A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

Background: Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results: We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions: We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins

Cyclic adenosine 3',5'-monophosphate regulates GLUT4 and GLUT1 glucose transporter expression and stimulates transcriptional activity of the GLUT1 promoter in muscle cells

We have previously reported that innervation-dependent basal contractile activity regulates in an inverse manner the expression of GLUT1 and GLUT4 glucose transporters in skeletal muscle. Based on the facts that muscle innervation decreases and muscle denervation increases cAMP levels, we investigated whether cAMP might mediate the effects of innervation/denervation on glucose transporter expression. Treatment of L6E9 myotubes with 8-bromo-cAMP, forskolin, or monobutyryl-8-bromo-cAMP led to a marked decrease in GLUT4 protein levels; 8-bromo-cAMP also diminished GLUT4 messenger RNA (mRNA), suggesting pretranslational repression. In contrast, L6E9 myoblasts and myotubes responded to 8-bromo-cAMP or forskolin by increasing the cell content of GLUT1 protein. Induction of GLUT1 protein was a consequence of the activation of different mechanisms in myoblast and myotube cells; whereas 8-bromo-cAMP treatment caused a substantial increase in GLUT1 mRNA in myoblasts, no change in GLUT1 mRNA was detected in myotubes. The increase in GLUT1 mRNA in L6E9 myoblasts induced by 8-bromo-cAMP was the result of transcriptional activation, as concluded from transfection analysis of 2.1 kilobases of the rat GLUT1 gene promoter fused to the bacterial chloramphenicol acetyltransferase gene. Furthermore, the stimulatory effect of 8-bromo-cAMP on the transcriptional activity of the GLUT1 promoter required a 33-bp sequence lying 5' upstream of the transcription start site. In all, cAMP inversely regulates GLUT4 and GLUT1 glucose transporter expression in muscle cells. Furthermore, our results suggest that down-regulation of GLUT4 expression and up-regulation of GLUT1 expression in muscle associated with denervation are partly attributable to cAMP

Developmental regulation of GLUT-1 (Erytroid/HepG2) and GLUT-4 (Muscle/Fat) glucose transporter expression in rat heart, skeletal muscle, and brown adipose tissue

The expression of GLUT-1 (erythroid/Hep G2) and GLUT-4 (muscle/fat) glucose transporters was assessed during development in rat heart, skeletal muscle, and brown adipose tissue. GLUT-4 protein expression was detectable in fetal heart by day 21 of pregnancy; it increased progressively after birth. attaining levels close to those of adults at day 15 post natal.'In contrast, GLUT-4 messenger RNA (mRNA)was already present in hearts from 17 day-old fetuses. GLUT-4 mRNA stayed low during early postnatal life in heart and brown adipose tissue and only increased after day 10 post natal. The expression pattern for GLUT-4 protein in skeletal muscle during development was comparable to that observed in heart. In contrast to heart and skeletal muscle, GLUT-4 protein in brown adipose tissue was detected in high levels (30% of adult) during late fetal life. During fetal life, GLUT-l presented a very high expression level in brown adipose tissue, heart, and skeletal muscle. Soon after birth, GLUT-1 protein diminished progressively, attaining adult levels at day 10 in heart and skeletal muscle. GLUT-1 mRNA levels in heart followed a similar pattern to the GLUT- 1 protein, being very high during fetal life and decreasing early in post natal life. GLUT-1 protein showed a complex pattern in brown adipose tissue: fetal levels were high, decreased after birth, and increased subsequently in post natal life, reaching a peak by day 9. Progesterone-induced postmaturity protected against the decrease in GLUT-1 protein associated with post natal life in skeletal muscle and brown adipose tissue. However, GLUT-4 induction was not blocked by postmaturity in any of the tissues subjected to study. These results indicate that: 1) during fetal and early post natal life, GLUT-1 is a predominant glucose transporter isotype expressed in heart, skeletal muscle, and brown adipose tissue; 2) during early post natal life there is a generalized GLUT-1 repression; 3) during development, there is a close correlation between protein and mRNA levels for GLUT-l, and therefore regulation at a pretranslational level plays a major regulatory role; 4) the onset of GLUT-4 protein induction occurs between days 20-21 of fetal life; based on data obtained in rat heart and brown adipose tissue, there is a dissociation during development between mRNA and protein levels for GLUT-4, suggesting modifications at translational or posttranslational steps; and 5) postmaturity blocks the decrease in GLUT-l expression but not the induction of GLUT-4. observed soon after birth. All these findings suggest that GLUT-1 repression and GLUT-4 induction are mediated by different mechanisms

Myogenesis and MyoD down-regulate Sp1. A mechanism for the repression of GLUT1 during muscle cell differentiation

Muscle cell differentiation caused a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels. A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drove transcriptional activity in myoblasts, and differentiation caused a decrease in transcription. Transient transfection of 5' and 3' deletion constructs showed that the fragment -99/-33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed the binding of Sp1 protein to the fragment -102/-37 in the myoblast state but not in myotubes, and Sp1 was found to transactivate the GLUT1 promoter. Western blot analysis indicated that Sp1 was drastically down-regulated during myogenesis. Furthermore, the forced over-expression of MyoD in C3H10T1/2 cells mimicked the effects observed during myogenesis, Sp1 down-regulation and reduced transcriptional activity of the GLUT1 gene promoter. In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in GLUT1 expression and glucose transport

Differential regulation of the muscle-specific GLUT4 enhancer in regenerating and adult skeletal muscle

We have reported a novel functional co-operation among MyoD, myocyte enhancer factor-2 (MEF2), and the thyroid hormone receptor in a muscle-specific enhancer of the rat GLUT4 gene in muscle cells. Here, we demonstrate that the muscle-specific enhancer of the GLUT4 gene operates in skeletal muscle and is muscle fiber-dependent and innervation-independent. Under normal conditions, both in soleus and in extensor digitorum longus muscles, the activity of the enhancer required the integrity of the MEF2-binding site. Cancellation of the binding site of thyroid hormone receptor enhanced its activity, suggesting an inhibitory role. Muscle regeneration of the soleus and extensor digitorum longus muscles caused a marked induction of GLUT4 and stimulation of the enhancer activity, which was independent of innervation. During muscle regeneration, the enhancer activity was markedly inhibited by cancellation of the binding sites of MEF2, MyoD, or thyroid hormone receptors. Different MEF2 isoforms expressed in skeletal muscle (MEF2A, MEF2C, and MEF2D) and all members of the MyoD family had the capacity to participate in the activity of the GLUT4 enhancer as assessed by transient transfection in cultured cells. Our data indicate that the GLUT4 enhancer operates in muscle fibers and its activity contributes to the differences in GLUT4 gene expression between oxidative and glycolytic muscle fibers and to the GLUT4 up-regulation that occurs during muscle regeneration. The activity of the enhancer is maintained in adult muscle by MEF2, whereas during regeneration the operation of the enhancer depends on MEF2, myogenic transcription factors of the MyoD family, and thyroid hormone receptors

Factors involved in GLUT1 glucose transporter gene transcription in cardiacmuscle

Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the -99/-33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator of GLUT-1 promoter, binds to the -102/-82 region of GLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E2 (PGE2) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE2 and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE2 production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE2, and suggest that the coordinated down-regulation of COX-1 facilitates PGE2 production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation

Biologia dels transportadors de glucosa de difusió facilitada. Redistribució cel.lular de transportadors de glucosa en resposta a hormones

La glucosa és la font d'energia més important per a les cèl·lules dels mamífers. La seva oxidació en condicions aeròbiques o anaeròbiques abasteix constantment d 'ATP les cèl·lules. Després de ]a ingesta, l'excés de glucosa s'emmagatzema al fetge i als músculs en forma de glicogen i al teixit adipós en forma de triacilglicèrids. La reserva de glucosa en forma de glicogen en el fetge és fonamental per a tamponar els nivells de glucosa a la sang i els dipòsits de glicogen dels músculs son una important font d'energia durant 1'exercici. D'altra banda, els triacilglicèrids del teixit adipós representen una forma compacta d'emmagatzemament d'energia, que es mobilitza en forma d'àcids grassos i glicerol en condicions catabòliques