[Content of C-reactive protein in patients in an acute period of a ruptured intracranial aneurysm].

A content of C-reactive protein (CRP) in the blood serum was determined in 36 patients in acute period of a ruptured intracranial arterial aneurysm (AA). It was significantly more, than in a control group, and have exceeded 10 mg/I in 1 - 4th day of the disease. The level of CRP have had differ, depending on severity of cerebral vasospasm (CVS), determined in accordance to the ultrasound investigation data. In a pronounced CVS in majority of patients the level of CRP in the blood serum have had exceed 10 mg/l, and have secured elevated in a spinal liquor on the 7 - 10th day of the disease, differing from this index in patients with moderately pronounced CVS or without it. In patients with severe invalidization or those, who have died, the level of CRP was trust-worthy higher.

Stimulation of the human RAD51 nucleofilament restricts HIV-1 integration in vitro and in infected cells.

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.

Incidence of injuries and factors related to injuries in combat soldiers.

INTRODUCTION: Musculoskeletal injuries to the lower extremities are major factors contributing to drop out from military tasks. The aim of the present study was to determine the incidence of musculoskeletal injuries and the parameters that differentiate between the soldiers who incurred these injuries and those who did not along 14 weeks of an infantry commanders course. METHODS: One-hundred and sixty-eight participants were recruited from an infantry commanders course. The soldiers were tested before (pre), in the middle (middle) and at the end (last) of the course for anthropometric measurements, proprioceptive ability and dynamic postural balance (DPB), and filled out an ankle stability questionnaire (Cumberland Ankle Instability Tool (CAIT). A physiotherapist followed and recorded all musculoskeletal injuries incurred by the participants during the course. RESULTS: Fifty-eight participants out of the 168 (34.5%) reported some pain/injury. Time effects were found for body mass index, DPB asymmetry, DPB in posterior-medial (P-M) direction and proprioception ability. Injury effects were found for DPB asymmetry, DPB in P-M direction, CAIT and proprioception ability. An interaction was found for proprioception ability. The Cox regression showed that the variables that are mostly effecting injuries were pretesting proprioception ability, DPB asymmetry and CAIT. CONCLUSIONS: More than one out of three participants incurred musculoskeletal injuries, with deficits in proprioception ability, DPB and ankle stability in pretesting as major factors contributing to injuries. Further studies should look at the effect of specific exercises such as proprioception, DPB and ankle stability exercises for prevention and treatment of musculoskeletal injuries among combat soldiers.

Generation of G-to-A and C-to-U changes in HIV-1 transcripts by RNA editing.

RNA editing involves posttranscriptional alterations of messenger RNA (mRNA) sequences modifying the information content encoded by the genetic message. Here, it is shown that, in chronically infected H9 cells, human immunodeficiency virus-type 1 (HIV-1) mRNAs undergo guanine-to-adenine (G-to-A) and cytosine-to-uracil (C-to-U) changes. G-to-A modification in the untranslated region of exon 1 was present only in spliced HIV-1 mRNAs. The creation of stop codons in HIV-1 mRNAs may function to control the translation of viral proteins, such as viral protein R, that are involved in the regulation of HIV-1 expression and the survival of chronically infected cells.

Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase.

The fundamental role played by reverse transcriptase in the replication of retroviruses has stimulated the study of the mechanism of action of this enzyme. The reverse transcriptase of the type 1 human immunodeficiency virus forms a stable complex with its cognate transfer RNA replication primer (tRNA(Lys3)). Here, we outline the role of this enzyme in the selection of its primer tRNA, the annealing of primer tRNA to the complementary region of the retroviral genome, and the first attempts to use the reverse-transcriptase-tRNA complex as a new target for antiviral agents.

Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51.

HIV-1 integrase (IN) is the key enzyme catalyzing the proviral DNA integration step. Although the enzyme catalyzes the integration step accurately in vitro, whether IN is sufficient for in vivo integration and how it interacts with the cellular machinery remains unclear. We set up a yeast cellular integration system where integrase was expressed as the sole HIV-1 protein and targeted the chromosomes. In this simple eukaryotic model, integrase is necessary and sufficient for the insertion of a DNA containing viral LTRs into the genome, thereby allowing the study of the isolated integration step independently of other viral mechanisms. Furthermore, the yeast system was used to identify cellular mechanisms involved in the integration step and allowed us to show the role of homologous recombination systems. We demonstrated physical interactions between HIV-1 IN and RAD51 protein and showed that HIV-1 integrase activity could be inhibited both in the cell and in vitro by RAD51 protein. Our data allowed the identification of RAD51 as a novel in vitro IN cofactor able to down regulate the activity of this retroviral enzyme, thereby acting as a potential cellular restriction factor to HIV infection.

Initiation of HIV-2 reverse transcription: a secondary structure model of the RNA-tRNA(Lys3) duplex.

Human immunodeficiency virus type 2 (HIV-2) reverse transcription is initiated from cellular tRNA(Lys3) partially annealed to the RNA viral genome at the primer binding site (PBS). This annealing involves interactions between two highly structured RNA molecules. In contrast to HIV-1, in which the reverse transcription initiation complex has been thoroughly studied, there is still little information regarding a possible model to describe the secondary structure of the template-primer complex in HIV-2. To determine whether HIV-2 RNA sequences flanking the PBS may specifically interact with the natural primer tRNA, we performed site-directed mutagenesis and enzymatic footprinting. An RNA fragment corresponding to the HIV-2 U5 RNA domain and tRNA(Lys3) were probed either in their free form or in the binary complex. Important reactivity changes to nucleases were obtained upon complex formation. In addition to the canonical contacts between the viral PBS and the 3' end acceptor stem of tRNA(Lys3), we identified two additional interacting domains: (i) the U-rich region of the anticodon loop with the A-rich sequence of the internal loop within the U5-prePBS region; (ii) nucleotides 48-54 from the TPsiC domain of tRNA(Lys3) and the 240-247 region of viral U5-RNA. In view of these experimental data and sequence comparison between different HIV-2 isolates, we propose a model for the secondary structure of the HIV-2 template-primer initiation complex.

Purification and properties of tRNA nucleotidyl transferase from Musca domestica.

The enzyme tRNA nucleotidyl transferase (EC 2.7.7.25) has been highly purified from whole adult houseflies. A molecular weight of 30 000 has been determined. The enzyme requires Mg2+ and tRNA deprived of the 3' terminal sequence CCA for activity in the incorporation of AMP and CMP onto the tRNA. UMP can be incorporated instead of CMP but the latter has a higher affinity than UMP as shown by competition experiments. A complex between the enzyme and tRNA has been shown by sucrose gradient centrifugation, nitrocellulose binding and protection by tRNA against enzyme denaturation at 50 degrees C. Comparative studies with tRNA nucleotidyl transferase purified from larvae, pupae and adult insects indicate that tRNA nucleotidyl transferase from these three developmental stages have the same molecular weight, sedimentation coefficient and optimum pH while the larval enzyme differs from the pupae and adult enzymes in the elution behaviour from a DEAE-cellulose column.

Specific inhibition of in vitro reverse transcription using antisense oligonucleotides targeted to the TAR regions of HIV-1 and HIV-2.

Antisense oligonucleotides (ODNs) overlapping the stem-loop structure of the trans-activating responsive (TAR) element at the 5' end of HIV-1 and HIV-2 viral RNAs were tested for their inhibitory effect on cDNA synthesis by HIV-1 and HIV-2 reverse transcriptases (RT). Inhibition of reverse transcription is sequence-specific and enhanced by the presence of the RT-associated RNase H activity. The degree of inhibition obtained with the anti-TAR antisense is significantly higher than with other HIV-1 targeted antisense ODNs used before [1]. Gel retardation showed a stable specific complex between the 16- and 25-mer anti-TAR HIV-1 selected ODNs and the target region. No complex was observed with a non-inhibitor 22-mer anti-TAR ODN and with the corresponding control sequences. Targeting of the first stem-loop in the 5' region of HIV-2 RNA by anti-TAR ODNs inhibited very strongly reverse transcription by HIV-2 RT. The structure of the antisense and the target sequence affect annealing efficiency and hence the degree of inhibition of reverse transcription.

2'-Fluoro-2'-deoxycytidine triphosphate as a substrate for RNA- and DNA-dependent DNA polymerases.

The ability of the analog 2'-fluoro-2'-deoxycytidine triphosphate (dCflTP) to be used as a substrate in the reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha and AMV reverse transcriptase has been studied. The apparent Km values for dCTP and dCflTP, using activated DNA as templates, were 0.6 microM and 7 mM with DNA polymerase alpha and 0.14 microM and 7 microM with AMV reverse transcriptase, respectively. As observed with dCTP, aphidicolin was a noncompetitive inhibitor in the DNA polymerase alpha-catalyzed DNA synthesis; the Ki values were about 2 microM for both substrates. dCflTP can also be incorporated into DNA synthetized by other eukaryotic DNA polymerases and by reverse transcriptase with RNA as a template, both in the presence or absence of (dT)12 primer.

Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase.

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.

Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA.

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.

2'-Fluoro-2'-deoxypolynucleotides as templates and inhibitors for RNA- and DNA-dependent DNA polymerases.

Poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) and poly(2'-deoxycytidylic acid) (poly(dCfl)) were tested as templates in DNA synthesis reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha, mouse cell DNA polymerase gamma and avian myeloblastis virus (AMV) reverse transcriptase. Poly(dAfl).(dT)12 can fully substitute for poly(rA).(dT)12 as template with DNA polymerase gamma, to 50% with reverse transcriptase, but was poorly recognized by DNA polymerase alpha. DNA synthesis by reverse transcriptase with poly(dCfl).(dG)12 as template was 50% of that with poly(rC).(dG).(dG)12. The use of 2'-fluoropolymers as templates was more efficient at 37 degrees C than at 25 degrees C. No appreciable differences on the fidelity of DNA synthesis by reverse transcriptase were observed when dCMP misincorporation was measured with poly(dAfl).(dT)12 or poly(rA).(dT)12 as template primers. Poly(C) and poly-2'-O-methylcytidylic acid had no significant effect on the reaction catalyzed by DNA polymerase gamma and reverse transcriptase, independent of the synthetic polynucleotide complex utilized as template. On the other hand, poly(dCfl) was an inhibitor when poly(rA).(dT)12 or poly(dA).(dT)12 were used as templates, but not when poly(dAfl).(dT)12 was employed. Analogous results have been obtained with activated DNA and AMV 70 S RNA as templates in the reverse transcriptase reaction. The inhibition by poly(dCfl) was noncompetitive with regard to TTP, poly(dA) and poly(rA). Xenopus laevis oocytes DNA polymerase alpha was not inhibited by poly(dCfl).

Human immunodeficiency virus type-1 reverse transcriptase copies very short templates: kinetic and crosslinking analysis.

We describe in this article some properties concerning the cDNA elongation activity of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT). The kinetic parameters of the polymerization reaction catalyzed by HIV-1 RT, using short templates, were studied. Values of Km and Vmax were measured as a function of the oligoadenylate template length: the logarithm of Km increased linearly, with an incremental factor of 2.2, when the template length differs by one nucleotide. Using short templates, olig(A)n (n = 7-14) and primers shorter or longer than the template, HIV-1 reverse transcriptase was able to synthesize polymer products longer than 200 nucleotides. We showed that an oligonucleotide as short as (pA)3 was long enough to serve as template for cDNA synthesis by RT. In the binding of RT to template of different lengths (5 to 14 nucleotides long), two constants were determined differing in each case by a factor of about 10. The three recombinant forms of HIV-1 RT (p66/p51, p66/p66 and p51/p51) were crosslinked to a short template, (pA)14, in the presence of cis-aquahydroxydiamminoplatinum. The efficiency of crosslink of [32P](pA)14 template with each of the subunits of RT correlated well with the affinity of this template to the different forms of RT. In the case of p66/p51, the crosslink occurred mainly with the p66 subunit. These results confirm the important catalytic role of the p66 subunit in the heterodimeric human retroviral polymerase.

Preferential interaction of human immunodeficiency virus reverse transcriptase with two regions of primer tRNA(Lys) as evidenced by footprinting studies and inhibition with synthetic oligoribonucleotides.

Primer tRNA regions involved in the interactions between human immunodeficiency virus reverse transcriptase (HIV RT) and tRNA(Lys) were studied by digestion of primer with pancreatic ribonuclease in the presence or absence of HIV RT. The acceptor stem of tRNA(Lys) is not noticeably protected against nuclease action in the presence of HIV RT, while this enzyme clearly protects part of the anticodon and dihydrouridine loops of tRNA(Lys). The acceptor stem of primer tRNA was digested by RNase A only in the presence of the retroviral enzyme, suggesting a partial destabilization of this region by the HIV RT. Synthetic oligoribonucleotides, corresponding to the anticodon and the dihydrouridine loops, inhibited strongly reverse transcription, confirming the strong interaction of these tRNA regions with the enzyme.

Cross-linking localization of a HIV-1 reverse transcriptase peptide involved in the binding of primer tRNALys3.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates the synthesis of DNA from the 3' end of its specific primer, tRNALys3. The regions of tRNALys3 in close contact with RT are well known, while a precise knowledge of the RT regions interacting with tRNALys3 is not yet available. To address this question we cross-linked the heterodimeric p66/p51 RT to tRNALys3 using cis-aquahydroxydiammino-platinum. Ribonucleoprotein complexes of molecular masses higher than the p66 subunit were obtained. After RNase A digestion of the RT-tRNA complex, a labeled oligoribonucleotide (ORN) was mainly found associated to the p66 subunit. This labeled p66-ORN complex was then proteolyzed with Staphylococcus aureus V8 protease. A highly purified radioactive peptide was obtained after two chromatographic purification steps. Its N-terminal sequence corresponded with amino acid residues 241VQPI244. Using the crystallographic structure of HIV-1 RT, this peptide was localized at the beta14-sheet end, near to the hairpin formed by beta12 and beta13-sheets ("primer grip") and the alphaH-helix. The so called "VQPI peptide" is in the border of the thumb and the palm subdomains of the p66 subunit. This study palliates the absence of a three- dimensional structure of the RT-tRNA complex and led to a peptide in interaction with tRNALys3 present in all HIV-1 RT isolates.

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants.

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.

DNA aptamers derived from HIV-1 RNase H inhibitors are strong anti-integrase agents.

HIV-1 integrase, the retroviral-encoded enzyme involved in the integration of the retrotranscribed viral genome into the host nuclear DNA, is an attractive and still unexploited target. To date, very few inhibitors of this enzyme with a potential therapeutic value have been described. During the search for new HIV-1 targets, we recently described DNA oligodeoxynucleotide aptamers (ODN 93 and ODN 112) that are strong inhibitors of the RNase H activity associated with HIV-1 reverse transcriptase. The striking structural homology between RNase H and integrase led us to study the effect of the RNase H inhibitors on the integrase. Shorter DNA aptamers derived from ODNs 93 and 112 (ODNs 93del and 112del) were able to inhibit HIV-1 integrase in the nanomolar range. They had G-rich sequences able to form G-quartets stabilized by the presence of K(+). The presence of these ions increased the inhibitory efficiency of these agents dramatically. Inhibition of enzymatic activities by ODN 93del and ODN 112del was observed in a cell-free assay system using a recombinant integrase and HIV-1 replication was abolished in infected human cells. Moreover, cell fusion assays showed that these agents do not block viral cell entry at concentrations where viral replication is stopped.

Selection of amino acid substitutions restoring activity of HIV-1 integrase mutated in its catalytic site using the yeast Saccharomyces cerevisiae.

The integration of proviral DNA into the genome of the host cell is an essential step in the replication of retroviruses. This reaction is catalyzed by a viral-encoded enzyme, the integrase (IN). We have previously shown that human immunodeficiency virus type 1 (HIV-1) IN causes a lethal effect when expressed in yeast cells. This system, called yeast lethal assay, was used as a tool to study IN activity in a cellular context. The yeast lethal assay allowed the selection and characterization of mutations affecting both the lethal phenotype and the in vitro IN activities. IN mutants were produced by random PCR mutagenesis in an IN gene bearing the inactivating D116A mutation in the catalytic site. The corresponding D116A substituted IN does not lead to lethality in yeast. Subsequent selection of mutants able to restore the lethal effect of IN was carried out using the yeast lethal assay. We isolated three mutants presenting a restored phenotype. The mutated IN genes were sequenced and the corresponding proteins were purified to characterize their in vitro activities. The three mutants presented restoration of the in vitro strand transfer activity, while 3' processing was only partially restored.The three mutants differ from D116A IN by at least one amino acid substitution located in the N-terminal domain of the protein, outside of the active site. These new mutated HIV-1 INs may therefore allow a better understanding of the N-terminal domain function in the integration reaction. In addition, these results support our hypothesis that explains the lethal effect as a consequence of the nuclear damage caused by wild-type IN in yeast cells. These data also indicate that the yeast lethal assay can be used as a tool to study the retroviral integration mechanism in a cellular context and to select specific inhibitors.

Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases).

DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants.