“Armed” oncolytic herpes simplex viruses for brain tumor therapy

Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for brain tumors and other solid cancers. They can replicate in situ, spread and exhibit oncolytic activity via a direct cytocidal effect. One of the advantages of HSV-1 is the capacity to incorporate large and/or multiple transgenes within the viral genome. Oncolytic HSV-1 can therefore be “armed” to add certain functions. Recently, the field of armed oncolytic HSV-1 has drastically advanced, due to development of recombinant HSV-1 generation systems that utilize bacterial artificial chromosome and multiple DNA recombinases. Because antitumor immunity is induced in the course of oncolytic activities of HSV-1, transgenes encoding immunomodulatory molecules have been most frequently used for arming. Other armed oncolytic HSV-1 include those that express antiangiogenic factors, fusogenic membrane glycoproteins, suicide gene products, and proapoptotic proteins. Provided that the transgene product does not interfere with viral replication, such arming of oncolytic HSV-1 results in augmentation of antitumor efficacy. Immediate-early viral promoters are often used to control the arming transgenes, but strict-late viral promoters have been shown useful to restrict the expression in the late stage of viral replication when desirable. Some armed oncolytic HSV-1 have been created for the purpose of noninvasive in vivo imaging of viral infection and replication. Development of a wide variety of armed oncolytic HSV-1 will lead to an establishment of a new genre of therapy for brain tumors as well as other cancers.Key words: oncolytic virus therapy, gene therapy, herpes simplex virus, viral vectors, G47Δ, G207, antitumor immunity     

Biochemical characterization of the Rho GTPase-regulated actin assembly by diaphanous-related formins, mDia1 and Daam1, in platelets

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.     

Collagen XVIII and corneal reinnervation following keratectomy

The significance of collagen XVIII in the regulation of corneal reinnervation remains largely unknown. We used whole-mount immunoconfocal microscopy to localize collagen XVIII to the nerve basement membrane of wild-type (WT) mouse corneas. Transmission electron microscopy showed corneal nerve disorganization in collagen XVIII knockout mice (col18a1(-/-)). Antibody 2H3-specific neurofilament colocalized with collagens XVIII and IV and laminin-2 in WT mouse corneas, but did not colocalize with collagen IV and laminin-2 in col18a1(-/-) mouse corneas. Following keratectomy, col18a1(-/-) mice displayed decreased corneal neurite extension compared to WT mice. Our data indicate that collagen XVIII may play an important role in corneal reinnervation after wounding.     

Mechanism of robust circadian oscillation of KaiC phosphorylation in vitro

By incubating the mixture of three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro, T. Kondo and his colleagues in recent work reconstituted the robust circadian rhythm of the phosphorylation level of KaiC. This finding indicates that protein-protein interactions and the associated hydrolysis of ATP suffice to generate the circadian rhythm. Several theoretical models have been proposed to explain the rhythm generated in this “protein-only” system, but the clear criterion to discern different possible mechanisms was not known. In this article, we discuss a model based on two basic assumptions: the assumption of the allosteric transition of a KaiC hexamer and the assumption of the monomer exchange between KaiC hexamers. The model shows a stable rhythmic oscillation of the phosphorylation level of KaiC, which is robust against changes in concentration of Kai proteins. We show that this robustness gives a clue to distinguish different possible mechanisms. We also discuss the robustness of oscillation against the change in the system size. Behaviors of the system with the cellular or subcellular size should shed light on the role of the protein-protein interactions in in vivo circadian oscillation.     

Long-term territoriality and mate acquisition in the lefteye flounder Engyprosopon grandisquama

Reproductive ecology and function of long-term territoriality in the lefteye flounder Engyprosopon grandisquama were investigated in the southwest of Kyushu Island, Japan. Field observations of marked individuals and monthly sampling suggested that the spawning season was from June to September with a peak in June–July. During the spawning season, males maintained their mating territories, although spawning behavior was not observed in August–September. Some males stayed until the next spawning season and then acquired more mates than newly appearing males, most of which could not acquire mates. Long-term retention of territory may be more advantageous for establishing a mating territory and acquiring mates in the next spawning season.     

Interaction of TIP26 from a hyperthermophilic archaeon with TFB/TBP/DNA ternary complex

Interactions of TBP-interacting protein (TIP26), TBP, and TFB from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with TATA-DNA were examined by electrophoretic mobility shift assay. Tk-TFB formed a ternary complex with Tk-TBP and TATA-DNA. Tk-TIP26 did not inhibit the formation of this ternary complex, but interacted with it to form a TIP26/TFB/TBP/DNA quaternary complex. This interaction is rather weak, and a large excess of Tk-TIP26 over Tk-TBP is required to fully convert the TFB/TBP/DNA ternary complex to the quaternary complex. However, determination of the concentration of Tk-TIP26 and Tk-TBP in KOD1 cells by Western blotting analysis indicated that the concentration of Tk-TIP26 is approximately ten times that of Tk-TBP, suggesting that the quaternary complex might also form in vivo.     

CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein

The ubiquitin–proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C-terminus of Hsc70-interacting protein (CHIP) with a U-box domain is an E3 ubiquitin-ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U-box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C-terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self-ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as ‘a quality-control E3’ that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.     

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl^–1 agar, different sucrose concentrations (10, 20, 30 or 40g l^–1), and with or without 1 mg l^–1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l^–1 agar and 20 gl^–1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l^–1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l^–1. The plantletswere transferred to soil and grew well under greenhouseconditions.