Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg-Gly-Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division in Escherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight different changes at two residues within this sequence. In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZ(D373G) failed to interact. Two mutant proteins examined restored this interaction significantly. In vivo, the alleles tested are significantly more toxic than the wild type ftsZ and cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZ(D373G), no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

Fatal familial insomnia and a subtype of Creutzfeldt-Jakob disease, two clinically and pathologically distinct diseases, are linked to the same mutation at codon 178 (Asp-178->>Asn) but segregate with different genotypes determined by this mutation and the methionine-valine polymorphism at codon 129 of the prion protein gene. The abnormal isoforms of the prion protein in these two diseases were found to differ both in the relative abundance of glycosylated forms and in the size of the protease-resistant fragments. The size difference was consistent with a different protease cleavage site, suggesting a different conformation of the protease-resistant prion protein present in the two diseases. These differences are likely to be responsible for the type and location of the lesions that characterize these two diseases. Therefore, the combination of the mutation at codon 178 and the polymorphism at codon 129 determines the disease phenotype by producing two altered conformations of the prion protein.

Pollen tube elongation is a rapid tip growth process that is driven by a dynamic actin cytoskeleton. A ubiquitous family of actin binding proteins, actin-depolymerizing factors (ADFs)/cofilins, bind to actin filaments, induce severing, enhance depolymerization from their slow-growing end, and are important for maintaining actin dynamics in vivo. ADFs/cofilins are regulated by multiple mechanisms, among which Rho small GTPase-activated phosphorylation at a terminal region Ser residue plays an important role in regulating their actin binding and depolymerizing activity, affecting actin reorganization. We have shown previously that a tobacco pollen-specific ADF, NtADF1, is important for maintaining normal pollen tube actin cytoskeleton organization and growth. Here, we show that tobacco pollen grains accumulate phosphorylated and nonphosphorylated forms of ADFs, suggesting that phosphorylation could be a regulatory mechanism for their activity. In plants, Rho-related Rac/Rop GTPases have been shown to be important regulators for pollen tube growth. Overexpression of Rac/Rop GTPases converts polar growth into isotropic growth, resulting in pollen tubes with ballooned tips and a disrupted actin cytoskeleton. Using the Rac/Rop GTPase-induced defective pollen tube phenotype as a functional assay, we show that overexpression of NtADF1 suppresses the ability of NtRac1, a tobacco Rac/Rop GTPase, to convert pollen tube tip growth to isotropic growth. This finding suggests that NtADF1 acts in a common pathway with NtRac1 to regulate pollen tube growth. A mutant form of NtADF1 with a nonphosphorylatable Ala substitution at its Ser-6 position [NtADF1(S6A)] shows increased activity, whereas the mutant NtADF1(S6D), which has a phospho-mimicking Asp substitution at the same position, shows reduced ability to counteract the effect of NtRac1. These observations suggest that phosphorylation at Ser-6 of NtADF1 could be important for its integration into the NtRac1 signaling pathway. Moreover, overexpression of NtRac1 diminishes the actin binding activity of green fluorescent protein (GFP)-NtADF1 but has little effect on the association of GFP-NtADF1(S6A) with actin cables in pollen tubes. Together, these observations suggest that NtRac1-activated activity regulates the actin binding and depolymerizing activity of NtADF1, probably via phosphorylation at Ser-6. This notion is further supported by the observation that overexpressing a constitutively active NtRac1 in transformed pollen grains significantly increases the ratio of phosphorylated to nonphosphorylated ADFs. Together, the observations reported here strongly support the idea that NtRac1 modulates NtADF1 activity through phosphorylation at Ser-6 to regulate actin dynamics.

Although (18)F-FDG PET is widely used for metastatic melanoma diagnosis, it is less accurate than desirable, particularly for small foci. Since both melanotic and amelanotic melanomas overexpress receptors for alpha-melanocyte-stimulating hormone (alpha-MSH; receptor name, melanocortin type 1 receptor [MC1R]), radiolabeled alpha-MSH analogs are potential candidates for melanoma diagnosis. The aim of this study was to develop a positron emitter-labeled alpha-MSH analog suitable for PET imaging of melanoma metastases.

METHODS

A short linear alpha-MSH analog, [Nle(4),Asp(5),D-Phe(7)]-alpha-MSH(4-11) (NAPamide), was newly designed and conjugated to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to enable radiometal incorporation. Compared with our previously reported DOTA-alpha-MSH analog, DOTA-MSH(oct) ([DOTA-betaAla(3),Nle(4),Asp(5),D-Phe(7),Lys(10)]-alpha-MSH(3-10)), the major modification lies in the conjugation of DOTA to the C-terminal end of the peptide via the epsilon-amino group of Lys(11), as opposed to the N-terminal alpha-amino group. After labeling with (111)In, (67)Ga, and the short-lived positron emitter (68)Ga, DOTA-NAPamide was characterized in vitro and in vivo using the mouse melanoma B16F1cell line.

RESULTS

DOTA-NAPamide exhibited an almost 7-fold higher MC1R binding potency as compared with DOTA-MSH(oct). In B16F1 melanoma-bearing mice, both (111)In-DOTA-NAPamide and (67)Ga-DOTA-NAPamide behaved more favorably than (111)In-DOTA-MSH(oct). Both radiopeptides exhibited higher tumor and lower kidney uptake leading to tumor-to-kidney ratios of the 4- to 48-h area under the curve that were 4.6 times ((111)In) and 7.5 times ((67)Ga) greater than that obtained with (111)In-DOTA-MSH(oct). In addition, the 4-h kidney uptake of (67)Ga-DOTA-NAPamide could be reduced by 64% by coinjection of 15 mg L-lysine, without affecting tumor uptake. Skin primary melanoma as well as lung and liver melanoma metastases could be easily visualized on tissue section autoradiographs after systemic injection of (67)Ga-DOTA-NAPamide. The melanoma selectivity of DOTA-NAPamide was confirmed by PET imaging studies using (68)Ga-DOTA-NAPamide. Tumor uptake was found to be highest when the smallest amount of peptide was administered.

CONCLUSIONS

DOTA-NAPamide labeled with either (111)In or (67)Ga/(68)Ga is in every way superior to (111)In-DOTA-MSH(oct) in murine models of primary and metastatic melanoma, which makes it a promising agent for melanoma targeting. High-contrast images obtained in PET studies with an experimental tumor model 1 h after injection augurs well for its clinical potential as an imaging tool.

The development of epitope-based vaccines, which have wide population coverage, is greatly complicated by MHC polymorphism. The grouping of alleles into supertypes, on the basis of common structural and functional features, addresses this problem directly. In the present study we applied a combined bioinformatics approach, based on analysis of both protein sequence and structure, to identify similarities in the peptide binding sites of 2225 human class II MHC molecules, and thus define supertypes and supertype fingerprints. Two chemometric techniques were used: hierarchical clustering using three-dimensional Comparative Similarity Indices Analysis fields and nonhierarchical k-means clustering using sequence-based z-descriptors. An average consensus of 84% was achieved, i.e., 1872 of 2225 class II molecules were classified in the same supertype by both techniques. Twelve class II supertypes were defined: five DRs, three DQs, and four DPs. The HLA class II supertypes and their fingerprints given in parenthesis are DR1 (Trp(9beta)), DR3 (Glu(9beta), Gln(70beta), and Gln/Arg(74beta)), DR4 (Glu(9beta), Gln/Arg(70beta), and Glu/Ala(74beta)), DR5 (Glu(9beta), Asp(70beta)), and DR9 (Lys/Gln(9beta)); DQ1 (Ala/Gly(86beta)), DQ2 (Glu(86beta), Lys(71beta)), and DQ3 (Glu(86beta), Thr/Asp(71beta)); DPw1 (Asp(84beta) and Lys(69beta)), DPw2 (Gly/Val(84beta) and Glu(69beta)), DPw4 (Gly/Val(84beta) and Lys(69beta)), and DPw6 (Asp(84beta) and Glu(69beta)). Apart from the good agreement between known binding motifs and our classification, several new supertypes, and corresponding thematic binding motifs, were also defined.

The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP gamma-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.

The heterotrimeric G protein alpha subunit (Galpha) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Galpha palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated Galpha(q), Galpha(s), and Galpha(i2) in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased Galpha(q/11) palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Galpha(q) rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous Galpha(q) shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the alpha(1A)-adrenergic receptor/Galpha(q/11)-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling Galpha localization to the plasma membrane.

We performed a two-stage genome screen to search for novel risk factors for late-onset Alzheimer disease (AD). The first stage involved genotyping 292 affected sibling pairs using 237 markers spaced at approximately 20 cM intervals throughout the genome. In the second stage, we genotyped 451 affected sibling pairs (ASPs) with an additional 91 markers, in the 16 regions where the multipoint LOD score was greater than 1 in stage I. Ten regions maintained LOD scores in excess of 1 in stage II, on chromosomes 1 (peak B), 5, 6, 9 (peaks A and B), 10, 12, 19, 21, and X. Our strongest evidence for linkage was on chromosome 10, where we obtained a peak multipoint LOD score (MLS) of 3.9. The linked region on chromosome 10 spans approximately 44 cM from D10S1426 (59 cM) to D10S2327 (103 cM). To narrow this region, we tested for linkage disequilibrium with several of the stage II microsatellite markers. Of the seven markers we tested in family-based and case control samples, the only nominally positive association we found was with the 167 bp allele of marker D10S1217 (chi-square=7.11, P=0.045, df=1).

Our application of transition path sampling to a complex biomolecular system in explicit solvent, the closing transition of DNA polymerase beta, unravels atomic and energetic details of the conformational change that precedes the chemical reaction of nucleotide incorporation. The computed reaction profile offers detailed mechanistic insights into, as well as kinetic information on, the complex process essential for DNA synthesis and repair. The five identified transition states extend available experimental and modeling data by revealing highly cooperative dynamics and critical roles of key residues (Arg-258, Phe-272, Asp-192, and Tyr-271) in the enzyme's function. The collective cascade of these sequential conformational changes brings the DNA/DNA polymerase beta system to a state nearly competent for the chemical reaction and suggests how subtle residue motions and conformational rate-limiting steps affect reaction efficiency and fidelity; this complex system of checks and balances directs the system to the chemical reaction and likely helps the enzyme discriminate the correct from the incorrect incoming nucleotide. Together with the chemical reaction, these conformational features may be central to the dual nature of polymerases, requiring specificity (for correct nucleotide selection) as well as versatility (to accommodate different templates at every step) to maintain overall fidelity. Besides leading to these biological findings, our developed protocols open the door to other applications of transition path sampling to long-time, large-scale biomolecular reactions.

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell-substrate interaction.

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.

Association and dissociation rate constants were measured for O2, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale myoglobins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and <em>Asp</em> using the synthetic gene and expression system developed by Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8961-8965). The His64----Gly substitution produced a sterically unhindered myoglobin that exhibited ligand binding parameters similar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant myoglobins was Gly64 (approximately 10(7) M-1 s-1) much greater than Val64 approximately Leu64 (approximately 10(6) M-1 s-1) greater than Met64 greater than Phe64 approximately His64 approximately Gln64 (10(5)-10(3) M-1 s-1) and indicates that the barrier to isocyanide entry into the distal pocket is primarily steric in nature. The bimolecular rates of methyl, ethyl, n-propyl, and n-butyl isocyanide binding to the His64----Arg and His64----<em>Asp</em> mutants were abnormally high (1-5 x 10(6) M-1 s-1), suggesting that Arg64 and <em>Asp</em>64 adopt conformations with the charged side chains pointing out toward the solvent creating a less hindered pathway for ligand binding. In contrast to the isocyanide data, the association rate constants for O2 and CO binding exhibited little dependence on the size of the E7 side chain. The values for all the mutants except His64----Gln approached or were larger than those for chelated model heme (i.e. approximately 1 x 10(8) M-1 s-1 for O2 and approximately 1 x 10(7) M-1 s-1 for CO), whereas the corresponding rate parameters for myoglobin containing either Gln64 or His64 were 5- to 10-fold smaller. This result suggests that a major kinetic barrier for O2 and CO binding to native myoglobin may involve disruption of polar interactions between His64 and water molecules found in the distal pocket of deoxymyoglobin. Finally, the rate and equilibrium parameters for O2 and CO binding to the His64----Gln, His64----Val, and His64----Leu mutants were compared to those reported previously for Asian elephant myoglobin (Gln-E7), Aplysia limacina myoglobin (Val-E7), and monomeric Hb II from Glycera dibranchiata (Leu-E7).

Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK(a) values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.

We determined the relationship between the activation state and phosphorylation state of the Na-K-Cl cotransport protein in tubules isolated from the shark rectal gland, a prototypic chloride-secreting epithelium. In response to cAMP-dependent secretagogues (e.g. vasoactive intestinal peptide, adenosine, and forskolin) or osmotically induced changes in cell volume, the activation state of the cotransport protein (assessed from measurements of loop diuretic binding) increased 5-10 fold. The response was temporally associated with a comparable increase (3-9 fold) in cotransport protein phosphorylation. Graded changes in cotransporter activation evoked proportional changes in cotransporter phosphorylation. Under the conditions of our experiments, the 195-kDa cotransporter was the only membrane protein whose phosphorylation state increased conspicuously in response to both cAMP and cell shrinkage. Both stimuli promoted phosphorylation of the cotransport protein at serine and threonine residues. One of the cAMP-sensitive phosphoacceptors was found within a segment of the cotransport protein comprised of a sequence (Phe-Gly-His-Asn-Thr*-Ile-Asp-Ala-Val-Pro) that corresponds to a segment of the Na-K-Cl cotransport protein predicted by cDNA analysis, where the phosphoacceptor (Thr*) is threonine 189. Incubation of rectal gland tubules with K-252a or H-8, structurally different protein kinase inhibitors, rendered the cotransporter insensitive to both cAMP and cell shrinkage. We conclude that the rectal gland Na-K-Cl cotransport protein is regulated by direct reversible phosphorylation at serine and threonine sites.

The enzyme-AMP reaction intermediate of the 102-kDa bovine DNA ligase I was digested with trypsin, and the adenylylated peptide was isolated by chromatography under conditions that maintain the acid-labile phosphoramidate bond. Microsequencing of the peptide showed that it contains an internal trypsin-resistant lysine residue, as expected for the site of adenylylation. Inhibition of DNA ligase I activity by pyridoxal 5'-phosphate also indicated the presence of a reactive lysine residue in the catalytic domain of the enzyme. Comparison of the known primary structures of several other DNA ligases with the adenylylated region of mammalian DNA ligase I allows their active sites to be tentatively assigned by sequence homology. The ATP-dependent DNA ligases of mammalian cells, fission yeast, budding yeast, vaccinia virus, and bacteriophages T3, T4, and T7 contain the active site motif Lys-Tyr/Ala-Asp-Gly-(Xaa)-Arg, with the reactive lysine residue flanked by hydrophobic amino acids. The distance between the postulated adenylylation site and the carboxyl terminus of the polypeptide is very similar in these ATP-dependent DNA ligases, whereas the size of the amino-terminal region is highly variable.

Apoptosis-inducing factor (AIF) triggers apoptosis in a caspase-independent manner. Here we report for the first time involvement of AIF in neuronal death induced by cerebral ischemia. Unilateral cerebral hypoxia-ischemia (HI) was induced in 7-day-old rats by ligation of the left carotid artery and hypoxia (7.7% O2) for 55 min. AIF release from mitochondria and AIF translocation to nuclei was detected immediately after HI, and only in damaged areas, as judged by the concurrent loss of MAP-2. AIF release was detected earlier than that of cytochrome c. Cells with AIF-positive nuclei displayed nuclear condensation and signs of DNA damage. The number of AIF-positive nuclei showed a positive correlation with the infarct volume 72 h post-HI, and this was not changed by treating the animals with boc-Asp-fmk (BAF), a multicaspase inhibitor. BAF treatment reduced the activity of caspase-3, -2 and -9 (78, 73 and 33%, respectively), and prevented caspase-dependent fodrin cleavage in vivo, but did not affect AIF release from mitochondria or the frequency of positive nuclear AIF or DNA damage 72 h post-HI, indicating that these processes occurred in a caspase-independent fashion. In summary, AIF-mediated cell death may be an important mechanism of HI-induced neuronal loss in the immature brain.

Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system. Under those conditions, the tripartite sensor kinase ArcB undergoes autophosphorylation at the expense of ATP and subsequently transphosphorylates its cognate response regulator ArcA through a His ->> Asp ->> His ->> Asp phosphorelay pathway. In this study we used various combinations of wild-type and mutant ArcB domains to analyze in vitro the pathway for signal decay. The results indicate that ArcA-P dephosphorylation does not occur by direct hydrolysis but by transfer of the phosphoryl group to the secondary transmitter and subsequently to the receiver domain of ArcB. This reverse phosphorelay involves both the conserved His-717 of the secondary transmitter domain and the conserved Asp-576 of the receiver domain of ArcB but not the conserved His-292 of its primary transmitter domain. This novel pathway for signal decay may generally apply to signal transduction systems with tripartite sensor kinases.

The group A colicins and the DNA of many single-stranded filamentous bacteriophage are able to use combinations of the Tol proteins to gain entrance into or across the membrane of Escherichia coli. The TolA protein is a 421-amino acid residue integral membrane protein composed of three domains. Domain I, consisting of the amino-terminal 47 amino acids, contains a 21-residue hydrophobic segment that anchors the protein in the inner membrane. The remaining 374 amino acids, containing the other two domains, reside in the periplasmic space. Domain III, consisting of the carboxyl-terminal 120 residues, is considered to be the functional domain based on the location of the tolA592 deletion mutation. The internal 262 amino acids comprise domain II, which connects domains I and III together via short regions of polyglycine. It contains a large number of 3- to 5-residue polyalanine stretches, many of which have a repeat of the sequence Lys-Ala-Ala-Ala-(Glu/Asp). Circular dichroism analysis of different portions of TolA show domain II to be predominantly alpha-helical in structure while domain III contains approximately 10% helical structure.

A new serine protease was encoded by a clone isolated from a murine cytotoxic T-lymphocyte complementary DNA library by an RNA-hybridization competition protocol. Complementary transcripts were detected in cytotoxic T lymphocytes, spleen cells from nude mice, a rat natural killer cell leukemia, and in two of eight T-helper clones (both cytotoxic), but not in normal mouse kidney, liver, spleen, or thymus, nor in several tested T- and B-cell tumors. T-cell activation with concanavalin A plus interleukin-2 induced spleen cells to express this gene with kinetics correlating with the acquisition of cytolytic capacity. The nucleotide sequence of this gene encoded an amino acid sequence of approximately 25,700 daltons, with 25 to 35 percent identity to members of the serine protease family. The active site "charge-relay" residues (His57, AspAsp (position 189 in trypsin). A Southern blot analysis indicated that this gene is conserved in humans, mouse, and chicken. This serine protease may have a role in lymphocyte lysis and a "lytic cascade."

PCR was used to amplify a 238-bp region from Helicobacter pylori which corresponded to the quinolone resistance-determining region in Escherichia coli. The gyrA gene of H. pylori was cloned and sequenced. An open reading frame of 2,478 nucleotides coded for a polypeptide of 826 amino acids with a calculated molecular mass of 92,508 Da. The amino acid sequence showed an overall 52% identity with other bacterial gyrA genes but was most closely related to the gyrA subunit of Campylobacter jejuni (76.5% identity). Sequencing of the amplification product from ciprofloxacin-resistant mutants of H. pylori revealed four classes of mutations with substitutions at amino acid 87 (Asn->>Lys), amino acid 88 (Ala->>Val), and amino acid 91 (Asp->>Gly, ->>Asn, or ->>Tyr) and a double substitution at amino acids 91 and 97 (Ala->>Val). Ciprofloxacin-susceptible strains of H. pylori could be transformed to ciprofloxacin resistance by using the amplified fragment from resistant strains as donor DNA. Of the 11 ciprofloxacin-resistant mutants examined, only one did not have an alteration within the quinolone resistance-determining region, suggesting that, in H. pylori, resistance to quinolones is primarily a result of alterations in gyrA.

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure-activity study from another laboratory using various effector-substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6beta-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by alpha-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6beta-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.