Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.     

Induction of localized auxin response during spontaneous nodule development in Lotus japonicus

In leguminous plants, rhizobial infection of the epidermis triggers proliferation of cortical cells to form a nodule primordium. Recent studies have demonstrated that two classic phytohormones, cytokinin and auxin, have important functions in nodulation. The identification of these functions in Lotus japonicus was facilitated by use of the spontaneous nodule formation 2 (snf2) mutation of the putative cytokinin receptor LOTUS HISTIDINE KINASE 1 (LHK1). Analyses using snf2 demonstrated that constitutive activation of cytokinin signaling causes formation of spontaneous nodule-like structures in the absence of rhizobia and that auxin responses are induced in proliferating cortical cells during such spontaneous nodule development. Thus, cytokinin signaling positively regulates the auxin response. In the present study, we further investigated the induction of the auxin response using a gain-of-function mutation of Ca²⁺/calmodulin-dependent protein kinase (CCaMK) that causes spontaneous nodule formation. We demonstrate that CCaMK^T265D-mediated spontaneous nodule development is accompanied by a localized auxin response. Thus, a localized auxin response at the site of an incipient nodule primordium is essential for nodule organogenesis.     

Environmental DNA monitoring method of the commercially important and endangered fish Gnathopogon caerulescens

Limnology - Gnathopogon caerulescens is an endangered but commercially important fish in Lake Biwa, Japan. The population size of G. caerulescens has drastically reduced in the past decades, and...     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

Processing and juxtacrine activity of membrane-anchored betacellulin

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells.     

Eribulin regresses a doxorubicin‐resistant Ewing's sarcoma with a FUS‐ERG fusion and CDKN2A‐deletion in a patient‐derived orthotopic xenograft (PDOX) nude mouse model

Ewing's sarcoma is a recalcitrant tumor greatly in need of more effective therapy. The aim of this study was to determine the efficacy of eribulin on a doxorubicin (DOX)‐resistant Ewing's sarcoma patient derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma PDOX model was previously established in the right chest wall of nude mice from tumor resected form the patient's right chest wall. In the previous study, the Ewing's sarcoma PDOX was resistant to doxorubicin (DOX) and sensitive to palbociclib and linsitinib. In the present study, the PDOX models were randomized into three groups when the tumor volume reached 60 mm³: G1, untreated control (n = 6); G2, DOX treated (n = 6), intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, Eribulin treated (n = 6, intravenous (i.v.) injection, weekly for 2 weeks). All mice were sacrificed on day 15. Changes in body weight and tumor volume were assessed two times per week. Tumor weight was measured after sacrifice. DOX did not suppress tumor growth compared to the control group (P = 0.589), consistent with the previous results in the patient and PDOX. Eribulin regressed tumor size significantly compared to G1 and G2 (P = 0.006, P = 0.017) respectively. No significant difference was observed in body weight among any group. Our results demonstrate that eribulin is a promising novel therapeutic agent for Ewing's sarcoma.     

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.