Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Photosynthetic Electron Transport Involved in PxcA-Dependent Proton Extrusion in Synechocystis sp. Strain PCC6803: Effect of pxcA Inactivation on CO2, HCO3−, and NO3− Uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na⁺-dependent proton extrusion. In the presence of 2,5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO₂ fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO₂ fixation is inhibited. At pH 8.0, NO₃⁻ uptake activity was very low in the pxcA mutant at low [Na⁺] (~100 μM). At pH 6.5, the pxcA strain did not take up CO₂ or NO₃⁻ at low [Na⁺] and showed very low CO₂ uptake activity even at 15 mM Na⁺. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO₂, HCO₃⁻, and NO₃⁻ is discussed.     

Albumin-binding PARACEST agents

Lanthanide complexes (Eu³⁺, Gd³⁺ and Yb³⁺) of two different 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tetraamide derivatives containing two (2) and four (3) O-benzyl-l-serine amide substituents were synthesized and their chemical exchange saturation transfer (CEST) and relaxometric properties were examined in the presence and absence of human serum albumin (HSA). Both Eu2 and Eu3 display a significant CEST effect from a single slowly exchanging Eu³⁺-bound water molecule, making these PARACEST complexes potentially useful as vascular MRI agents. Yb2 also showed a detectable CEST effect from both the Yb³⁺-bound water protons and the exchangeable NH amide protons, making it potentially useful as a vascular pH sensor. Fluorescence displacement studies using reporter molecules indicate that both Gd2 and Gd3 displace dansylsarcosine from site II of HSA with inhibition constants of 32 and 96 μM, respectively, but neither complex significantly displaces warfarin from site I. Water proton relaxation enhancements of 135 and 171% were observed upon binding of Gd2 and Gd3 to HSA, respectively, at 298 K and pH 7.4.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

HMGB‐1 induces c‐kit+ cell microvascular rolling and adhesion via both toll‐like receptor‐2 and toll‐like receptor‐4 of endothelial cells

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1–mediated adhesion and rolling of c-kit⁺ cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit⁺ cells are implicated in the activation of downstream migration signals to peripheral c-kit⁺ cells. Effects of HMGB-1 on the c-kit⁺ cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (−/−) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c-kit⁺ cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm² in ‘WT’ versus 1.1 ± 1.0 cells/mm² in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (−/−) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm² in ‘TLR-2 (−/−)’ and 2.4 ± 1.4 cells/mm² in ‘Tlr4 (LPS-del)’ versus 14.6 ± 5.1 cells/mm² in ‘WT’, P < 0.05). TLR-2 (−/−) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit⁺ cell recruitment via endothelial TLR-2 and TLR-4.     

The effect of regioisomerism on the coordination chemistry and CEST properties of lanthanide(III) NB-DOTA-tetraamide chelates

Chemical exchange saturation transfer (CEST) offers many advantages as a method of generating contrast in magnetic resonance images. However, many of the exogenous agents currently under investigation suffer from detection limits that are still somewhat short of what can be achieved with more traditional Gd³⁺ agents. To remedy this limitation we have undertaken an investigation of Ln³⁺ DOTA-tetraamide chelates (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that have unusually rigid ligand structures: the nitrobenzyl derivatives of DOTA-tetraamides with (2-phenylethyl)amide substituents. In this report we examine the effect of incorporating hydrophobic amide substituents on water exchange and CEST. The ligand systems chosen afforded a total of three CEST-active isomeric square antiprismatic chelates; each of these chelates was found to have different water exchange and CEST characteristics. The position of a nitrobenzyl substituent on the macrocyclic ring strongly influenced the way in which the chelate and Ln³⁺ coordination cage distorted. These differential distortions were found to affect the rate of water proton exchange in the chelates. But, by far the greatest effect arose from altering the position of the hydrophobic amide substituent, which, when forced upwards around the water binding site, caused a substantial reduction in the rate of water proton exchange. Such slow water proton exchange afforded a chelate that was 4.5 times more effective as a CEST agent than its isomeric counterparts in dry acetonitrile and at low temperatures and very low presaturation powers.     

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, GdnH⁺, and Ca²⁺), identity. The Q* decreased in the order F⁻ > Cl⁻ > Br⁻ > NO₃⁻ ∼ I⁻ > SCN⁻ > ClO₄⁻ ≫ SO₄²⁻, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO₄²⁻ anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.     

Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation: A NOVEL MECHANISM FOR INCREASING TENSION AND REDUCING TENSION COST*

Nebulin is a giant filamentous F-actin-binding protein (∼800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (−log[Ca²⁺]) and force-ATPase relations, as well as the rate of tension re-development (k_tr) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons⁻¹ mm⁻¹ s⁻¹) and reductions in k_tr (4.7 versus 7.3 s⁻¹), calcium sensitivity (pCa₅₀ 5.74 versus 5.90), and cooperativity of activation (n_H 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.     

Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO₃⁻ catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO₂; the values of the second order catalytic rate constant (k_on) are 4.1 × 10⁵ and 4.5 × 10⁵ m⁻¹ s⁻¹, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k_on (pK_a = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO₂. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO₂, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10⁻⁴ and 9.7 × 10⁻⁴ m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.     

Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice

Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1^−/− mice. Four-month-old male Srd5a1 ^−/− mice had reduced trabecular bone mineral density (−36%, p<0.05) and cortical bone mineral content (−15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1 ^−/− mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1 ^−/− mice. Male Srd5a1 ^−/− mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1 ^−/− mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1 ^−/− mice, is an indirect effect mediated by elevated circulating androgen levels.     

Ion‐specific modulation of protein interactions: Anion‐induced,reversible oligomerization of a fusion protein

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s_w) and hydrodynamic diameter (D_H) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s_w and D_H, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D_H and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M_z) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I⁻ > Br⁻ > Cl⁻ > F⁻) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.     

Characterization of Lung Cancer by Amide Proton Transfer (APT) Imaging: An In-Vivo Study in an Orthotopic Mouse Model

Amide proton transfer (APT) imaging is one of the chemical exchange saturation transfer (CEST) imaging methods which images the exchange between protons of free tissue water and the amide groups (−NH) of endogenous mobile proteins and peptides. Previous work suggested the ability of APT imaging for characterization of the tumoral grade in the brain tumor. In this study, we tested the feasibility of in-vivo APT imaging of lung tumor and investigated whether the method could differentiate the tumoral types on orthotopic tumor xenografts from two malignant lung cancer cell lines. The results revealed that APT imaging is feasible to quantify lung tumors in the moving lung. The measured APT effect was higher in the tumor which exhibited more active proliferation than the other. The present study demonstrates that APT imaging has the potential to provide a characterization test to differentiate types or grade of lung cancer noninvasively, which may eventually reduce the need invasive needle biopsy or resection for lung cancer.     

The ClC-3 Cl?/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl⁻/H⁺ antiport current (I_ClC-3) in HEK293 cells. The outward rectification and time dependence of I_ClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl⁻ current (ICl_acid) seen at extracellular pH ≤ 5.5. ICl_acid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl_acid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E_rev) of I_ClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl⁻ and H⁺ transport become uncoupled at low pH. At pH 4.0 E_rev was completely Cl⁻ dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native ICl_acid; 1) RNA interference directed at ClC-3 message reduced native ICl_acid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type I_ClC-3 and ICl_acid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I_ClC-3 and from native ICl_acid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl_acid. Low pH uncouples Cl⁻/H⁺ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl⁻/H⁺ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.     

Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease

Chemical Exchange Saturation Transfer (CEST) is an MRI approach that can indirectly detect exchange broadened protons that are invisible in traditional NMR spectra. We modified the CEST pulse sequence for use on high-resolution spectrometers and developed a quantitative approach for measuring exchange rates based upon CEST spectra. This new methodology was applied to the rapidly exchanging Hδ1 and Hε2 protons of His57 in the catalytic triad of bovine chymotrypsinogen-A (bCT-A). CEST enabled observation of Hε2 at neutral pH values, and also allowed measurement of solvent exchange rates for His57-Hδ1 and His57-Hε2 across a wide pH range (3–10). Hδ1 exchange was only dependent upon the charge state of the His57 (k _ex,Im+ = 470 s⁻¹, k _ex,Im = 50 s⁻¹), while Hε2 exchange was found to be catalyzed by hydroxide ion and phosphate base ( k_{\textOH ^-} k_{{{\text{OH}}^{ - } }}  = 1.7 × 10¹⁰ M⁻¹ s⁻¹, k_{\textHPO4^{2 -}} k_{{{\text{HPO}}_{4}^{2 - } }}  = 1.7 × 10⁶ M⁻¹ s⁻¹), reflecting its greater exposure to solute catalysts. Concomitant with the disappearance of the Hε2 signal as the pH was increased above its pK _a, was the appearance of a novel signal (δ = 12 ppm), which we assigned to Hγ of the nearby Ser195 nucleophile, that is hydrogen bonded to Nε2 of neutral His57. The chemical shift of Hγ is about 7 ppm downfield from a typical hydroxyl proton, suggesting a highly polarized O–Hγ bond. The significant alkoxide character of Oγ indicates that Ser195 is preactivated for nucleophilic attack before substrate binding. CEST should be generally useful for mechanistic investigations of many enzymes with labile protons involved in active site chemistry.     

Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1^−/− skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K_1/2) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K_1/2 for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K_1/2 for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K_1/2 for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in “parallel” at alkaline pH. In the second “serial” model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.     

Genome-wide Association Study Reveals Multiple Nasopharyngeal Carcinoma-Associated Loci within the HLA Region at Chromosome 6p21.3

Nasopharyngeal carcinoma (NPC) is a multifactorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. To identify the common genetic variants linked to NPC susceptibility, we conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3. Associations were replicated in two independent sets of case-control samples. Two of the most significant SNPs (rs2517713 and rs2975042; p_combined = 3.9 × 10⁻²⁰ and 1.6 × 10⁻¹⁹, respectively) were located in the HLA-A gene. Moreover, we detected significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 (GABBR1) (rs29232; p_combined = 8.97 × 10⁻¹⁷) and HLA-F (rs3129055 and rs9258122; p_combined = 7.36 × 10⁻¹¹ and 3.33 × 10⁻¹⁰, respectively). Notably, the association of rs29232 remained significant (residual p < 5 × 10⁻⁴) after adjustment for age, gender, and HLA-related SNPs. Furthermore, higher GABA_B receptor 1 expression levels can be found in the tumor cells in comparison to the adjacent epithelial cells (p < 0.001) in NPC biopsies, implying a biological role of GABBR1 in NPC carcinogenesis. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Although some of these relationships may be attributed to linkage disequilibrium between the loci, the findings clearly provide a fresh direction for the study of NPC development.     

Pressure-accelerated dissociation of amyloid fibrils in wild-type hen lysozyme

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml⁻¹ fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (k_obs = 0.00193 min⁻¹ at 0.1 MPa, 0.0348 min⁻¹ at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and the activation compressibility Δκ^‡ = −0.0075 ± 0.0006 ml mol(monomer)⁻¹ bar⁻¹ for the dissociation reaction. The negative ΔV^0‡ and Δκ^‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril.     

The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway

The NO reductase from Paracoccus denitrificans reduces NO to N₂O (2NO + 2H⁺ + 2e⁻ → N₂O + H₂O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O₂-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O₂ reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O₂. We further show that electron transfer during single-turnover reduction of O₂ is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.     

Oxygen-binding characteristics of three extra-cellular haemoglobins of Artemia salina

The oxygen-binding characteristics of the three extracellular haemoglobins of brine shrimp (Artemia salina) were studied in vitro by using highly purified preparations. Haemoglobin I is induced last in the development of brine shrimps when functional gills are formed. It has the lowest oxygen affinity (p₅₀ 5.34mmHg), an intermediate Bohr effect (ø −0.09 at 20°C) above pH8 and a temperature-sensitivity (ΔH −44.8 to −45.6kJ/mol at pH8–9) comparable with those observed with other invertebrate haemoglobins [Weber & Heidemann (1977) Comp. Biochem. Physiol. A 57, 151–155]. Haemoglobin II, which is the first to be induced, soon after hatching of nauplius larvae, persists generally throughout the whole adult life. It has an intermediate oxygen affinity (p₅₀ 3.7mmHg), the highest Bohr effect (ø −0.21 at 20°C) above pH8 and a similar temperature-sensitivity (ΔH −46.0 to −54.8kJ/mol at pH8–9) as haemoglobin I. However, haemoglobin III, which is induced second several hours after the induction of haemoglobin II but disappearing from the haemolymph in the middle of adult life, has the highest oxygen affinity (p₅₀ 1.8mmHg), the lowest Bohr effect (ø −0.03 at 20°C) above pH8.5 and a high resistance against temperature variation between 10 and 25°C at pH8.5–9 (ΔH −22.6 to −23.0kJ/mol). At pH7.5–8, haemoglobin III exhibits a similar temperature-sensitivity under 30°C as do other haemoglobins. All three haemoglobins have a rather low co-operativity, with Hill coefficients (h 1.6–1.9 at pH8.5), which are dependent on both pH and temperature. The highest co-operativity was observed at 20°C and pH9 for haemoglobins I and II, whereas it was at 27°C and pH8.5 for haemoglobin III. Thus the oxygen-binding behaviour of haemoglobin III in vitro is significantly different from those of haemoglobins I and II and indicates possibly its specific physiological role in vivo in the adaptive process in the natural environment.     

Unique Extracellular Matrix Heparan Sulfate from the Bivalve Nodipecten nodosus (Linnaeus, 1758) Safely Inhibits Arterial Thrombosis after Photochemically Induced Endothelial Lesion

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing ∼4.6 mg g⁻¹ of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional ¹H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg⁻¹, 5-fold lower than porcine heparin (180 IU mg⁻¹), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC₅₀ = 0.835 μg ml⁻¹) and thrombin (IC₅₀ = 9.3 μg ml⁻¹) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg⁻¹, the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.