Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.

Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of the HLA-DQ2 (alpha1(*)0501, beta1(*)0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several size-variants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClalpha46 - 63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClalpha46 - 63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides.

The crystal structures of the molecular complexes between two serine proteinases and two of their protein inhibitors have been determined: subtilisin Carlsberg with the recombinant form of eglin-c from the leech Hirudo medicinalis and subtilisin Novo with chymotrypsin inhibitor 2 from barley seeds. The structures have been fully refined by restrained-parameter least-squares methods to crystallographic R factors (sigma[[Fo[ - [Fc[[/sigma[Fo[) of 0.136 at 1.8-A resolution and 0.154 at 2.1-A resolution, respectively. The 274 equivalent alpha-carbon atoms of the enzymes superpose with an rms deviation of 0.53 A. Sequence changes between the enzymes result in localized structural adjustments. Functional groups in the active sites superpose with an rms deviation of 0.19 A for 161 equivalent atoms; this close similarity in the conformation of active-site residues provides no obvious reason for known differences in catalytic activity between Carlsberg and Novo. Conformational changes in the active-site region indicate a small induced fit of enzyme and inhibitor. Some conformational differences are observed between equivalent active-site residues of subtilisin Carlsberg and alpha-chymotrypsin. Despite differences in tertiary architecture, most enzyme-substrate (inhibitor) interactions are maintained. Subtilisin Carlsberg has a rare cis-peptide bond preceding Thr211 (Gly211 in Novo). Both enzymes contain tightly bound Ca2+ ions. Site 1 is heptacoordinate with the oxygen atoms at the vertices of a pentagonal bipyramid. Site 2 in Carlsberg is probably occupied by a K+ ion in Novo. Conserved water molecules appear to play important structural roles in the enzyme interior, in the inhibitor beta-sheet, and at the enzyme-inhibitor interface. The 62 equivalent alpha-carbon atoms of the inhibitors superpose with an rms deviation of 1.68 A. Sequence changes result in somewhat different packing of the alpha-helix, beta-sheet, and reactive-site loop relative to each other. Hydrogen bonds and electrostatic interactions supporting the conformation of the reactive-site loop are conserved. The 24 main-chain plus C beta atoms of P4 to P1' overlap with an rms deviation of 0.19 A. Features contributing to the inhibitory nature of eglin-c and CI-2 are discussed.

Many of the anomalies observed in studies or properdin may be explained on the basis of its ability to form a series of multi-subunit polymers and by differences in the functions of these forms of properdin. Dimers (P2), trimers (P3), tetramers (P4), and higher Mr polymers (Pn) of the 46,000-Da subunit were separated by gel filtration or by cation exchange chromatography of purified properdin. The specific activity of each form was measured in two assays. The native properdin activity of P4 was 10 times that of P2 (on a molar basis) with the order: P4 greater than P3 greater than P2 greater than Pn. During C activation P4 was found to be consumed first, P3 second, and P2 last, consistent with their measured specific activities. Assays for activated properdin showed that only Pn caused fluid phase C consumption when incubated in serum at normal concentrations. Pn accumulated during long term storage of purified P and freezing rapidly converted the smaller oligomers to Pn. The isolated oligomers were extremely stable, but did redistribute after denaturation-renaturation cycles by using low pH or guanidine. Renaturation after exposure of any species to denaturing conditions yielded mixtures of 20:54:26 (P4:P3:P2). This distribution was almost identical to that found in fresh normal human serum or plasma, suggesting that a distinct distribution of oligomers exists in blood that provides the C system with an apparently advantageous range of specific activities.

17ß-Estradiol and progesterone exert a number of physiological effects throughout the brain due to interactions with several types of receptors belonging to the traditional family of intracellular hormonal receptors as well as to membrane-bound receptors. In particular, both hormones elicit rapid modifications of neuronal excitability that have been postulated to underlie their effects on synaptic plasticity and learning and memory. Likewise, both hormones have been shown to be neuroprotective under certain conditions, possibly due to the activation of pro-survival pathways and the inhibition of pro-apoptotic cascades. Because of the similarities in their cellular effects, there have been a number of questions raised by numerous observations that progesterone inhibits the effects of estrogen. In this manuscript, we first review the interactions between 17ß-estradiol (E2) and progesterone (P4) in synaptic plasticity, and conclude that, while E2 exerts a clear and important role in long-term potentiation of synaptic transmission in hippocampal neurons, the role of P4 is much less clear, and could be accounted by the direct or indirect regulation of GABAA receptors. We then discuss the neuroprotective roles of both hormones, in particular against excitotoxicity. In this case, the neuroprotective effects of these hormones are very similar to those of the neurotrophic factor BDNF. Interestingly, P4 antagonizes the effects of E2, possibly through the regulation of estrogen receptors or of proteins associated with the receptors or interactions with signaling pathways activated by E2. Overall, this review emphasizes the existence of common molecules and pathways that participate in the regulation of both synaptic plasticity and neurodegeneration.

Hashimoto's thyroiditis (HT) is associated with HLA, but the associated allele is still controversial. We hypothesized that specific HLA-DR pocket-sequence variants are associated with HT and that similar variants in the murine I-E locus (homologous to HLA-DR) predispose to experimental autoimmune thyroiditis (EAT), a classical mouse model of HT. Therefore, we sequenced the polymorphic exon 2 of the HLA-DR gene in 94 HT patients and 149 controls. In addition, we sequenced exon 2 of the I-E gene in 22 strains of mice, 12 susceptible to EAT and 10 resistant. Using logistic regression analysis, we identified a pocket amino acid signature, Tyr-26, Tyr-30, Gln-70, Lys-71, strongly associated with HT (P = 6.18 x 10(-5), OR = 3.73). Lys-71 showed the strongest association (P = 1.7 x 10(-8), OR = 2.98). This association was seen across HLA-DR types. The 5-aa haplotype Tyr-26, Tyr-30, Gln-70, Lys-71, Arg-74 also was associated with HT (P = 3.66 x 10(-4)). In mice, the I-E pocket amino acids Val-28, Phe-86, and Asn-88 were strongly associated with EAT. Structural modeling studies demonstrated that pocket P4 was critical for the development of HT, and pockets P1 and P4 influenced susceptibility to EAT. Surprisingly, the structures of the HT- and EAT-susceptible pockets were different. We conclude that specific MHC II pocket amino acid signatures determine susceptibility to HT and EAT by causing structural changes in peptide-binding pockets that may influence peptide binding, selectivity, and presentation. Because the HT- and EAT-associated pockets are structurally different, it is likely that distinct antigenic peptides are associated with HT and EAT.

BACKGROUND

Progesterone (P4) inhibits human granulosa/luteal cell apoptosis by an unknown mechanism.

OBJECTIVE

Our objective was to assess the role of the nuclear P4 receptor (PGR) and PGR membrane component 1 (PGRMC1) in mediating P4's antiapoptotic action in human granulosa/luteal cells.

METHODS

In vitro laboratory studies were designed in which human granulosa/luteal cells were harvested from in vitro fertilization patients from 2004-2006.

METHODS

Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays and DNA staining. Protein expression was observed by Western blot and immunocytochemistry.

RESULTS

PGR was detected in 20% of the human granulosa/luteal cells, and 25 and 50 microM RU486 induced at least 70% of the cells to undergo apoptosis. Five micromolar RU486 neither induced apoptosis nor attenuated the antiapoptotic action of 1 microM P4. PGRMC1 and its binding partner, plasminogen activator inhibitor RNA-binding protein-1 (PAIRBP1), were detected in human granulosa/luteal cells. Antibodies to either PGRMC1 or PAIRBP1 completely attenuated P4's action.

CONCLUSIONS

PGR does not exclusively mediate P4's action because 1) 5 microM RU486 should have been able to override the antiapoptotic action of 1 microM P4 because RU486 binds to the PGR at a greater affinity than P4; 2) 25 and 50 microM RU486 induce three to four times more cells to undergo apoptosis than express PGR; 3) P4 must be continuously present to prevent apoptosis, which implies a rapid, possibly membrane-initiated mechanism of action; and 4) expression and blocking antibody studies suggest that PGRMC1 and PAIRBP1 account in part for P4's action in human granulosa/luteal cells.

Progesterone receptor membrane component-1 (PGRMC1) is present in both the cytoplasm and nucleus of spontaneously immortalized granulosa cells (SIGCs). PGRMC1 is detected as a monomer in the cytoplasm and a DTT-resistant PGRMC1 dimer in the nucleus. Transfected PGRMC1-GFP localizes mainly to the cytoplasm and does not form a DTT-resistant dimer. Moreover, forced expression of PGRMC1-GFP increases the sensitivity of the SIGCs to progesterone (P4)'s anti-apoptotic action, indicating that the PGRMC1 monomer is functional. However, when endogenous PGRMC1 is depleted by siRNA treatment and replaced with PGRMC1-GFP, P4 responsiveness is not enhanced, although overall levels of PGRMC1 are increased. P4's anti-apoptotic action is also attenuated by actinomycin D, an inhibitor of RNA synthesis, and P4 activation of PGRMC1 suppresses Bad and increases Bcl2a1d expression. Taken together, the present studies suggest a genomic component to PGRMC1's anti-apoptotic mechanism of action, which requires the presence of the PGRMC1 dimer.

The solute carrier family 10 (SLC10) comprises two sodium-dependent bile acid transporters, i.e. the Na(+)/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2). These carriers are essentially involved in the maintenance of the enterohepatic circulation of bile acids mediating the first step of active bile acid transport through the membrane barriers in the liver (NTCP) and intestine (ASBT). Recently, four new members of the SLC10 family were described and referred to as P3 (SLC10A3), P4 (SLC10A4), P5 (SLC10A5) and sodium-dependent organic anion transporter (SOAT; SLC10A6). Experimental data supporting carrier function of P3, P4, and P5 is currently not available. However, as demonstrated for SOAT, not all members of the SLC10 family are bile acid transporters. SOAT specifically transports steroid sulfates such as oestrone-3-sulfate and dehydroepiandrosterone sulfate in a sodium-dependent manner, and is considered to play an important role for the cellular delivery of these prohormones in testes, placenta, adrenal gland and probably other peripheral tissues. ASBT and SOAT are the most homologous members of the SLC10 family, with high sequence similarity ( approximately 70%) and almost identical gene structures. Phylogenetic analyses of the SLC10 family revealed that ASBT and SOAT genes emerged from a common ancestor gene. Structure-activity relationships of NTCP, ASBT and SOAT are discussed at the amino acid sequence level. Based on the high structural homology between ASBT and SOAT, pharmacological inhibitors of the ASBT, which are currently being tested in clinical trials for cholesterol-lowering therapy, should be evaluated for their cross-reactivity with SOAT.

The term P4 medicine is used to denote an evolving field of medicine that uses systems biology approaches and information technologies to enhance wellness rather than just treat disease. Its four components include predictive, preventive, personalized, and participatory medicine. In the current paper, it is argued that in order to fulfill the promise of P4 medicine, a "fifth P" must be integrated-the population perspective-into each of the other four components. A population perspective integrates predictive medicine into the ecologic model of health; applies principles of population screening to preventive medicine; uses evidence-based practice to personalize medicine; and grounds participatory medicine on the three core functions of public health: assessment, policy development, and assurance. Population sciences-including epidemiology; behavioral, social, and communication sciences; and health economics, implementation science, and outcomes research-are needed to show the value of P4 medicine. Balanced strategies that implement both population- and individual-level interventions can best maximize health benefits, minimize harm, and avoid unnecessary healthcare costs.

Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic β-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4' to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease.

We have cloned cDNAs coding for human type 2 and type 3 and part of type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs). The complete nucleotide sequences for type 2 and type 3 receptors were determined and the pharmacological properties of the latter were characterized. Human type 2 and type 3 IP3Rs are 2701 amino acids and 2671 amino acids long, respectively, and have significant sequence homologies as well as structural similarities including the six membrane-spanning regions near the C-termini when compared with the rat or mouse counterpart. COS-7 cells transfected with human type 3 IP3R showed characteristic inositol 1,4,5-trisphosphate (IP3)-binding properties with Kd values of 28.8 nM. The order of potency of competition with IP3 was Ins(1,4,5)P3 (IP3)>> Ins(2,4,5)P3>> Ins(1,3,4,5)P4>> Ins(1,2,3,4,5,6)P6. Type 2 and type 3 IP3Rs were mapped to human chromosomes 12p11 and 6p21, respectively, by in situ hybridization. cDNA cloning of the human IP3Rs allowed us to identify the types of the receptor expressed in various human hematopoietic and lymphoma cell lines. The type 3 receptor was present in all of cell lines tested, while the type 1 or 2 receptor was expressed in only particular cell types. The differential expression of the IP3R types could confer the cell-specific regulation on the IP3/Ca2+ signalling.

alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.

Synaptogenesis during early development is thought to follow a canonical program whereby synapses increase rapidly in number and individual axons multiply-innervate nearby targets. Typically, a subset of inputs then out-competes all others through experience-driven processes to establish stable, long-lasting contacts. We investigated the formation of the calyx of Held, probably the largest nerve terminal in the mammalian CNS. Many basic functional and morphological features of calyx growth have not been studied previously, including whether mono-innervation, a hallmark of this system in adult animals, is established early in development. Evoked postsynaptic currents, recorded from neonatal mice between postnatal day 1 (P1) and P4, increased dramatically from -0.14 +/- 0.04 nA at P1 to -6.71 +/- 0.65 nA at P4 with sharp jumps between P2 and P4. These are the first functional assays of these nascent synapses for ages less than P3. AMPA and NMDA receptor-mediated currents were prominent across this age range. Electron microscopy (EM) revealed a concomitant increase, beginning at P2, in the prevalence of postsynaptic densities (16-fold) and adhering contacts (73-fold) by P4. Therefore, both functional and structural data showed that young calyces could form within 2 d, well before the onset of hearing around P8. Convergence of developing calyces onto postsynaptic targets, indicative of competitive processes that precede mono-innervation, was rare (4 of 29) at P4 as assessed using minimal stimulation electrophysiology protocols. Serial EM sectioning through 19 P4 cells further established the paucity (2 of 19) of convergence. These data indicate that calyces of Held follow a noncanonical program to establish targeted innervation that occurs over a rapid time course and precedes auditory experience.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3]. In order to study the effects of PtdIns(3,4,5)P3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P3, inositol 1, 3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, 3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 283+/-32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.

Protein-disulfide isomerase (PDI) has been shown to be a multifunctional enzyme catalyzing the formation of disulfide bonds, as well as being a component of the enzymes prolyl 4-hydroxylase (P4-H) and microsomal triglyceride transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate the role of this multifunctional protein within a cellular context, we have established a semi-permeabilized cell system that reconstitutes the synthesis, folding, modification, and assembly of procollagen as they would occur in the cell. We demonstrate here that P4-H associates transiently with the triple helical domain during the assembly of procollagen. The release of P4-H from the triple helical domain coincides with assembly into a thermally stable triple helix. However, if triple helix formation is prevented, P4-H remains associated, suggesting a role for this enzyme in preventing aggregation of this domain. We also show that PDI associates independently with the C-propeptide of monomeric procollagen chains prior to trimer formation, indicating a role for this protein in coordinating the assembly of heterotrimeric molecules. This demonstrates that PDI has multiple functions in the folding of the same protein, that is, as a catalyst for disulfide bond formation, as a subunit of P4-H during proline hydroxylation, and independently as a molecular chaperone during chain assembly.

By equilibrium dialysis a disadenosine 5',5'''-P1,P2-tetraphosphate (Ap4A) binding activity is shown to be present in mammalian cells. The Ap4A binding activity copurifies with DNA polymerase alpha during the isolation procedure, which includes chromatography on phospho-, DEAE-, and DNA-cellulose; gel filtration; sucrose gradient centrifugation; and electrophoresis in nondenaturing polyacrylamide gels. After these purification steps, DNA polymerase alpha appears to be homogeneous in nondenaturing polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of such a purified DNA polymerase alpha preparation reveals seven distinct protein bands with apparent Mrs of 64,000, 63,000, 62,000, 60,000, 57,000, 55,000, and 52,000. By affinity labeling, the protein with Mr 57,000 has been shown to be the Ap4A-binding constituent of DNA polymerase alpha. The binding activity of DNA polymerase alpha for Ap4A is highly specific because neither structural analogs nor several other adenine nucleotides compete effectively with Ap4A for its binding site. The Ap4A binding site is lost in neuronal cells during maturation of rat brains concomitantly with the loss of DNA polymerase alpha and mitotic activity in those cells. From these results, DNA polymerase seems to be the intracellular target of Ap4A. This is discussed in respect to the recently reported of Ap4A to trigger DNA replication in quiescent mammalian cells [Grummt, F. (1978) Proc. Natl. Acad. Sci. USA 75, 371-375].

Some strains of Escherichia coli contain retroelements (retrons) that encode genes for reverse transcriptase and branched, multicopy, single-stranded DNA (msDNA) linked to RNA. However, the origin of retrons is unknown. A P4-like cryptic prophage was found that contains a retroelement (retron Ec73) for msDNA-Ec73 in an E. coli clinical strain. The entire genome of this prophage, named phi R73, is 12.7 kilobase pairs and is flanked by 29-base pair direct repeats derived from the 3' end of the selenocystyl transfer RNA gene (selC). P2 bacteriophage caused excision of the phi R73 prophage and acted as a helper to package phi R73 DNA into an infectious virion. The newly formed phi R73 closely resembled P4 as a virion and in its lytic growth. Retronphage phi R73 lysogenized a new host strain, reintegrating its genome into the selC gene of the host chromosome and enabling the newly formed lysogens to produce msDNA-Ec73. Hence, retron Ec73 can be transferred intercellularly as part of the genome of a helper-dependent retronphage.

A model for the kinetic folding pathway of the Tetrahymena ribozyme has been proposed where the two main structural domains, P4-P6 and P3-P7, form in a hierarchical manner with P4-P6 forming first and P3-P7 folding on the minute timescale. Recent studies in our laboratory identified a set of mutations that accelerate P3-P7 formation, and all of these mutations appear to destabilize a native-like kinetic trap. To better understand the microscopic details of this slow step in the Tetrahymena ribozyme folding pathway, we have used a previously developed kinetic oligonucleotide hybridization assay to characterize the folding of several fast folding mutants. A comparison of the temperature dependence of P3-P7 folding between the mutant and wild-type ribozymes demonstrates that a majority of the mutations act by decreasing the activation enthalpy required to reach the transition state and supports the existence of the native-like kinetic trap. In several mutant ribozymes, P3-P7 folds with biphasic kinetics, indicating that only a subpopulation of molecules can evade the kinetic barrier. The rate of folding of the wild-type increases in the presence of urea, while for the mutants urea merely shifts the distribution between the two folding populations. Small structural changes or changes in solvent can accelerate folding, but these changes lead to complex folding behavior, and do not give rise to rapid two-state folding transitions. These results support the recent view of folding as an ensemble of molecules traversing a rugged energy landscape to reach the lowest energy state.

Due to the many similarities in mechanisms of action, targets and effects, progesterone (P4), estrogen and neurotrophins have been implicated in synaptic plasticity as well as in neuroprotection and neurodegeneration. In this study, we examined the interactions between 17beta-estradiol (E2) and P4 and brain-derived neurotrophic factor (BDNF) on both plasticity and excitotoxicity in rat cultured hippocampal slices. First, we evaluated the neuroprotective effects of E2 and P4 against N-methyl-D-aspartate (NMDA) toxicity in cultured rat hippocampal slices. As previously reported, pretreatment with 10 nm E2 (24 h) was neuroprotective against NMDA toxicity. However, P4 (10 nm) added 20 h after E2 treatment for 4 h reversed its protective effect. In addition, the same E2 treatment resulted in an increase in BDNF protein levels as well as in activation of its receptor, TrkB, while addition of P4 attenuated E2-mediated increase in BDNF and TrkB levels. Furthermore, E2-mediated neuroprotection was eliminated by a BDNF scavenger, TrkB-Fc. Our results indicate that E2 neuroprotective effects are mediated through the BDNF pathway and that, under certain conditions, P4 antagonizes the protective effect of estrogen.

During induction of the Caenorhabditis elegans hermaphrodite vulva, a signal from the anchor cell activates the LET-23 epidermal growth factor receptor (EGFR)/LET-60 Ras/MPK-1 MAP kinase signaling pathway in the vulval precursor cells. We have characterized two mechanisms that limit the extent of vulval induction. First, we found that gap-1 may directly inhibit the LET-60 Ras signaling pathway. We identified the gap-1 gene in a genetic screen for inhibitors of vulval induction. gap-1 is predicted to encode a protein similar to GTPase-activating proteins that likely functions to inhibit the signaling activity of LET-60 Ras. A loss-of-function mutation in gap-1 suppresses the vulvaless phenotype of mutations in the let-60 ras signaling pathway, but a gap-1 single mutant does not exhibit excess vulval induction. Second, we found that let-23 EGFR prevents vulval induction in a cell-nonautonomous manner, in addition to its cell-autonomous role in activating the let-60 ras/mpk-1 signaling pathway. Using genetic mosaic analysis, we show that let-23 activity in the vulval precursor cell closest to the anchor cell (P6.p) prevents induction of vulval precursor cells further away from the anchor cell (P3.p, P4.p, and P8.p). This result suggests that LET-23 in proximal vulval precursor cells might bind and sequester the inductive signal LIN-3 EGF, thereby preventing diffusion of the inductive signal to distal vulval precursor cells.

CsrA of Escherichia coli is an RNA-binding protein that globally regulates gene expression by repressing translation and/or altering the stability of target transcripts. Here we explored mechanisms that control csrA expression. Four CsrA binding sites were predicted upstream of the csrA initiation codon, one of which overlapped its Shine-Dalgarno sequence. Results from gel shift, footprint, toeprint and in vitro translation experiments indicate that CsrA binds to these four sites and represses its own translation by directly competing with 30S ribosomal subunit binding. Experiments were also performed to examine transcription of csrA. Primer extension, in vitro transcription and in vivo expression studies identified two σ⁷⁰-dependent (P2 and P5) and two σ(S) -dependent (P1 and P3) promoters that drive transcription of csrA. Additional primer extension studies identified a fifth csrA promoter (P4). Transcription from P3, which is indirectly activated by CsrA, is primarily responsible for increased csrA expression as cells transition from exponential to stationary-phase growth. Taken together, our results indicate that regulation of csrA expression occurs by a variety of mechanisms, including transcription from multiple promoters by two sigma factors, indirect activation of its own transcription, as well as direct repression of its own translation.

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating strategies against the transmission of the virus. A chimeric simian/human immunodeficiency virus (SHIV), SHIV(CHN19), was generated with a primary, non-syncytium-inducing HIV-1 subtype C envelope from a Chinese strain in the background of SHIV(33). Unlike R5-tropic SHIV(162), SHIV(CHN19) was not found to replicate in rhesus CD4(+) T lymphocytes. SHIV(CHN19) does, however, replicate in CD4(+) T lymphocytes of pig-tailed macaques (Macaca nemestrina). The observed replication competence of SHIV(CHN19) requires the full tat/rev genes and partial gp41 region derived from SHIV(33). To evaluate in vivo infectivity, SHIV(CHN19) was intravenously inoculated, at first, into two pig-tailed and two rhesus macaques. Although all four animals became infected, the virus replicated preferentially in pig-tailed macaques with an earlier plasma viral peak and a faster seroconversion. To determine whether in vivo adaptation would enhance the infectivity of SHIV(CHN19), passages were carried out serially in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. The passages greatly enhanced the infectivity of the virus as shown by the increasingly elevated viral loads during acute infection in animals with each passage. Moreover, the doubling time of plasma virus during acute infection became much shorter in passage 4 (P4) animals (0.2 day) in comparison to P1 animals (1 to 2 days). P2 to P4 animals all became seropositive around 2 to 3 weeks postinoculation and had a decline in CD4/CD8 T-cell ratio during the early phase of infection. In P4 animals, a profound depletion of CD4 T cells in the lamina propria of the jejunum was observed. Persistent plasma viremia has been found in most of the infected animals with sustained viral loads ranging from 10(3) to 10(5) per ml up to 6 months postinfection. Serial passages did not change the viral phenotype as confirmed by the persistence of the R5 tropism of SHIV(CHN19) isolated from P4 animals. In addition, the infectivity of SHIV(CHN19) in rhesus peripheral blood mononuclear cells was also increased after in vivo passages. Our data indicate that SHIV(CHN19) has adapted well to grow in macaque cells. This established R5-tropic SHIV(CHN19)/macaque model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.

The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.