Paradoxical Effect of Quercetin Antioxidant on Goat Sperm Parameters After Cryopreservation.

BACKGROUND: Some antioxidants have been used in semen extenders to reduce adverse effects caused by excessive reactive oxygen species (ROS) production. The study was carried out to assess the effect of quercetin (QC) antioxidant therapy on goat semen submitted to cryopreservation. OBJECTIVE: To evaluate the effect of quercetin incorporation in different phases of the cryopreservation process of goat spermatozoa. MATERIALS AND METHODS: Five ejaculates from each of four goats (n= 20) were collected and split into four groups: Control (G1), without QC; G2, 15 µM of QC added to semen before centrifugation; G3, 15 µM QC added to semen after centrifugation; G4, 15 µM QC added to semen before centrifugation and 15 µM of QC added to semen after centrifugation (total of 30 µM of QC); and cryopreserved. All semen samples were evaluated after thawing for sperm kinetics, plasma membrane integrity, and ROS levels. RESULTS: Although lower concentrations of ROS were associated with groups that received antioxidant supplementation (P=0.0213), linear and dose dependent (P<0.05) reductions of the total and progressive sperm motility, velocity and percentage of fast cells were related to the QC groups. Likewise, plasma membrane integrity was better preserved (P=0.0154) in the control group (35.5%) than in groups that received QC (G2=32.6%, G3=32.4% and G4=26.7%). CONCLUSION: Although quercetin was efficient at reducing the oxidative stress related to sperm cryopreservation, it exerted a deleterious dose-dependent effect on the kinetics and integrity of the frozen goat semen, contradicating its use in the tested concentrations.

The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs / O uso da glutationa reduzida (GSH) como antioxidante na criopreservação seminal em cães

The aim of this study was to evaluate the effect of supplementation with different concentrations of reduced glutathione GSH (0; 5; 7.5; 10mM) in the extender for cryopreservation in dogs with evaluations performed after glycerolization (chilled) and thawing (thawed). For this purpose, we used 8 dogs and two semen collections were performed in a weekly interval, totaling 16 semen samples. The sperm were analyzed by automatic sperm motility (CASA) and flow cytometry analysis of mitochondrial potential (JC1 dye) and membrane/acrosome integrity (FITC-PI dyes). We evaluated subjectively the membrane and acrosome integrity, mitochondrial activity and DNA integrity. Seminal plasma was evaluated for lipid peroxidation (TBARS concentration). Chilled and thawed samples supplemented with 7.5 and 10mM of GSH had lower percentage of sperm with high (DAB - Class I) and medium (DAB - Class II) mitochondrial activity. And 10mM of GSH had higher percentage of low mitochondrial activity (DAB - Class III). Moreover, thawed samples of 10mM of GSH had high DNA fragmentation rates. Probably by a reductive stress effect on mitochondria which lead to an increase in reactive oxygen species, and a mitochondrial malfunction.(AU)

O objetivo deste estudo foi avaliar o efeito da suplementação com diferentes concentrações de glutationa reduzida (GSH - 0; 5; 7,5; 10mM) para criopreservação em cães com avaliações realizadas após glicerolização (refrigeração) e descongelação. Para tal, foram utilizados oito cães e foram realizadas duas coletas de sêmen em intervalo semanal, totalizando 16 amostras de sêmen. Foram avaliadas a motilidade espermática computadorizada (CASA) e a análise de citometria de fluxo do potencial mitocondrial (sonda JC-1) e integridade da membrana/acrossomal (sonda FITC-PI). Subjetivamente foi avaliada a integridade da membrana plasmática e do acrossomal, atividade mitocondrial e integridade do DNA. O plasma seminal foi avaliado quanto à peroxidação lipídica (concentração de TBARS). As amostras refrigeradas e descongeladas suplementadas com 7,5 e 10mM de GSH apresentaram menor porcentagem de espermatozoides com alta atividade mitocondrial (DAB - Classe I) e média (DAB - Classe II). Na concentração de 10mM de GSH, apresentaram maior porcentagem de baixa atividade mitocondrial (DAB - Classe III). Além disso, amostras descongeladas de 10mM de GSH apresentaram taxas de fragmentação de DNA elevadas, provavelmente por efeito de estresse redutivo sobre as mitocôndrias que elevam as espécies reativas de oxigênio e disfunção mitocondrial.(AU)

Growth suppression and mitotic defect induced by JNJ-7706621, an inhibitor of cyclin-dependent kinases and aurora kinases.

Aurora kinases and cyclin-dependent kinases, which play critical roles in the cell cycle and are frequently overexpressed in a variety of tumors, have been suggested as attractive targets for cancer therapy. JNJ-7706621, a recently identified dual inhibitor of these kinases, is reported to induce cell cycle arrest, endoreduplication, and apoptosis. In the present study, we further investigated the molecular mechanisms underlying these effects. The inhibitor arrested various cells at G2 phase at low concentration, and at both G1 and G2 phases at high concentration. JNJ-7706621 did not prevent localization of Aurora A to the spindle poles, but did inhibit other centrosomal proteins such as TOG, Nek2, and TACC3 in early mitotic phase. Similarly, the drug did not prevent localization of Aurora B to the kinetochore, but did inhibit other chromosomal passenger proteins such as Survivin and INCENP. In the cells exposed to JNJ-7706621 after nocodazole release, Aurora B, INCENP, and Survivin became relocated to the peripheral region of chromosomes, but Plk1 and Prc1 were localized on microtubules in later mitotic phase. Treatment of nocodazole-synchronized cells with JNJ-7706621 was able to override mitotic arrest by preventing spindle checkpoint signaling, resulting in failure of chromosome alignment and segregation. Injection of the drug significantly inhibited the growth of TC135 Ewing's sarcoma cells transplanted into athymic mice by cell cycle arrest and apoptosis. JNJ-7706621 is a unique inhibitor regulating cell cycle progression at multiple points, suggesting that it could be useful for cell cycle analysis and therapy of various cancers, including Ewing's sarcoma.

Consultant attitudes to undertaking undergraduate teaching duties: perspectives from hospitals serving a large medical school.

OBJECTIVE: To explore attitudes among National Health Service consultants responsible for delivering basic clinical teaching to medical students. DESIGN: Postal questionnaire. SUBJECTS AND SETTING: A total of 308 acute hospital trust consultants working in 4 'new' and 4 'established' teaching hospitals in the West Midlands metropolitan area, and involved in the delivery of clinical teaching to Year 3 medical students at the University of Birmingham Medical School during 2002-03. MAIN OUTCOME MEASURE(S): The questionnaire explored contractual requirements, actual teaching commitments and perceptions of medical students' knowledge and attitudes. Responses from doctors and surgeons and from respondents working in established and new teaching hospitals were compared. RESULTS: A total of 249 responses were received (response rate 80.8%). Although many consultants enjoy teaching students, their enjoyment and their ability to deliver high standards of teaching are compromised by time and resource constraints. For many the situation is aggravated by the perceived inappropriate organisation of the clinical teaching curriculum and the inadequate preparation of students for clinical practice. Linking these themes is the overarching perception among teachers that neither service nor educational establishments afford teaching the levels of recognition and reward associated with clinical work or research. CONCLUSION: To overcome barriers to teaching requires more reciprocal links between hospital staff and medical schools, opportunities for consultants to understand and to comment on curricular and timetable developments, and, perhaps most importantly, recognition (in contractual, financial, managerial and personal terms) of the importance of undergraduate teaching in the competing triad of service, research and education.

Two basic regions of NCp7 are sufficient for conformational conversion of HIV-1 dimerization initiation site from kissing-loop dimer to extended-duplex dimer.

Nucleocapsid (NC) protein possesses nucleotide-annealing activities, which are used in various processes in retroviral life cycle. As conserved characters, the NC proteins have one or two zinc fingers of CX(2)CX(4)HX(4)C motif surrounded by basic amino acid sequences. Requirement of the zinc fingers for the annealing activities of NC protein remains controversial. In this study, we focused the requirement in the process of maturation of dimeric viral RNA. Discrimination between immature and mature dimers of synthetic RNA corresponding to the dimerization initiation site of human immunodeficiency virus type 1 (HIV-1) genomic RNA was performed based on their Mg(2+)-dependent stability in gel electrophoreses and on their distinct signal pattern from NMR analysis of imino protons. Chaperoning activity of the HIV-1 NC protein, NCp7, and its fragments for maturation of dimeric RNA was investigated using these experimental systems. We found that the two basic regions flanking the N-terminal zinc finger of NCp7, which are connected by two glycine residues instead of the zinc finger, were sufficient, although about 10 times the amounts of peptide were needed in comparison with intact NCp7. Further, it was found that the amount of basic residues rather than the amino acid sequence itself is important for the activity. The zinc fingers may involve the binding affinity and/or such a possible specific binding of NCp7 to dimerization initiation site dimer that leads to the maturation reaction.

Minimal functional structure of Escherichia coli 4.5 S RNA required for binding to elongation factor G.

Escherichia coli cells contain abundant amounts of metabolically stable 4.5 S RNA. Consisting of 114 nucleotides, 4.5 S RNA is structurally homologous to mammalian 7 S RNA, and it plays an essential role in targeting proteins containing signal peptide to the secretory apparatus by forming an signal recognition-like particle with Ffh protein. It also binds independently to protein elongation factor G (EF-G) and functions in the translation process. This RNA contains a phylogenetically conserved RNA domain, the predicted secondary structure of which consists of a hairpin motif with two bulges. We examined the binding activity of mutants with systematic deletions to define the minimal functional interaction domain of 4.5 S RNA that interacts with EF-G. This domain consisted of 35-nucleotides extending from 36 to 70 nucleotides of mature 4.5 S RNA and contained two conserved bulges in which mutations of A47, A60, G61, C62, A63, and A67 diminished binding to EF-G, whereas those at A39, C40, C41, A42, G48, and G49 did not affect binding. These data suggested that the 10 nucleotides in 4.5 S RNA, which are conserved between 4.5 S RNA and 23 S rRNA, have a key role for EF-G binding. Based on the NMR-derived structure of mutant A47U, we further verified that substituting U at A47 causes striking structural changes and the loss of the symmetrical bulge. These results indicate the mechanism by which EF-G interacts with 4.5 S RNA and the importance of the bulge structure for EF-G binding.

An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria.

We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans. Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu. Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs. This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.

Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase sigma 28 subunit.

The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines. Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.

Conformational change of dimerization initiation site of HIV-1 genomic RNA by NCp7 or heat treatment.

Dimerization initiation site (DIS) of the human immunodeficiency virus type 1 (HIV-1) genome has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. A DIS RNA fragment spontaneously forms a kissing dimer and is converted into an extended-duplex dimer by supplement of nucleocapsid protein NCp7. This two-step dimerization reaction can be also executed by a heat treatment instead of the binding proteins. However, it has not identified whether mechanisms of the conformational conversion by two different treatments are identical or not. In the present study, we used a series of DIS RNA oligonucleotides and the conformations of two extended-duplex dimers produced by the two different treatments were compared by the analysis of NMR spectra in the imino proton region. It was found that the effects of the two kinds of treatments are quite similar and the conformations of the two extended-duplex dimers are identical. These findings suggest that the conversion mechanisms DIS RNA by NCp7 and heat treatments are similar.

Interactions of a didomain fragment of the Drosophila sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions.

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sx1) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-2H]uridine phosphoramidite, and synthesized a series of 2H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-2H]uridine in the target sequence, GU8C. By observing the H5-H6 TOCSY cross peaks of the series of 2H-labeled RNAs complexed with the Sx1 RBDI-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU2GU8, AU8, and UAU8, were assigned by comparison with those of GU8C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.

NMR analysis of intra- and inter-molecular stems in the dimerization initiation site of the HIV-1 genome.

Two positive-strand HIV-1 genomic RNAs form a dimer in virion particles through interaction of the dimerization initiation sites (DIS). The DIS RNA fragment spontaneously formed a "loose-dimer" and was converted into a "tight-dimer" by supplementation with nucleocapsid protein NCp7. This two-step dimerization reaction requires the whole DIS sequence [Takahashi et al. (2000) RNA 6, 96-102]. In the present study, we measured imino proton resonances to investigate the secondary structures of the two types of dimers in a 39-mer RNA covering the entire DIS (DIS39), including discrimination between intra- and inter-molecular base pairing. Both the presence and absence of inter-molecular NOE between (15)N-labeled and unlabeled DIS39 were unambiguously detected in an equimolar mixture of (15)N-labeled and unlabeled DIS39. The stem-bulge-stem structures in both dimers were confirmed and found to be very close to each other from clear superimposition of the NMR spectra in the two dimeric states. Nevertheless, the modes of base pairing in the stems of the loose- and tight-dimers were intra- and inter-molecular, respectively. Our results suggest a large structural alteration of genomic RNA occurs during virion maturation.

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome.

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.

Classification of 3D structural character of RNA by hydrogen bond and base stacking.

We are developing a computational system to classify RNA structures by its structural character. Here, an improved grouping algorithm was introduced to the system and the base-stacking pattern (BSP) is used as a criterion for the classification in addition to hydrogen-bond pattern (HBP). 279 conformers of 15 mer RNA hairpin were classified into 89 and 36 groups by HBP and BSP, respectively, suggesting that HBP represents conformational character better than BSP.

Self-cleavage of p2Sp1 RNA with Mg2+ and non-ionic detergent (Brij 58).

The precursor of an RNA molecule from T4-infected E. coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions [(UpA (139-140) and CpA (170-171)], within a putative loop and stem structure. This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents. We studied the self-cleavage reaction of an RNA fragment (GUUUCGUACAAAC) (R1) with the sequence corresponding to the p2Sp1 RNA in the presence of Mg2+ and non-ionic detergents. It requires Mg2+ and is aided by a non-ionic detergent, Brij 58. The cleavage reaction is time, temperature, and pH-dependent. The cleavage occurs at the phosphodiester bond between UpA and CpA on the RNA fragment (GUUUCGUACAAAC) (R1). Furthermore, the maximum of cleavage of R1 occurs at a very low Mg2+ concentration (< or = 5 mM).

An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in trans-translation.

Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+). Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.

Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA). Involvement of a structural change of the Shine-Dalgarno sequence and the initiation codon aug in oppa mRNA.

We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro. The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T(1), specific for single-stranded RNA, and RNase V(1), which recognizes double-stranded or stacked RNA. In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.

The guanosine binding mechanism of the Tetrahymena group I intron.

The Tetrahymena group I intron catalyzes self-splicing through two consecutive transesterification reactions, using a single guanosine-binding site (GBS). In this study, we constructed a model RNA that contains the GBS and a conserved guanosine nucleotide at the 3'-terminus of the intron (omegaG). We determined by NMR the solution structure of this model RNA, and revealed the guanosine binding mechanism of the group I intron. The G22 residue, corresponding to omegaG, participates in a base triple, G22 xx G3 x C12, hydrogen-bonding to the major groove edge of the Watson-Crick G3 x C12 pair. The G22 residue also interacts with A2, which is semi-conserved in all sequenced group I introns.

NMR signal assignment of the polyuridine tract of the single-stranded RNA complexed with Sxl RBD1-RBD2 by using residue selective [5-(2)H]uridine substitutions.

Using [5-(2)H]uridine phosphoramidite, we synthesized a series of 2H-labeled Drosophila Sex-lethal (Sxl) target RNAs, in which all the uridine residues except one were specifically replaced by [5-(2)H]uridine. By observing the H5-H6 cross peaks of RNA in the TOCSY spectra, we unambiguously assigned all the base proton resonances of the target RNA in a Sxl x RNA complex. Furthermore, it was shown that Sxl differently recognizes A and G in a position prior to the polyuridine tract.

Phosphorothioate G3T4G3 motifs inhibits the early stage of HIV-1 infection.

It is well known that G-rich phosphorothioate oligonucleotides (G-rich PS) bind to the V3 loop of HIV-1 gp120 and inhibit HIV-1 infection. In this study, we investigated the inhibitory mechanism of a new type of G-rich PS (PS-G3T4G3) on the replication cycle of HIV-1. PS-G3T4G3 inhibits both cell to cell and cell free infections. Binding and entry assays revealed that the inhibitory step of PS-G3T4G3 occurs at the early stage of HIV-1 infection. V3 loop-specific mAb test showed that PS-G3T4G3 binds to the V3 loop and prevents its interaction with chemokine receptors. These results suggest that PS-G3T4G3 may be a novel candidate for an HIV-1 inhibitor.

Development of a system to classify 3D structural character of RNA.

We are developing a computational system to extract structural character of RNA. We made a program that classifies conformers by recognizing the hydrogen bonding patterns. The program was applied to a set of 279 conformers and they were classified into about 40 groups. The system is expected to be useful for searching structural motifs of RNA and classifying large number of generated conformers in structural modeling process.