Synthesis of the β-amyloid fragment <Superscript>5</Superscript>RHDSGY<Superscript>10</Superscript> and its isomers

The peptide RHDSGY, a fragment of the human β-amyloid Zn-binding site, and its isomers RH(D-Asp)SGY and RH(β-Asp)SGY have been obtained as amides by means of solid-phase synthesis and analyzed by HPLC and various mass spectrometric methods. The problem of low yield of the RHDSGY peptide and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of such side-products as RH(β-Asp)SGY (or RHDSGY during synthesis of RH(β-Asp)SGY) and RH(Asp-imide) SGY was solved via selection of individual reagents for removal of Fmoc groups from α-amino groups of the growing peptide chain.     

A Voltage-Dependent Ca<Superscript>2+</Superscript> Influx Pathway Regulates the Ca<Superscript>2+</Superscript>-Dependent Cl<Superscript>−</Superscript> Conductance of Renal IMCD-3 Cells

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca²⁺-dependent Cl⁻ current (I_CLCA). Here we report that I_CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al³⁺-sensitive, voltage-dependent, Ca²⁺ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), I_CLCA was found to be inactive and resting currents were predominantly K⁺ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I_CLCA (T _0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in I_CLCA (T _0.5 ~ 100 s). The activation of I_CLCA by depolarization was reduced by lowering extracellular Ca²⁺ and completely inhibited by buffering cytosolic Ca²⁺ with EGTA, suggesting a role for Ca²⁺ influx in the activation of I_CLCA. However, raising bulk cytosolic Ca²⁺ at −60 mV did not produce sustained I_CLCA activity. Therefore I_CLCA is dependent on both an increase in intracellular Ca²⁺ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca²⁺ influx pathway that displays equal permeability to Ca²⁺ and Ba²⁺ ions and that is blocked by extracellular Al³⁺ and La³⁺. Furthermore, Al³⁺ completely and reversibly inhibited depolarization-induced activation of I_CLCA, thereby directly linking Ca²⁺ influx to activation of I_CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I_CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

Accuracy of the StressEraser<Superscript>®</Superscript> in the Detection of Cardiac Rhythms

StressEraser is a commercially marketed biofeedback device designed to enhance heart rate variability. StressEraser makes its internal calculations on beat-to-beat measures of finger pulse intervals. However, the accuracy and precision of StressEraser in quantifying interbeat intervals using finger pulse intervals has not been evaluated against standard laboratory equipment using R-R intervals. Accuracy was assessed by simultaneously recording interbeat intervals using StressEraser and a standard laboratory ECG system. The interbeat intervals were highly correlated between the systems. The average deviation in interbeat interval recordings between the systems was approximately 6 ms. Moreover, correlations approached unity between the systems on estimates of heart period, heart rate, and heart rate variability. Feedback from StressEraser is based on an interbeat time series that provides sufficient information to provide an excellent estimate of the dynamic changes in heart rate and heart rate variability. The slight variations between StressEraser and the laboratory equipment in quantifying heart rate and heart rate variability are due to features related to monitoring heart rate with finger pulse: (1) a lack in precision in the peak of the finger pulse relative to the clearly defined inflection point in the R-wave, and (2) contribution of variations in pulse transit time.     

Anionic Selectivity Sequence of the Cl<Superscript>−</Superscript>–H<Superscript>+</Superscript> Symporter in the Synaptosomal Preparation from Rat Brain Cortex

The Na⁺/H⁺ exchanger has been the only unequivocally demonstrated H⁺-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl⁻–H⁺ symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl⁻–H⁺ symporter is NO₃⁻ > Br⁻ > Cl⁻ >> I⁻ = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 μM inhibits the gluconate-dependent alkalinization by 30 ± 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization. Special issue article in honor of Dr. Ricardo Tapia.     

Structure of the “Peroxy-Y Base” from Liver tRNA<Superscript>Phe</Superscript>

WE wish to report that reconstituted sperm whale myoglobin prepared by the method of Breslow¹ (except that pH 2 was found sufficient to remove all the haem) (I) crystallizes² in a different habit from those prepared by the method of Rossi-Fanelli et al.³ (II) using haemin of Sigma lot 77B-0220 and our own ⁵⁷Fe photoporphyrin preparation and the native myoglobin (III). Although all three form type A³ monoclinic prisms, the best developed plane is [001] for II and III, it is [100] for I. There seems to be great interest in reconstituted haemoproteins^4,5, so it is important that crystallization habit may be a sensitive test for subtle changes in protein structures.     

A <Superscript>13</Superscript>C-detected <Superscript>15</Superscript>N double-quantum NMR experiment to probe arginine side-chain guanidinium <Superscript>15</Superscript>N<Superscript>η</Superscript> chemical shifts

Arginine side-chains are often key for enzyme catalysis, protein–ligand and protein–protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine ¹³C^ζ-detected NMR experiment in which a double-quantum coherence involving the two ¹⁵N^η nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two ¹⁵N^η; this new approach is shown to eliminate the previously deleterious line broadenings of ¹⁵N^η resonances caused by the partially restricted rotation about the C^ζ–N^ε bond. Consequently, sharp and well-resolved ¹⁵N^η resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine ¹⁵N^η nuclei becomes possible using the double-quantum experiment.     

Volume-sensitive Cl<Superscript>−</Superscript> Current in Bovine Adrenocortical Cells: Comparison with the ACTH-induced Cl<Superscript>−</Superscript> Current

In a previous study performed on zona fasciculata (ZF) cells isolated from calf adrenal glands, we identified an ACTH-induced Cl⁻ current involved in cell membrane depolarization. In the present work, we describe a volume-sensitive Cl⁻ current and compare it with the ACTH-activated Cl⁻ current. Experiments were performed using the whole-cell patch-clamp recording method, video microscopy and cortisol-secretion measurements. In current-clamp experiments, hypotonic solutions induced a membrane depolarization to −22 mV. This depolarization, correlated with an increase in the membrane conductance, was sensitive to different Cl⁻ channel inhibitors. In voltage-clamp experiments, hypotonic solution induced a membrane current that slowly decayed and reversed at −21 mV. This ionic current displayed no time dependence and showed a slight outward rectification. It was blocked to variable extent by different conventional Cl⁻-channel inhibitors. Under hypotonic conditions, membrane depolarizations were preceded by an increase in cell volume that was not detected under ACTH stimulation. It was concluded that hypotonic solution induced cell swelling, which activated a Cl⁻ current involved in membrane depolarization. Although cell volume change was not observed in the presence of ACTH, biophysical properties and pharmacological profile of the volume-sensitive Cl⁻ current present obvious similarities with the ACTH-activated Cl⁻ current. As compared to ACTH, hypotonic solutions failed to trigger cortisol production that was weakly stimulated in the presence of high-K⁺ solution. This shows that in ZF cells, membrane depolarization is not a sufficient condition to fully activate secretory activities.This revised version was published online in August 2005 with a corrected cover date.     

Tracking the fate of digesta <Superscript>13</Superscript>C and <Superscript>15</Superscript>N compositions along the ruminant gastrointestinal tract: Does digestion influence the relationship between diet and faeces?

Faecal stable isotope compositions reflect wildlife diets, if digestive processes along the gastrointestinal tract (GIT) do not alter diet–faeces isotopic relationships in an unpredictable way. We investigated ¹³C and ¹⁵N compositions of digesta along the ruminant GIT, using Saanen dairy goats kept on pure grass hay or browse for >20 days. Isotopic changes occurred in the ventral rumen, and in the small intestine, where digesta had significantly higher δ¹³C and δ¹⁵N (associated with lower C or higher N content, respectively) values relative to other GIT sites. However, effects on isotope fractionation were small (∼1.0‰ for δ¹³C and ∼ 2.0‰ for δ¹⁵N), and were reversed in the hindgut such that faecal isotope compositions did not differ from the foregut. No other substantial isotopic changes occurred across GIT sites, despite the morphophysiological complexity of the ruminant GIT. We found similarly small differences across GIT components of rheem gazelles (Gazella leptoceros) fed a mixture of C₃ lucerne and C₄ grass, although in this case faeces were ¹⁵N-depleted relative to other GIT components. Along with differences in δ¹⁵N between goats fed browse or grass, this result implies a systematic difference in diet–faeces δ¹⁵N relationships, contingent on the botanical composition of ruminant diets. Thus, while our results support faecal δ¹³C as a reliable proxy for wildlife diets, further work on factors influencing faecal ¹⁵N abundance is needed. Finally, we note high levels of isotopic variability between individuals fed the same diets, even accounting for the relatively short duration of the experiments, suggesting an important influence of stochasticity on isotope fractionation.     

Investigation of the encapsulation of metal cations (Cu<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript>) by the dipeptide Phe–Phe using natural bond orbital theory and molecular dynamics simulation

Complexes of the dipeptide phenylalanine–phenylalanine (Phe–Phe) with divalent metal cations (Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺) were studied at the B3LYP and MP2 levels of theory with the basis sets 6-311++G(d,p) and 6-31 + G(d) in the gas phase. The relative energies of these complexes indicated that cation–π bidentate/tridentate conformations are more favourable than other conformations with uncoordinated rings. These findings were confirmed by the calculated values of thermodynamic parameters such as the Gibbs free energy. Natural bond orbital (NBO) analysis was carried out to explore the metal–ligand coordination in Phe–Phe–Cu²⁺/Zn²⁺ complexes. Possible orbital transitions, types of orbitals and their occupancies were determined for a range of Phe–Phe–Cu²⁺/Zn²⁺ complexes. The charge transfer involved in various orbital transitions was explored by considering the second-order perturbation energy. NBO analysis revealed that the change transfer is stronger when the metal cation uses both the 4s + 4p subshells rather than just its 4p subshell. We also performed molecular dynamics (MD) simulations to check the stability and consistency of the most favourable binding motifs of Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺ with Phe–Phe over time. The structures of the Phe–Phe–Cu²⁺/Zn²⁺/Ca²⁺/Ba²⁺ complexes obtained using MD simulation were found to be in good agreement with those obtained in the DFT-based calculations.

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Graphical Abstract Conformational search on encapsulation of divalent metal cations (Ca²⁺, Zn²⁺, Ca²⁺, Ba²⁺) by the Phe-Phe dipeptide

     

Rapid measurement of <Superscript>3</Superscript>J(H<Superscript>N</Superscript>–H<Superscript>α</Superscript>) and <Superscript>3</Superscript>J(N–H<Superscript>β</Superscript>) coupling constants in polypeptides

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.     

H<Superscript>N</Superscript>, N,C<Superscript>α</Superscript> and C<Superscript>β</Superscript> assignments of the two periplasmic domains of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> DsbD

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H^N, N, C^α and C^β) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using ¹³C^αβ shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.     

Increased Cell Proliferation and Neurogenesis in the Hippocampal Dentate Gyrus of Old GFAP<Superscript>−/−</Superscript>Vim<Superscript>−/−</Superscript> Mice

In response to central nervous system (CNS) injury, and more discretely so also during aging, astrocytes become reactive and increase their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Studies of mice deficient in astrocytic intermediate filaments have provided insights into the function of reactive gliosis. Recently we demonstrated robust integrationof retinal transplants (1) and increased posttraumatic synaptic regeneration (2) in GFAP^–/–Vim^–/– mice, suggesting that modulation of astrocyte activity affects the permissiveness of the CNS environment for regeneration. Neurogenesis in the adult mammalian CNS is restricted to essentially two regions, the hippocampus and the subventricular zone. Here, we assessed neurogenesis in the hippocampus of 18-month-old GFAP^–/–Vim^–/– mice. In the granular layer of the dentate gyrus, cell proliferation/survival was 34% higher and neurogenesis 36% higher in GFAP^–/–Vim^–/– mice than in wildtype controls. These findings suggest that the adult hippocampal neurogenesis in healthy old mice can be increased by modulating astrocyte reactivity.Special issue dedicated to Lawrence. F. Eng.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.