Next-generation sequencing reveals the mitogenomic heteroplasmy in the topmouth culter (Culter alburnus Basilewsky, 1855).

BACKGROUND: The mitogenomic heteroplasmy is the presence of multiple haplotypes in the mitochondria, which could cause genetic diseases and is also associated with many critical biological functions. The topmouth culter (Culter alburnus Basilewsky, 1855) is one of the most important freshwater fish in the family of Cyprinidae in China. At present, there are no reports on the topmouth culter's mtDNA heteroplasmy and the existence of which is not known. METHODS AND RESULTS: This study aimed to analyze the mitogenomic heteroplasmy in the topmouth culter by the next-generation sequencing of the fins' total DNA. The results confirmed the existence of the heteroplasmy and indicated the presence of the extensive heteroplasmy in the topmouth culter's mitogenome. There were 38 heteroplasmic variations in the protein-coding genes from the three specimens, with 33 non-synonymous substitutions accounting for 86.84% and five synonymous substitutions accounting for 13.16%. Among them, the ND6 had the most heteroplasmic variations but only one synonymous substitution. After removing the putative nuclear mitochondrial DNA fragments, the ratio of primary haplotype in the three specimens was 43.89%, 74.72%, and 32.76%, respectively. The three specimens contained 21, 7, and 21 haplotypes of the mitogenomes, respectively. Due to the extensive heteroplasmy, we reconstructed the phylogenetic tree of the topmouth culter using the RY-coding method, which improved the performance of the phylogenetic tree to some extent. CONCLUSIONS: This study reported the mitogenomic heteroplasmy in the topmouth culter and enhanced the knowledge regarding the mitogenomic heteroplasmy in phylogenetic studies. As the topmouth culter is a commercial species, the mitogenomic heteroplasmy is crucial for the fisheries management of the topmouth culter.

First mitogenome of subfamily Langiinae (Lepidoptera: Sphingidae) with its phylogenetic implications.

To date, a relatively complete classification of Sphingidae (Lepidoptera) has been generated, but the phylogeny of the family remains need to be fully resolved. Some phylogenetic relationships within Sphingidae still remains uncertain, especially the taxonomic status of the subfamily Langiinae and its sole included genus and species, Langia zenzeroides. To begin to address this problem, we generated nine new complete mitochondrial genomes, including that of Langia, and together with that of Theretra oldenlandiae from our previous study and 25 other Sphingidae mitogenomes downloaded from GenBank, analyzed the phylogenetic relationships of Sphingidae and investigated the mitogenomic differences among members of the Langiinae, Sphinginae, Smerinthinae and Macroglossinae. The mitogenomes of Sphingidae varied from 14995 bp to 15669 bp in length. The gene order of all newly sequenced mitogenomes was identical, containing 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and the A + T-rich region. Nucleotide composition was A + T biased, and all the protein-coding genes exhibited a positive AT-skew, which was reflected in the nucleotide composition, codon, and amino acid usage. The A + T-rich region was comprised of nonrepetitive sequences, which contained regulatory elements related to the control of replication and transcription. We analyzed concatenated gene sequences, with third codon positions of protein coding genes and rRNAs excluded, using Maximum Likelihood and Bayesian Inference techniques. All four currently recognized subfamilies were recovered as monophyletic but in contrast to the most recent studies, our preferred tree placed Langiinae as the first subfamily to diverge within Sphingidae rather as sister to Smerinthinae + Sphinginae. Our results also support the removal of the genus Barbourion from the smerinthine tribe Ambulycini to an unresolved position in "Smerinthinae incertae sedis".

Complete mitogenome of endemic plum-headed parakeet Psittacula cyanocephala - characterization and phylogenetic analysis.

Psittacula cyanocephala is an endemic parakeet from the Indian sub-continent that is widespread in the illegal bird trade. Previous studies on Psittacula parakeets have highlighted taxonomic ambiguities, warranting studies to resolve the issues. Since the mitochondrial genome provides useful information concerning the species evolution and phylogenetics, we sequenced the complete mitogenome of P. cyanocephala using NGS, validated 38.86% of the mitogenome using Sanger Sequencing and compared it with other available whole mitogenomes of Psittacula. The complete mitogenome of the species was 16814 bp in length with 54.08% AT composition. P. cyanocephala mitogenome comprises of 13 protein-coding genes, 2 rRNAs and 22 tRNAs. P. cyanocephala mitogenome organization was consistent with other Psittacula mitogenomes. Comparative codon usage analysis indicated the role of natural selection on Psittacula mitogenomes. Strong purifying selection pressure was observed maximum on nad1 and nad4l genes. The mitochondrial control region of all Psittacula species displayed the ancestral avian CR gene order. Phylogenetic analyses revealed the Psittacula genus as paraphyletic nature, containing at least 4 groups of species within the same genus, suggesting its taxonomic reconsideration. Our results provide useful information for developing forensic tests to control the illegal trade of the species and scientific basis for phylogenetic revision of the genus Psittacula.

Characterization and Comparative Analyses of Mitochondrial Genomes in Single-Celled Eukaryotes to Shed Light on the Diversity and Evolution of Linear Molecular Architecture.

Determination and comparisons of complete mitochondrial genomes (mitogenomes) are important to understand the origin and evolution of mitochondria. Mitogenomes of unicellular protists are particularly informative in this regard because they are gene-rich and display high structural diversity. Ciliates are a highly diverse assemblage of protists and their mitogenomes (linear structure with high A+T content in general) were amongst the first from protists to be characterized and have provided important insights into mitogenome evolution. Here, we report novel mitogenome sequences from three representatives (Strombidium sp., Strombidium cf. sulcatum, and Halteria grandinella) in two dominant ciliate lineages. Comparative and phylogenetic analyses of newly sequenced and previously published ciliate mitogenomes were performed and revealed a number of important insights. We found that the mitogenomes of these three species are linear molecules capped with telomeric repeats that differ greatly among known species. The genomes studied here are highly syntenic, but larger in size and more gene-rich than those of other groups. They also all share an AT-rich tandem repeat region which may serve as the replication origin and modulate initiation of bidirectional transcription. More generally we identified a split version of ccmf, a cytochrome c maturation-related gene that might be a derived character uniting taxa in the subclasses Hypotrichia and Euplotia. Finally, our mitogenome comparisons and phylogenetic analyses support to reclassify Halteria grandinella from the subclass Oligotrichia to the subclass Hypotrichia. These results add to the growing literature on the unique features of ciliate mitogenomes, shedding light on the diversity and evolution of their linear molecular architecture.

Of mitogens and morphogens: modelling Sonic Hedgehog mechanisms in vertebrate development.

Sonic Hedgehog (Shh) Is a critical protein in vertebrate development, orchestrating patterning and growth in many developing systems. First described as a classic morphogen that patterns tissues through a spatial concentration gradient, subsequent studies have revealed a more complex mechanism, in which Shh can also regulate proliferation and differentiation. While the mechanism of action of Shh as a morphogen is well understood, it remains less clear how Shh might integrate patterning, proliferation and differentiation in a given tissue, to ultimately direct its morphogenesis. In tandem with experimental studies, mathematical modelling can help gain mechanistic insights into these processes and bridge the gap between Shh-regulated patterning and growth, by integrating these processes into a common theoretical framework. Here, we briefly review the roles of Shh in vertebrate development, focusing on its functions as a morphogen, mitogen and regulator of differentiation. We then discuss the contributions that modelling has made to our understanding of the action of Shh and highlight current challenges in using mathematical models in a quantitative and predictive way. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

Purification of recombinant human fibroblast growth factor 13 in E. coli and its molecular mechanism of mitogenesis.

Fibroblast growth factor (FGF) 13, a member of the FGF11 subfamily, is a kind of intracrine protein similar to other family members including FGF11, FGF12, and FGF14. Unlike classical FGF, FGF13 exerts its bioactivities independent of fibroblast growth factor receptors (FGFRs). However, the effect of exogenous administration of FGF13 still remains further investigated. In the present study, we established an Escherichia coli expression system for the large-scale production of FGF13 and then obtained two isoform proteins including recombinant human FGF13A (rhFGF13A) and rhFGF13B with a purity greater than 90% by column chromatography, respectively. Otherwise, soluble analysis indicated that both rhFGF13A and rhFGF13B expressed in E. coli BL21 (DE3) pLysS were soluble. Furthermore, cellular-based experiments demonstrated that rhFGF13A, rather than rhFGF13B, could promote the proliferation of NIH3T3 cells in the presence of heparin. Mechanistically, the mitogenic effect of FGF13 was mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), but not p38. Moreover, blockage of FGFRs also significantly attenuated the mitogenic effects of rhFGF13A, implying that FGFRs are still related to FGF13. Thus, our research shows that exogenous FGF13 can act as secreted FGF to participate in cell signal transmission and heparin is still required as an ancillary cofactor for the mitogenic effects of FGF13, which may help people to discover more potential functions of FGF13 in cell life activities.

Identification of two new core chromosome-encoded superantigens in Streptococcus pyogenes; speQ and speR.

Superantigens are ubiquitous within the Streptococcus pyogenes genome, which suggests that superantigen-mediated T-cell activation provides a significant selective advantage. S. pyogenes can carry a variable complement of the 11 known superantigens. We have identified two novel S. pyogenes superantigens, denoted speQ and speR, adjacent to each other in the core-chromosome of isolates belonging to eleven different emm-types. Although distinct from other superantigens, speQ and speR were most closely related to speK and speJ, respectively. Recombinant SPEQ and SPER were mitogenic towards human peripheral blood mononuclear cells at ng/ml concentrations, and SPER was found to be more mitogenic than SPEQ.

Nutrient sensing, growth and senescence.

Cell growth is dictated by a wide range of mitogenic signals, the amplitude and relative contribution of which vary throughout development, differentiation and in a tissue-specific manner. The ability to sense and appropriately respond to changes in mitogens is fundamental to control cell growth, and reduced responsiveness of nutrient sensing pathways is widely associated with human disease and ageing. Cellular senescence is an important tumour suppressor mechanism that is characterised by an irreversible exit from the cell cycle in response to replicative exhaustion or excessive DNA damage. Despite the fact that senescent cells can no longer divide, they remain metabolically active and display a range of pro-growth phenotypes that are supported in part by the mTORC1-autophagy signalling axis. As our understanding of the basic mechanisms of controlling mTORC1-autophagy activity and cell growth continues to expand, we are able to explore how changes in nutrient sensing contribute to the acquisition and maintenance of cellular senescence. Furthermore, while the protective effect of senescence to limit cellular transformation is clear, more recently, the age-related accumulation of these pro-inflammatory senescent cells has been shown to contribute to a decline in organismal fitness. We will further discuss whether dysregulation of nutrient sensing pathways can be targeted to promote senescent cell death which would have important implications for healthy ageing.

A comprehensive model for the proliferation-quiescence decision in response to endogenous DNA damage in human cells.

Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation-quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.

Uncoupling the Mitogenic and Metabolic Functions of FGF1 by Tuning FGF1-FGF Receptor Dimer Stability.

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1ΔHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1ΔHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.

Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure.

Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest.

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.

Pro- and anti-mitogenic actions of pituitary adenylate cyclase-activating polypeptide in developing cerebral cortex: potential mediation by developmental switch of PAC1 receptor mRNA isoforms.

During corticogenesis, pituitary adenylate cyclase-activating polypeptide (PACAP; ADCYAP1) may contribute to proliferation control by activating PAC1 receptors of neural precursors in the embryonic ventricular zone. PAC1 receptors, specifically the hop and short isoforms, couple differentially to and activate distinct pathways that produce pro- or anti-mitogenic actions. Previously, we found that PACAP was an anti-mitogenic signal from embryonic day 13.5 (E13.5) onward both in culture and in vivo and activated cAMP signaling through the short isoform. However, we now find that mice deficient in PACAP exhibited a decrease in the BrdU labeling index (LI) in E9.5 cortex, suggesting that PACAP normally promotes proliferation at this stage. To further define mechanisms, we established a novel culture model in which the viability of very early cortical precursors (E9.5 mouse and E10.5 rat) could be maintained. At this stage, we found that PACAP evoked intracellular calcium fluxes and increased phospho-PKC levels, as well as stimulated G1 cyclin mRNAs and proteins, S-phase entry, and proliferation without affecting cell survival. Significantly, expression of hop receptor isoform was 24-fold greater than the short isoform at E10.5, a ratio that was reversed at E14.5 when short expression was 15-fold greater and PACAP inhibited mitogenesis. Enhanced hop isoform expression, elicited by in vitro treatment of E10.5 precursors with retinoic acid, correlated with sustained pro-mitogenic action of PACAP beyond the developmental switch. Conversely, depletion of hop receptor using short-hairpin RNA abolished PACAP mitogenic stimulation at E10.5. These observations suggest that PACAP elicits temporally specific effects on cortical proliferation via developmentally regulated expression of specific receptor isoforms.

Oxidized LDL enhances stretch-induced smooth muscle cell proliferation through alterations in nuclear protein import.

Mechanical stress contributes to hypertension and atherosclerosis partly through the stimulation of vascular smooth muscle cell (VSMC) proliferation. Oxidized low density lipoprotein (oxLDL) is another important atherogenic factor that can increase VSMC proliferation. The purpose of this study was to investigate whether oxLDL could further enhance the proliferative action of mechanical stretch on VSMC, and to determine the mechanism responsible for this interaction. Because nuclear protein import is critical in regulating gene expression, transcription, and cell proliferation, its involvement in the mitogenic effects of oxLDL and mechanical stress was studied. OxLDL enhanced the proliferative effects of mechanical stretch on its own in rabbit aortic VSMC, and induced increases in the expression of HSP60 in an additive manner. Adenoviral-mediated overexpression of HSP60 induced increases in cell proliferation compared with uninfected VSMC. Mechanical stretch and oxLDL stimulated the rate of nuclear protein import in VSMC and increased the expression of nucleoporins. These effects were sensitive to inhibition of the MAPK pathway. We conclude that oxLDL and mechanical stretch have a synergistic effect on VSMC proliferation. This synergistic effect is induced through a stimulation of nuclear protein import via HSP60 and an activation of the MAPK pathway.

Analysis of mutants with altered shh activity and posterior digit loss supports a biphasic model for shh function as a morphogen and mitogen.

Sonic hedgehog (Shh) controls the number and type of digits formed. Using a conditional genetic approach for timed removal of Shh, we previously proposed a biphasic model of Shh function: a transient patterning phase, during which digit progenitors are specified, and an extended proliferative phase, during which expansion of progenitor pools enables digit formation. Other models favor a close integration of digit patterning and expansion, with sequential promotion to more posterior identity over time, apparently supported by some mutants with selective posterior digit loss. To further test these models, we analyzed the dynamics of Shh activity in several oligodactylous mutants with different types of digit loss. The profile of Shh activity and phenotypic outcome in these mutants supports a biphasic over an integrated temporal model. Eomesodermin expression, as an independent marker of posterior digit identity, confirmed that proper digit 4 specification requires only the transient phase of Shh activity.

Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen.

BACKGROUND: Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification. RESULTS: We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037). CONCLUSIONS: Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.

Mitogenic signaling from apoptotic cells in Drosophila.

Apoptotic cells of Drosophila not only activate caspases, but also are able to secrete developmental signals like Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) before dying. Since Dpp and Wg are secreted in growing tissues and behave as growth factors, it was proposed that they play a role in compensatory proliferation, the process by which a growing blastema can restore normal size after massive apoptosis. We discuss recent results showing that there is normal compensatory proliferation in the absence of Dpp/Wg signaling, thus indicating it has no significant role in the process. Furthermore, we argue that Dpp/Wg signaling is not a resident feature of apoptotic cells, but a side effect of the necessary activation of the JNK pathway. Nevertheless, the ectopic JNK/Dpp/Wg signaling may have an important role in tissue regeneration. Recent work in other organisms suggests that paracrine signaling from apoptotic cells may be of general significance in wound healing and tissue regeneration in metazoans.

Quantitative assessment of AKAP12 promoter methylation in human prostate cancer using methylation-sensitive high-resolution melting: correlation with Gleason score.

OBJECTIVES: To quantitatively investigate the A kinase anchoring protein 12 (AKAP12) gene promoter methylation and its association with clinicopathologic variables in human prostate cancer (PCa). The AKAP12 gene has shown reduced expression and marked hypermethylation in a variety of cancers. METHODS: The percentage levels of DNA methylation were measured in 78 PCa, 22 benign prostatic hyperplasia, and 22 normal adjacent tissue samples using an AKAP12 methylation-sensitive high-resolution melting assay. AKAP12 gene expression was also examined in 4 human prostate carcinoma cell lines, PC-3, DU145, LNCaP, and 22RV1, using quantitative reverse transcriptase-polymerase chain reaction and methylation-sensitive high-resolution melting analysis and after DNA methyltransferase inhibition with 5-aza-2'-deoxycytidine. RESULTS: Methylation (>1%) of the AKAP12 promoter region was present in 47 (60.2%) of the 78 PCa, 5 (22.7%) of the 22 benign prostatic hyperplasia, and 2 (9.1%) of the 22 adjacent normal tissue samples. AKAP12 methylation was significantly greater in the PCa than in the benign prostatic hyperplasia or adjacent tissue samples (P < .01). AKAP12 methylation was significantly greater in the PCa samples with higher Gleason scores (P = .03); however, no correlation was found with age, pT category, or serum prostate-specific antigen level. Reverse transcriptase-polymerase chain reaction demonstrated that PC-3 and DU-145 cells expressed AKAP12 RNA and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in the LNCaP and 22RV1 cells. Treatment of LNCaP cells with 5-aza-2'-deoxycytidine markedly decreased the methylation levels and increased the expression of AKAP12. CONCLUSIONS: The results of the present study have demonstrated that AKAP12 promoter methylation is a frequent event in human PCa. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of PCa.

Regulation and function of Cav3.1 T-type calcium channels in IGF-I-stimulated pulmonary artery smooth muscle cells.

Arterial smooth muscle cells enter the cell cycle and proliferate in conditions of disease and injury, leading to adverse vessel remodeling. In the pulmonary vasculature, diverse stimuli cause proliferation of pulmonary artery smooth muscle cells (PASMCs), pulmonary artery remodeling, and the clinical condition of pulmonary hypertension associated with significant health consequences. PASMC proliferation requires extracellular Ca(2+) influx that is intimately linked with intracellular Ca(2+) homeostasis. Among the primary sources of Ca(2+) influx in PASMCs is the low-voltage-activated family of T-type Ca(2+) channels; however, up to now, mechanisms for the action of T-type channels in vascular smooth muscle cell proliferation have not been addressed. The Ca(v)3.1 T-type Ca(2+) channel mRNA is upregulated in cultured PASMCs stimulated to proliferate with insulin-like growth factor-I (IGF-I), and this upregulation depends on phosphatidylinositol 3-kinase/Akt signaling. Multiple stimuli that trigger an acute rise in intracellular Ca(2+) in PASMCs, including IGF-I, also require the expression of Ca(v)3.1 Ca(2+) channels for their action. IGF-I also led to cell cycle initiation and proliferation of PASMCs, and, when expression of the Ca(v)3.1 Ca(2+) channel was knocked down by RNA interference, so were the expression and activation of cyclin D, which are necessary steps for cell cycle progression. These results confirm the importance of T-type Ca(2+) channels in proper progression of the cell cycle in PASMCs stimulated to proliferate by IGF-I and suggest that Ca(2+) entry through Ca(v)3.1 T-type channels in particular interacts with Ca(2+)-dependent steps of the mitogenic signaling cascade as a central component of vascular remodeling in disease.

Crystal structure of the Mycoplasma arthritidis-derived mitogen in apo form reveals a 3D domain-swapped dimer.

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vbeta elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-A resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the "reconstituted" MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM(wt) molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.