions (41%). In conclusion, a high JVP not only at rest but also on inspiration was associated with a poor prognosis. The response of JVP to inspiration using this simple technique of physical examination may be a new approach in the management of HF.

Hydration-controlled excited-state relaxation in protonated dopamine studied by cryogenic ion spectroscopy.

Ultraviolet (UV) and infrared (IR) spectra of protonated dopamine (DAH+) and its hydrated clusters DAH+(H2O)1-3 are measured by cryogenic ion spectroscopy. DAH+ monomer and hydrated clusters with up to two water molecules show a broad UV spectrum, while it turns to a sharp, well-resolved one for DAH+-(H2O)3. Excited state calculations of DAH+(H2O)3 reproduce these spectral features. The conformer-selected IR spectrum of DAH+(H2O)3 is measured by IR dip spectroscopy, and its structure is assigned with the help of quantum chemical calculations. The excited state lifetime of DAH+ is much shorter than 20 ps, the cross correlation of the ps lasers, revealing a fast relaxation dynamics. The minimal energy path along the NH → π proton transfer coordinate exhibits a low energy barrier in the monomer, while this path is blocked by the high energy barrier in DAH+(H2O)3. It is concluded that the excited state proton transfer in DAH+ is inhibited by water-insertion.

Frontal affinity chromatography: An excellent method of analyzing weak biomolecular interactions based on a unique principle.

BACKGROUND: Not only strong biomolecular interactions but also weak interactions play important roles in ensuring appropriate operations of many biological systems. Although a thorough investigation of the latter is essential in understanding life science, few suitable research tools are available because of inherent difficulties. SCOPE OF REVIEW: Frontal affinity chromatography (FAC) is a versatile method that overcomes the inherent difficulties to provide accurate information on weak interactions. Since its concept and merit are not widely recognized, a comprehensive interpretation of FAC is provided in this review to encourage its application among researchers. MAJOR CONCLUSION: FAC is based on a unique principle of measuring the binding strength by the delayed migration of an analyte through an affinity column. Its utility was elucidated via the lectin-glycan interactions. GENERAL SIGNIFICANCE: FAC has a great potential as a research tool to solve many difficult problems in general bioscience that are relevant to almost all researchers.

Frontal Affinity Chromatography: A Highly Suitable Retardation Phenomenon-Based Research Tool for Analyzing Weak Interactions Between Biomolecules.

The greatest advantage of frontal affinity chromatography (FAC) is that the analyte concentration does not need to be taken into consideration, and this renders FAC an extremely favorable analytical tool for weak interactions. In this short review, we propose a straightforward explanation of the underlying mechanism. When FAC is performed using analyte solutions at relatively high concentrations, concentration-dependent retardation is observed due to competition among analyte molecules, and the elution volume changes depending on the degree of saturation of the immobilized ligand.However, when the analyte concentration is very low, no competition occurs among the analytes, and the elution volume reaches a constant value, which reflects the proportion of bound state to free state of a single analyte molecule. Therefore, the binding strength can be determined using a minimum analyte concentration.

Response of Jugular Venous Pressure to Exercise in Patients With Heart Failure and Its Prognostic Usefulness.

Jugular venous pressure (JVP) has been established for the assessment of central venous pressure in patients with heart failure (HF), but data are limited regarding the response of JVP to exercise because of its complicated methods. Simplifying the estimation of JVP may be applied in such situations. JVP was assessed before and after the 6-minute walk test (6MWT) in 81 patients with HF using a simple method by which the JVP was considered high when the internal jugular venous pulsation on the right side was visually identified above the right clavicle in the sitting position. The primary outcome was a composite of cardiovascular death and hospitalization for worsening HF. None of the patients exhibited high JVP before the 6MWT and 11 patients (14%) had a high JVP after. The 6MWT distances were lower in patients with a high JVP after the 6MWT (338 ± 114 m) than those in patients without a high JVP (417 ± 78 m, p = 0.04). During a follow-up period of 13.4 ± 6.9 months, 11 patients died and 8 patients were hospitalized for worsening HF. The incidence of adverse cardiac events was higher in patients with a high JVP after the 6MWT (64%) than in patients without a high JVP after (64% vs 17%; hazard ratio, 7.52; 95% confidence interval, 2.69 to 20.83; p <0.001). In conclusion, high JVP after exercise was associated with exercise intolerance and poor prognosis. The response of JVP to exercise using this simple technique of physical examination may be a new approach for patients with HF for risk assessment.

Galactoseß1-4fucose: A unique disaccharide unit found in N-glycans of invertebrates including nematodes.

Galactoseß1-4fucose (Galß1-4Fuc), a unique disaccharide unit found only on the N-glycans of Protostomia, has been intensively studied, particularly in Nematoda. Galß1-4Fuc attached to the 6-OH of the innermost GlcNAc of N-glycans has been identified as an endogenous target recognized by Caenorhabditis elegans galectin LEC-6 and might function as an endogenous ligand for other galectins as well. Interactions between galectins and N-glycans might be subject to fine-tuning through modifications of the penultimate GlcNAc and the Galß1-4Fuc unit. Similar fine-tuning is also observable in vertebrate galectins, although their major recognition unit is a Galß1-4GlcNAc. In Protostomia, it can be postulated that glycan-binding proteins and their ligands have coevolved; however, epitopes such as Galß1-4Fuc were then hijacked as targets by other organisms. Fungal (Coprinopsis cinerea) galectin 2, CGL2, binds the Galß1-4Fuc on C. elegans glycans to exert its nematotoxicity. Some human and mouse galectins bind to synthesized Galß1-4Fuc; as some parasitic nematodes express this motif, its recognition by mammalian galectins could hypothetically be involved in host defense, similar to fungal CGL2. In this review, we discuss the Galß1-4Fuc unit in Protostomia as a possible equivalent for the Galß1-4GlcNAc unit in vertebrates and a potential non-self glycomarker useful for pathogen recognition.

Preparation of a polyclonal antibody that recognizes a unique galactoseß1-4fucose disaccharide epitope.

Galactoseß1-4fucose (Galß1-4Fuc) is a unique disaccharide unit that has been found only in the N-glycans of protostomia. We demonstrated that this unit has a role as an endogenous ligand for Caenorhabditis elegans galectins. This unit is also recognized by fungal and mammalian galectins possibly as a non-self glycomarker. In order to clarify its biological function, we made a polyclonal antibody using (Galß1-4Fuc)n-BSA as the antigen, which was prepared by crosslinking Galß1-4Fuc-O-(CH2)2-SH and BSA. The binding specificity of the antibody was analyzed by frontal affinity chromatography, and it was confirmed that it recognizes naturally occurring N-glycans containing the Galß1-4Fuc unit linked to the reducing-end GlcNAc via α1-6 linkage. By western blotting analysis, the antibody was also found to bind to (Galß1-4Fuc)n-BSA but not to BSA or asialofetuin, which has N-glycan chains containing Galß1-4GlcNAc. Western blotting experiments also revealed presence of stained proteins in crude extracts of C. elegans, the parasitic nematode Ascaris suum, and the allergenic mite Dermatophagoides pteronyssinus, while those from Drosophila melanogaster, Mus musculus, and the allergenic mites Dermatophagoides farinae and Tyrophagus putrescentiae were negative. This antibody should be a very useful tool for research on the distribution of the Galß1-4Fuc disaccharide unit in glycans in a wide range of organisms.

Frontal affinity chromatography (FAC): theory and basic aspects.

Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory.

Frontal affinity chromatography: a unique research tool for biospecific interaction that promotes glycobiology.

Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described.

Crosslinking of Cys-mutated human galectin-1 to the model glycoprotein ligands asialofetuin and laminin by using a photoactivatable bifunctional reagent.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys(28), forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.

Affinity probe capillary electrophoresis of insulin using a fluorescence-labeled recombinant Fab as an affinity probe.

Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence-labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti-insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol-reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab-gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral-polymer coated fused silica capillary (50 µm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser-induced fluorescence under focusing conditions. The insulin-AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first-order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.

Galectin LEC-1 plays a defensive role against damage due to oxidative stress in Caenorhabditis elegans.

LEC-1 is a major galectin in Caenorhabditis elegans and contains two carbohydrate recognition domains (CRDs), N-CRD and C-CRD. To determine the role of LEC-1, we examined the phenotypes of a mutant C. elegans strain lacking lec-1. We observed negligible differences in embryogenesis, morphogenesis and egg laying at 20 °C between the mutant and the wild-type. Furthermore, the life spans of the mutant and the wild-type were equivalent at either 20 °C or 25 °C. However, the lec-1 mutant showed a greater susceptibility to H2O2 and paraquat than the wild-type. This result suggests an increased susceptibility to oxidative stress, with the phenotypes being similar to those of lec-10 deletion mutants as previously described. To understand the molecular mechanism underlying this phenotype, C. elegans proteins bound by either the LEC-1 N-CRD or C-CRD were isolated and identified using a nano liquid chromatography-tandem mass spectrometry technique. MIG-6 was identified as a major binding partner of LEC-1 with both N- and C-CRD. From these results and previous reports, we speculate that interaction of LEC-1 and MIG-6 in the pharynx may affect susceptibility to paraquat and that LEC-10 has different functions from LEC-1 in regulating H2O2 and paraquat resistance in the intestine.

Mammalian galectins bind galactoseß1-4fucose disaccharide, a unique structural component of protostomial N-type glycoproteins.

Galactoseß1-4Fucose (Galß1-4Fuc) is a unique disaccharide exclusively found in N-glycans of protostomia, and is recognized by some galectins of Caenorhabditis elegans and Coprinopsis cinerea. In the present study, we investigated whether mammalian galectins also bind such a disaccharide. We examined sugar-binding ability of human galectin-1 (hGal-1) and found that hGal-1 preferentially binds Galß1-4Fuc compared to Galß1-4GlcNAc, which is its endogenous recognition unit. We also tested other human and mouse galectins, i.e., hGal-3, and -9 and mGal-1, 2, 3, 4, 8, and 9. All of them also showed substantial affinity to Galß1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galß1-4Fuc, Galß1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galß1-4Fuc is the most effective. Although the biological significance of galectin-Galß1-4Fuc interaction is obscure, it might be possible that Galß1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galß1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors.

Structural basis of preferential binding of fucose-containing saccharide by the Caenorhabditis elegans galectin LEC-6.

Galectins are a group of lectins that can bind carbohydrate chains containing ß-galactoside units. LEC-6, a member of galectins of Caenorhabditis elegans, binds fucose-containing saccharides. We solved the crystal structure of LEC-6 in complex with galactose-ß1,4-fucose (Galß1-4Fuc) at 1.5 Å resolution. The overall structure of the protein and the identities of the amino-acid residues binding to the disaccharide are similar to those of other galectins. However, further structural analysis and multiple sequence alignment between LEC-6 and other galectins indicate that a glutamic acid residue (Glu67) is important for the preferential binding between LEC-6 and the fucose moiety of the Galß1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galß1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galß1-4Fuc unit, are drastically reduced relative to that of the wild-type protein. We conclude that the Glu67 in the oligosaccharide-binding site assists the recognition of the fucose moiety by LEC-6.

Estimation of the deamidation rates of major deamidation sites in a Fab fragment of mouse IgG1-κ by capillary isoelectric focusing of mutated Fab fragments.

The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modification. The identification of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such analysis becomes difficult and sometimes impossible. In this report, a quantitative method for estimating the deamidation rate of a specific amide in a protein is presented without using peptide mapping. Five Asn residues of a recombinant fragment antigen binding (rFab) (mouse IgG1, κ) were mutated to a serine (Ser) residue, one by one, through site-directed mutagenesis, and the single-residue deamidation rates of the original rFab and the mutants were determined using capillary isoelectric focusing. The difference of the rate between the original rFab and the mutant was assumed to be equal to the deamidation rate of the specific Asn residue, which had been mutated. Among five mutants established, three major deamidation sites-H chain Asn135, L chain Asn157, and L chain Asn161, using the Kabat numbering system-were identified, accounting for 66%, 29%, and 7% of the single-residue deamidation of the original rFab, respectively. Although the former two have been known by peptide mapping, the last one, which resides on the same tryptic peptide that carries one of the former two, previously has not been identified. For the first time, the deamidation rate constants of the three sites were estimated to be 10.5 × 10(-3) h(-1), 4.6 × 10(-3) h(-1), and 1.1 × 10(-3) h(-1) in 0.1 M phosphate buffer, pH 7.5 at 37 °C, respectively, with corresponding half-life of 2.8 days, 6.3 days, and 27 days. The method should be applicable to any recombinant proteins.

Glycerol-enhanced detection of a preferential structure latent in unstructured 1SS-variants of lysozyme.

Four species of 1SS-varinats of lysozyme were almost unstructured in water, judged from their near-UV CD and (1) H-(15) N-HSQC spectra. Some preferential structure might exist in such a disordered state, but the population of molecules in such a conformation must have been too small to be detected by spectroscopic methods. Indeed, our previous study showed that the addition of 30% glycerol induced the unstructured 2SS-variant of lysozyme to form a native-like structure. To extend this method to more disordered proteins, we attempted to detect some preferential structure latent in unstructured 1SS-variants by the glycerol-enhanced detection. Only in one molecular species of the four 1SS-variants, 1SS[6-127] containing a single disulfide bridge of Cys6-Cys127, a preferential structure was found in the presence of 50% glycerol. It was detected by near-UV CD measurements and the H/D exchange method combined with the NMR spectroscopy. The glycerol-induced structure in 1SS[6-127] was not localized only in the vicinity of Cys6-Cys127, and largely protected regions distributed themselves among A-, B-, and C-helices and Ile55 and Leu56. It was similar to the glycerol-induced structure in 2SS[6-127, 64-80] containing two disulfide bridges of Cys6-Cys127 and Cys64-Cys80, although the former was less rigid than the latter. The role of A-helix (residues 4-15) is proposed as an origin of excellent potential of Cys6-Cys127 for inducing a tertiary structure in the α-domain.

The DC2.3 gene in Caenorhabditis elegans encodes a galectin that recognizes the galactoseß1→4fucose disaccharide unit.

Galectins comprise a large family of ß-galactoside-binding proteins in animals and fungi. We previously isolated cDNAs of 10 galectin and galectin-like genes (lec-1 to lec-6 and lec-8 to lec-11) from Caenorhabditis elegans and characterized the carbohydrate-binding properties of their recombinant proteins. In the present study, we isolated cDNA corresponding to an open reading frame of the DC2.3a gene from C. elegans total RNA; this cDNA encodes another potential galectin. A recombinant DC2.3a protein was expressed in Escherichia coli and used for analysis. The protein displayed hemagglutinating activity against rabbit erythrocytes, bound to an asialofetuin-Sepharose column, and was eluted with lactose. Furthermore, frontal affinity chromatography (FAC) analysis confirmed that DC2.3a recognized oligosaccharides with a non-reducing terminal galactose. According to these results, we designated DC2.3 as lec-12. The carbohydrate-binding property of the recombinant DC2.3a/LEC-12a was essentially similar to that of LEC-6. Additionally, DC2.3a/LEC-12a and LEC-6 showed higher affinities for the galactoseß1→4fucose (Galß1→4Fuc) disaccharide than for N-acetyllactosamine. This suggests that the principal recognition unit is the Galß1→4Fuc disaccharide as in the case of the C. elegans galectins. However, the recombinant DC2.3a/LEC-12a showed weak affinity for N-glycan E3, which was previously shown to be a preferential endogenous ligand for LEC-6. The DC2.3a/LEC-12a endogenous ligand structures appear to be somewhat different but contain the same galactose-fucose recognition motif.