Circulating antiplasmin-cleaving enzyme (APCE) has a role in fibrinolysis and appears structurally similar to fibroblast activation protein (FAP), a cell-surface proteinase that promotes invasiveness of certain epithelial cancers. To explore this potential relationship, we performed comparative structure/function analyses of the 2 enzymes. APCE from human plasma and recombinant FAP (rFAP) exhibited identical pH optima of 7.5, extinction coefficients (in(280 nm)(1%)) of 20.2 and 20.5, common sequences of tryptic peptides, and cross-reactivity with FAP antibody. APCE and rFAP are homodimers with monomeric subunits of 97 and 93 kDa. Only homodimers appear to have enzymatic activity, with essentially identical kinetics toward Met-alpha2-antiplasmin (Met-alpha2AP) and peptide substrates. APCE and rFAP cleave both Pro3-Leu4 and Pro12-Asn13 bonds of Met-alpha2AP, but relative kcat/Km values for Pro12-Asn13 are about 16-fold higher than for Pro3-Leu4. APCE and rFAP demonstrate higher kcat/Km values toward a peptide modeled on P4-P4' sequence surrounding the Pro12-Asn13 primary cleavage site than for Z-Gly-Pro-AMC and Ala-Pro-AFC substrates. These data support APCE as a soluble derivative of FAP and Met-alpha2AP as its physiologic substrate. Conversion of Met-alpha2AP by membrane or soluble FAP to the more easily fibrin-incorporable form, Asn-alpha2AP, may increase plasmin inhibition within fibrin surrounding certain neoplasms and have an impact on growth and therapeutic susceptibility.

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.

Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P(4)-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P(4)-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P(4)-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Although a definite diagnosis is only possible by necropsy, a differential diagnosis with other types of dementia and with major depression should be attempted. The aim of this study was to analyse the electroencephalogram (EEG) background activity of AD patients to test the hypothesis that the regularity of the AD patients' EEG is higher than that of age-matched controls. We recorded the EEG from 19 scalp electrodes in 11 AD patients and 11 age-matched controls. Two different methods were used to estimate the regularity of the EEG background activity: spectral entropy (SpecEn) and sample entropy (SampEn). We did not find significant differences between AD patients and control subjects' EEGs with SpecEn. On the other hand, AD patients had significantly lower SampEn values than control subjects (p < 0.01) at electrodes P3, P4, O1 and O2. Our results show an increase of EEG regularity in AD patients. These findings suggest that nonlinear analysis of the EEG with SampEn could yield essential information and may contribute to increasing the insight into brain dysfunction in AD in ways which are not possible with more classical and conventional statistical methods.

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.

Chronic obstructive pulmonary disease (COPD) is a complex disease at the clinical, cellular, and molecular levels. However, its diagnosis, assessment, and therapeutic management are based almost exclusively on the severity of airflow limitation. A better understanding of the multiple dimensions of COPD and its relationship to other diseases is very relevant and of high current interest. Recent theoretical (scale-free networks), technological (high-throughput technology, biocomputing), and analytical improvements (systems biology) provide tools capable of addressing the complexity of COPD. The information obtained from the integrated use of those techniques will be eventually incorporated into routine clinical practice. This review summarizes our current knowledge in this area and offers an insight into the elements needed to progress toward an integrated, multilevel view of COPD based on the novel scientific strategy of systems biology and its potential clinical derivative, P4 medicine (Personalized, Predictive, Preventive, and Participatory).

Throughout its complex morphogenesis, the vertebrate skull must at once protect the brain and expand to accommodate its growth. A key structural adaptation that allows this dual role is the separation of the bony plates of the skull with sutures, fibrous joints that serve as growth centers and allow the calvarial bones to expand as the brain enlarges. Craniosynostosis, the premature fusion of one or more calvarial bones with consequent abnormalities in skull shape, is a common developmental anomaly that disrupts this process. We found previously that a single amino acid substitution in the homeodomain of the human MSX2 gene is associated with the autosomal dominant disorder craniosynostosis, Boston type. This mutation enhances the affinity of Msx2 for its target sequence, suggesting that the mutation acts by a dominant positive mechanism. Consistent with this prediction, we showed that general overexpression of Msx2 under the control of the broadly expressed CMV promoter causes the calvarial bones to invade the sagittal suture. Here we use tissue-specific overexpression of Msx2 within the calvarial sutures to address the developmental mechanisms of craniosynostosis and skull morphogenesis. We demonstrate that a segment of the Msx2 promoter directs reporter gene expression to subsets of cells within the sutures. In late embryonic and neonatal stages, this promoter is expressed in undifferentiated mesenchymal cells medial to the growing bone. By P4, promoter activity is reduced in the suture, exhibiting a punctate pattern in undifferentiated osteoblastic cells in the outer margin of the osteogenic front. Overexpression of Msx2 under the control of this promoter is sufficient to enhance parietal bone growth into the sagittal suture by P6. This phenotype is preceded by an increase in both the number and the BrdU labeling of osteoblastic cells in the osteogenic fronts of the calvarial bones. These findings suggest that an important early event in MSX2-mediated craniosynostosis in humans is a transient retardation of osteogenic cell differentiation in the suture and a consequent increase in the pool of osteogenic cells.

The dynamics of ovarian and pituitary hormone changes during the midcycle period were evaluated. Changes in hormone levels were determined at 2-h intervals for 5 consecutive days during the periovulatory phase of the cycle in five women. During the 50 h preceding the onset of the surge, the rates of increments for estradiol (E2), progesterone (P4), and LH were similar, with doubling times of 57-61 h. The onset of LH and FSH surges was found to occur abruptly (LH doubled within 2 h). They were temporally associated with the attainment of peak E2 levels and occurred 12 h after the initiation of a rapid rise of P4. The mean duration of the surge was 48 h, with a rapidly ascending limb (doubling time, 5.2 h) lasting 14 h accompanied by a rapid decline of E2 and a continued rise of P4. The surge was followed by a peak plateau of gonadotropin levels lasting for 14 h and a transient leveling of P4. The longer descending limb (half-time, 9.6 h), lasting for 20 h, was associated with a second rapid rise of P4, beginning 36 h after surge onset or 12 h before termination of the surge. By using the onset of the LH surge as a reference point, our data provide a relatively precise picture of the hormonal changes preceding the onset of the gonadotropin surge and the temporal relationship between the multiphasic P4 rise and pituitary-ovarian function.

Identification of mechanisms underlying endometriosis pathogenesis will facilitate understanding and treatment of infertility and pain associated with this disorder. Herein, we investigated the expression of steroidogenic pathway enzymes and key decidualization biomarkers in endometrial tissue and in eutopic endometrial stromal fibroblasts (hESFs) from women with vs. those without endometriosis, and subsequently treated in vitro with 8-bromo-cAMP (8-Br-cAMP) or progesterone (P4). Real-time quantitative PCR, immunohistochemistry, ELISA, and radiometric aromatase activity assay were used. The results demonstrate significantly increased (14.5-fold; P=0.037) expression of aromatase in eutopic endometrium of women with disease. In 8-Br-cAMP-treated hESF from eutopic endometrium of women with endometriosis, the balance in estradiol (E2) and P4 biosynthetic and metabolizing enzymes is disturbed (decreased HSD3B1 and HSD17B2, and increased HSD17B1 and aromatase), with the equilibrium being shifted towards an E2-enriched milieu. However, hESF from the same group of women treated with P4 did not demonstrate such responsiveness. Lower expression of IGFBP1 and prolactin mRNA and protein was observed in hESF from women with vs. those without endometriosis in response to 8-Br-cAMP, but not P4, suggesting a blunted response of these decidual biomarkers to activation of the PKA pathway in eutopic endometrium in women with disease. The dichotomy of 8-Br-cAMP regulation of select steroidogenic enzymes leading to an enriched E2 milieu within the endometrium and a blunted response of decidual biomarkers to this decidualizing agent of hESF from women with endometriosis suggests resistance to full decidualization of the stromal fibroblasts and mechanisms underlying implantation failure and the pathophysiology of this disorder.

The folding and catalytic function of RNA molecules depend on their interactions with divalent metal ions, such as magnesium. As with every molecular process, the most basic knowledge required for understanding the close relationship of an RNA with its metal ions is the stoichiometry of the interaction. Unfortunately, inventories of the numbers of divalent ions associated with unfolded and folded RNA states have been unattainable. A common approach has been to interpret Hill coefficients fit to folding equilibria as the number of metal ions bound upon folding. However, this approach is vitiated by the presence of diffusely associated divalent ions in a dynamic ion atmosphere and by the likelihood of multiple transitions along a folding pathway. We demonstrate that the use of molar concentrations of background monovalent salt can alleviate these complications. These simplifying solution conditions allow a precise determination of the stoichiometry of the magnesium ions involved in folding the metal ion core of the P4-P6 domain of the Tetrahymena group I ribozyme. Hill analysis of hydroxyl radical footprinting data suggests that the P4-P6 RNA core folds cooperatively upon the association of two metal ions. This unexpectedly small stoichiometry is strongly supported by counting magnesium ions associated with the P4-P6 RNA via fluorescence titration and atomic emission spectroscopy. By pinpointing the metal ion stoichiometry, these measurements provide a critical but previously missing step in the thermodynamic dissection of the coupling between metal ion binding and RNA folding.

Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349-34358), later termed "glycosynapse 3" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin alpha3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the alpha3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of alpha3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.

Iron uptake systems which are critical for bacterial survival and which may play important roles in bacterial virulence are often carried on mobile elements, such as plasmids and pathogenicity islands (PAIs). In the present study, we identified and characterized a ferric dicitrate uptake system (Fec) in Shigella flexneri serotype 2a that is encoded by a novel PAI termed the Shigella resistance locus (SRL) PAI. The fec genes are transcribed in S. flexneri, and complementation of a fec deletion in Escherichia coli demonstrated that they are functional. However, insertional inactivation of fecI, leading to a loss in fec gene expression, did not impair the growth of the parent strain of S. flexneri in iron-limited culture media, suggesting that S. flexneri carries additional iron uptake systems capable of compensating for the loss of Fec-mediated iron uptake. DNA sequence analysis showed that the fec genes are linked to a cluster of multiple antibiotic resistance determinants, designated the SRL, on the chromosome of S. flexneri 2a. Both the SRL and fec loci are carried on the 66,257-bp SRL PAI, which has integrated into the serX tRNA gene and which carries at least 22 prophage-related open reading frames, including one for a P4-like integrase. This is the first example of a PAI that carries genes encoding antibiotic resistance and the first report of a ferric dicitrate uptake system in Shigella.

We have identified and purified six subforms of peroxisomes, designated P1 to P6, from the yeast, Yarrowia lipolytica. An analysis of trafficking of peroxisomal proteins in vivo suggests the existence of a multistep peroxisome assembly pathway in Y. lipolytica. This pathway operates by conversion of peroxisomal subforms in the direction P1, P2->>P3->>P4->>P5->>P6 and involves the import of various peroxisomal proteins into distinct vesicular intermediates. We have also reconstituted in vitro the fusion of the earliest intermediates in the pathway, small peroxisomal vesicles P1 and P2. Their fusion leads to the formation of a larger and more dense peroxisomal vesicle, P3. Fusion of P1 and P2 in vitro requires cytosol and ATP hydrolysis and is inhibited by antibodies to two membrane-associated ATPases of the AAA family, Pex1p and Pex6p. We provide evidence that the fusion in vitro of P1 and P2 peroxisomes reconstructs an actual early step in the peroxisome assembly pathway operating in vivo in Y. lipolytica.

The metalloprotease ADAMTS13 efficiently cleaves only the Tyr(1605)-Met(1606) bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal alpha-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln(1624) and Arg(1668), and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln(1624)-Arg(1641) minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4'-P18' residues on either side of the Tyr(1605)-Met(1606) bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.

The biological behaviors of ribozymes, riboswitches, and numerous other functional RNA molecules are critically dependent on their tertiary folding and their ability to sample multiple functional states. The conformational heterogeneity and partially folded nature of most of these states has rendered their characterization by high-resolution structural approaches difficult or even intractable. Here we introduce a method to rapidly infer the tertiary helical arrangements of large RNA molecules in their native and non-native solution states. Multiplexed hydroxyl radical (.OH) cleavage analysis (MOHCA) enables the high-throughput detection of numerous pairs of contacting residues via random incorporation of radical cleavage agents followed by two-dimensional gel electrophoresis. We validated this technology by recapitulating the unfolded and native states of a well studied model RNA, the P4-P6 domain of the Tetrahymena ribozyme, at subhelical resolution. We then applied MOHCA to a recently discovered third state of the P4-P6 RNA that is stabilized by high concentrations of monovalent salt and whose partial order precludes conventional techniques for structure determination. The three-dimensional portrait of a compact, non-native RNA state reveals a well ordered subset of native tertiary contacts, in contrast to the dynamic but otherwise similar molten globule states of proteins. With its applicability to nearly any solution state, we expect MOHCA to be a powerful tool for illuminating the many functional structures of large RNA molecules and RNA/protein complexes.

The development of the chamber angle was studied in the eyes of heterozygous Pax6(lacZ/+) mutant mice (Nature 387 (1997) 406). Mutations in PAX6 cause aniridia, a condition that is frequently associated with glaucoma, a blinding disease that may be associated with chamber angle defects. Mesenchymal cells were seen in the chamber angle at P1-P5. In wild-type mice, these cells differentiated into typical trabecular meshwork (TM) cells next to Schlemm's canal. In Pax6(lacZ/+) mice, TM cells remained undifferentiated and Schlemm's canal was absent. From P1 to P4, staining for beta-galactosidase and immunoreactivity for Pax6 were observed in chamber angle mesenchyme, but were absent later. Cultured murine TM cells expressed Pax6. The defects in chamber angle and TM differentiation were associated with a wide spectrum of other anterior eye defects, which included various degrees of iris hypoplasia and corneal haze, isolated iridocorneal adhesions and atypical coloboma, and a vascularized cornea in all adult animals. A third of the animals showed Peters' anomaly including corneal opacity and iridocorneal adhesions. The separation of the lens from the cornea was incomplete, and epithelial layers of lens and cornea were continuous. Pax6 activity is directly required for differentiation of the chamber angle. Variations in phenotype of Pax6(lacZ/+) mice appear not to involve direct dominant-negative or dose-dependent effects.

The solution structure of a 22 nt RNA hairpin and its complex with Co(NH(3))(6)(3+) bound to the GAAA tetraloop has been determined by NMR spectroscopy. Co(NH(3))(6)(3+) has a similar geometry to Mg(H(2)O)(6)(2+) and can be used as a probe for binding sites of completely solvated magnesium ions. The hairpin contains tandem G.A mismatches, similar to the P5abc region of a group I intron, and is closed by a GAAA tetraloop. The tandem G.A mismatches are imino hydrogen bonded in contrast with the sheared G.A mismatches found in a different context in the crystal structure of the P4-P6 domain. Chemical shift changes of the imino protons upon titration of the RNA hairpin with Mg(2+) and with Co(NH(3))(6)(3+) were used to identify ion-binding sites. Paramagnetic resonance broadening upon titration with Mn(2+) was also used. The titration curves gave dissociation binding constants for the magnesium ions in the millimolar range, similar to the binding in the major groove of RNA at tandem G.U base-pairs. Although the largest chemical shift change occurred at an imino proton of one of the G.A base-pairs, no nuclear Overhauser enhancement cross-peaks between the cobalt ligand and neighboring RNA protons were seen, presumably due to the high mobility of the Co(NH(3))(6)(3+) at this site. Nuclear Overhauser enhancement cross-peaks between Co(NH(3))(6)(3+) and the GAAA tetraloop were observed, which allowed the determination of the structure of the tetraloop binding site. The Co(NH(3))(6)(3+) is bound in the major groove of the GAAA tetraloop with hydrogen bonds to guanine base N7 and to phosphate oxygen atoms of the tetraloop.

Large excitatory synapses in the auditory system, such as the calyx of Held, faithfully transmit trains of action potentials up to a frequency of a few hundred hertz, and these synapses are thought to display a limited repertoire of synaptic plasticity. Here, we show that brief trains of 100 Hz stimulation induce posttetanic potentiation (PTP) of transmitter release at the calyx of Held. In young rats [postnatal day 4 (P4) to P6], PTP could be induced with shorter 100 Hz trains compared with older age groups (P8-P10 and P12-P14), but the maximal amount of PTP was similar, with 200% of control EPSC amplitude. The size of the readily releasable pool of vesicles was not increased significantly during PTP. Bath application of the membrane-permeable Ca2+ chelator EGTA-AM suppressed PTP, indicating a role for presynaptic Ca2+ in PTP at the calyx of Held. Presynaptic Ca2+ imaging showed that the intracellular Ca2+ concentration, [Ca2+]i, was increased by 40-120 nM at the peak of PTP, and this "residual" [Ca2+]i decayed in parallel with PTP, with time constants in the range of 10-60 s. During whole-cell recording of the presynaptic calyx of Held, PTP was absent, and the decay of residual [Ca2+]i was strongly accelerated. The data show that the calyx of Held expresses a mechanism of transmitter release potentiation in which a small, sustained elevation of basal [Ca2+]i increases the transmitter release probability after trains of high-frequency stimulation.

Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a kcat/KM of 4.6x10(5) M-1 s-1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin.

Pseudomonas sp. strain B13 carries the clcRABDE genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb clc element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (glyV) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (int-B13) was located near the right end of the clc element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa). The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1. Similar to the bacteriophages phiR73 and P4, the clc element integrates into the 3' end of the target tRNA gene. This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence specificity of the integration. In Pseudomonas sp. strain B13, a circular form of the clc element, which carries an 18-bp DNA sequence identical to the 3'-end portion of glyV as part of its attachment site (attP), could be detected. Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstructed at the right end of the element. The integration process could be demonstrated in RecA-deficient Escherichia coli with two recombinant plasmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1.

BACKGROUND

We tested the hypothesis that in newborn rats, sevoflurane may cause seizures, neurotoxicity, and impairment in synaptic plasticity-effects that may be diminished by the Na-K-2Cl cotransporter 1 inhibitor, bumetanide.

METHODS

Electroencephalography, activated caspase-3, and hippocampal long-term potentiation were measured in rats exposed to 2.1% sevoflurane for 0.5-6 h at postnatal days 4-17 (P4-P17).

RESULTS

Arterial blood gas samples drawn at a sevoflurane concentration of 2.1% showed no evidence of either hypoxia or hypoventilation in spontaneously breathing rats. Higher doses of sevoflurane (e.g., 2.9%) caused respiratory depression. During anesthesia maintenance, the electroencephalography exhibited distinctive episodes of epileptic seizures in 40% of P4-P8 rats. Such seizure-like activity was not detected during anesthesia maintenance in P10-P17 rats. Emergence from 3 h of anesthesia with sevoflurane resulted in tonic/clonic seizures in some P10-P17 rats but not in P4-P8 rats. Bumetanide (5 micromol/kg, intraperitoneally) significantly decreased seizures in P4-P9 rats but did not affect the emergence seizures in P10-P17 rats. Anesthesia of P4 rats with sevoflurane for 6 h caused a significant increase in activated caspase-3 and impairment of long-term potentiation induction measured at 1 and 14-17 days after exposure to sevoflurane, respectively. Pretreatment of P4 rats with bumetanide nearly abolished the increase in activated caspase-3 but did not alleviate impairment of long-term potentiation.

CONCLUSIONS

These results support the possibility that excitatory output of sevoflurane-potentiated gamma-aminobutyric acid type A/glycine systems may contribute to epileptogenic and neurotoxic effects in early postnatal rats.

Although the structure of the 20 S proteasome from Thermoplasma acidophilum has been elucidated, its enzymatic properties have not been explored in depth. Thermoplasma proteasomes, which contain one type of active site, exhibit not only "chymotrypsin-like" activity (as reported), but also some "post-glutamyl" and "trypsin-like" activities. Like eukaryotic proteasomes, its activity can be stimulated by SDS, Mg2+, and also guanidine HCl, but not urea. The enzyme was strongly inhibited by novel peptide aldehydes with hydrophobic P4 residues, and was rapidly inactivated by 3, 4-dichloroisocoumarin (DCI). DCI modified the N-terminal threonine of the catalytic beta-subunit, the presumed active site nucleophile. To define how proteins are degraded, casein was derivatized with fluorescein isothiocyanate to facilitate detection of released products by the proteasome. Many fluorescent peptides were generated, but the relative amounts of different peptides were independent of the duration of the reaction. The rate of disappearance of protein substrates paralleled the rate of appearance of small products. Unlike conventional proteases, proteasome degrades proteins processively without release of polypeptide intermediates. Upon activation by SDS, guanidine, heat (55 degrees C), or partial inhibition with DCI, proteasomes still functioned processively, but generated a different pattern of peptides under each condition. Thus, processivity is an inherent feature of the 20 S proteasome, not requiring all active sites or ATP hydrolysis.