Structural evidence for non-canonical binding of Ca2+ to a canonical EF-hand of a conventional myosin

We have previously identified a single inhibitory Ca2+-binding site in the first EF-hand of the essential light chain of Physarum conventional myosin (Farkas, L., Malnasi-Csizmadia, A., Nakamura, A., Kohama, K., and Nyitray, L. (2003) J. Biol. Chem. 278, 27399-27405). As a general rule, conformation of the EF-hand-containing domains in the calmodulin family is "closed" in the absence and "open" in the presence of bound cations; a notable exception is the unusual Ca2+-bound closed domain in the essential light chain of the Ca2+-activated scallop muscle myosin. Here we have reported the 1.8 A resolution structure of the regulatory domain (RD) of Physarum myosin II in which Ca2+ is bound to a canonical EF-hand that is also in a closed state. The 12th position of the EF-hand loop, which normally provides a bidentate ligand for Ca2+ in the open state, is too far in the structure to participate in coordination of the ion. The structure includes a second Ca2+ that only mediates crystal contacts. To reveal the mechanism behind the regulatory effect of Ca2+, we compared conformational flexibilities of the liganded and unliganded RD. Our working hypothesis, i.e. the modulatory effect of Ca2+ on conformational flexibility of RD, is in line with the observed suppression of hydrogen-deuterium exchange rate in the Ca2+-bound form, as well as with results of molecular dynamics calculations. Based on this evidence, we concluded that Ca2+-induced change in structural dynamics of RD is a major factor in Ca2+-mediated regulation of Physarum myosin II activity.     

A continuous process for the synthesis of hexyl β-D-glucoside in aqueous phase using immobilized β-glucosidase and extractive product recovery with 1-hexanol

Hexyl -d-glucoside was synthesized at 1 mM using immobilized -glucosidase with 2.0 M glucose solution with 1-hexanol. The continuous production of hexyl glucoside was achieved by its extractive recovery with 1-hexanol, and 4.9 g of product was obtained during a one week operation using a 26.5 ml reactor.     

Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting

The ribosomal frameshifting signal of the mouse embryonal carcinoma differentiation regulated (Edr) gene represents the sole documented example of programmed -1 frameshifting in mammalian cellular genes [Shigemoto,K., Brennan,J., Walls,E,. Watson,C.J., Stott,D., Rigby,P.W. and Reith,A.D. (2001), Nucleic Acids Res., 29, 4079-4088]. Here, we have employed site-directed mutagenesis and RNA structure probing to characterize the Edr signal. We began by confirming the functionality and magnitude of the signal and the role of a GGGAAAC motif as the slippery sequence. Subsequently, we derived a model of the Edr stimulatory RNA and assessed its similarity to those stimulatory RNAs found at viral frameshift sites. We found that the structure is an RNA pseudoknot possessing features typical of retroviral frameshifter pseudoknots. From these experiments, we conclude that the Edr signal and by inference, the human orthologue PEG10, do not represent a novel 'cellular class' of programmed -1 ribosomal frameshift signal, but rather are similar to viral examples, albeit with some interesting features. The similarity to viral frameshift signals may complicate the design of antiviral therapies that target the frameshift process.     

Effects of adipokines on expression of adrenomedullin and endothelin-1 in cultured vascular endothelial cells

Obesity is a major risk factor for the development of hypertension. Adipokines may cause hypertension by acting both centrally and directly on the vascular vessels. We wished to clarify whether three adipokines, leptin, resistin and tumor necrosis factor-alpha, affect expression of adrenomedullin and endothelin-1 in vascular endothelial cells. Human umbilical vein endothelial cells were cultured for 24 h with leptin (1-10 nmol/l), resistin (1-10 nmol/l) or tumor necrosis factor-alpha (1-10 ng/ml). Expression of adrenomedullin and endothelin-1 was examined by radioimmunoassay and northern blot analysis. Immunoreactive-adrenomedullin in the medium and adrenomedullin mRNA expression levels were decreased by treatment of tumor necrosis factor-alpha time- and dose-dependently, whereas endothelin-1 secretion was not significantly changed by it. Leptin or resistin had no significant effects on expression of adrenomedullin or endothelin-1 in human umbilical vein endothelial cells. Under hypoxic conditions (1% O2), expression of both adrenomedullin and endothelin-1 was induced in these cells. Immunoreactive-adrenomedullin levels in the medium were decreased by treatment of tumor necrosis factor-alpha under hypoxia. Leptin or resistin had no significant effects on adrenomedullin or endothelin-1 expression also in hypoxia. These findings have raised the possibility that decreased expression of adrenomedullin by tumor necrosis factor-alpha may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects.     

Enhanced antinociception by intracerebroventricularly and intrathecally-administered orexin A and B (hypocretin-1 and -2) in mice

Orexins are neuropeptides located exclusively in neurons of the lateral hypothalamic area, which send projections to most monoaminergic nuclei, such as noradrenergic locus coeruleus, dopaminergic ventral tegmental areas, and histaminergic tuberomammillary nuclei. The present work was carried out to examine the role of orexins in nociception in mice. C57BL/6 mice were administered with orexin A and B intracerebroventricularly (i.c.v.), intrathecally (i.t.) and subcutaneously (s.c.) to reveal the sites of action of these peptides and to examine the pain thresholds using four kinds of nociceptive tasks. Orexins showed antinociceptive effects in all four types of assays for thermal (hot-plate, tail-flick, paw-withdrawal), mechanical (tail-pressure), chemical (formalin, capsaicin and abdominal stretch) nociceptions and nociceptin-induced behavioral responses, when administered i.c.v. or i.t., whereas the s.c. administration was ineffective. The antinociceptive effects of orexin A were more remarkable than those of orexin B. The i.c.v. administration of orexin A was as effective as, or more potent than the i.t. administration. The effects of orexin A were completely blocked by adenosine type 1 receptor antagonists, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and theophylline, but not by naloxone, suggesting a possible involvement of the adenosine-containing neurons and/or the adenosine pathway in these orexin actions. The i.c.v. administration of nociceptin had no significant effects on orexin expression in the brain and spinal cord. The present findings suggest that orexins have an antinociceptive role in at least four different types of pains, probably acting on both the brain and spinal cord.     

Diagnosis of cell death induced by methylglyoxal, a metabolite derived from glycolysis, in Saccharomyces cerevisiae

Methylglyoxal (MG) is a ubiquitous metabolite derived from glycolysis; however, this aldehyde kills all types of cell. We analyzed the properties of MG-induced cell death of the budding yeast Saccharomyces cerevisiae. The MCA1 gene encodes a caspase homologue that is involved in H2O2-induced apoptosis in yeast, although the disruption of MCA1 did not repress sensitivity to MG. In addition, the intracellular oxidation level did not increase under conditions in which MG kills the cell. Furthermore, the disruption of genes encoding antioxidant enzymes did not affect the susceptibility to MG. Here, we demonstrate that yeast cells killed by MG do not exhibit the characteristics of apoptosis in a TUNEL assay or an annexin V staining, but show those of necrosis upon propidium iodide staining. We demonstrate that MG at high concentrations provokes necrotic cell death without the generation of reactive oxygen species in S. cerevisiae.     

In vitro fertilization and subsequent development of porcine oocytes using cryopreserved and liquid-stored spermatozoa from various boars

We had previously developed a porcine IVF system using a chemically defined medium, i.e., porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac). In the present study, we investigated the utility of this IVF system using different types of semen: (1) cryopreserved ejaculated (n = 8); (2) cryopreserved epididymal (n = 4); and (3) liquid-stored ejaculated (n = 5). Cryopreserved spermatozoa were prepared by three methods. In vitro-matured porcine oocytes were fertilized for 20 h in PGMtac using each type of semen, and the presumptive zygotes were cultured in porcine zygote medium (PZM)-4 for 5 days. In the case of frozen-thawed spermatozoa, the number of spermatozoa per penetrated oocyte (1.1-1.7), rate of blastocyst formation (26-56%), and total number of cells per blastocyst (34-49) differed (P < 0.05) among freezing methods. However, blastocysts were produced using all types of cryopreserved spermatozoa (14-75%). When spermatozoa were liquid-stored for 1-14 days after semen collection, the rate of sperm penetration (P < 0.05) decreased as storage time increased, although there was no significant reduction in sperm motility during storage. In all groups, semen that had been stored within 10 days after collection enabled blastocyst production in vitro (20-48%). In conclusion, this IVF system, which uses a chemically defined medium, had widespread utility with both frozen-thawed and liquid-stored spermatozoa.     

The non-selective cation-permeable channel TRPC3 is a tetrahedron with a cap on the large cytoplasmic end

TRPC3 plays important roles in neuronal differentiation and immune cell maturation by mediating the cationic current in response to phospholipase C activation, Ca2+ depletion, and diacylglycerol stimulation. Here, we purified the TRPC3 channel using a glycosylated tetramer and observed the structure using electron microscopy. Negatively stained specimens demonstrate homogeneous protein particles containing an internal cavity-like structure. These particle images were picked up by automated pick-up programs, aligned, and classified by the growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. The averaged images progress from the top view to the side views, which are representative of their raw images. The top view confirmed the hypothesis of a four-domain structure, and the side view demonstrates a large cytoplasmic domain with a capped structure at the bottom, which is near a predicted locus of ion release. The total image of the protein is a blunt-edged trapezoid of 200 x 200 x 235 A. This large dimension of TRPC3 is also supported by the Stokes radius (92 A) obtained from gel filtration chromatography.     

Genomic differentiation of Hordeum chilense from H. vulgare as revealed by repetitive and EST sequences

Hordeum vulgare, cultivated barley, and its wild relative, H. chilense, have several important traits that might be useful for wheat improvement. Here, in situ hybridization and barley expressed sequence tag (EST) markers were used to characterize and compare the chromosomes of H. chilense with those of H. vulgare. FISH with four repetitive DNA sequences, AG, AAG, 5S rDNA and 45S rDNA, was applied to the mitotic chromosomes of H. vulgare, H. chilense and available wheat-H. chilense addition and substitution lines. FISH with the AAG repeat differentiated the individual chromosomes of H. chilense and H. vulgare. The patterns of FISH signals in the two species differed greatly. The 45S rDNA signals were observed on two pairs of chromosomes in both species, while the 5S rDNA signals were observed on four pairs of chromosomes in H. vulgare and on one pair in H. chilense. The AG repeat showed FISH signals at the centromeric regions of all chromosomes of H. vulgare but none of the chromosomes of H. chilense. These results indicate that the chromosomes of the two species are highly differentiated. To study the homoeology between the two species, 209 EST markers of H. vulgare were allocated to individual chromosomes of H. chilense. One hundred and forty of the EST markers were allocated to respective chromosomes of H. chilense using the wheat-H. chilense addition and substitution lines. Twenty-six EST markers on average were allocated to each chromosome except to the chromosome 2H(ch)S, to which only 10 markers were allocated. Ninety percent of the allocated EST markers in H. chilense were placed on H. vulgare chromosomes of the same homo-eologous group, indicating that the expressed sequences of the two species were highly conserved. These EST markers would be useful for detecting chromatin introgressed from these species into the wheat genome.