Blood gene expression profiling in liver transplant recipients with hepatitis C virus and posttransplantation diabetes mellitus.

BACKGROUND: Hepatitis C virus (HCV) is a risk factor for developing posttransplantation diabetes mellitus (PTDM) after liver transplantation; little is known about the biological mechanisms involved with this risk. This study investigated gene expression differences to provide insight into potential mechanisms. PATIENTS AND METHODS: Gene expression profiles of blood samples obtained from 6 HCV+ liver transplant recipients were determined using Affymetrix U133 Plus 2.0 microarrays. Differential gene expression was assessed between HCV+ recipients with PTDM (n = 3) and without PTDM (n = 3) using the GeneSpring 7.3 software package. The Welch t test was used to identify significant differences (P < .05) between groups. Gene expression profiles for 6 HCV- liver transplant recipients (with PTDM = 3, without PTDM = 3) were used as a blind test set to evaluate a subset of genes to predict PTDM. RESULTS: Expression levels of 347 genes were significantly different between recipients with PTDM and those without PTDM. Seventy-four genes were up-regulated and 270 were down-regulated in PTDM. Genes were categorized into functional classes: apoptosis (n = 69 genes); immune function (n = 110); diabetes (n = 17); hepatitis C (n = 12); liver transplant (n = 69). The expression profile of a subset of genes was evaluated for predicting PTDM in 6 HCV- transplant recipients. We accurately predicted the presence or absence of PTDM in 5/6 recipients. CONCLUSIONS: PTDM in HCV+ liver transplant recipients was associated with down-regulated expression of a large number of genes. A subset of these genes was useful to predict PTDM in HCV- recipients. Most genes were associated with apoptosis and immune function. HCV may act as a primer by affecting a group of genes involved in developing diabetes.

Disabled-1 interacts with a novel developmentally regulated protocadherin.

Disabled-1 (Dab1) is an intracellular adapter protein that mediates the effect of Reelin on neuronal migration and cell positioning during mammalian brain development. To identify components of the Reelin-Dab1 signaling pathway, we searched for proteins that interact with Dab1 using a yeast two-hybrid strategy. We found that the Dab1 phosphotyrosine binding (PTB) domain interacts with a novel protocadherin, orthologous to human protocadherin 18. Mouse Pcdh18 (mPcdh18), which consists of four exons similar to other protocadherin family members, maps to chromosome 3. The deduced amino acid sequence of mPcdh18 contains six extracellular cadherin motifs, a single transmembrane region, and a large intracellular domain. The site of Dab1 interaction was localized to the C-terminal 243 residues of mPcdh18. Expression analyses revealed that mPcdh18 is present in a variety of tissues in the embryo, but in adult mice it is primarily expressed in lung and kidney. In embryonic brain, mPcdh18 expression is temporally and spatially regulated. Our results indicate that mPcdh18 participates in signaling pathways involving PTB-containing proteins and suggest that it may play a role during brain development.

Cortical development: Cdk5 gets into sticky situations.

Cyclin-dependent kinase 5 (Cdk5) is much more than its name implies; it plays a role in neuronal migration, neurite outgrowth and degeneration. Recent evidence suggests that Cdk5 regulates neuronal adhesion and cytoskeletal dynamics.

Reelin is a ligand for lipoprotein receptors.

A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.

Disabled-1 binds to the cytoplasmic domain of amyloid precursor-like protein 1.

Disruption of the disabled-1 gene (Dab1) results in aberrant migration of neurons during development and disorganization of laminar structures throughout the brain. Dab1 is thought to function as an adapter molecule in signal transduction processes. It contains a protein-interaction (PI) domain similar to the phosphotyrosine-binding domain of the Shc oncoprotein, it is phosphorylated by the Src protein tyrosine kinase, and it binds to SH2 domains in a phosphotyrosine-dependent manner. To investigate the function of Dab1, we searched for binding proteins using the yeast two-hybrid system. We found that the PI domain of Dab1 interacts with the amyloid precursor-like protein 1 (APLP1). The association of Dab1 with APLP1 was confirmed in biochemical assays, and the site of interaction was localized to a cytoplasmic region of APLP1 containing the amino acid sequence motif Asn-Pro-x-Tyr (NPxY). NPxY motifs are involved in clathrin-mediated endocytosis, and they have been shown to bind to PI domains present in several proteins. This region of APLP1 is conserved among all members of the amyloid precursor family of proteins. Indeed, we found that Dab1 also interacts with amyloid precursor protein (APP) and APLP2 in biochemical association experiments. In transiently transfected cells, Dab1 and APLP1 colocalized in membrane ruffles and vesicular structures. Cotransfection assays in cultured cells indicated that APP family members increased serine phosphorylation of Dab1. Dab1 and APLP1 are expressed in similar cell populations in developing and adult brain tissue. These results suggest that Dab1 may function, at least in part, through association with APLP1 in the brain.

Identification of specific mRNAs affected by treatments producing long-term facilitation in Aplysia.

Neural correlates of long-term sensitization of defensive withdrawal reflexes in Aplysia occur in sensory neurons in the pleural ganglia and can be mimicked by exposure of these neurons to serotonin (5-HT). Studies using inhibitors indicate that transcription is necessary for production of long-term facilitation by 5-HT. Several mRNAs that change in response to 5-HT have been identified, but the molecular events responsible for long-term facilitation have not yet been fully described. To detect additional changes in mRNAs, we investigated the effects of 5-HT (1.5 hr) on levels of mRNA in pleural-pedal ganglia using in vitro translation. Four mRNAs were affected by 5-HT, three of which were identified as calmodulin (CaM), phosphoglycerate kinase (PGK), and a novel gene product (protein 3). Using RNase protection assays, we found that 5-HT increased all three mRNAs in the pleural sensory neurons. CaM and protein 3 mRNAs were also increased in the sensory neurons by sensitization training. Furthermore, stimulation of peripheral nerves of pleural-pedal ganglia, an in vitro analog of sensitization training, increased the incorporation of labeled amino acids into CaM, PGK, and protein 3. These results indicate that increases in CaM, PGK, and protein 3 are part of the early response of sensory neurons to stimuli that produce long-term facilitation, and that CaM and protein 3 could have a role in the generation of long-term sensitization.

Identification of two phosphoproteins affected by serotonin in Aplysia sensory neurons.

Protein phosphorylation appears to play important roles in the mechanisms responsible for presynaptic facilitation in Aplysia. To screen for phosphoproteins that may be involved in facilitation, we previously examined protein phosphorylation in pleural sensory neurons as a function of different durations (2 min, 25 min and 1.5 h) of serotonin treatments. Different durations of serotonin had unique effects on the phosphorylation of different sets of proteins. To determine the functions of these phosphoproteins, we have begun to obtain their amino acid sequences using protein microsequencing techniques. We report here partial sequencing of 2 such proteins. One protein (S6), whose phosphorylation was affected by 2 min treatments with serotonin, appeared to be an intermediate filament protein. Another protein (L55), whose phosphorylation was affected by 1.5-h treatments with serotonin, appeared to be a calmodulin-like Ca(2+)-binding protein. Although the exact cellular functions for S6 and L55 are not known, obtaining partial sequences of these proteins sets the stage for future studies that will examine their regulation and their specific roles in facilitation.

Octamer-primed cycle sequencing: design of an optimized primer library.

This paper describes a novel method of primer walking using octamer oligonucleotides to prime DNA sequencing reactions. Octamer sequencing is compatible with isotopic and fluorescent sequencing chemistry, reaction conditions are optimized such that the samples can be processed in parallel, and the procedure has the potential to be automated. This strategy is faster than the traditional primer walking sequencing strategy, as the existence of a primer library allows immediate access to a primer for the next sequencing reaction, eliminating delays associated with designing and synthesizing gene-specific primers. The octamer library is comprised of optimized sequencing primers, such that octamer sequencing yields results equivalent to or better than traditional primer walking. This technology is more economical because gene-specific sequencing primers, the major cost in the reaction, are replaced by an optimized subset of frequently occurring octamers that are able to prime multiple reactions.

Dynamics of protein phosphorylation in sensory neurons of Aplysia.

Protein phosphorylation plays important roles in the mechanisms underlying serotonin (5-HT)-induced presynaptic facilitation of Aplysia sensory neurons. To study mechanisms involved in facilitation, we investigated the pattern of protein phosphorylation in sensory neurons as a function of different durations of 5-HT. Two minutes and 1.5 hr treatments with 5-HT altered the phosphorylation of 5 and 10 proteins, respectively. These different duration treatments with 5-HT produced unique effects on the phosphorylation of different sets of proteins. This result suggests that cells may encode and measure the duration of a stimulus by the pattern of specific proteins that are phosphorylated or dephosphorylated. In addition, because the changes in phosphorylation produced by 2 min treatments with 5-HT were not observed after 25 min treatments with 5-HT, mechanisms must exist for the transient phosphorylation of some proteins even when the 5-HT treatment persists. Anisomycin, an inhibitor of protein synthesis, blocked the effect of 1.5 hr treatments with 5-HT on the phosphorylation of six proteins but had no effect on the phosphorylation change of four other proteins. Both CPT-cAMP (an activator of protein kinase A) and PDAc (an activator of protein kinase C) mimicked the effects of 5-HT on four proteins. Interestingly, the effect of 5-HT on these four proteins did not require protein synthesis. CPT-cAMP, but not PDAc, mimicked the effect of 5-HT on one protein (L55) and, the effect of 5-HT on this protein appeared to require protein synthesis. Because both activation of PKA and protein synthesis are involved in the induction of long-term facilitation, protein L55 is a good candidate for a protein that might play a key role in long-term facilitation. Finally, the effects of 5-HT on four proteins were not mimicked by either CPT-cAMP or PDAc. This finding raises the interesting possibility that some effects of 5-HT are mediated by second-messenger systems other than PKA or PKC.