The VD1/RPD2 α1-neuropeptide is highly expressed in the brain of cephalopod mollusks

In certain gastropod mollusks, the central neurons VD(1) and RPD(2) express a distinct peptide, the so-called VD(1)/RPD(2) α1-neuropeptide. In order to test whether this peptide is also present in the complex cephalopod central nervous system (CNS), we investigated several octopod and squid species. In the adult decapod squid Idiosepius notoides the α1-neuropeptide is expressed throughout the CNS, with the exception of the vertical lobe and the superior and inferior frontal lobes, by very few immunoreactive elements. Immunoreactive cell somata are particularly abundant in brain lobes and associated organs unique to cephalopods such as the subvertical, optic, peduncle, and olfactory lobes. The posterior basal lobes house another large group of immunoreactive cell somata. In the decapod Idiosepius notoides, the α1-neuropeptide is first expressed in the olfactory organ, while in the octopod Octopus vulgaris it is first detected in the olfactory lobe. In prehatchlings of the sepiolid Euprymna scolopes as well as the squids Sepioteuthis australis and Loligo vulgaris, the α1-neuropeptide is expressed in the periesophageal and posterior subesophageal mass. Prehatchlings of L. vulgaris express the α1-neuropeptide in wide parts of the CNS, including the vertical lobe. α1-neuropeptide expression in the developing CNS does not appear to be evolutionarily conserved across various cephalopod taxa investigated. Strong expression in different brain lobes of the adult squid I. notoides and prehatching L. vulgaris suggests a putative role as a neurotransmitter or neuromodulator in these species; however, electrophysiological evidence is still missing.     

The gene expression of the myocardial lipid droplet protein is highly regulated by PPARγ in adipocytes differentiated from MEFs or SVCs

We demonstrate that expression of the myocardial lipid droplet protein (MLDP) and ERα observed in adipose tissues is undetectable in 3T3-L1 cells but detectable in mouse embryonic fibroblasts (MEFs) and stromal-vascular cells (SVCs) during adipocyte differentiation. MLDP gene expression in MEFs or SVCs is induced by treatment with a PPARγ agonist or forced expression of PPARγ, indicating that PPARγ enhances MLDP expression during adipogenesis. PCR analyses reveal the dual expression of SREBP-1a and SREBP-1c in MEFs and SVCs as well as white adipose tissues unlike the predominant expression of SREBP-1a in 3T3-L1 cells. These results suggest that MEFs and SVCs are useful model cells for examining function of MLDP in lipid droplet formation and adipocyte differentiation.     

PANP is a novel O-glycosylated PILRα ligand expressed in neural tissues

Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.     

How random is a highly denatured protein?

There has been renewed interest in determining the physicochemical properties of denatured states of proteins. In many denatured states there is evidence for the existence of nonrandom configurational distributions. Here we examine the small-angle neutron scattering profile of yeast phosphoglycerate kinase in the native state and in highly denaturing conditions. We show that the denatured protein scattering profile can be interpreted using a model developed for synthetic polymers in which the chain behaves as a random coil in a good solvent, i.e. with excluded volume interactions. The implications of this result for our appreciation of the protein folding process are discussed.     

A novel gene constitutively expressed in human lymphoid cells is inducible with interferon-γ in myeloid cells

A cluster of at lest six interferon- (IFN)-inducible genes designated Ifi201-204 and located on mouse chromosome 1 has recently been described. Here , we report a human IFN--inducible gene, IFI 16, which has nucleotide sequence similarity with portions of two of the mouse genes, Ifi202 and Ifi204. A full-length cDNA clone derived from IFI 16 [2.709 kilobases (kb)] contained a single open reading frame of 2.187 kb which encoded a putative polypeptide of 729 amino acids and a predicted non-glycosylated M _r of 80020. IFI 16 mRNA was found to be constitutively expressed in lymphoid cells and in cell lines of both the T and B lineages. By contrast, the mRNA was not expresed by the cell lines HL-60, U937, and K562, which represent early stages of myeloid development, but was strongly inducible in HL-60 and U937 with IFN-. The IFI 16 protein demonstrated a putative domain structure with patchy similarity to the proteins expressed from gene Ifi202 and Ifi204. The mouse and human proteins each contain two analogous 200 amino acid domains which are imperfect copies, but IFI 16 demonstrated additional unique regions, including a Lys-rich N-terminal portion and a spacer region between the reiterated domains, analogous to spacer regions in the CD5 and CD8 molecules. Using a panel of inter-species somatic cell hybrid cell lines, IFI 16 was localized to the chromosomal region 1q121qter, a region systenic between mouse an man. DNA blotting indicated that, in contrast to the mouse, IFI 16 is present as a single copy gene in the human genome.The authors are pleased to make the cDNA clones described in this paper available to interested investigators.The nucleotide sequence data reported in this paper have been submitted to the Genbank database and have been assigned the accession number M63838. Correspondence to: J. A. Trapani.     

HEP-COP,a novel human gene whose product is highly homologous to the α-subunit of the yeast coatomer protein complex

A 4333-bp novel human cDNA sequence designated HEP-COP was isolated from the Hep3B hepatocellular carcinoma cell line by the RACE technique. Within HEP-COP was identified an ORF of 3672 bp encoding a deduced 1224-amino-acid (aa) sequence which exhibited striking homology with the 1201-aa sequence of RET1P, the α-subunit of the coatomer complex (α-COP) in Saccharomyces cerevisiae which participates in membrane transport between the endoplasmic reticulum and Golgi apparatus. The aa homology was highest in their N-terminal regions which each contained six WD-40 repeat motifs [Van der Voorn and Ploegh, FEBS Lett. 307 (1992) 131–134], and both proteins were predicted to be hydrophilic with similar estimated molecular masses of 138 324 and 135599 Da, respectively. Northern blot hybridization demonstrated that HEP-COP was expressed in a wide range of human adult and fetal tissues. RT-PCR analysis revealed no differential expression of HEP-COP in 14 human cancer cell lines, as compared with normal control cells. Considering the close similarities between HEP-COP and yeast α-COP, and the ubiquitous expression of HEP-COP implying an essential cellular role, it is likely that HEP-COP is the human homologue of α-COP     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

LSR/angulin-1 is a tricellular tight junction protein involved in blood–brain barrier formation

The blood–brain barrier (BBB) is a term used to describe the unique properties of central nervous system (CNS) blood vessels. One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs). Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues. We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis. We further demonstrate that the BBB does not seal during embryogenesis in Lsr knockout mice with a leakage to small molecules. Finally, in mouse models in which BBB was disrupted, including an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and a middle cerebral artery occlusion (MCAO) model of stroke, LSR was down-regulated, linking loss of LSR and pathological BBB leakage.     

Conventional and novel Gγ protein families constitute the heterotrimeric G-protein signaling network in soybean

Heterotrimeric G-proteins comprised of Gα, Gβ and Gγ proteins are important signal transducers in all eukaryotes. The Gγ protein of the G-protein heterotrimer is crucial for its proper targeting at the plasma membrane and correct functioning. Gγ proteins are significantly smaller and more diverse than the Gα and Gβ proteins. In model plants Arabidopsis and rice that have a single Gα and Gβ protein, the presence of two canonical Gγ proteins provide some diversity to the possible heterotrimeric combinations. Our recent analysis of the latest version of the soybean genome has identified ten Gγ proteins which belong to three distinct families based on their C-termini. We amplified the full length cDNAs, analyzed their detailed expression profile by quantitative PCR, assessed their localization and performed yeast-based interaction analysis to evaluate interaction specificity with different Gβ proteins. Our results show that ten Gγ genes are retained in the soybean genome and have interesting expression profiles across different developmental stages. Six of the newly identified proteins belong to two plant-specific Gγ protein families. Yeast-based interaction analyses predict some degree of interaction specificity between different Gβ and Gγ proteins. This research thus identifies a highly diverse G-protein network from a plant species. Homologs of these novel proteins have been previously identified as QTLs for grain size and yield in rice.     

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.