P1 Site Research Articles

Early Insight into Mitralign Direct Annuloplasty for Treatment of Functional Mitral Regurgitation

The Mitralign system (Mitralign, Inc., Tewksbury, Massachusetts, US) is a set of devices approaching the posterior mitral annulus through the left ventricle (LV). Two pairs of surgical pledgets are delivered at the P1 and P3 site of the annulus. The paired pledgets are pulled together to decrease the annulus circumference, and the achieved plication is locked in place. In swine hearts the permanent implants, pledgets and locks, lack any adverse pathology such as necrosis, thrombosis, haemorrhage, mineralisation or exuberant inflammation, and the overall tissue response at 30-day follow-up is considered benign. The clinical experience is limited to a few patients enrolled in a first-in-man study. In a perfect candidate with relatively preserved LV function, moderately enlarged ventricle and limited leaflet tethering, Mitralign annuloplasty may reduce mitral regurgitation significantly from grade 4+ to grade 1+. More research and clinical trials are needed to understand this novel technique for percutaneous mitral valve repair.

Structural and Mutational Analysis of Canonical Loop Residues In Protease Nexin 2 Involved In Factor XIa and Trypsin Inhibition.

AbstractAbstract 1147Factor XIa (FXIa) activates FIX and is regulated by platelet-secreted protease nexin 2 (PN2) that contains a Kunitz-type protease inhibitor (KPI) domain. Trypsin is regulated by basic pancreatic trypsin inhibitor (BPTI). The primary and tertiary structures of trypsin and the catalytic domain of FXIa are highly homologous, and KPI and BPTI are nearly identical structurally. We have previously identified two loop structures (loops 1 and 2) in the KPI domain of PN2 that interact with residues in the FXIa catalytic domain. Based on the structure of the FXIa/KPI complex crystal structure, residues within loops 1 and 2 were mutated for experiments examining the inhibition of FXIa and trypsin.Results show that the loop-1-region P1 site residue Arg15 of PN2KPI plays a major role in FXIa inhibition by protruding into the S1 specificity pocket of FXIa. Ala mutation at this site renders PN2KPI non-inhibitory for both FXIa and trypsin. BPTI has Lys15 at the P1 site. BPTI inhibits both FXIa and trypsin significantly less effectively than PN2KPI. PN2KPI-R15K lost FXIa inhibitory activity, whereas BPTI-K15R substantially gained affinity for FXIa. Like FXIa, trypsin preferred BPTI-K15R showing a significant enhancement in affinity. Thus, a major determinant of the inhibitory activity of PN2KPI and BPTI against FXIa and trypsin is the P1 residue, with Arg being preferred over Lys for both inhibitors and both proteases. In addition, loop 1 residues Pro13 and Arg20 make important contributions to both FXIa and trypsin inhibition as demonstrated by significantly elevated Ki values for Ala mutations (P13A, and R20A) at these sites. In contrast, Ser19 makes no significant contribution to inhibition of either FXIa or trypsin whereas Met17 makes a significant contribution to the inhibition of trypsin, but not FXIa. In loop 2, only Phe34 is identified as a residue making significant contributions to the inhibition of both FXIa and trypsin, since the PN2KPI-F34A mutant displayed reduced inhibitory activity for both FXIa (6-fold) and for trypsin (3-fold). To rationalize these findings, we examined the crystal structures of the FXIa(catalytic domain)/PN2KPI complex, and the trypsin/BPTI complex. Structurally, the PN2KPI loop-1, P1-site residue Arg15 makes a complex primary interaction with Asp189 of both FXIa and trypsin. Disruption of this site by R15A mutation renders PN2KPI non-inhibitory because it preempts salt bridge interactions from two nitrogen atoms of the guanidinium group of Arg15 with Asp189 and Gly218 in FXIa. In addition, the Arg15 carbonyl oxygen forms hydrogen bonds with main-chain nitrogen atoms of one of the catalytic triad residues, Ser195, and with Gly193. The other important interaction in FXIa/PN2KPI or trypsin/BPTI is hydrophobic, between PN2KPI-Phe34 and FXIa-Tyr143 and between BPTI-Val34 and trypsin-Tyr151. This intermolecular interaction is further strengthened by an intramolecular interaction in which the side chain of Phe34 packs closely with the side chain of Met17 within PN2KPI, altogether forming a strong hydrophobic patch in FXIa-PN2KPI and trypsin-PN2KPI. PN2KPI-F34A disrupts both inter- and intramolecular hydrophobic interactions, leading to discernable reductions in affinity for both FXIa and trypsin. Despite occupying extreme positions in the autolysis loops, (143YRKLRDKI151 in FXIa and 143NTKSSGTSY151 in trypsin), Tyr143/151 residues still orient themselves in close proximity to Phe34. Thus, loop-1 residues of PN2KPI establish complex ionic interactions that play a major role, which is supplemented by the loop-2 residue, Phe34 (in PN2KPI) or Val34 (in BPTI), which establish hydrophobic interactions with residues in FXIa and trypsin leading to very high-affinity enzyme-inhibitor complexes. Disclosures:No relevant conflicts of interest to declare.

Docking and molecular dynamics study on the inhibitory activity of N, N-disubstituted-trifluoro-3-amino-2-propanols-based inhibitors of cholesteryl ester transfer protein

Extensive studies suggest direct links between cholesteryl ester transfer protein (CETP), high-density lipoproteins-cholesterol level and cardiovascular diseases. Many therapeutic approaches are aimed at the CETP. A series of N, N-disubstituted-trifluoro-3-amino-2-propanol analogues are among the most highly potent and selective inhibitors of CETP described to date. For in-depth investigation into the structural and chemical features responsible for exploring the binding pocket of these compounds, as well as for the binding recognition mechanism concerned, we performed a series of automated molecular docking operations. Moreover, the docking results were quite robust as further validated by molecular dynamics. The docking results reveal that the binding site mainly consists of two hydrophobic regions (P1 and P2 site) which are able to accommodate the lipophilic arms of the compounds investigated. Val421 in P1 site and Met194 in P2 site could be considered to be two important residues in forming the two hydrophobic regions. The presence of residues Phe197 and Phe463 in P2 site may be responsible for the binding recognition through π-π stacking interactions. The hydrophobic 3-phenoxy substituent may be important in creating the preferable inhibitive capability for increasing the binding potency. The hydrophobic character of the tetrafluoroethoxybenzyl group at position 3 displays better hydrophobicity than a shorter hydrophobic substituent. An interaction model of CETP-inhibitors is derived that can be successfully used to explain the different biologic activities of these inhibitors. It is anticipated that the findings reported here may provide very useful information or clues for designing effective drugs for the therapeutic treatment of CETP-related cardiovascular diseases.

Diversity and abundance of anammox bacterial community in the deep-ocean surface sediment from equatorial Pacific

The community structure and diversity of anaerobic ammonium oxidation (anammox) bacteria in the surface sediments of equatorial Pacific were investigated by phylogenic analysis of 16S rRNA and hydrazine oxidoreductase (hzo) genes and PCoA (principal coordinates analysis) statistical analysis. Results indicated that 16S rRNA and hzo sequences in the P2 (off the center of western Pacific warm pool) and P3 (in the eastern equatorial Pacific) sites all belong to the Candidatus "Scalindua", the dominate anammox bacteria in the low-temperature marine environment proved by previous studies. However, in the P1 site (in center of warm pool of western Pacific), large part of 16S rRNA gene sequences formed a separated cluster. Meanwhile, hzo gene sequences from P1 sediment also grouped into a single cluster. PCoA analysis demonstrated that the anammox community structure in the P1 has significant geographical distributional difference from that of P2, P3, and other marine environments based on 16S rRNA and hzo genes. The abundances of anammox bacteria in surface sediments of equatorial Pacific were quantified by q-PCR analysis of hzo genes, which ranged from 3.98 × 10(3) to 1.17 × 10(4) copies g(-1) dry sediments. These results suggested that a special anammox bacteria phylotypes exist in the surface sediment of the western Pacific warm pool, which adapted to the specific habitat and maybe involved in the nitrogen loss process from the fixed inventory in the habitat.

Substituted 4-Carboxymethylpyroglutamic Acid Diamides as Potent and Selective Inhibitors of Fibroblast Activation Protein

Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

Novel macrocyclic HCV NS3 protease inhibitors derived from α-amino cyclic boronates

A novel series of P2–P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.

Understanding the Mechanism of Deacylation Reaction Catalyzed by the Serine Carboxyl Peptidase Kumamolisin-As: Insights from QM/MM Free Energy Simulations

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics and free energy simulations are performed to study the process of the deacylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism. The results given here suggest that Asp-164 acts as a general acid/base catalyst not only for the acylation reaction but also for the deacylation reaction. It is shown that the electrostatic oxyanion hole interactions may be less effective in transition state stabilization for the kumamolisin-As catalyzed reaction compared to the general acid/base mechanism involving the proton transfer from or to Asp-164. The dynamic substrate-assisted catalysis (DSAC) involving His at the P1 site of the substrate is found to be less important for the deacylation reaction than for the acylation reaction in the kumamolisin-As catalyzed reaction. The proton transfer processes during the enzyme-catalyzed process are examined and their role in the catalysis is discussed.

Slow Magnetic Relaxation from Hard‐Axis Metal Ions in Tetranuclear Single‐Molecule Magnets

We report the synthesis of the novel heterometallic complex [Fe(3)Cr(L)(2)(dpm)(6)]⋅Et(2)O (Fe(3)CrPh) (Hdpm=dipivaloylmethane, H(3)L=2-hydroxymethyl-2-phenylpropane-1,3-diol), obtained by replacing the central iron(III) atom by a chromium(III) ion in an Fe(4) propeller-like single-molecule magnet (SMM). Structural and analytical data, high-frequency EPR (HF-EPR) and magnetic studies indicate that the compound is a solid solution of chromium-centred Fe(3)Cr (S=6) and Fe(4) (S=5) species in an 84:16 ratio. Although SMM behaviour is retained, the |D| parameter is considerably reduced as compared with the corresponding tetra-iron(III) propeller (D=-0.179 vs. -0.418 cm(-1)), and results in a lower energy barrier for magnetisation reversal (U(eff)/k(B)=7.0 vs. 15.6 K). The origin of magnetic anisotropy in Fe(3)CrPh has been fully elucidated by preparing its Cr- and Fe-doped Ga(4) analogues, which contain chromium(III) in the central position (c) and iron(III) in two magnetically distinct peripheral sites (p1 and p2). According to HF-EPR spectra, the Cr and Fe dopants have hard-axis anisotropies with D(c)=0.470(5) cm(-1), E(c)=0.029(1) cm(-1), D(p1)=0.710(5) cm(-1), E(p1)=0.077(3) cm(-1), D(p2)=0.602(5) cm(-1), and E(p2)=0.101(3) cm(-1). Inspection of projection coefficients shows that contributions from dipolar interactions and from the central chromium(III) ion cancel out almost exactly. As a consequence, the easy-axis anisotropy of Fe(3)CrPh is entirely due to the peripheral, hard-axis-type iron(III) ions, the anisotropy tensors of which are necessarily orthogonal to the threefold molecular axis. A similar contribution from peripheral ions is expected to rule the magnetic anisotropy in the tetra-iron(III) complexes currently under investigation in the field of molecular spintronics.

Mechanisms and specificity of factor XIa and trypsin inhibition by protease nexin 2 and basic pancreatic trypsin inhibitor

Factor XIa (FXIa) inhibition by protease nexin-2 (PN2KPI) was compared with trypsin inhibition by basic pancreatic trypsin inhibitor (BPTI). PN2KPI was a potent inhibitor of FXIa (K(i) ∼ 0.81 nM) and trypsin (K(i) ∼ 0.03 nM), but not of other coagulation proteases (thrombin, FVIIa, FIXa, FXa, FXIIa, plasmin, kallikrein, K(i) > 185 nM). PN2KPI was ∼775-fold more potent than BPTI in FXIa inhibition, but both exhibited similar potencies against trypsin. Studies of FXIa and trypsin inhibition by PN2KPI and BPTI and P1 site swap mutants (PN2KPI-R15 K, BPTI-K15 R) demonstrated that FXIa inhibition by PN2KPI and P1 site swap mutants and trypsin inhibition by PN2KPI and BPTI conform to a single-step, slow equilibration inhibitory mechanism, whereas FXIa-inhibition by BPTI follows a classical, competitive inhibitory mechanism. Mutation of P1 impaired FXIa inhibition by PN2KPI-R15 K ∼14-fold, enhanced FXIa inhibition by BPTI-K15 R ∼150-fold, and had no effect on trypsin inhibition. Arginine at the P1 site of either PN2KPI or BPTI confers high affinity and specificity for FXIa, whereas either arginine or lysine suffices for trypsin inhibition. Thus, PN2KPI is a highly specific inhibitor of FXIa among coagulation enzymes, but the flexibility of trypsin renders it susceptible to inhibition by both wild-type and mutant forms of PN2KPI and BPTI.

Identifying potential sites for artificial groundwater recharge in sub-watershed of River Kanhan, India

Groundwater is an important decentralized source of drinking water. Being underground, it is relatively less susceptible to contamination. In addition to domestic needs, it is extensively used for irrigation and industrial purposes. It is therefore necessary to implement groundwater recharge systems by capturing the rainwater runoff. In the present study, GIS-based hydrological assessment of watershed has been used to identify the potential sites for locating the groundwater recharge structures. Based on land use, soil and topography, rainfall runoff modelling was carried out in GIS for a sub-watershed of River Kanhan, in Nagpur District, Maharashtra State, India. Five potential sites with maximum flow accumulation were delineated using the rational method for peak runoff estimation. As the groundwater recharge potential also depends on the geological and geomorphological characteristics of land, analytic hierarchy process (AHP) with expert’s judgement was used for ranking the sites. The criteria considered in AHP were geological features, i.e. lineament density, depth to bedrock and soil cover; geomorphological features, i.e. drainage density, slope, landforms and land use/land cover; and water table level fluctuation. The site P5 with maximum flow accumulation and sandstone rock formation was ranked first. The site P1, where catchment has well-developed drainage and geological formation shows rock with secondary porosity, was ranked second.

ChemInform Abstract: Protease Inhibitors. Part 3. Synthesis of Non-basic Thrombin Inhibitors Incorporating Pyridinium-Sulfanilylguanidine Moieties at the P1 Site.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Identification of folding preferences of cleavage junctions of HIV-1 precursor proteins for regulation of cleavability

Human immunodeficiency virus type 1 protease (HIV-1 PR) cleaves two viral precursor proteins, Gag and Gag-Pol, at multiple sites. Although the processing proceeds in the rank order to assure effective viral replication, the molecular mechanisms by which the order is regulated are not fully understood. In this study, we used bioinformatics approaches to examine whether the folding preferences of the cleavage junctions influence their cleavabilities by HIV-1 PR. The folding of the eight-amino-acid peptides corresponding to the seven cleavage junctions of the HIV-1(HXB2) Gag and Gag-Pol precursors were simulated in the PR-free and PR-bound states with molecular dynamics and homology modeling methods, and the relationships between the folding parameters and the reported kinetic parameters of the HIV-1(HXB2) peptides were analyzed. We found that a folding preference for forming a dihedral angle of Cβ (P1)-Cα (P1)- Cα (P1')-Cβ (P1') in the range of 150 to 180 degrees in the PR-free state was positively correlated with the 1/K(m) (R = 0.95, P = 0.0008) and that the dihedral angle of the O (P2)-C (P2)- C (P1)- O (P1) of the main chains in the PR-bound state was negatively correlated with k(cat) (R = 0.94, P = 0.001). We further found that these two folding properties influenced the overall cleavability of the precursor protein when the sizes of the side chains at the P1 site were similar. These data suggest that the dihedral angles at the specific positions around the cleavage junctions before and after binding to PR are both critical for regulating the cleavability of precursor proteins by HIV-1 PR.

Osteomyelitis and Osteonecrosis after Intraosseous Perfusion with Gentamicin

To describe and discuss previously unreported complications associated with intraosseous perfusion with gentamicin in horses. Case report. Ten-year-old Warmblood gelding. Intraosseous perfusion with gentamicin into the proximal phalanx (P1) was used as part of the treatment regimen for distal interphalangeal joint and navicular bursa synovial sepsis. Although the sepsis responded favorably complications developed at the perfusion site, including persistent osteomyelitis, progressive osteonecrosis, and ultimately pathologic fracture of P1. The progression of the clinical signs and findings at necropsy are suggestive of a toxic osteonecrosis secondary to intraosseous perfusion. Further work is needed to investigate the effects of high dose gentamicin on equine mesenchymal cells that may be achieved during intraosseous perfusion. Lower doses of perfusate within the medullary canal of P1 or alternative perfusion sites should be considered.

Inhibitory Properties of the P1 Tyr Variant of Antithrombin

Antithrombin (AT) and protein Z-dependent protease inhibitor (ZPI) are among two physiological serpin inhibitors in plasma that are involved in the regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has narrower protease specificity, inhibiting only factors Xa (fXa) and XIa. Unlike an Arg at the P1 site of the AT reactive center loop (RCL), this residue is a Tyr in ZPI. To investigate the contribution of P1 Tyr in restricting the specificity of ZPI, we engineered an AT mutant in which the P1 Arg of the RCL was replaced with the P1 Tyr of ZPI (AT-R393Y). The reactivity of AT-R393Y with fXa and thrombin was decreased 155- and 970-fold, respectively. However, the serpin mutant inhibited chymotrypsin with an efficiency higher by >4 orders of magnitude. By contrast, chymotrypsin did not exhibit any reactivity with ZPI. The substitution of Asp-189 of fXa with the corresponding residue of chymotrypsin (Ser) did not improve the reactivity of the protease mutant with AT-R393Y; however, the fXa mutant reacted normally with ZPI. These results suggest that the contribution of P1 Tyr to restricting the protease specificity of ZPI is RCL context-dependent and that in addition to P1 Tyr, other structural features within and/or outside the ZPI RCL are involved in determining the protease specificity of the serpin. The results further suggest that thrombin is less tolerant than fXa in accommodating the nonoptimal P1 Tyr of the AT mutant in its active-site pocket.

Boron/nitrogen pairs doping in armchair single-walled carbon nanotubes

By performing first principles electronic structure calculations, we have revealed the effect of boron/nitrogen pairs doping in armchair single-walled carbon nanotubes (SWCNT). It is shown that for two kinds of sites in the armchair SWCNT, the doping of B/N pairs can more easily happen on the P1 site which is at 30° angle to the tube axis. An energy gap is opened in metallic SWCNT by doping B/N pairs, and the energy gap increases with raising the axial concentration of the B/N pairs. Moreover, when two couples of B/N pairs are doped in SWCNT, the electronic structure is sensitive to the relative positions of B/N pairs in the couple along the circumference of tubes. It’s due to that the original charge distribution is changed by B/N pairs doping, and the effect of B/N pairs is localized. When the distance between B/N pairs increases, the extent of the effect is increased. This result may contribute to preparing pure semiconductor and effectively controlling the electronic structure.

Early Growth Response Protein-1 (Egr-1) Is Preferentially Expressed in T Helper Type 2 (Th2) Cells and Is Involved in Acute Transcription of the Th2 Cytokine Interleukin-4

The early growth response gene product Egr-1 has been shown to have great impact on growth, proliferation, and differentiation in a wide variety of cells, including T cells. In this study, we show that Egr-1 is rapidly induced upon T cell stimulation and is expressed predominantly in T helper type 2 (Th2) compared with type 1 (Th1) cells. We further investigate the role of Egr-1 in regulation of the Th2 cytokine interleukin-4 (IL-4) expression. IL-4 is a key Th2 cytokine that regulates humoral immunity and also causes allergic inflammation. Regulation of IL-4 gene transcription in Th2 cells has been shown to be controlled by multiple T cell receptor (TCR)-induced transcription factors. However, only a few transcription factors were shown to be selectively induced in differentiated Th2 cells in response to TCR stimulation. Chromatin immunoprecipitation analysis demonstrates that Egr-1 binds to the IL-4 promoter in vivo upon T cell stimulation. Ectopic expression of Egr-1 enhances endogenous IL-4 mRNA expression and elevates IL-4 promoter activity. We also show that Egr-1, nuclear factor of activated T cell, and NF-kappaB cooperatively bind to an NFAT/NF-kappaB-overlapping IL-4 enhancer element and activate the IL-4 promoter synergistically. Furthermore, we show that antisense oligonucleotides that knock down Egr-1 expression attenuate IL-4 transcription. Our study provides the first evidence that Egr-1 protein is differentially expressed in Th1 and Th2 cells and is involved in the acute phase of the IL-4 transcription in response to TCR stimulation.

Single-crystalP31NMR studies of the frustrated square-lattice compoundPb2(VO)(PO4)2

The static and dynamic properties of ${\text{V}}^{4+}$ spins $(S=1/2)$ in the frustrated square-lattice compound ${\text{Pb}}_{2}(\text{VO}){({\text{PO}}_{4})}_{2}$ were investigated by means of magnetic susceptibility $\ensuremath{\chi}$ and $^{31}\text{P}$ nuclear magnetic resonance (NMR) shift $(K)$ and $^{31}\text{P}$ nuclear spin-lattice relaxation rate $1/{T}_{1}$ measurements on a single crystal. This compound exhibits long-range antiferromagnetic order below ${T}_{\text{N}}\ensuremath{\simeq}3.65\text{ }\text{K}$. NMR spectra above ${T}_{\text{N}}$ show two distinct lines corresponding to two inequivalent P sites present in the crystal structure. The observed asymmetry in hyperfine coupling constant for the in-plane (P1) P site directly points toward a distortion in the square lattice at the microscopic level, consistent with the monoclinic crystal structure. The nearest- and next-nearest-neighbor exchange couplings were estimated by fitting $K$ versus temperature $T$ by a high-temperature series expansion for the spin susceptibility of the frustrated square lattice to be ${J}_{1}/{k}_{\text{B}}=(\ensuremath{-}5.4\ifmmode\pm\else\textpm\fi{}0.5)\text{ }\text{K}$ (ferromagnetic) and ${J}_{2}/{k}_{\text{B}}=(9.3\ifmmode\pm\else\textpm\fi{}0.6)\text{ }\text{K}$ (antiferromagnetic), respectively. $1/({T}_{1}T\ensuremath{\chi})$ is almost $T$ independent at high temperatures due to random fluctuation of spin moments. Below 20 K, the compound shows an enhancement of $1/({T}_{1}T\ensuremath{\chi})$ which arises from a growth of antiferromagnetic spin correlations above ${T}_{\text{N}}$. Below ${T}_{\text{N}}$ and for the field applied along the $c$ axis, the NMR spectrum for the P1 site splits into two satellites and the spacing between them increases monotonically with decreasing $T$ which is a direct evidence of a columnar antiferromagnetic ordering with spins lying in the $ab$ plane. This type of magnetic ordering is consistent with expectation from the ${J}_{2}/{J}_{1}\ensuremath{\simeq}\ensuremath{-}1.72$ ratio. The critical exponent $\ensuremath{\beta}=0.25\ifmmode\pm\else\textpm\fi{}0.02$ estimated from the temperature dependence of the sublattice magnetization as measured by $^{31}\text{P}$ NMR at 11.13 MHz is close to the value (0.231) predicted for the two-dimensional XY model.

Purification and Characterization of a Salt-Activated and Organic Solvent-Stable Heterotrimer Proteinase from Virgibacillus sp. SK33 Isolated from Thai Fish Sauce

A NaCl-activated proteinase produced by Virgibacillus sp. SK33 was purified to homogeneity using phenyl-Sepharose and Sephadex G-75 with a yield of 12% and purification of 2.6-fold. A single protein was detected at approximately 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, three subunits with molecular weights of 27,858, 33,918, and 35,368 Da were obtained from MALDI-TOF mass spectra, implying that the enzyme was a heterotrimer. The isoelectric point of the proteinase was 5.4. Optimum catalytic activity was at 55 degrees C and pH 7.5. The enzyme showed serine characteristics as it was completely inhibited by phenylmethanesulfonyl fluoride. The purified proteinase showed broad specificity toward oxidized insulin B including Gln4, Cys7, Glu13, Ala14, Leu15,17, Tyr16,26, Arg22, Phe24,25, and Lys29. Dominant cleavage sites of the enzyme were Tyr16-Leu17 and Phe25-Tyr26, indicating that it preferably hydrolyzed aromatic amino acids located on the P1 site. Among various substrates studied, the enzyme hydrolyzed anchovy protein to the greatest extent at 4 M NaCl. Activity increased with either CaCl2 or NaCl concentration with the maximum 2-fold increase at either 50 mM CaCl2 or 4 M NaCl. The enzyme was also highly stable up to 500 mM CaCl2 or 4 M NaCl. The proteinase showed high stability in various organic solvents (25%, v/v) including dimethylsulfoxide, methanol, acetonitrile, and ethanol. Results of peptide mass fingerprint and de novo peptide sequencing showed that the purified proteinase is a novel proteinase. The proteinase from Virgibacillus sp. SK33 could have a potential application in high ionic strength environments and aqueous-organic solvent systems.

Residues Surrounding Arg372, Arg740, and Arg1689 Contribute to the Rates of Thrombin-Catalyzed Cleavage of Factor VIII.

Abstract 847Hemophilia A results from defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor that must be cleaved by thrombin or factor Xa at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction) to yield the active cofactor, factor VIIIa. Activation of factor VIII by thrombin is exosite-dependent yielding rates of cleavage at Arg740 ∼20-fold faster than Arg372, while cleavage at Arg1689 appears intermediary to Arg740 and Arg372. The contribution of P3-P3' residues flanking each cleavage site to the mechanism of thrombin-catalyzed cleavage of factor VIII has not been extensively studied. The P3-P3' residues for the 372, 1689, and 740 factor VIII sites are 370QIR*↓SVA375, 1687SPR*↓SFQ1692, and 738EPR*↓SFS743, respectively. Residues flanking Arg372 are considered non-optimal for thrombin cleavage with only two residues optimal (in bold type) for cleavage in the P3-P3' sequence, while residues flanking at the two other P1 sites are considered near-optimal with four out of six residues optimal (in bold type). Therefore, we investigated whether the P3-P3'residues surrounding Arg740, Arg372, and Arg1689 affect activation of factor VIII by thrombin. We constructed, stably transfected, and expressed four recombinant P3-P3' factor VIII mutants designated 372(P3-P3')740, 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740/1689(P3-P3')372. For example, the 372(P3-P3')740 variant has replaced the non-optimal P3-P3' residues flanking Arg372 with the near-optimal P3-P3' residues flanking Arg740. The specific activities of the 372(P3-P3')740 and 372(P3-P3')740/740(P3-P3')372 mutants were 98% and 122% the wild-type factor VIII value, respectively. In comparison, the 372(P3-P3')1689 and 372(P3-P3')740/1689(P3-P3')372 showed reductions in specific activity with values that were 14% and 17% of wild-type factor VIII, consistent with possible impaired rates of activation by thrombin. SDS-PAGE and Western blotting of the three variants possessing the 372(P3-P3')740 mutation showed cleavage rates at Arg372 increased 11- to 14-fold compared with wild-type factor VIII as judged by rates of generation of the A1 subunit. Furthermore, these variants revealed 11-21-fold rate increases in the generation of the A2 subunit as compared to wild-type factor VIII. The rates of A1 and A2 subunit generation were moderately increased from 2-3-fold for the 372(P3-P3')1689 mutant. These results indicate that replacing the non-optimal residues flanking Arg372 with near-optimal residues enhances rates of cleavage at this site. Furthermore, since the P2-P2' residues flanking Arg740 and Arg1689 are identical, these results also suggest that the P3 and/or P3' residues from the Arg740 cleavage site make a greater contribution to the enhanced cleavage rate when inserted at Arg372 than the equivalent residues from the Arg1689 site. Thrombin cleavage of light chain showing the largest effect was obtained for the 372(P3-P3')740/1689(P3-P3')372 mutant which yielded a reduced rate of A3-C1-C2 subunit generation by 33-fold. This result suggests that replacing near-optimal P3-P3' residues at Arg1689 with non-optimal residues at Arg372 significantly reduces the rate of thrombin cleavage at Arg1689, an effect that may contribute to its low specific activity. There was no observed defect in Arg1689 cleavage in the 372(P3-P3')740 mutant and moderate 2-3-fold reductions in thrombin-catalyzed cleavage rates at Arg1689 in the 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740 variants. Overall, these results suggest that faster cleavage rates at Arg740 and Arg1689 can be attributed to more optimal residues in the P3-P3' region, while the relatively slower cleavage rate at Arg372 can be accelerated by replacement with more optimal residues for thrombin cleavage. Thus, the P3-P3' residues surrounding Arg740, Arg1689, and Arg372 in factor VIII impact rates of thrombin proteolysis at each site and contribute to the mechanism for thrombin activation of the procofactor. Disclosures:No relevant conflicts of interest to declare.

The i-Motif in the bcl-2 P1 Promoter Forms an Unexpectedly Stable Structure with a Unique 8:5:7 Loop Folding Pattern

Transcriptional regulation of the bcl-2 proto-oncogene is highly complex, with the majority of transcription driven by the P1 promoter site and the interaction of multiple regulatory proteins. A guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to bcl-2 promoter activity, as deletion or mutation of this region significantly increases transcription. This GC-rich element consists of six contiguous runs of guanines and cytosines that have the potential to adopt DNA secondary structures, the G-quadruplex and i-motif, respectively. Our laboratory has previously demonstrated that the polypurine-rich strand of the bcl-2 promoter can form a mixture of three different G-quadruplex structures. In this current study, we demonstrate that the complementary polypyrimidine-rich strand is capable of forming one major intramolecular i-motif DNA secondary structure with a transition pH of 6.6. Characterization of the i-motif folding pattern using mutational studies coupled with circular dichroic spectra and thermal stability analyses revealed an 8:5:7 loop conformation as the predominant structure at pH 6.1. The folding pattern was further supported by chemical footprinting with bromine. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. The potential of the GC-rich element within the bcl-2 promoter region to form DNA secondary structures suggests that the transition from the B-DNA to non-B-DNA conformation may play an important role in bcl-2 transcriptional regulation. Furthermore, the two adjacent large lateral loops in the i-motif structure provide an unexpected opportunity for protein and small molecule recognition.