Evaluation of 2LHERP in preventing recurrences of genital herpes. Institut International 3IDI.

The objective of the study was to evaluate the effectiveness of a homeopathic complex in terms of intensity of attacks and duration of remission between attacks of genital herpes. Fifty three patients aged 18 or over with a minimum of four attacks annually were followed in this open multicentre study in a primary care setting. The principal parameters analysed were: frequency of attacks; intensity of symptoms, during treatment and/or after stopping treatment; treatment tolerance. Eighty-two percent of patients treated for recurrent genital herpes benefited. In 41% of cases, there was no recurrence after the first treatment with follow-up of between 8 and 50 months. In 32% of patients, one or two relapses, in 9% of patients recurrences continued but with reduced frequency and intensity.

Evaluation of 2LHERP in preventing recurrences of genial hespes

The objective of the study was to evaluate the effectiveness of a homeopathic complex in terms of intensity of attacks and duration of remission between attacks of genital herpes. Fifty three patients aged 18 or over with a minimum of four attacks annually were followed in this open multicentre study in... (AU)

The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway monitors premature translation termination and degrades aberrant mRNAs. In yeast, it has been proposed that a surveillance complex searches 3' of a nonsense codon for a downstream sequence element (DSE) associated with RNA-binding proteins. An interaction between the complex and the DSE-binding protein(s) triggers NMD. Here we describe the identification and characterization of the Hrp1/Nab4 protein as a DSE-binding factor that activates NMD. Mutations in HRP1 stabilize nonsense-containing transcripts without affecting the decay of wild-type mRNAs. Hrp1p binds specifically to a DSE-containing RNA and interacts with Upf1p, a component of the surveillance complex. A mutation in HRP1 that stabilizes nonsense-containing mRNAs abolishes its affinity for the DSE and fails to interact with Upf1p. We present a model describing how Hrp1p marks a transcript for rapid decay.

The mitochondrial protein targeting suppressor (mts1) mutation maps to the mRNA-binding domain of Npl3p and affects translation on cytoplasmic polysomes.

In all eukaryotic organisms, messenger RNA (mRNA) is synthesized in the nucleus and then exported to the cytoplasm for translation. The export reaction requires the concerted action of a large number of protein components, including a set of shuttle proteins that can exit and re-enter the nucleus through the nuclear pore complex. Here, we show that, in Saccharomyces cerevisiae, the shuttle protein Npl3p leaves the nuclear pore complex entirely and continues to function in the cytoplasm. A mutation at position 219 in its RNA-binding domain leaves Npl3p lingering in the cytoplasm associated with polysomes. Yeast cells expressing the mutant Npl3(L-219S) protein show alterations in mRNA stability that can affect protein synthesis. As a result, defects in nascent polypeptide targeting to subcellular compartments such as the mitochondria are also suppressed.

Biochemical analysis of the arginine methylation of high molecular weight fibroblast growth factor-2.

The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) has been shown to affect the nuclear accumulation of the growth factor. In this study, we determined the extent and position of methyl groups in HMW FGF-2. Using mass spectrometry and amino acid sequence analysis, we have shown that the 22- and 22.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -22, -24, -26, -36, and -38 using the methionine residue normally used to initiate the 18-kDa form as position 0. The 24-kDa form of HMW FGF-2 contains seven to eight dimethylated arginines located at positions -48, -50, and -52, in addition to positions -22, -24, -26, -36, and -38. In vitro methylation reactions demonstrate that the N-terminal extension of HMW FGF-2 acts as a specific substrate for yeast Hmt1p and human HRMT1L2 arginine methyltransferases. These findings indicate that HMW FGF-2, with the presence of five or more dimethylated Gly-Arg-Gly repeats, contains an RGG box-like domain, which may be important for protein-protein and/or protein-RNA interactions.

Arginine methylation facilitates the nuclear export of hnRNP proteins.

Eukaryotic mRNA processing and export is mediated by various heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated on arginine residues. In the yeast, Saccharomyces cerevisiae, the predominant enzyme responsible for arginine methylation is Hmt1p. Hmt1p methylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved in mRNA processing and export. Here, we employ an in vivo nuclear export assay to show that arginine methylation is important for the nuclear export of these hnRNPs. Both Npl3p and Hrp1p fail to exit the nucleus in cells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export. The export of a novel hnRNP-like protein, Hrb1p, which does not bind poly(A)+ RNA, however, is not affected by the lack of methylation. Furthermore, we find a genetic relationship between Hmt1p and cap-binding protein 80 (CBP80). Together, these findings establish that one biological role for arginine methylation is in facilitating the export of certain hnRNPs out of the nucleus.

Identification and characterization of two putative human arginine methyltransferases (HRMT1L1 and HRMT1L2).

RNA-binding proteins such as heterogeneous nuclear ribonucleoproteins (hnRNPs), which contain the bulk of methylated arginine residues in eukaryotic cells, play many essential roles in the metabolism of nuclear pre-mRNA. Arginine methyltransferase activity has also been implicated in signal transduction events with components of the cellular growth and viral response pathways. We recently characterized a single yeast hnRNP methyltransferase (HMT1). We now present the identification and characterization of two putative human arginine methyltransferases termed HRMT1L1 and HRMT1L2. In addition to methyltransferase similarities, the N-terminal region of the HRMT1L1 protein contains an Src homology 2 domain. HRMT1L1 maps to a YAC containing the telomere of chromosome 21q. Three alternatively spliced HRMT1L2 transcripts with variable 5'-ends were observed, encoding proteins of 343, 347, and 361 amino acids, respectively. HRMT1L2 maps to human chromosome 19q. Recombinant HRMT1L2 protein encoded by the most common 5'-variant exhibited methyltransferase activity in vitro. Furthermore, in vivo activity was demonstrated by complementation of a yeast HMT1 mutant strain. The identification of highly conserved Hmt1p human homologues that function in yeast indicates that analyses of this class of enzymes in yeast may be directly applicable to higher eukaryotes. The possible roles of HRMT1L1 and HRMT1L2 in human disease are currently unknown.

Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast.

In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. Here we show that CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3'-end processing and mRNA export from the nucleus.

A novel methyltransferase (Hmt1p) modifies poly(A)+-RNA-binding proteins.

RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA. As such, they demonstrate a myriad of dynamic behaviors and modifications. In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells. We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A)+-RNA-binding protein termed Npl3p. We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus. New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele. Four alleles of the same gene were identified in this manner. Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase. HMT1 is not required for normal cell viability except when NPL3 is also defective. The Hmt1 protein is located in the nucleus. We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro. These findings now allow further exploration of the function of this previously uncharacterized class of enzymes.

A new mechanism of transcriptional regulation: release of an activator triggered by small molecule binding.

The FadR protein of E. coli activates transcription of the fabA gene, a key enzyme of fatty acid synthesis. We report that FadR binds to a DNA sequence positioned at -40 relative to the start site of the FadR-regulated fabA transcript (the location favored by positive activators). This binding was found to be specifically antagonized by long chain acyl-CoAs. The chain length specificity of the disassociation of the FadR-DNA complex by acyl-CoAs observed in vitro reflects that seen in the repression of fabA transcription observed upon addition of fatty acids to bacterial cultures. Acyl-CoA antagonism of FadR-DNA interactions is readily reversible. These data indicate that repression of fabA transcription by fatty acids is the first reported example of a repression system mediated by positive control.

Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation.

The fadR gene of Escherichia coli encodes a protein that acts as a negative regulator (repressor) of the inducible beta-oxidation pathway. We report that the FadR protein also functions as a positive transcriptional activator of the fabA gene, which encodes the enzyme introducing the double bond of the unsaturated fatty acids of E. coli.

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H.

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).

Isolation and characterization of a protein with cyanide-sensitive superoxide dismutase activity from the prokaryote, Paracoccus denitrificans.

1. A protein with cyanide-sensitive superoxide dismutase activity was isolated from the prokaryote Paracoccus denitrificans. 2. This enzyme, present in low amount in the cell, represented not more than 10% of the total cellular superoxide dismutase activity. It was obtained in a form which was 20-40-times less active than the main superoxide dismutase of P. denitrificans which is a manganese-containing enzyme. 3. It was a soluble monomeric enzyme, highly negatively charged (pI = 4.8), with an apparent molecular weight of 33,000. 4. Cyanide sensitivity was observed by NMR assay, enzyme assay and by staining the protein for superoxide dismutase activity on polyacrylamide electrophoretogram. KCN was shown to be a competitive inhibitor of this dismutase, with an inhibitor constant of 0.15 mM. 5. From the amino acid analysis, S delta Q values lower than 100 were obtained with copper-containing proteins such as the subunit II of cytochrome oxidase from P. denitrificans (69), the azurin from P. denitrificans (77), the bacteriocuprein from Photobacter leiognathi (71); with iron and manganese superoxide dismutases (40-88), and with some eukaryotic copper/zinc dismutases of fish origin (55-82).