Embryonic Isoform Research Articles

Splice variants of Enigma homolog, differentially expressed during heart development, promote or prevent hypertrophy

Proteins with a PDZ (for PSD-95, DLG, ZO-1) and one to three LIM (for Lin11, Isl-1, Mec-3) domains are scaffolding sarcomeric and cytoskeletal elements that form structured muscle fibres and provide for the link to intracellular signalling by selectively associating protein kinases, ion channels, and transcription factors with the mechanical stress-strain sensors. Enigma homolog (ENH) is a PDZ-LIM protein with four splice variants: ENH1 with an N-terminal PDZ domain and three C-terminal LIM domains and ENH2, ENH3, and ENH4 without LIM domains. We addressed the functional role of ENH alternative splicing. We studied the expression of the four ENH isoforms in the heart during development and in a mouse model of heart hypertrophy. All four isoforms are expressed in the heart but the pattern of expression is clearly different between embryonic, neonatal, and adult stages. ENH1 appears as the embryonic isoform, whereas ENH2, ENH3, and ENH4 are predominant in adult heart. Moreover, alternative splicing of ENH was changed following induction of heart hypertrophy, producing an ENH isoform pattern similar to that of neonatal heart. Next, we tested a possible causal role of ENH1 and ENH4 in the development of cardiac hypertrophy. When overexpressed in rat neonatal cardiomyocytes, ENH1 promoted the expression of hypertrophy markers and increased cell volume, whereas, on the contrary, ENH4 overexpression prevented these changes. Antagonistic splice variants of ENH may play a central role in the adaptive changes of the link between mechanical stress-sensing and signalling occurring during embryonic development and/or heart hypertrophy.

Roles of Oral Bacteria in Cardiovascular Diseases — From Molecular Mechanisms to Clinical Cases: Involvement of Porphyromonas gingivalis in the Development of Human Aortic Aneurysm

Accumulating evidence suggests the involvement of Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen, in cardiovascular diseases. Clinical specimens of aneurysmal tissue and dental plaque collected from patients infected with or without P. gingivalis were analyzed. The number of aneurysms in the distal aorta in the P. gingivalis –infected group was significantly higher than that in the non-infected group. Cellular accumulation of adipocytes in aneurysms was less frequently identified in the infected group. The expression of embryonic myosin heavy chain isoform, a phenotypic marker for proliferative smooth muscle cells, was higher in the P. gingivalis –infected group than the non-infected group. Clinical and histopathological features of aortic aneurysms associated with P. gingivalis infection are different from those present in non-infected patients. The major characteristic of P. gingivalis infection associated with aneurysms is smooth muscle cell proliferation in the distal aorta.

Roles of Oral Bacteria in Cardiovascular Diseases — From Molecular Mechanisms to Clinical Cases: Porphyromonas gingivalis Is the Important Role of Intimal Hyperplasia in the Aorta

It has been reported that DNA of oral bacterial species, such as Porphyromonas gingivalis and Streptococcus mutans, was detected frequently in specimens of arteriosclerotic vessels. However, the source of DNA, whether from live intact bacteria or a part of the bacteria, has not been identified yet. Moreover, there was no precise evidence concerning involvement of oral bacteria in the progression of arteriosclerosis. We tried to clarify the involvement of P. gingivalis on the mechanisms of development of aortic intimal hyperplasia. Intravenous administration of P. gingivalis dramatically induced intimal hyperplasia in the mouse model with photochemical impairment of the femoral artery. However there were no changes identified in the mice without aortic impairment, even with the P. gingivalis infection. Concomitantly, S100 calcium–binding protein A9 (S100A9) and the embryonic isoform of myosin heavy chain (SMemb), a proliferative phenotypic marker of smooth muscle cells, were significantly overexpressed on the surfaces of smooth muscle cells present in the injured blood vessels. Similarly, increased expressions of S100A9 and SMemb proteins were observed in aneurismal specimens obtained from P. gingivalis – infected patients. We found that bacteremia induced by P. gingivalis leads to intimal hyperplasia associated with overexpressions of S100A9 and SMemb. Our results strongly suggest that oral-hematogenous spreading of P. gingivalis is a causative event in the development of aortic hyperplasia in periodontitis patients.

HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer

When oxygen is abundant, quiescent cells efficiently extract energy from glucose primarily by oxidative phosphorylation, whereas under the same conditions tumour cells consume glucose more avidly, converting it to lactate. This long-observed phenomenon is known as aerobic glycolysis, and is important for cell growth. Because aerobic glycolysis is only useful to growing cells, it is tightly regulated in a proliferation-linked manner. In mammals, this is partly achieved through control of pyruvate kinase isoform expression. The embryonic pyruvate kinase isoform, PKM2, is almost universally re-expressed in cancer, and promotes aerobic glycolysis, whereas the adult isoform, PKM1, promotes oxidative phosphorylation. These two isoforms result from mutually exclusive alternative splicing of the PKM pre-mRNA, reflecting inclusion of either exon 9 (PKM1) or exon 10 (PKM2). Here we show that three heterogeneous nuclear ribonucleoprotein (hnRNP) proteins, polypyrimidine tract binding protein (PTB, also known as hnRNPI), hnRNPA1 and hnRNPA2, bind repressively to sequences flanking exon 9, resulting in exon 10 inclusion. We also demonstrate that the oncogenic transcription factor c-Myc upregulates transcription of PTB, hnRNPA1 and hnRNPA2, ensuring a high PKM2/PKM1 ratio. Establishing a relevance to cancer, we show that human gliomas overexpress c-Myc, PTB, hnRNPA1 and hnRNPA2 in a manner that correlates with PKM2 expression. Our results thus define a pathway that regulates an alternative splicing event required for tumour cell proliferation.

Molecular analysis of aortic intimal hyperplasia caused byPorphyromonas gingivalisinfection in mice with endothelial damage

Porphyromonas gingivalis infection is thought to be a significant etiological factor in the development of cardiovascular diseases. However, scant definitive evidence has been presented concerning the pathological molecular mechanisms of these disorders. In the present study, we performed a molecular analysis of the developmental mechanisms of aortic intimal hyperplasia induced by P. gingivalis. The effects of P. gingivalis-induced bacteremia on intimal hyperplasia were evaluated using a mouse model of aortic hyperplasia created by photochemical-induced endothelial cell injury. Alterations of gene expression profiles in injured blood vessels of the mice were extensively analyzed using DNA microarray assays to identify the key molecules involved in P. gingivalis-induced hyperplasia. In addition, human aneurismal specimens from patients with or without P. gingivalis infection were analyzed histochemically. Intravenous administration of P. gingivalis dramatically induced intimal hyperplasia in the mouse model. Concomitantly, S100 calcium-binding protein A9 (S100A9) and embryonic isoform of myosin heavy chain (SMemb), a proliferative phenotypic marker of smooth muscle cells, were significantly overexpressed on the surfaces of smooth muscle cells present in the injured blood vessels. Similarly, increased expressions of S100A9 and SMemb proteins were observed in aneurismal specimens obtained from P. gingivalis-infected patients. We found that bacteremia induced by P. gingivalis leads to intimal hyperplasia associated with overexpressions of S100A9 and SMemb. Our results strongly suggest that oral-hematogenous spreading of P. gingivalis is a causative event in the development of aortic hyperplasia in periodontitis patients.

Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1) Interaction Reveals Regulatory and Activating Domains of Neurofascin

Fibroblast growth factor receptors (FGFRs) are important for many different mechanisms, including cell migration, proliferation, differentiation, and survival. Here, we show a new link between FGFR1 and the cell adhesion molecule neurofascin, which is important for neurite outgrowth. After overexpression in HEK293 cells, embryonal neurofascin isoform NF166 was able to associate with FGFR1, whereas the adult isoform NF186, differing from NF166 in additional extracellular sequences, was deficient. Pharmacological inhibitors and overexpression of dominant negative components of the FGFR signaling pathway pointed to the activation of FGFR1 after association with neurofascin in neurite outgrowth assays in chick tectal neurons and rat PC12-E2 cells. Both extra- and intracellular domains of embryonal neurofascin isoform NF166 were able to form complexes with FGFR1 independently. However, the cytosolic domain was both necessary and sufficient for the activation of FGFR1. Cytosolic serine residues 56 and 100 were shown to be essential for the neurite outgrowth-promoting activity of neurofascin, whereas both amino acid residues were dispensable for FGFR1 association. In conclusion, the data suggest a neurofascin intracellular domain, which activates FGFR1 for neurite outgrowth, whereas the extracellular domain functions as an additional, regulatory FGFR1 interaction domain in the course of development.

Drebrin a knockout eliminates the rapid form of homeostatic synaptic plasticity at excitatory synapses of intact adult cerebral cortex

Homeostatic synaptic plasticity (HSP) is important for maintaining neurons' excitability within the dynamic range and for protecting neurons from unconstrained long-term potentiation that can cause breakdown of synapse specificity (Turrigiano [2008] Cell 135:422-435). Knowledge of the molecular mechanism underlying this phenomenon remains incomplete, especially for the rapid form of HSP. To test whether HSP in adulthood depends on an F-actin binding protein, drebrin A, mice deleted of the adult isoform of drebrin (DAKO) but retaining the embryonic isoform (drebrin E) were generated. HSP was assayed by determining whether the NR2A subunit of N-methyl-D-aspartate receptors (NMDARs) can rise rapidly within spines following the application of an NMDAR antagonist, D-APV, onto the cortical surface. Electron microscopic immunocytochemistry revealed that, as expected, the D-APV treatment of wild-type (WT) mouse cortex increased the proportion of NR2A-immunolabeled spines within 30 minutes relative to basal levels in hemispheres treated with an inactive enantiomer, L-APV. This difference was significant at the postsynaptic membrane and postsynaptic density (i.e., synaptic junction) as well as at nonsynaptic sites within spines and was not accompanied by spine size changes. In contrast, the D-APV treatment of DAKO brains did not augment NR2A labeling within the spine cytoplasm or at the synaptic junction, even though basal levels of NR2A were not significantly different from those of WT cortices. These findings indicate that drebrin A is required for the rapid (<30 minutes) form of HSP at excitatory synapses of adult cortices, whereas drebrin E is sufficient for maintaining basal NR2A levels within spines.

Evolutionary changes in myosin isoform expression in the tongue by quantitative PCR

Tongue and cranial muscles experience unique mechanical demands, particularly in the complex speech of humans, and may require specialized contractile properties. Cranial muscles, derived from somitomeres, display uncommon myosin heavy chain (MHC) isoforms, including cardiac‐α (Ca) and extraocular (EOM). Tongue muscles derive from somites and do not appear to express these isoforms by protein analysis. mRNA analysis may reveal expression of Ca and EOM. We hypothesize that MHC isoform expression will reflect the functional demands on the tongue, including expression of Ca and EOM. Expression of 8 MHC isoforms in intrinsic muscles of the tongues of human, monkey, and rat were determined by QPCR. Rats express mostly 2x (67 ± 4%) with substantial 2b (31 ± 4%) and no slow MHC. Humans and monkeys express primarily conventional MHCs: 2x (20 ± 6% human, 28± 6% monkey), 2a (50 ± 7%, 58 ± 7%) and slow MHC (24 ± 6%, 10 ± 2%). The expression of faster MHC isoforms in rat than primate tongue is similar to limb MHC protein expression patterns. Of the uncommon isoforms, Ca constitutes 5 ± 3% of total MHC in humans, and individual monkey samples contained up to 7.5% EOM MHC. Neonatal and embryonic isoforms contribute less than 1% of MHC in all 3 species. We conclude that the properties of conventional MHC isoforms may not provide sufficient functional diversity for tongue action. Supported by DC05017

L-Carnitine supplementation during suckling intensifies the early postnatal skeletal myofiber formation in piglets of low birth weight1

Piglets of low birth weight exhibit a reduced total number of skeletal myofibers at birth and throughout life compared with piglets of middle and heavy birth weight, which is associated with impaired (lean) growth and quality of carcass and meat at market weight. We investigated the effect of L-carnitine supplementation to suckling piglets of different birth weights on early postnatal myofiber formation, muscle growth, and body composition. A total of 48 piglets of low (LW) and middle (MDW) birth weight from 9 German Landrace gilts received 400 mg of L-carnitine (carnitine, n = 25) or a placebo (control, n = 23) once daily from d 7 to 27 of age and were slaughtered on d 28 of age (weaning). Carnitine-supplemented piglets deposited less fat as indicated by a reduced proportion of perirenal (P = 0.1) and intramuscular fat (P = 0.05). Circulating glucose concentrations tended to be greater in supplemented LW piglets (P = 0.13). The concentration of carnitine in semitendinosus (STN) muscle was approximately doubled (P < 0.001) by supplementation, with emphasis on the proportion of esterified carnitine. The ratio of lactate dehydrogenase to isocitrate dehydrogenase tended (P = 0.12) to be smaller in STN muscle of supplemented piglets, indicating a more oxidative muscle metabolism. The total number of STN myofibers was increased by 13% (P = 0.02) in supplemented LW piglets, thereby reaching the unchanged level of MDW littermates. In addition, supplemented LW piglets displayed a 2.4-fold mRNA expression of the gene encoding the embryonic isoform of the myosin heavy chain in STN muscle than control piglets (P = 0.05), but there were no differences in the proportion of fibers positively staining for the embryonic myosin isoform. L-carnitine-supplemented piglets exhibited a greater DNA:protein ratio (P = 0.02) in STN muscle, which resulted from a greater DNA concentration (P = 0.04). However, the STN muscle of L-carnitine-supplemented piglets was not less mature as indicated by unchanged myofiber size, creatine kinase activity, and protein concentration. The results indicate that energy balance has been improved through intensified fatty acid oxidation. As a consequence, myogenic proliferation appears to be stimulated, which in LW piglets may have contributed to a compensatory increase in myofiber number. Thus, piglets, particularly those of low birth weight, could profit from an early postnatal L-carnitine supplementation, which may attenuate the negative consequences of low birth weight on body composition and meat quality at market weight.

The Mechanical Properties of Drosophila Jump Muscle Expressing Wild-type and an Embryonic Myosin Isoform

Transgenic Drosophila are highly useful for muscle protein structure-function studies, particularly myosin isoform diversity. However, our ability to mechanically analyze mutant proteins in Drosophila muscle has been limited to the skinned indirect flight muscle (IFM) preparation. We have developed a new preparation using the Drosophila tergal depressor of trochanter muscle (TDT) that increases our experiments to include maximum shortening velocity (Vmax), force-velocity relations, and steady-state power generation, which are not possible using IFM fibers. As with the IFM, we can replace the native TDT myosin with our myosin of choice. When expressing its native isoform (P2), the TDT is equivalent to a very fast vertebrate muscle, with a Vmax of 6.1±0.3 muscle lengths/second at 15°C, a steep tension-pCa curve, a Hill coefficient of 11±2, a high active isometric tension of 37±3 mN/mm2, and maximum power production (Pmax) at 43% of Vmax and 42% of maximum tension. Expressing an embryonic myosin isoform (EMB) in the TDT muscle decreased Vmax, isometric tension and Pmax by 50%, and the tension-pCa Hill coefficient decreased to 6±2. Varying ATP concentration, while measuring Vmax, revealed a higher ATP affinity for EMB than P2. Increasing Pi concentration reduced isometric tension of TDT expressing either isoform. A slight decrease in TDT Vmax with increasing Pi concentration suggests TDT Vmax may be influenced by Pi release rate. TDT Vmax was not influenced by [Pi] when expressing EMB. With our advances in the TDT preparation we will now be able to test a wider speed range of myosin isoforms, including the superfast IFM myosin, to test our hypothesis that a step associated with Pi release is rate limiting for Vmax of very fast myosins, while a step associated with ADP release is limiting for slower isoforms.

Alternative Versions of the Myosin Relay Domain Differentially Respond to Load to Influence Drosophila Muscle Kinetics

We measured the influence of alternative versions of the Drosophila melanogaster myosin heavy chain relay domain on muscle mechanical properties. We exchanged relay domain regions (encoded by alternative versions of exon 9) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin. Previously, we observed no effect of exchanging the EMB relay domain region into the flight muscle isoform (IFI-9b) on in vitro actin motility velocity or solution ATPase measurements compared to IFI. However, in indirect flight muscle fibers, IFI-9b exhibited decreased maximum power generation ( P max) and optimal frequency of power generation ( f max) to 70% and 83% of IFI fiber values. The decrease in muscle performance reduced the flight ability and wing-beat frequency of IFI-9b Drosophila compared to IFI Drosophila. Previously, we found that exchanging the flight muscle specific relay domain into the EMB isoform (EMB-9a) prevented actin movement in the in vitro motility assay compared to EMB, which does support actin movement. However, in indirect flight muscle fibers EMB-9a was a highly effective motor, increasing P max and f max 2.5-fold and 1.4-fold, respectively, compared to fibers expressing EMB. We propose that the oscillatory load EMB-9a experiences in the muscle fiber reduces a high activation energy barrier between two strongly bound states of the cross-bridge cycle, thereby promoting cross-bridge cycling. The IFI relay domain's enhanced sensitivity to load increases cross-bridge kinetics, whereas the EMB version is less load-sensitive.

Identification of Candidate SNP (Single Nucleotide Polymorphism) for Growth and Carcass Traits Related to QTL on Chromosome 6 in Hanwoo (Korean Cattle)

As genetic markers, single nucleotide polymorphisms (SNP) are very appropriate for the development of genetic tests for economic traits in livestock. Several microsatellite markers have been identified as useful markers for the genetic improvement of Hanwoo. Among those markers, ILSTS035 was recently mapped at a similar position with four SNPs (AH1_11, AH1_9, 31465_446, and 12273_165) in a linkage map of EST-based SNP in BAT6. Among the four SNPs, two SNPs (31465_446 and 12273_165) were analyzed using BLAST at the NCBI web site. The sequences including the 12273_165 SNP were identified at the intron region within the LOC534614 gene on the gene sequence map (Bos taurus NCBI Map view, build 3.1). The LOC534614 gene represents a protein similar to myosin heavy chain, fat skeletal muscle, embryonic isoform 1 in the dog, and myosin_1 (Myosin heavy chain D) in Macaca mulatta. In cattle, the myosin heavy chain was associated with muscle development. The phenotypic data for growth and carcass traits in the 415 animals were analyzed by the mixed ANCOVA (analysis of covariance) linear model using PROC GLM module in SAS v9.1. By the genotyping of Hanwoo individuals (n = 415) to evaluate the association of SNP with growth and carcass traits, it was shown that the 12273_165 SNP region within LOC534614 may be a candidate marker for growth. The results of the statistical analyses suggested that the genotype of the 12273_165 SNP significantly affected birth weight, weight of the cattle at 24 months of age, average daily gain and carcass cold weight (p<0.05). Consequently, the 12273_165 SNP polymorphisms at the LOC534614 gene may be associated with growth in Hanwoo, and functional validation of polymorphisms in LOC534614 should be performed in the future. (Key Words : SNP, QTL, BTA6, Growth, Hanwoo)

Embryonic Myosin Heavy-Chain Mutations Cause Distal Arthrogryposis and Developmental Myosin Myopathy That Persists Postnatally

Myosin is a molecular motor and the essential part of the thick filament of striated muscle. The expression of myosin heavy-chain (MyHC) isoforms is developmentally regulated. The embryonic isoform encoded from MYH3 (OMIM *160720) is expressed during fetal life. Recently, mutations in MYH3 were demonstrated to be associated with congenital joint contractures, that is, Freeman-Sheldon and Sheldon-Hall syndromes, which are both distal arthrogryposis syndromes. Mutations in other MyHC isoforms cause myopathy. It is unknown whether MYH3 mutations cause myopathy because muscle tissue has not been studied. To determine whether novel MYH3 mutations are associated with distal arthrogryposis and to demonstrate myopathic changes in muscle biopsy specimens from 4 patients with distal arthrogryposis and MYH3 mutations. In a cohort of patients with distal arthrogryposis, we analyzed the entire coding sequence of MYH3. Muscle biopsy specimens were obtained, and in addition to morphologic analysis, the expression of MyHC isoforms was investigated at the protein and transcript levels. We identified patients from 3 families with novel MYH3 mutations. These mutations affect developmentally conserved residues that are located in different regions of the adenosine triphosphate-binding pocket of the MyHC head. The embryonic (MYH3) isoform was not detected in any of the muscle biopsy samples, indicating a normal developmental downregulation of MYH3 in these patients. However, morphologic analysis of muscle biopsy specimens from the 4 patients revealed mild and variable myopathic features and a pathologic upregulation of the fetal MyHC isoform (MYH8) in 1 patient. Distal arthrogryposis associated with MYH3 mutations is secondary to myosin myopathy, and postnatal muscle manifestations are variable.

RNAi Targeting Embryonic Myosin Heavy Chain Isoform Inhibited Bound Thrombin-Induced Migration of Vascular Smooth Muscle Cells

To investigate the effect of bound thrombin, a complex of α-thrombin with fibrin fragments derived from clots, on proliferation and migration of cultured rabbit vascular smooth muscle cells, cell proliferation was measured by WST-1 reagent and migration was evaluated by counting migrated cells through pores of cell culture insert (8 μm size) after 48-hour treatment with bound thrombin (10 U/ml). To examine the role of an embryonic myosin heavy chain isoform (SMemb) in these effects by bound thrombin, the cells were subsequently treated for 48 h with an siRNA expression vector (ORF-2/pSilencer) directed against the open reading frame of SMemb mRNA. SMemb and plasminogen activator inhibitor-1 mRNA expressions were measured by Northern blot analysis. Bound thrombin significantly increased SMemb mRNA expression by 1.4 ± 0.01-fold and significantly increased plasminogen activator inhibitor-1 mRNA expression by 2.65 ± 0.69-fold (p < 0.01 vs. PBS treatment for each), which were abolished by treatment with ORF-2/pSilencer. Although bound thrombin had no effect on cell proliferation, bound thrombin significantly increased migration by 1.93 ± 0.20-fold (p < 0.05). ORF-2/pSilencer treatment significantly reduced the bound thrombin-stimulated migration activity by 1.28 ± 0.15-fold (p < 0.05). Thus, SMemb plays an important role in bound thrombin-induced cell migration activity of cultured vascular smooth muscle cells.

Alternative Relay Domains of Drosophila melanogaster Myosin Differentially Affect ATPase Activity, in Vitro Motility, Myofibril Structure and Muscle Function

The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.

The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth

Many tumour cells have elevated rates of glucose uptake but reduced rates of oxidative phosphorylation. This persistence of high lactate production by tumours in the presence of oxygen, known as aerobic glycolysis, was first noted by Otto Warburg more than 75 yr ago. How tumour cells establish this altered metabolic phenotype and whether it is essential for tumorigenesis is as yet unknown. Here we show that a single switch in a splice isoform of the glycolytic enzyme pyruvate kinase is necessary for the shift in cellular metabolism to aerobic glycolysis and that this promotes tumorigenesis. Tumour cells have been shown to express exclusively the embryonic M2 isoform of pyruvate kinase. Here we use short hairpin RNA to knockdown pyruvate kinase M2 expression in human cancer cell lines and replace it with pyruvate kinase M1. Switching pyruvate kinase expression to the M1 (adult) isoform leads to reversal of the Warburg effect, as judged by reduced lactate production and increased oxygen consumption, and this correlates with a reduced ability to form tumours in nude mouse xenografts. These results demonstrate that M2 expression is necessary for aerobic glycolysis and that this metabolic phenotype provides a selective growth advantage for tumour cells in vivo.

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter of the adult nervous system and its biosynthetic enzyme glutamic acid decarboxylase (GAD) are abundantly expressed in the embryonic nervous system and are involved in the modulation of cell proliferation, migration, and differentiation. Here we describe for the first time the expression of GABA and embryonic and adult GAD isoforms in the developing mouse lens. We show that the GAD isoforms are sequentially induced with specific spatiotemporal profiles: GAD65 and embryonic GAD isoforms prevail in primary fibers, while GAD67 is the predominant GAD expressed in the postnatal secondary fibers. This pattern correlates well with the expression of Dlx2 and Dlx5, known as upstream regulators of GAD. GABA and GAD are most abundant at the tips of elongating fibers and are absent from organelle-free cells, suggesting their involvement is primarily in shaping of the cytoskeleton during fiber elongation stages.

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with α4β1 integrins

BackgroundThe regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for α4β1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the α4 integrin cytoplasmic domain in neurite outgrowth.ResultsExpression of α4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type α4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal α4-mediated migration in leucocytes requires spatial regulation of α4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this α4(S988D), which mimics phosphorylated α4, did not promote neurite outgrowth. However, α4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable α4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin.ConclusionWe establish the importance of the α4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of α4 for extension of neuronal growth cones and migration of non-neural cells.

Neurofascin regulates the formation of gephyrin clusters and their subsequent translocation to the axon hillock of hippocampal neurons

Little is known about the role of cell adhesion molecules (CAMs) in inhibitory synapse development. In particular, a functional link between CAMs and the clustering of postsynaptic scaffold component gephyrin, which is a critical determinant of γ-aminobutyric acid A (GABA) receptor clustering, still needs to be elaborated. At early stages of inhibitory synapse formation, gephyrin and CAM neurofascin are diffusely expressed in the soma of hippocampal neurons. Subsequently, gephyrin clusters become localized to the axon hillock and neurofascin is observed all over the soma including the axon hillock suggesting a function for neurofascin in gephyrin clustering. Transfection of expression vectors for different isoforms and mutants of neurofascin revealed that neurofascin is required for the formation of gephyrin clusters presumably dependent on extracellular interactions. Furthermore, expression of neurofascin is necessary for the translocation of gephyrin clusters to the axon hillock of hippocampal neurons as shown by shRNA-mediated knockdown. In addition, overexpression of an embryonic neurofascin isoform is sufficient for functional rescue after knockdown of endogenous neurofascin.

Research Article: Expression pattern of deoxyguanosine kinase3 during mouse development

The mitochondrial salvage pathway supplies deoxyribonucleotide triphosphates for mitochondrial DNA synthesis. Its rate limiting enzymes are deoxyguanosine kinase (dGK) and thymidine kinase2. Mutations in these enzymes lead to several mitochondrial diseases in infants. Deoxyguanosine kinase3 (dGK3) is an isoform of dGK that was cloned from gestational day 8 mouse embryos. The objective of this study was to characterize the developmental expression of dGK3 in mouse embryos. Non-radioactive Northern blot and reverse transcription-polymerase chain reaction analyses were used. Expression analyses showed that the smaller sized transcript of dGK3 was expressed in embryos from gestational day 8 through day 17. Expression levels were uniform from gestational day 8 to 15 with the lowest expression on gestational day 17. Further analyses showed expression of the gene in multiple adult tissues indicating the dGK3 is not an embryonic isoform of dGK. This study is the first analysis of dGK3 expression during early development.