Herpes simplex virus type 1 UL51 protein is involved in maturation and egress of virus particles

The UL51 gene of herpes simplex virus type 1 (HSV-1) encodes a phosphoprotein whose homologs are conserved throughout the herpes virus family. Recently, we reported that UL51 protein colocalizes with Golgi marker proteins in transfected cells and that targeting of UL51 protein to the Golgi apparatus depends on palmitoylation of its N-terminal cysteine at position 9 (N. Nozawa, T. Daikoku, T. Koshizuka, Y. Yamauchi, T. Yoshikawa, and Y. Nishiyama, J. Virol. 77:3204-3216, 2003). However, its role in the HSV replication cycle was unknown. Here, we generated UL51-null mutants (FDL51) in HSV-1 to uncover the function of UL51 protein. We show that the mutant plaques were much smaller in size and that maximal titers were reduced nearly 100-fold compared to wild-type virus. Electron microscopy indicated that the formation of nucleocapsids was not affected by the deletion of UL51 but that viral egress from the perinuclear space was severely compromised. In FDL51-infected cells, a large number of enveloped nucleocapsids were observed in the perinuclear space, but enveloped mature virions in the cytoplasm, as well as extracellular mature virions, were rarely detected. These defects were fully rescued by reinsertion of the UL51 gene. These results indicate that UL51 protein is involved in the maturation and egress of HSV-1 virus particles downstream of the initial envelopment step.     

Structural analysis of isosteviol and related compounds as DNA polymerase and DNA topoisomerase inhibitors

Isosteviol (ent-16-ketobeyeran-19-oic acid) is a hydrolysis product of stevioside, which is a natural sweetener produced in the leaves of Stevia rebaudiana (Bertoni) Bertoni. In this report, we prepared isosteviol and related compounds from stevioside by microbial transformation and chemical conversion and assayed the inhibitory activities toward DNA metabolic enzymes and human cancer cell growth. Among twelve compounds obtained, only isosteviol (compound 3) potently inhibited both mammalian DNA polymerases (pols) and human DNA topoisomerase II (topo II), and IC50 value for pol alpha was 64.0 microM. This compound had no inhibitory effect on higher plant (cauliflower) pols, prokaryotic pols, human topo I, and DNA metabolic enzymes such as human telomerase, T7 RNA polymerase, and bovine deoxyribonuclease I. With pol alpha, isosteviol acted non-competitively with the DNA template-primer and nucleotide substrate. Isosteviol prevented the growth of human cancer cells, with LD50 values of 84-167 microM, and 500 microg of the compound caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 53.0%). The relationship between the structure of stevioside-based compounds and these activities were discussed.     

The opsins

The photosensitive molecule rhodopsin and its relatives consist of a protein moiety - an opsin - and a non-protein moiety - the chromophore retinal. Opsins, which are G-protein-coupled receptors (GPCRs), are found in animals, and more than a thousand have been identified so far. Detailed molecular phylogenetic analyses show that the opsin family is divided into seven subfamilies, which correspond well to functional classifications within the family: the vertebrate visual (transducin-coupled) and non-visual opsin subfamily, the encephalopsin/tmt-opsin subfamily, the G_q-coupled opsin/melanopsin subfamily, the G_o-coupled opsin subfamily, the neuropsin subfamily, the peropsin subfamily and the retinal photoisomerase subfamily. The subfamilies diversified before the deuterostomes (including vertebrates) split from the protostomes (most invertebrates), suggesting that a common animal ancestor had multiple opsin genes. Opsins have a seven-transmembrane structure similar to that of other GPCRs, but are distinguished by a lysine residue that is a retinal-binding site in the seventh helix. Accumulated evidence suggests that most opsins act as pigments that activate G proteins in a light-dependent manner in both visual and non-visual systems, whereas a few serve as retinal photoisomerases, generating the chromophore used by other opsins, and some opsins have unknown functions.     

Distinct effects of 4-nonylphenol and estrogen-17 beta on expression of estrogen receptor alpha gene in smolting sockeye salmon

Xenoestrogens such as 4-nonylphenol (4-NP) have been shown to affect the parr-smolt transformation, but their mechanisms of action are not known. We therefore examined effects of 4-NP and estradiol-17beta (E2) on expression of estrogen receptor (ER) alpha gene in the liver, gill, pituitary and brain of sockeye salmon to elucidate molecular mechanisms of 4-NP and E2 and developmental differences in response during smolting. Fish were treated twice within a week with 4-NP (15 and 150 mg/kg BW), E2 (2 mg/kg BW) or only vehicle at three stages of smolting, pre-smolting in March, early smolting in April and late smolting in May. The absolute amounts of ERalpha mRNA were determined by real-time PCR. The basal amounts of ERalpha mRNA peaked in April in the liver, gill and pituitary. In March, E2 extensively increased the amounts in the liver, while 4-NP had no effects at this stage. In contrast, 4-NP (but not E2) decreased liver ERalpha mRNA in April. 4-NP also decreased the amount of ERalpha mRNA in the gill in April. In the pituitary, 4-NP increased ERalpha mRNA in March but decreased it in May. There were no significant effects in the brain. Changes in basal ERalpha mRNA observed in this study indicate that estrogen responsiveness of tissues may change during salmon smolting. Furthermore, 4-NP and E2 have different effects on expression of ERalpha gene in the liver and gill during smolting, and the response is dependent on smolt stage.     

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.     

Remnant lipoprotein particles are taken up into myocardium through VLDL receptor--a possible mechanism for cardiac fatty acid metabolism

The VLDL (very low-density lipoprotein) receptor is a peripheral lipoprotein receptor expressing in fatty acid active tissues abundantly. In the Balb/c fasting mice, VLDL receptor as well as LPL (lipoprotein lipase), FAT (fatty acid translocase)/CD36, H-FABP (heart-type fatty acid-binding protein), ACS (acyl-CoA synthetase) and LCAD (long-chain acyl-CoA dehydrogenase) expressions increased. An electron microscopic examination indicated the lipid droplets that accumulated in the hearts of fasting Balb/c mice. During the development of SD (Sprague-Dawley) rats, VLDL receptor, LPL, FAT/CD36, H-FABP, ACS, and LCAD mRNAs concomitantly increased with growth. However, PK (pyruvate kinase) mRNA expression was negligible. In cultured neonatal rat cardiomyocytes, VLDL receptor expression increased with days in culture. Oil red-O staining showed that cardiomyocytes after 7 days in culture (when the VLDL receptor protein is present) accumulated beta-migrating VLDL. Thereby, we showed that the cardiac VLDL receptor pathway for delivery of remnant lipoprotein particles might be part of a cardiac fatty acid metabolism.     

A new species of the genusamblyeleotris (pisces: Gobiidae) from Japan

A new shrimp-associated goby,Amblyeleotris melanocephala, is described on the basis of specimens from Okinoshima Island. Kochi Prefecture, and Okinawa Island, Okinawa Prefecture, Japan. The species is distinguished from other members of the genus by the following combination of characters: head dark brown, a few yellow spots on pectoral fin base and opercular margin, 13 second dorsal and 13 anal fin soft rays, 20 pectoral fin rays, longitudinal scales 92–101, proportional length of interpelvic connecting membrane relative to longest pelvic fin ray (CM-value) 0.46–0.55, presence of a ventral frenum, midline of nape naked, sides scaled above midpoint between preopercle and opercle.     

Antennatus flagellatus (Teleostei: Antennariidae), a new species of frogfish from southern Japan

A new species of frogfish,Antennatus flagellatus, is described on the basis of two specimens from Kashiwajima Island, Kochi Pref., southern Japan. It is distinguished from congeneric species as follows: illicium long, more than 40% SL and about 3.6 times length of second dorsal spine; caudal peduncle present.     

Examination of a Viral Infection Mimetic Model in Human iPS Cell-Derived Insulin-Producing Cells and the Anti-Apoptotic Effect of GLP-1 Analogue

Aims

Viral infection is associated with pancreatic beta cell destruction in fulminant type 1 diabetes mellitus. The aim of this study was to investigate the acceleration and protective mechanisms of beta cell destruction by establishing a model of viral infection in pancreatic beta cells.

Methods

Polyinosinic:polycytidylic acid was transfected into MIN6 cells and insulin-producing cells differentiated from human induced pluripotent stem cells via small molecule applications. Gene expression was analyzed by real-time PCR, and apoptosis was evaluated by caspase-3 activity and TUNEL staining. The anti-apoptotic effect of Exendin-4 was also evaluated.

Results

Polyinosinic:polycytidylic acid transfection led to elevated expression of the genes encoding IFNα, IFNβ, CXCL10, Fas, viral receptors, and IFN-inducible antiviral effectors in MIN6 cells. Exendin-4 treatment suppressed the elevated gene expression levels and reduced polyinosinic:polycytidylic acid-induced apoptosis both in MIN6 cells and in insulin-producing cells from human induced pluripotent stem cells. Glucagon-like peptide-1 receptor, protein kinase A, and phosphatidylinositol-3 kinase inhibitors counteracted the anti-apoptotic effect of Exendin-4.

Conclusions

Polyinosinic:polycytidylic acid transfection can mimic viral infection, and Exendin-4 exerted an anti-apoptotic effect both in MIN6 and insulin-producing cells from human induced pluripotent stem cells.