Frequent overexpression of CADM1/IGSF4 in lung adenocarcinoma

We reported comprehensive screening for antigens (Ags) overexpressed on various carcinomas via isolation of human monoclonal antibodies (mAbs) that may be therapeutic in a previous paper (Proc. Natl. Acad. Sci. USA 105, 7287-7292, 2008). Twenty-one distinct Ags highly expressed on several carcinomas were identified and 356 mAbs with unique sequences turned out to bind to one of the 21 Ags. Among them CADM1/IGSF4 which had been originally referred to as tumor suppressor lung cancer 1 (TSLC1) was included. Therefore we examined the expression of CADM1 in lung cancers in this study. Eight different anti CADM1 mAbs were used for immunohistochemical analysis of 29 fresh lung cancer specimens. Staining patterns were categorized to six groups based on the extent of positive staining and the localization of stained portions. While overexpression of CADM1 was observed on the cell surface of adenocarcinomas at a high frequency, around 60%, positive stainings were rarely observed on that of other lung carcinomas including squamous cell carcinomas. Moreover, some clones among the eight mAbs gave different staining patterns from those by the other clones against the same fresh specimen, suggesting presence of variant forms of CADM1 differentiated by mAbs.     

Oligodendrocytes regulate formation of nodes of Ranvier via the recognition molecule OMgp

The molecular mechanisms underlying the involvement of oligodendrocytes in formation of the nodes of Ranvier (NORs) remain poorly understood. Here we show that oligodendrocyte-myelin glycoprotein (OMgp) aggregates specifically at NORs. Nodal location of OMgp does not occur along demyelinated axons of either Shiverer or proteolipid protein (PLP) transgenic mice. Over-expression of OMgp in OLN-93 cells facilitates process outgrowth. In transgenic mice in which expression of OMgp is down-regulated, myelin thickness declines, and lateral oligodendrocyte loops at the node-paranode junction are less compacted and even join together with the opposite loops, which leads to shortened nodal gaps. Notably, each of these structural abnormalities plus modest down-regulation of expression of Na(+) channel alpha subunit result in reduced conduction velocity in the spinal cords of the mutant mice. Thus, OMgp that is derived from glia has distinct roles in regulating nodal formation and function during CNS myelination.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).     

Complete nucleotide sequence of pTZ12, a chloramphenicol-resistance plasmid of Bacillus subtilis

The complete nucleotide sequence of pTZ12, a chloramphenicol-resistance (CmR) plasmid (2517 bp) derived from Corynebacterium xerosis plasmid pTZ10, has been determined after propagation in Bacillus subtilis. The nucleotide sequence of pTZ12 suggests that a recombination event may have occurred naturally within the open reading frames for the Rep protein of pT181 (or a pT181-like plasmid) and pC221 (or a pC221-like plasmid).     

Formation of 5 alpha-products as major C19-steroids in estrogen-responsive mouse Leydig cell tumor lines

Homogenates of estrogen-responsive mouse Leydig cell tumors (T 124958-R and T 22137) or 28- and 120-day-old mouse testes were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione in the presence of NADPH, and progesterone metabolism and enzyme activities were estimated. The growth of T 124958-R tumor transplanted in BALB/c mice was markedly stimulated by estrogenization of host mice, but the growth of T 22137 tumor was evidently suppressed by the estrogenization. The major C21-17-OH-steroids and C19-steroids formed from progesterone by both tumors and the testes of immature mice were 5 alpha-steroids, such as 3 alpha,17-dihydroxy-5 alpha-pregnan-20-one, 5 alpha-androstane-3,17-dione, androsterone, 3 beta-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha,17 beta-diol. In contrast, the major steroids formed by the testes of adult mice were testosterone and 4-androstene-3,17-dione, and no or little 5 alpha-steroids were produced. 5 alpha-Reductase activities in both tumor cells (40-50 nmol/l X 10(8) cells per h) were also found to be approx. 5-6 times higher than that in Leydig cells of adult mouse testes (8 nmol/l X 10(8) Leydig cells per h), though 17-hydroxylase activity was much higher in the Leydig cells of adult testes (730 nmol/l X 10(8) Leydig cells per h) than in both tumor cells (1-7 nmol/l X 10(8) cells per h). Furthermore, the presence of significant amounts of endogenous androsterone and/or 5 alpha-androstane-3 alpha,17 beta-diol was demonstrated in both tumors by radioimmunoassay. The present results demonstrate for the first time that C19-5 alpha-steroids are major C19-steroid products (immature type of testicular androgen production) in Leydig cell tumor lines.     

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.     

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells.