Biol Reprod, 2001 Sep, 65(3), 944 - 50 Cloning and characterization of a complementary DNA encoding a human epididymis-associated disintegrin and metalloprotease 7 protein; Lin YC et al.; Mammalian spermatozoa interact with the proteins secreted by the epididymis to develop fertility . Transmembrane proteins that possess a disintegrin and metalloprotease (ADAM) domains are shown to be closely related to spermatogenesis and fertilization . Our previous study demonstrated that GP-83, a glycoprotein secreted by the epididymis, was conjugated to mature sperm . In this study, a 2.1-kilobase (kb) GP-83-expressing insert was isolated from a cDNA library of human epididymis by immunoscreening using GP-83-specific antiserum . The 5' end rapid amplification of cDNA ends (RACE) and 3'-RACE of the 2.1-kb insert elucidated two isoforms of GP-83-encoding cDNA sequences, an alpha-form of 3451 base pairs (bp) and beta-form of 2643 bp . Both forms exhibit the same open reading frame of 2262 bp predicting a peptide of 754 amino acid residues . Deduced amino acid sequence revealed signal sequence, prodomain, metalloproteinase, disintegrin, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains . The GP-83-encoding sequence was recognized as human ADAM7 due to significant homology to other ADAM7s . According to the DNA sequences elucidated in the Human Genome Project, h-ADAM7 was located at chromosome 8p22 . Ex vivo expression confirmed that h-ADAM7 cDNA did encode GP-83 . Northern blot analysis revealed two transcripts of 4 kb and 3 kb in the epididymis, but not in testis or other major tissues . These results indicate that the GP-83-encoding gene is a human epididymis-associated ADAM7 gene (human ADAM7, h-ADAM7) and may be involved in the sperm-egg interaction. Mol Cell Endocrinol, 2001 Sep, 182(2), 249 - 63 Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists; Muyan M et al.; Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators . Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective . Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers . We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population . This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers . Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha . We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor . When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha . These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists. Biochim Biophys Acta, 2001 Aug 13, 1548(2), 265 - 77 Expression and characterisation of the thrombospondin type I repeats of human properdin; Perdikoulis MV et al.; Properdin, an upregulator of the alternative complement pathway, is central to deposition of the activated complement fragment C3b on the surfaces of the pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes . It is also known to bind sulphated glycoconjugates, such as sulphatides . Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyclic dimers, trimers and tetramers . The monomer (approximately 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids (based on exon/intron boundaries), called 'thrombospondin type I repeats' or TSR modules . We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coli . Although the individual recombinant TSRs, after a denaturation-renaturation cycle, appeared to be correctly folded modules, as judged by the one-dimensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide . Polyclonal antibodies were raised against each TSR and were found to be module-specific . The anti-TSR5 polyclonal antibody was found to inhibit binding of native human properdin to solid-phase C3b, or sulphatides . It could also block properdin-dependent haemolysis of rabbit erythrocytes . These results are consistent with the view that the TSR5 contains the major site in properdin which is involved in both C3b and sulphatide binding . It also suggests that a co-operative intramolecular interaction between TSRs, as found in the native molecule, is required for TSR5 to bind either C3b or sulphatides. FEBS Lett, 2001 Aug 10, 503(1), 80 - 4 Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-acetylglucosamine 2-epimerase; Mantey LR et al.; Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems . N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid . N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid . Thus, UDP-N-acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids. Biochim Biophys Acta, 2001 Sep 3, 1514(1), 33 - 50 Probing of the substrate binding domain of lactose permease by a proton pulse; Nachliel E et al.; The lactose permease of Escherichia coli coupled proton transfer across the bacterial inner membrane with the uptake of beta-galactosides . In the present study we have used the cysteine-less C148 mutant that was selectively labeled by fluorescein maleimide on the C148 residue, which is an active component of the substrate transporting cavity . Measurements of the protonation dynamics of the bound pH indicator in the time resolved domain allowed us to probe the binding site by a free diffusing proton . The measured signal was reconstructed by numeric integration of differential rate equations that comply with the detailed balance principle and account for all proton transfer reactions taking place in the reaction mixture . This analysis yields the rate constants and pK values of all residues participating in the fast proton transfer reaction between the bulk and the protein's surface, revealing the exposed residues that react with free protons in a diffusion controlled reaction and how they transfer protons among themselves . The magnitudes of these rate constants were finally evaluated by comparison with the rate predicted by the Debye-Smoluchowski equation . The analysis of the kinetic and pK values indicated that the protein-fluorescein adduct assumes two conformation states . One is dominant above pH 7.4, while the other exists only below 7.1 . In the high pH range, the enzyme assumes a constrained configuration and the rate constant of the reaction of a free diffusing proton with the bound dye is 10 times slower than a diffusion controlled reaction . In this state, the carboxylate moiety of residue E126 is in close proximity to the dye and exchanges a proton with it at a very fast rate . Below pH 7.1, the substrate binding domain is in a relaxed configuration and freely accessed by bulk protons, and the rate of proton exchange between the dye and E126 is 100,000 times slower . The relevance of these observations to the catalytic cycle is discussed. Biochem J, 2001 Sep 1, 358(Pt 2), 505 - 10 Local and spatial factors determining HIV-1 protease substrate recognition; Hazebrouck S et al.; Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context . By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease-substrate recognition . A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS . This result supports a dynamic interpretation of HIV-1 protease specificity . Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence . Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site . To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS . In both environments, insertion of only two amino acids could determine specific cleavage . We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts. Biochem J, 2001 Sep 1, 358(Pt 2), 473 - 80 Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine; Ishii Y et al.; SulA protein, a cell division inhibitor in Escherichia coli, is degraded by Lon protease . The C-terminal eight residues of SulA have been shown to be recognized by Lon; however, it remains to be elucidated which amino acid in the C-terminus of SulA is critical for the recognition of SulA by Lon . To clarify this point, we constructed mutants of SulA with changes in the C-terminal residues, and examined the accumulation and stability of the resulting mutant SulA proteins in vivo . Substitution of the extreme C-terminal histidine residue with another amino acid led to marked accumulation and high stability of SulA in lon(+) cells . A SulA mutant in which the C-terminal eight residues were deleted (SulAC161) showed high accumulation and stability, but the addition of histidine to the C-terminus of SulAC161 (SulAC161+H) made it labile . Similarly, SulAC161+H fused to maltose-binding protein (MBP-SulAC161+H) formed a tight complex with and was degraded rapidly by Lon in vitro . Histidine competitively inhibited the degradation of MBP-SulA by Lon, while other amino acids did not . These results suggest that the histidine residue at the extreme C-terminus of SulA is recognized specifically by Lon, leading to a high-affinity interaction between SulA and Lon. Biochem J, 2001 Sep 1, 358(Pt 2), 457 - 64 Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C . 31461; Videira P et al.; Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs) . Few studies on the mechanism of action for this type of GT are available . Sphingomonas paucimobilis A.T.C.C . 31461 produces the industrially important polysaccharide gellan gum . We have cloned the gelK gene from S . paucimobilis A.T.C.C . 31461 . GelK belongs to family 1 of the GT classification {Campbell, Davies, Bulone, Henrissat (1997) Biochem . J . 326, 929-939} . Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity . The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli . GelK and GelK(NH2) were present in both the soluble and membrane fractions of E . coli, whereas GelK(COOH) was only present in the soluble fraction . GelK catalysed the transfer of {(14)C}glucuronic acid from UDP-{(14)C}glucuronic acid into a glycolipid extracted from S . paucimobilis or E . coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase . GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity . Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity . Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily. Biochemistry, 2001 Aug 28, 40(34), 10402 - 10 The 4,4'-dipyridyl disulfide-induced formation of GroEL monomers is cooperative and leads to increased hydrophobic exposure; Panda M et al.; The molecular chaperone, GroEL, is completely disassembled into monomers by the addition of 4,4'-dipyridyl disulfide . The dissociation leads to monomers in a kinetically controlled process . The additions of functional ligands of GroEL such as Mg(2+) or adenine nucleotides produced differences in the observed rates, but at the end of the kinetics, the dissociation was complete . In addition to the information obtained from native gels, the fluorescent probe bis-ANS was utilized to follow the monomer formation . The results demonstrate that the formation of monomers was associated with the exposure of hydrophobic surfaces . This assessment was possible without the use of added chaotropes, such as urea, to dissociate GroEL . Dissociation kinetics were also followed by light scattering . The kinetics of dissociation of the 14mer are cooperative with respect to the concentration of 4,4'-DPDS . Thermodynamic parameters for the kinetic process gave a free energy of activation (DeltaG) of 19.3 +/- 1.2 kcal mol(-1), which was decomposed to an enthalpy of activation (DeltaH) of 19.30 +/- 1.2 kcal mol(-1) and an entropy of activation (DeltaS) of -8.2 +/- 3.9 cal mol(-1) K(-1) . We conclude that the dissociation of GroEL observed in this investigation is an enthalpy-controlled process. Biochemistry, 2001 Aug 28, 40(34), 10326 - 33 Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface; Koide A et al.; It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity . We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4 . Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a) . These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH . Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability . The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins . Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride . These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions. Biochemistry, 2001 Aug 28, 40(34), 10187 - 96 On the multiple functional roles of the active site histidine in catalysis and allosteric regulation of Escherichia coli glucosamine 6-phosphate deaminase; Montero-Moran GM et al.; The active site of glucosamine-6-phosphate deaminase (EC 3.5.99.6, formerly 5.3.1.10) from Escherichia coli was first characterized on the basis of the crystallographic structure of the enzyme bound to the competitive inhibitor 2-amino-2-deoxy-glucitol 6-phosphate . The structure corresponds to the R allosteric state of the enzyme; it shows the side-chain of His143 in close proximity to the O5 atom of the inhibitor . This arrangement suggests that His143 could have a role in the catalysis of the ring-opening step of glucosamine 6-phosphate whose alpha-anomer is the true substrate . The imidazole group of this active-site histidine contacts the carboxy groups from Glu148 and Asp141, via its Ndelta1 atom {Oliva et al . (1995) Structure 3, 1323-1332} . These interactions change in the T state because the side chain of Glu148 moves toward the allosteric site, leaving at the active site the dyad Asp141-His143 {Horjales et al . (1999) Structure 7, 527-536} . In this research, a dual approach using site-directed mutagenesis and controlled chemical modification of histidine residues has been used to investigate the role of the active-site histidine . Our results support a multifunctional role of His143; in the forward reaction, it is involved in the catalysis of the ring-opening step of the substrate, glucosamine 6-P . In the reverse reaction, the substrate fructose 6-P binds in its open chain, carbonylic form . The role of His143 in the binding of both glucosamine 6-P and reaction intermediates in their extended-chain forms was demonstrated by binding experiments using the reaction intermediate analogue, 2-amino-2-deoxy-D-glucitol 6-phosphate . His143 was also shown to be a critical residue for the conformational coupling between active and allosteric sites . From the pH dependence of the reactivity of the active site histidine to diethyl dicarbonate, we observed a pK(a) change of 1.2 units to the acid side when the enzyme undergoes the allosteric T to R transition during which the side chain of Glu148 moves toward the active site . The kinetic study of the Glu148-Gln mutant deaminase shows that the loss of the carboxy group and its replacement with the corresponding amide modifies the k(cat) versus pH profile of the enzyme, suggesting that the catalytic step requiring the participation of His143 has become rate-limiting . This, in turn, indicates that the interaction Glu148-His143 in the wild-type enzyme in the R state contributes to make the enzyme functional over a wide pH range. Biochemistry, 2001 Aug 28, 40(34), 10161 - 8 Crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP(+) and ursodeoxycholate; Jin Y et al.; The crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP(+) and 3alpha,7beta-dihydroxy-5beta-cholanic acid (ursodeoxycholate) at 3.0 A resolution is presented . Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily . AKR1C2 is implicated in the prostatic production of the potent androgen 5alpha-dihydrotestosterone and the hepatic transport of bile acids . It also catalyzes the formation of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria . Like other members of the superfamily, AKR1C2 folds into an alpha/beta-barrel and binds NADP(+) in an extended conformation . The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site . More interestingly, the orientation of ursodeoxycholate is essentially "backwards" and "upside-down" from that observed for testosterone in the related rat 3alpha-HSD.NADP(+).testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate . The orientation of ursodeoxycholate is thus similar to that expected of a 17beta-HSD substrate . The ternary structure explains the ability of AKR1C2 to catalyze 3alpha-, 17beta-, and 20alpha-HSD reactions . Comparison of the steroid binding pocket of AKR1C2 with that of rat 3alpha-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3alpha-HSD isoforms but not in the rat isoform. Biochemistry, 2001 Aug 28, 40(34), 10150 - 60 Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity; Domanski TL et al.; Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity . The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione . The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha-naphthoflavone (ANF) as substrates . CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation . Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6beta-hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4 . 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6beta-hydroxylation . Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6beta-hydroxylation . These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site . Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket. Biochemistry, 2001 Aug 28, 40(34), 10140 - 9 Hydrophobic core manipulations in ribonuclease T1; De Vos S et al.; Differential scanning calorimetry, urea denaturation, and X-ray crystallography were combined to study the structural and energetic consequences of refilling an engineered cavity in the hydrophobic core of RNase T1 with CH(3), SH, and OH groups . Three valines that cluster together in the major hydrophobic core of T1 were each replaced with Ala, Ser, Thr, and Cys . Compared to the wild-type protein, all these mutants reduce the thermodynamic stability of the enzyme considerably . The relative order of stability at all three positions is as follows: Val > Ala approximately equal to Thr > Ser . The effect of introducing a sulfhydryl group is more variable . Surprisingly, a Val --> Cys mutation in a hydrophobic environment can be as or even more destabilizing than a Val --> Ser mutation . Furthermore, our results reveal that the penalty for introducing an OH group into a hydrophobic cavity is roughly the same as the gain obtained from filling the cavity with a CH(3) group . The inverse equivalence of the behavior of hydroxyl and methyl groups seems to be crucial for the unique three-dimensional structure of the proteins . The importance of negative design elements in this context is highlighted. Biochemistry, 2001 Aug 28, 40(34), 10078 - 86 Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps; Leon BC et al.; Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v-Fps enhances the phosphoryl transfer reaction without influencing substrate, ATP, or metal ion binding affinities {Saylor, P., et al . (1998) Biochemistry 37, 17875-17881} . A structural model of v-Fps, generated from the insulin receptor, indicates that pTyr-1073 chelates two arginines . Mutation of these residues to alanine (R1042A and R1066A) results in weakly phosphorylated enzymes, indicating that one electropositive center is insufficient for attaining maximum loop phosphorylation and concomitant high catalytic activity . While the turnover rate for R1066A is similar to that for a mutant lacking a phosphorylatable residue in the activation loop, the rate for R1042A is 50-fold slower . While solvent perturbation studies suggest that the former is due to a slow phosphoryl transfer step, the latter effect results from a slow conformational change in the mutant, potentially linked to motions in the catalytic loop . Binding of a stoichiometric quantity of Mg(2+) is essential for ATP binding and catalysis, while binding of an additional Mg(2+) ion activates further the wild-type enzyme . The affinity of the R1066A enzyme for the second Mg(2+) ion is 23-fold higher than that of the phosphorylated or unphosphorylated form of wild-type v-Fps, with substrate binding unaffected . Conversely, the affinity of R1066A for a substrate mimic lacking a phosphorylation site is 12-fold higher than that for the phosphorylated or unphosphorylated form of wild-type v-Fps, with binding of the second Mg(2+) ion unaffected . A comparison of these enzyme-independent parameters indicates that Arg-1042 and Arg-1066 induce strain in the active site in the repressed form of the enzyme . While this strain is not relieved in the phosphorylated form, the improvements in catalysis in activated v-Fps compensate for reduced metal and substrate binding affinities. Biochemistry, 2001 Aug 28, 40(34), 10063 - 77 Structure, dynamics, and thermodynamics of the structural domain of troponin C in complex with the regulatory peptide 1-40 of troponin I; Mercier P et al.; The structure of the calcium-saturated C-domain of skeletal troponin C (CTnC) in complex with a regulatory peptide comprising residues 1-40 (Rp40) of troponin I (TnI) was determined using nuclear magnetic resonance (NMR) spectroscopy . The solution structure determined by NMR is similar to the structure of the C-domain from intact TnC in complex with TnI(1)(-)(47) determined by X-ray crystallography {Vassylyev, D . G., Takeda, S., Wakatsuki, S., Maeda, K., and Maeda, Y . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 4847-4852} . Changes in the dynamic properties of CTnC.2Ca2+ induced by Rp40 binding were investigated using backbone amide (15)N NMR relaxation measurements . Analysis of NMR relaxation data allows for extraction of motional order parameters on a per residue basis, from which the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy associated with complex formation can be estimated . The results indicate that binding of Rp40 decreases backbone flexibility in CTnC, particularly at the end of the C-terminal helix . The backbone conformational entropy change (-TDeltaS) associated with binding of Rp40 to CTnC.2Ca2+ determined from (15)N relaxation data is 9.6 +/- 0.7 kcal mol(-1) at 30 degrees C . However, estimation of thermodynamic quantities using a structural approach {Lavigne, P., Bagu, J . R., Boyko, R., Willard, L., Holmes, C . F., and Sykes, B . D . (2000) Protein Sci . 9, 252-264} reveals that the change in solvation entropy upon complex formation is dominant and overcomes the thermodynamic "cost" associated with "stiffening" of the protein backbone upon Rp40 binding . Additionally, backbone amide (15)N relaxation data measured at different concentrations of CTnC.2Ca2+.Rp40 reveal that the complex dimerizes in solution . Fitting of the apparent global rotational correlation time as a function of concentration to a monomer-dimer equilibrium yields a dimerization constant of approximately 8.3 mM. Biochemistry, 2001 Aug 28, 40(34), 10054 - 62 Evolution of enzymatic activities in the enolase superfamily: identification of the general acid catalyst in the active site of D-glucarate dehydratase from Escherichia coli; Gulick AM et al.; D-Glucarate dehydratase from Escherichia coli (GlucD), a member of the enolase superfamily, catalyzes the dehydration of both D-glucarate and L-idarate to form 5-keto-4-deoxy-D-glucarate (KDG) . Previous mutagenesis and structural studies identified Lys 207 and the His 339-Asp 313 dyad as the general basic catalysts that abstract the C5 proton from L-idarate and D-glucarate, respectively, thereby initiating the reaction by formation of a stabilized enediolate anion intermediate {Gulick, A . M., Hubbard, B . K., Gerlt, J . A., and Rayment, I . (2000) Biochemistry 39, 4590-4602} . The vinylogous elimination of the 4-OH group from this intermediate presumably requires a general acid catalyst . The structure of GlucD with KDG and 4-deoxy-D-glucarate bound in the active site revealed that only His 339 and Asn 341 are proximal to the presumed position of the 4-OH leaving group . The N341D and N341L mutants of GlucD were constructed and subjected to both mechanistic and structural analyses . The N341L but not N341D mutant catalyzed the dehydrofluorination of 4-deoxy-4-fluoro-D-glucarate, demonstrating that in this mutant the initial proton abstraction from C5 can be decoupled from elimination of the leaving group from C4 . The kinetic properties and structures of these mutants suggest that either Asn 341 participates in catalysis as the general acid that facilitates the departure of the 4-leaving group or is essential for proper positioning of His 339 . In the latter scenario, His 339 would function not only as the general base that abstracts the C5 proton from D-glucarate but also as the general acid that catalyzes both the departure of the 4-OH group and the stereospecific incorporation of solvent hydrogen with retention of configuration to form the KDG product . The involvement of a single functional group in this reaction highlights the plasticity of the active site design in members of the enolase superfamily. Biochemistry, 2001 Aug 28, 40(34), 10047 - 53 Polar residues in the protein core of Escherichia coli thioredoxin are important for fold specificity; Bolon DN et al.; Most globular proteins contain a core of hydrophobic residues that are inaccessible to solvent in the folded state . In general, polar residues in the core are thermodynamically unfavorable except when they are able to form intramolecular hydrogen bonds . Compared to hydrophobic interactions, polar interactions are more directional in character and may aid in fold specificity . In a survey of 263 globular protein structures, we found a strong positive correlation between the number of polar residues at core positions and protein size . To probe the importance of buried polar residues, we experimentally tested the effects of hydrophobic mutations at the five polar core residues in Escherichia coli thioredoxin . Proteins with single hydrophobic mutations (D26I, C32A, C35A, T66L, and T77V) all have cooperative unfolding transitions like the wild type (wt), as determined by chemical denaturation . Relative to wt, D26I is more stable while the other point mutants are less stable . The combined 5-fold mutant protein (IAALV) is less stable than wt and has an unfolding transition that is substantially less cooperative than that of wt . NMR spectra as well as amide deuterium exchange indicate that IAALV is likely sampling a number of low-energy structures in the folded state, suggesting that polar residues in the core are important for specifying a well-folded native structure. Biochemistry, 2001 Aug 28, 40(34), 10038 - 46 Crystal structure of thrombin-ecotin reveals conformational changes and extended interactions; Wang SX et al.; The protease inhibitor ecotin fails to inhibit thrombin despite its broad specificity against serine proteases . A point mutation (M84R) in ecotin results in a 1.5 nM affinity for thrombin, 10(4) times stronger than that of wild-type ecotin . The crystal structure of bovine thrombin is determined in complex with ecotin M84R mutant at 2.5 A resolution . Surface loops surrounding the active site cleft of thrombin have undergone significant structural changes to permit inhibitor binding . Particularly, the insertion loops at residues 60 and 148 in thrombin, which likely mediate the interactions with macromolecules, are displaced when the complex forms . Thrombin and ecotin M84R interact in two distinct surfaces . The loop at residue 99 and the C-terminus of thrombin contact ecotin through mixed polar and nonpolar interactions . The active site of thrombin is filled with eight consecutive amino acids of ecotin and demonstrates thrombin's preference for specific features that are compatible with the thrombin cleavage site: negatively charged-Pro-Val-X-Pro-Arg-hydrophobic-positively charged (P1 Arg is in bold letters) . The preference for a Val at P4 is clearly defined . The insertion at residue 60 may further affect substrate binding by moving its adjacent loops that are part of the substrate recognition sites. Acc Chem Res, 2001 Aug, 34(8), 681 - 9 Cobalamin-dependent methyltransferases; Matthews RG; Cobalamin cofactors play critical roles in radical-catalyzed rearrangements and in methyl transfers . This Account focuses on the role of methylcobalamin and its structural homologues, the methylcorrinoids, as intermediaries in methyl transfer reactions, and particularly on the reaction catalyzed by cobalamin-dependent methionine synthase . In these methyl transfer reactions, the cobalt(I) form of the cofactor serves as the methyl acceptor . Biological methyl donors to cobalamin include N5-methyltetrahydrofolate, other methylamines, methanol, aromatic methyl ethers, acetate, and dimethyl sulfide . The challenge for chemists is to determine the enzymatic mechanisms for activation of these unreactive methyl donors and to mimic these amazing biological reactions. Arch Microbiol, 2001 Sep, 176(3), 231 - 5 Molecular characterization and heterologous expression of hypCD, the first two {NiFe} hydrogenase accessory genes of Thermococcus litoralis; Takacs M et al.; The hypCD genes, encoding the counterparts of mesophilic proteins involved in the maturation of {NiFe} hydrogenases, were isolated from the hyperthermophilic archaeon Thermococcus litoralis . The deduced gene products showed 30-40% identity to the corresponding mesophilic proteins . HypC and HypD were synthesized by the T7 expression system . Heterologous complementation experiments were done in Escherichia coli and Ralstonia eutropha strains lacking functionally active hypC and hypD genes . Only the cytoplasmic hydrogenase of R . eutropha could be processed by HypD from T . litoralis . This was the first demonstration of mesophilic hydrogenase processing using a hyperthermophilic archaeal accessory protein to produce an active enzyme. J Korean Med Sci, 2001 Aug, 16(4), 489 - 97 Immunochemical characterization of brain and pineal tryptophan hydroxylase; Chung YI et al.; Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escherichia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis . This purified enzymatically active TPH protein was used for production of a specific antiserum . This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, and the rat pineal gland . However, this antiserum recognized an additional protein band of lower molecular weight (48 kDa) in pineal extract . It is not clear whether the 48 kDa TPH band represents an isozyme or a protease cleavage product of TPH . Since the pineal gland contains higher TPH mRNA and lower TPH activity when it is compared with dorsal raphe nucleus enzyme, this lower molecular weight TPH may participate in the reduced TPH specific activity . In addition, there are no specific TPH inhibitors in the pineal gland and this lower molecular weight TPH is inactive or has a very low specific activity . This antiserum specifically immunostained serotonergic cell bodies in the dorsal raphe nuclei, some large caliber serotonergic processes in the dorsal raphe area as well as terminals in the olfactory bulb . It also immunolabeled the pineal gland and immunoprecipitated equally well TPH protein from the dorsal raphe nucleus and the pineal gland in a concentration-dependent manner. Mol Cell, 2001 Jul, 8(1), 159 - 68 Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB; de Zamaroczy M et al.; Colicin D is known to kill target cells by cleaving tRNA(Arg) . A colicin D-resistant mutant was selected that was altered in the inner membrane leader peptidase, LepB . The substituted residue (Asn274Lys) is located close to the catalytic site . The mutation abolishes colicin D cleavage but not the processing of exported proteins . LepB is required for colicin D cleavage, releasing a small C-terminal fragment that retains full tRNase activity . The immunity protein was found to prevent colicin D processing and furthermore masks tRNase activity, thus protecting colicin D against LepB-mediated cleavage during export . Catalytic colicins share a consensus sequence at their putative processing site . Mutations affecting normal processing of colicin D abolish cytotoxicity without affecting the in vitro tRNase activity. Mol Cell, 2001 Jul, 8(1), 21 - 31 Binding of the initiation factor sigma(70) to core RNA polymerase is a multistep process; Gruber TM et al.; The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation . To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and sigma(70), the Escherichia coli initiation factor . We identified nine mutationally verified interaction sites between sigma(70) and specific domains of RNA polymerase and provide evidence that sigma(70) and RNA polymerase interact in at least a two-step process . We propose that a cycle of changes in the interface of sigma(70) with core RNA polymerase is associated with progression through the process of transcription initiation. Mol Cell, 2001 Jul, 8(1), 4 - 6 The importance of being cleaved: an essential step in killing by enzymatic colicins; Jakes KS; In this issue, de Zamaroczy et al . show that cleavage of the bacterial toxin colicin D is required for its ability to kill cells . Surprisingly, the cleavage requires the inner membrane peptidase LepB that normally functions in protein secretion. Cell, 2001 Jul 27, 106(2), 243 - 52 Allosteric binding of nucleoside triphosphates to RNA polymerase regulates transcription elongation; Foster JE et al.; The regulation of transcription elongation and termination appears to be governed by the ability of RNA polymerase elongation complexes to adopt multiple conformational states; however, the factors controlling the distribution between these states remain elusive . We used transient-state kinetics to investigate the incorporation of single nucleotides . We demonstrate that E . coli RNA polymerase contains an allosteric binding site in addition to the catalytic site . Binding of the templated nucleoside triphosphate (NTP), but not nontemplated NTPs, to this site increases the rate of nucleotide incorporation . The data suggest that RNA polymerase can exist in a state that catalyzes synthesis slowly (unactivated) and one that catalyzes synthesis rapidly (activated), with the transition from the slow to the fast state being induced by binding of the templated NTP to the allosteric site. Cell, 2001 Jul 27, 106(2), 233 - 41 The path of messenger RNA through the ribosome; Yusupova GZ et al.; Using X-ray crystallography, we have directly observed the path of mRNA in the 70S ribosome in Fourier difference maps at 7 A resolution . About 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit . The Shine-Dalgarno helix is bound in a large cleft between the head and the back of the platform . At the interface, only about eight nucleotides (-1 to +7), centered on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA . The mRNA enters the ribosome around position +13 to +15, the location of downstream pseudoknots that stimulate -1 translational frame shifting. Chem Res Toxicol, 2001 Aug, 14(8), 1082 - 9 Effect of benzo-ring hydroxyl groups on site-specific mutagenesis by tetrahydrobenzo{a}pyrene adducts at N(6) of deoxyadenosine; Ramos LA et al.; We have previously investigated the mutations induced on replication in Escherichia coli of the M13mp7L2 genome containing each of the eight possible adducts derived from the four optically active 7,8-diol 9,10-epoxide metabolites of benzo{a}pyrene (B{a}P) by alkylation of a specific deoxyadenosine (dAdo) residue at N(6) . Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups in these adducts . To determine how the presence or absence of these hydroxyl groups affects mutational response, we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously studied with the diol epoxide adducts, but containing B{a}P-dAdo adducts in which two or all three of the adduct hydroxyl groups were replaced by hydrogen . Transfection of the adducted M13 constructs into SOS-induced Escherichia coli consistently gave fewer infective centers than the control construct, with viabilities ranging from 8.4 to 44.9% relative to control . In general, decreasing the number of adduct hydroxyls decreased the total frequency of substitution mutations induced . For all but one of the present adducts, the total mutational frequency was lower than that for any of the previously reported diol epoxide adducts in the same sequence . Remarkably, this (9S,10R)-adduct with cis orientation of the dAdo residue and the 9-OH group gave the highest mutational frequency of all the B{a}P adducts studied in this sequence, including the diol epoxide adducts . With the present adducts, A --> T transversions predominated, with smaller numbers of A --> G transitions and even fewer A --> C transversions. Gene Ther, 2001 Aug, 8(15), 1174 - 9 Successful and optimized in vivo gene transfer to rabbit carotid artery mediated by electronic pulse; Matsumoto T et al.; Several gene transfer methods, including viral or nonviral vehicles have been developed, however, efficacy, safety or handling continue to present problems . We developed a nonviral and plasmid-based method for arterial gene transfer by in vivo electronic pulse, using a newly designed T-shaped electrode . Using rabbit carotid arteries, we first optimized gene transfer efficiency, and firefly luciferase gene transfer via electronic pulse under 20 voltage (the pulse length: P(on)time 20 ms, the pulse interval: P(off) time 80 ms, number of pulse: 10 times) showed the highest gene expression . Exogenous gene expression was detectable for at least up to 14 days . Electroporation-mediated gene transfer of E . coli lacZ with nuclear localizing signal revealed successful gene transfer to luminal endothelial cells and to medial cells . Histological damage was recognized as the voltage was increased but neointima formation 4 weeks after gene transfer was not induced . In vivo electroporation-mediated arterial gene transfer is readily facilitated, is safe and may prove to be an alternative form of gene transfer to the vasculature. Intervirology, 2001, 44(2-3), 176 - 97 Development of new vaccines against dengue fever and Japanese encephalitis; Kinney RM et al.; Mosquito-borne dengue (DEN) and Japanese encephalitis (JE) viruses are the leading causes of arthropod-transmitted viral disease in humans . A licensed tetravalent vaccine that provides effective, long-term immunity against all four serotypes of DEN virus is needed, but is currently unavailable . Improvements to currently available JE vaccines are also needed . Past and recent strategies for the development of new DEN and JE vaccines include inactivated and live attenuated viruses, engineered viruses and chimeric viruses derived from infectious cDNA clones of DEN or JE virus, recombinant poxviruses, recombinant baculoviruses, protein expression in Escherichia coli, and naked DNA vaccines . This report summarizes some of the recent developments in DEN and JE vaccinology, particularly vaccine strategies that involve live attenuated viruses, engineered viruses derived from infectious cDNA clones, and naked DNA vaccines . Intervirology, 2001, 44(2-3), 154 - 61 Vaccination against hepatitis delta virus infection: studies in the woodchuck (Marmota monax) model; Fiedler M et al.; Hepatitis delta virus (HDV) superinfection of hepatitis B virus carriers causes severe liver disease and results in a high rate of chronicity . So far, neither sufficient therapy nor vaccines to prevent HBV carriers from superinfection are available . A good model to test vaccine candidates is the woodchuck chronically infected with the woodchuck hepatitis virus (WHV); the woodchuck can be superinfected with HDV and shows a course of infection similar to that of patients . Different strategies have been investigated to establish a protective vaccine against HDV superinfection . Both proteins of HDV (HDAg p24 and p27), which differ only in the C-terminal amino acid sequence, have been used as vaccine candidates . Synthetic peptides derived from B cell epitopes of HDAg and HDAg p24 expressed in Escherichia coli, yeast, or baculovirus have been used to immunize woodchucks . The protein immunization induced a specific antibody response, however, no protection from HDV superinfection was achieved . Vaccinations with vaccinia virus expressing HDAg p24 or p27 and DNA immunization with vectors expressing p24 were also not able to induce a protective immune response, but seemed to modulate the course of HDV superinfection . Thus, new strategies to develop a vaccine to prevent HDV superinfection are needed . Toxicol Sci, 2001 Sep, 63(1), 74 - 81 Methylmercury-induced decrement in neuronal migration may involve cytokine-dependent mechanisms: a novel method to assess neuronal movement in vitro; Sass JB et al.; A major toxic effect associated with methylmercury (MeHg) exposure in developing humans is damage to the nervous system, which involves inhibition of cell migration, particularly in the cerebellum . The mechanisms by which MeHg impairs neural migration are not fully known, especially at low doses . In this paper we report on a novel method for observing and quantitating the movement of individual cells in primary cultures of murine neonatal cerebellar cells, which offers an opportunity to assess the role of endogenous and exogenous factors on neural migration . We have used this system to test the hypothesis that treatment with methylmercury would inhibit movement of granule cell neurons, possibly via a cytokine-mediated mechanism . We demonstrate that LPS (50 ng/ml) increases movement of neurons, concomitant with increased levels of TNF-alpha and IL-6 secreted protein, and IL-1alpha mRNA . Treatment with LPS did not increase the number of neurons that moved, but, of the cells that did move, exposure to LPS significantly increased the total distances moved . Treatment with methylmercury (0.1 microM) decreased the number of moving cells and inhibited overall distance traveled by granule cells. J Biol Chem, 2001 Nov 2, 276(44), 41263 - 9 Epub 2001 Aug 16. Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation; Shah K et al.; The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene encodes a receptor-like protein kinase that is transiently expressed during embryogenesis . To determine the intrinsic biochemical properties of the AtSERK1 protein, we have expressed the intracellular catalytic domain as a glutathione S-transferase fusion protein in Escherichia coli . The AtSERK1-glutathione S-transferase fusion protein mainly autophosphorylates on threonine residues (K(m) for ATP, 4 x 10(-6) m), and the reaction is Mg(2+) dependent and inhibited by Mn(2+) . A K330E substitution in the kinase domain of AtSERK1 abolishes all kinase activity . The active AtSERK1(kin) can phosphorylate inactive AtSERK1(K330E) protein, suggesting an intermolecular mechanism of autophosphorylation . The AtSERK1 kinase protein was modeled using the insulin receptor kinase as a template . On the basis of this model, threonine residues in the AtSERK1 activation loop of catalytic subdomain VIII are potential targets for phosphorylation . AtSERK1 phosphorylation on myelin basic protein and casein showed tyrosine, serine, and threonine as targets, demonstrating that AtSERK1 is a dual specificity kinase . Replacing Thr-468 with either alanine or glutamic acid not only obliterated the ability of the AtSERK1 protein to be phosphorylated but also inhibited phosphorylation on myelin basic protein and casein, suggesting that Thr-468 is essential for AtSERK-mediated signaling. J Biol Chem, 2001 Dec 14, 276(50), 47217 - 26 Epub 2001 Aug 16. Backbone dynamics of plastocyanin in both oxidation states . Solution structure of the reduced form and comparison with the oxidized state; Bertini I et al.; A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp . PCC6803 . The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale . The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments . In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms . A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state . Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed . The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes. Biophys J, 2001 Sep, 81(3), 1570 - 9 Stability and folding rates of domains spanning the large A-band super-repeat of titin; Head JG et al.; Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension . A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains . Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli . Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain . Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units . The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains . Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1) . This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin. Biophys J, 2001 Sep, 81(3), 1345 - 59 Molecular dynamics simulations of wild-type and mutant forms of the Mycobacterium tuberculosis MscL channel; Elmore DE et al.; The crystal structure of the Mycobacterium tuberculosis homolog of the bacterial mechanosensitive channel of large conductance (Tb-MscL) provides a unique opportunity to consider mechanosensitive signal transduction at the atomic level . Molecular dynamics simulations of the Tb-MscL channel embedded in an explicit lipid bilayer and of its C-terminal helical bundle alone in aqueous solvent were performed . C-terminal calculations imply that although the helix bundle structure is relatively unstable at physiological pH, it may have been stabilized under low pH conditions such as those used in the crystallization of the channel . Specific mutations to the C-terminal region, which cause a similar conservation of the crystal structure conformation, have also been identified . Full channel simulations were performed for the wild-type channel and two experimentally characterized gain-of-function mutants, V21A and Q51E . The wild-type Tb-MscL trajectory gives insight into regions of relative structural stability and instability in the channel structure . Channel mutations led to observable changes in the trajectories, such as an alteration of intersubunit interactions in the Q51E mutant . In addition, interesting patterns of protein-lipid interactions, such as hydrogen bonding, arose in the simulations . These and other observations from the simulations are relevant to previous and ongoing experimental studies focusing on characterization of the channel. Curr Biol, 2001 Jul 24, 11(14), R563 - 5 Protein synthesis: twenty three amino acids and counting; Ibba M et al.; The genetic code can be interpreted during translation as 21 amino acids and three termination signals . Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis. Zhonghua Gan Zang Bing Za Zhi, 2001 Jul, 9 Suppl, 30 - 3 {Cloning and sequence analysis of light chain gene of human antibody against HBV pre-S2}; Shi X et al.; OBJECTIVE: To obtain the gene of light chain of human antibody against HBV pre-S2 . METHODS: A human antibody against HBV pre-S2 displayed on phage was obtained from the constructed human immunoglobulin phage displaying library through four rounds panning of "adherence-elution-amplification" with Pre-S2 (120aa-145aa) short peptide synthesized and help phage . The positive clone of anti-pre-s2 was characterized by ELISA competitive inhibition assay . The plasmid from the positive clone was amplified by PCR with designed oligonucleotide primers of kaph light chain . The light chain fragment was amplified with PCR and plasmid pUC18 digested by restriction endonuclease SacI & XbaI, respectively, then it was subcloned into the plasmid pUC18 and was transformed into E.coli XL1-blue sensitized by CaCl2 . The recombinant plasmid pUC18-kaph was sequenced . RESULTS: The competitive inhibition rate of the positive clone of anti-pre-S2 was 65% at 1:50 dilution . Sequence was compared with data in gene bank through internet and analyzed with Blast . According to the sequence and ELISA competitive inhibition assay the integral gene of kaph light chain of human antibody against HBV-PreS2 was obtained . Its variable region located on 1-328bp including three hypervariable regions (82-105bp, 145~177bp, 274~309bp) and four frame regions (1-81bp, 106-144bp, 178-273bp, 310-328bp) . Its constant region was located on 329-645bp . CONCLUSIONS: The HBV pre-S2 kaph light chain is screened from phage antibody displaying library by antigen-antibody special response. Zhonghua Gan Zang Bing Za Zhi, 2001 Jul, 9 Suppl, 6 - 8 {Studies on cloning and expression of hepatitis B virus x gene}; Sun Z et al.; OBJECTIVE: To express and purify the x gene of hepatitis B virus (HBV), and then the purity of the protein could specifically be analysed by the human antibodies against HBV x antigen (protein) . METHODS: HBV x gene was amplified from the complete genome by PCR, and cloned in the pGEX-2T fused expression vector . After transforming the plasmid into E.coli, the recombinant with HBx gene was obtained . The fusion protein was generated with E.coli fused expression system and purified with affinity chromatography . RESULTS: The HBx protein could be applied to detecting specific antibody of hepatitis B virus patients . CONCLUSIONS: The purity of the HBx protein can specifically be recognized by the human antibodies against HBV x antigen. Jpn J Cancer Res, 2001 Aug, 92(8), 848 - 53 Inhibition of DNA adduct formation and mutagenic action of 3-amino-1-methyl-5h-pyrido{4,3-b}indole by chlorophyllin-chitosan in rpsL transgenic mice; Anzai N et al.; We have studied the inhibitory effect of chlorophyllin-chitosan (Chl-Chi) complex, an insoluble form of chlorophyllin, on the DNA adduct formation and mutagenesis by a heterocyclic food mutagen-carcinogen, 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2), in mice carrying the E . coli rpsL gene as a mutagenesis reporter . Upon administration of a diet containing 0.002% or 0.01% Trp-P-2, DNA adducts were formed in various tissues in a dose-dependent manner, with the maximum level observed in the liver . Addition of 3% Chl-Chi to the diet reduced the Trp-P-2 adduct by up to 90% . The rpsL mutant frequencies increased significantly in both the liver and spleen upon administration of a 0.01% Trp-P-2 diet . Addition of Chl-Chi to the diet decreased these induced mutant frequencies to the background level . No harmful effect of Chl-Chi was detected during these experiments . The results show that Chl-Chi may be a candidate chemopreventive agent against the genotoxic action of Trp-P-2, and possibly also other aromatic carcinogens in the diet. Shock, 2001 Aug, 16(2), 143 - 7 Ex vivo fluorescence microscopy provides simple and accurate assessment of neutrophil-endothelial adhesion in the rat lung; Han I et al.; Neutrophil adhesion to the pulmonary endothelium is prerequisite to neutrophil transmigration and activation, both of which may lead to lung injury . A simple method to evaluate neutrophil adherence in the lung would be useful for developing new strategies for neutrophil-mediated lung injury . The purpose was to establish a simple method to evaluate neutrophil adhesion in the lung using ex vivo fluorescence microscopy . Rats were anesthetized, and the right jugular veins were catheterized . Neutrophils were isolated from another set of rats and labeled with 5,(6)-carboxyfluorescein diacetate . Animals were killed 120 s after a 1 x 10(6) labeled neutrophil injection . The pulmonary labeled neutrophil number was counted under a fluorescence microscope . In the first experiment, rats were given 0, 20, 200, or 2000 microg/kg lipopolysaccharide (LPS) i.p . At 4 h after challenge, the pulmonary labeled neutrophil number was determined . Kinetic studies were also performed at 0, 1, 4, and 8 h after 200 microg/kg LPS . Finally, anti-ICAM-1 Ab was injected i.v . before LPS 200 microg/kg, and the labeled neutrophil number in the lung was determined at 4 h . The number of pulmonary labeled neutrophils was higher after LPS 200 or 2000 microg/kg than after the other doses . The pulmonary labeled neutrophil number was increased at 4 h compared with the other time points . ICAM-1 blocking normalized the pulmonary labeled neutrophil number in the LPS group . In conclusion, our method seems to reflect ICAM-1-mediated neutrophil adherence to the endothelium in the present setting . This simple technique may be useful for evaluating neutrophil adhesion. Shock, 2001 Aug, 16(2), 130 - 6 Effects of combined selective iNOS inhibition and peroxynitrite blockade during endotoxemia in pigs; Ploner F et al.; We investigated the effect of mercaptoethylguanidine (MEG, 3 mg kg(-1)h(-1)), a combined selective inducible nitric oxide synthase (iNOS) inhibitor, a peroxynitrite and oxygen free radical scavenger with cyclooxygenase-inhibitor properties on intestinal and hepatic perfusion, O2 exchange, and metabolism during long-term hyperdynamic porcine endotoxemia . MEG was started 12 h after onset of endotoxemia . At baseline and after 12, 18, and 24 h of endotoxemia, hepatic arterial and portal venous blood flow, ileal mucosal-arterial PCO2 gap, portal and hepatic venous lactate/pyruvate ratio, free glutathione (GSH), and 8-isoprostanes were measured . Expired NO and plasma nitrate levels were assessed as well . MEG blunted the endotoxin-induced increase in expired NO and prevented the progressive fall in blood pressure without affecting cardiac output . It attenuated both systemic and regional venous acidosis without influencing the impairment of hepatosplanchnic metabolism nor counteracting the increase in GSH levels . In our model MEG failed to beneficially affect variables of oxidative stress. Eur Biophys J, 2001 Jul, 30(3), 186 - 97 Ionization properties of titratable groups in ribonuclease T1 . I . pKa values in the native state determined by two-dimensional heteronuclear NMR spectroscopy; Spitzner N et al.; pKa values of amino acid side chains of ribonuclease T1 have been determined from the pH dependence of 13C and 15N resonances . It was possible to derive pKa values of single protonation or deprotonation sites of carboxylate and imidazole groups . Deviations from pKa values of free amino acids could be interpreted with electrostatic interactions of corresponding side chains with the protein environment . In particular, the interaction between H27 and E82 led to an increase of the H27 pKa and a decrease of the E82 pKa . The pKa of E28 at the C-terminal end of the alpha-helix was increased because of the dipolar character of the alpha-helix . D76 did not titrate in the investigated pH range of about 2-9 . From the chemical shift value this buried side chain seems to be protonated . The pKa values of side chains in the active site deviate from a normal behaviour . The lower pKa value of E58 may be interpreted with the close proximity of this side chain with positively charged H40 and R77 . A novel two-dimensional 1H(13Cdelta)13Cgamma correlation experiment was developed to observe the pH dependence of the chemical shifts of the Cgamma resonances of histidine residues . From the inspection of the Cgamma chemical shift-pH profiles it was possible to determine the predominant tautomeric form for the histidine residues at higher pH values. J Nutr Sci Vitaminol (Tokyo), 2001 Apr, 47(2), 132 - 8 Expression and properties of human liver beta-ureidopropionase; Sakamoto T et al.; A cDNA encoding beta-ureidopropionase (BUP) was isolated from a human liver cDNA library, expressed in E . coli, and purified from the culture extract . The 2,006 bp cDNA contained a 1,152 bp open reading frame encoding a protein of 384 amino acids with a molecular weight of 43,165 Da . The subunit molecular weight of the enzyme expressed was about 43,000 Da . The enzyme was inhibited by 1 mM propionate, but not by 10 mM beta-alanine . Chemical analysis of the purified human BUP showed 0.54 zinc atoms per subunit, and the sequence of BUP cDNA contained one putative zinc-binding site motif . The purified enzyme had a pI of 5.65, and exhibited positive cooperativity with N-carbamoyl-beta-alanine as the substrate with a Hill coefficient 2.0 . These properties of human BUP, except the inhibition by beta-alanine, were similar to the rat liver purified enzyme . Beta-alanine inhibits rats BUP activity . The complex regulatory function and the negative cooperative mechanism of BUP by beta-alanine have been observed in rats . This kind of mechanism may not exist in humans, because beta-alanine did not inhibit human BUP. J Rheumatol, 2001 Aug, 28(8), 1874 - 80 Chlamydial nucleic acids in synovium in osteoarthritis: what are the implications? Olmez N, Wang GF, Li Y, Zhang H, Schumacher HR. OBJECTIVE: To study whether there is evidence of bacterial DNA in some osteoarthritic (OA) joint tissues, and the clinical implications of finding bacterial DNA in this relatively noninflammatory disease . METHODS: Polymerase chain reaction (PCR) was used to detect DNA of Chlamydia trachomatis, Chlamydia pneumoniae, and other bacteria using panbacterial primers in synovial membranes and other articular tissues of 32 consecutive patients undergoing surgery for hip and knee OA . Patients were interviewed and examined postoperatively . Operative reports were reviewed and followup examinations were accomplished on all patients . RESULTS: Nine of 32 patients with OA (28.1%) had evidence for bacterial DNA in joint tissues with at least one set of primers for Chlamydia: 7 for C . trachomatis (21.9%), 2 for C . pneumoniae (6.2%) . Five of 32 (15.6%) patients had postoperative complications; 3 of these were in patients who showed amplified DNA of C . trachomatis in joints and one in a patient in whom we detected Escherichia coli . CONCLUSION: C . trachomatis and C . pneumoniae nucleic acids can be present in joints in some cases of apparently classical OA . Whether chlamydial or other difficult to culture bacterial presence is associated with complications is suggested, but remains to be determined . Simple presence of C . trachomatis by PCR does not define a clinical syndrome or disease course. Am J Trop Med Hyg, 2001 Aug, 65(2), 159 - 61 Short report: Antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine; Simmons M et al.; Recombinant proteins containing the B domain of dengue virus serotypes 1-4 fused to the maltose binding protein (MBP) of Escherichia coli were evaluated individually and as a tetravalent vaccine candidate in mice . Sera from mice immunized with monovalent DEN-MBP recombinant protein vaccines developed high titers of serotype homologous antibody in the enzyme-linked immunosorbent assay and the plaque-reduction neutralization test . Cross-reactive antibody titers were either several dilutions lower or not detectable . Sera from mice immunized with the tetravalent DEN subunit vaccine neutralized all 4 DEN viruses in the plaque-reduction neutralization test . The neutralizing antibody titers to each individual serotype were significantly greater than any cross-reactive neutralizing antibody titers induced by the monovalent vaccines, providing evidence that the tetravalent DEN recombinant subunit vaccine produced specific neutralizing antibody to all 4 serotypes of dengue virus. Mutagenesis, 2001 Sep, 16(5), 431 - 7 Mutations induced by 2-amino-1-methyl-6-phenylimidazo {4,5-b}pyridine (PhIP) in cecum and proximal and distal colon of lacI transgenic rats; Stuart GR et al.; 2-Amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) is a food-borne mutagen and carcinogen that induces tumors of the colon and the prostate gland in male rats and of the mammary gland in female rats . In this study we describe the frequency and specificity of PhIP-induced mutations in the cecum, proximal colon and distal colon of male and female lacI transgenic rats . This is the first report of mutational data from discrete regions of the colon . After 61 days of treatment with 200 p.p.m . PhIP mixed into the diet, PhIP-induced mutant frequencies were elevated 7-fold in the cecum and 14- to 21-fold in the colon of male and female rats compared with untreated controls . PhIP-induced mutant frequencies increased significantly (overall trend, P < 10(-4)) along the length of the colon of both males and females, with cecum < proximal colon < distal colon . A total of 754 PhIP mutants (363 male, 391 female) were sequenced to provide the mutational spectra for each of the three tissue sections from males and females . These mutational spectra consisted predominantly of G:C-->T:A and G:C-->C:G transversions and deletions of G:C base pairs . There were no significant differences between the mutational spectra with respect to sex or position in the colon . Therefore, we surmise that following induction of mutations by PhIP in male and female colons, non-mutagenic factors, possibly hormonal, preferentially influence the formation of tumors in the colon of male rats. J Biol Chem, 2001 Oct 19, 276(42), 38602 - 9 Epub 2001 Aug 15. Purification and characterization of membrane-associated CooC protein and its functional role in the insertion of nickel into carbon monoxide dehydrogenase from Rhodospirillum rubrum; Jeon WB et al.; The accessory protein CooC, which contains a nucleotide-binding domain (P-loop) near the N terminus, participates in the maturation of the nickel center of carbon monoxide dehydrogenase (CODH) . In this study, CooC was purified from the chromatophore membranes of Rhodospirillum rubrum with a 3,464-fold purification and a 0.8% recovery, and its biochemical properties were characterized . CooC is a homodimer with a molecular mass of 61-63 kDa, contains less than 0.1 atom of Ni(2+) or Fe(2+) per dimer, and has a lambda(max) at 277.5 nm (epsilon(277.5) 32.1 mm(-1) cm(-1)) with no absorption peaks at the visible region . CooC catalyzes the hydrolysis of ATP and GTP with K(m) values of 24.4 and 26.0 microm and V(max) values of 58.7 and 3.7 nmol/min/mg protein for ATP and GTP hydrolysis, respectively . The P-loop mutated form of K13Q CooC was generated by site-specific replacement of lysine by glutamine and was purified according to the protocol for wild-type CooC purification . The K13Q CooC was inactive both in ATP hydrolysis and in vivo nickel insertion . In vitro nickel activation of apoCODH in the cell extracts from UR2 (wild type) and UR871 (K13Q CooC) showed that activation of nickel-deficient CODH was enhanced by CooC and dependent upon ATP hydrolysis . The overall results suggest that CooC couples ATP hydrolysis with nickel insertion into apoCODH . On the basis of our results and models for analogous systems, the functional roles of CooC in nickel processing into the active site of CODH are presented. Vet Microbiol, 2001 Oct 1, 82(4), 311 - 20 Characteristics of necrotoxigenic Escherichia coli isolated from septicemic and diarrheic calves between 1958 and 1970; Van Bost S et al.; A total of 434 Escherichia coli isolated from septicemic calves between 1958 and 1965 and 430 E . coli isolated from diarrheic calves between 1967 and 1970 were studied by colony hybridisation and PCR assays for the presence of the cnf1- and the cnf2-like genes . They were also studied for the presence of genes coding for putative virulence factors associated with the CNF toxins including F17-, Pap- and Sfa-fimbrial adhesins and the recently described CDT-III toxin and AfaVIII-afimbrial adhesin . Thirty (7%) of the 434 septicemic strains were positive for CNF by colony hybridisation . Twenty-six were confirmed as necrotoxigenic E . coli type 2 (NTEC2) and four as NTEC1 by PCR . Thirty-five (8%) of the 430 diarrheic strains were positive for CNF by colony hybridisation . Five of them were studied by PCR and confirmed as NTEC1 . The 26 septicemic NTEC2 strains and 20 of the 35 diarrheic NTEC including three of the five NTEC1 were positive for CDT-III . All adhesins studied were present in NTEC as well as in non-NTEC . NTEC1 were mainly Pap-, Sfa- and/or Afa8-positive, whereas NTEC2 were mainly F17- and/or Afa8-positive . This study shows that necrotoxigenic E . coli with their associated adhesins and toxins were present in calves as early as 1958, but their prevalence seems to have increased since that time. FEMS Microbiol Lett, 2001 Aug 7, 202(1), 145 - 8 Characterization of a new feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase AroF of Escherichia coli; Jossek R et al.; Tyrosine feedback-inhibits the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli . Here we show that an Asn-8 to Lys-8 substitution in AroF leads to a tyrosine-insensitive DAHP synthase . This mutant enzyme exhibited similar activities (v=30-40 U mg(-1)) and substrate affinities (K(m)(erythrose-4-phosphate)=0.5 mM, positive cooperativity with respect to phospho(enol)pyruvate) as the wild-type AroF, but showed decreased thermostability . An engineered AroF enzyme lacking the seven N-terminal residues also was tyrosine-resistant . These results strongly suggest that the N-terminus of AroF is involved in the molecular interactions occurring in the feedback-inhibition mechanism. FEMS Microbiol Lett, 2001 Aug 7, 202(1), 121 - 4 H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors; Laurinavichene TV et al.; Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2 . Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite . Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential . Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations . Hydrogenase 2 catalyzed this reaction only at low air concentrations . Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2. FEMS Microbiol Lett, 2001 Aug 7, 202(1), 85 - 90 Cloning, expression and characterisation of a Family B ATP-dependent phosphofructokinase activity from the hyperthermophilic crenarachaeon Aeropyrum pernix; Ronimus RS et al.; We have cloned a Family B sugar kinase gene from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix and have subsequently expressed the protein in Escherichia coli . The enzyme was purified with its associated histidine-tag by affinity chromatography with a nickel-nitrilotriacetic acid column followed by cation exchange chromatography and possesses a high degree of thermostable ATP-dependent phosphofructokinase activity . The enzyme has an estimated apparent K(m) for ATP and fructose-6-phosphate of 0.027 and 1.212 mM, respectively, that were determined in discontinuous assays at 95 degrees C . The Family B ATP-dependent phosphofructokinase has a half-life of approximately 30 min at 95 degrees C and is indicated to be monomeric . The implications of the presence of a Family B phosphofructokinase in the Crenarchaea are discussed with reference to the origins of the Embden-Meyerhof pathway. FEMS Microbiol Lett, 2001 Aug 7, 202(1), 45 - 50 Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae; Tola S et al.; Mycoplasma agalactiae, the causative agent of contagious agalactia in small ruminants, produces a protein, named P80, that is detectable in all wild-type isolates examined to date and that appears expressed during the early phase of infection . We describe here the identification, cloning and expression of the gene encoding P80 (ma-mp81) . The deduced amino acid sequence is consistent with a hydrophobic and basic protein that possesses a lipoprotein signal peptide . Sequence analysis of gene ma-mp81 suggests that P80 is a membrane lipoprotein that shows significant homology with other putative lipoproteins of M . pneumoniae . An internal 1-kb fragment of ma-mp81 was expressed in Escherichia coli as a 6xHis-tagged protein . The purified recombinant protein greatly reacted with polyclonal anti-P80 sera raised in lamb. Trends Neurosci, 2001 Sep, 24(9), 498 - 500 To be or not to be: a question of B-Raf? Kolch W. A recent study has identified the B-Raf gene as essential for the survival of explanted sensory and motor neurons in culture in response to neurotrophic factors . This finding sheds new light on the control of neuronal development and poses new questions with regard to Raf-isozyme-specific functions. Obstet Gynecol, 2001 Aug, 98(2), 284 - 8 Interleukin-10 inhibition of gelatinases in fetal membranes: therapeutic implications in preterm premature rupture of membranes; Fortunato SJ et al.; OBJECTIVE: To examine the effect of interleukin-10 on production and regulation of gelatinases by amniochorion in an in vitro model of infection . METHODS: We placed amniochorionic membranes collected from eight women who had elective repeat cesareans at term in an organ explant culture system . After 48 hours in culture, the membranes were stimulated with lipopolysaccharide (50 ng/mL), and some were costimulated with interleukin-10 (500 ng/mL) . Tissue and media samples were collected after 24-hour stimulation . Quantitative polymerase chain reactions and enzyme-linked immunosorbent assays were used to evaluate matrix metalloproteinase 2 and matrix metalloproteinase 9 messenger RNA and proteins, respectively . RESULTS: Lipopolysaccharide stimulation induced 55.14 transcripts of matrix metalloproteinase 9, compared with 0.83 in control tissues (P <.001) . Costimulation with interleukin-10 and lipopolysaccharide significantly reduced matrix metalloproteinase 9 messenger RNA levels to 10 transcripts (P <.001) . Lipopolysaccharide stimulation produced 29.25 ng/mL of immunoreactive matrix metalloproteinase 9, which was reduced to 6.3 ng/mL (P(adj) =.016) after costimulation with interleukin-10 . Although not significant, matrix metalloproteinase 2 messenger RNA levels were higher in lipopolysaccharide-stimulated tissues (4.37 x 10(6) transcripts) compared with control (2.8 x 10(5) transcripts; P(adj) =.08), with a significant decrease in matrix metalloproteinase 2 messenger RNA levels in interleukin-10- costimulated tissues (2.9 x 10(6); P(adj) =.007) . Interleukin-10 costimulation resulted in a significant decrease in matrix metalloproteinase 2 protein production (203.1 {lipopolysaccharide} and 149.75 {with interleukin-10}; P(adj) <.001) . CONCLUSION: Interleukin-10 eliminated lipopolysaccharide induction of matrix metalloproteinase 2 and 9 in amniochorion. Mutat Res, 2001 Sep 1, 480-481, 277 - 84 The HAP1 protein stimulates the turnover of human mismatch-specific thymine-DNA-glycosylase to process 3,N(4)-ethenocytosine residues; Privezentzev CV et al.; When present in DNA, 3,N(4)-ethenocytosine (epsilon C) residues are potentially mutagenic and carcinogenic in vivo . The enzymatic activity responsible for the repair of the epsilon C residues in human cells is the hTDG protein, the human thymine-DNA-glycosylase that removes thymine in a T/G base pair {Proc . Natl . Acad . Sci., U.S.A., 95 (1998) 8508} . One of the distinctive properties of the hTDG protein is that it remains tightly bound to the AP-site resulting from its glycosylase activity . In this paper we report that the human AP endonuclease, the HAP1 (Ape1, APEX Ref-1) protein, stimulates the processing of epsilon C residues by the hTDG protein in vitro, in a dose-dependent manner . This property of HAP1 protein is specific since E.coli Fpg, Nfo and Nth proteins, all endowed with an AP nicking activity, do not show similar features . The results suggest that the HAP1 protein displaces the hTDG protein bound to the AP-site and therefore increases the turnover of the hTDG protein . However, using a variety of techniques including gel retardation assay, surface plasmon resonance and two-hybrid system, it was not possible to detect evidence for a complex including the substrate, the hTDG and HAP1 proteins. APMIS, 2001 Jun, 109(6), 447 - 53 Genotypic detection of enterotoxigenic Escherichia coli colonisation factors; Grewal HM et al.; We have developed a nonradioactive colony hybridisation assay for the detection of enterotoxigenic Escherichia coli (ETEC) that harbor the structural genes for CFA/I, CS1, CS2, CS4, CS17, or PCFO166 . Thus, a polynucleotide probe derived from the colonisation factor antigen I (CFA/I) operon hybridised under very low stringency conditions to total DNA from CFA/I-producing (CFA/I), coli-surface antigen 1 and 3 (CS1 CS3-), CS2 CS3-, CS4 CS6-, CS17-, and putative colonisation factor O166 (PCFO166)-producing enterotoxigenic Escherichia coli (ETEC) . The probe did not hybridise to DNA from CS3, CFA/III CS6, CS5 CS6, CS6, CS7, or PCFO159 ETEC . Visual registration of colour intensity could be used to differentiate between CFA/I, CS4 and PCFO166-positive strains on the one hand and strains with the genetic potential to express CS1, CS2, or CS17 on the other . As a confirmatory test, restriction fragment patterns obtained from Sau3AI-digested ETEC plasmid DNA could be used to distinguish between CFA/I, CS1, CS4, CS17, and PCFO166 ETEC in nonradioactive Southern blot hybridisation . The simultaneous genotypic detection of several ETEC colonisation factors will prove useful in vaccine-oriented studies of ETEC disease. Clin Chem Lab Med, 2001 Jun, 39(6), 491 - 3 The effect of Escherichia coli-derived lipopolysaccharides on plasma levels of malondialdehyde and 3-nitrotyrosine; Unlu A et al.; The aim of this study was to determine the effect of Escherichia coli (E . coli)-derived lipopolysaccharide on rat plasma low density lipoprotein (LDL), malondialdehyde and 3-nitrotyrosine levels (an indicator of protein nitration) . Six hours after intraperitoneal administration of E.coli, plasma LDL was measured electrophoretically and malondialdehyde level was measured by spectrophotometric method . Plasma malondialdehyde was significantly (p<0.001) elevated in E . coli-injected rats (4.97 +/- 1.33; n=10) in comparison to control animals (1.83 +/- 0.5; n=10) . In addition, plasma 3-nitrotyrosine level, determined by reverse-phase HPLC, was also increased in the infected group (2.84 +/- 1.17 to 0.22 +/- 0.13; n=10) . This increase was statistically significant (p<0.001) . An increased level of oxidation of lipids and 3-nitrotyrosine was observed as a result of free radical-mediated damage in plasma . In conclusion, asymptomatic infections may increase the risk of atherosclerosis by inducing free radical formation and a consequent increase in the oxidation of LDL. Planta, 2001 Jul, 213(3), 483 - 7 First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli; Miller B et al.; For the first time, the complete functional gene for isoprene synthase has been isolated from poplar (Populus alba x Populus tremula) . The gene was quite similar to known limonene and other monoterpene synthases, but was found to specifically catalyze the formation of isoprene from the precursor dimethylallyl diphosphate with only a marginal activity for the formation of the monoterpene limonene from geranyl diphosphate as compared with limonene synthases . Omitting the part of the gene that putatively encoded the signal peptide necessary for transport into the chloroplast led to an enhanced rate of isoprene formation by the recombinant protein. Planta, 2001 Jul, 213(3), 402 - 10 Cloning, characterization and mRNA expression analysis of PVAS1, a type I asparagine synthetase gene from Phaseolus vulgaris; Osuna D et al.; A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.) . A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0 . The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS . The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes . Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean . Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes. Carbohydr Lett, 2001, 4(2), 71 - 6 Synthesis and enzymatic evaluation of mucin type core 4 O-glycan; Misra AK et al.; Lactosylaminylated core-4 tetrasaccharide found in mucin type O-glycans has been synthesized . The non-reducing galactose residue of the deblocked tetrasaccharide was removed by beta-galactosidase from E . coli to produce the corresponding GlcNAc terminated compound . The core-2 and core-4 tetrasaccharides were evaluated as acceptors for the beta-1,3-N-acetylglucosaminyltransferase (iGnT), beta-1,4-Galactosyltransferase IV(beta4GalTIV) and beta-1,4-Galactosyltransferase I(beta4GalTI). Anesthesiology, 2001 Aug, 95(2), 428 - 36 Continuous venovenous hemofiltration improves arterial oxygenation in endotoxin-induced lung injury in pigs; Ullrich R et al.; BACKGROUND: Hypoxemia is common in septic acute lung failure . Therapy is mainly supportive, and most trials using specific inhibitors of key inflammatory mediators (ie., tumor necrosis factor alpha, interleukin 1) have failed to prove beneficial . The authors investigated if a nonspecific blood purification technique, using zero-balanced high-volume continuous venovenous hemofiltration (CWH), might improve arterial oxygenation in a fluid-resuscitated porcine model of endotoxin-induced acute lung injury . METHODS: Piglets of both sexes weighing 25-30 kg were anesthetized and mechanically ventilated . After baseline measurements, animals received an intravenous infusion of 0.5 mg/kg endotoxin (Escherichia coli lipopolysaccharide) . One hour after endotoxin, animals were randomly assigned to either treatment with CWH (endotoxin + hemofiltration, n = 6) or spontaneous course (endotoxin, n = 6) . At 4 h after randomization, animals were killed . Hemofiltration was performed from femoral vein to femoral vein using a standard circuit with an EF60 polysulphone hemofilter . RESULTS: Endotoxin challenge induced arterial hypoxemia, an increase in peak inspiratory pressure, pulmonary hypertension, and systemic hypotension . Treatment with CWH did not improve systemic or pulmonary hemodynamics . However, arterial oxygenation was increased in endotoxin-challenged animals at 5 h after completion of endotoxin infusion, as compared with animals not receiving CVVH (arterialoxygen tension, 268+/-33 vs . 176+/-67 mm/Hg, respectively, P < 0.01) . In addition, treatment with CWH attenuated the endotoxin-induced increase in peak inspiratory pressure and increased lung compliance . CONCLUSION: These results suggest that nonspecific blood purification with high-volume CWH improves arterial oxygenation and lung function in endotoxin-induced acute lung injury in pigs, independent of improved hemodynamics, fluid removal, or body temperature. Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9517 - 20 Propagating conformational changes over long (and short) distances in proteins; Yu EW et al.; The problem of the propagation of conformational changes over long distances or through a closely packed protein is shown to fit a model of a ligand-induced conformational change between two protein states selected by evolution . Moreover, the kinetics of the pathway between these states is also selected so that the energy of ligand binding and the speed of the transition between conformational states are physiologically appropriate . The crystallographic data of a wild-type aspartate receptor that has negative cooperativity and a mutant that has no cooperativity but has native transmembrane signaling are shown to support this model. Nucleic Acids Res, 2001 Aug 15, 29(16), 3433 - 8 Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA; Murata-Kamiya N et al.; Methylglyoxal, a known endogenous and environmental mutagen, is a reactive alpha-ketoaldehyde that can modify both DNA and proteins . To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a 'primed template' DNA and the exonuclease-deficient Klenow fragment (KF(exo-)) of DNA polymerase I with methylglyoxal in vitro . When the reaction mixtures were analyzed by SDS-PAGE, we found that methylglyoxal induced a DNA-KF(exo-) crosslink . The specific binding complex of KF(exo-) and 'primed template' DNA was necessary for formation of the DNA-KF(exo-) crosslink . Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA . When 2'-deoxyguanosine was incubated with Nalpha-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed . No other amino acid derivatives tested could generate a crosslinked product . These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA-DNA polymerase crosslink. Nucleic Acids Res, 2001 Aug 15, 29(16), 3394 - 403 Effects of base change mutations within an Escherichia coli ribosomal RNA leader region on rRNA maturation and ribosome formation; Schaferkordt J et al.; The effects of base change mutations in a highly conserved sequence (boxC) within the leader of bacterial ribosomal RNAs (rRNAs) was studied . The boxC sequence preceding the 16S rRNA structural gene constitutes part of the RNase III processing site, one of the first cleavage sites on the pathway to mature 16S rRNA . Moreover, rRNA leader sequences facilitate correct 16S rRNA folding, thereby assisting ribosomal subunit formation . Mutations in boxC cause cold sensitivity and result in 16S rRNA and 30S subunit deficiency . Strains in which all rRNA operons are replaced by mutant transcription units are viable . Thermodynamic studies by temperature gradient gel electrophoresis reveal that mutant transcripts have a different, less ordered structure . In addition, RNA secondary structure differences between mutant and wild-type transcripts were determined by chemical and enzymatic probing . Differences are found in the leader RNA sequence itself but also in structurally important regions of the mature 16S rRNA . A minor fraction of the rRNA transcripts from mutant operons is not processed by RNase III, resulting in a significantly extended precursor half-life compared to the wild-type . The boxC mutations also give rise to a new aberrant degradation product of 16S rRNA . This intermediate cannot be detected in strains lacking RNase III . Together the results indicate that the boxC sequence, although important for RNase III processing, is likely to serve additional functions by facilitating correct formation of the mature 16S rRNA structure . They also suggest that quality control steps are acting during ribosome biogenesis. Mol Biol Evol, 2001 Sep, 18(9), 1694 - 702 Scale-free behavior in protein domain networks; Wuchty S; Several technical, social, and biological networks were recently found to demonstrate scale-free and small-world behavior instead of random graph characteristics . In this work, the topology of protein domain networks generated with data from the ProDom, Pfam, and Prosite domain databases was studied . It was found that these networks exhibited small-world and scale-free topologies with a high degree of local clustering accompanied by a few long-distance connections . Moreover, these observations apply not only to the complete databases, but also to the domain distributions in proteomes of different organisms . The extent of connectivity among domains reflects the evolutionary complexity of the organisms considered. J Biol Chem, 2001 Oct 19, 276(42), 38494 - 501 Epub 2001 Aug 14. Translational repression of the Escherichia coli alpha operon mRNA: importance of an mRNA conformational switch and a ternary entrapment complex; Schlax PJ et al.; Ribosomal protein S4 represses synthesis of the four ribosomal proteins (including itself) in the Escherichia coli alpha operon by binding to a nested pseudoknot structure that spans the ribosome binding site . A model for the repression mechanism previously proposed two unusual features: (i) the mRNA switches between conformations that are "active" or "inactive" in translation, with S4 as an allosteric effector of the inactive form, and (ii) S4 holds the 30 S subunit in an unproductive complex on the mRNA ("entrapment"), in contrast to direct competition between repressor and ribosome binding ("displacement") . These two key points have been experimentally tested . First, it is found that the mRNA pseudoknot exists in an equilibrium between two conformers with different electrophoretic mobilities . S4 selectively binds to one form of the RNA, as predicted for an allosteric effector; binding of ribosomal 30 S subunits is nearly equal in the two forms . Second, we have used S4 labeled at a unique cysteine with either of two fluorophores to characterize its interactions with mRNA and 30 S subunits . Equilibrium experiments detect the formation of a specific ternary complex of S4, mRNA pseudoknot, and 30 S subunits . The existence of this ternary complex is unambiguous evidence for translational repression of the alpha operon by an entrapment mechanism. J Biol Chem, 2001 Oct 19, 276(42), 38570 - 81 Epub 2001 Aug 14. DNA pairing and strand exchange by the Escherichia coli RecA and yeast Rad51 proteins without ATP hydrolysis: on the importance of not getting stuck; Rice KP et al.; The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them . Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51 . However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different . When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51 . For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis . Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, I.e . the reaction can get stuck . Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate. J Biol Chem, 2001 Oct 19, 276(42), 39186 - 91 Epub 2001 Aug 14. Timely release of both replication forks from oriC requires modulation of origin topology; Smelkova N et al.; Initiation of DNA replication at oriC occurs bidirectionally both in vivo and in vitro . Although the proteins involved in establishing the replication forks are known, little is known about the events that ensure that initiation is bidirectional . We show here that in the absence of DNA gyrase, replication fork progression from oriC on a plasmid template in vitro is unidirectional, although both replication forks have formed at the origin . There was no bias in the release of one fork or the other, ruling out protein blockage of one fork as a possible reason for the asymmetric release . Timely release of both forks required the presence of either DNA gyrase or topoisomerase IV, suggesting that modulation of the topology of the origin region is the governing factor. BMC Microbiol . 2001;1(1):12 . Epub 2001 Jul 19. Evidence that the RNAseH activity of the duck hepatitis B virus is unable to act on exogenous substrates; Gong Y et al.; BACKGROUND: The hepadnaviral reverse transcriptase can synthesize DNA on its native RNA template within viral cores but it is usually unable to synthesize DNA employing exogenous nucleic acids as a template . The mechanism of this template commitment is unknown . Here we provide evidence that the RNAseH activity of duck hepatitis B virus reverse transcriptase may also be unable to act on exogenous substrates . RESULTS: RNAseH assays were performed under a wide variety of conditions employing substrate RNAs of Duck Hepatitis B Virus sequence annealed to complementary DNA oligonucleotides and permeabilized intracellular viral core particles . Temperature, pH, cation type, salt concentration, substrate concentration, and the sequences of the cleavage sites were varied, and the effects of ATP and dNTPs on RNAseH activity were examined . duck hepatitis B virus RNAseH activity was not detected under any of these conditions, although E . coli or Avian Myeloblastosis Virus RNAseH activity could be detected under all conditions . Access of the RNA substrate to the enzyme within the viral cores was confirmed . CONCLUSIONS: These results imply that the RNAseH activity of the DHBV reverse transcriptase may not be able to degrade exogenous RNA:DNA heteroduplexes, although it can degrade heteroduplexes of the same sequence generated during reverse transcription of the endogenous RNA template . Therefore, the RNAseH activity appears to be "substrate committed" in a manner similar to the template commitment observed for the DNA polymerase activity. Virology, 2001 Aug 15, 287(1), 40 - 8 Effects of alanine cluster mutations in the D12 subunit of vaccinia virus mRNA (guanine-N7) methyltransferase; Saha N et al.; The (guanine-N7)-methyltransferase domain of the vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit D1(498-844) bound to a stimulatory subunit D12 . To identify structural elements of the 287-amino-acid D12 subunit that participate in binding and activation of the catalytic subunit, we introduced 12 double-alanine mutations at vicinal residues that are conserved in the D12 homologs of other vertebrate poxviruses . His-tagged D12 mutants were coexpressed in bacteria with the D1(498-544) subunit, and the recombinant D1(498-844)/His-D12 heterodimers were purified . Eight of the mutants (K111A-R112A, N120A-N121A, N126A-N127A, F141A-R142A, K223A-D224A, H260A-S261A, E275A-N276A, and R280A-R281A) had no significant effect on methyltransferase activity . Three of the mutants (L61A-K62A, F176A-K177A, and F245A-L246A) displayed an intermediate level of cap methylation (35-50% of wild-type activity) . Only one mutation, N42A-Y43A, elicited a significant loss of the methyltransferase activation function (<20% of the wild-type activity) . Nine of the D12-Ala/Ala proteins were produced individually in bacteria and tested for reconstitution of methyltransferase activity in vitro by mixing with the catalytic subunit . K111A-R112A, N120A-N121A, F176A-K177A, F245A-L246A, and L61A-K62A displayed diminished affinity for the D1 catalytic subunit . N42A-Y43A was uniquely defective in its ability to activate cap methylation by the catalytic subunit . Our results suggest that the methyltransferase activation function of D12, though clearly dependent on the physical interaction with D1, also requires constituents of D12 that are engaged specifically in catalysis . Am J Trop Med Hyg, 2001 Jul, 65(1), 13 - 4 Short report: characterization of enteroaggregative Escherichia coli isolates from Iranian children; Bouzari S et al.; We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Iranian infants and children . To better understand the characteristics of EAEC in Iran, we analyzed EAEC isolates for the presence of pAA plasmid-borne factors . Ninety-eight E . coli strains that displayed the aggregative adherence (AA) pattern on HeLa cells were hybridized with the CVD432 (AA) probe and with genes encoding enteroaggregative heat-stable enterotoxin-1 and aggregative adherence fimbriae (AAF) I and II . Our data suggest that AAF/II is common in this population and that AAF/I and AAF/II can sometimes be detected in the same E . coli isolate . Surprisingly, we have found that AA probe-negative strains in Iran share virulence factors with AA probe-positive isolates and therefore may be more similar to probe-positive strains than previously believed. J Wildl Dis, 2001 Jul, 37(3), 617 - 20 Enterotoxigenic Escherichia coli infection in captive black-footed ferrets; Bradley GA et al.; Enterotoxigenic Escherichia coli with genes for heat stabile toxins Sta and STb was isolated from the gastrointestinal tract and multiple visceral organs of three adult and three juvenile black-footed ferrets (Mustela nigripes) that died in a captive breeding colony between 24 May 1998 and 2 July 1998 . Similar isolates were obtained from rectal swabs of one adult and one juvenile that were clinically ill . All were fed a diet composed of mink chow, raw rabbit meat, beef liver powder, blood meal and lard . Escherichia coli of the same toxin genotype was isolated from the mixed ration . Clinical signs included sudden death, dehydration, anorexia and diarrhea . Necropsy lesions included acute enteritis with large numbers of rod shaped bacteria microscopically visible on intestinal villi. J Wildl Dis, 2001 Jul, 37(3), 614 - 6 Granulocytic ehrlichiosis in a roe deer calf in Norway; Stuen S et al.; A case of granulocytic ehrlichiosis is described in a roe deer (Capreolus capreolus) calf from Norway . The calf was heavily infested with Ixodes ricinus and died from Escherichia coli septicemia . Granulocytic Ehrlichia sp . was detected by polymerase chain reaction (PCR) from several organs and sequence determination identified a variant of human granulocytic ehrlichiosis (HGE) agent . This is the first report of a possible clinical granulocytic Ehrlichia sp . infection in a roe deer. Vet Rec, 2001 Jul 28, 149(4), 105 - 8 Serological, colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and Escherichia coli F5 (K99); Crouch CF et al.; Twenty-five Ayrshire/Friesian cows were vaccinated once with a new combined vaccine against rotavirus, coronavirus and Escherichia coli F5 (K99) or given a saline placebo 31 days before the first expected calving date . Blood samples were taken from the cows at intervals from vaccination until seven days after calving and from their calves up to 28 days after birth, and colostrum and milk samples were collected from the cows at intervals for 28 days after calving . There was a significant increase in the mean specific antibody titre against all three antigens in the serum of the vaccinated animals (even in the presence of pre-existing antibody) which was accompanied by increased levels of protective antibodies to rotavirus, coronavirus and E coli F5 (K99) in their colostrum and milk for at least 28 days. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2000 Jun, 14(2), 121 - 4 {Generation of monoclonal antibody Fab fragments to parathyroid hormone related protein by phage display technology}; Dou L et al.; OBJECTIVE: To develop monoclonal antibody Fab fragments using display technique . METHODS: The mouse IgG Fab genes of heavy and light chains were amplified from spleen cells of a parathyroid hormone-related protein (PTHrP) immunized mouse . The combinatorial phage antibody library was prepared by inserting both heavy and light chain Fab genes into phagemid vector pComb3 and followed by infection of helper phage . The library was selected by purified recombinant PTHrP . RESULTS: The combinatorial phage antibody library was constructed successfully and the specific mouse Fabs to PThrP were selected and expressed in E . coli . CONCLUSIONS: The selected specific mouse Fabs can recognize PTHrP with high specificity. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2000 Mar, 14(1), 80 - 4 {Preparation of hepatitis C virus nonstructural region antigens and detection of their relative antibodies}; Peng X et al.; OBJECTIVE: To investigate the relationship between HCV NS3 and the pathogenesis of HCV and research a method to assess the therapeutic effect of interferon (lEN) in clinic . METHODS: In a retrospective research, we used HCV-NS3 antigens of different terminals expressed in E . coli which contained the recombinant plasmids involving amino terminal region (N') or carboxyl terminal region (C') of HCV-NS3, to detect the antibodies by Western blot in sera of 63 hepatitis C patients who were treated with IFN . RESULTS: It proved that antibodies against HCV-NS3 N' and/or HCV NS3 C' terminals in hepatitis C patients may predict the treatment result of IFN, especially in the patients with chronic hepatitis C . CONCLUSIONS: The detection of antibodies against HCV-NS3 N' and/or C' terminals may be used for evaluation of IFN treatment, especially in chronic hepatitis C ones. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2000 Mar, 14(1), 69 - 72 {Expression, purification and identification of human IFN-alpha 2 b/HBV Pre S2 fusion protein}; Xu D et al.; OBJECTIVE: To express a fusion protein of human interferon-a2b and HBV Pre S2 in E.coli for the purpose of investigating anti-HBV immunomodulatory protein . METHODS: Human interferon-a2b and HBV Pre S2 encoding genes were amplified from plasmid templates through PCR, then fused and cloned into plasmid pBV220 through engineering technique to generate expression plasmid pBV-IFN-Pre S2 . The plasmid was transfected into E . coli to produce fusion protein . RP-HPLC and ion-exchange chromatography were employed to purify fusion protein . RESULTS: 27 kDa fusion protein was expressed up to 15% of total bacterial protein in E . coli . After purification, the purity of fusion protein reached 95% of total protein . Anti-viral assay showed that IFN-Pre S2 protein induced a VSV-resistant activity of 1.25 x 10(8) lU/mg protein in Wish cell line, similar to the bioactivity of original recombinant human IFN-alpha 2b . ELISA data showed that IFN-Pre S2 protein had antigenicities of both human IFN-alpha 2b and HBV Pre S2 . In addition, fusion protein showed the feature of binding to polymeric human serum albumin (PHSA) . CONCLUSIONS: The bifunctional fusion protein was efficiently expressed in E . coli . The work provided initial evidence for studying PHSA-receptor-targeting IFN-alpha 2b,which might have potential application for the treatment of HBV infection. Antimicrob Agents Chemother, 2001 Sep, 45(9), 2638 - 42 Pretreatment of mice with clindamycin improves survival of endotoxic shock by modulating the release of inflammatory cytokines; Hirata N et al.; Suppression of endotoxin release and subsequent production of inflammatory cytokines is crucial in the treatment of septic shock . We investigated the effect of clindamycin (CLI) on endotoxic shock induced in mice by Escherichia coli lipopolysaccharide (LPS) . Mice were treated with CLI (160 to 600 mg/kg) or saline and then injected with E . coli LPS and D-(+)-galactosamine intraperitoneally 0.5 h after CLI administration . Pretreatment with CLI significantly improved survival in a dose-dependent manner (CLI, at 160, 300, and 440 mg/kg) and significantly lowered the peak concentrations of tumor necrosis factor alpha and interleukin-1beta (IL-1beta) in serum . However, the peak concentrations of IL-6 in the sera of CLI-treated mice were higher than in control mice . Our findings suggest that CLI alters LPS-induced inflammatory cytokine production and suppresses endotoxin-induced mortality in this murine model. Eur J Biochem, 2001 Aug, 268(16), 4527 - 36 A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF; Kotova I et al.; Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH . Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits . The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity . We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae . The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells . When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro . Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter. Eur J Biochem, 2001 Aug, 268(16), 4468 - 76 A further clue to understanding the mobility of mitochondrial yeast cytochrome c: a (15)N T1rho investigation of the oxidized and reduced species; Barker PD et al.; A new approach was developed to overproduce 15N-enriched yeast iso-1-cytochrome c in the periplasm of Escherichia coli in order to perform a study of the motions in the ms-micros time scale on the oxidized and reduced forms through rotating frame 15N relaxation rates and proton/deuterium exchange studies . It is confirmed that the reduced protein is rather rigid whereas the oxidized species is more flexible . The regions of the protein that display increased internal mobility upon oxidation are easily identified by the number of residues experiencing conformational equilibria and by their exchange rates . These data complement the information already available in the literature and provide a comprehensive picture of the mobility in the protein . In particular, oxidation mobilizes the loop containing Met80 and, through specific contacts, affects the mobility of helix 3 and possibly of helix 5, and of a section of protein connecting the heme propionates to helix 2 . The relevance of internal motions to molecular recognition and to the early steps of the unfolding process of the oxidized species is also discussed . In agreement with the reported data, subnanosecond mobility is found to be less informative than the ms-micros with respect to redox dependent properties. Biochemistry, 2001 Aug 21, 40(33), 9977 - 82 Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein; Hayakawa H et al.; 8-Oxoguanine, an oxidized form of guanine, has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors . To prevent such an outcome, organisms probably have a mechanism for recognizing RNA molecules carrying 8-oxoguanine and prevent them from entering into the cellular translational machinery . We now report that the Escherichia coli cell possesses proteins that bind specifically to RNA carrying 8-oxoguanine . On incubation with a cell-free extract, 8-oxoguanine-containing RNA is stable while normal RNA is degraded by cellular nucleases . The RNase protection assay and gel shift assay revealed that some proteins bind specifically to 8-oxoguanine-containing RNA, hence preventing nuclease attacks . Among the complexes that were detected, one with a 77 kDa protein exhibits tight binding between RNA and protein components . This protein was identified as polynucleotide phosphorylase, encoded by the pnp gene . pnp(-)() mutants are hyperresistant to paraquat, a drug that induces oxidative stress in the cell . Binding of Pnp protein to 8-oxoguanine-containing RNA would inhibit cell growth, probably due to withdrawal of such RNA from the translational machinery . The Pnp protein may, therefore, discriminate between an oxidized RNA molecule and a normal one, thus contributing a high fidelity of translation. Biochemistry, 2001 Aug 21, 40(33), 9927 - 34 Membrane binding and self-association of alpha-synucleins; Narayanan V et al.; Although its function is unknown, alpha-synuclein is widely distributed in neural tissue and is the major component in the pathological aggregates found in patients with Parkinson's disease, Alzheimer's disease, Down's syndrome, and multiple system atrophy . In this report, we have quantified the binding alpha-synucleins to lipid membranes . In contrast to previous studies, we find, using real time equilibrium fluorescence methods, that alpha-synuclein binds strongly to large, unilamellar vesicles with either anionic or zwitterionic headgroups . Membrane binding is also strong for beta-synuclein, phosphorylated alpha-synuclein, and a synuclein mutant that is associated with familial Parkinson's disease .