Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

Impact of polymorphism of the regulatory subunit of the ��-calpain (CAPN1S) on the proteolysis process and meat tenderness of young cattle

The objective of this study was to estimate the impact of the polymorphism of ??-calpain (CAPN1S) gene on protein changes of the cattle muscle tissue and its tenderness during 10-day cold storage. The analysis was performed on the longest dorsal and lumbar muscles collected from 76 bulls 6 to 12 months of age. Polymorphism identification of the above-mentioned gene was conducted using the PCR-RFLP technique. Its effect on the course of the proteolysis process was assessed by monitoring changes in proportions of tissue proteins during 10-day process of meat ageing. Special attention was focused on changes in native titin (T1) share and products of its degradation (proteins of molecular weight (m.w.) of 2400 and 200 kDa), ??-actinin and protein of 37 kDa as well as myosin heavy chains (MHC). In the case of the last proteins, their polymorphism was evaluated as well. Meat tenderness was estimated measuring the value of shear force and sensorially. The highest tenderness was ascertained for the heterozygote. Its improvement was associated with a significant decrease in proportions of proteins of molecular weight of approximately 37 kDa accompanied by an increase of those with 200 kDa molecular weight. Muscles derived from cattle of CT genotype were characterised by the highest proportions of type 2a MHC isoform. Value differences between proportions determined for the heterozygote and CC and TT homozygotes of the CAPN1S gene were statistically significant. Therefore, it can be presumed that the process of meat tenderisation was especially connected with MHC polymorphism.     

Assignment of the 1H, 13C and 15N resonances of the calponin homology-2 domain of α-actinin-4

We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology domains of α-actinin combine to regulate F-actin binding.     

Solution conformations of the pituitary opioid peptide dynorphin-(1–13)

Circular dichroism spectra have been measured for dynorphin-(1–13) in water and in solutions of sodium dodecyl sulfate and L-α-lysophosphatidylcholine (palmitoyl). Spectra in water have the features expected for a peptide containing little, if any, order. Small changes are brought about by L-α-lysophosphatidylcholine (palmitoyl), but the resulting spectrum retains the characteristics expected for a random coil. In contrast, sodium dodecyl sulfate produces significant changes which are those expected for induction of α helical content. Quantitative analysis of the circular dichroism spectra suggests the conformation changes from about 5% helix in water to 17% helix in sodium dodecyl sulfate. These results from experiment are in excellent agreement with those obtained from our formulation of the configuration partition function. This formulation predicts a change in helical content from 1% to 19%. The ordering influence is felt most strongly by those residues immediately following the enkephalin sequence.     

Photochemistry and photophysics of the endoperoxide of mesodiphenylhelianthrene: a contribution to the localization of the S1(π*σ*) state of aromatic endoperoxides

The endoperoxide of mesodiphenylhelianthrene MDHPO has been studied in detail with respect to fluorescence and photo-induced rearrangement. MDHPO proved to be non-fluorescent, although its absorption spectrum is dominated at the low energy side by a strong ππ* band with a maximum at 429.5 nm. Irradiation of that band effects rearrangement to the corresponding diepoxide MDHDO, a reaction typical for S(1)(π*σ*) excited endoperoxides (EPOs). The absorption spectrum of the product MDHDO is blue shifted by only 3.5 nm. MDHDO has the same extended planar aromatic system like its precursor MDHPO, but MDHDO fluoresces strongly. These results set the excitation energy of the S(1)(π*σ*) state of MDHPO to ≤23?000 cm(-1), which is considered to be a generally realistic value of the S(1)(π*σ*) state energy of aromatic EPOs. The main reaction of S(1)(π*σ*) excited MDHPO is, however, chemical deactivation to ground state MDHPO via an oxygen biradical. The sequence of O-O bond opening and closing is the general way of repopulation of the S(0) state of aromatic EPOs from S(1)(π*σ*) excited states.     

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

The dependency of the stereoselectivity of penicillin amidases-enzymes with R-specific S1- and S-specific S′1-subsites- on temperature and primary structure

Summary The stereoselectivity of penicillin amidase (PA, EC 3.5.1.11) from E coli and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis. The stereoselectivity of these reactions decreased almost by one order of magnitude from 5 to 45°C. It increased with the substrate (k _cat/K _m) and nucleophile (k _T/k _H) specificity, and was found to differ in the S₁- (R-specific) and S₁-(S-specific)-binding subsites of the active site. The S₁-stereoselectivity was determined mainly by differences in the activation energy, i.e. the turnover number. The stereoselectivity of PA from different sources differed by almost an order of magnitude for the same substrate.     

Assignment of the ferriheme resonances of high- and low-spin forms of the symmetrical hemin-reconstituted nitrophorins 1–4 by 1H and 13C NMR spectroscopy: the dynamics of heme ruffling deformations

The four major nitrophorins (NPs) of the adult blood-sucking insect Rhodnius prolixus have been reconstituted with the "symmetrical hemin" 2,4-dimethyldeuterohemin, and their NMR spectra have been investigated as the high-spin (S = 5/2) aqua and low-spin (S = 1/2) N-methylimidazole (NMeIm) and cyanide complexes. The NMeIm complexes allow assignment of the high-spin hemin resonances by saturation transfer difference spectroscopy. The cyanide complexes were investigated as paramagnetic analogues of the NO complexes. It is shown that the hemin ring is highly distorted from planarity, much more so for NP2 than for NP1 and NP4 (with ruffling being the major distortion mode), for both high- and low-spin forms. For the cyanide complexes, the conformation of the distorted ring changes on the NMR timescale to yield chemical exchange (exchange spectroscopy, EXSY) cross peaks for NP1sym(CN), NP3sym(CN) and NP4sym(CN) but not for NP2sym(CN). These changes in nonplanar conformation are visualized as a "rolling" of the ruffled macrocycle ridges through some number of degrees, the lowest-energy ruffling mode. This probably occurs in response to slow protein dynamics that cause the I120 and L132 side chains in the distal heme pocket to move in opposite directions (up and away vs. down and toward the hemin ring). This in turn changes the out-of-plane displacements of the 2M and 3M of the symmetrical hemin on the NMR timescale. Two other types of dynamics, i.e., changes in heme seating and NMeIm rotation, are also observed. The highly distorted heme and the dynamics it causes are unique to the NPs and a few other heme proteins with highly distorted macrocycles.     

Trypanosoma cruzi: molecular identification and characterization of new members of the Tc13 family. Description of the interaction between the Tc13 antigen from Tulahuén strain and the second extracellular loop of the beta(1)-adrenergic receptor

Trypanosoma cruzi Tc13 antigens belong to the trans-sialidase superfamily. Their sequences have been described only partially and, up to now, their physiological activity has not been elucidated. Here we present two new members of this family from the Tulahuén strain (Tc13 Tul) and the CL Brener clone (Tc13 CL), being the latter the first Tc13 sequence fully described. Alignment of all Tc13 sequences allowed us to define two sub-families that differ in the number of repeats and the presence or absence of the GPI addition site. Chromoblots demonstrate that Tc13 antigens are mainly located in chromosome III and its homologous. Pull down assays suggest that recombinant MBP-Tc13 Tul interacts with the second extracellular loop of the beta(1)-adrenergic receptor. This is the first evidence that a Tc13 antigen acts as a ligand interacting with a neurotransmitter receptor. These observations might add some light to the development of chagasic pathology.     

Arrangement of the Template 3′ of the A-Site Codon on the Human 80S Ribosome

The arrangement of the template sequence 3′ of the A-site codon on the 80S ribosome was studied using mRNA analogs containing Phe codon UUU at the 5′ end and a photoreactive perfluoroarylazido group linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which directed the UUU codon to the P site, bringing a modified nucleotide to position +9 or +12 relative to the first nucleotide of the P-site codon. Upon mild UV irradiation of ribosome complexes, the analogs of both types crosslinked to the 18S rRNA and proteins of the 40S subunit. Comparisons were made with the crosslinking patterns of complexes in which an mRNA analog contained a modified nucleotide in position +7 (the crosslinking to 18S rRNA in such complexes has been studied previously). The efficiency of crosslinking to ribosomal components depended on the nature of the modified nucleotide of an mRNA analog and its position on the ribosome. The extent of crosslinking to the 18S rRNA drastically decreased as the modified nucleotide was transferred from position +7 to position +12. The 18S rRNA nucleotides involved in crosslinking were identified. A modified nucleotide in position +9 crosslinked to the invariant dinucleotide A1824/A1825 and variable A1823 in the 3′ minidomain of the 18S rRNA and to S15. The same ribosomal components have earlier been shown to crosslink to modified nucleotides in positions +4 to +7. In addition, all mRNA analogs crosslinked to invariant C1698 in the 3′ minidomain and to conserved region 605–620, which closes helix 18 in the 5′ domain.     

Accurate measurements of the effects of deuteration at backbone amide positions on the chemical shifts of 15N, 13Cα, 13Cβ, 13CO and 1Hα nuclei in proteins

An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins (¹⁵N, ¹³C_α, ¹³CO, ¹H_α) and ¹³C_β nuclei arising from ¹H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond ¹³C_α isotope shifts, ²ΔC_α(N_iD) and ³ΔC_α(N_i+1D), and three-bond ¹³C_β isotope shifts, ³ΔC_β(N_iD), are useful reporters of the local geometry of the protein backbone.     

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β

Introduction

Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.

Methods

Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO₂) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs).

Results

Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1β versus 52.91% ± 9.35% (SD) with carprofen + IL-1β.

Conclusions

Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.     

The Suppressive Effect of β-Glucan on the Production of Tumor Necrosis Factor-Alpha in BV2 Microglial Cells

β-Glucan was recently shown to have the ability to enhance and stimulate the immune system in humans, but little is known about its the anti-inflammatory effects. We investigated the effect of β-glucan on the production of tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory mediator, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. β-Glucan decreased the production and expression of TNF-α. In addition, it blocked LPS-stimulated activation of nuclear factor kappa B (NF-κB). Hence β-glucan might suppress LPS-stimulated TNF-α production by inhibiting NF-κB in BV2 microglial cells.     

Age-dependent Expression of S100β in the Brain of Mice

S100β is a soluble calcium binding protein released by glial cells. It has been reported as a neurotrophic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and in long term potentiation in the adult brain. The ability of S100β to modulate neuronal morphology raises the important question whether there is an age-related difference in the expression of S100β in the cerebral and cerebellar cortices of AKR strain mice and is this change is region specific. Our RT–PCR and Western blotting experiments show that the expression of S100β gene in the cerebral and cerebellar cortices starts from 0 day, peaks at about 45 days. However, in 70-week old mice its expression is significantly up-regulated as compared to that of 20-week old mice. S100β follows the same age-related pattern in both cerebral and cerebellar cortices. These results suggest that S100β is important for brain development and establishment of proper brain functions. Up-regulation of S100β in old age may have some role in development of age-related pathological systems in the brain.