Protein Science

BACKGROUNDProstate cancer (PCa) is the second leading cause of cancer-related death in men in the United States. Many men have implemented purported chemopreventive agents into their daily diet in an attempt to delay the early onset of a PCa. Green tea polyphenols, one such agent, has been shown to be chemopreventive in skin, breast, and prostate cancers. We hypothesized that Epigallocatechin-3-Gallate (EGCG), the major polyphenol found in green tea, will exert its chemopreventive effect in the prostate via regulation of sex steroid receptor, growth factor-signaling, and inflammatory pathways.Prostate cancer (PCa) is the second leading cause of cancer-related death in men in the United States. Many men have implemented purported chemopreventive agents into their daily diet in an attempt to delay the early onset of a PCa. Green tea polyphenols, one such agent, has been shown to be chemopreventive in skin, breast, and prostate cancers. We hypothesized that Epigallocatechin-3-Gallate (EGCG), the major polyphenol found in green tea, will exert its chemopreventive effect in the prostate via regulation of sex steroid receptor, growth factor-signaling, and inflammatory pathways.METHODSFive-week-old male TRAMP (Transgenic Adenocarcinoma Mouse Prostate) offspring were fed AIN-76A diet and 0.06% EGCG in tap water. Animals were sacrificed at 28 weeks of age and the entire prostates were scored histopathologically. In addition, animals were sacrificed at 12 weeks of age and ventral (VP) and dorsolateral (DLP) prostates were removed for histopathological evaluation and immunoblot analyses or ELISA.Five-week-old male TRAMP (Transgenic Adenocarcinoma Mouse Prostate) offspring were fed AIN-76A diet and 0.06% EGCG in tap water. Animals were sacrificed at 28 weeks of age and the entire prostates were scored histopathologically. In addition, animals were sacrificed at 12 weeks of age and ventral (VP) and dorsolateral (DLP) prostates were removed for histopathological evaluation and immunoblot analyses or ELISA.RESULTSEGCG, inhibited early but not late stage PCa in the current study. In the VP, EGCG significantly reduced cell proliferation, induced apoptosis, and decreased androgen receptor (AR), insulin-like growth factor-1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-extracellular signal-regulated kinases 1 and 2 (phospho-ERKs 1 and 2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS).EGCG, inhibited early but not late stage PCa in the current study. In the VP, EGCG significantly reduced cell proliferation, induced apoptosis, and decreased androgen receptor (AR), insulin-like growth factor-1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-extracellular signal-regulated kinases 1 and 2 (phospho-ERKs 1 and 2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS).CONCLUSIONSThe attenuation of the AR, the down-regulation of potent growth factor IGF-1, modulation of inflammation biomarkers, and decrease in the MAPK signaling may contribute to the reduction in cell proliferation and induction of apoptosis and hence provide a biochemical basis for EGCG suppressing PCa without toxicity. Prostate 67: 1576–1589, 2007. © 2007 Wiley-Liss, Inc.The attenuation of the AR, the down-regulation of potent growth factor IGF-1, modulation of inflammation biomarkers, and decrease in the MAPK signaling may contribute to the reduction in cell proliferation and induction of apoptosis and hence provide a biochemical basis for EGCG suppressing PCa without toxicity. Prostate 67: 1576–1589, 2007. © 2007 Wiley-Liss, Inc.

Calculating protein–protein interaction energies is crucial for understanding protein–protein associations. On the basis of the methodology of mean-field potential, we have developed an empirical approach to estimate binding free energy for protein–protein interactions. This knowledge-based approach has been used to derive distance-dependent free energies of protein complexes from a nonredundant training set in the Protein Data Bank (PDB), with a careful treatment of homology. We calculate atom pair potentials for 16 pair interactions, which can reflect the importance of hydrophobic interactions and specific hydrogen-bonding interactions. The derived potentials for hydrogen-bonding interactions show a valley of favorable interactions at a distance of ≈3 Å, corresponding to that of an established hydrogen bond. For the test set of 28 protein complexes, the calculated energies have a correlation coefficient of 0.75 compared with experimental binding free energies. The performance of the method in ranking the binding energies of different protein–protein complexes shows that the energy estimation can be applied to value binding free energies for protein–protein associations. Proteins 2002;46:190–196. © 2001 Wiley-Liss, Inc.

Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single-polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single-chain PGA not dependent on autoproteolytic processing. The mature sequence of the β-domain was expressed as the N terminus of a new polypeptide, connected by a random tetra-peptide to the α-domain, to afford a permuted protein. We found several active enzymes among variants differing in their linker peptides. Protein expression analysis showed that the functional single-chain variants were produced when using a Sec-dependent leader peptide, or when expressed inside the bacterial cytoplasm. Active-site titration experiments showed that the single-chain proteins displayed similar kcat values to the ones obtained with the wild-type enzyme. Interestingly, the single-chain proteins also displayed close to 100% of functional active sites compared to 40% to 70% functional yield usually obtained with the heterodimeric protein.

We present a study of the competition between protein refolding and aggregation for simple lattice model proteins. The effect of solvent conditions (i.e., the denaturant concentration and the protein concentration) on the folding and aggregation behavior of a system of simple, two-dimensional lattice protein molecules has been investigated via dynamic Monte Carlo simulations. The population profiles and aggregation propensities of the nine most populated intermediate configurations exhibit a complex dependence on the solution conditions that can be understood by considering the competition between intra- and interchain interactions. Some of these configurations are not even seen in isolated chain simulations; they are observed to be highly aggregation prone and are stabilized primarily by the aggregation reaction in multiple-chain systems. Aggregation arises from the association of partially folded intermediates rather than from the association of denatured random-coil states. The aggregation reaction dominates over the folding reaction at high protein concentration and low denaturant concentration, resulting in low refolding yields at those conditions. However, optimum folding conditions exist at which the refolding yield is a maximum, in agreement with some experimental observations.

The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30°C-90°C. We found that predominantly ␤-sheet structure of both proteins was stable up to 70°C, and that a reversible conformational change toward ␣-helix was apparent at temperatures ranging from 70°C to 90°C. The ability of both proteins to inhibit complement was not impaired after incubation at 95°C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in ␤-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.

The native conformation of host-encoded cellular prion protein (PrP C ) is metastable. As a result of a post-translational event, PrP C can convert to the scrapie form (PrP Sc ), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a ␤-sheet form. Article and publication are at http://www.proteinscience.org/cgi/doi/ 10.1110/ps.04989405. Protein Science (2005), 14:956-967.

The C-terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43-residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence-specific assignments for *H-, "N-, and I3C-NMR resonances have been obtained from 2D 'H NOESY and TOCSY spectra, and from 3D HNCA, I5N NOESY-HSQC, and HCCH-TOCSY spectra. A stretch of a-helix encompassing five heptad repeats (35 residues) has been identified from intra-chain HN-HN and HN-Ha NOE connectivities, 3J~~~o coupling constants, and chemical shift indices. The a-helix begins immediately downstream of inter-chain disulfide bonds between residues Cys zyxw 5 and Cys 7, and extends to near the C-terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter-chain disulfide bonds that flank the N-terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOES between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N-terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed.

The X-ray structure of an oxygenated hemocyanin molecule, subunit I1 of Limulus polyphemus hemocyanin, was determined at 2.4 A resolution and refined to a crystallographic R-factor of 17.1%. The 73-kDa subunit crystallizes with the symmetry of the space group R32 with one subunit per asymmetric unit forming hexamers with 32 point group symmetry. Molecular oxygen is bound to a dinuclear copper center in the protein's second domain, symmetrically between and equidistant from the two copper atoms. The coppercopper distance in oxygenated Limulus hemocyanin is 3.6 2 0.2 A, which is surprisingly 1 A 0 1994 WILEY-LISS. INC.

BACKGROUNDProstate cancer has been reported to occur more commonly in neutered than intact male dogs in several case series. This study was undertaken to evaluate risk of prostate cancer in a large population database. The hypothesis was that castration is a risk factor for prostate cancer in male companion dogs.Prostate cancer has been reported to occur more commonly in neutered than intact male dogs in several case series. This study was undertaken to evaluate risk of prostate cancer in a large population database. The hypothesis was that castration is a risk factor for prostate cancer in male companion dogs.METHODSData were derived from recorded visits to North American veterinary teaching hospitals. The Veterinary Medical Databases (VMDB) were queried to yield male dogs with urinary bladder transitional cell carcinoma (TCC), prostate adenocarcinoma (ACA), prostate TCC, prostate carcinoma (CA), and prostate tumors. A second query yielded all male dogs over the age of 4 years without a diagnosis of urinary tract cancer. These populations were compared to determine relative risks for developing each disease, singly and collectively, associated with neutering status. Odds ratios were calculated for breed as a risk factor.Data were derived from recorded visits to North American veterinary teaching hospitals. The Veterinary Medical Databases (VMDB) were queried to yield male dogs with urinary bladder transitional cell carcinoma (TCC), prostate adenocarcinoma (ACA), prostate TCC, prostate carcinoma (CA), and prostate tumors. A second query yielded all male dogs over the age of 4 years without a diagnosis of urinary tract cancer. These populations were compared to determine relative risks for developing each disease, singly and collectively, associated with neutering status. Odds ratios were calculated for breed as a risk factor.RESULTSNeutered males had a significantly increased risk for each form of cancer. Neutered males had an odds ratio of 3.56 (3.02–4.21) for urinary bladder TCC, 8.00 (5.60–11.42) for prostate TCC, 2.12 (1.80–2.49) for prostate adenocarcinoma, 3.86 (3.13–4.16) for prostate carcinoma, and 2.84 (2.57–3.14) for all prostate cancers. Relative risks were highly similar when cases were limited to those with a histologically confirmed diagnosis.Neutered males had a significantly increased risk for each form of cancer. Neutered males had an odds ratio of 3.56 (3.02–4.21) for urinary bladder TCC, 8.00 (5.60–11.42) for prostate TCC, 2.12 (1.80–2.49) for prostate adenocarcinoma, 3.86 (3.13–4.16) for prostate carcinoma, and 2.84 (2.57–3.14) for all prostate cancers. Relative risks were highly similar when cases were limited to those with a histologically confirmed diagnosis.CONCLUSIONSBreed predisposition suggests that genetic factors play a role in the development of prostate cancer. The risk associated with being neutered is highest for TCC, supporting previous work identifying the urothelium and ductular rather than acinar epithelium as the source of these tumors. Prostate 67: 1174–1181, 2007. © 2007 Wiley-Liss, Inc.Breed predisposition suggests that genetic factors play a role in the development of prostate cancer. The risk associated with being neutered is highest for TCC, supporting previous work identifying the urothelium and ductular rather than acinar epithelium as the source of these tumors. Prostate 67: 1174–1181, 2007. © 2007 Wiley-Liss, Inc.

Protein structure alignment methods are essential for many different challenges in protein science, such as the determination of relations between proteins in the fold space or the analysis and prediction of their biological function. A number of different pairwise and multiple structure alignment (MStA) programs have been developed and provided to the community. Prior knowledge of the expected alignment accuracy is desirable for the user of such tools. To retrieve an estimate of the performance of current structure alignment methods, we compiled a test suite taken from literature and the SISYPHUS database consisting of proteins that are difficult to align. Subsequently, different MStA programs were evaluated regarding alignment correctness and general limitations. The analysis shows that there are large differences in the success between the methods in terms of applicability and correctness. The latter ranges from 44 to 75% correct core positions. Taking only the best method result per test case this number increases to 84%. We conclude that the methods available are applicable to difficult cases, but also that there is still room for improvements in both, practicability and alignment correctness. An approach that combines the currently available methods supported by a proper score would be useful. Until then, a user should not rely on just a single program.

The chapter gives more information about the application of integrated nutrigenomics approach in nutritional sciences. It also emphasized for accelerated implementation of mechanistic
knowledge in food design. It gives an application and modification approaches of proteomic to analyze the complexity of food protein modification in the area of general food science and quality assurance

An elementary introduction into the Seiberg-Witten theory is given. Many efforts are made to get it as pedagogical as possible, and within a reasonable size. The selection of the relevant material is heavily oriented towards graduate students. The basic ideas about solitons, monopoles, supersymmetry and duality are reviewed from first principles, and they are illustrated on the simplest examples. The exact Seiberg-Witten solution to the low-energy effective action of the four-dimensional N=2 supersymmetric pure Yang-Mills theory with the gauge group SU(2) is the main subject of the review. Other gauge groups are also discussed. Some related issues (like adding matter, confinement, string dualities) are outlined. 1 Supported in part by the 'Deutsche Forschungsgemeinschaft' and the 'Volkswagen Stiftung' 2 On leave of absence from: High Current Electronics Institute of the Russian Academy of Sciences, Siberian Branch, Akademichesky 4, Tomsk 634055, Russia 10

The free radical-mediated damage to proteins results in the modification of amino acid residues, cross-linking of side chains and fragmentation. L-tyrosine and protein bound tyrosine are prone to attack by various mediators and reactive nitrogen intermediates to form 3-nitrotyrosine (3-NT). 3-NT formation is also catalyzed by a class of peroxidases utilizing nitrite and hydrogen peroxide as substrates. Evidence supports the formation of 3-NT in vivo in diverse pathologic conditions and 3-NT is thought to be a relatively specific marker of oxidative damage. The formation of nitrotyrosine represents a specific peroxynitrite-mediated protein modification; thus, detection of nitrotyrosine in proteins is considered as a biomarker for endogenous peroxynitrite activity. Formation of tyrosine nitrated proteins is considered to be a post-translational modification with important pathophysiological consequences and is one of the markers of nitrosative stress that have been reported in neurodegeneration, inflammatory and other pathological conditions. -). The latter is considered to be the direct oxidant of peroxynitrite-mediated reagents in biological environments [43]. Peroxynitrite-mediated tyrosine nitration is also accelerated in the presence of transition metal ions, either in free form (Cu 2+ , Fe 3+ , Fe 2+ ) or as complexes involving protoporphyrin IX (heme) or certain chelators -ethylene diamine tetraacetic acid (EDTA) [15,16,44]. Hence, metal ions catalysis plays an important role in the nitration of protein residues in proteins [45].

The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANSapomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.

These lectures give an elementary introduction to the subject of four dimensional black holes (BHs) in supergravity and the Attractor Mechanism in the extremal case. Some thermodynamical properties are discussed and some relevant formulae for the critical points of the BH effective potential are given. The case of Maxwell-Einstein-axion-dilaton (super)gravity is discussed in detail. Analogies among BH entropy and multipartite entanglement of qubits in quantum information theory, as well moduli spaces of extremal BH attractors, are also discussed.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of G-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry52:2888–2906) suggested that there are high- and low-affinity subunit conformations of G, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of G-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the “strong” allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (<10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol12:88–118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the α and β subunits. This was shown by calculating the separate α and β subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol256:775–792).

The  mineral  element  content  of  coconut  water  from  three  (3)
different species was determined in samples gotten form Rivers State.
Ca,  Na,  K,  Fe,  Cu,  S,  P  and  Cl  were  determined  using the  Atomic
Absorption  Spectrometry  (AAS)  excluding  chloride  which  was
determined  by  titration  method.  The  concentration  of  this  mineral
element  studied  lies  within  the  ranges  (ppm)  with  Ca  (25-55),  Na
(40-85), K (110-165), Fe (0.542-1.625), Cu (0.000-0.018), S (10.35-30.00), P (0.01-0.53), Cl (2050.00-2565.00). The protein content (i.e.
%  total  protein)  was  determined  as  crude  protein  using  the  macrokjeldahl  Markham  distillation  method.  The  values  ranged  between
0.30-0.40,  0.10-0.20,  and  0.49-0.55  for  the  three  different  species
respectively.

The proximate composition of Bryophyllum pinnatum leaves was evaluated in both dry and fresh samples to determine the ash, carbohydrate, fat, fibre, protein, moisture and mineral contents using the method of Association of Analytical Chemist (A OAC) and Atomic Absorption Spectrophotometer (AAS).The result shows that Bryophyllum pinnatum contain (1.21 ± 0.07 and 0.8 ± 0.03%) ash, (72.92 ± 1.08 and 4.46 ± 0.52%) carbohydrate, (1.38 ± 0.06 and 1. 15 ± 0.05%) fat, (6.02 ± 1. 06 and 0.95 ± 0.06%) fibre, (5. 38 ± 0.10 and 1.61 ± 0.02%) protein, and (13.01 ± 1.03 and 91.03± 0.55%) moisture in dry and fresh samples respectively. The result of the mineral content revealed that Bryophyllum pinnatum is good sources of minerals such as ‘Fe, Zn, Cu, Mg, Na, K, Ca, Ni, Cd and Pd. This shows that Bryophyllum pinnatum leaf is a good source of human nutrition and should be included as dietary supplement.

The key to the keys to immortality and eternal youth lies in the correct answer to the main question: How to naively discover new essential – but still hidden – features required for properly training novel adaptive supervised machine learning algorithms (SMLA)

A rare γ-pyranopyrazole skeleton: design, one-pot synthesis and computational study †
Drawing upon a consecutive amide coupling and intramolecular cyclisation pathway, a one-pot, straightforward synthetic route has been developed for a range of pyrazole fused γ-pyrone derivatives. The reaction mechanism proposed for the chemoselective formation of γ-pyranopyrazole is furthermore fully supported by experimental and computational studies.

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pK, of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 'HN, I5N, I3Ca, "CO, 'Ha, and I3Cp NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple ( 'H-'3C-'5N) and double resonance ('H-'5N or 'H-I3C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, ? T H~H U and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro15 1, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large I3Ca chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.

B-cell epitopes play a vital role in the development of peptide vaccines, in diagnosis of diseases, and also for allergy research. Experimental methods used for characterizing epitopes are time consuming and demand large resources. The availability of epitope prediction method(s) can rapidly aid experimenters in simplifying this problem. The standard feed-forward (FNN) and recurrent neural network (RNN) have been used in this study for predicting B-cell epitopes in an antigenic sequence. The networks have been trained and tested on a clean data set, which consists of 700 non-redundant B-cell epitopes obtained from Bcipep database and equal number of non-epitopes obtained randomly from Swiss-Prot database. The networks have been trained and tested at different input window length and hidden units. Maximum accuracy has been obtained using recurrent neural network (Jordan network) with a single hidden layer of 35 hidden units for window length of 16. The final network yields an overall prediction accuracy of 65.93% when tested by fivefold cross-validation. The corresponding sensitivity, specificity, and positive prediction values are 67.14, 64.71, and 65.61%, respectively. It has been observed that RNN (JE) was more successful than FNN in the prediction of B-cell epitopes. The length of the peptide is also important in the prediction of B-cell epitopes from antigenic sequences. The webserver ABCpred is freely available at www.imtech.res.in/raghava/abcpred/. Proteins 2006; 65:40-48. V V C 2006 Wiley-Liss, Inc.

Because Tyr35␤ is located at the convergence of the ␣1␤1, ␣1␤2, and ␣1␣2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins ␤Y35F and ␤Y35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35␤ results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in ␤Y35F and increases in ␤Y35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in ␤Y35F or ␤Y35A. X-ray crystal structures of deoxy ␤Y35F and ␤Y35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The ␤Y35F mutation repositions the carboxyl group of Asp126␣1 so that it may form a more favorable interaction with the guanidinium group of Arg141␣2. The ␤Y35A mutation results in increased mobility of the Arg141␣ side chain, implying that the interactions between Asp126␣1 and Arg141␣2 are weakened. Therefore, the changes in the functional properties of these 35␤ mutants appear to correlate with subtle structural differences at the C terminus of the ␣-subunit. . Abbreviations: Bis-tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Tris, tris(hydroxymethyl)aminomethane; EDTA, disodium ethylenediaminetetraacetate; PEG, polyethylene glycol; r.m.s., root mean square; IHP, inositol hexaphosphate; HbA, human hemoglobin major component; ␤V1M, recombinant hemoglobin with the Val 1␤ → Met mutation; ␤Y35F, recombinant hemoglobin with the Tyr 35␤ → Phe and Val 1␤ → Met mutations; ␤Y35A, recombinant hemoglobin with the Tyr 35␤ → Ala and Val 1␤ → Met mutations; met-hemoglobin, hemoglobin with iron oxidized to Fe(III). Article and publication are at

A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.

The fourth EGF-like domain of thrombomodulin (TM4), residues E346-F389 in the TM sequence, has been synthesized. Refolding of the synthetic product under redox conditions gave a single major product. The disulfide bonding pattern of the folded, oxidized domain was (1-3, 2-4, 5-6), which is the same as that found in EGF protein. TM4 was tested for TM anticoagulant activity because deletion and substitution mutagenesis experiments have shown that the fourth EGF-like domain of TM is essential for TM cofactor activity. TM4 showed no TM-like activity in two assay systems, both for inhibition of fibrin clot formation, and for cofactor activity in thrombin activation of protein C.

In this study, the chemical composition of 32 samples coming from 29 different biomass species including a gymnosperm, 2 dicotyledonous angiosperms, 17 monocotyledonous angiosperms and 9 algae species was successfully determined using an established method applicable to analyze various biomass species. The obtained data allowed a direct comparison of the biomass in their chemical composition. It was, thus, revealed that although the chemical composition differed from one species to another, and even from different parts of the same plants, similar trends were found in the composition of biomass species belonging to the same taxonomic group. Based on those results, it was clarified that the chemical composition of a biomass sample is related to its taxonomy. Therefore, typical chemical composition for each taxonomic group was proposed.

In the last decade enormous advances in life sciences have been possible due to the information obtained from DNA sequencing projects. The optimal interpretation and analysis of genome sequence data requires the precise annotation and classification of proteins deduced ...

Hydropathy plot methods form a cornerstone of membrane protein research, especially in the early stages of biochemical and structural characterization. Membrane Protein Explorer (MPEx), described in this article, is a refined and versatile hydropathy-plot software tool for analyzing membrane protein sequences. MPEx is highly interactive and facilitates the characterization and identification of favorable protein transmembrane regions using experimentbased physical and biological hydrophobicity scales. Besides allowing the consequences of sequence mutations to be examined, it provides tools for aiding the design of membrane-active peptides. MPEx is freely available as a Java Web Start application from our web site at

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by coincubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and Abbreviations: Pep (pET29b) , peptide encoded in pET29b plasmid; Trx (pET32b) , thioredoxin protein and connector peptide from pET32b plasmid; TrxFNR, mature region of pea ferredoxin-NADP + reductase fused to thioredoxin and thrombin recognition site encoded in pET205 plasmid; GST (pGEX-3X) , glutathione S-transferase protein and connector peptide encoded in pGEX-3X plasmid; GST (pGEX-2T) , glutathione S-transferase protein and connector peptide encoded in pGEX-2T plasmid; E. coli ΔdnaK, E. coli BB1553 strain (deficient in DnaK chaperone); GST-DnaK-free, GST protein encoded in pGEX-2T plasmid purified from E. coli BB1553 cells; TN buffer, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl; TN-ATP buffer, TN buffer containing freshly prepared 5 mM ATP-Mg.

BACKGROUNDProstate cancer (PCa) is the second leading cause of cancer-related death in men in the United States. Many men have implemented purported chemopreventive agents into their daily diet in an attempt to delay the early onset of a PCa. Green tea polyphenols, one such agent, has been shown to be chemopreventive in skin, breast, and prostate cancers. We hypothesized that Epigallocatechin-3-Gallate (EGCG), the major polyphenol found in green tea, will exert its chemopreventive effect in the prostate via regulation of sex steroid receptor, growth factor-signaling, and inflammatory pathways.Prostate cancer (PCa) is the second leading cause of cancer-related death in men in the United States. Many men have implemented purported chemopreventive agents into their daily diet in an attempt to delay the early onset of a PCa. Green tea polyphenols, one such agent, has been shown to be chemopreventive in skin, breast, and prostate cancers. We hypothesized that Epigallocatechin-3-Gallate (EGCG), the major polyphenol found in green tea, will exert its chemopreventive effect in the prostate via regulation of sex steroid receptor, growth factor-signaling, and inflammatory pathways.METHODSFive-week-old male TRAMP (Transgenic Adenocarcinoma Mouse Prostate) offspring were fed AIN-76A diet and 0.06% EGCG in tap water. Animals were sacrificed at 28 weeks of age and the entire prostates were scored histopathologically. In addition, animals were sacrificed at 12 weeks of age and ventral (VP) and dorsolateral (DLP) prostates were removed for histopathological evaluation and immunoblot analyses or ELISA.Five-week-old male TRAMP (Transgenic Adenocarcinoma Mouse Prostate) offspring were fed AIN-76A diet and 0.06% EGCG in tap water. Animals were sacrificed at 28 weeks of age and the entire prostates were scored histopathologically. In addition, animals were sacrificed at 12 weeks of age and ventral (VP) and dorsolateral (DLP) prostates were removed for histopathological evaluation and immunoblot analyses or ELISA.RESULTSEGCG, inhibited early but not late stage PCa in the current study. In the VP, EGCG significantly reduced cell proliferation, induced apoptosis, and decreased androgen receptor (AR), insulin-like growth factor-1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-extracellular signal-regulated kinases 1 and 2 (phospho-ERKs 1 and 2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS).EGCG, inhibited early but not late stage PCa in the current study. In the VP, EGCG significantly reduced cell proliferation, induced apoptosis, and decreased androgen receptor (AR), insulin-like growth factor-1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-extracellular signal-regulated kinases 1 and 2 (phospho-ERKs 1 and 2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS).CONCLUSIONSThe attenuation of the AR, the down-regulation of potent growth factor IGF-1, modulation of inflammation biomarkers, and decrease in the MAPK signaling may contribute to the reduction in cell proliferation and induction of apoptosis and hence provide a biochemical basis for EGCG suppressing PCa without toxicity. Prostate 67: 1576–1589, 2007. © 2007 Wiley-Liss, Inc.The attenuation of the AR, the down-regulation of potent growth factor IGF-1, modulation of inflammation biomarkers, and decrease in the MAPK signaling may contribute to the reduction in cell proliferation and induction of apoptosis and hence provide a biochemical basis for EGCG suppressing PCa without toxicity. Prostate 67: 1576–1589, 2007. © 2007 Wiley-Liss, Inc.

Techniques from graph theory are applied to analyze the bond networks in proteins and identify the flexible and rigid regions. The bond network consists of distance constraints defined by the covalent and hydrogen bonds and salt bridges in the protein, identified by geometric and energetic criteria. We use an algorithm that counts the degrees of freedom within this constraint network and that identifies all the rigid and flexible substructures in the protein, including overconstrained regions (with more crosslinking bonds than are needed to rigidify the region) and underconstrained or flexible regions, in which dihedral bond rotations can occur. The number of extra constraints or remaining degrees of bond-rotational freedom within a substructure quantifies its relative rigidity/flexibility and provides a flexibility index for each bond in the structure. This novel computational procedure, first used in the analysis of glassy materials, is approximately a million times faster than molecular dynamics simulations and captures the essential conformational flexibility of the protein main and side-chains from analysis of a single, static threedimensional structure. This approach is demonstrated by comparison with experimental measures of flexibility for three proteins in which hinge and loop motion are essential for biological function: HIV protease, adenylate kinase, and dihydrofolate reductase. Proteins 2001;44:150 -165.