Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins and initiate G-protein-independent signalling. Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs. Structures of the inactive state of arrestin are available, but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a ∼21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor-binding loops in the central crest region include the finger loop, loop 139 (refs 8, 10, 11) and the sequence Asp 296-Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system.

Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes/macrophages by means of a bacterial surface-associated protein, filamentous hemagglutinin (FHA) and the leukocyte integrin, complement receptor 3 (CR3, alpha M beta 2, CD11b/CD18). We show that an FHA Arg-Gly-Asp site induces enhanced B. pertussis binding to monocytes, and that this enhancement is blocked by antibodies directed against CR3. Enhancement requires a monocyte signal transduction complex, composed of leukocyte response integrin (alpha? beta 3) and integrin-associated protein (CD47). This complex is known to upregulate CR3 binding activity. Thus, a bacterial pathogen enhances its own attachment to host cells by coopting a host cell signaling pathway.

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 ->> <em>Asp</em>) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the <em>Asp</em>137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of <em>Asp</em>137 in the homologous position in acetylcholinesterase suggests that <em>Asp</em>137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

Orotidine 5'-monophosphate decarboxylase catalyzes the conversion of orotidine 5'-monophosphate to uridine 5'-monophosphate, the last step in biosynthesis of pyrimidine nucleotides. As part of a Structural Genomics Initiative, the crystal structures of the ligand-free and the6-azauridine 5'-monophosphate-complexed forms have been determined at 1.8 and 1.5 A, respectively. The protein assumes a TIM-barrel fold with one side of the barrel closed off and the other side binding the inhibitor. A unique array of alternating charges (Lys-Asp-Lys-Asp) in the active site prompted us to apply quantum mechanical and molecular dynamics calculations to analyze the relative contributions of ground state destabilization and transition state stabilization to catalysis. The remarkable catalytic power of orotidine 5'-monophosphate decarboxylase is almost exclusively achieved via destabilization of the reactive part of the substrate, which is compensated for by strong binding of the phosphate and ribose groups. The computational results are consistent with a catalytic mechanism that is characterized by Jencks's Circe effect.

The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain. The catalytic domain is subdivided into two subdomains comprising four- and six-stranded beta-sheets sandwiched by alpha-helices. Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1. The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor.

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor-product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.

Integrins are heterodimeric adhesion receptors that mediate cell-matrix interaction. Osteoclast exhibits high expression of the alpha(v)beta(3) integrin, which binds to a variety of extracellular matrix proteins including vitronectin, osteopontin, and bone sialoprotein. Arg-Gly-Asp (RGD)-containing peptides, RGD-mimetics, and blocking antibodies to alpha(v)beta(3) integrin were shown to inhibit bone resorption in vitro and in vivo, suggesting that this integrin may play an important role in regulating osteoclast function. Several lines of evidence have demonstrated that a number of signaling molecules are involved in the alpha(v)beta(3) integrin-dependent signaling pathway, including c-Src, Pyk2, c-Cbl, and p130(Cas). In this article, we review the history of "alpha(v)beta(3) integrin and osteoclasts" and discuss the involvement of alpha(v)beta(3) integrins in osteoclast function at tissue, cellular, and molecular levels. A better understanding of the role of alpha(v)beta(3) integrin in osteoclastic bone resorption would provide opportunities for developing new therapeutics to treat human bone diseases, including rheumatoid arthritis, osteoporosis, and periodontal disease.

OBJECTIVE

Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides have great potential for the early tumor detection and noninvasive monitoring of tumor metastasis and therapeutic response. (18)F-labeled RGD analogs ([(18)F]-AH111585 and [(18)F]Galacto-RGD) have been investigated in clinical trials for positron emission tomography (PET) imaging of integrin expression in cancer patients. To develop new RGD radiotracers with higher tumor accumulation, improved in vivo kinetics, easy availability and low cost, we developed two new RGD peptides and labeled them with generator-eluted (68)Ga (t(1/2) = 68 min) for PET imaging of integrin alpha(v)beta(3) expression in tumor xenograft models.

METHODS

The two new cyclic RGD dimers, E[PEG(4)-c(RGDfK)](2) (P(4)-RGD2, PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and E[Gly(3)-c(RGDfK)](2) (G(3)-RGD2, G(3) = Gly-Gly-Gly) were designed, synthesized and conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) for (68)Ga labeling. The microPET imaging and biodistribution of the (68)Ga labeled RGD tracers were investigated in integrin alpha(v)beta(3)-positive tumor xenografts.

RESULTS

The new RGD dimers with the Gly(3) and PEG(4) linkers showed higher integrin alpha(v)beta(3) binding affinity than no-linker RGD dimer (RGD2). NOTA-G(3)-RGD2 and NOTA-P(4)-RGD2 could be labeled with (68)Ga within 30 min with higher purity (>98%) and specific activity (8.88-11.84 MBq/nmol). Both (68)Ga-NOTA-P(4)-RGD2 and (68)Ga-NOTA-G(3)-RGD2 exhibited significantly higher tumor uptake and tumor-to-normal tissue ratios than (68)Ga-NOTA-RGD2.

CONCLUSIONS

Because of their high affinity, high specificity and excellent pharmacokinetic properties, further investigation of the two novel RGD dimers for clinical PET imaging of integrin alpha(v)beta(3) expression in cancer patients is warranted.

This article describes the fundamental cleavage reactions of (M-H)(-) anions of underivatized peptides that contain up to 25 amino acid residues. The experimental observations of these cleavages have been backed up by molecular modeling, generally at the AM1 level of theory. The basic cleavages are the ubiquitous alpha- and beta-backbone cleavage reactions, which provide information similar to that of the B and Y + 2 cleavages of MH(+) ions of peptides. The residues Asp and Asn also effect cleavages of the backbone (called delta- and gamma-cleavages), by reactions initiated from side chain enolate anions, causing elimination reactions that cleave the backbone between the Asp (Asn) N bond;C backbone bond. Glu and Gln also direct analogous delta- and gamma-cleavages of the backbone, but in this case the processes are initiated by attack of the side chain CO(2) (-) (CONH(-)) to form a lactone (lactam). Ser and Thr residues undergo characteristic fragmentations of the side chain. These processes, losses of CH(2)O (Ser) and MeCHO (Thr), convert these residues into Gly. In larger peptides, Ser and Thr can effect two backbone cleavage reactions, called gamma- and epsilon -processes. The C-terminal CO(2) (-) (or CONH(-)) forms a hydrogen bond with the side chain OH (of Ser or Thr), placing the C-terminal residue in a position where it may affect S(N) (2) attack at the electrophilic backbone CH of Ser, with concomitant cleavage of the backbone. All of the above negative ion cleavages require the peptide backbone to be conformationally flexible. However, there is a backbone cleavage that requires the peptide to have an alpha-helical conformation in order for the two reacting centers to approach. This cleavage is illustrated for the Glu 23-initiated backbone cleavage at Ile 21 for the (M-H)(-) anion of the antimicrobial peptide caerin 1.1.

In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-to-Asp phosphorelay mechanisms are involved presumably in propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we identified and characterized a set of novel Arabidopsis genes whose products considerably resemble the authentic response regulators (ARR-series) of Arabidopsis in the sense that they have a phospho-accepting receiver-like domain. However, they should be discriminated from the classical ones in the strict sense that they lack the invariant phospho-accepting aspartate site. They were thus named APRRs (Arabidopsis pseudo-response regulators). Two such representatives, APRR1 and APRR2, were characterized extensively through cloning of the corresponding cDNAs, in terms of their structural designs, biochemical properties, subcellular localization in plant cells, and expression profiles at the transcriptional level. The result of in vitro phosphorylation experiment with the Arabidopsis AHP phosphotransmitter suggested that the pseudo-receivers have no ability to undergo phosphorylation. The result of transient expression assay with onion epidermal cells showed that the GFP-APRR1 fusion protein has an ability to enter into the nuclei. The C-terminal domain of APRR1, termed CONSTANS-motif, appears to be responsible for the nuclear-localization. The most intriguing result was that the accumulation of APRR1 transcript is subjected to a circadian rhythm. The APRR1 protein is identical to the one that was recently suggested to interact with the ABI3 (ABISCISIC ACID INSENSITIVE3) protein. These are discussed with special reference to the His-to-Asp phosphorelay signal transduction and circadian rhythm in Arabidopsis thaliana.

In fleshy fruit, levels of indole-3-acetic acid (IAA), the most abundant auxin, decline towards the onset of ripening. The application of auxins to immature fruit can delay the ripening processes. However, the mechanisms by which the decrease in endogenous IAA concentrations and the maintenance of low auxin levels in maturing fruit are achieved remain elusive. The transcript of a GH3 gene (GH3-1), encoding for an IAA-amido synthetase which conjugates IAA to amino acids, was detected in grape berries (Vitis vinifera L.). GH3-1 expression increased at the onset of ripening (veraison), suggesting that it might be involved in the establishment and maintenance of low IAA concentrations in ripening berries. Furthermore, this grapevine GH3 gene, responded positively to the combined application of abscisic acid and sucrose and to ethylene, linking it to the control of ripening processes. Levels of IAA-aspartic acid (IAA-Asp), an in vitro product of recombinant GH3-1, rose after veraison and remained high during the following weeks of the ripening phase when levels of free IAA were low. A similar pattern of changes in free IAA and IAA-Asp levels was detected in developing tomatoes (Solanum lycopersicum Mill.), where low concentrations of IAA and an increase in IAA-Asp concentrations coincided with the onset of ripening in this climacteric fruit. Since IAA-Asp might be involved in IAA degradation, the GH3 catalysed formation of this conjugate at, and after, the onset of ripening could represent a common IAA inactivation mechanism in climacteric and non-climacteric fruit which enables ripening.

Tubular damage by cisplatin leads to acute renal failure, which limits its use in cancer therapy. In tubular cells, a primary target for cisplatin is presumably the genomic DNA. However, the pathway relaying the signals of DNA damage to tubular cell death is unclear. In response to DNA damage, the tumor suppressor gene p53 is induced and is implicated in subsequent DNA repair and cell death by apoptosis. The current study was designed to examine the role of p53 in cisplatin-induced apoptosis in cultured rat kidney proximal tubular cells. Cisplatin at 20 microM induced apoptosis in approximately 70% of cells, which was partially suppressed by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (VAD), a general caspase inhibitor. Of interest, cisplatin-induced apoptosis was also suppressed by pifithrin-alpha, a pharmacological inhibitor of p53. Cisplatin-induced caspase activation was completely inhibited by VAD, but only partially by pifithrin-alpha. Early during cisplatin treatment, p53 was phosphorylated and upregulated. The p53 activation was blocked by pifithrin-alpha, but not by VAD. Bcl-2 expression abolished cisplatin-induced apoptosis without blocking p53 phosphorylation or induction. The results suggest that p53 activation might be an early signal for apoptosis during cisplatin treatment. To further determine the role of p53, tubular cells were stably transfected with a dominant-negative mutant of p53 with diminished transcriptional activity. Expression of the mutant attenuated cisplatin-induced apoptosis and caspase activation. In conclusion, the results support an important role for p53 in cisplatin-induced apoptosis in renal tubular cells. p53 May regulate apoptosis through the transcription of apoptotic genes.

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.

BACKGROUND

Dubowitz syndrome (DS) is an autosomal recessive disorder characterized by the constellation of mild microcephaly, growth and mental retardation, eczema and peculiar facies. Over 140 cases have been reported, but the genetic basis is not understood.

METHODS

We enrolled a multiplex consanguineous family from the United Arab Emirates with many of the key clinical features of DS as reported in previous series. The family was analyzed by whole exome sequencing. RNA splicing was evaluated with reverse-transcriptase PCR, immunostaining and western blotting was performed with specific antibodies, and site-specific cytosine-5-methylation was studied with bisulfite sequencing.

RESULTS

We identified a homozygous splice mutation in the NSUN2 gene, encoding a conserved RNA methyltransferase. The mutation abolished the canonical splice acceptor site of exon 6, leading to use of a cryptic splice donor within an AluY and subsequent mRNA instability. Patient cells lacked NSUN2 protein and there was resultant loss of site-specific 5-cytosine methylation of the tRNA(Asp GTC) at C47 and C48, known NSUN2 targets.

CONCLUSIONS

Our findings establish NSUN2 as the first causal gene with relationship to the DS spectrum phenotype. NSUN2 has been implicated in Myc-induced cell proliferation and mitotic spindle stability, which might help explain the varied clinical presentation in DS that can include chromosomal instability and immunological defects.

PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.

BACKGROUND

Delay discounting is associated with impulsivity and drug abuse, and this study evaluated the relationship between discounting of delayed rewards and antisocial personality disorder (ASP).

OBJECTIVE

To determine whether discounting is increased in substance abusers with ASP relative to those without; both groups were compared with non-substance-abusing controls.

METHODS

Subjects ( n=166) chose between hypothetical monetary amounts available after various delays or immediately. Under one condition, a US $1000 reward was delayed at intervals ranging from 6 h to 25 years. At each delay interval, the immediately available rewards varied from US $1 to US $999 until choices reflected indifference between the smaller immediate and larger delayed rewards. Under a second condition, the delayed reward was US $100, and immediate rewards varied from US $0.10 to US $99.90.

RESULTS

In all three groups, hyperbolic discounting functions provided a good fit of the data, and the smaller reward was discounted at a higher rate than the larger reward. Substance abusers discounted delayed rewards at significantly greater rates than controls, and substance abusers with ASP discounted delayed rewards at higher rates than their non-ASP substance-abusing counterparts. Discounting rates were correlated with impulsivity scores on a personality questionnaire.

CONCLUSIONS

These results provide further evidence of more rapid discounting of delayed rewards in substance abusers, especially among substance abusers with a co-morbid diagnosis of ASP.

Synthetic peptides were prepared which corresponded to the carboxy termini of the human adenovirus type 5 early region 1B (E1B) 58,000-molecular-weight (58K) protein (Tyr-Ser-Asp-Glu-Asp-Thr-Asp) and of the E1A gene products (Tyr-Gly-Lys-Arg-Pro-Arg-Pro). Antisera raised against these peptides precipitated polypeptides from adenovirus type 5-infected KB cells; serum raised against the 58K carboxy terminus was active against the E1B 58K phosphoprotein, whereas serum raised against the E1A peptide immunoprecipitated four major and at least two minor polypeptides. These latter proteins migrated with apparent molecular weights of 52K, 50K, 48.5K, 45K, 37.5K, and 35K, and all were phosphoproteins. By using tryptic phosphopeptide analysis, the four major species (52K, 50K, 48.5K, and 45K) were found to be related, as would be expected if all were products of the E1A region. The ability of the antipeptide sera to precipitate these viral proteins thus confirmed that the previously proposed sequence of E1 DNA and mRNA and the reading frame of the mRNA are correct. Immunofluorescent-antibody staining with the antipeptide sera indicated that the 58K E1B protein was localized both in the nucleus and in the cytoplasm, especially in the perinuclear region. The E1A-specific serum also stained both discrete patches in the nucleus and diffuse areas of the cytoplasm. These data suggest that both the 58K protein and the E1A proteins may function in or around the nucleus. These highly specific antipeptide sera should allow for a more complete identification and characterization of these important viral proteins.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes alpha3beta1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBdeltaTM) and a gBdeltaTM mutant (gBdeltaTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBdeltaTM, gBdeltaTM-RGA, and deltaTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBdeltaTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBdeltaTM-RGA and DeltaTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBdeltaTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBdeltaTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBdeltaTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBdeltaTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBdeltaTM protein to the target cells but not by gBdeltaTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBdeltaTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.

We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli.

The human proto-oncogene bcl-2 and its Caenorhabditis elegans homologue ced-9 inhibit programmed cell death. In contrast, members of the human interleukin-1beta converting enzyme (ICE) family of cysteine proteases and their C. elegans homologue CED-3 promote the death program. Genetic experiments in C. elegans have shown that ced-9 is formally a negative regulator of ced-3 function, but neither those studies nor others have determined whether CED-9 or Bcl-2 proteins act biochemically upstream or downstream of CED-3/ICE proteases. CPP32, like all known members of the CED-3/ICE family, is synthesized as a proenzyme that is subsequently processed into an active protease with specificity for cleavage at Asp-X peptide bonds. In this report, we demonstrate that the CPP32 proenzyme is proteolytically processed and activated in Jurkat cells induced to die by Fas ligation. CPP32 activation is blocked by cell-permeable inhibitors of aspartate-directed, cysteine proteases, suggesting that pro-CPP32 is cleaved by active CPP32 or by other ICE family members. Heterologous expression of Bcl-2 in Jurkat cells prevents Fas-induced cell death as well as proteolytic processing and activation of CPP32. Thus, Bcl-2 acts at or upstream of the CPP32 activation step to inhibit apoptosis induced by Fas stimulation.

OBJECTIVE

We tested the hypotheses that Src tyrosine kinase overactivity represents a chemoresistance mechanism and that Src inhibition may enhance gemcitabine cytotoxicity in pancreatic adenocarcinoma cells.

METHODS

Pancreatic adenocarcinoma cells PANC1, MiaPaCa2, Capan2, BxPC3, and PANC1(GemRes), a stably gemcitabine-resistant subline of PANC1, were exposed to combinations of gemcitabine and Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Src expression, phosphorylation (Tyr-416), and activity were analyzed by immunoblotting and in vitro kinase assay. Expression of the M2 subunit of ribonucleotide reductase (RRM2), a putative chemoresistance enzyme, was quantified by Northern and Western blot. Cellular proliferation was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was characterized by YO-PRO-1/propidium iodide staining, fluorometric caspase profiling, and caspase inhibition (Z-Val-Ala-Asp-fluoromethyl ketone). The effects of constitutively active and dominant negative Src were determined. The therapeutic efficacy of PP2 in combination with gemcitabine was tested in nude mice orthotopically xenografted with PANC1(GemRes).

RESULTS

Greater gemcitabine resistance was associated with higher Src phosphorylation and activity, both of which were higher in PANC1(GemRes), relative to PANC1; total Src levels were alike. PANC1(GemRes) overexpressed RRM2. PP2 enhanced inherent gemcitabine chemosensitivity and attenuated gemcitabine resistance in PANC1(GemRes). Constitutively active Src increased gemcitabine chemoresistance; dominant negative Src impaired gemcitabine chemoresistance. PP2 augmented gemcitabine-induced caspase-mediated apoptosis, suppressed RRM2 expression, and decreased activity of the RRM2-regulating transcription factor E2F1 in PANC1(GemRes). PP2 and gemcitabine in combination substantially decreased tumor growth and inhibited metastasis in vivo.

CONCLUSIONS

Increased Src tyrosine kinase activity represents a potential chemoresistance mechanism and a promising therapeutic target warranting further investigation in gemcitabine-resistant pancreatic adenocarcinoma.

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca(2+)-activated K(+) channel (BK channel) contains Ca(2+)-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca(2+) binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His(6)-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a (45)Ca(2+)-overlay protein blot assay to detect Ca(2+) binding. Dslo-C280 exhibits specific binding of (45)Ca(2+) in comparison with various control proteins and known EF-hand Ca(2+)-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the "Ca-bowl" motif are changed to Asn, reduces (45)Ca(2+)-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca(2+) sensitivity for activation and a shift of approximately +80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca(2+) and a reduction in apparent Ca(2+) affinity, suggesting the loss of one Ca(2+)-binding site per monomer. These results demonstrate a functional correlation between Ca(2+) binding to a specific region of the BK protein and Ca(2+)-dependent activation, thus providing a biochemical approach to study this process.