Role of water in structural changes of poly(AVGVP) and poly(GVGVP) Studied by FTIR and Raman spectroscopy and ab initio calculations

Two elastin-like poly(pentapeptides), poly(AV1GV2P) and poly(G1V1G2V2P), have been studied in water and in solid state by ATR FTIR and Raman spectroscopy in combination with model ab initio calculations. In aqueous solutions below the transition temperature T(t), a part of the amide groups and of the methyl groups of both polypentapeptides interacts with neighboring water molecules, whereas the other part of amide groups mutually interacts forming a beta-sheetlike structure. Below T(t), poly(AV1GV2P) is dissolved more perfectly, and the water shells around the polymer chains are more closely structured. The suspension of poly(AV1GV2P) formed above T(t) is more compact and, on cooling, resists more to the reverse dissolution, whereas the suspension of poly(G1V1G2V2P) contains more water molecules bound to the carbonyl of amide groups and on backward cooling dissolves fairly reversibly. The measured poly(pentapeptides) tend to form beta-turns due to the conformational transition on the residue between P and V1.     

Structural and serological studies on the O-antigen of Proteus mirabilis O14, a new polysaccharide containing 2-[(R)-1-carboxyethylamino]ethyl phosphate.

An O-specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain D-galactose, 2-acetamido-2-deoxy-D-glalactose, phosphate, N-(2-hydroxyethyl)-D-alanine (D-AlaEtn), and O-acetyl groups. Studies of the initial and O-deacetylated polysaccharides using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, NOESY, H-detected 1H,13C heteronuclear multiple-quantum coherence, and heteronuclear multiple-bond correlation experiments, demonstrated the following structure of the repeating unit: [equation: see text] This is the second bacterial polysaccharide reported to contain alpha-D-Galp6PAlaEtn, whereas the first one was the O-antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska-Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem., 188, 645-651]. Anti-(P. mirabilis O14) serum cross-reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O-antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O-antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.     

Functional and structural interactions of the transmembrane domain X of NhaA, Na+/H+ antiporter of Escherichia coli, at physiological pH

The 3D structure of Escherichia coli NhaA, determined at pH 4, provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 7.0-8.5), many questions pertaining to the active state of NhaA have remained open, including the physiological role of helix X. Using a structural-based evolutionary approach in silico, we identified a segment of most conserved residues in the middle of helix X. These residues were then used as targets for functional studies at physiological pH. Cysteine-scanning mutagenesis showed that Gly303, in the middle of the conserved segment, is an essential residue and Cys replacement of Lys300 retains only Li+/H+ antiporter activity, with a 20-fold increase in the apparent KM for Li+. Cys replacements of Leu296 and Gly299 increase the apparent KM of the Na+/H+ antiporter for both Na+ and Li+. Accessibility test to N-ethylmaleimide and 2-sulfonatoethyl methanethiosulfonate showed that G299C, K300C, and G303C are accessible to the cytoplasm. Suppressor mutations and site-directed chemical cross-linking identified a functional and/or structural interaction between helix X (G295C) and helix IVp (A130C). While these results were in accordance with the acid-locked crystal structure, surprisingly, conflicting data were also obtained; E78C of helix II cross-links very efficiently with several Cys replacements of helix X, and E78K/K300E is a suppressor mutation of K300E. These results reveal that, at alkaline pH, the distance between the conserved center of helix X and E78 of helix II is drastically decreased, implying a pH-induced conformational change of one or both helices.     

PKC delta phosphorylates p52ShcA at Ser29 to regulate ERK activation in response to H2O2

Both PKC delta and ShcA have been implicated in cell response to oxidative stress [Y. Hu, X. Wang, L. Zeng, D.Y. Cai, K. Sabapathy, S.P. Goff, E.J. Firpo, B. Li, Mol Biol Cell., 16 (2005) 3705-3718, B. Li, X. Wang, N. Rasheed, Y. Hu, S. Boast, T. Ishii, K. Nakayama, K.I. Nakayama, S.P., Goff, Genes Dev, 18 (2004) 1824-1837, E. Migliaccio, M. Giorgio, S. Mele, G. Pelicci, P. Reboldi, P.P. Pandolfi, L. Lanfrancone, P.G. Pelicci, Nature, 402 (1999) 309-313], yet their relationship in the response has not been studied. Here we report that PKC delta interacts with ShcA and this interaction is promoted by H(2)O(2). PKC delta and ShcA are also colocalized in the cytoplasm and displayed co-translocation in response to H(2)O(2). Activated PKC delta was able to phosphorylate ShcA at Ser29, as determined by mass spectrometry. These results suggest that ShcA, p66 and p52, are substrates that interact with PKC delta. This phosphorylation is critical in H(2)O(2) induced ERK activation as reconstitution with ShcA Ser29A failed to rescue ERK activation of ShcA-/- MEFs, while ShcA could. In line with this conclusion, inhibition of PKC delta with inhibitors is able to diminish H(2)O(2) induced ERK activation in MEFs. These results suggest that the interaction between PKC delta and ShcA and the phosphorylation of ShcA at Ser29 play important roles in ERK activation in cell response to H(2)O(2).     

Conformations of nucleoside analogue 1-(2'-deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide in different DNA sequence contexts

The concept of using a dynamic base-pairing nucleobase as a mode for degenerate recognition presents a unique challenge to analysis of DNA structure. Proton and phosphorus NMR studies are reported for two nine-residue DNA oligodeoxyribonucleotides, d(CATGGGTAC).d(GTACNCATG) (1) and d(CATGTGTAC).(GTACNCATG) (2), which contained 1-(2'-deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (N) in the center of the helix at position 14. The duplexes were compared to the canonical Watson-Crick duplexes, d(CATGGGTAC).d(GTACCCATG) (3) and d(CATGTGTAC).d(GTACACATG) (4). Two-dimensional NOESY spectra of 1-4 in H(2)O and D(2)O solutions collected at 5 degrees C allowed assignment of the exchangeable and nonexchangeable protons for all four oligodeoxyribonucleotides. Thermodynamic and circular dichroism data indicated that 1-4 formed stable, B-form duplexes at 5 degrees C. Two-dimensional (1)H-(31)P correlation spectra indicated that there were minor perturbations in the backbone only near the site of the triazole base. Strong NOESY cross-peaks were observed between the H5 and H1' of N14 in 1 and, unexpectedly, 2, which indicated that, in both duplexes, N14 was in the syn(chi)() conformation about the glycosidic bond. NOESY spectra of 1 and 2 recorded in 95% H(2)O, 5% D(2)O indicated that the imino proton of the base opposite N14, G5, or T5, formed a weak hydrogen bond with N14. These conformations place the polar carboxamide functional group in the major groove with motional averaging on the intermediate time scale.     

Structure of the O-polysaccharide and serological cross-reactivity of the Providencia stuartii O33 lipopolysaccharide containing 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the following structure of the tetrasaccharide repeating unit was established: -->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp)-(1-->, where d-Qui4N(Ac-D-Asp) is 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D 1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O-D2O mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related also to P. stuartii O4, whose O-polysaccharide contains a lateral beta-D-Qui4N(Ac-L-Asp) residue.     

Ca(2+)-activated K+ channel inhibition by reactive oxygen species

We studied the effect of H(2)O(2) on the gating behavior of large-conductance Ca(2+)-sensitive voltage-dependent K(+) (K(V,Ca)) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevis oocytes membranes expressing the human Slowpoke (hSlo) alpha-subunit. Exposure of the intracellular side of K(V,Ca) channels to H(2)O(2) (4-23 mM) leads to a time-dependent decrease of the open probability (P(o)) without affecting the unitary conductance. H(2)O(2) did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H(2)O(2) action. Desferrioxamine (60 microM) and cysteine (1 mM) completely inhibited the effect of H(2)O(2), indicating that the decrease in P(o) was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H(2)O(2). However, DTT did completely reverse the decrease in P(o) induced by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of K(V,Ca) channel activity promoted by DTT suggests that H(2)O(2) treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing hSlo channels showed that H(2)O(2) induced a decrease in current mediated by a decrease both in the number of active channels and P(o).     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

A water network within a protein: temperature-dependent water ligation in H64V-metmyoglobin and relaxation to deoxymyoglobin

The sperm whale myoglobin mutant H64V, where the distal histidine is mutated to valine, is known to be five coordinated in the ferric state at room temperature and physiological pH. A change of the ligation in this H64V-Mbmet has been observed by optical absorption spectroscopy as a function of temperature from 20 K to 300 K. Above the dynamical transition at about 180 K one observes the temperature-dependent equilibrium between five- and six-ligated heme. Below the dynamical transition the equilibrium is frozen-in at about 50% of six-coordinate molecules. The water ligation of the iron occurs at temperatures where protein-specific motions are present, as monitored by M?ssbauer spectroscopy. The X-ray structures of H64V-Mbmet at 300 K and 110 K are reported with a resolution of 1.5 A and 1.3 A, respectively. The measurements at high resolutions are possible owing to crystallization in the space group P2(1), whereas all mutant myoglobins studies up to now have been carried out with crystals in the space group P6. The overall structure at both temperatures is very close to the native myoglobin. The binding of water at the sixth coordination site at lower temperatures is possible owing to a stabilizing water network extending from the protein surface to the active centre. The reduction of the H64V-Mbmet by electrons obtained by X-ray irradiation of the water-glycerol solvent at 85 K produces an intermediate low-spin state of the water-ligated molecules where Fe(II) retains the six-fold coordination. M?ssbauer spectroscopy shows that the relaxation of the metastable low-spin state to high-spin H64V-Mbdeoxy with dissociation of the Fe(II)-H(2)O bond starts at about 115 K and is completed at about 170 K. Differences in the dynamics properties of the native and mutant myoglobin and the connection to the dynamical transition around 180 K are discussed.     

Two-dimensional 1H NMR of gene 5 protein indicates that only two aromatic rings interact significantly with oligodeoxynucleotide bases

The interaction of gene 5 protein (G5P) with oligodeoxynucleotides is investigated by 1H NMR methods, principally two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Aromatic resonances of G5P are specifically assigned from crystallographic data, while the low-field resonances of nucleotides are assigned with sequential or other procedures. Chemical shift changes that accompany binding of d(pA)4, d(A)4, d(pT)4, and d(pA)8, combined with specific protein-nucleotide nuclear Overhauser effects (NOEs) obtained from NOESY spectra, suggest that Phe-73 and Tyr-26 are the only aromatic residues that stack significantly with nucleotide bases. Chemical shift data also imply a role for Leu-28, though this has not been confirmed with intermolecular NOEs. Binding of all four oligonucleotides causes marked upfield movements (0.1-0.6 ppm) of G5P NOESY cross peaks belonging to Tyr-26, Leu-28, and Phe-73. Most other G5P spin systems, notably those of Tyr-34 and Tyr-41, do not appear to be significantly affected. In the d(pA)4-G5P complex an intermolecular NOE is observed between Tyr-26 and H1' of Ade-1, while Phe-73 has NOEs with the H2, H8, and H1' protons of Ade-2 and -3. Intramolecular NOEs seem to follow a similar pattern in the partially cooperative d(pA)8-G5P complex, though specific nucleotide resonance assignments are not possible in this case. Binding causes relatively small chemical shift changes for the base resonances in adenylyl nucleotides, suggesting that there is some, but not complete, unstacking of the bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of highly hydroxylated aromatics by evolved biphenyl dioxygenase and subsequent dihydrodiol dehydrogenase

The evolved bphA1 (2049) gene, in which nine amino acids from the Pseudomonas pseudoalcaligenes KF707 BphA1 were changed to those from the Burkholderia xenovorans LB400 BphA1 (M247I, H255Q, V258I, G268A, D303E, -313G, S324T, V325I, and T376N), was expressed in Escherichia coli along with the bphA2A3A4 and bphB genes derived from strain KF707. This recombinant E. coli cells converted biphenyl and several heterocyclic aromatic compounds into the highly hydroxylated products such as biphenyl-2,3,2′,3′-tetraol (from biphenyl), 2-(2,3-dihydroxyphenyl)benzoxazole-4,5-diol (from 2-phenylbenzoxazole), and 2-(2,5-dihydroxyphenyl)benzoxazole-4,5-diol [from 2-(2-hydroxyphenyl)benzoxazole]. The antioxidative activity of these generated compounds was markedly higher than that of the original substrate used.     

New structural proteins of Halobacterium salinarum gas vesicle revealed by comparative proteomics analysis

The Halobacterium salinarum gas vesicle (GV) is an extremely stable intracellular organelle with air trapped inside a proteinaceous membrane. Reported here is a comparative proteomics analysis of GV and GV depleted lysate (GVD) to reveal the membrane structural proteins. Ten proteins encoded by gvp-1 (gvpMLKJIHGFED-1 and gvpACNO-1) and five proteins encoded by gvp-2 (gvpMLKJIHGFED-2 and gvpACNO-2) gene clusters for the biogenesis of spindle- and cylindrical-, respectively, shaped GV were identified by LC-MS/MS. The peptides of GvpA1, I1, J1, A2, and J2 were exclusively identified in purified GV, GvpD1, H1, L1, and F2 only in GVD, and GvpC1, N1, O1, F1, H2, and O2 in both samples. The identification of GvpA1, C1, F1, J1, and A2 in GV is in agreement with their previously known structural function. In addition, the detection of GvpI1, N1, O1, H2, J2, and O2 in GV suggested they are new structural proteins. Among these, the structural role of GvpI1 and N1 in GV was further validated by immuno-detection of protein A-tagged GvpI1 and N1 fusion proteins in purified GV. Thus, LC-MS/MS could reveal at least a half dozen gas vesicle structural proteins in the predominant spindle-shaped GV that may be helpful for studying its biogenesis.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Temperature-dependent osmotic behavior of germinal vesicle and metaphase II stage bovine oocytes in the presence of Me2SO in relationship to cryobiology

Plasma membrane permeability coefficients and their activation energies (Ea) for water (Lp) and dimethyl sulfoxide (PMe2SO) as well as the reflection coefficient (sigma) were determined for germinal vesicle (GV) and metaphase II (MII) bovine oocytes. A micropipette perfusion technique was used with a temperature controlled circulation chamber, which was adapted to a micromanipulator. Experiments were performed at five different temperatures (30, 20, 10, 4 and -3 degrees C). The Kedem and Katchalsky model was assumed and L(p), P(Me2SO) and sigma were estimated. Estimated permeability values from the experimental temperatures were then applied to Arrhenius plots In(Lp) or In(PMe2SO) vs 1/Temperature (K) to estimate the activation energies (Ea) for L(p)Me2SO and P(Me2SO). The estimated E(a) for L(p)Me2SO for GV and MII oocytes were 23.84 Kcal/mol and 8.46 Kcal/mol, respectively. The E(a) for P(Me2SO) were 21.0 Kcal/mol and 23.20 Kcal/mol, respectively. The correlation (r2) for these linear regression plots for GV oocytes were 0.83 and 0.95 for L(p)Me2SO and P(Me2SO), respectively. For MII oocytes, r2 values were 0.95 and 0.99 for L(p)Me2SO and P(Me2SO), respectively. There was a possible discontinuity detected in the Arrhenius plot for L(p)Me2SO for GV oocytes. A significant decrease of the reflection coefficient was observed at 10 degrees C compared to other experimental temperatures. These data provide a fundamental basis that should be taken into account for low temperature preservation of bovine oocytes in the presence of Me2SO.     

H2O2 activates redox- and 4-aminopyridine-sensitive Kv channels in coronary vascular smooth muscle

Previously, we demonstrated that coronary vasodilation in response to hydrogen peroxide (H(2)O(2)) is attenuated by 4-aminopyridine (4-AP), an inhibitor of voltage-gated K(+) (K(V)) channels. Using whole cell patch-clamp techniques, we tested the hypothesis that H(2)O(2) increases K(+) current in coronary artery smooth muscle cells. H(2)O(2) increased K(+) current in a concentration-dependent manner (increases of 14 +/- 3 and 43 +/- 4% at 0 mV with 1 and 10 mM H(2)O(2), respectively). H(2)O(2) increased a conductance that was half-activated at -18 +/- 1 mV and half-inactivated at -36 +/- 2 mV. H(2)O(2) increased current amplitude; however, the voltages of half activation and inactivation were not altered. Dithiothreitol, a thiol reductant, reversed the effect of H(2)O(2) on K(+) current and significantly shifted the voltage of half-activation to -10 +/- 1 mV. N-ethylmaleimide, a thiol-alkylating agent, blocked the effect of H(2)O(2) to increase K(+) current. Neither tetraethylammonium (1 mM) nor iberiotoxin (100 nM), antagonists of Ca(2+)-activated K(+) channels, blocked the effect of H(2)O(2) to increase K(+) current. In contrast, 3 mM 4-AP completely blocked the effect of H(2)O(2) to increase K(+) current. These findings lead us to conclude that H(2)O(2) increases the activity of 4-AP-sensitive K(V) channels. Furthermore, our data support the idea that 4-AP-sensitive K(V) channels are redox sensitive and contribute to H(2)O(2)-induced coronary vasodilation.     

ISO-DALT characterization of 12 ''new'' equine plasma protease inhibitor (Pi) alleles

Twelve equine protease inhibitory alleles, PiE, H, J, K, L2, O, P, Q, R, V, X, Z, have been characterized in terms of isoelectric point, molecular mass and inhibitory activity to bovine trypsin and chymotrypsin by ISO-DALT electrophoresis. Protein maps for 20 Pi alleles including those of the eight 'Thoroughbred' alleles (PiF, G, I, L, N, S1, S2, U) have now been determined. Five pairs of alleles, S1/S2, G/K, L/L2, P/R and U/Z, possessed varying numbers of common proteins ranging from one protein in the case of G/K and L/L2 to six in the case of U/Z. Based on these results and studies of the abnormal expressions of PiF, PiL and PiS1, a theory of at least three closely linked loci has been postulated to account for the marked heterogeneity of the equine protease inhibitory system.     

Structural studies of the O6meG.C interaction in the d(C-G-C-G-A-A-T-T-C-O6meG-C-G) duplex

One- and two-dimensional nuclear magnetic resonance (NMR) experiments have been undertaken to investigate the conformation of the d(C1-G2-C3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) self-complementary dodecanucleotide (henceforth called O6meG.C 12-mer), which contains C3.O6meG10 interactions in the interior of the helix. We observe intact base pairs at G2.C11 and G4.C9 on either side of the modification site at low temperature though these base pairs are kinetically destabilized in the O6meG.C 12-mer duplex compared to the G.C 12-mer duplex. One-dimensional nuclear Overhauser effects (NOEs) on the exchangeable imino protons demonstrate that the C3 and O6meG10 bases are stacked into the helix and act as spacers between the flanking G2.C11 and G4.C9 base pairs. The nonexchangeable base and H1', H2', H2', H3', and H4' protons have been completely assigned in the O6meG.C 12-mer duplex at 25 degrees C by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) experiments. The observed NOEs and their directionality demonstrate that the O6meG.C 12-mer is a right-handed helix in which the O6meG10 and C3 bases maintain their anti conformation about the glycosidic bond at the modification site. The NOEs between the H8 of O6meG10 and the sugar protons of O6meG10 and adjacent C9 exhibit an altered pattern indicative of a small conformational change from a regular duplex in the C9-O6meG10 step of the O6meG.C 12-mer duplex. We propose a pairing scheme for the C3.O6meG10 interaction at the modification site. Three phosphorus resonances are shifted to low field of the normal spectral dispersion in the O6meG.C 12-mer phosphorus spectrum at low temperature, indicative of an altered phosphodiester backbone at the modification site. These NMR results are compared with the corresponding parameters in the G.C 12-mer, which contains Watson-Crick base pairs at the same position in the helix.     

Thermodynamics of single-stranded RNA binding to oligolysines containing tryptophan.

The equilibrium binding to the synthetic RNA poly(U) of a series of oligolysines containing one, two, or three tryptophans has been examined as a function of pH, monovalent salt concentration (MX), temperature, and Mg2+. Oligopeptides containing lysine (K) and tryptophan (W) of the type KWKp-NH2 and KWKp-CO2 (p = 1-8), as well as peptides containing additional tryptophans or glycines, were studied by monitoring the quenching of the peptide tryptophan fluorescence upon binding poly(U). Equilibrium association constants, K(obs), and the thermodynamic quantities delta G(o)obs, delta H(o)obs, and delta S(o)obs describing peptide-poly(U) binding were measured as well as their dependences on monovalent salt concentration, temperature, and pH. In all cases, K(obs) decreases significantly with increasing monovalent salt concentration, with (delta log K(obs)/delta log [K+]) = -0.74 (+/- 0.04)z, independent of temperature and salt concentration, where z is the net positive charge on the peptide. The origin of these salt effects is entropic, consistent with the release of counterions from the poly(U) upon formation of the complex. Upon extrapolation to 1 M K+, the value of delta G(o)obs is observed to be near zero for all oligolysines binding to poly(U), supporting the conclusion that these complexes are stabilized at lower salt concentrations due to the increase in entropy accompanying the release of monovalent counterions from the poly(U). Only the net peptide charge appears to influence the thermodynamics of these interactions, since no effects of peptide charge distribution were observed. The binding of poly(U) to the monotryptophan peptides displays interesting behavior as a function of the peptide charge. The extent of tryptophan fluorescence quenching, Qmax, is dependent upon the peptide charge for z less than or equal to +4, and the value of Qmax correlates with z-dependent changes in delta H(o)obs and delta S(o)obs(1 M K+), whereas for z greater than or equal to +4, Qmax, delta H(o)obs, and delta S(o)obs (1 M K+) are constant. The correlation between Qmax and delta H(o)obs and delta S(o)obs(1 M K+) suggests a context (peptide charge)-dependence of the interaction of the peptide tryptophan with poly(U). However the interaction of the peptide tryptophan does not contribute substantially to delta G(o)obs for any of the peptides, independent of z, due to enthalpy-entropy compensations. Each of the tryptophans in multiple Trp-containing peptides appear to bind to poly(U) independently, with delta H(o)Trp = -2.9 +/- 0.7, although delta G(o)Trp is near zero due to enthalpy-entropy compensations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrogen peroxide mediates Rac1 activation of S6K1

We previously reported that hydrogen peroxide (H2O2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H2O2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H2O2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H2O2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H2O2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H2O2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H2O2 as a mediator in the activation of S6K1 by Rac1. However, H2O2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H2O2 could be produced by arsenite, which has been shown to be a stimulator of H2O2 production. Taken together, these results suggest that H2O2 plays a pivotal role as a mediator in Rac1 activation of S6K1.