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.

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.

Molecular species of cerebrosides in fruiting bodies of Lentinus edodes and their biological activity.

Cerebroside fraction was obtained from fresh fruiting bodies of Lentinus edodes and separated into ten molecular species by reverse-phase high-performance liquid chromatography. The species were identified by GLC, GC-MS and NMR. Their component glycosides and sphingoids were uniformly glucose and (4E,8E)-9-methyl-4,8-sphingadienine, respectively. The component fatty acids were 2-hydroxy acids with the carbon chain length of 16, 15, 14, 18, 24, 17, 25, 26, 22 and 23 (from major to minor). The cerebrosides with the C14-18 fatty acids showed strong fruiting-inducing activity in Schizophyllum commune. Those with the C22 and C23 ones had one-eighth and one-sixteenth of the activity, respectively, and those with C24-26 had no detectable activity. 22 and 23 must be the carbon chain lengths of the component fatty acid of the sphingolipids critical for expression of biological activity.

Structural analysis of mutant hen egg-white lysozyme preferring a minor binding mode.

Trp62 in hen egg-white lysozyme has general features observed in protein-carbohydrate interactions, a stacking interaction toward nonpolar surface of the substrate sugar residue B and a hydrogen bonding network with the residue C. Our previous report (I. Kumagai, K. Maenaka, F. Sunada, S. Takeda, K. Miura, Eur. J. Biochem. 212 (1993) 151-156.) showed that the substitution of Trp62 changed the substrate binding modes; especially, the Trp62His mutant exhibited the drastic change of the binding mode and preferred to a minor binding mode of the wild-type enzyme. In order to clarify the relationship between functional and structural changes of the Trp62His mutant, we analyzed the structure of the Trp62His mutant hen lysozyme complexed with the substrate analogue, (GlcNAc)3, by X-ray crystallography. The overall protein structure in the mutant lysozyme complex was almost identical to that in the wild-type. His62 shared almost the same plane as the indole ring of Trp62 of the wild-type. Although the (GlcNAc)3 molecule which is an inhibitor against the wild-type lysozyme was cocrystallized, the Trp62His mutant did not put it in the sites A-B-C but hydrolyzed it as a substrate. One of the products, (GlcNAc)2, whose reducing end is alpha-anomer, was bound in another binding mode sticking out from the active-site cleft. The hydrolytic activity against the synthetic substrate showed that the mutant was a beta-anomer retaining enzyme, so the alpha-anomer product was converted from the beta-anomer product. Therefore, the Trp62His mutant showed the remarkable change of the substrate binding modes not by alteration of the catalytic system but possibly by subtle rearrangement of general features of protein-carbohydrate interactions between His62 and the sugar residues B and C.

The stem hairpin loop structure of p2Sp1 RNA is required for RNA-cleaving activity.

We studied the hairpin-loop structure of an RNA fragment (GUUUCGUACAAAC) (R13) with the sequence corresponding to the self-cleavage domain in the precursor of an RNA molecule from bacteriophage T4-infected Escherichia coli cells (p2Sp1 RNA). In order to determine the influence of the hairpin-loop structure on these sequence-specific cleavage reactions, we have synthesized oligoribonucleotides containing hairpin-loop, double-helical stem-loop, and single-stranded RNA structures. The cleavage was affected by the hairpin-loop structure. Furthermore, the helix-stem, which retains the thermodynamically extrastable stem hairpin-loop structures, is also important for the cleavage activity. However, the thermodynamically extrastable helix-stem structure reduced the cleavage activity of the adjacent UA and CA sequences at the helix-stem site. For the cleavage reactions of the RNA cleavage products, the R6 (ACAAAC), R7 (GUUUCGU), and R9 (GUUUCGUAC) mers from the parent RNA, R13 (GUUUCGUACAAAC), a very slight amount of cleavage product (2%) from the RNA 9 was observed, but no reaction occurred for the R6 and R7. We also describe the influences of the sequences (UA and CA) on the cleavage activity.

Higher-order structure and thermal instability of bovine mitochondrial tRNASerUGA investigated by proton NMR spectroscopy.

Although mammalian mitochondrial serine-specific tRNA with the anticodon UGA (tRNASerUGA) appears to possess an almost normal cloverleaf secondary structure, it exhibits an extraordinarily low melting temperature (tm). An in vitro tRNASerUGA transcript without modified nucleosides had an even lower tm and slightly less hyperchromicity, but its tertiary structure was apparently very similar to that of the native counterpart judging from its aminoacylation activity and the body of experimental evidence so far obtained for canonical tRNAs. The transcript was therefore used to investigate the higher-order structure and thermal instability of tRNASerUGA. 1H-NMR analysis of the transcript showed that it takes a nearly L-shaped tertiary structure with similar tertiary base-pairings to those found in yeast tRNAPhe, which is representative of canonical tRNAs. However, magnesium ion titration revealed that Mg2+ affected the chemical shifts of the tRNASerUGA transcript differently than those of canonical tRNAs so far studied; the former was less sensitive toward Mg2+, especially in the D-arm region. This observation was confirmed by NMR analysis with paramagnetic manganese ion titration. Hill plots derived from the CD spectral changes caused by titration with Mg2+ suggested that the tRNASerUGA transcript had fewer Mg2+ binding sites than those of yeast tRNAPhe as well as its transcript, a finding that was consistent with the NMR data. We thus surmise that the thermal instability of both the transcript and tRNASerUGA itself originated from a reduction in the number of the divalent ion binding sites within the tRNA molecule. These results suggest a new type of thermal instability for mitochondrial tRNA.

A 15N-1H nuclear magnetic resonance study on the interaction between isoleucine tRNA and isoleucyl-tRNA synthetase from Escherichia coli.

Imino 15N and 1H resonances of Escherichia coli tRNA(lIle) were observed in the absence and presence of E coli isoleucyl-tRNA synthetase. Upon complex formation of tRNA(lIle) with isoleucyl-tRNA synthetase, some imino 15N-1H resonances disappeared, and some others were significantly broadened and/or shifted in the 1H chemical shift, while the others were observed at the same 15N-1H chemical shifts. It was indicated that the binding of tRNA(lIle) with IleRS affect the following four regions: the anticodon stem, the junction of the acceptor and T stems, the middle of the D stem, and the region where the tertiary base pair connects the T, D, and extra loops. This result is consistent with those of chemical footprinting and site-directed mutagenesis studies. Taken together, these three independent results reveal the recognition mechanism of tRNA(lIle) by IleRS: IleRS recognizes all the identity determinants distributed throughout the tRNA(lIle) molecule, which induces changes in the secondary and tertiary structures of tRNA(lIle).

Stable isotope-edited NMR analysis of Ascaris suum mitochondrial tRNAMet having a TV-replacement loop.

Most nematode mitochondrial (mt) tRNAs have a TV-replacement loop (TV loop) which replaces the normal T arm and the variable loop in standard tRNAs with a less-structured loop. The tertiary structure of such tRNAs has been discussed theoretically with reference to the crystal structure of yeast tRNAPhe [Wolstenholme et al. (1994) Nucleic Acids Res. 22, 4300-4306] and examined experimentally by chemical and enzymatic probing [Watanabe et al. (1994) J. Biol. Chem. 269, 22902-22906]. The results suggest that most regions of the tRNA other than the TV loop are folded in a similar manner to yeast tRNAPhe. To confirm this notion more clearly, the tertiary structure of Ascaris suum mt tRNAMet was analyzed by NMR using various synthetic tRNAs site-specifically labeled with stable isotopes, which were prepared by a combination of chemical synthesis and enzymatic ligation. Tertiary interactions involving G(L2), G(L3), U(L4), and U8 were observed in the NMR spectra of the labeled tRNAs, but those relating to G(L5) were not. On the basis of these results, a possible tertiary structural model of nematode mitochondrial tRNAMet was constructed.

Assignment of imino proton signals of G-C base pairs and magnesium ion binding: an NMR study of bovine mitochondrial tRNA(SerGCU) lacking the entire D arm.

The mammalian mitochondrial tRNA(SerGCU) (mt tRNA[SerGCU]) has a unique structure in that it lacks the whole D arm. To elucidate its higher-order structure, we synthesized unmodified bovine mt tRNA(SerGCU) using T7 RNA polymerase and measured its 1H-NMR spectrum in the imino proton region. Although the imino proton signals heavily overlapped, we succeeded in assigning all the seven imino proton signals of the G-C base pairs by a combination of base replacement and 15N-labeling of the G residues of a whole tRNA molecule or of the 3'-half fragment. The results indicate that the tRNA possesses the secondary structure that has been supposed on the basis of biochemical studies. Analysis of the effect of the magnesium concentration on the G-C pairs suggests that the acceptor and T stems do not form a co-axial helix, and that the core region of the tRNA does not interact with magnesium ions. These features are significantly different from those of canonical tRNAs. Despite this, it is very likely that the tRNA as a whole takes a nearly L-shape tertiary structure.

Automated chemical synthesis of biologically active tRNA having a sequence corresponding to Ascaris suum mitochondrial tRNA(Met) toward NMR measurements.

RNA samples corresponding to Ascaris suum mitochondrial tRNA(Met) were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same 1H-NMR spectrum in the iminoproton region as the ligated tRNA. This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.

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.

Hairpin structure of an RNA 28-mer, which contains a sequence of the enzyme component of a hammerhead ribozyme system: evidence for tandem G:A pairs that are not of side-by-side type.

An RNA 28-mer (Rz28) was obtained as a major product by in vitro transcription with T7 RNA polymerase of a promoter-template DNA, which contains a sequence for the enzyme component, RNA 24-mer (Rz24), of a mutant hammerhead ribozyme system. Sequence analysis and enzymatic probing study showed that Rz28 has 4 extra nucleotides at the 3'-terminus, the sequence of which is complementary to that of the 5'-terminal sequence of Rz24, and forms a stable hairpin structure. NMR studies using a 15N-guanine-labeled derivative suggested that Rz28 contains tandem G:A pairs that are not of the side-by-side type which is found in the crystal structure of hammerhead ribozyme complexes. Comparison of the HMQC spectra of 15N-guanine-labeled Rz28 and Rz24 suggested that Rz24 also contains the same type of tandem G:A pairs.

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).

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.

Structure of biologically active and inactive cerebrosides prepared from Schizophyllum commune.

A cerebroside fraction prepared from the mycelia of Schizophyllum commune was further fractionated into five components (I-V) by reverse-phase high-performance liquid chromatography. Fruiting-inducing activity was found in I-IV but not in V. By gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses it was shown that these fractions contained: I, a mixture of N-2'-hydroxypentadecanoyl-1-O-glucosyl-nonadecasphingadienine++ + and N-2'-hydroxyhexadecanoyl-1-O-glucosyl-sphingadienine; II, (4E,8E)-N-D-2'-hydroxyhexadecanoyl-1-O-beta-D-glucopyr anosyl-9-methyl-4,8- sphingadienine (Kawai and Ikeda. 1983. Biochim. Biophys. Acta. 754: 243-248); III, N-2'-hydroxyheptadecanoyl-1-O-glucosyl-nonadecasphingadienine++ +; IV, N-2'-hydroxyoctadecanoyl-1-O-glucosyl-nonadecasphinadienine; V, (4E,8E)-N-2'-hydroxytetracosanoyl-1-O-beta-glucopyrano syl-9-methyl-4,8- sphingadienine. The only structural difference observed between biologically active and inactive cerebrosides was the chain length of acyl moiety; the cerebroside having an acyl chain of 24 carbon atoms was inactive.

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.