Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein

Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.     

Establishment and characterization of CAG/EGFP transgenic rabbit line

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.     

Cyclic RGD peptide-conjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins

A cyclic RGD peptide-conjugated block copolymer, cyclo[RGDfK(CX-)]-poly(ethylene glycol)-polylysine (c(RGDfK)-PEG-PLys), was synthesized from acetal-PEG-PLys under mild acidic conditions and spontaneously associated with plasmid DNA (pDNA) to form a polyplex micelle in aqueous solution. The cyclic RGD peptide recognizes alphavbeta3 and alphavbeta5 integrin receptors, which play a pivotal role in angiogenesis, vascular intima thickening, and the proliferation of malignant tumors. The c(RGDfK)-PEG-PLys/pDNA polyplex micelle showed a remarkably increased transfection efficiency (TE) compared to the PEG-PLys/pDNA polyplex micelle for the cultured HeLa cells possessing alphavbeta3 and alphavbeta5 integrins. On the other hand, in the transfection against the 293T cells possessing no alphavbeta3 and a few alphavbeta5 integrins, the TE of the c(RGDfK)-PEG-PLys/pDNA micelle showed no increase compared to the TE of the PEG-PLys/pDNA micelle. Flow cytometric analysis revealed a higher uptake of the c(RGDfK)-PEG-PLys/pDNA micelle than the PEG-PLys/pDNA micelle against HeLa cells, consistent with the transfection results. Furthermore, a confocal laser scanning microscopic observation revealed that the pDNA in the c(RGDfK)-PEG-PLys micelle preferentially accumulated in the perinuclear region of the HeLa cells within 3 h of incubation. No such fast and directed accumulation of pDNA to the perinuclear region was observed for the micelles without c(RGDfK) ligands. These results indicate that the increase in the TE induced by the introduction of the c(RGDfK) peptide ligand was due to an increase in cellular uptake as well as facilitated intracellular trafficking of micelles toward the perinuclear region via alphavbeta3 and alphavbeta5 integrin receptor-mediated endocytosis, suggesting that the cyclic RGD peptide-conjugated polyplex micelle has promising feasibility as a site-specifically targetable gene delivery system.     

Hypermetabolism of fat in V1a vasopressin receptor knockout mice

[Arg8]Vasopressin (AVP) has an antilipolytic action on adipocytes, but little is known about the mechanisms involved. Here, we examined the involvement of the V1a receptor in the antilipolytic effect of AVP using V1a receptor-deficient (V1aR-/-) mice. The levels of blood glycerol were increased in V1aR-/- mice. The levels of ketone bodies, such as acetoacetic acid and 3-hydroxybutyric acid, the products of the lipid metabolism, were increased in V1aR-/- mice under a fasting condition. Triacylglyceride and free fatty acid levels in blood were decreased in V1aR-/- mice. Furthermore, measurements with tandem mass spectrometry determined that carnitine and acylcarnitines in serum, the products of beta-oxidation, were increased in V1aR-/- mice. Most acylcarnitines were increased in V1aR-/- mice, especially in the case of 2-carbon (C2), C10:1, C10, C14:1, C16, C18:1, and hydroxy-18:1-carbon (OH-C18:1)-acylcarnitines under feeding rather than under fasting conditions. The analysis of tissue C2-acylcarnitine level showed that beta-oxidation was promoted in muscle under the feeding condition and in liver under the fasting condition. An in vitro assay using brown adipocytes showed that the cells of V1aR-/- mice were more sensitive to isoproterenol for lipolysis. These results suggest that the lipid metabolism is enhanced in V1aR-/- mice. The cAMP level was enhanced in V1aR-/- mice in response to isoproterenol. The phosphorylation of Akt by insulin stimulation was reduced in V1aR-/- mice. These results suggest that insulin signaling is suppressed in V1aR-/- mice. In addition, the total bile acid, taurine, and cholesterol levels in blood were increased, and an enlargement of the cholecyst was observed in V1aR-/- mice. These results indicated that the production of bile acid was enhanced by the increased level of cholesterol and taurine. Therefore, these results indicated that AVP could modulate the lipid metabolism by the antilipolytic action and the synthesis of bile acid via the V1a receptor.     

Time-resolved X-ray diffraction study on poly[(R)-3-hydroxybutyrate] films during two-step-drawing: generation mechanism of planar zigzag structure

Uniaxially oriented films with high tensile strength were processed from ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] (P(3HB)) by a method combining hot-drawing near the melting point of the polymer and two-step-drawing at room temperature. In a two-step-drawn and subsequently annealed film, P(3HB) molecular chains fall into two states: 2/1 helix (alpha-form) and planar zigzag (beta-form) conformations. The mechanism for generating the beta-form during two-step-drawing was investigated by time-resolved synchrotron wide- and small-angle X-ray scattering measurements (WAXD and SAXS), together with the measurement of stress-strain curves. It was found that the improvement of mechanical properties is due to not only the orientation of molecular chains but also the generation of the beta-form during the drawing. The crystal and molecular structures of the alpha-form remained unchanged until the yield point of the stress-strain curve. At the yield point, the long period obtained from SAXS doubled and a new reflection indicative of the beta-form was observed on the equatorial line in WAXD. The intensity of the reflection from the beta-form increased with an increase in the two-step-drawing ratio at room temperature. The SAXS pattern changed from a two-point reflection along the meridian to a cross pattern with streaking on the equatorial line, demonstrating the close alignment of shish-kebab structures. The reflection intensity, crystal orientation and crystal size of the alpha-form decreased during two-step-drawing. Based on these results, the beta-form is mainly introduced from the orientation of free molecular chains in the amorphous regions between alpha-form lamellar crystals, but the structural transformation of molecular chains also occurs from the alpha-form to the beta-form at the deformed lamellar crystals.     

Movements generated by intraspinal microstimulation in the intermediate gray matter of the anesthetized, decerebrate, and spinal cat

The intermediate laminae of the lumbosacral spinal cord are suggested to contain a small number of specialized neuronal circuits that form the basic elements of movement construction ("movement primitives"). Our aim was to study the properties and state dependence of these hypothesized circuits in comparison with movements elicited by direct nerve or muscle stimulation. Microwires for intraspinal microstimulation (ISMS) were implanted in intermediate laminae throughout the lumbosacral enlargement. Movement vectors evoked by ISMS were compared with those evoked by stimulation through muscle and nerve electrodes in cats that were anesthetized, then decerebrated, and finally spinalized. Similar movements could be evoked under anesthesia by ISMS and nerve and muscle stimulation, and these covered the full work space of the limb. ISMS-evoked movements were associated with the actions of nearby motoneuron pools. However, after decerebration and spinalization, ISMS-evoked movements were dominated by flexion, with few extensor movements. This indicates that the outputs of neuronal networks in the intermediate laminae depend significantly on descending input and on the state of the spinal cord. Frequently, the outputs also depended on stimulus intensity. These experiments suggest that interneuronal circuits in the intermediate and ventral regions of the spinal cord overlap and their function may be to process reflex and descending activity in a flexible manner for the activation of nearby motoneuron pools.     

In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.     

Crystal structure of the endonuclease domain encoded by the telomere-specific long interspersed nuclear element, TRAS1

The telomere-specific long interspersed nuclear element, TRAS1, encodes an endonuclease domain, TRAS1-EN, which specifically cleaves the telomeric repeat targets (TTAGG)n of insects and (TTAGGG)n of vertebrates. To elucidate the sequence-specific recognition properties of TRAS1-EN, we determined the crystal structure at 2.4-A resolution. TRAS1-EN has a four-layered alpha/beta sandwich structure; its topology is similar to apurinic/apyrimidinic endonucleases, but the beta-hairpin (beta10-beta11) at the edge of the DNA-binding surface makes an extra loop that distinguishes TRAS1-EN from cellular apurinic/apyrimidinic endonucleases. A protein-DNA complex model suggests that the beta10-beta11 hairpin fits into the minor groove, enabling interaction with the telomeric repeats. Mutational studies of TRAS1-EN also indicated that the Asp-130 and beta10-beta11 hairpin structure are involved in specific recognition of telomeric repeats.     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.