Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to the alpha(3)beta(3) hexagon of F(1). To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)). With five different F(1) enzymes, K(d)(b(ST34-156)) ranged from 91 to 157 nm. Binding was strongly Mg(2+)-dependent; in EDTA buffer, K(d)(b(ST34-156)) was increased to 1.25 microm. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of delta subunit to delta-depleted F(1). The apparent K(d)(b(ST34-156)) for this effect was increased from 150 nm in Mg(2+) buffer to 1.36 microm in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F(1) contributes to stator resistance and emphasizes the importance of Mg(2+) in stator interactions.     

The affinity-enhancing roles of flexible linkers in two-domain DNA-binding proteins.

Recently many attempts have been made to design high-affinity DNA-binding proteins by linking two domains. Here a theory for guiding these designs is presented. Flexible linkers may play three types of roles: (a) linking domains which by themselves are unfolded and bind to DNA only as a folded dimer (as in a designed single-chain Arc repressor), (b) connecting domains which can separately bind to DNA (as in the Oct-1 POU domain), and (c) linking a DNA-binding domain with a dimerization domain (as in the lambda repressor). In (a), the linker keeps the protein as a folded dimer so that it is always DNA-binding-competent. In (b), the linker is predicted to enhance DNA-binding affinity over those of the individual domains (with dissociation constants K(A) and K(B)) by p(d(0))/K(B) or p(d(0))/K(A), where p(d(0)) = (3/4pil(p)bL)(3/2) exp(-3d(0)(2)/4l(p)bL)(1 - 5l(p)/4bL +...) is the probability density for the end-to-end vector of the linker with L residues to have a distance d(0). In (c), the linker is predicted to enhance the binding affinity by K(d)(C)/p(d(0)), where K(d)(C) is the dimer dissociation constant for the dimerization domain. The predicted affinity enhancements are found to be actually reached by the Oct-1 POU domain and lambda repressor. However, there is room for improvement in many of the recently designed proteins. The theoretical limits presented should provide a useful guide for current efforts of designing DNA-binding proteins.     

Structural basis for alpha1 versus alpha2 isoform-distinct behavior of the Na,K-ATPase

We showed earlier that the kinetic behavior of the alpha2 isoform of the Na,K-ATPase differs from the ubiquitous alpha1 isoform primarily by a shift in the steady-state E(1)/E(2) equilibrium of alpha2 in favor of E(1) form(s). The aim of the present study was to identify regions of the alpha chain that confer the alpha1/alpha2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K(+) sensitivity of Na-ATPase measured at micromolar ATP, under which condition E(2)(K(+)) --> E(1) + K(+) becomes rate-limiting, (ii) changes in K'(ATP) for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E(1)P --> E(2)P. We first confirmed that interactions between the cytoplasmic domains of alpha2 that modulate conformational shifts are fundamentally similar to those of alpha1, suggesting that the predilection of alpha2 for E(1) state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the alpha1/alpha2 chimeras indicates that the difference in E(1)/E(2) poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the alpha1/alpha2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E(1) shift of alpha2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E(2)(K(+)) --> E(1) + K(+)), rather than phosphoenzyme (E(1)P --> E(2)P).     

Quantitative structure-selectivity relationship for M2 selectivity between M1 and M2 of piperidinyl piperidine derivatives as muscarinic antagonists

Muscarinic M2 receptor antagonists with high subtype selectivity (M2/M1) will decrease the toxicity in central nervous system in treatment of AD. The exploration of quantitative structure-selectivity relationship (QSSR) to muscarinic M2 receptor antagonists will provide design information for drug with fewer side effects. In this paper, CoMFA models of pK(i)(M1), pK(i)(M2) and p[K(i)(M2)/K(i)(M1)] (pK(i)(M2)-pK(i)(M1)) were used to study the subtype selectivity (M2/M1) of piperidinyl piperidine derivatives as muscarinic M2 subtype receptor antagonists. The parameters of the three models are: 0.633, 0.636 and 0.726 for cross-validated r(2) (r(cv)(2)), 0.109, 0.204 and 0.09 for the Standard error of estimate (SD), respectively. The results show the model of p[K(i)(M2)/K(i)(M1)] is the best one for design of piperidinyl piperidine derivatives as muscarinic antagonists with high subtype selectivity (M2/M1).     

Peptide-plane flipping in proteins

A peptide-plane flip is a large-scale rotation of the peptide plane that takes the phi,psi angles at residues i and i + 1 to different structural regions in the Ramachandran plot with a comparatively small effect on the relative orientation of their side chains. This phenomenon, which is expected to play an important role during the early stages of protein folding, has been investigated using 76 proteins for which two high-resolution X-ray conformations are available. Peptide-plane flips are identified by looking for those cases where changes in /psi(i)/ + /phi(i + 1)/ are large (>200 degrees), but changes in /psi(i) + phi(i + 1)/ are comparatively small (<50 degrees). Of a total of 23 cases, the most common peptide-plane flip was identified to be the type I to type II beta-turn interconversion. Although individually rarer, there are many other types of flips that are collectively more common. Given the four main accessible regions alpha(R), alpha(L), beta and epsilon, identified from the phi,psi distribution corresponding to non-hydrogen-bonded peptide planes, 32 main types of peptide-plane flip are identified. Only 8 of these are "passive," in that they require only relatively minor adjustments in the orientation of adjacent peptide planes. Of these, only the type I to type II beta-turn interconversion, denoted, beta(i) + alpha(L)(i + 1) <--> alpha(R)(i) + alpha(R)(i + 1), and the rarer alpha(R)(i) + alpha(L)(i + 1) <--> beta(i) + alpha(R)(i + 1), do not involve the epsilon region. "Active" peptide-plane flips affect the orientation of adjacent peptide planes. The flip, alpha(L)(i) + alpha(L)(i + 1) <--> beta(i) + beta(i + 1), of which one example was found, shows how concerted peptide-plane flips can convert the alpha(L) structure to the beta structure without affecting the relative orientations of the side chains.     

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.     

Activation of Rap1B by G(i) family members in platelets

It has become increasingly appreciated that receptors coupled to G(alpha)(i) family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by G(alpha)(i) family members in platelets. First, we show that platelets from mice lacking the G(alpha)(i) family member G(alpha)(z) (which couples to the alpha(2A) adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking G(alpha)(i2), which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack G(alpha)(q) show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the G(alpha)(i)-coupled P2Y12 receptor and the G(alpha)(q)-coupled P2Y1 receptor. G(alpha)(i)-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the G(alpha)(i2)-/- mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the G(beta)(gamma)-activated form of his enzyme (PI3Kgamma), we show that G(i)-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by G(alpha)(i)- and PI3K-dependent mechanisms in platelets and by G(q)- and Ca(2+)-dependent mechanisms, both of which may play a role in promoting platelet activation.     

1,2,4-Benzothiadiazine derivatives as alpha1 and 5-HT1A receptor ligands

A series of new 1,2,4-benzothiadiazine derivatives with an arylpiperazine mojety linked at position 3 of the heterocyclic ring were synthesized and assessed for their pharmacological profiles at alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) by functional experiments and by in vitro binding studies at human cloned 5-HT(1A) receptor. Compound 1 was identified as a novel alpha(1D) antagonist (pK(b)alpha(1D)=7.59; alpha(1D)/alpha(1A)>389; alpha(1D)/alpha(1B)=135) with high selectivity over 5-HT(1A) receptor (5-HT(1A)/alpha(1D)<0.01), while compound 6, a 3,4-dihydro-derivative, was characterized as a novel 5-HT(1A) receptor ligand, highly selective over alpha(1D)-adrenoceptor subtype (pK(i)5-HT(1A)=8.04; 5-HT(1A)/alpha(1D)=1096). Further pharmacological studies demonstrated that 6 is a partial agonist at 5-HT(1A) receptor (E(max)=23, pD(2)=6.92).     

Evaluation of C-2-substituted 19-nor-1alpha,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1alpha,25(OH)(2)D(3) can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized (3)H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1alpha,25(OH)(2)D(3) analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2alpha- or 2beta-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1alpha,25(OH)(2)D(3). The (3)H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1alpha,25(OH)(2)D(3), 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) or 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) for 7 days. 19-Nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) and 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) were also shown to be about 10-fold more active than 1alpha,25(OH)(2)D(3) in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1alpha,25(OH)(2)D(3) molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.     

The role of Pre-H2 domains of alpha- and delta-epithelial Na+ channels in ion permeation, conductance, and amiloride sensitivity

Epithelial Na(+) channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na(+) selectivity, unitary Na(+) conductance (gamma(Na)), and amiloride sensitivity of delta-ENaC are strikingly different from those of alpha-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of delta- and alpha-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li(+) and Na(+) (P(Li)/P(Na)) for the swap alpha chimeras co-expressed with betagamma-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of P(Li)/P(Na) for the swap delta constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the gamma(Na) of alpha-ENaC but decreased the gamma(Na) of delta-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (K(i)(amil)) was observed in the alpha chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of K(i)(amil) was obtained in the chimeric delta constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the P(Li)/P(Na) ratio, single channel Na(+) conductance, and amiloride sensitivity of alpha- and delta-ENaCs.     

Inactivation of cysteine proteases by (acyloxy)methyl ketones using S'-P' interactions

We have synthesized (acyloxy)methyl ketone inactivators of papain, cathepsin B, and interleukin-1beta conversion enzyme (ICE) that interact with both the S and S' subsites. The value of k(inact)/K(i) for these inactivators is strongly dependent on the leaving group. For example, Z-Phe-Gly-CH(2)-X is a poor inactivator of papain when X is OCOCH(3) (k(inact)/K(i) = 2.5 M(-)(1) s(-)(1)) but becomes a potent inactivator when X is OCO-L-Leu-Z (k(inact)/K(i) = 11 000 M(-)(1) s(-)(1)). Since these leaving groups have similar chemical reactivities, the difference in potency must be attributed to interactions with the S' sites. The potency of the leaving group correlates with the P' specificity of papain. Similar results are also observed for the inactivation of cathepsin B by these compounds. A series of inactivators with the general structure Fmoc-L-Asp-CH(2)-X were designed to inactivate ICE. No inhibition was observed when X was OCOCH(3). In contrast, ICE is inactivated when X is OCO-D-Pro-Z (k(inact)/K(i) = 131 M(-)(1) s(-)(1)). These results demonstrate that S'-P' interactions can be utilized to increase the efficacy and selectivity of (acyloxy)methyl ketone inactivators.     

The immunodominant,Ld-restricted T cell response to hepatitis B surface antigen (HBsAg) efficiently suppresses T cell priming to multiple Dd-, Kd-, and Kb-restricted HBsAg epitopes

MHC-I-restricted CTL responses of H-2(d) (L(d+) or L(d-)) and F(1) H-2(dxb) mice to hepatitis B surface Ag (HBsAg) are primed by either DNA vaccines or HBsAg particles. The D(d)/S(201-209) and K(d)/S(199-208) epitopes are generated by processing endogenous HBsAg; the K(b)/S(208-215) epitope is generated by processing exogenous HBsAg; and the L(d)/S(28-39) epitope is generated by exogenous as well as endogenous processing of HBsAg. DNA vaccination primed high numbers of CTL specific for the L(d)/S(28-39) HBsAg epitope, low numbers of CTL specific for the D(d)/S(201-209) or K(d)/S(199-208) HBsAg epitopes in BALB/c mice, and high numbers of D(d)/S(201-209)- and K(d)/S(199-208)-specific CTL in congenic H-2(d)/L(d-) dm2 mice. In F(1)(dxb) mice, the K(d)-, D(d)-, and K(b)-restricted CTL responses to HBsAg were strikingly suppressed in the presence but efficiently elicited in the absence of L(d)/S(28-39)-specific CTL. Once primed, the K(d)- and D(d)-restricted CTL responses to HBsAg were resistant to suppression by immunodominant L(d)/S(28-39)-specific CTL. The L(d)-restricted immunodominant CTL reactivity to HBsAg can thus suppress priming to multiple alternative epitopes of HBsAg, independent of the processing pathway that generates the epitope, of the background of the mouse strain used, and of the presence/absence of different allelic variants of the K and D MHC class I molecules.     

5Alpha-androstane-3beta,7alpha,17beta-triol and 5alpha-androstane-3beta,7beta,17beta-triol as substrates for the human 11beta-hydroxysteroid dehydrogenase type 1

Several studies have shown that the native 7alpha-hydroxy-dehydroepiandrosterone (7alpha-hydroxy-DHEA) is a substrate for the human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which converts the 7alpha- into the 7beta-epimer through an oxido-reduction process. Research on the 11beta-HSD1 has investigated its function and structure through using native glucocorticoid substrates and known inhibitors. Other steroid substrates are also of interest. Among testosterone metabolites, 5alpha-androstane-3beta,17beta-diol (Adiol) is a substrate for the cytochrome P450 7B1 which produces 5alpha-androstane-3beta,7alpha,17beta-triol (7alpha-Adiol). This steroid may be a substrate for the 11beta-HSD1. We used recombinant yeast-expressed 11beta-HSD1 with NADP(H)-regenerating systems for examining the products obtained after incubation with 7alpha-Adiol, 7beta-Adiol or 7-oxo-Adiol. Oxidative conditions for the 11beta-HSD1 provided no trace of 7-oxo-Adiol but the inter-conversion of 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) (pmol min(-1) microg(-1)/microM) values of 2 and 0.5, respectively. This state was maintained under reductive conditions. The use of a 7-oxo-Adiol substrate under reductive conditions led to the production of both 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) values of 3.43 and 0.22, respectively. These findings support the hypothesis that the oxido-reductase and epimerase activities of 11beta-HSD1 depend on the positioning of the steroid substrates within the active site and may provide insight into its fine structure and mechanism of action.     

Mouse Ae1 E699Q mediates SO42-i/anion-o exchange with [SO42-]i-dependent reversal of wild-type pHo sensitivity

The SLC4A1/AE1 gene encodes the electroneutral Cl(-)/HCO(3)(-) exchanger of erythrocytes and renal type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl(-)/HCO(3)(-) exchange or (36)Cl(-) efflux but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO(4)(2-)(i)/SO(4)(2-)(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO(4)(2-)(i)/Cl(-)(o) exchange). Whereas wild-type AE1 mediates 1:1 H(+)/SO(4)(2-) cotransport in exchange for either Cl(-) or for the H(+)/SO(4)(2-) ion pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wild-type AE1-mediated SO(4)(2-) transport, both SO(4)(2-)(i)/Cl(-)(o) exchange and SO(4)(2-)(i)/SO(4)(2-)(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pH(o) and activated by alkaline pH(o). hAE1 E681OH displays a similarly altered pH(o) dependence of SO(4)(2-)(i)/Cl(-)(o) exchange. Elevated [SO(4)(2-)](i) increases the K(1/2) of Ae1 E699Q for both extracellular Cl(-) and SO(4)(2-), while reducing inhibition of both exchange mechanisms by acid pH(o). The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO(4)(2-). Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide and should continue to provide valuable paths toward understanding substrate selectivity and self-inhibition in SLC4 anion transporters.     

In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display

The G protein regulatory (GPR) motif is a approximately 20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G(i/o)(alpha) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G(i)(alpha)(1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(i)(alpha)(1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G(i)(alpha)(1), inhibiting both the exchange of GDP in GTPgammaS binding assays and the AlF(4)(-)-stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G(betagamma) subunits for binding to G(i)(alpha)(1), suggesting their use as activators of G(betagamma) signaling.     

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.     

Structure-affinity studies for a novel series of homochiral naphtho and tetrahydronaphtho analogues of alpha 1 antagonist WB-4101

A number of enantiomeric pairs of naphthodioxane, tetrahydronaphthodioxane and naphthoxy analogues of WB-4101 (1) were designed and synthesized in order to improve the selectivity profile of the parent compound, hopefully in favour of the alpha(1a)-AR with respect to the other two alpha(1) subtypes and the 5-HT(1A) receptor. The new compounds 2-8 and, in addition, the two enantiomers of 1 were tested in binding assays on the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR, and the 5-HT(1A) receptor. Two of them, namely the naphtho- and tetrahydronaphthodioxane derivatives (S)-2 and (S)-3, showed lower, but significantly more specific alpha(1a) affinity than (S)-1, while the two enantiomers of the 2-methoxy-1-naphthoxy analogue 6 maintained most of the very high alpha(1a) affinity of (S)-1 and its alpha(1a) versus alpha(1b) selectivity slightly increasing the alpha(1a)/alpha(1d) and alpha(1a)/5HT(1A) affinity ratios. The SAR data were evaluated in the light of known alpha(1) subtype pharmacophores and of the alpha(1a)-AR binding mode of WB-4101 resultant from literature mutagenesis studies disclosing some interesting consonances with these models.     

Evidence for the intertwined dimer of the cytochrome bc(1) complex in solution

To confirm that the cytochrome bc(1) complex exists as a dimer with intertwining Rieske iron-sulfur proteins in solution, four Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes containing two pairs of cysteine substitutions, one in the interface between the head domain of iron-sulfur protein (ISP) and cytochrome b and the other between the tail domain of ISP and cytochrome b, were generated and characterized. They are: K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb), K70C(ISP)/A185C(cytb).P33C(ISP)/M92C (cytb), K70C (ISP)/A185C(cytb).L34C(ISP)/V64C(cytb), and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb). The K70C(ISP)/A185C(cytb) cysteine pair cross-links the head domain of ISP and cytochrome b; the P33C(ISP)/G89C(cytb), P33C(ISP)/M92C (cytb), L34C(ISP)/V64C(cytb), and N36C(ISP)/G89C(cytb) cysteine pairs cross-link the tail domain of ISP and cytochrome b. An adduct protein with an apparent molecular mass of 128 kDa containing two cytochrome b and two ISP proteins is detected in the K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb) and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb) mutant complexes, confirming that the bc(1) complex exists as a dimer with intertwining ISPs. The loss of activity in these two double-cysteine-pair mutant complexes was attributed to the disulfide bond between the head domain of ISP and cytochrome b and not the one between the tail domain of ISP and cytochrome b.     

Noncovalent association with stress protein facilitates cross-priming of CD8+ T cells to tumor cell antigens by dendritic cells.

A viral oncogene carrying well-defined K(b)/D(b)-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Ag-expressing tumor cells are taken up and processed by immature dendritic cells (DC), and the K(b)/D(b)-binding epitopes T1, T2/3, and T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-gamma-producing CD8+ T cells were elicited by a DNA vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to 5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, D(b)/K(b)-restricted IFN-gamma-producing CD8+ T cells were primed during the growth of transfected H-2(d) Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F(1)(b x d) hosts. Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo of CTL by DC that process material from apoptotic cells.