Factors controlling denitrification of mudflat sediments in Ariake Bay, Japan.

To investigate the seasonal variation of denitrification rate (DR) and clarify the controlling factors of denitrification in the mudflat sediments of Ariake Bay, we conducted field surveys biweekly each month from April 2006 to January 2008. NH4(+)-N porewater concentration increased from summer to autumn due to the organic material mineralization under higher sediment temperatures. The seasonal pattern of NH4 (+)-N flux between sediments and overlying water interface indicated that the mudflat sediments were a source of NH4(+)-N in summer. NO3(-)+NO2(-)-N porewater concentrations were low, ranging from 0.53 to 11.46 µM, and mudflat sediments were sinks of NO3(-)+NO2(-)-N throughout the year. The mean number of denitrifiers tended to increase in July-September (2188-75,057 MPN g(-1)) and to decrease in March-May (500-3740 MPN g(-1)). DR tended to increase in summer, ranging from 76.03 to 990.21 µmol m(-2) day(-1), and to decrease in winter, ranging from 25.01 to 206.07 µmol m(-2) day(-1). There was no significant correlation between DR and denitrifier number. Environmental factors influencing DR during the investigation period were determined by multiple regression analysis with the stepwise method. The results indicated that NO3(-)+NO2(-)-N flux was an important factor in denitrification of mudflat sediments in Ariake Bay. Denitrification was depended on nitrate diffusing from overlying water into sediments under reduced sediment conditions during summer-mid-autumn. On the other hand, in late autumn-winter at Eh>+200 mV and sediment temperature >10 °C, nitrate produced by sediment nitrification was thought to be denitrified subsequently; that is, the coupled nitrification-denitrification may have taken place in the upper layer of mudflat sediments.

Repetitive sequences originating from the centromere constitute large-scale heterochromatin in the telomere region in the siamang, a small ape.

Chromosomes of the siamang Symphalangus syndactylus (a small ape) carry large-scale heterochromatic structures at their ends. These structures look similar, by chromosome C-banding, to chromosome-end heterochromatin found in chimpanzee, bonobo and gorilla (African great apes), of which a major component is tandem repeats of 32-bp-long, AT-rich units. In the present study, we identified repetitive sequences that are a major component of the siamang heterochromatin. Their repeat units are 171 bp in length, and exhibit sequence similarity to alpha satellite DNA, a major component of the centromeres in primates. Thus, the large-scale heterochromatic structures have different origins between the great apes and the small ape. The presence of alpha satellite DNA in the telomere region has previously been reported in the white-cheeked gibbon Nomascus leucogenys, another small ape species. There is, however, a difference in the size of the telomere-region alpha satellite DNA, which is far larger in the siamang. It is not known whether the sequences of these two species (of different genera) have a common origin because the phylogenetic relationship of genera within the small ape family is still not clear. Possible evolutionary scenarios are discussed.

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans.

Tol1 is a DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes, and has been proven to be transposed in various vertebrate species, including mammals. This element belongs to the hAT (hobo/Activator/Tam3) transposable element family, whose members are distributed in a wide range of organisms. It is thus possible that Tol1 is mobile in organisms other than vertebrates. We here show that transposition of this element occurs in the nematode Caenorhabditis elegans. A donor plasmid containing a Tol1 element and a helper plasmid carrying the transposase gene were delivered into gonad cells and, after several generations of culturing, were recovered from worms. PCR analysis of the donor plasmid, using primers that encompassed the Tol1 element, revealed excision of the Tol1 portion from the plasmid. Analysis of genomic DNA of the worms by the inverse PCR method provided evidence that Tol1 had been integrated into the C. elegans chromosomes. Vertebrates and C. elegans are phylogenetically distantly related organisms in that the former are deuterostomes and the latter a protostome animal. Our results indicate (1) the transposition reaction of the Tol1 element requires, besides the transposase, no factors from host cells, or (2) the host factors, even if required, are those that are common to protostomes and deuterostomes. The results also have significance for the development of a gene transfer vector and other biotechnology tools for C. elegans.

Tramadol, but not its major metabolite (mono-O-demethyl tramadol) depresses compound action potentials in frog sciatic nerves.

BACKGROUND AND PURPOSE: Although tramadol is known to exhibit a local anaesthetic effect, how tramadol exerts this effect is not understood fully. EXPERIMENTAL APPROACH: The effects of tramadol and its metabolite mono-O-demethyl-tramadol (M1) on compound action potentials (CAPs) were examined by applying the air-gap method to frog sciatic nerves, and the results were compared with those of other local anaesthetics, lidocaine and ropivacaine. KEY RESULTS: Tramadol reduced the peak amplitude of the CAP in a dose-dependent manner (IC50=2.3 mM). On the other hand, M1 (1-2 mM), which exhibits a higher affinity for mu-opioid receptors than tramadol, did not affect CAPs. These effects of tramadol were resistant to the non-selective opioid receptor antagonist naloxone and the mu-opioid receptor agonist, DAMGO, did not affect CAPs. This tramadol action was not affected by a combination of the noradrenaline uptake inhibitor, desipramine, and the 5-hydroxytryptamine uptake inhibitor, fluoxetine. Lidocaine and ropivacaine also concentration-dependently reduced CAP peak amplitudes with IC50 values of 0.74 and 0.34 mM, respectively. CONCLUSIONS AND IMPLICATIONS: These results indicate that tramadol reduces the peak amplitude of CAP in peripheral nerve fibres with a potency which is less than those of lidocaine and ropivacaine, whereas M1 has much less effect on CAPs. This action of tramadol was not produced by activation of mu-opioid receptors nor by inhibition of noradrenaline and 5-hydroxytryptamine uptake. It is suggested that the methyl group present in tramadol but not in M1 may play an important role in producing nerve conduction block.

Beneficial effect of concomitant induction with antilymphoblast globulin, cyclosporine, and steroids on long-term renal allograft outcome.

BACKGROUND: The addition of induction therapy with antilymphocytic antibodies to cyclosporine (CsA) based immunosuppression, has reduced acute rejection incidence and improved short-term survivals, but has not had well-established effects on long-term renal transplant survival. PATIENTS: We analyzed the long-term allograft outcome of patients included in a prospective randomized clinical study conducted in our center 15 years ago by comparing two strategies: (A) horse antilymphoblast globulin (ALG) given at 10 mg/kg on alternate days to a maximum of 6 doses with low-dose CsA started at 8 mg/kg per day and prednisone at 0.25 mg/kg per day, versus (B) CsA started at 15 mg/kg per day and prednisone at 0.5 mg/kg per day. Diabetic and highly sensitized patients (PRA > 70%) were excluded from the study. RESULTS: The characteristics of the 50 patients enrolled in each group were not different. Although patient survival was not different (88% in group A vs 77% in group B), recipients treated with ALG showed a lower incidence of acute rejection episodes (20% vs 44%, P = .01) and better death-censored renal allograft survival (57% vs 41%, P = .03). Among rejection-free patients, graft survival was 15% higher in group A (60% vs 45%, P = .12). Multivariate Cox regression analysis showed that an acute rejection episode (relative risk [RR]: 2.44, 95% confidence interval [CI] 1.36-4.39; P = .0029) rather than ALG immunosuppression (RR 0.74, 95% CI 0.41-1.33; P = NS) was an independent predictor of death-censored graft survival. CONCLUSIONS: In summary, we confirmed that concomitant induction therapy with ALG, CsA, and steroids improves long-term renal allograft survival.

Superconductivity without inversion symmetry: MnSi versus CePt3Si.

Superconductivity in materials without spatial inversion symmetry is studied. We show that in contrast to common belief, spin-triplet pairing is not entirely excluded in such systems. Moreover, paramagnetic limiting is analyzed for both spin-singlet and -triplet pairing. The lack of inversion symmetry reduces the effect of the paramagnetic limiting for spin-singlet pairing. These results are applied to MnSi and CePt3Si.

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer.

We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blastocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated.

Functional interference between estrogen-related receptor alpha and peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha heterodimer complex in the nuclear receptor response element-1 of the medium chain acyl-coenzyme A dehydrogenase gene.

We undertook a study of molecular interference of nuclear orphan receptors. Nuclear receptor response element-1 (NRRE-1) from the human medium-chain acyl coenzyme A dehydrogenase (MCAD) gene promoter was shown to contain three hexamer elements (site 1 through 3) that are known to interact with a number of nuclear receptors including chicken ovalbumin upstream promoter transcription factor (COUP-TF) and estrogen-related receptor alpha (ERRalpha). We demonstrated that the peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha (PPARalpha/RXRalpha) heterodimer complex can also bind to the two hexamer repeat sequences (between site 1 and site 3) arranged as an everted imperfect repeat separated by 14 bp (ER14). Mutations of the putative core elements have shown that these three sites are differentially involved in ERRalpha and PPARalpha/RXRalpha binding. Homodimer of ERRalpha was shown to interact between site 1 and site 3 (ER14). To date, no nuclear receptor is known to bind to response elements over such long intervals. Interestingly, site 1 was shown to be essential for ERRalpha binding while site 3 supports its binding only in the presence of site 1. Furthermore, it was shown that the binding profile of ERRalpha and PPARalpha/RXRalpha are competitive rather than making a high order complex within NRRE-1. At the cellular level, transcriptional activation driven by the PPARalpha/RXRalpha complex was counteracted by the expression of ERRalpha in HeLa cells. These results suggest that ERRalpha and PPARalpha/RXRalpha could interfere with each other's function through binding to similar DNA elements, thereby finetuning the transcriptional outcome of the target gene. Our findings suggest a mechanism whereby multiple nuclear receptors can activate or repress DNA binding or transcription via a single pleiotropic regulatory element.

Trypanocidal activity of extracts from Brazilian Atlantic Rain Forest plant species.

The trypanocidal activity of crude hydro alcoholic extracts and several fractions of 13 plants from Brazilian Atlantic Rain Forest were tested in vitro against epimastigote and trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. Crude ethanol extracts with promising in vitro activity (DL50 between 5-10 microg/ml) against epimastigotes were fractionated by solvent partition and further tested against bloodstream form of the parasite. Activity against bloodstream parasites was observed in both dichloromethane and hexane fractions of Polygala sabulosa and P. paniculata.

Gamera, a family of LINE-like repetitive sequences widely distributed in medaka and related fishes.

A family of repetitive sequences, designated Gamera, has been identified in the genome of the Hainan medaka fish Oryzias curvinotus, a closely related species to the common medaka fish O. latipes. Sequencing and Southern blot analyses of this family revealed: (1) amino acid sequence similarity to reverse transcriptase domains of long interspersed nuclear elements (LINEs); (2) 5' truncation of dispersed copies; and (3) the disruption of another genetic element, indicating a past transposition event. These results suggest that Gamera belongs to the LINE superfamily. Gamera is widely distributed in the genus Oryzias, and the phylogenetic relationship might indicate its presence in the common ancestor of the genus.

Contribution of trabecular and cortical components to the mechanical properties of bone and their regulating parameters.

To evaluate the mechanical contributions of the spongiosa and cortex to the whole rat vertebra, we developed a finite element analysis (FEA) system linked to three-dimensional data from microcomputed tomography (micro-CT). Twenty-eight fifth lumbar vertebrae (L-5) were obtained from 10-month-old female rats, comprised of ovariectomized (ovx, n = 6), sham operated (n = 7), and alfacalcidol-treated after ovx (0.1 microg/kg [n = 8] and 0.2 microg/kg [n = 7]) groups. The trabecular microstructure of L-5 was measured by micro-CT. Yield strength at the tissue level (YS), defined as the value at which 0.034% of all elements reached yield stress, was calculated by the FEA. Then, the ultimate compressive load of each specimen was measured by mechanical testing. The YS of the whole bone (YSw) showed a significant correlation with ultimate load (r = 0.91, p < 0.0001). The YS values of the isolated spongiosa (YSs) and cortex (YSc) were calculated in models with varying amounts of trabecular or cortical bone mass. The mechanical contribution of the spongiosa showed a nonlinear relationship with bone mass, and ovx reduced the mean mechanical contribution of the spongiosa to the whole bone by 13% in comparison to the sham group. YSs had a strong relationship with trabecular microstructure, especially with trabecular bone pattern factor (TBPf) and structure model index (SMI), and YSc had a strong relationship with cortical bone volume. The structural parameters most strongly related to YSw were BV/TV and TBPf. Our micro-FEA system was validated to assess the mechanical properties of bone, including the individual properties of the spongiosa and cortex, in the osteoporotic rat model. We found that the mechanical property of each component had a significant relationship with the respective bone mass, volume, or structure. Although trabecular microstructure has a significant relationship with bone strength, in ovx bone with deteriorated trabecular microstructure, the strength depended mainly on the cortical component.

The Tol2 transposable element of the medaka fish: an active DNA-based element naturally occurring in a vertebrate genome.

Several DNA-based transposable elements are known to be present in vertebrate genomes, but few of them have been demonstrated to be active. The Tol2 element of the medaka fish is one such element and, therefore, is potentially useful for developing a gene tagging system and other molecular biological tools applicable to vertebrates. Towards this goal, analyses of the element at the molecular, cellular and population levels are in progress. Results so far obtained are described here.

Detection of de novo insertion of the medaka fish transposable element Tol2.

Tol2 is a terminal-inverted-repeat transposable element of the medaka fish Oryzias latipes. It is a member of the hAT (hobo/Activator/Tam3) transposable element family that is distributed in a wide range of organisms. We here document direct evidence for de novo insertion of this element. A Tol2 clone marked with the bacterial tetracycline-resistance gene was microinjected into fertilized eggs together with a target plasmid, and the plasmid was recovered from embryos. The screening of plasmid molecules after transformation into Escherichia coli demonstrated transposition of tet into the plasmid and, by inference, precise insertion of Tol2 in medaka fish cells. De novo excision of Tol2 has previously been demonstrated. The present study provides direct evidence that the Tol2 element has the entire activity necessary for cut-and-paste transposition. Some elements of the mariner/Tc1 family, another widespread group, have already been applied to development of gene tagging systems in vertebrates. The Tol2 element of the hAT family, having different features from mariner/Tc1 family elements, also has potential as an alternative gene tagging tool in vertebrates.

Quantum phase transitions in the shastry-sutherland model for SrCu2(BO3)(2)

We investigate quantum phase transitions in the frustrated antiferromagnetic Heisenberg model for SrCu2(BO3)(2) by using the series expansion method. It is found that a novel spin-gap phase, adiabatically connected to the plaquette-singlet phase, exists between the dimer and the magnetically ordered phases known thus far. When the ratio of the competing exchange couplings alpha( = J'/J) is varied, this spin-gap phase exhibits a first- (second-) order quantum phase transition to the dimer (the magnetically ordered) phase at the critical point alpha(c1) = 0.677(2) [ alpha(c2) = 0. 86(1)]. Our results shed light on some controversial arguments about the nature of quantum phase transitions in this model.

Chromosome mapping and expression of human tip49 family genes.

TBP-interacting protein 49 (TIP49) was originally identified as a TBP-binding protein, and two related proteins are encoded by individual genes, tip49a and b. Although the function of this gene family has not been elucidated, they are supposed to play a critical role in nuclear events because they interact with various kinds of nuclear factors and have DNA helicase activities. At least, TIP49a has been suggested to act as an autoantigen in some patients with autoimmune diseases. In this study, we investigated the chromosome positions of this family of genes. Human tip49a and tip49b genes were mapped on 3q21 and 19q13.2, respectively. Consistent with the notion that tip49 family genes are essential for cell growth, Northern blot analysis demonstrated that both genes are expressed ubiquitously in human tissues. It is worthy of notice that the testes contained large amounts of the both transcripts. These results are consistent with our previous results from tissue distribution analysis for of TIP49 proteins.

Evidence for recent invasion of the medaka fish genome by the Tol2 transposable element.

Tol2 is a transposable element of the terminal-inverted-repeat class, residing in the genome of the medaka fish Oryzias latipes. The genus Oryzias contains more than 10 species for which phylogenetic relationships have previously been estimated. To infer the history of Tol2 in this genus we performed genomic Southern blots and PCR analyses of 10 of the species. It was revealed that Tol2 occurs in 2 of the 10 species (O. curvinotus and O. latipes) and that the length and the restriction map structure of Tol2 are identical in the two cases. Further, sequencing analysis revealed an extremely low level of divergence compared with that in a nuclear gene. These results suggest recent incorporation of Tol2 into one or both of the two species, implying horizontal transfer of Tol2 from one species to the other or into them both from a common source.

Identification of a novel 70 kDa protein that binds to the core promoter element and is essential for ribosomal DNA transcription.

Mammalian ribosomal RNA genes (rDNA) are transcribed by RNA polymerase I and at least two auxiliary factors, UBF and SL1/TFID/TIF-IB. It has also been reported that an additional factor(s) is required to reconstitute efficient initiation of rDNA transcription in vitro, depending upon the procedures of chromatographic separation. In an attempt to elucidate the molecular identity of such yet uncertain activities, we have developed agarose gel shift and UV cross-linking assays to detect proteins directly bound to the core promoter region of murine rDNA. With these techniques, we identified a 70 kDa protein (p70) in the flow-through fraction of a phosphocellulose column (TFIA-fraction). Interestingly, the binding of p70 to the rDNA core promoter was observed only in the presence of the SL1-containing fraction. The probable human orthologue of p70 was also detected in HeLa cells. Consistent with the observation that p70 bound to the core promoter only in the presence of the TFIA- and SL1-fractions, alteration of DNase I footprint pattern over the core promoter element was demonstrated by cooperative action of the TFIA- and SL1-fractions. A reconstituted in vitro transcription assay with further purified p70 indicated that p70 was required for accurate initiation of rDNA transcription. These results indicate that the p70 identified recently by the current DNA-binding experiments represents a novel transcription factor in rDNA transcription.

Single-fiber analysis of mitochondrial A3243G mutation in four different phenotypes.

Five unrelated patients harboring the A3243G mutation in the mitochondrial DNA (mtDNA) but presenting with different clinical phenotype were studied for their percentage of mutation at the single muscle fiber levels. One patient had a clinically and pathologically defined Leigh syndrome (LS), two showed mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), another showed progressive external ophthalmoplegia (PEO), and the other showed mitochondrial diabetes mellitus (MDM). The mutation load was greater in the muscle from the patient with LS (92%), who showed more than 80% even in the non-ragged red fibers (RRF) and also presented the highest proportion of RRF. The patients with MELAS had lower mutation levels as well as a lower proportion of RRF, and these two parameters were even lower in the PEO and MDM patients. These results were consistent with the concept that differences in the mutation load and in the somatic distribution of the mutation among different cells and tissues are responsible for the differences in phenotypical expression of the disease.