Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrp1p is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using-functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrp1p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrp1p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.

Structure of tick anticoagulant peptide at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at t.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 Å. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (CyS5(T)- Cys 15(T)) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 Å to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 Å with the guanidinium group forming a cation-π-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 Å in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N- terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (elF2), absent from elF5, mediate interaction with elF2γ

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2B homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues, eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

Transposon Tn1721 distribution among strains of Xylella fastidiosa

Transposons are mobile genetic elements found within the genomes of various organisms including bacteria, fungi, plants and animals. Fragments of the transposon Tn1721 were found included in the genome of Xylella fastidiosa strain 9a5c. Regions from such fragments were PCR-amplified using specially designed primers (TNP1 and TNP2). In order to detect insertions of the Tn1721 element, both primers were used and one of them included a region of the transposon (TNP1) and the other one had the right repeat and part of the bacterial chromosome (TNP2). The PCR products obtained from strain 9a5c were used as a pattern for fragment size comparisons when DNA samples from other X. fastidiosa strains were used as template for the PCR assays. Differences were observed concerning the PCR products of such amplifications when some X. fastidiosa strains isolated from grapevine and plum were used. For the citrus-derived strains only the strains U187d and GP920b produced fragments with different sizes or weak band intensity. Such variations in the X. fastidiosa genome related to disrupted Tn1721 copies are probably due to the possibility of such a transposon element being still able to duplicate even after deletion events might have taken place and also because the bacterial strains in which the main differences were detected are derived from different host plants cultivated under different climate conditions from the one used as reference. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Structural basis for inhibition of cyclin-dependent kinase 9 by flavopiridol

Flavopiridol has been shown to potently inhibit CDK1 and 2 (cyclin-dependent kinases 1 and 2) and most recently it has been found that it also inhibits CDK9. The complex CDK9-cyclin T1 controls the elongation phase of transcription by RNA polymerase II. The present work describes a molecular model for the binary complex CDK9-flavopiridol. This structural model indicates that the inhibitor strongly binds to the ATP-binding pocket of CDK9 and the structural comparison of the complex CDK2-flavopiridol correlates the structural differences with differences in inhibition of these CDKs by flavopiridol. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known leading structures such as flavones and adenine derivatives. © 2002 Elsevier Science (USA). All rights reserved.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5′ sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Chemical constituents from the infusion of Zollernia ilicifolia vog. and comparison with Maytenus species

The new flavonoid glycoside kaempferol-3-O-α-L-rhamnopyranosyl(1→2)-O-[α-L- rhamnopyranosyl(1→6)]-O-β-D-galactopyranoside-7-O-α-L- rhamnopyranoside was isolated together with (S)-zierin from the leaves of Zollernia ilicifolia (Fabaceae), a medicinal plant used as analgesic and antiulcerogenic effects in Brazilian Tropical Atlantic Rain Forest. The structures were established on the basis of 1H, 13C NMR and 2D NMR (COSY, HMBC, HMQC), UV, MS and IV spectra. The infusion of Zollernia ilicifolia was qualitatively compared to the infusion of the espinheiras-santas (Maytenus aquifolium and Maytenus ilicifolia) by HPLC-DAD.

Araçatuba virus: A vaccinialike virus associated with infection in humans and cattle

We describe a vaccinialike virus, Araçatuba virus, associated with a cowpoxlike outbreak in a dairy herd and a related case of human infection. Diagnosis was based on virus growth characteristics, electron microscopy, and molecular biology techniques. Molecular characterization of the virus was done by using polymerase chain reaction amplification, cloning, and DNA sequencing of conserved orthopoxvirus genes such as the vaccinia growth factor (VGF), thymidine kinase (TK), and hemagglutinin. We used VGF-homologous and TK gene nucleotide sequences to construct a phylogenetic tree for comparison with other poxviruses. Gene sequences showed 99% homology with vaccinia virus genes and were clustered together with the isolated virus in the phylogenetic tree. Araçatuba virus is very similar to Cantagalo virus, showing the same signature deletion in the gene. Araçatuba virus could be a novel vaccinialike virus or could represent the spread of Cantagalo virus.

Two Digestive Trypsins Occur in Three Species of Neotropical Anophelines

Trypsin activity increases in the midgut of Anopheles aquasalis, Anopheles albitarsis, and Anopheles darlingi after a bloodmeal. The activity returns to basal levels at the time the blood is completely digested. Affinity chromatography, reversed-phase high performance liquid chromatography (HPLC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to sequentially purify the mosquito trypsins found in the midguts at 24 h after feeding. Amino-terminal sequencing of the purified trypsins showed the occurrence of two distinct trypsins in the midgut of each of the mosquitoes studied. The sequences obtained are similar to those of the trypsins of other hematophagous insects.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugarcane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrux paradixi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the α and β subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding α and β subunits of C. paradisi. A high degree of similarity was also observed among the PFK β subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum, it appears that α and β are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of β PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the β subunit of the pyrophosphate-dependent enzyme.

p38 MAPK regulates IL-1β induced IL-6 expression through mRNA stability in osteoblasts

Osteoblast-derived IL-6 functions in coupled bone turnover by supporting osteoclastogenesis favoring bone resorption instead of bone deposition. Gene regulation of IL-6 is complex occurring both at transcription and post-transcription levels. The focus of this paper is at the level of mRNA stability, which is important in IL-6 gene regulation. Using the MC3T3-E1 as an osteoblastic model, IL-6 secretion was dose dependently decreased by SB203580, a p38 MAPK inhibitor. Steady state IL-6 mRNA was decreased with SB203580 (2 μM) ca. 85% when stimulated by IL-1β (1-5 ng/ ml). These effects require de novo protein synthesis as they were inhibited by cycloheximide. p38 MAPK had minor effects on proximal IL-6 promoter activity in reporter gene assays. A more significant effect on IL-6 mRNA stability was observed in the presence of SB203580. Western blot analysis confirmed that SB203580 inhibited p38 MAP kinase, in response to IL-1β in a dose dependent manner in MC3T3-E1 cells. Stably transfected MC3T3-E1 reporter cell lines (MC6) containing green fluorescent protein (GFP) with the 3′untranslated region of IL-6 were constructed. Results indicated that IL-1β, TNFα, LPS but not parathyroid hormone (PTH) could increase GFP expression of these reporter cell lines. Endogenous IL-6 and reporter gene eGFP-IL-6 3′UTR mRNA was regulated by p38 in MC6 cells. In addition, transient transfection of IL-6 3′UTR reporter cells with immediate upstream MAP kinase kinase-3 and -6 increased GFP expression compared to mock transfected controls. These results indicate that p38 MAPK regulates IL-1β-stimulated IL-6 at a post transcriptional mechanism and one of the primary targets of IL-6 gene regulation is the 3′UTR of IL-6.