cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

Collisionally activated dissociation (CAD) of the protonated polyalanines Ala-Ala,Ala-Ala-Ala, and Ala-Ala-Ala-Ala causes breakup of the peptide bonds leading to sequence-indicative fragment ions. The neutral molecules eliminated during these reactions are identified here using neutralization-reionization mass spectrometry (NRMS). N-terminal acylium ions (bn) arise after the C-terminus is lost as an intact amino acid or peptide; further loss of CO leads to immonium ions (an). Upon generation of C-terminal sequence ions (yn), a hydrogen atom attached to a nitrogen rearranges from the N-terminal to the C-terminal side yielding a protonated amino acid (y1) or peptide (y>> or = 2) as the ionic fragment; the complementary neutral fragment is an aziridinone if the N-terminal amino acid is cleaved and a diketopiperazine if two N-terminal amino acid units are eliminated. Detection of neutral dissociation products can reveal valuable structure information, as demonstrated with the tetrapeptides Val-Gly-Ser-Glu and Val-Gly-Asp-Glu.

The structural gene for DNA polymerase II was cloned by using a synthetic inosine-containing oligonucleotide probe corresponding to 11 amino acids, which were determined by sequencing the amino terminus of the purified protein. The labeled oligonucleotide hybridized specifically to the lambda clone 7H9 from the Kohara collection as well as to plasmid pGW511 containing the SOS-regulated dinA gene. Approximately 1400 base pairs of dinA sequence were determined. The predicted amino-terminal sequence of dinA demonstrated that this gene encoded DNA polymerase II. Sequence analysis of the upstream region localized a LexA binding site overlapping the -35 region of the dinA promoter, and this promoter element was found to be only two nucleotides downstream from the 3' end of the araD gene. These results demonstrate that the gene order is thr-dinA (pol II)-ara-leu on the Escherichia coli chromosome and that the DNA polymerase II structural gene is transcribed in the same direction as the araBAD operon. Based on the analysis of the predicted protein, we have identified a sequence motif Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro-Ser in DNA polymerase II that is highly conserved among a diverse group of DNA polymerases, which include those from humans, yeast, Herpes and vaccinia viruses, and phages T4 and PRD1. The demonstration that DNA polymerase II is a component of the SOS response in E. coli suggests that it plays an important role in DNA repair and/or mutagenesis.

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.

Protein-tyrosine phosphatases (PTPases) contain an evolutionarily conserved segment of 250 amino acids referred to as the PTPase catalytic domain. The recombinant PTPase domain from Yersinia enterocolitica enhances the rate of hydrolysis of p-nitrophenyl phosphate, a phosphate monoester, by approximately 10(11) over the non-enzyme-catalyzed rate by water. Specific amino acid residues responsible for the catalytic rate acceleration have been examined by site-directed mutagenesis. Our results suggest that Asp-356 (D356) and Glu-290 (E290) are the general acid and the general base catalysts responsible for Yersinia PTPase-catalyzed phosphate ester hydrolysis. The PTPase with both E290Q and D356N mutations shows no pH dependence for catalysis but displays a rate enhancement of 2.6 x 10(6), compared to the noncatalyzed hydrolysis of p-nitrophenyl phosphate by water. This rate enhancement probably occurs via transition-state stabilization. Our results suggest that all PTP-ases use a common mechanism that depends upon formation of a thiol-phosphate intermediate and general acid-general base catalysis.

Breast cancer frequently spreads to bone and is almost always associated with osteolysis. This tumor-induced osteolysis is caused by increased osteoclastic bone resorption. Bisphosphonates are used successfully to inhibit bone resorption in tumor bone disease and may prevent development of new osteolytic lesions. The classical view is that bisphosphonates only act on bone cells. We investigated their effects on breast cancer cells using three human cell lines, namely, MCF-7, T47D, and MDA.MB.231, and we tested four structurally different bisphosphonates: clodronate, pamidronate, ibandronate, and zoledronate. We performed time course studies for each bisphosphonate at various concentrations and found that all four compounds induced a nonreversible growth inhibition in both MCF-7 and T47D cell lines in a time- and dose-dependent manner. The MDA.MB.231 cell line was less responsive. Bisphosphonates induced apoptosis in MCF-7 and cell necrosis in T47D cells. The inhibition of MCF-7 cell proliferation could be reverted almost completely by the benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk) inhibitor of caspases, suggesting that the apoptotic process observed in the MCF-7 cell line is mediated, at least partly, by the caspase system. Caspase activity was little changed by bisphosphonates in T47D cells and the inhibitor of caspase did not modify bisphosphonates effects. In summary, we found that bisphosphonates inhibit breast cancer cell growth by inducing cell death in vitro. Such effects could contribute to the beneficial role of bisphosphonates in the treatment and the prevention of tumor-induced osteolysis.

We investigated the ability of caspases (cysteine proteases with aspartic acid specificity) to induce cytochrome c release from mitochondria. When Jurkat cells were induced to undergo apoptosis by Fas receptor ligation, cytochrome c was released from mitochondria, an event that was prevented by the caspase inhibitor, zVAD-fmk (zVal-Ala-Asp-CH2F). Purified caspase-8 triggered rapid cytochrome c release from isolated mitochondria in vitro. The effect was indirect, as the presence of cytosol was required, suggesting that caspase-8 cleaves and activates a cytosolic substrate, which in turn is able to induce cytochrome c release from mitochondria. The cytochrome c releasing activity was not blocked by caspase inhibition, but was antagonized by Bcl-2 or Bcl-xL. Caspase-8 and caspase-3 cleaved Bid, a proapoptotic Bcl-2 family member, which gains cytochrome c releasing activity in response to caspase cleavage. However, caspase-6 and caspase-7 did not cleave Bid, although they initiated cytochrome c release from mitochondria in the presence of cytosol. Thus, effector caspases may cleave and activate another cytosolic substrate (other than Bid), which then promotes cytochrome c release from mitochondria. Mitochondria significantly amplified the caspase-8 initiated DEVD-specific cleavage activity. Our data suggest that cytochrome c release, initiated by the action of caspases on a cytosolic substrates, may act to amplify a caspase cascade during apoptosis.

Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate-adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features.

Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities. At positive voltages, gating is similar in Cx46 and Cx50 hemichannels, primarily showing increased transitioning to long-lived substates. At negative voltages, Cx46 currents deactivate completely and the underlying single hemichannels exhibit transitions to a fully closed state. In contrast, Cx50 currents do not deactivate completely at negative voltages and the underlying single hemichannels predominantly exhibit transitions to various substates. Transitions to a fully closed state occur, but are infrequent. In the respective GJ channels, both forms of gating contribute to the reduction in conductance by V(j). However, examination of gating of mutant hemichannels and GJ channels in which the Asp at position 3 was replaced with Asn (D3N) showed that the positive hemichannel gate predominantly closes Cx50 GJs, whereas the negative hemichannel gate predominantly closes Cx46 GJs in response to V(j). We also report, for the first time, single Cx50 hemichannels in oocytes to be inwardly rectifying, high conductance channels (gamma = 470 pS). The antimalarial drug mefloquine, which selectively blocks Cx50 and not Cx46 GJs, shows the same selectivity in Cx50 and Cx46 hemichannels indicating that the actions of such uncoupling agents, like voltage gating, are intrinsic hemichannel properties.

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.

Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectable products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by gamma-rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys>> Met>> Trp>> Tyr>> Phe>> cystine>> His>> Leu, Ile>> Arg, Lys, Val>> Ser, Thr, Pro>> Gln, Glu>> Asp, Asn>> Ala>> Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectable. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise approximately 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.

Suppression of the host defense is one of the hallmarks of Yersinia enterocolitica infection. This enteric pathogen resists phagocytosis and interferes with macrophage functions from an extracellular localization (oxidative-burst generation and tumor necrosis factor alpha production). In this study, we investigated the fate of the Y. enterocolitica-infected macrophage. We found that murine J774A.1 macrophages and macrophages derived from human monocytes were killed by infection with Y. enterocolitica. Analysis of cellular morphology and DNA fragmentation revealed that macrophage cell death occurs through the induction of apoptosis. A total of 92% +/- 5% (mean +/- standard deviation) of murine J774A.1 macrophages and 74% +/- 6% of human monocyte-derived macrophages underwent apoptosis upon Yersinia infection after 4 and 20 h, respectively. The broad-spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone blocked completion of the Yersinia-induced apoptotic program but not the surface exposure of phosphatidylserine as an early-stage apoptotic event. Analysis of different Yersinia mutants showed that macrophage apoptosis depends on a functional Y. enterocolitica type III protein secretion system. Apoptotic cell death of macrophages was not related to the YopE-mediated cytotoxic effect of Yersinia, since disruption of actin microfilaments by a Y. enterocolitica strain expressing a restricted repertoire of yop genes, including YopE, did not result in macrophage apoptosis. Furthermore, Yersinia-induced cytotoxic alterations in epithelial HeLa cells, which are conferred by YopE, did not lead to apoptosis. Our data demonstrate for the first time that Y. enterocolitica promotes the apoptosis of macrophages, an effect which is clearly distinct from the morphological alterations mediated by Yersinia on epithelial HeLa cells.

Fibronectin (Fn) binding to the integrins alpha IIb beta 3 and alpha v beta 3 involves the Arg-Gly-<em>Asp</em> sequence. The identification of other regions of Fn that interact with alpha IIb beta 3 suggests a potential mechanism for differential ligand recognition by integrins. We report here the identification of an 11-residue peptide sequence from the 9th type III repeat of Fn (3Fn9), which inhibits ligand binding to alpha IIb beta 3 by interacting directly with this receptor. Mutational analysis demonstrated that this same region was involved in the formation of epitopes for two anti-Fn mAbs that inhibit Fn binding to alpha IIb beta 3, thus emphasizing the role of this site in the macromolecule. Molecular modeling of the 3Fn9-10 modules suggested that Fn residues <em>Asp</em>1373-Thr1383 are at least 25 A distant from the Arg-Gly-<em>Asp</em> site and therefore does not directly interact with it. The 3Fn9 site was differentially recognized by the beta 3 integrin family. The <em>Asp</em>1373-Thr1383 peptide failed to inhibit ligand binding to alpha v beta 3, a recombinant Fn Ala1235-Ser1436 fragment was not recognized by alpha v beta 3, and addition of the 3Fn9 module to the amino terminus of the 3Fn10 did not affect the potency of inhibition of Fn binding to alpha v beta 3. Thus, a novel integrin recognition site in the 3Fn9 module of Fn that is differentially recognized by the beta 3 integrins has been localized within the residues <em>Asp</em>1373-Thr1383.

To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4.

The group B Streptococcus (GBS) causes the majority of life-threatening bacterial infections in newborn children. Most GBS strains isolated from such infections express a surface protein, designated Rib, that confers protective immunity and therefore is of interest for analysis of pathogenetic mechanisms. Sequence analysis demonstrated that Rib has an exceptionally long signal peptide (55 amino acid residues) and 12 repeats (79 amino acid residues each) that account for >80% of the sequence of the mature protein. The repeats are identical even at the DNA level, indicating that an efficient mechanism operates to maintain a highly repetitive structure in Rib. The structure of Rib is similar to that of alpha, a previously characterized surface protein that is common among GBS strains lacking Rib. However, highly purified preparations of Rib and alpha did not cross-react immunologically, although the two proteins show extensive amino acid residue identity (47% in the repeat region). When analyzed in Western blots, Rib and alpha give rise to a regularly spaced ladder pattern, apparently due to hydrolysis of acid-labile Asp-Pro bonds in the repeats. We conclude that Rib and alpha are members of a novel family of streptococcal surface proteins with unusual repetitive structure.

Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis, especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.

Vascular Endothelial Growth Factor (VEGF) is a potent mitogen for vascular endothelial cells that has been implicated in tumor neovascularization. We show that, in hamster fibroblasts (CCL39 cells), VEGF mRNAs are expressed at low levels in serum-deprived or exponentially growing cells, whereas it is rapidly induced after stimulation of quiescent cells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the p42/p44 mitogen-activated protein (MAP) kinase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val12), MKK1 in which Ser218 and Ser222 were mutated to Asp (MKK1-SS/DD)), express very high levels of VEGF mRNA. To analyze the contribution of the p42/p44MAP kinase in this induction, we used the CCL39-derived cell line (Raf-1:ER) expressing an estradiol-activable Raf-1. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clearly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-1 is reverted by an inhibitor of MKK1, PD 098059, highlighting a specific role for the p42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an additive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-1:ER cells stimulated by estradiol. In contrast to VEGF, the isoforms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic factors. We have identified a GC-rich region of the VEGF promoter between -88 and -66 base pairs which contains all the elements responsible of its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutation of the putative binding sites and electrophoretic mobility supershift experiments, we showed that the GC-rich region constitutively binds Sp1 and AP-2 transcription factors. Furthermore, following activation of the p42/p44 MAP kinase module, the binding of Sp1 and AP-2 is increased in the complexes formed in this region of the promoter. Altogether, these data suggest that hypoxia and p42/p44 MAP kinase independently play a key role in the regulation of the VEGF expression.

OBJECTIVE

Until recently, human leucocyte antigen (HLA) class II-independent associations with type 1 diabetes (T1D) in the Major Histocompatibility Complex (MHC) region were not adequately characterized owing to insufficient map coverage, inadequate statistical approaches and strong linkage disequilibrium spanning the entire MHC. Here we test for HLA class II-independent associations in the MHC using fine mapping data generated by the Type 1 Diabetes Genetics Consortium (T1DGC).

METHODS

We have applied recursive partitioning to the modelling of the class II loci and used stepwise conditional logistic regression to test approximately 1534 loci between 29 and 34 Mb on chromosome 6p21, typed in 2240 affected sibpair (ASP) families.

RESULTS

Preliminary analyses confirm that HLA-B (at 31.4 Mb), HLA-A (at 30.0 Mb) are associated with T1D independently of the class II genes HLA-DRB1 and HLA-DQB1 (P = 6.0 x 10(-17) and 8.8 x 10(-13), respectively). In addition, a second class II region of association containing the single-nucleotide polymorphism (SNP), rs439121, and the class II locus HLA-DPB1, was identified as a T1D susceptibility effect which is independent of HLA-DRB1, HLA-DQB1 and HLA-B (P = 9.2 x 10(-8)). A younger age-at-diagnosis of T1D was found for HLA-B*39 (P = 7.6 x 10(-6)), and HLA-B*38 was protective for T1D.

CONCLUSIONS

These analyses in the T1DGC families replicate our results obtained previously in approximately 2000 cases and controls and 850 families. Taking both studies together, there is evidence for four T1D-associated regions at 30.0 Mb (HLA-A), 31.4 Mb (HLA-B), 32.5 Mb (rs9268831/HLA-DRA) and 33.2 Mb (rs439121/HLA-DPB1) that are independent of HLA-DRB1/HLA-DQB1. Neither study found evidence of independent associations at HLA-C, HLA-DQA1 loci nor in the UBD/MAS1L or ITPR3 gene regions. These studies show that to find true class II-independent effects, large, well-powered sample collections are required and be genotyped with a dense map of markers. In addition, a robust statistical methodology that fully models the class II effects is necessary. Recursive partitioning is a useful tool for modelling these multiallelic systems.

OBJECTIVE

To study the pharmacokinetic and pharmacodynamic profile of insulin aspart (a new fast-acting human insulin analog) after subcutaneous administration in the deltoid, abdominal, and thigh sites and to compare this profile with regular human insulin (Novolin; Novo Nordisk A/S, Copenhagen).

METHODS

A total of 20 healthy subjects were studied in a single-center six-period double-blind randomized crossover trial with 6 study days and a washout period of 1 week between each single daily dose of the trial drug. Subjects were randomized to receive a single dose of 0.2 U/kg of insulin aspart or regular insulin on each of the 6 study days in three different sites (the deltoid, the abdomen, and the thigh) during a 10-h euglycemic clamp (two drugs and three injection sites). Pharmacokinetic and pharmacodynamic measurements were derived from blood sample measurements of glucose, insulin, and C-peptide during these clamps.

RESULTS

The pharmacodynamic data from the euglycemic clamp study showed that, regardless of injection site, the maximal glucose infusion rate (GIR Cmax) was greater and occurred at an earlier time (GIR Tmax) after administration of insulin aspart than regular insulin (GIR Cmax: abdomen 813 vs. 708, deltoid 861 vs. 736, and thigh 857 vs. 720 g/min, P < 0.05 for all; GIR Tmax: abdomen 94 vs. 173, deltoid 111 vs. 192, and thigh 145 vs. 193 g/min, P < 0.05 for all). Pharmacokinetic parameters were also consistent with faster absorption and higher peak insulin concentrations after insulin aspart administration. From all sites, the peak insulin concentration (Cmax) was higher and occurred earlier (Tmax) after administration of insulin aspart than of regular insulin (Cmax: abdomen 501 vs. 260, deltoid 506 vs. 252, thigh 422 vs. 220 pmol/l, P < 0.001 for all sites; Tmax: abdomen 52 vs. 109, deltoid 54 vs. 98, and thigh 60 vs. 107 min, P < 0.01 for all sites). The absorption and glucose-lowering action of insulin aspart did not differ between sites (similar GIR Cmax, Tmax, and area under the curve parameters). However, the duration of the glucose-lowering effect was up to 34 min shorter (P < 0.01) for the abdomen injections than for the deltoid or thigh injections (lower time of 50% glucose disposal). In addition, the amount of glucose infused was significantly lower by 10-14% in the abdomen than in other sites.

CONCLUSIONS

Subcutaneous administration of insulin aspart causes a more rapid and intense maximal effect compared with regular insulin during euglycemic clamp studies in nondiabetic subjects. Abdominal administration of insulin aspart has a shorter duration of glucose-lowering effect compared with administration in the deltoid or thigh.

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deléage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989-14000). The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues. All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-[alpha-32P]ATP. However, and in contrast to the wild-type enzyme, the use of 8-N3-[gamma-32P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.

Animal development and homeostasis is a balance between cell proliferation and cell death. Physiologic, and sometimes pathologic, cell death - apoptosis - is driven by activation of a family of proteases known as the caspases, present in almost all nucleated animal cells. The enzymatic properties of these proteases are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side chain for catalyzing peptide bond cleavage. The primary specificity for Asp turns out to be very rare among proteases, and currently the only other known mammalian proteases with the same primary specificity is the physiological caspase activator granzyme B. Like most other proteases, the caspases are synthesized as inactive zymogens whose activation requires limited proteolysis or binding to co-factors. To transmit the apoptotic execution signal, caspase zymogens are sequentially activated through either an intrinsic or an extrinsic pathway. The activation of caspases at the apex of each pathway, the initiators, occurs by recruitment to specific adapter molecules through homophilic interaction domains, and the activated initiators directly process the executioner caspases to their catalytically active forms. In the present communication we review the different mechanisms underlying the selective activation of the caspases.