A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained. (c) 2006 Elsevier Inc. All rights reserved.

Rapid parallel expression in E-coli and insect cells: Analysis of five lef gene products of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)

A number of strategies are emerging for the high throughput (HTP) expression of recombinant proteins to enable structural and functional study. Here we describe a workable HTP strategy based on parallel protein expression in E. coli and insect cells. Using this system we provide comparative expression data for five proteins derived from the Autographa californica polyhedrosis virus genome that vary in amino acid composition and in molecular weight. Although the proteins are part of a set of factors known to be required for viral late gene expression, the precise function of three of the five, late expression factors (lefs) 6, 7 and 10, is unknown. Rapid expression and characterisation has allowed the determination of their ability to bind DNA and shown a cellular location consistent with their properties. Our data point to the utility of a parallel expression strategy to rapidly obtain workable protein expression levels from many open reading frames (ORFs).

Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis ( 2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a > 1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha 2 delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S(35)methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

Background: Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results: Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion: The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response.

Regulation of expression of the rat orthologue of mouse double minute 2 (MDM2) by H2O2-induced oxidative stress in neonatal rat cardiac myocytes.

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress.

Fully automated software solution for protein quantitation by global metabolic labeling with stable isotopes

Metabolic stable isotope labeling is increasingly employed for accurate protein (and metabolite) quantitation using mass spectrometry (MS). It provides sample-specific isotopologues that can be used to facilitate comparative analysis of two or more samples. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has been used for almost a decade in proteomic research and analytical software solutions have been established that provide an easy and integrated workflow for elucidating sample abundance ratios for most MS data formats. While SILAC is a discrete labeling method using specific amino acids, global metabolic stable isotope labeling using isotopes such as (15)N labels the entire element content of the sample, i.e. for (15)N the entire peptide backbone in addition to all nitrogen-containing side chains. Although global metabolic labeling can deliver advantages with regard to isotope incorporation and costs, the requirements for data analysis are more demanding because, for instance for polypeptides, the mass difference introduced by the label depends on the amino acid composition. Consequently, there has been less progress on the automation of the data processing and mining steps for this type of protein quantitation. Here, we present a new integrated software solution for the quantitative analysis of protein expression in differential samples and show the benefits of high-resolution MS data in quantitative proteomic analyses.

Activation of protein kinase cascades in the heart by hypertrophic G protein-coupled receptor agonists.

Cardiac myocyte hypertrophy involves changes in cell structure and alterations in protein expression regulated at both the transcriptional and translational levels. Hypertrophic G protein-coupled receptor (GPCR) agonists such as endothelin-(ET-1) and phenylephrine stimulate a number of protein kinase cascades in the heart. Mitogen-activated protein kinase (MAPK) cascades stimulated include the extracellularly regulated kinase cascade, the stress-activated protein kinase/c-Jun N-terminal kinase cascade, and the p38 MAPK cascade. All 3 pathways have been implicated in hypertrophy, but recent ex vivo evidence also suggests that there may be additional effects on cell survival. ET-1 and phenylephrine also stimulate the protein kinase B pathway, and this may be involved in the regulation of protein synthesis by these agonists. Thus, protein kinase-mediated signaling may be important in the regulation of the development of myocyte hypertrophy.

Regulation of Bcl-xL expression by H2O2 in cardiac myocytes.

Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.

Signaling pathways mediating cardiac myocyte gene expression in physiological and stress responses

The contractile cells in the heart (the cardiac myocytes) are terminally differentiated. In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis, responses associated with the development of cardiac pathologies. There has been much effort expended in gaining an understanding of the stimuli which promote these responses, and in identifying the intracellular signaling pathways which are activated and potentially involved. These signaling pathways presumably modulate gene and protein expression to elicit the end-stage response. For the regulation of gene expression, the signal may traverse the cytoplasm to modulate nuclear-localized transcription factors as occurs with the mitogen-activated protein kinase or protein kinase B/Akt cascades. Alternatively, the signal may promote translocation of transcription factors from the cytoplasm to the nucleus as is seen with the calcineurin/NFAT and JAK/STAT systems. We present an overview of the principal signaling pathways implicated in the regulation of gene expression in cardiac myocyte pathophysiology, and summarize the current understanding of these pathways, the transcription factors they regulate and the changes in gene expression associated with the development of cardiac pathologies. Finally, we discuss how intracellular signaling and gene expression may be integrated to elicit the overall change in cellular phenotype.

Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts

Yano Y, Cesar KR, Araujo M, Rodrigues Jr. AC, Andrade LC, Magaldi AJ. Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts. Am J Physiol Renal Physiol 296: F54-F59, 2009. First published October 1, 2008; doi: 10.1152/ajprenal.90367.2008.-It is well known that Glucagon (Gl) is released after a high protein diet and participates in water excretion by the kidney, principally after a protein meal. To study this effect in in vitro perfused inner medullary collecting ducts (IMCD), the osmotic water permeability (Pf; mu m/s) at 37 degrees C and pH 7.4 in normal rat IMCDs (n = 36) perfused with Ringer/HCO(3) was determined. Gl (10(-7) M) in absence of Vasopressin (AVP) enhanced the Pf from 4.38 +/- 1.40 to 11.16 +/- 1.44 mu m/s (P < 0.01). Adding 10(-8), 10(-7), and 10(-6) M Gl, the Pf responded in a dose-dependent manner. The protein kinase A inhibitor H8 blocked the Gl effect. The specific Gl inhibitor, des-His(1)-[Glu(9)] glucagon (10(-7) M), blocked the Gl-stimulated Pf but not the AVP-stimulated Pf. There occurred a partial additional effect between Gl and AVP. The cAMP level was enhanced from the control 1.24 +/- 0.39 to 59.70 +/- 15.18 fm/mg prot after Gl 10(-7) M in an IMCD cell suspension. The immunoblotting studies indicated an increase in AQP2 protein abundance of 27% (cont 100.0 +/- 3.9 vs. Gl 127.53; P = 0.0035) in membrane fractions extracted from IMCD tubule suspension, incubated with 10(-6) M Gl. Our data showed that 1) Gl increased water absorption in a dose-dependent manner; 2) the anti-Gl blocked the action of Gl but not the action of AVP; 3) Gl stimulated the cAMP generation; 4) Gl increased the AQP2 water channel protein expression, leading us to conclude that Gl controls water absorption by utilizing a Gl receptor, rather than a AVP receptor, increasing the AQP2 protein expression.

Na plus -Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

Akt Expression is Diminished by Physical Inactivity in Early Stages of Development in Soleus Muscle of Rats

Metabolic Syndrome is a group of conditions related to obesity and physical inactivity. Little is known about the role of physical inactivity, in early stages of development, in the susceptibility to insulin resistant phenotype induced by high fat diet. Akt plays a key role in protein synthesis and glucose transport in skeletal muscle and has been regulated by muscle activity. The objective of present study was to determine the effect of early physical inactivity on muscle growth and susceptibility to acquire a diabetic phenotype and to assess its relationship with Akt expression. Forty Wistar male rats were distributed in two groups (standard group, Std) and movement restriction (RM). Between days 23 and 70 after birth, RM group was kept in small cages that did not allow them to perform relevant motor activity. From day 71 to 102 after birth, 10 rats of each group were fed with hyperlipidic diet (groups Std-DAG and RM-DAG). No differences were observed in total body weight although DAG increased epididymal fat pad weight. RM decreased significantly the soleus weight. Insulin-mediated glucose uptake was lower in RM-DAG group. Akt protein levels were lower in RM groups. Real time RT-PCR analysis showed that movement restriction decreased mRNA levels of AKT1 in soleus muscle, regardless of supplied diet. These findings suggest that early physical inactivity limits muscle`s growth and contributes to instauration of insulin resistant phenotype, which can be partly explained by dysregulation of Akt expression.

Glucose-Induced Regulation of NHEs Activity and SGLTs Expression Involves the PKA Signaling Pathway

The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 +/- A 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (similar to 40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (similar to 50%, P < 0.05) and the plasma membrane (similar to 100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.

TRPV1 receptors are involved in protein nitration and Muller cell reaction in the acutely axotomized rat retina

We report here the protein expression of TRPV1 receptor in axotomized rat retinas and its possible participation in mechanisms involved in retinal ganglion cell (RGC) death. Adult rats were subjected to unilateral, intraorbital axotomy of the optic nerve, and the retinal tissue was removed for further processing. TRPV1 total protein expression decreased progressively after optic nerve transection, reaching 66.2% of control values 21 days after axotomy. The number of cells labeled for TRPV1 in the remnant GCL decreased after 21 days post-lesion (to 63%). Fluoro-jade B staining demonstrated that the activation of TRPV1 in acutely-lesioned eyes elicited more intense neuronal degeneration in the GCL and in the inner nuclear layer than in sham-operated retinas. A single intraocular injection of capsazepine (100 mu M), a TRPV1 antagonist, 5 days after optic nerve lesion, decreased the number of GFAP-expressing Muller cells (72.5% of control values) and also decreased protein nitration in the retinal vitreal margin (75.7% of control values), but did not affect lipid peroxidation. Furthermore, retinal explants were treated with capsaicin (100 mu M), and remarkable protein nitration was then present, which was reduced by blockers of the constitutive and inducible nitric oxide synthases (7-NI and aminoguanidine, respectively). TRPV1 activation also increased GFAP expression, which was reverted by both TRPV1 antagonism with capsazepine and by 7-NI and aminoguanidine. Given that Muller cells do not express TRPV1, we suppose that the increased GFAP expression in these cells might be elicited by TRPV1 activation and by its indirect effect upon nitric oxide overproduction and peroxynitrite formation. We incubated Fluorogold pre-labeled retinal explants in the presence of capsazepine (1 mu M) during 48 h. The numbers of surviving RGCs stained with fluorogold and the numbers of apoptotic cells in the GCL detected with TUNEL were similar in lesioned and control retinas. We conclude that TRPV1 receptor expression decreased after optic nerve injury due to death of TRPV1-containing cells. Furthermore, these data indicate that TRPV1 might be involved in intrinsic protein nitration and Muller cell reaction observed after optic nerve injury. (C) 2010 Elsevier Ltd. All rights reserved.