Multiple nodal-related genes act coordinately in Xenopus embryogenesis.

Four nodal-related genes (Xnr1-4) have been isolated in Xenopus to date, and we recently further identified two more, Xnr5 and Xnr6. In the present functional study, we constructed cleavage mutants of Xnr5 (cmXnr5) and Xnr6 (cmXnr6) which were expected to act in a dominant-negative manner. Both cmXnr5 and cmXnr6 inhibited the activities of Xnr5 and Xnr6 in co-overexpression experiments. cmXnr5 also inhibited the activity of Xnr2, Xnr4, Xnr6, derrière, and BVg1, but did not inhibit the activity of Xnr1 or activin. Misexpression of cmXnr5 led to a severe delay in initiation of gastrulation and phenotypic changes, including defects in anterior structures, which were very similar to those seen in maternal VegT-depleted embryos. Further, although the expression of Xnr1, Xnr2, and Xnr4 was not delayed in these embryos, it was markedly reduced. Injection of cmXnr5 had no notable effect on expression of Xnr3, Xnr6, derrière, or siamois. Several mesodermal and endodermal markers also showed delayed and decreased expression during gastrulation in cmXnr5-injected embryos. These results suggest that, in early Xenopus embryogenesis, nodal-related genes may heterodimerize with other TGF-beta ligands, and further that one nodal-related gene alone is insufficient for mesendoderm formation, which may require the cooperative interaction of multiple nodal-related genes.     

Microdetermination of unbound tryptophan in plasma by a combination of ultrafiltration and high-performance liquid chromatography

Previous methods for measuring unbound plasma tryptophan are not completely satisfactory, and therefore we have developed an improved method for this purpose. Unbound tryptophan is separated from bound tryptophan by centrifugation through Amicon ultrafiltration membrane cones in a short period (within 2 min). The first fraction of filtrate is obtained in 30 s by centrifugation at 3000g at controlled pH and is assayed with a high-performance liquid chromatography system. The first filtrate has a higher tryptophan concentration than that obtained by prolonged centrifugation. We propose that the tryptophan concentration in the first filtrate is the value nearest that of the plasma, since a change of equilibrium between bound and unbound tryptophan during the separation procedure is quite small. The method is also simple and convenient for clinical application.     

Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching

To investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go.     

Matured Hop Bittering Components Induce Thermogenesis in Brown Adipose Tissue via Sympathetic Nerve Activity

Obesity is the principal symptom of metabolic syndrome, which refers to a group of risk factors that increase the likelihood of atherosclerosis. In recent decades there has been a sharp rise in the incidence of obesity throughout the developed world. Iso-α-acids, the bitter compounds derived from hops in beer, have been shown to prevent diet-induced obesity by increasing lipid oxidation in the liver and inhibition of lipid absorption from the intestine. Whereas the sharp bitterness induced by effective dose of iso-α-acids precludes their acceptance as a nutrient, matured hop bittering components (MHB) appear to be more agreeable. Therefore, we tested MHB for an effect on ameliorating diet-induced body fat accumulation in rodents. MHB ingestion had a beneficial effect but, compared to iso-α-acids and despite containing structurally similar compounds, acted via different mechanisms to reduce body fat accumulation. MHB supplementation significantly reduced body weight gain, epididymal white adipose tissue weight, and plasma non-esterified free fatty acid levels in diet-induced obese mice. We also found that uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) was significantly increased in MHB-fed mice at both the mRNA and protein levels. In addition, MHB administration in rats induced the β-adrenergic signaling cascade, which is related to cAMP accumulation in BAT, suggesting that MHB could modulate sympathetic nerve activity innervating BAT (BAT-SNA). Indeed, single oral administration of MHB elevated BAT-SNA in rats, and this elevation was dissipated by subdiaphragmatic vagotomy. Single oral administration of MHB maintained BAT temperature at a significantly higher level than in control rats. Taken together, these findings indicate that MHB ameliorates diet-induced body fat accumulation, at least partly, by enhancing thermogenesis in BAT via BAT-SNA activation. Our data suggests that MHB is a useful tool for developing functional foods or beverages to counteract the accumulation of body fat.     

Salicylic Acid Signaling Controls the Maturation and Localization of the Arabiclopsis Defense Protein ACCELERATED CELL DEATH6

ACCELERATED CELL DEATH6 （ACD6） is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid （SA）. This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum （ER）- and plasma membrane （PM）-Iocalized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 （FLS2） and BRI1-ASSOClATED RECEPTOR KINASE 1 （BAK1）. FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses.     

Identification of Fasciola species isolated from Egypt based on sequence analysis of genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers

Fascioliasis has a negative impact on the farming industry in both developed and developing countries, rather than a public health challenge. This study was performed to identify Fasciola sp. from different definitive hosts (buffalo, cattle, and sheep) based on the molecular parameters and spermatogenesis. Ninety-one adult flukes were collected from livers of slaughtered animals at abattoirs in different prefectures in Egypt. Microscopic examination of the analyzed flukes showed many normal spermatozoa in the seminal vesicles (spermic), suggesting that they have the ability of spermatogenesis. This study showed that no parthenogenic Fasciola species occurred in Egypt. Molecular analysis was performed utilizing genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers. Whereas 16 animals proved to have infection with a single Fasciola species, 2 were infected with both F. hepatica and F. gigantica. The results indicated that sheep were prone to F. hepatica (8 out of 10 animals) more than F. gigantica infection. Sequences of ITS1 and ITS2 ribosomal region indicated that the flukes were categorized into 3 groups F. hepatica-type (47), F. gigantica-type (42) and 2 flukes possessed sequences of both types indicating an existence of different alleles at the same loci. Unique overlapping of T/C bases were detected in both ITS1 (Position 96) and ITS2 (Position 416). Based on results of mitochondrial gene markers (NDI and COI), flukes were classified into F. hepatica-type and F. gigantica-type. Extensive intra-sequence polymorphism was detected at both markers. NDI and COI sequences of Egyptian strain of F. gigantica showed pronounced diversity compared with relevant sequences at database.     

Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation

Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: l-arginine (l-Arg) →N-hydroxy-l-arginine →citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retard NO synthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the l-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498–33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the l-Arg reaction but also during the N-hydroxy-l-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS (Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L., and Yeh, S. R. (2007) J. Am. Chem. Soc. 129, 6943–6951).Nitric-oxide synthase (NOS) is a heme-containing flavoenzyme that synthesizes nitric oxide (NO) from l-arginine (l-Arg) in a two-step process (Scheme 1). In the first step of the reaction, one molecule of O₂ and two electrons from NADPH are consumed for the conversion of l-Arg to N-hydroxy-l-arginine (NOHA).² In the second step of the reaction, another molecule of O₂ and an additional electron from NADPH are used to convert NOHA to l-citrulline and NO. Previous studies suggest that the two steps of the reaction follow distinct mechanisms meditated by a compound I (Cmpd I) type of ferryl intermediate and a peroxyl intermediate, respectively (1–7). These mechanisms, however, remain elusive, as none of the putative intermediates have been experimentally observed under solution conditions, although (hydro)peroxo intermediates have been identified at cryogenic temperatures by radiolytic reduction methods (8, 9); in addition, a Cmpd I intermediate has been observed after peroxyacid treatment (10).Three isoforms of NOS have been identified in mammals: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). Similar to the P450 class of enzymes, the heme prosthetic group in all three isoforms of NOS is coordinated by a thiolate sidechain group of an intrinsic cysteine residue in the proximal heme pocket. In P450s, the thiolate ligand forms a H-bond with a peptide NH group (11), whereas in NOSs the analogous thiolate ligand accepts a H-bond from the side chain of a conserved tryptophan residue (Trp-188 in iNOS). It is believed that the H-bonding interaction with the tryptophan residue reduces the electron donating capability of the thiolate ligand in NOSs, thereby modulating the oxygen chemistry occurring in the distal heme pocket of the enzymes (1, 12–15). The mutation of the conserved tryptophan (Trp-409) in neuronal NOS to Phe or Tyr was shown to increase the rate of NO synthesis during multiple turnover conditions by decreasing the heme reduction rate and the degree of NO autoinhibition (15, 16). Comparable mutants of iNOS, W188F, and W188Y, could not be overexpressed as stable recombinant forms (17); however, the W188H mutant was successfully expressed, purified, and studied (18).It was shown that the W188H mutation slowed down the l-Arg hydroxylation reaction by stabilizing a new intermediate with a Soret maximum at 420 nm, which had never been observed during the wild type reaction, and that the formation of the 420-nm intermediate coincides with the disappearance of the ternary complex of the enzyme and the formation of a H₄B radical, whereas its decay was concurrent with the recovery of the resting ferric enzyme. Tejero et al. (18) postulated that the 420-nm species is a catalytically competent oxygen-containing intermediate, such as a Cmpd I type of ferryl species. Regardless of the identity of the intermediate, the data demonstrated that the mutation modulates the structural properties and biochemical reactivity of the enzyme. However, the crystallographic data of the W188H mutant of the oxygenase domain of iNOS (iNOS_oxy) revealed that its active site structure is strikingly similar to that of the wild type enzyme (18). In particular, the side chain of His-188, like that of Trp-188 in the wild type enzyme, formed a H-bond with the thiolate ligand of the heme.Open in a separate windowTo determine how the W188H mutation modulates the oxygen chemistry of iNOS_oxy without significantly perturbing the active site structure of the enzyme, we carried out a series of studies of the W188H mutant with optical absorption, resonance Raman, and EPR spectroscopic methods under steady-state and single turnover conditions. We discovered that the mutation introduced a unique out-of-plane distortion to the heme macrocycle that stabilizes the 420-nm intermediate populated during both the l-Arg and NOHA reactions and at the same time destabilizes the NO bound to the ferric heme during the NOHA reaction. The results are summarized and discussed in the context of the previously postulated NOS mechanism (1).     

Gene expression in the vascular wall of the aortic arch in spontaneously hypertensive hyperlipidemic model rats using DNA microarray analysis

Aims

In recent years, there has been an increase in patients with arteriosclerosis and the risk of lifestyle-related diseases. However, the pathogenesis and medication of atherosclerosis have not been elucidated. We developed a rat model of lifestyle-related diseases by feeding a high-fat diet and 30% sucrose solution (HFDS) to spontaneously hypertensive hyperlipidemic rats (SHHR) and reported that this model is a useful model of early atherosclerosis. In order to elucidate the pathogenesis of early atherosclerosis, we searched for atherosclerosis-related genes by microarray analysis using the aortic arch rat model of lifestyle-related diseases.

Main methods

Four-month-old male Sprague–Dawley rats and SHHR were each divided into two normal diet (ND) groups and two HFDS groups. After a four-month treatment, the expression of mRNA in the aortic arch was detected using the oligo DNA microarray one-color method and quantified using real-time PCR.

Key findings

In this study, we detected 39 genes in microarray analysis. Esm1, Retnlb Mkks, and Grem2 showed particularly marked changes in gene expression in the SHHR-HFDS group. Compared with the SD-ND group, the SHHR-HFDS group had an increase in Mkks gene expression of about 26-fold and an approximately 22-fold increase in the expression of Grem2. Similarly, the expression of Esm1 increased by about 12-fold and that of Retnlg by about 10-fold as shown by quantitative real-time PCR.

Significance

This study suggested that these four genes might be important in early atherosclerosis development.     

Comparative characterization of GPRC5B and GPRC5CLacZ knockin mice; behavioral abnormalities in GPRC5B-deficient mice

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor.