Myogenesis is a complex and tightly regulated process, the end result of which is the formation of a multinucleated myofibre with contractile capability. Typically, this process is described as being regulated by a coordinated transcriptional hierarchy. However, like any cellular process, myogenesis is also controlled by members of the protein kinase family, which transmit and execute signals initiated by promyogenic stimuli. In this review, we describe the various kinases involved in mammalian skeletal myogenesis: which step of myogenesis a particular kinase regulates, how it is activated (if known) and what its downstream effects are. We present a scheme of protein kinase activity, similar to that which exists for the myogenic transcription factors, to better clarify the complex signalling that underlies muscle development. Embryonic myogenesis is a multistep process that begins with the commitment of an embryonic precursor to the myogenic lineage, followed by the proliferation of these committed myoblasts, the differentiation of myoblasts into postmitotic myocytes, and finally fusion of myocytes to form a multinucleated myotube. As the myotube matures, the syncytial cell becomes specialized for its particular function, with the bulk of the cytoplasm occupied by the contractile apparatus, and where the myotube/myofibre can further grow or hypertrophy in response to appropriate stimuli. Postnatal myogenesis is a similar process, except that fusion occurs primarily between myoblasts and preexisting myotubes, and where the role of the embryonic precursor is played by the quiescent satellite cell.The process of myogenesis is controlled by several myogenic transcription factors that act as terminal effectors of signalling cascades and produce appropriate developmental stage-specific transcripts. The concerted roles that these transcription factors play is well known and well reviewed , or cAMP-dependent protein kinase, was discovered over 40 years ago, the second protein kinase to be described . It is iPax3, MyoD and Myf5 in cells of the dermomyotome that will transition to form the myotome proper [Pax3 and Myf5. Chen et al. [Pax3, MyoD and Myf5 and that this phosphorylation is critical for the induction of these genes. What is interesting about PKA with regards to myogenesis is that although it triggers the expression of several myogenic transcription factors, it also acts to suppress their transcription activity. It has been shown that PKA can inhibit the activity of Myf5, MyoD, myogenin and MEF2D without affecting their ability to bind DNA [PKA is required at multiple stages during myogenesis, but an initial requirement is found during embryogenesis and the formation of myogenic precursors (i.e. myoblasts) within the dermomyotome Figure . PKA acte proper . This acn et al.  showed tbind DNA -10. In tIt is not clear what happens at the onset of differentiation with regards to PKA, although previous results suggest that PKA activity ultimately drops as differentiation proceeds, at least in C2C12 cells . Obvious1 cyclins, which regulate progression through G1 and entry into S phase; the mitotic cyclins, which regulate entry into mitosis; and the non-cell cycle cyclins, which have cell cycle-independent roles. This last group is not a typical classification, but we introduce it here because this group of cyclins/CDKs is important for myogenesis. The G1 cyclins include cyclins D and E, and are responsible for activating CDK4 (cyclin D), CDK6 (cyclin D) and CDK2 (cyclins D and E). The mitotic cyclins A and B activate CDK2 (cyclin A) and CDK1 (cyclin B). Levels of these cyclins are regulated by intrinsic cell cycle-derived signals, with the exception of cyclin D, which is regulated largely by extrinsic signals such as growth factors [The cyclin-dependent kinases (CDKs) take their name from a catalytic dependence on the cyclin family of regulatory proteins. There are several cyclins and CDKs that collectively control cell cycle progression as well as other processes. The cyclins, and by extension the CDKs, can be divided into three major groups: the G factors . The fin1 and mitotic cyclins/CDKs regulate cell cycle progression and proliferation, and myoblasts are no different  and the cyclin-dependent kinase-interacting protein/kinase-inhibitory protein family (CIP/KIP). The INK4 family includes p15, p16, p18 and p19, and, despite the family name, these members also inhibit CDK6. The CIP/KIP members include p21, p27 and p57, and these members inhibit all G1 CDKs. There is substantial evidence demonstrating the importance of the CKIs for myoblast differentiation in vitro and in vivo. The expression levels of p16, p18, p19, p21, p27 and p57 all increase with differentiation [1 and mitotic CDKs [For myoblasts to differentiate, the cell cycle must be exited and the restraints the CDKs place on differentiation must be removed. Differentiation cues, such as serum withdrawal or cell-cell contact in cultured cells, elicit several effects that ultimately cause a decrease in Gdecrease ,26,32-41ntiation ,39,41-46ntiation , and micntiation . When mytic CDKs ,36,39,41tic CDKs ,27,35-38tic CDKs ,39,41,49tic CDKs ,35,37-39tic CDKs ,36,38, cin vitro differentiation and in vivo regeneration following injury [Once the cell cycle CDKs have been effectively silenced and the cell cycle exited, the non-cell cycle CDKs are important for promoting and establishing differentiation Figure . These ig injury -57. Overg injury . Cyclin g injury ,57. The g injury . NeedlesExtracellular signal-regulated kinase (ERK) was first identified as an insulin-sensitive kinase that could phosphorylate the microtubule-associated protein 2, hence its original name 'MAP2 kinase' or 'MAP2K' . It was ERK1 and ERK2 (ERK1/2) belong to a well-defined pathway  [At the turn of the century, several groups reported a critical role for one of or both the p38α and β isoforms during myoblast differentiation ,122-125. (MLC3F) . Most re (MLC3F) -129. It  (MLC3F) ,131, higet al. [Cell-cell contact in myoblast cultures triggers precocious differentiation, and contact is at least one mechanism by which p38α is activated  that code for a protein kinase with an N-terminal pleckstrin homology (PH) domain [The protein kinase Akt first became known as the product of the oncogenic trovirus . The ret) domain -153. Owi) domain ,151.2) and phosphatidylinositol -trisphosphate P3) from PI(4)P and PIP2, respectively. These phosphoinositide products localise PH domain-containing proteins to the plasma membrane, including Akt, the constitutively active phosphoinositide-dependent kinase 1 (PDK1), and "PDK2". The colocalisation of these kinases allows for PDK1 and PDK2 to phosphorylate Akt at distinct sites, with both phosphorylations necessary for activation. PDK2 is not a single kinase but rather a group of kinases [Akt forms part of a well-studied pathway Figure , and for kinases , any oneet al. [et al. [This pathway, with Akt at its heart, is activated by IGF or insulin stimulation, but there is evidence to suggest that Akt can be activated by other mechanisms in muscle cell lines. Elia et al.  showed t [et al.  showed t [et al. ,160, indin vivo, as IGF overexpression in mice triggers myoblast differentiation, myofibre hypertrophy and regeneration [in vivo [While the pathway of Akt activation requires additional elaboration, the importance of the IGF-Akt axis to myogenesis cannot be debated. It has been demonstrated in culture that IGF is critical to, and a potent stimulator of, myoblast differentiation and hypertrophy, and that muscle cell lines upregulate IGF2 upon differentiation ,161-167.neration -170. Sev[in vivo ,171-175.[in vivo ,177, altin vivo or in primary cells, but there is certainly strong evidence to support distinct roles for Akt1 and Akt2 during myogenesis.IGF can activate any of the three Akt isoforms, and currently both Akt1 and Akt2 have been implicated in myogenesis, while Akt3 has not. There is very strong evidence to suggest that isoforms 1 and 2 are required at different stages, although how their activation is differentially controlled is unknown. Protein levels of Akt1 remain constant from proliferating to differentiating cells, whereas the levels and activity of Akt2 increase with differentiation ,178-181.p27 [Akt2 transcript levels [When myoblasts are initially treated with IGF, there is a proliferative response and differentiation is prevented ,185-187.p27 ,99,177. p27 -191, andt levels . Contrarcyclin D1 transcript levels to maintain proliferation, while Akt2 phosphorylation of Pitx2 causes dissociation of this complex and degradation of cyclin D1 mRNA. Once cell cycle exit has occurred, Akt's phosphorylation (and inhibition) of the FoxO family of transcription factors now allows differentiation to occur [Once Akt2 becomes activated, it triggers myoblast cell cycle exit Figure . It doesto occur , as thesto occur , are inhto occur ,181,194.to occur . In myobto occur ,195, allto occur ,196-198 to occur ,198, resto occur ,198. TheFollowing commitment to differentiation, Akt is further required for the growth/hypertrophy of myotubes Figure . Its mulA substantial amount of research on the IGF-Akt signalling axis has been conducted, and we have briefly summarized it here. Further research regarding the distinct roles of Akt1 and Akt2 is required: if, in fact, there is a distinction; how they are differentially regulated; and the similarities and differences between downstream targets. Over 100 Akt substrates are known, but very few have been studied during myogenesis. We have described the handful of substrates that have been studied, but this must be a very incomplete picture, and so there is still much room for further exploration.As we have reviewed here, the differential activation (and inhibition) of distinct protein kinases acts to control the formation of a mature myotube from a population of embryonic precursors or satellite cells. Although there is more to be discovered, a synthesis of the available information reveals a kinase hierarchy that coordinates myogenesis in a fashion analogous to the myogenic transcription factors. Initially, during development, and likely via analogous mechanisms during juvenile and adult myogenesis, the presence of specific Wnts induces PKA activity and the myogenic commitment of precursors to form a pool of dividing myoblasts. Growth factors, and likely other extrinsic components, then activate ERK1/2, Akt1 and cyclin D/CDK2, 4 and 6 to promote proliferation, which, along with PKA, act simultaneously to restrict differentiation. Intrinsic cell cycle-derived signals regulate the levels of cyclins A, B and E, which, together with their respective CDKs, promote cell cycle progression and inhibit differentiation. As the myoblast population expands to the threshold of available space, cell-cell contact can turn off ERK, and a decline in certain growth factors can further silence ERK and downregulate cyclin D/CDK2, 4 and 6 activity, to inhibit additional proliferation. With the silencing of ERK and cell cycle CDK activity, and proliferation lessening, cell-cell contact can promote differentiation through p38 and possibly Akt2 as well. Certain unknown cues may also relocalise PKA activity to remove its restraint on differentiation while allowing it to have a positive effect elsewhere in the cell. Similarly, undefined signals lead to an upregulation of p35 and cyclin T levels to induce the activity of CDK5 and 9, respectively. An increasing concentration of IGF, which is released during differentiation, stimulates Akt2 activity to drive differentiation and hypertrophy, and elevated IGF levels may also induce ERK2 activity at later time points to promote fusion. Finally, as differentiation progresses, p38 activity relocalises to the cytoplasm, where it promotes the mid to late stages of differentiation.The protein kinase scheme we present herein demonstrates how a number of kinases are used by myogenic cells to transition from state to state, and the intricate signalling used to coordinate the proper development of muscle tissue. As complex as is the picture we offer, it is only partial. Not only does more need to be known about the kinases we have discussed, but there are many other kinases that have been implicated in controlling some aspect of myogenesis Table . As resecyclin-dependent kinase-interacting protein/kinase-inhibitory protein; CKI: CDK inhibitor; CREB: cAMP response element-binding protein; ERK: extracellular signal-regulated kinase, a.k.a. microtubule-associated protein 2 kinase (MAP2K); EZH2: enhancer of zeste homologue 2; FGF: fibroblast growth factor; FGFR: fibroblast growth factor receptor; FoxO1: forkhead box protein O1; GFR: growth factor receptor; GRB2: growth factor receptor-bound protein 2; GRIP-1: glucocorticoid receptor-interacting protein 1; GSK3β: glycogen synthase kinase 3β; HDAC: histone deacetylase; HGF: hepatocyte growth factor; HuR: human antigen R; IGF: insulin-like growth factor; IGFR: insulin-like growth factor receptor; INK4: inhibitor of cyclin-dependent kinase 4; IRS: insulin-receptor substrate; KSRP: KH type-splicing regulatory protein; LIF: leukaemia-inhibitory factor; MAFbx: muscle atrophy F box; MAPK: mitogen-activated protein kinase; MAP3K: mitogen-activated protein kinase kinase kinase; MEF2: myocyte enhancer factor 2; MEK: dual-specificity MAP or ERK kinase; MKK3/6: mitogen-activate protein kinase kinase 3/6; MRF4: muscle-specific regulatory factor 4; mTOR: mammalian target of rapamycin; MuRF1: muscle RING finger 1; Myf5: myogenic factor 5; MyoD: myoblast determination protein; NFAT3: nuclear factor of activated T cell 3; p38: p38 mitogen-activated protein kinase; p65/RelA: v-rel reticuloendotheliosis viral oncogene cellular homologue A; Pax7: paired-box protein 7; PDGF: platelet-derived growth factor; PDK1: phosphoinositide-dependent kinase; PH: pleckstrin homology; PHB2: prohibitin 2; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol -bisphosphate; PIP3: phosphatidylinositol -trisphosphate; PI(4)P: phosphatidylinositol 4-phosphate; PIP2: phosphatidylinositol -bisphosphate; Pitx2: pituitary homeobox 2; PKA: protein kinase A, a.k.a. cAMP-dependent protein kinase; PRC2: polycomb repressive complex 2; Rb: retinoblastoma protein; RSK2: 90-kDa ribosomal S6 kinase 2; S6K: ribosomal protein S6 kinase 1; SH2: Src homology 2; SHH: Sonic hedgehog; SOS: son of sevenless; STAT: signal transducer and activator of transcription; SWI-SNF: switch/sucrose nonfermentable; TAK1: transforming growth factor β-activated kinase 1; TCF/LEF: T-cell factor/lymphoid enhancer-binding factor; TGF: transforming growth factor; Wnt: wingless/int; YY1: yin and yang 1.Akt: v-akt murine thymoma viral oncogene cellular homologue, a.k.a. protein kinase B (PKB), a.k.a. the related to the A and C kinases (RAC-PK); APPL1: adapter protein containing PH domain, PTB domain and leucine zipper motif 1; Ash2l: Ash2-like methyltransferase; BAF60: BRG1-associated factor 60; Cdc42: cell division control protein 42; CDK: cyclin-dependent kinase; Cdo: cell adhesion molecule-related/downregulated by oncogenes; CIP/KIP: The authors declare that they have no competing interests.JDRK wrote the manuscript. RK revised and edited the manuscript. Both authors read and approved the final manuscript.


SAG), and lung glycoprotein-340 (DMBT1GP340) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1 InteracThe oral tissues, a part of the mucosal immune system, are constantly covered by saliva, which harbors a similar set of antimicrobial proteins as other mucosal fluids . A major2.SAG) [As a result of the continuous flow of saliva in our mouth, planktonic bacteria are swallowed, making microbial adherence one of the key selective criteria in the oral cavity ,8. SalivSAG) ,13.3.Streptococcus mutans is considered the major cause for dental caries. In search for S. mutans agglutinating substances, Ericson and Rundegren isolated a 300–400 kDa glycoprotein from parotid saliva [S. mutans. This protein was named salivary agglutinin (DMBT1SAG). It also binds a number of both Gram-positive and Gram-negative bacteria, as well as to host proteins including IgA, complement factor C1q, bovine and human lactoferrin, albumin and lysozyme [SAG is identical with lung glycoprotein 340 (DMBT1GP340) and Deleted in Malignant Brain Tumors-1 (DMBT1) [DMBT1 gene [SAG and DMBT1gp-340 cross react with monoclonal antibodies raised against the DMBT1s isolated from the different sources. Both proteins show calcium-dependent binding to S. mutans and SP-D [GP340 was purified from bronchoalveolar lavage fluid of patients with alveolar proteinosis [SAG and DMBT1GP340 thus represent DMBT1 isoforms that are encoded by the DMBT1 gene on chromosome 10q26.13. The DMBT1 gene shows frequent genomic rearrangements and/or loss of expression in two common types of malignant brain tumors, but also in diverse epithelial cancer types, including lung, gastric, esophageal, colon, breast, and skin cancer [d saliva  that sholysozyme –19. Seve (DMBT1) –22. The BT1 gene ,24. DMBTand SP-D . DMBT1GPteinosis . It bindteinosis , membersteinosis . DMBT1SAn cancer –36. A se4.N-terminal end of the DMBT1s, are short serine-threonine-rich amino acid motifs of 20–24 amino acids in length, called SRCR interspersed domains (SIDs). The largest naturally occurring allele known to date contains 13 of these SRCR-SID units, while the shortest known displays only eight. This genetic variability in normal individuals raises the question if size variations of DMBT1GP340, DMBT1SAG and variants from other tissues are due to alternative splicing and post-translational processing or are alternatively determined by genetic differences.The most typical feature of DMBT1 is that it consists of protein domains . DMBT1 iC-terminal end of the gene is a Zona Pellucida domain. Except for the SIDs, these domains are all known to be involved in ligand binding [The stretch of tandem repeated SRCR domains with their interspersed SIDs are followed by two CUB domains separated by a 14th SRCR domain, and at the  binding –39. DMBT5.Cryptosporidium, Toxoplasma and Plasmodium and algae of the genus Chlamidomonas [Strongylocentrotus purpuratus, which contains 218 genes comprising altogether 1,095 SRCR domains [The most prevalent domains in DMBT1 are the SRCR domains . The SRCidomonas ,44. The  domains . For the domains , SRCR do domains . The pos domains .6.Complement subcomponents (C1s/C1r), embryonic sea urchin protein , and bone morphogenetic protein 1 (Bmp1). CUB domains are found in numerous proteins that are mainly involved in developmental processes [The CUB domain is a 100–110 residue-spanning extracellular domain named after the three proteins, in which it was first recognized: rocesses . Almost rocesses .7.C-terminal of the polypeptide, which is also the case for DMBT1s. The functions of proteins containing a ZP domain vary tremendously. They are found in glycoproteins involved in development, acoustic perception, immunity, and cancer [SAG forms aggregates [The zona pellucida (ZP) domain was first recognized in the sperm receptor proteins ZP2 and ZP3 ,47. Thesd cancer . ZP domad cancer . This isgregates ,52.8.SAG accounts for 25–40% of the molecular weight [N-linked glycosylation sites [O-linked sites situated primarily in the SIDs. The SRCR domains, however, contain only few potential O-glycosylation sites (Ser and Thr residues). Presumably, the high density of glycans forces the SIDs in an extended conformation, as has been demonstrated for mucins [The carbohydrate part of DMBT1r weight ,52. DMBTon sites , and numr mucins , therebyO-linked structures in DMBT1SAG are extended core 1 and core 2 oligosaccharides, either neutral or monosialylated  [RVEVLYRGSW). Of these eight residues, four were always present in the binding motif (GRVEVLYRGSW). Of the 14 SRCR domains in the full length molecule, 12 contain the eight amino acids involved in binding giving bacterial binding of DMBT1 a multivalent character. One of the two deviating SRCR domains is the 14th SRCR domain (GRVEIYHGGTW), located between two CUB domains and as such probably not involved in bacterial binding.BT1pbs1) . AlthougRGRAEVYYSG) [SAG bacteria binding site were synthesized for all nine SRCR domains and tested for bacteria binding [SRCR domains in other proteins have previously been implicated in bacteria binding –70. The AEVYYSG) ,72. SimiAEVYYSG) ,74. CD 1 binding . One of 9.2.SAG-mediated agglutination of S.mutans is inhibited by high concentrations of fucose and lactose. In line with these results, it was shown that the Lea-antigen GlcNAc) was involved in S.mutans binding, harboring an essential role for the terminal fucose [SAG to S. sanguis and S. mutans [H. pylori can bind Leb structures and sialic acid on DMBT1SAG with its BabA and SabA adhesins, respectively [Studies of bacterial binding sites on SAG were first mainly focused on carbohydrate ligands, following the concept that bacteria primarily recognize carbohydrate ligands on mucosal surfaces ,77. Thesl fucose . Other s. mutans ,78. H. pectively .10.10.1.SAG was first described as an agglutinating agent for S.mutans and other streptococci [SAG on the dental surface promoted S. mutans colonization, but high concentrations in saliva inhibit its colonization [DMBT1ptococci . Severalptococci ,79–81. Hnization .SAG binds in a calcium-dependent manner to antigen I/II polypeptides, a group of surface receptors on S. mutans and related streptococci. Antigen I/II polypeptides have been characterized under different names in a variety of streptococci, S. mutans , S. sobrinus , S. gordonii , S. intermedius (Pas), S. pyogenes (aspA) and S. agalactiae (bspD) [C-terminal region of about 615 amino acids shows 64% or more sequence homology between the I/II polypeptides.DMBT1e (bspD) ,83. Antie (bspD) . These pSAG were identified on antigen I/II polypeptides and other proteins . DMBT1SAG binds to lipoteichoic acid, a component on the membrane of Gram-positive bacteria, and to lipopolysaccharide (LPS), a common component on the cell membrane of Gram-negative bacteria. LPS has been found to bind to SRCRP2, suggesting DMBT1 recognizes LPS with its SRCR domains [Dmbt1−/− mice were more susceptible to low doses of DSS than Dmbt1+/+ mice. However, since this is a commensal bacteria induced colitis model, this effect is likely to be attributed to Dmbt1’s ability to control the natural intestinal microflora.Bacteria that do not possess antigen I/II polypeptides, like DMBT1SAG ,65. CRP-eumoniae . As many domains . Binding11.SAG also binds viruses, including HIV-1 [SAG shows a calcium-dependent interaction with the virus gp120. The DMBT1SAG binding region on gp120 is localized to a linear, highly conserved sequence (CTRPNYNKRKR) near the stem of the V3 loop that is critical for chemokine receptor interaction during viral binding and infection [SAG most probably blocks the access of gp120 to the chemokine receptor. The N-terminal sequence of DMBT1, including the first SRCR domain and one-half of the first SID, binds in a Ca2+-dependent manner to the same HIV-1 V3 sequences previously identified to interact with full-length SAG [Besides bacteria, DMBT1ng HIV-1 ,103 and ng HIV-1 ,104. HIVng HIV-1 ,106. DMBnfection . Thus, Dngth SAG .SAG and DMBT1GP340 also inhibit the hemagglutination activity and infectivity of Influenza A virus (IAV) [SAG had significantly greater inhibitory activity against avian-like IAV strains which preferentially bound sialic acids in α linkage, than DMBT1SAG from another donor or three lung preparations of DMBT1GP340 [SAG from this donor as compared with the other samples. Hence, the specificity of sialic acid linkages on DMBT1 is an important determinant of anti-IAV activity. In addition to these direct virus-neutralizing properties, DMBT1GP340 displays cooperative interactions with SP-D in viral neutralization and aggregation assays. On the other hand, in some cases DMBT1SAG strongly antagonizes the antiviral activities of SP-D by binding to its carbohydrate recognition domain, which is involved in virus binding [DMBT1us (IAV) , which ius (IAV) . A variaBT1GP340 . In line binding .12.SAG binds to secretory IgA in a calcium-dependent manner, resulting in a cooperative effect on bacterial aggregation [SAG to IgA is mediated by the same 11-mer peptide that is responsible for the broad-spectrum bacteria binding of DMBT1SAG [SAG-IgA complex to the Pac molecule of S. mutans was demonstrated [SAG and concomitantly binding to Pac disappeared.DMBT1 binds to a number of endogenous proteins such as secretory IgA, MUC5B, surfactant proteins A and D, complement factor C1q, lactoferrin and albumin ,108,109.regation ,110. BinDMBT1SAG . Bindingnstrated . After cSAG also binds to C1q of the complement system, a system of plasma proteins that may react with one another to opsonize or permeabilize pathogens and induce a series of inflammatory responses [SAG is secreted in saliva and C1q is a serum component, these two fluids may mix in the oral cavity under conditions of oral inflammation, e.g., periodontal disease, or mucosal damage, thus enabling a local complement activation.DMBT1esponses . By bindesponses . AlthougPseudomonas biofilms [SAG binding to S. mutans protein antigen Pac, which belongs to the antigen I/II family [SAG that is responsible for S. mutans binding also mediates binding of lactoferrin [SAG binding. Although the homologous sequence in human lactoferrin is slightly different (SCKFDEYFSQSCA), human lactoferrin also binds to DMBT1 [DMBT1 also binds to bovine and human lactoferrin, an 80 kDa iron binding protein belonging to the transferrin family ,19. Lactbiofilms . In addibiofilms . Bovine I family . The SRCtoferrin ,89. BoviGP340, which originally was isolated as a soluble receptor for SP-D, also binds SP-A [N-terminal ends of the chains. Each polypeptide consists of a short N-terminal region, followed by a collagen-like sequence, a short α-helical sequence and the carbohydrate recognition domain. DMBT1GP340 binds to the carbohydrate recognition domain of SP-D through a calcium-dependent protein-protein interaction [DMBT1nds SP-A ,25. SP-Ands SP-A . SP-D foeraction . SP-A an13.Due to their broad pathogen binding spectrum, DMBT1 and its mouse homolog CRP-ductin are considered pattern recognition receptors (PRRs) , an anci14.Conclusively, DMBT1 plays a role in innate immunity by binding to a large panel of exogenous and endogenous ligands. SRCRP2 on the SRCR domains and sialic acids on the glycan chains have been identified as ligand binding sites, but DMBT1 may harbor many other potential ligand binding sites. These many ligand binding sites and its structural heterogeneity—both in the different tissues and between different individuals—are only two of the many surprises hidden in this molecule. Future research should focus on the precise role of DMBT1 in the innate immune system and the physiological consequences of its heterogeneity.


Systemic sclerosis (scleroderma: SSc) is a multisystem, connective tissue disease of unknown aetiology characterized by vascular dysfunction, autoimmunity, and enhanced fibroblast activity resulting in fibrosis of the skin, heart, and lungs, and ultimately internal organ failure, and death. One of the most important and early modulators of disease activity is thought to be oxidative stress. Evidence suggests that the free radical nitric oxide (NO), a key mediator of oxidative stress, can profoundly influence the early microvasculopathy, and possibly the ensuing fibrogenic response. Animal models and human studies have also identified dietary antioxidants, such as epigallocatechin-3-gallate (EGCG), to function as a protective system against oxidative stress and fibrosis. Hence, targeting EGCG may prove a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc. The free radical nitric oxide (NO) is an important physiological signalling molecule, potent vasodilator, and mediator of oxidative stress. Nitric oxide is synthesised from L-arginine by NO synthase (NOS), and three main isoforms of NOS have been identified with a constitutive expression in neuronal (nNOS or NOS 1), endothelial (eNOS or NOS 3), and several other cell types including fibroblasts. Furthermore, an inducible expression (iNOS or NOS 2) in response to a variety of inflammatory stimuli is possible [2.−) and peroxynitrite (ONOO−) [In the disease scleroderma (SSc: systemic sclerosis), the metabolism of NO appears to be profoundly disturbed. There is considerable evidence implicating overproduction of NO –5 and re (ONOO−) , 4, 6–8  (ONOO−) . Major f (ONOO−) –12. Ther (ONOO−) , 14. End (ONOO−) . The nat (ONOO−) , cell ad (ONOO−) , anti-en (ONOO−) , and the (ONOO−) , 5. Whil (ONOO−) .β) [In SSc the excessive connective tissue fibrosis is the most characteristic pathological manifestation of the disease . The fibβ) –26 and cβ) . Myofibrβ) , 29. The2.−, hydrogen peroxide (H2O2), hydroxyl radicals (.OH), and ONOO−. The sources of oxidative stress in SSc are complex and interactive and likely to be due to ischaemic-reperfusion injury, dysregulated metabolism of the free radical NO [α-tocopherol, β-carotene, and selenium were found to be lower in patients than in controls [The scleroderma phenotype at the cellular level has also been shown to be characterized by oxidative stress, an imbalance between the elevated level of ROS and/or the impaired function of the antioxidant defence system . ROS usudical NO , and gendical NO  and actidical NO . ROS havdical NO , collagedical NO , 8, 21. dical NO , 33, incdical NO –37, and dical NO , 7. In Scontrols , 32, 38.controls . Subsequcontrols , 38, 39 controls , 41 with3−) and nitrite (NO2−), with the measurement of total nitrate and nitrite NOx)( production in SSc have shown conflicting results [x) mouse, which is predisposed to SSc and often used as an experimental animal model for fibrosis, reported that while type I collagen protein expression was elevated in Tsk-1/+ skin tissue, eNOS protein and gene expressions were reduced compared to wild-type controls [+ skin tissue [+ skin, while there was also less total antioxidant activity [+ mouse, particularly in the skin, while expression and activity of protective antioxidants were reduced. NO has also been reported to act as an antifibrotic effector in animal models of experimental fibrosis , 59. Forcontrols . Furthern tissue . Correspactivity . The fin−/− knock-out mice are significantly less resistant to the cytotoxicity induced by H2O2 and paraquat than wild-type controls [−/− knock-out mice reported increased bleomycin-induced pulmonary fibrosis [−/− mice secondarily triggers regulation of extracellular matrix genes for repair responses [Several studies have gained insights into the intracellular mechanisms by which NO might further modulate a fibrotic phenotype. Nitric oxide can induce HO-1, which is also induced by a variety of cellular stress, including free-radical-mediated stresses and oxygen deprivation . HO-1 incontrols , 65. Concontrols . Indeed,fibrosis  and exprfibrosis  when comesponses . Interesesponses .β pathway [2+ and ascorbate and generates free radicals, all of which are sensitive to NO [κB, AP-1, SP-1) [Other studies suggest that NO influences fibrosis through modulatory effects on the TGF- pathway –71, init pathway , 72, 73, pathway , 54   which ar1, SP-1) , 89, 90 2.− [α2-adrenoceptors accounted for the supersensitivity [α1-adrenoceptors appears to predominate in the physiological control of cold-induced digital vasoconstriction whereas both α1- and α2-adrenoceptors play an equal role in Raynaud's subjects [In systemic sclerosis clear evidence for oxidative stress has been shown by increased levels of O2.− , antibod2.− , enhance2.− , 33, inc2.− –37, and 2.− , 7. Addi2.− , 92, 93.2.− , 92, 93.2.− . The pat2.− , 92. Inisitivity . Furthersubjects . Howeversubjects , digitalsubjects , as wellsubjects  of primaα-tocopherol or vitamin C, which did not decrease urinary markers of oxidative stress such as F(2)-isoprostanes nor improved microvascular perfusion after cold exposure [Although the vasospastic condition in Raynaud's Phenomenon may arise as a result of a functional disturbance at the level of the vessel wall, it is possible that circulating factors in the blood that alter the release of endothelial cell mediators, such as prostacyclin, NO, endothelin, or increase ROS and oxidative stress may contribute to the disease . Early cexposure , 99. Morexposure , 101. Th2.− and ONOO−, and reduce oxidative stress [α-tocopherol or vitamin C [Natural antioxidants, such as polyphenols from green tea extracts, are now being considered and investigated in particular (-)-epigallocatechin-3-gallate (EGCG). Other less active polyphenol constituents of green tea are believed to be (-)-epigallocatechin (EGC), (-)-epicatechin (EC), and (-)-epicatechin-3-gallate (ECG). The prominent antioxidant effects of EGCG derive from the phenol rings that can act as electron traps to scavenge free radicals, inhibit the formation of ROS such as Oe stress , 103. EGitamin C , and hasitamin C , 105.κB, AP-1) and/or induce antioxidant enzymes such as HO-1 [β and PDGF-induced α1(I) collagen, fibronectin, α-smooth muscle actin (α-SMA), and proliferation in activated human and rat hepatic stellate cells [β-induced ROS in human dermal fibroblasts from healthy controls, SSc patients, and in a dermal fibroblast cell line, indicating its potential effectiveness as an antioxidant to reduce oxidant stress in the disease scleroderma [Studies have also shown that EGCG may directly inhibit molecular targets and regulate multiple signal transduction pathways such as MAP kinases  and transcription factors (Nrf2, NF- as HO-1 , 107. Inte cells –110, ratte cells , 112, hute cells , and SScte cells . Additioeroderma . Intereseroderma , 116. Fueroderma , 118. Paeroderma –121. In summary, it is clear that further studies are needed to delineate the key NO-mediated signal transduction and transcription pathways that facilitate type I collagen production and fibrosis in the disease scleroderma. Studies such as these will help define key targets and candidates for therapy. Furthermore the dietary antioxidant EGCG, with its long history of safe beverage consumption in green tea together with its demonstrated potent antioxidant capability, is a good candidate for therapeutic treatment targeting oxidative stress and fibrogenesis in patients with SSc. Further clinical studies, to confirm its efficacy, determine optimal dosage and duration of use, and treatment indicators are required.


Cytosolic pattern recognition receptors (PRRs) sense a wide range of endogenous danger-associated molecular patterns as well as exogenous pathogen-associated molecular patterns. In particular, Nod-like receptors containing a pyrin domain (PYD), called NLRPs, and AIM2-like receptors (ALRs) have been shown to play a critical role in host defense by facilitating clearance of pathogens and maintaining a healthy gut microflora. NLRPs and ALRs both encode a PYD, which is crucial for relaying signals that result in an efficient innate immune response through activation of several key innate immune signaling pathways. However, mutations in these PRRs have been linked to the development of auto-inflammatory and autoimmune diseases. In addition, they have been implicated in metabolic diseases. In this review, we summarize the function of PYD-containing NLRPs and ALRs and address their contribution to innate immunity, host defense, and immune-linked diseases. The innate immune system relies on germline-encoded pattern recognition receptors (PRRs) to detect threats against tissue homeostasis. In response to pathogen infection, tissue damage or environmental stress, inflammatory mediators including cytokines, type I interferons (IFNs), and anti-microbial factors are produced. While Toll-like receptors (TLRs) utilize their TIR domain and RIG-I-like receptors (RLRs) and NLRCs their CARD for downstream signaling upon activation, NLRPs and AIM2-like receptors (ALRs) recruit signaling adaptors through their PYRIN domain (PYD). Active NLRPs and ALRs trigger multiple innate immune effector pathways, but by far the best established function of these PYD-containing proteins is the assembly of inflammasomes, which are large multiprotein platforms that form in response to infection and tissue damage and are responsible for the activation of inflammatory caspases, in particular caspase-1 . Active The PYD, also referred to as PAAD or DAPIN, is a protein binding domain belonging to the death domain superfamily . The strOnly 14 NLRPs and 4 ALRs are encoded in humans, while both families are amplified to 34 and 13 members, respectively, in mice Figures –39. BesiNLRP1 Figure .in vivo function of NLRP1 however remains largely elusive, at least partially due to several key differences between mice and human, which limits the relevance of in vivo mouse models. In contrast to human NLRP1, mouse NLRP1 lacks the PYD and exists in three tandem paralog genes  , a metalloproteinase composed of the pore-forming antigen (PA) and a lethal factor (LF), also cleaves NLRP1, but within the N-terminal domain, suggesting that NLRP1 potentially has several protease cleavage sites , confer susceptibility to LTx Figure  48). Th. Th48). e linked . Furthere linked . A similng cells .Several studies observed NLRP1/NLRP1b sensing of MDP , 25, 52.L were reported to specifically inhibit NLRP1 activation by blocking ATP binding     , 67–71, evisiae) , RNA andevisiae) –75, as walariae) –78. The alariae) –83, asbealariae) , 85, silalariae) , 84, 85,alariae) , 86, andalariae) . An incralariae) . Other kalariae) , 89, hydalariae) , and hyaalariae) . In addialariae) , high glalariae) , and satalariae) . The mecin vivo function of the LRR in NLRP activation has only been shown for NLRP3. In contrast to many in vitro studies showing that deletion of the LRR renders the NLRP constitutively active, likely because of a lack of autoinhibition, the absence of the LRR in vivo renders NLRP3 unresponsive to MSU and ameliorates MSU-induced inflammation in mice  triggers NLRP3 activation and assembly of the NLRP3 inflammasome. Three main activating mechanisms have been proposed: (1) K+ efflux, (2) mitochondrial dysfunction and generation of mitochondria-derived reactive-oxygen species (ROS), and (3) phagolysosomal destabilization in response to particulates 7R, which is an ATP-gated K+ ion channel, that facilitates K+ efflux, which activates the NLRP3 inflammasome , phospholipase C-mediated generation of inositol-1,4,5-trisphosphate, IP3R mediated release of Ca2+ from the ER, and store-operated Ca2+ entry (SOCE) mediated influx of extracellular Ca2+, which is important for NLRP3 inflammasome activation by extracellular ATP. Hence, caspase-1 and IL-1β processing and release are also controlled by PLC, IP3R, and SOCE phox, which is essential for the proper function of the NADPH oxidase Nox1-4, but CGD macrophages showed either no defect in IL-1β release (397 and C362), which is in contrast to an earlier study. Furthermore, the crystal structure of the NLRP3 PYD revealed that it is unique in containing a disulfide bond between C8 and C108, which could be important for redox potential-dependent regulation + into the cytosol, either from lysosomal rupture or from the activation of a proton-selective ion channel, such as the M2 channel upon infection with Influenza virus ATP + efflux and subsequent Na+ influx, and that K+-free medium alone is sufficient to activate NLRP3 in the absence of any other agonist  infection, NLRP4 inhibits the initiation of autophagy through interaction with beclin-1. Interestingly, all other tested NLRs, including NLRC4, NLRP3, and NLRP10 also interacted with beclin-1, potentially indicating this is a more common mechanism of NLRs  Figure . Recentl of NLRs . NLRP4 f of NLRs , suggest of NLRs . NLRP4 t of NLRs .Prevotellaceae and TM7 species in the steady state and upon DSS treatment through induction of CCL5 and IL-6  Figure . A subseand IL-6 , 146. Thand IL-6 . In addinfection . Mechaninfection . A potenS. aureus and L. monocytogenes in human macrophages  Figure , severalrophages . Similar+ efflux . Thus, N+ efflux .in vitro studies. Thus, one may predict that NLRP10 might not respond in a stimuli-dependent manner. Over expression studies proposed that NLRP10 oligomerizes with ASC and inhibits ASC-mediated NF-κB activation and apoptosis, as well as caspase-1-dependent IL-1β release  mouse model and Th2-dependent OVA- and Alum-driven asthma models  Figure , while d release . Direct  release . In cont in vivo . In contf NLRP10 . Mechaninfection . Howevera models . These desponses . In bothpression , 157. Alpression , 159, an−/− mice recently revealed immune defects. NLRP12 is predominately expressed in DCs and neutrophils, and mice lacking NLRP12 display less severe inflammation in models of contact hypersensitivity  Figure . It alsositivity . Howevere levels . Similare Figure  165). I. I−/− mis Figure , 167. Alis study . Thus, dPYHIN genes exist, this gene cluster is amplified in mice and contains at least 13 predicted and diverse members with only AIM2 being conserved between man and mice  domains Figure . While Aand mice –38. Howeand mice .Francisella tularensis  mutagenesis screening in mice revealed that NLRP1aQ593P, an activating mutation located within the linker connecting the NACHT and LRR, causes lethal systemic neutrophilia, thus linking NLRP1 mutations to hyper-inflammation  in the pathogenesis of Sjogren’s syndrome and systemic lupus erythematosus (SLE) . In part−/− mice also show reduced hepatic steatosis and are protected against the accumulation of lipid deposits in the liver (Chronic low-grade metabolic inflammation (metaflammation) is an underlying cause for metabolic diseases. In obesity an excess of nutrients triggers inflammation, since the metabolic surplus induces the expression of inflammatory cytokines, including IL-1β. Hence, there are numerous obesity-related diseases, which include cardiovascular disease, atherosclerosis, insulin resistance, and type 2 diabetes mellitus (T2DM), which are linked to the NLRP3 inflammasome. The NLRP3 inflammasome can be triggered by oligomers of IAPP, which commonly form amyloid deposits in the pancreas during T2DM. In response to IAPP, inflammasome priming, which causes the transcriptional up-regulation of IL-1β, requires a sufficient glucose metabolism and can be facilitated by minimally oxidized low-density lipoprotein . Subsequhe liver . Thus, NPrevotellaceae (Prevotellaceae and TM7 (−/− and NLRP6−/− mice are more susceptible to colon inflammation and colon cancer (Increasing evidence supports a profound impact of the intestinal microbiota to metabolic health and the intestinal microbiota of obese individuals differs from that of lean people and shows increased prevalence of ellaceae . NLRP3 a and TM7 , 230. Re and TM7 , 230. Thn cancer , 146.Similar to NLRP6, NLRP12 dampens gastrointestinal inflammation and associated tumorigenesis, albeit through a distinct mechanism. Rather than through inflammasome-mediated IL-18 production, NLRP12 prevents intestinal inflammation through dampening NF-κB, ERK, and AKT activation and release of pro-inflammatory cytokines, chemokines, and tumorigenic factors from macrophages and intestinal epithelial cells , 167.By now, the crucial role of PYD-containing PRRs in host defense is well-established. Although, these PRRs trigger many key innate immune pathways, their contribution to inflammasome activation is currently best understood. Nevertheless, it becomes increasingly recognized that not all PYD-containing PRRs assembly inflammasomes or even promote a pro-inflammatory response. However, the precise signaling mechanisms and in particular, the stimuli that trigger their activation, are largely elusive for most members. The tight affiliation of these PRRs with immune-based diseases further underscores their critical function in maintaining homeostasis, while at the same time opening up exciting avenues for developing novel therapies targeting these PRRs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ATL is a distinct peripheral T-lymphocytic malignancy associated with human T-cell lymphotropic virus type I (HTLV-1). The diversity in clinical features and prognosis of patients with this disease has led to its subtype-classification into four categories, acute, lymphoma, chronic, and smoldering types, defined by organ involvement, and LDH and calcium values. In case of acute, lymphoma, or unfavorable chronic subtypes (aggressive ATL), intensive chemotherapy like the LSG15 regimen (VCAP-AMP-VECP) is usually recommended if outside of clinical trials, based on the results of a phase 3 trial. In case of favorable chronic or smoldering ATL (indolent ATL), watchful waiting until disease progression has been recommended, although the long-term prognosis was inferior to those of, for instance, chronic lymphoid leukemia. Retrospective analysis suggested that the combination of interferon alpha and zidovudine was apparently promising for the treatment of ATL, especially for types with leukemic manifestation. Allogeneic hematopoietic stem cell transplantation  is also promising for the treatment of aggressive ATL possibly reflecting graft versus ATL effect. Several new agent trials for ATL are ongoing and in preparation, including a defucosylated humanized anti-CC chemokine receptor 4 monoclonal antibody, IL2-fused with diphtheria toxin, histone deacetylase inhibitors, a purine nucleoside phosphorylase inhibitor, a proteasome inhibitor, and lenalidomide. Adult T-cell leukemia-lymphoma (ATL) was first described in 1977 by Uchiyama et al. as a distinct clinico-pathological entity with a suspected viral etiology because of the clustering of the disease in the southwest region of Japan –5. In thATL patients show a variety of clinical manifestations because of various complications of organ involvement by ATL cells, opportunistic infections and/or hypercalcemia –14. ThesATL cells are usually detected quite easily in the blood of affected individuals except for the smoldering type with mainly skin manifestations and lymphoma type . These sHypercalcemia is the most distinctive laboratory abnormality in ATL as compared to other lymphoid malignancies and is observed in 31% of patients  at onset . Individβ2-microglobulin, and serum thymidine kinase levels reflecting disease bulk/activity, the level of the soluble form of interleukin (IL)-2 receptor alpha-chain is elevated in the order of acute/lymphoma-type ATL, smoldering/chronic-type ATL, and HTLV-1 carriers as compared with normal individuals, perhaps with better accuracy than the other markers .–45.40–45P < .01). Patients with ATL still showed a poor prognosis, with a median survival time of 8 months and a 4-year survival rate of 12%.In 1987, JCOG initiated a multicenter phase II study (JCOG8701) of a multiagent combination chemotherapy (LSG4) for advanced aggressive NHL (including ATL). LSG4 consisted of three regimens: (1) VEPA-B (VEPA plus bleomycin), (2) M-FEPA , and (3) VEPP-B,  . The CR The disappointing results with conventional chemotherapies have led to a search for new active agents. Multicenter phase I and II studies of pentostatin  were conducted against ATL in Japan . The pha2 vincristine on days 1 and 8, 40 mg/m2 doxorubicin on day 1, 100 mg/m2 etoposide on days 1 through 3, 40 mg/m2 prednisolone (PSL) on days 1 and 2, and 5 mg/m2 pentostatin on days 8, 15, and 22 was administered every 28 days for 10 cycles. Among the 61 patients evaluable for toxicity, four patients (7%) died of infections, two from septicemia, and two from cytomegalovirus pneumonia. Among the 60 eligible patients, there were 17CRs (28%) and 14 partial responses (PRs) . The median survival time was 7.4 months, and the estimated 2-year survival rate was 17%. The prognosis in patients with ATL remained poor, even though they were treated with a pentostatin-containing combination chemotherapy. These encouraging results prompted the investigators to conduct a phase II trial (JCOG9109) with a pentostatin-containing combination (LSG11) as the initial chemotherapy . PatientIn 1994, JCOG initiated a phase II trial (JCOG9303) of an eight-drug regimen (LSG15) consisting of vincristine, cyclophosphamide, doxorubicin, prednisone, ranimustine, vindesine, etoposide, and carboplatin for untreated ATL . Dose inDose intensification of CHOP with prophylactic use of G-CSF was expected to improve survival among patients with aggressive NHL, and our randomized phase II study (JCOG9505) comparing CHOP-14 (LSG19) and dose-escalated CHOP (LSG20) to treat aggressive NHL excluding ATL revealed biweekly CHOP to be more promising . TherefoTo confirm whether the LSG15 regimen is a new standard for the treatment of aggressive ATL, JCOG conducted a phase III trial comparing modified (m)-LSG15 with biweekly CHOP , both supported with G-CSF and intrathecal prophylaxis .2, ADM 50 mg/m2,VCR 1.4 mg/m2(maximum 2 mg) on day 1 and PSL 100 mg on days 1 to 5] every 2 weeks  on day 1, AMP  on day 8, and VECP  on days 15–17, and the next course was to be started on day 29 (P = .020). As shown in P = .169, and the hazard ratio was 0.75; 95% confidence interval (CI), 0.50 to 1.13]. A Cox regression analysis with performance status (PS 0 versus 1 versus 2–4) as the stratum for baseline hazard functions was performed to evaluate the effect on overall survival of age, B-symptoms, subtypes of ATL, LDH, BUN, bulky mass, and treatment arms. According to this analysis, the hazard ratio and two-sided P value for the treatment arms were 0.62  and  .056, respectively. The difference between the crude analysis and this result was because of unbalanced prognostic factors, such as PS 0 versus 1, and the presence or absence of bulky lesions between the treatment arms. The progression-free survival rate at 1 year was 28% in the mLSG15 arm compared with 16% in the biweekly CHOP arm (two-sided P = .20).mLSG15 in JCOG9801 was a modified version of LSG15 in JCOG9303, consisting of three regimens: VCAP ), an interleukin-2-diphtheria toxin fusion protein targeting IL-2 receptor-expressing malignant T lymphocytes, shows efficacy as a single agent against CTCL and peripheral T-cell lymphoma (PTCL) . Also thCD52 antigen is present on normal and pathologic B and T cells. In PTCL, however, CD52 expression varies among patients, with an overall expression rate lower than 50% in one study but not in another , 79. ATLATL cells express CD52, the target of alemtuzumab, which was active in a preclinical model of ATL and toxic to p53-deficient cells, and several ATL cases successfully treated with this agent have been reported –83.Siplizumab is a humanized MoAb targeting CD2 and showed efficacy in a murine ATL model. P1 dose-escalating study of this agent in 22 patients with several kinds of T/NK-cell malignancy revealed 6 responses . However, 4 patients developed EBV-associated LPD . The broγ receptor on effecter cells [n = 10), neutropenia (n = 3), leukopenia (n = 2), herpes zoster (n = 1), and acute infusion reaction/cytokine release syndrome (n = 1). Neither the frequency nor severity of these effects increased with dose escalation or the plasma concentration of the agent. The maximum tolerated dose was not reached. No patients had detectable levels of anti-KW-0761 antibody. Five patients  achieved objective responses: 2 complete  and 3 partial  responses. Three out of 13 patients with ATL (31%) achieved a response (2 CR and 1 PR). Responses in each lesion were diverse, that is, good in PB , intermediate in skin , and poor in LN . KW-0761 was well tolerated at all the doses tested, demonstrating potential efficacy against relapsed CCR4-positive ATL or PTCL. Recently, results of subsequent phase II studies at the 1.0 mg/kg in relapsed ATL, showing 50% of response rate with acceptable toxicity profiles, reported [CC chemokine receptor 4 (CCR4) is expressed on normal T helper type 27 and regulatory T (Treg) cells and on certain types of T-cell neoplasms , 21, 35.er cells . A phaseer cells . Other treported . A phaseA proteasome inhibitor, bortezomib (Velcade), and an immunomodulatory agent, lenalidomide (Revlimid), both have potent preclinical and clinical activity in T-cell malignancies including ATL, are now under clinical trials for relapsed ATL in Japan –90. OtheTwo steps should be considered for the prevention of HTLV-1-associated ATL. The first is the prevention of HTLV-1 infections. This has been achieved in some endemic areas in Japan by screening for HTLV-1 among blood donors and asking mothers who are carriers to refrain from breast feeding. For several decades, before initiation of the interventions, the prevalence of HTLV-1 has declined drastically in endemic areas in Japan, probably because of birth cohort effects . The eliClinical trials have been paramount to the recent advances in ATL treatment, including assessments of chemotherapy, AZT/IFN, and allo-HSCT. Recently, a strategy for ATL treatment, stratified by subtype-classification, prognostic factors, and the response to initial treatment as well as response criteria, was proposed . The rec


Enterobacter sp. strain R4-368 is one of the few characterized Jatropha endophytic diazotrophic bacteria and was isolated from surface-sterilized roots. This bacterium shows strong growth-promoting effects, being able to increase plant biomass and seed yields. Enterobacter sp. R4-368 is the second fully sequenced diazotrophic Enterobacter species. The sequence information shall facilitate the elucidation of the molecular mechanisms of plant growth promotion, nitrogen fixation in nonlegume plant species, and evolution of biological nitrogen fixation systems. Jatropha curcas is a biofuel crop targeted to marginal land where soil nutrients are low  is a plasmid, as it shares high sequence homology with the E. cloacae plasmid pEC-IMPQ (GenBank accession number EU855788). This assumption was confirmed by extensive sequencing of PCR products spanning two open ends. Scaffold 10  is apparently an rRNA gene consensus sequence encoding the 23S rRNA and 5S rRNA. The remaining 8 scaffolds were tentatively plotted to the genome sequence of Enterobacter sp. 638 (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/). A total of 4,948 genes were assigned though the PGAAP and categorized into 4,827 CDS, 7 pseudogenes, 27 rRNAs , 87 tRNAs, and 6 frameshifted genes.The genomic DNA sample of strain R4-368 was sequenced on a FLX Titanium platform at Macrogen, Inc. (Republic of Korea). A shotgun and 3-kb paired-end library sequencing reads, each with about 30× sequence coverage, were assembled using the GS De Novo assembler (v 2.6). Assembly of both libraries yielded 10 scaffolds covering 5,210,394 bases, with an average size of 521,039 bases, an  sp. 638 . The seqSeveral gene clusters related to plant growth promotion were found in the genome, including a complete set of genes for nitrogen fixation and synthesis and efflux of volatile compounds , auxin, and siderophore.Enterobacter sp. strain R4-368 have been deposited in GenBank under the accession numbers CP005991 and CP005992, respectively.The genome sequences of the chromosome and plasmid of


Plant mitochondria, semiautonomous organelles that function as manufacturers of cellular ATP, have their own genome that has a slow rate of evolution and rapid rearrangement. Cytoplasmic male sterility (CMS), a common phenotype in higher plants, is closely associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce F1 hybrid seeds in a variety of valuable crop species. Novel chimeric genes deduced from mtDNA rearrangements causing CMS have been identified in several plants, such as rice, sunflower, pepper, and rapeseed, but there are very few reports about mtDNA rearrangements in wheat. In the present work, we describe the mitochondrial genome of a wheat K-type CMS line and compare it with its maintainer line.Aegilops kotschyi) CMS line, Ks3, was assembled into a master circle (MC) molecule of 647,559 bp and found to harbor 34 known protein-coding genes, three rRNAs , and 16 different tRNAs. Compared to our previously published sequence of a K-type maintainer line, Km3, we detected Ks3-specific mtDNA  and repeats  as well as genes that are unique to each line: rpl5 was missing in Ks3 and trnH was absent from Km3. We also defined 32 single nucleotide polymorphisms (SNPs) in 13 protein-coding, albeit functionally irrelevant, genes, and predicted 22 unique ORFs in Ks3, representing potential candidates for K-type CMS. All these sequence variations are candidates for involvement in CMS. A comparative analysis of the mtDNA of several angiosperms, including those from Ks3, Km3, rice, maize, Arabidopsis thaliana, and rapeseed, showed that non-coding sequences of higher plants had mostly divergent multiple reorganizations during the mtDNA evolution of higher plants.The complete mtDNA sequence of a wheat K-type (with cytoplasm of The complete mitochondrial genome of the wheat K-type CMS line Ks3 is very different from that of its maintainer line Km3, especially in non-coding sequences. Sequence rearrangement has produced novel chimeric ORFs, which may be candidate genes for CMS. Comparative analysis of several angiosperm mtDNAs indicated that non-coding sequences are the most frequently reorganized during mtDNA evolution in higher plants. Plasmodium to 200-2000 kb in higher plants [cis-/trans-splicing, RNA editing, and use of the universal genetic code [Mitochondria, as semiautonomous organelles, function as manufacturers of cellular ATP through the process of oxidative phosphorylation in all eukaryotes. It is believed that mitochondria originated from a free-living eubacterial ancestor and became an endosymbiotic organelle through engulfment by a eukaryotic host cell ,2. The sr plants ,4. Due ttic code . In hightic code -8, and ttic code . The dyntic code . Gene shtic code .CMS is a common phenotype in higher plants, and is closely associated with mutations in mtDNAs that cause pollen abortion. CMS systems have been widely used as a convenient way to produce F1 hybrid seeds in a variety of valuable crop species, including rice, maize, sugar beet, and cotton. In addition, CMS is exploited to study nucleocytoplasmic interactions . mtDNAs Triticum aestivum cv. Yumai 3, Km3), which is a maintainer line of K-type CMS [Aegilops kotschyi, Boiss.Wheat K-type CMS, which lacks adverse cytoplasmic effects and has more restoration line resources than other types of CMS, has been widely used in the production of hybrid seeds. Moreover, we recently sequenced the complete mtDNA genome for fertile Yumai 3  of more than 100 bp in Ks3 mtDNA that were not maintained in Km3 mtDNA  in U23, U26, and U30 were found to be significantly homologous to wheat chloroplast DNA. Five segments, U8, U11, U14, U24, and U32, partially matched several nuclear genome sequences of different higher plants. Nevertheless, Ks3-specific regions, which accounted for about 47,353 bp and 7.3% of Ks3 mtDNA, were novel to the current NCBI databases.We annotated these 38 Ks3-specific sequences using BlastN and BlastX searching against NCBI databases. Four integrated segments, U17, U18, U19, and U28, were found in the databases with a total of 6727 bp, while 10 segments  could not be detected, and 24 segments were partially annotated in 37 pieces . As a result, 26,317 bp were explained, accounting for 35.7% of Ks3-specific sequences and 4% of the Ks3 mitochondrial genome. Furthermore, 21 Ks3-specific segments , ranging from 33 to 3301 bp, were homologous to several previously determined mitochondrial sequences in higher plants, e.g., We analyzed homology between Ks3 mtDNA and the wheat chloroplast genome (ctDNA) using BLAST2, and revealed 123 segments  with more than 81% identity . The other 50 fragments were classified into 10 mtDNA-derived genes : atp1, rrn18-1, rrn18-2, rrn18-3, rrn18-4, rrn26-1, rrn26-2, trnM-1, and trnM-2, corresponding to wheat ctDNA genes atpA, rrn16, rrn23, and ct-trnM.We noticed that some segments in Ks3 mtDNA were homologous to wheat ctDNA with multiple copies; Ct7, Ct11, Ct16, and Ct17 were duplicated and Ct3 has four copies. Fifty-six segments covered the full length or parts of known genes; six segments contained tRNA genes derived from ctDNA  between wheat ctDNA and Km3 mtDNA were not shared with Ks3 mtDNA. Similarly, four segments (3991 bp) in Ks3 mtDNA were uniquely homologous with wheat ctDNA, and were located in Ks3-specific mtDNA regions, U23, U26, and U30 , comprising both direct (DR) and inverted repeats (IR)  and 10 indels (insertions and deletions) [Moreover, we also compared repeats between Ks3 mtDNA and Km3 mtDNA. It is known that the Km3 mtDNA sequence is almost identical to the previously reported sequence of letions) ,20. As aAegilops kotschyi belongs to two different genera, Triticum and Aegilops, respectively. Most of the protein-coding genes are highly conserved, especially in size, except atp6, nad6, nad9, and rps19-p  upstream of KSnad9, its ORF is 291 bp shorter than that of KMnad9. An extreme case is rpsl5, which is absent in Ks3 but present in Km3. A DNA exchange between mtDNA and nuclear DNA might have occurred, resulting in a nuclear rps15 protein, if the rps15 protein is proven to exist; otherwise, we have a defective Ks3 mitochondrial ribosome without rpl5.The cytoplasm of Km3 and Ks3 originated from common wheat and KSapt1, KSmatR, KSrps13, and KSnad4) and 20 were non-synonymous  were novel; a database search performed with the Blast network service using default parameters revealed no homologies to other sequences in the NCBI databases. Two ORFs (KSorf1292 and KSorf778), which were situated in two Ks3-specific mtDNA regions (U23 and U30), showed significant homology to wheat chloroplast DNA. As mentioned above, two Ks3 mtDNA fragments homologous to wheat ctDNA were unique and not found in Km3 mtDNA. This indicates that KSorf1292 and KSorf778 were probably derived from an extraneous wheat chloroplast genome. Another pair of ORFs, KSorf1321 and KSorf1319, located in a Ks3 mtDNA unique region, U36, exhibited homology to a DNA polymerase in rye mtDNA. This result indicates that KSorf1321 and KSorf1319 likely encode proteins with similar function in Ks3 mtDNA, but further empirical data are needed. It is notable that KSorf249 in Ks3 mtDNA is homologous to orf256, a candidate for a sterile gene associated with wheat T-CMS, which originated from the transfer of the wheat nuclear counterpart into Triticum timopheevii cytoplasm. In wheat T-CMS, the chimeric gene orf256 is situated upstream of cox1, is transcribed together with cox1, and expresses a 7-kDa protein that is not found in fertile lines [KSorf249 resembled orf256 upstream of KScox1, and deserves further study. Previous studies have shown that the ORFs involved in CMS are usually located in the vicinity of known genes or form a chimeric gene by overlapping with parts of known genes in the plant mitochondrial genome. For example, urf13-T which leads to CMS in maize T-CMS is located downstream of atp6, which provides the regulatory sequence, and the two are co-transcribed [orf107, the CMS gene in sorghum A3-CMS, forms a chimeric sequence by partially overlapping the 5'-end with that of atp9 [KSorfs have similar structures to known ORFs involved in CMS in other plant mtDNAs  were located in corresponding Ks3-specific regions, whereas another sequence of these ORFs was homologous to Km3 mtDNA  had remarkable homology to Km3 mtDNA, but homologous Km3 mtDNA were divided into two discrete segments in the Km3 mitochondrial genome, which indicates that they are likely derived from different parts of Km3 mtDNA. It is notable that five ORFs were not situated in repeat regions of Ks3 mtDNA , which has 24 SNPs in 11 protein-coding genes compared to the fertile form [Frequent recombination events have distorted the synteny between Ks3 and Km3 mtDNAs Figure , as alsoile form . FinallyMsh1, in the nuclear genome homologous to the Escherichia coli MutS mismatch repair component, RecA3, affects structural diversity in A. thaliana [Msh1 in tobacco and tomato is knocked out by RNAi, novel mitochondrial genome organizations are observed, and plants show a male sterility phenotype [Our analysis of structural diversity is necessary to understanding the sequence diversity among plant mtDNAs . We detethaliana . In maizthaliana . Researchenotype .We conducted extensive sequence comparison between Ks3 and Km3 mtDNAs to search for functional alterations of genes that were responsible for the CMS phenotype in plants. We noticed that Ks3 mtDNA encodes several partial subunits of the respiratory chain complex, including ATP4, ATP6, NAD3, NAD6, NAD9, COX1, and COX3 . Another functional scenario is that these novel ORFs may be involved in CMS by damaging mitochondrial membrane structure. In maize, URF13, encoded by T-urf13, assembles into a tetramer that penetrates the mitochondrial membrane, and the resulting permeability change affects normal mitochondrial function [Research on the expression of novel ORFs in Ks3 mtDNA is also necessary , as the in maize , orf224 rapeseed , orf522 unflower , orf138 n radish , orf107  sorghum , and orf in rice . The pro petunia  and orf4function ,47.Previous studies have shown that the process of anther abortion in K-type CMS occurs in the two-cell stage or the late period of the three-cell state of anther development, and the development of pollen is regulated by multiple genes . Thereforpl5 is missing, and 32 SNPs are involved in 13 protein-coding, albeit functionally irrelevant, genes, and 22 ORFs are unique in Ks3. All these sequence variations are candidates for CMS. Comparative analysis of the mtDNA of several angiosperms including Ks3, Km3, rice, maize, Arabidopsis thaliana, and rapeseed, indicates that non-coding sequences are the most frequently reorganized part of the mitochondrial genome during mtDNA evolution in higher plants.The complete mitochondrial genome of the wheat K-type CMS line Ks3 is very different from that of its maintainer line, Km3, especially in non-coding sequences. The Ks3 mtDNA is 647,559 bp and harbors 34 known protein-coding genes, three rRNAs , 16 different tRNAs, Ks3-specific mtDNA , and repeats . In addition, Aegilops kotschyi was designated as K-type Yumai 3 CMS line (abbreviated Ks3), and its isonuclear line with normal male-fertile cytoplasm was designated as K-type Yumai 3 (Triticum aestivum cv. Yumai 3) maintainer line (abbreviated Km3) [A wheat CMS line with male-sterile cytoplasm from ted Km3) ; both weMitochondria were isolated from etiolated 2-week-old seedlings of Km3 and Ks3 according to a previously published procedure . MitochoSau3AI, size-fractioned by pulsed-field gel electrophoresis, and ligated to PIndigoBAC-5 BamHI cloning-ready vector . The ligation mix was transformed into DH10B-competent cells through electroporation. High-density nylon filters (eight 384-well plates) were screened for a tiling path that covers the entire genome. Shotgun plasmid libraries were made from minimal tiling clones in the pUC-18 vector, and used for sequencing on ABI-3730xl DNA analyzers.Mitochondrial genome BAC libraries for Ks3 and Km3 were constructed following a previously published procedure with minor modifications . MitochoThe entire nucleotide sequences of Km3 mtDNA (accession number EU534409) and Ks3 mtDNA (accession number GU985444) were determined at the Beijing Institute of Genomics, Chinese Academy of Sciences, and DNA sequences were assembled using the software package phred/phrap/consed ,51 on a http://blast.ncbi.nlm.nih.gov/Blast.cgi with default parameters.The mitochondrial sequences were annotated with Glimmer 3.0 and BLAST tools, and tRNA genes and their secondary structures were identified according to tRNA scan-SE . The Paihttp://bio.cse.psu.edu/pipmaker[Triticum aestivum cv. Yumai 3; EU534409), rice , maize NB , Arabidopsis , and rapeseed Alignments were obtained using MultiPipMaker, a web-based tool for genomic sequence alignments /pipmaker,54. The atp1, atp4, atp6, atp8, and atp9: ATP synthase subunits 1, 4, 6, 8, and 9 genes; cob: apocytochrome b gene; cox1-3: cytochrome c oxidase subunits 1-3 genes; nad1-7, 9 and nad4L: NADH dehydrogenase subunits 1-7, 9, and 4L genes; rpl2-p, rpl5, rpl16, rps1-4, rps7, rps12-13, and rps19-p: ribosomal protein large and small subunit genes; rrnS and rrnL: small and large subunit ribosomal RNA (rRNA) genes; trnX: transfer RNA (tRNA) genes, where X is the one-letter abbreviation of the corresponding amino acid; SNPs: single nucleotide polymorphisms; aa: amino acids; nt: nucleotides; bp: base pairs; kb: kilobase.CMS: cytoplasmic male sterility; mtDNA: mitochondrial DNA; ctDNA: chloroplast DNA; MC: master circle; DR: direct repeats; IR: inverted repeats; ORF: open reading frame; Supplementary data are available at BMC Genomics Online.HL, PC, QL, GZ, WY, and DL carried out the molecular experiments. KZ, XG, SH, JY, and AZ designed and coordinated all experiments. HL, PC, QL, and FD performed the genomic analyses. All authors contributed to the manuscript and then read and approved the final version.Genes in the Ks3 mitochondrial genome. The file contains the list of size and MC coordinates of genes in the Ks3 mitochondrial genome, including Known protein-coding genes, rRNAs genes and tRNAs genes.Click here for fileTransposons in the Ks3 mitochondrial genome. The file contains the list of size and MC coordinates of transposons in the Ks3 mitochondrial genome. These transposons were identical to known those of rice or wheat with different identity, respectively.Click here for fileList of Ks3 mtDNA unique regions (compared with Km3 mtDNA). Compared with Km3 mtDNA, Ks3 mtDNA has 38 specific regions. The file contains the list of size and MC coordinates of those Ks3-specific mtDNA regions. Some unique regions in Ks3 mtDNA were homologous to previously determined sequences in NCBI databases, while others could not be detected in NCBI databases.Click here for fileMap of homologous fragments in Ks3 mtDNA unique regions. These fragments homologous to sequences in NCBI databases mapped to Ks3-specific mtDNA regions. Black bars show Ks3 mtDNA unique regions, and fragments of Ks3 mtDNA unique regions homologous to NCBI databases are indicated by broad red bars. The vertical numbers show the coordinates of the homologous fragments in Ks3 mtDNA unique regions. The letters A, B, and C indicate different annotated sequences in NCBI databases, shown in Additional File Click here for fileList of Ks3 mtDNA sequences showing homology to ctDNA sequences. The file contains the list of size and MC coordinates of Ks3 mtDNA sequences showing homology to ctDNA sequences with more than 81% identity and a size range of 24 to 2790 bp.Click here for fileList of Ks3 mtDNA sequences of more than 100 bp showing homology to ctDNA sequences. The file contains the list of Ks3 mtDNA sequences of more than 100 bp showing homology to ctDNA sequences. These Ks3 mtDNA sequences showed homology to the ctDNA sequences bringing the corresponding chloroplast genes.Click here for fileList of wheat ctDNA sequences uniquely homologous to Km3 mtDNA and those uniquely homologous to Ks3 mtDNA. The file contains the list of size and MC coordinates of wheat ctDNA sequences uniquely homologous to Km3 mtDNA and those uniquely homologous to Ks3 mtDNA with different identity.Click here for fileDot matrix representation of the Ks3 MC molecule. Repeats of more than 100 bp are marked on the map. Red and blue dots and lines represent direct and inverted repeats, respectively. Nine repeats of more than 500 bp, R1-R9, are marked with arrows.Click here for fileList of repeats larger than 100 bp found in Km3 mtDNA. The file contains the list of type, size and MC coordinates of 16 repeats larger than 100 bp in Km3 mtDNA.Click here for fileHomology of mtDNA repeats between Ks3 and Km3. The file contains the list of homology of mtDNA repeats between Ks3 and Km3. Four repeats were almost identical in the Ks3 mtDNA and Km3 mtDNA, while the relationship between the large repeats in two mitochondrial genomes is complicated.Click here for fileComparison of mtDNA repeats between Ks3 and Km3. (A) Blue bars above or below the horizontal lines indicate one direct or inverted copy of repeats of Ks3 mtDNA, respectively. Netted bars show repeats of Km3 mtDNA homologous to Ks3 mtDNA, and numbers in brackets indicate coordinates of partial homologous fragments in repeats of Km3 mtDNA. Red bars indicate that the partial fragments in repeats of Km3 mtDNA did not show significant homology to repeats of Ks3 mtDNA. (B) The specific mtDNA repeats of Km3 and Ks3 and mtDNA repeats shared by Ks3 and Km3. A pair of identical repeats is indicated by a left-right arrow.Click here for fileAlignments of atp6 (A), nad6 (B), nad9 (C), and rps19-p (D) between Ks3 and Km3. the file contains the alignments of atp6 (A), nad6 (B), nad9 (C), and rps19-p (D) between Ks3 and Km3. The identical sequences are highlighted with a dark background. The vertical arrow in (D) indicates deletion in Ks3 mtDNA (Ks3_seq) and STOP shows the location of the terminal codon (TAG).Click here for fileMultiPipMaker analysis of the mtDNA of several angiosperms. Ks3 mtDNA was used as the reference genome for comparison with those of Km3, rice, maize, Arabidopsis thaliana, and rapeseed. (A) Positions of genes or exons of Ks3 mtDNA are indicated with black bars, and their orientations are shown with arrows. The percentage identity is shown at the right side. (B) A schematic representation of (A) in which only representative genes of Ks3 mtDNA are marked. Red and green bars indicate identity with Km3 mtDNA and other mtDNAs, respectively.Click here for fileThe number of copies of mitochondrial genes in Ks3, Km3, maize, and rice. The file contains the list of the number of copies of mitochondrial genes in Ks3, Km3, maize, and rice. The identity of genes between Ks3 mtDNA and Km3 mtDNA was also shown.Click here for fileCorrelation of gene order between the mitochondrial gene maps of Ks3 and Km3 (A), maize (B), and rice(C). The file contains the comparison of gene order in Ks3 mtDNA to that in Km3, maize, and rice mtDNA. The protein-coding and rRNA-coding genes are arranged from top to bottom for Ks3, and from left to right for Km3, rice, and maize, based on their order in the respective gene maps. Genes of Km3, rice, and maize are indicated by code numbers representing the corresponding Ks3 genes on the left margin of figures.Click here for fileAlignments of ATP6, NAD6, and NAD9 between Km3 and Ks3. the file contains the alignments of (A) KMATP6 and KSATP6, (B) KMNAD6 and KSNAD6, and (C) KMNAD9 and KSNAD9. The identical sequences are highlighted with a dark background.Click here for file


The right tibia and kidney were removed for histologic examination and renal immunohistochemical analysis for endothelial nitric oxide synthase (eNOS) was performed. The results revealed that omega-3 reduced SBP, DBP, and MAP and plasma levels of sodium, potassium, Pi, creatinine, urea, and TGF-β1, but increased plasma levels of calcium, ALP, and 1,25(OH)2D3 as well as renal eNOS. Omega-3 increased cortical and trabecular bone thickness, decreased osteoclast number, and increased newly formed osteoid bone. Renal morphology was found preserved. In conclusion, omega-3 prevents the disturbed bone status imposed by salt loading. This osteoprotective effect is possibly mediated by attenuation of alterations in Ca2+, Pi, and ALP, and improvement of renal function and arterial blood pressure.There is evidence that dietary fats are important components contributing in bone health and that bone mineral density is inversely related to sodium intake. Salt loading is also known to impose negative effects on renal function. The present study aimed to determine the effect of the polyunsaturated fatty acid omega-3 on bone changes imposed by salt loading, highlighting the role of kidney as a potential mechanism involved in this effect. Male Wistar rats were divided into three groups: control group, salt-loaded group consuming 2% NaCl solution as drinking water for 8 weeks, and omega-3-treated salt-loaded group receiving 1 g/kg/day omega-3 by gavage with consumption of 2% NaCl solution for 8 weeks. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate (HR) were recorded. Plasma levels of sodium, potassium, calcium, inorganic phosphorus (Pi), alkaline phosphatase (ALP), creatinine, urea, 1,25-dihydroxyvitamin D [1,25(OH) The acquisition and maintenance of bone mass and strength are influenced by environmental factors, including physical activity and nutrition , proteins (12–13%), and fats (4–5%) according to the normal dietary requirements  consisting of rats consuming tap water.Salt-loaded group (n = 8) consisting of rats consuming 2% NaCl solution as drinking water  consisting of rats receiving omega-3 fatty acid  daily at a dose of 1 g/kg b.w. , diastolic blood pressure (DBP), and mean arterial pressure (MAP) as well as heart rate (HR) of rats in the three groups were measured at the beginning of the study  and at the end of the study period . Tail SBP, DBP, MAP, and HR were measured using rat tail noninvasive blood pressure system .2D3], and transforming growth factor-beta1 (TGF-β1). The right tibia and the right kidney were dissected out, fixed in 10% neutral buffered formaldehyde, and subjected to histological studies.On the day of experiment, overnight fasted rats, except with free access to water, were weighed and anesthetized i.p. with 40 mg/kg b.w. thiopental sodium . A midline abdominal incision was made, and the abdominal aorta was exposed and cannulated. Blood samples were collected in heparinized tubes, and centrifuged at 3000 rpm for 15 min. to separate plasma. The obtained plasma was subjected to measurement of sodium, potassium, calcium, inorganic phosphorus (Pi), alkaline phosphatase (ALP) enzyme activity, creatinine, urea, 1,25-dihydroxyvitamin D [1,25 (OH)+, K+, Ca2+, Pi, ALP activity, creatinine, and urea were measured by colorimetric methods using kits supplied by Bio-diagnostic, Egypt. Plasma 1,25-dihydroxyvitamin D was measured using 1,25-dihydroxyvitamin D 125I RIA  and plasma total TGF-β1was determined by TGF-β1 ELISA kit . All measurements were performed according to the manufacturer's instructions.Plasma levels of NaThe right tibia specimens were dissected out and fixed in 10% neutral buffered formaldehyde for 2 days. Then, they were decalcified using the chelating agent, formalin-EDTA  for 4 weeks. The solution was changed every day. The specimens were then processed for light microscopic study to get paraffin sections of 5-μm thickness. They were stained with Hematoxylin and Eosin (H&E) and Masson's trichrome to examine the proximal metaphysises of tibia   at pH = 7.4. To unmask the antigenic sites, sections were put into 10 mmol/L citrate buffer (pH = 6) in the microwave for 10 min followed by cooling at room temperature for 20 min. The slides were incubated in 1% bovine serum albumin dissolved in PBS for 30 min at 37°C in order to prevent the nonspecific background staining. The monoclonal primary antibody with dilution of 1:50 of eNOS  was applied to sections except for negative control. Then, they were incubated for 60 min at room temperature. The slides were rinsed with PBS, then incubated for 60 min with anti-mouse immunoglobulins (secondary antibody) conjugated to peroxidase labeled dextran polymer . In order to detect the reaction, the slides were incubated in 3,3-diaminobenzidine (DAB) for 15 min. The slides were counterstained by Hematoxylin, then dehydrated, cleared, and mounted by a mixture of distyrene, a plasticizer and xylene (DPX).Kidney changes as glomerular hypercellularity, renal tubular affection (dilatation of lumen and/or degenerated cells), lesions of renal cortical vessels (thickening of media), and interstitial cellular infiltration were estimated in H&E-stained sections of all animals using Zeiss microscope in Histology department, Faculty of Medicine, Ain Shams University. They were scored from 0 to 3 as: score 0 = no abnormalities, score 1 = changes affecting less than one-third of the specimen, score 2 = changes affecting one to two-thirds of the specimen, and score 3 = changes affecting more than two-thirds of the specimen  in μm, the mean cortical bone thickness (CBT) in μm, the mean of osteoclast number/ high-power field (HPF), and the area percent (%) of eNOS immunoreactivity in renal cortex were measured in five fields from three serial sections of each animal, using Digimizer version 4.1.1.0, copyright © 2005–2011 MedCalc software . The CBT was measured by drawing a vertical line from just beneath the periosteum to the endosteum, and the TBT was measured in cancellous bone of metaphysises at their midpoint away from their branching areas.U-tests were used to compare renal histological nonparametric data. Pearson's correlation was used to assess relationships between variables. A probability of P < 0.05 was considered statistically significant.The statistical data and statistical significance were performed using SPSS statistical package  version 16.0.1. The statistical significance was determined by one-way analyses of variance (ANOVA) for differences among means of different groups followed by least significant difference test (LSD) to find intergroupal significance. Chi-squared and Mann–Whitney The body weight showed insignificant differences among the control (249.4 ± 4.57 g), salt-loaded (241.6 ± 9.14 g), and omega-3 fatty acid-treated salt-loaded (234.4 ± 5.74 g) groups.Table +, K+, Pi, creatinine, urea, and TGF-β1, and significant decrease in plasma levels of Ca2+, ALP activity, and 1,25 (OH)2D3. These changes were alleviated by omega-3 supplementation  of eNOS immunoreactivity was significantly decreased in salt-loaded group compared to control group and significantly increased in omega-3 salt-loaded group compared to salt-loaded group Table .2+, 1,25(OH)2D3 and ALP activity, the bone formation marker, and elevation of plasma Pi in rats exposed to high-salt intake. Such changes were accompanied by and correlated with the altered bone integrity, denoted by enhanced bone resorption, and decreased bone formation, that is observed histologically in the form of thinning of trabecular and cortical bones, many irregular resorption areas on bone surface together with increased number of osteoclasts and decreased new bone formation. These findings are in agreement with previous studies showing deleterious effect of high salt on bone 2D3 levels are known to stimulate secretion of parathyroid hormone (PTH), with secondary hyperparathyroidism; the accumulating PTH stimulating osteoclastic activity 2+ reabsorption, which in turn leads to a greater urinary calcium excretion and loss of bone Ca2+ 2D3 levels and decreased the Pi level, thereby promoting increased bone mass. This confirms the positive influence of omega-3 on bone formation. In concordance with the present results, increased calcium absorption after dietary intake of n-3 fatty acids has been reported in humans  negative correlations with calcium, 1,25(OH)2D3, and ALP plasma levels and positive relations with plasma Pi.Omega-3-induced prevention of excess salt-related renal functional and structural derangements could contribute in its bone-preserving effect, through restoration of renal Ca+ and TGF-β1, findings which are in consistence with previous studies (Medeiros et al. The suppression of the hypertension following treatment with omega-3 could provide an additional mechanism for the beneficial effect of omega-3 on bone and could be explained by omega-3-induced increase in eNOS and NO bioavailability and decrease in Na2+, Pi, and ALP and improvement of renal function and arterial blood pressure. Thus, omega-3 is a potentially useful natural agent in the preservation of bone integrity in high-salt consumers.It could, thus, be concluded that omega-3 supplementation prevented the disturbed bone status imposed by salt loading. This osteoprotective effect might be possibly mediated by attenuation of alterations in CaSeveral limitations should be mentioned for the present study. First, the exact effect of omega-3 fatty acids alone on the different studied parameters was not determined as a control group ingesting omega-3 fatty acids alone was not included in the study. Second, the used salt load induced intrinsic renal damage which could have interfered with the bone changes; it remains questionable if the use of a moderate salt load might have enabled to specify the effect of salt alone on bone histology. Third, although the decalcified specimens used in the histology provided obvious and significant changes that were very satisfactory, the question arises if the use of calcified specimens could have allowed us to comment on more range of bone changes. Therefore, future studies are recommended in order to provide answers to these inquires.


Proteinuria is an independent risk factor for end-stage renal disease (ESRD) . Recent studies highlighted the mechanisms of podocyte injury and implications for potential treatment strategies in proteinuric kidney diseases . Reactive oxygen species (ROS) are cellular signals which are closely associated with the development and progression of glomerular sclerosis. NADPH oxidase is a district enzymatic source of cellular ROS production and prominently expressed in podocytes . In the last decade, it has become evident that NADPH oxidase-derived ROS overproduction is a key trigger of podocyte injury, such as renin-angiotensin-aldosterone system activation , epithelial-to-mesenchymal transition , and inflammatory priming . This review focuses on the mechanism of NADPH oxidase-mediated ROS in podocyte injury under different pathophysiological conditions. In addition, we also reviewed the therapeutic perspectives of NADPH oxidase in kidney diseases related to podocyte injury. Chronic kidney disease (CKD) is a major public health problem worldwide. Proteinuria is a common clinical signature and a potent predictor for the progression of CKD. Podocytes are highly differentiated glomerular epithelial cells which lie in the outmost layer of the glomerular filtration barrier. Podocyte foot processes interdigitate with the counterparts of neighboring cells to form the slit diaphragm, which constitutes the final barrier to prevent protein loss from vascular to urinary space. Podocyte injury has been considered as the most important early event initiating glomerulosclerosis in many proteinuric kidney diseases . The sli2•−), hydroxyl (•OH), hypochlorous acid (HOCl), ozone (O3), singlet oxygen (1O2), and hydrogen peroxide (H2O2) [Oxidative stress is a characteristic feature in many chronic and inflammatory diseases and indeed associated with the development and progression of CKD . The imbe (H2O2) . ROS plaSeveral oxidoreductases have been identified as potential sources of ROS, including cyclooxygenase, lipoxygenase, cytochrome P450 enzymes, nitric-oxide synthase, xanthine oxidase, mitochondrial NADH: ubiquinone oxidoreductase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Unlike other enzymes that produce ROS secondary to their specific catalytic process, NADPH oxidase is the only enzyme known to produce ROS as the primary function. And a previous study showed that the primary source of ROS generation in the renal cortex is NADPH oxidase [ROS overproduction was found in many podocyte injury models in vitro and in experimental diseases such as diabetic nephropathy (DN), membranous nephropathy (MN), minimal change disease (MCD), and focal segmental glomerulo-sclerosis (FSGS) . Oxidantphox, NOXO1 localized in the membrane and cytosolic submits p40phox, p47phox, p67phox, NOXA1, and Rac GTPases. It has long been accepted that the translocation and binding of p47phox with membrane complex of NOX2 and p22phox are the key events leading to the activation of NADPH oxidase and generation of ROS.NADPH oxidase is a cytoplasmic enzyme consisting of at least one catalytic, transmembrane-spanning NOX subunit, which produces ROS by transferring electrons from NADPH to molecular oxygen. To date, the NOX family consists of seven members, NOX1–5, and two NOX5-like dual oxidases, Duox1-2. All NOX subunits have been reported to bind one or more regulatory subunits, including p22phox, is the prototypical catalytic subunit of NADPH oxidase. And the backbone of the enzyme is cell membrane-bound cytochrome b558, which consists of NOX2 and p22phox. NOX1, NOX3, and NOX4 share some similarities with NOX2, such as structural organization and molecular weight. NOX5 possess an extra aminoterminal calmodulin-like domain that contains four Ca2+-binding EF-hands structures. Duox possess an extracellular peroxidase domain that uses the H2O2 generated by its NOX catalytic moiety in addition to the NOX5-based structure. All the NOX1–4 isoforms require the p22phox subunit, whereas NOX5 and Duox are activated directly by Ca2+.The phagocyte NADPH oxidase NOX2, also named gp91phox, p47phox, p67phox, and Rac1/2 protein have been indentified within the kidney [phox, p47phox, NOX2, and p67phox were expressed [phox, p47phox, and Rac1 are expressed immortalized murine podocyte cells [NADPH oxidase plays a fundamental role in maintaining normal cell functions and can be activated by diverse stimuli, such as chemical, physical, environmental, and biological factors. Various NADPH oxidase components are found in the kidney and glomerular cells. NOX1, NOX2, NOX4, p22xpressed . In addite cells .Growing evidence reveals that NADPH oxidase-derived ROS overproduction is importantly involved in the progression of podocyte injury associated with the upregulation of various NOX subtypes . NADPH ophox in podocytes and showed its translocation to the plasma membrane after C5b-9 assembly [ROS-mediated glomerular injury was proved in many experimental studies, and ROS may play important roles in the pathogenesis of primary glomerular nephritis . ROS genassembly . It implphox, p22phox, and NOX4 was found upregulated in glomeruli [2O2, the surface expression of the NADPH oxidase, NOX4, and TRPC6 channels in cultured podocytes [It is widely accepted that oxidative stress is a key component in the development of diabetic nephropathy. NADPH oxidases are the main source of ROS in the diabetic kidney. In rat models of streptozotocin-induced diabetic nephropathy, the expression of p47lomeruli . Podocytlomeruli –17, 28. lomeruli . Interesodocytes . These rphox expression, NADPH oxidase activation, and superoxide production in immortalized murine podocytes [Li's team and others have demonstrated that oxidative stress mediated by NADPH oxidase is importantly involved in the progression of glomerular injury associated with hyperhomocysteinemia (hHcys) , 32. Recodocytes , 33, 34.odocytes .phox and TRPC6 channels and reduced total and cell surface expression of podocyte slit diaphragm proteins nephrin and podocin without loss of cells [2+ concentration may also activate NADPH oxidase in podocytes.It was confirmed that NADPH oxidase-derived ROS was induced by NMDA receptor activation in neurons , and latof cells . Since Nof cells , it is p1R, and total NADPH oxidase activity submits  expression, ROS production, podocyte effacement, and loss of the slit-pore diaphragm integrity in Ren2 rats compared with age-matched Sprague-Dawley (S-D) rats [1R blockade, and MR antagonism could attenuate increased NADPH oxidase activity and submit expression, accompanied by restore podocyte slit diaphragm protein nephrin expression and ultrastructural changes in Ren2 rats [phox, Rac1, NOX2, p47phox, and p22phox gene expressions, ROS overproduction, decreased nephrin and podocin expressions, and enhanced expression of aldosterone effector kinase Sgk1 in aldosterone-infused rats could be prevented by treatment of eplerenone, a selective aldosterone receptor blocker [phox translocation and oxidative stress generation in cultured podocytes with consistent expression of MR [It is noteworthy that podocytes express a functional intrinsic renin-angiotensin-aldosterone system (RAAS) characterized by neprilysin, aminopeptidase A, angiotensin converting enzyme (ACE), prorenin receptor (PRR), renin, and mineralocorticoid receptor (MR), implying a local RAAS in the glomerulus , 37. Pre-D) rats , 42. Furen2 rats , 44. In en2 rats , 46. Theen2 rats . To stud blocker . Furtheron of MR . Data frβ1 (TGF-β1) [α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide production, which was substantially suppressed by NADPH oxidase inhibition. In wild wildtype (gp91+/+) mice, hHcys induced enhanced expression of mesenchymal markers but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in mice lacking NOX2 (gp91−/−) [Studies from different laboratories have demonstrated that podocytes could undergo an epithelial-to-mesenchymal transition (EMT) process when challenged by different injurious stimuli, such as transforming growth factor-(TGF-β1) , 48, hig(TGF-β1) , adriamy(TGF-β1) , hcys [1(TGF-β1) , 20, and(TGF-β1) . Recent (TGF-β1) , NOX1 [5(TGF-β1) , NOX4 [5(TGF-β1) , and Rac(TGF-β1)  were rel(TGF-β1) . EMT in gp91−/−) . Recentlphox-dificient mice, found that LPS-induced systemic inflammatory responses were significantly enhanced by genetic deficiency of NOX [α (TNF-α) and interleukin 6 (IL-6); oxidative stress index NOX2 and p22phox were upregulated in kidney, which could be partly restored by farnesoid X receptor (FXR) agonist chenodeoxycholic acid (CDCA) treatment [The phagocyte NADPH oxidase NOX2 was initially implicated in innate immunity and inflammation. NOX is recognized as a critical mediator of inflammatory responses in nonimmune systems, such as neuroinflammation, gastric inflammation, colon inflammation, lung inflammation, and vascular inflammation. An anti-inflammatory activity of NOX enzymes, also mentioned in NOX2- and p47y of NOX . Howevery of NOX . Moreovereatment . Inductireatment . Recent reatment , 59. SinPharmacological NADPH oxidase inhibitors have been used for many years, such as apocynin, diphenylene iodonium (DPI), and 4-(2-aminoethyl)-benzenesulfonylfluorid (AEBSF). NADPH oxidase inhibition by Apocynin or DPI could attenuate podocyte injury , 59, 60.phox) and ROS generation in cultured podocytes [Statins, also known as 3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors, exert beneficial actions on oxidative stress independent cholesterol-lowering effects. Statins have an indirect NOX inhibitory action by preventing the isoprenylation of Rac1 . In the odocytes . Pretreaodocytes .Accumulating evidence reveals that drugs inhibit the RAAS system, including rennin inhibitors, ACE inhibitors (ACEI), AT1R blockers (ARB), aldosterone receptor blocker, and MR antagonist, reduce kidney oxidative stress by the inhibition of NADPH oxidase. Notably, recent study showed that losartan metabolite EXP3179 blocked NADPH oxidase-mediated ROS production . The metphox migration to the podocyte cellular plasma after C5b-9 treatment in vitro [Triptolide, a major active component of Tripterygium wilfordii Hook F, has potent anti-inflammatory  and podoin vitro . It implIn conclusion, NADPH oxidase appears to be one of the most promising targets to attenuate podocyte injury. Oxidative stress is a common mechanism of CKD . Restore