Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons.

R483, a plasmid of the Ialpha incompatibility group, contained a deoxyribonucleic acid (DNA) sequence encoding resistance to trimethoprim (TpR) and streptomycin (SmR) that could be transposed to other replicons, i.e., to the Escherichia coli chromosome and to related and unrelated plasmids. Each transposition resulted in the acquisition by the recipient replicon of a segment of DNA of about 9 X 10(6) daltons, both resistance genes, but never the colicin Ia or pilus genes of R483. Transposition took place at a single chromosomal site between dnaA and ilv and did not suppress the DnaA phenotype, in contrast to integration of the whole R483 plasmid. The chromosome, having received the transposition, could secondarily act as a transposition donor to another plasmid. Such a plasmid was indistinguishable from one having received a direct transposition from R483. TpR SmR transposition was very site specific and did not require a functional recA+ gene. We postulate that the TpR SmR segment of R483 is a transposon (TnC) with specific boundary sequences.     

Temperature-dependent changes in hydrogen bonds in cellulose Ialpha studied by infrared spectroscopy in combination with perturbation-correlation moving-window two-dimensional correlation spectroscopy: comparison with cellulose Ibeta

Our recent IR study demonstrated that hydrogen-bond structure in cellulose Ibeta drastically changes around 220 degrees C (Watanabe et al. Biomacromolecules 2006, 7, 3164). In the present study, temperature-dependent IR spectra of cellulose Ialpha from 30 to 260 degrees C were analyzed by use of perturbation-correlation moving-window two-dimensional correlation spectroscopy. It was observed that as in the case of cellulose Ibeta abrupt changes in the hydrogen-bond structure occur around 220 degrees C in cellulose Ialpha. It was also revealed that although weakly hydrogen-bonded OH groups in Ibeta are stable below 230 degrees C thermal oxidation of those in Ialpha is accelerated around 220 degrees C. In this way, the present study has clarified a difference between the thermal behavior of Ialpha and that of Ibeta at the functional group level. Our result suggests that the drastic change in the hydrogen-bond structure around 220 degrees C makes cellulose Ialpha much more unstable than Ibeta.     

Kinetic evidence of an on-pathway intermediate in the folding of lysozyme

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Ialpha) of hen lysozyme with alpha-domain folded and beta-domain unfolded is on the folding pathway under the classical definition. Ialpha folds to the native (N) state directly (unfolded (U) <==> Ialpha <==> N) without having to unfold to U and then refold to N through alternative folding pathways as in Ialpha <==> U <==> N.     

Type Ialpha phosphatidylinositol 4-phosphate 5-kinase is a putative target for increased intracellular phosphatidic acid

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4-phosphate 5-kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5-bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)-tagged type Ialpha PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected PAE cells were stimulated with lyso-PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1-butanol, thereby implicating phospholipase D (PLD) as the in vivo generator of PtdOH. In order to determine if the PtdOH-dependent activation of type Ialpha PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine alpha isoforms of diacylglycerol kinase (DGK) were individually co-transfected along with type Ialpha PIPkin. Under these conditions an increase in type Ialpha PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type Ialpha PIPkin was co-transfected with either the human DGKepsilon isoform or a kinase-dead mutant of the murine DGKalpha isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type Ialpha PIPkin by this lipid second messenger.     

Identification of a novel spliceoform of inositol polyphosphate 4-phosphatase type Ialpha expressed in human platelets: structure of human inositol polyphosphate 4-phosphatase type I gene

Inositol polyphosphate 4-phosphatases (IP4Ps) are enzymes involved in the regulation of phosphoinositide 3-kinase (PI3K) signaling. IP4Ps catalyze the hydrolysis of the D-4 position phosphoester of the PI3K generated lipid second messenger, phosphatidylinositol 3,4-bisphosphate. Western blot analysis detected the expression of a novel 110 kDa form of IP4P type Ialpha in mouse spleen, heart, lung, and uterus. In addition, the 110 kDa form of IP4P type Ialpha was found to be the major form of this enzyme expressed in human platelets, MEG-01 megakaryocytes and Jurkat T-cells. RT-PCR analysis of MEG-01 megakaryocytes and Jurkat T-cells indicates that the 110-kDa form of IP4P Ialpha is derived from an alternatively spliced mRNA that encodes an additional internal domain of 40 amino acids not present in the two previously described brain IP4P Ialpha spliceoforms. The predicted molecular mass of this spliceoform is 109,968 Da, consistent with its apparent molecular mass estimated by Western blot analysis. The novel domain is proline rich and contains a PEST sequence characteristic of proteins that are rapidly degraded by the calpain family of proteases. Analysis of genomic DNA sequence indicates that the IP4P type I gene consists of 25 exons and that this novel spliceoform is obtained as a result of an unusual type of differential splicing involving the use of an alternative 5'-GU donor splice site during the excision of intron 15. In addition, we show that all three known spliceoforms of IP4P Ialpha result from alternative splicing involving exon 15 and 16 indicating that structural variability in this region of the enzyme may be important for its function.     

Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Ialpha and beta

We isolated a constitutively active form of cGMP-dependent protein kinase Ialpha (cGK Ialpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Ialpha, and Ile-78 of cGK Ibeta is also required for the autoinhibitory property. Furthermore, cGK Ialpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.     

Quantitative analysis for the cellulose I alpha crystalline phase in developing wood cell walls

FT-IR and X-ray analyses were employed to determine the relative ratio of cellulose Ialpha and Ibeta crystalline phases present in each developmental stage of coniferous tracheid cell wall formation. The IR spectra showed that initially the Ialpha phase occupies 50% of the crystalline regions in the primary cell wall cellulose and this value drops to 20% after ceasing of the cell enlarging growth for the formation of the secondary wall cellulose (the remaining regions are composed of the Ibeta phase). Although it is reasonable that the content for Ibeta, which is stress-reduced crystalline form, was higher in the secondary wall formation (Kataoka Y, and Kondo T. Macromolecules 1996;29:6356 6358) it is more interesting that during the crystallization of stress-induced Ialpha cellulose for the primary wall the stress-reduced Ibeta, is also possible to be crystallized in an alternative way. This means that throughout the period the Ialpha-causing stress may not be necessarily kept loaded. In light of our previously reported hypothesis (Kataoka Y. and Kondo T. Macromolecules 1998;31:760-764) for the formation of Ialpha phase due to cellular growing stresses in the primary wall cellulose, such an alternating on-off stress effect to account for the occurrence of both Ialpha and Ibeta phases might be related to a biological growth system in coniferous wood cells.     

Structural details of crystalline cellulose from higher plants

It is commonly assumed that cellulose from higher plants contains the Ialpha and Ibeta crystalline allomorphs together with surface and disordered chains. For cellulose Ialpha, the evidence for its presence in higher plants is restricted to the C-4 signals in the solid-state (13)C NMR spectrum, which match those of crystalline cellulose Ialpha from algal sources. Algal cellulose Ialpha can be converted to the Ibeta form by high-temperature annealing. We used this approach to generate cellulose samples differing in Ibeta content from flax fibers and celery collenchyma, which respectively are representative of textile (secondary-wall) and primary-wall cellulose. It was then possible to isolate the detailed spectral contributions of the surface, Ibeta and Ialpha-like phases from linear combinations of the observed (13)C NMR and FTIR spectra. The (13)C NMR spectra resembled those of highly crystalline tunicate or algal cellulose Ibeta and Ialpha, with slight differences implying increased disorder and minor conformational discrepancies. The FTIR spectrum of the Ibeta form was closely similar to its more crystalline counterparts, but the FTIR spectrum of the Ialpha form was not. In addition to increased bandwith indicative of lower order, it showed substantial differences in the profile of hydroxyl stretching bands. These results confirm that higher plants synthesize cellulose Ibeta but show that the Ialpha-like chains, although conformationally quite similar to crystalline algal cellulose Ialpha, sit in a different hydrogen-bonding environment in higher plants. The differences are presumably occasioned by the small diameter of the crystallites and the influence of the crystallite surface on chain packing.     

Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.     

PAP IB, a new member of the Reg gene family: cloning, expression, structural properties, and evolution by gene duplication

Reg proteins are expressed in various organs and are involved in cancers and neurodegenerative diseases. They display a typical C-type lectin-like domain but possess additional highly conserved amino acids. By studying human databases and Expressed Sequence Tags library, we identified a new member called PAP IB. Using probabilistic approaches, we established a phylogenetic tree of eighteen Reg proteins. The dendogram showed that they constitute a superfamily composed of three distinct families (FI to FIII) of paralogues that resulted from duplication. We therefore focused on two proteins, REG Ialpha and PAP IB, belonging to the more closely related FI and FII families, respectively. REG Ialpha and PAP IB share 50% sequence identity. After cloning PAP IB, however, we found that it was expressed almost only in pancreas, unlike REG Ialpha, whose expression is ubiquitous. In addition, by building a model of the structure of PAP IB based on the X-ray structure of REG Ialpha, we observed that the two proteins displayed distinctive surface charge distribution, which may lead to different ligands binding. In spite of their common fold that should result in closely related functions, REG Ialpha and PAP IB are a good example of duplication and divergence, probably with the acquisition of new functions, thus participating in the evolution of the protein repertoire.     

A novel pathway of cellular phosphatidylinositol(3,4,5)-trisphosphate synthesis is regulated by oxidative stress

BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a key second messenger found ubiquitously in higher eukaryotic cells. The activation of Class I phosphoinositide 3-kinases and the subsequent production of PtdIns(3,4,5)P(3) is an important cell signaling event that has been causally linked to the activation of a variety of downstream cellular processes, such as cell migration and proliferation. Although numerous proteins regulating a variety of biological pathways have been shown to bind PtdIns(3,4,5)P(3), there are no data to demonstrate multiple mechanisms for PtdIns(3,4,5)P(3) synthesis in vivo. RESULTS: In this study, we demonstrate an alternative pathway for the in vivo production of PtdIns(3,4,5)P(3) mediated by the action of murine Type Ialpha phosphatidylinositol 4-phosphate 5-kinase (Type Ialpha PIPkinase), an enzyme best characterized as regulating cellular PtdIns(4,5)P(2) levels. Analysis of this novel pathway of PtdIns(3,4,5)P(3) synthesis in cellular membranes leads us to conclude that in vivo, Type Ialpha PIPkinase also acts as a PtdIns(3,4)P(2) 5-kinase. We demonstrate for the first time that cells actually contain an endogenous PtdIns(3,4)P(2) 5-kinase, and that during oxidative stress, this enzyme is responsible for PtdIns(3,4,5)P(3) synthesis. Furthermore, we demonstrate that by upregulating the H(2)O(2)-induced PtdIns(3,4,5)P(3) levels using overexpression studies, the endogenous PtdIns(3,4)P(2) 5-kinase is likely to be Type Ialpha PIPkinase. CONCLUSIONS: We describe for the first time a novel in vivo activity for Type Ialpha PIPkinase, and a novel pathway for the in vivo synthesis of functional PtdIns(3,4,5)P(3), a key lipid second messenger regulating a number of diverse cellular processes.     

Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor

Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the beta2-adrenergic receptor (beta2-AR), a prototypical 7TMR, beta-arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Ialpha, a PIP(2)-producing enzyme. Furthermore, beta-arrestin2 is required to form a complex with PIP5K Ialpha and agonist-occupied beta2-AR, and beta-arrestins synergize with the kinase to produce PIP(2) in response to isoproterenol stimulation. Interestingly, beta-arrestins themselves bind PIP(2), and a beta-arrestin mutant deficient in PIP(2) binding no longer internalizes 7TMRs, fails to interact with PIP5K Ialpha, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Ialpha has been fused to the same beta-arrestin mutant rescues internalization of beta2-ARs. Collectively, these data support a model in which beta-arrestins direct the localization of PIP5K Ialpha and PIP(2) production to agonist-activated 7TMRs, thereby regulating receptor internalization.     

Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol.

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.     

Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase alpha (Type Ialpha PIPkinase). We find that both PLD1 and PLD2 interact with the Type Ialpha PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ialpha PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.     

Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination

Intramuscular injection of Japanese flounder, Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 microg of a plasmid DNA vaccine encoding the hirame rhabdovirus (HIRRV) glycoprotein gene (pCMV-HRVg) was found to provide strong protection against HIRRV. We also conducted a real-time PCR analysis to quantify immune-related genes, e.g. MHC class Ialpha, IIalpha, IIbeta, TCR-alpha, beta1, beta2 and delta, to characterize the immune response at 1 and 7 days after DNA vaccination. In general, the copy numbers were at least 2-fold higher than those of the non-vaccinated fish. Interestingly, the gene expression of TCR beta1 and beta2 increased 1 day post-DNA vaccination, after which their copy numbers returned to levels similar to those before vaccination. These results suggest that the immune system of Japanese flounder was activated immediately after DNA immunization.     

A novel interaction of cGMP-dependent protein kinase I with troponin T

cGMP-dependent protein kinase (cGK) is a major intracellular receptor of cGMP and is implicated in several signal transduction pathways. To identify proteins that participate in the cGMP/cGK signaling pathway, we employed the yeast two-hybrid system with cGK Ialpha as bait. cDNAs encoding slow skeletal troponin T (skTnT) were isolated from both mouse embryo and human skeletal muscle cDNA libraries. The skTnT protein interacted with cGK Ibeta but not with cGK II nor cAMP-dependent protein kinase. The yeast two-hybrid and in vitro binding assays revealed that the N-terminal region of cGK Ialpha, containing the leucine zipper motif, is sufficient for the association with skTnT. In vivo analysis, mutations in cGK Ialpha, which disrupted the leucine zipper motif, were shown to completely abolish the binding to skTnT. Furthermore, cGK I also interacted with cardiac TnT (cTnT) but not with cardiac troponin I (cTnI). Together with the observations that cTnI is a good substrate for cGK I and is effectively phosphorylated in the presence of cTnT in vitro, these findings suggest that TnT functions as an anchoring protein for cGK I and that cGK I may participate in the regulation of muscle contraction through phosphorylation of TnI.     

Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode

The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group.