Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation

Lymphocyte antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG endonuclease introduces DNA DSBs at two recombining gene segments to form broken DNA coding end pairs and signal end pairs. These paired DNA ends are joined by proteins of the nonhomologous end-joining (NHEJ) pathway of DSB repair to form a coding joint and signal joint, respectively. RAG DSBs are generated in G1-phase developing lymphocytes, where they activate the ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases to orchestrate diverse cellular DNA damage responses including DSB repair. Paradoxically, although Atm and DNA-PKcs both function during coding joint formation, Atm appears to be dispensible for signal joint formation; and although some studies have revealed an activity for DNA-PKcs during signal joint formation, others have not. Here we show that Atm and DNA-PKcs have overlapping catalytic activities that are required for chromosomal signal joint formation and for preventing the aberrant resolution of signal ends as potentially oncogenic chromosomal translocations.     

Gibberellin A1 is required for stem elongation in spinach

The effects of the growth retardants 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (AMO-1618) and calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate (BX-112) on stem elongation were investigated in the rosette plant spinach (Spinacia oleracea L.) under long-day (LD) conditions. Stem growth induced by a LD treatment was prevented by both retardants. The inhibition caused by AMO-1618 was reversed by gibberellin A1 (GA1) and GA20, whereas the effects of BX-112 were reversed by GA1 only. Six GAs (GA53, GA44, GA19, GA20, GA1, and GA8) were quantified by gas chromatography-selected ion monitoring using internal standards. Plants treated with BX-112 had reduced levels of GA1 and GA8 and accumulated GA53, GA44, GA19, and GA20. The relative levels of four additional GAs (3-epi-GA1, GA29, GA60, and GA81) were compared by ion intensities only. Relative to GA81, the level of GA29 was decreased by BX-112, whereas the levels of GA60 and 3-epi-GA1 were increased. Transfer of spinach from short-day conditions to LD conditions caused an increase in all identified GAs of the early 13-hydroxylation pathway with GA20, GA1, and GA8 showing the largest increases. These findings support the position that, of the GAs belonging to the early 13-hydroxylation pathway, GA1 is the primary GA active per se for stem elongation in spinach. The increase in endogenous GA1 in plants in LD conditions is most likely the primary factor for stem elongation.     

The catalytic subunit of DNA-protein kinase (DNA-PKcs) is not required for Ig class-switch recombination

The joining of DNA ends during Ig class-switch recombination (CSR) is thought to involve the same nonhomologous end-joining pathway as used in V(D)J recombination. However, we reported earlier that CSR can readily occur in Ig transgenic SCID mice lacking DNA-dependent protein kinase (DNA-PK) activity, a critical enzymatic activity for V(D)J recombination. We were thus led to question whether the catalytic subunit of DNA-PK (DNA-PKcs) is essential for CSR. To address this issue, we asked whether class switching to different Ig isotypes could occur in a line of Ig transgenic mice lacking detectable DNA-PKcs protein. The answer was affirmative. We conclude that joining of DNA ends during CSR does not require DNA-PKcs and can occur by an alternative repair pathway to that used for V(D)J recombination.     

Beyond average: potential for measurement of short telomeres

The length of telomeres, and in particular the abundance of short telomeres, has been proposed as a biomarker of aging and of general health status. A wide variety of studies show the association of short telomeres with age related pathologies and cancer, as well as with lifespan and mortality. These facts highlight the importance of measuring telomere length in human populations and by using reliable methods to uncover the association between telomere length and human disease. This review discusses the advantages and drawbacks of current telomere length measurement methods. Most of these methods provide mean telomere length values per cell or per sample and very few of them are able to measure the abundance of short telomeres, which are the ones indicative of telomere dysfunction. The information provided by each method and their suitability for different studies is discussed here.     

T-cell P/E-selectin ligand alpha(1,3)fucosylation is not required for graft-vs-host disease induction

OBJECTIVE: Recognition of E- and P-selectins on vascular endothelium by their leukocyte glycoprotein counterreceptor P-selectin glycoprotein ligand-1 (PSGL-1) initiates and sustains leukocyte rolling, culminating in extravasation of lymphocytes from blood into organs. PSGL-1 is rendered functional by terminal glycosylation steps, which occur mainly in activated Th1 but not Th2 cells. alpha(1,3)Fucosyltransferases IV and VII control this glycosylation pathway. Mice lacking these transferases (Fuc-TIV(-/-)/Fuc-TVII(-/-)) lack functional E- and P-selectin ligands. We hypothesized that Fuc-TIV(-/-)/Fuc-TVII(-/-) donor T cells might have reduced capacity to roll on vessels of inflamed target tissues and mediate graft-vs-host disease (GVHD). MATERIALS AND METHODS: We compared the ability of Fuc-TIV(-/-)/Fuc-TVII(-/-) and wild-type (WT) C57BL/6 (B6) spleen cells (SPCs) to produce GVHD in lethally irradiated major histocompatibility complex (MHC) haplotype-mismatched B6D2F1 recipients. Clinical GVHD, GVHD pathology in target organs, memory phenotype conversion, proliferation of donor T cells, and tissue and serum cytokine expression were examined. RESULTS: Surprisingly, clinical GVHD was not reduced in lethally irradiated mice receiving full haplotype MHC mismatched or matched Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared to those receiving WT SPCs. GVHD pathology in target organs, memory phenotype conversion, and proliferation of donor T cells were similar in both groups. However, reduced interferon-gamma was detected in liver and lung, and serum levels of tumor necrosis factor-alpha were higher in mice receiving Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared with WT SPCs. CONCLUSIONS: These results suggest that donor T cells, including Th1, are capable of trafficking to GVHD target tissues independently of P- and E- selectin ligand in conditioned hosts.     

Apoptosis is not required for mammalian neural tube closure

Apoptotic cell death occurs in many tissues during embryonic development and appears to be essential for processes including digit formation and cardiac outflow tract remodeling. Studies in the chick suggest a requirement for apoptosis during neurulation, because inhibition of caspase activity was found to prevent neural tube closure. In mice, excessive apoptosis occurs in association with failure of neural tube closure in several genetic mutants, but whether regulated apoptosis is also necessary for neural tube closure in mammals is unknown. Here we investigate the possible role of apoptotic cell death during mouse neural tube closure. We confirm the presence of apoptosis in the neural tube before and during closure, and identify a correlation with 3 main events: bending and fusion of the neural folds, postfusion remodeling of the dorsal neural tube and surface ectoderm, and emigration of neural crest cells. Both Casp3 and Apaf1 null embryos exhibit severely reduced apoptosis, yet neurulation proceeds normally in the forebrain and spine. In contrast, the mutant embryos fail to complete neural tube closure in the midbrain and hindbrain. Application of the apoptosis inhibitors z-Vad-fmk and pifithrin-α to neurulation-stage embryos in culture suppresses apoptosis but does not prevent initiation or progression of neural tube closure along the entire neuraxis, including the midbrain and hindbrain. Remodeling of the surface ectoderm to cover the closed tube, as well as delamination and migration of neural crest cells, also appear to be normal in the apoptosis-suppressed embryos. We conclude that apoptosis is not required for neural tube closure in the mouse embryo.     

Placenta growth factor is not required for exercise-induced angiogenesis

Angiogenesis is a tightly regulated process, both during development and adult life. Animal models with mutations in the genes coding for placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, or the tyrosine kinase domain of the PlGF receptor (Flt-1) have revealed differences between normal physiological angiogenesis and pathological angiogenesis associated with conditions such as tumor growth, arthritis and atherosclerosis. In the present paper, we investigated the potential role of PlGF in regulating physiological angiogenesis by analyzing vascular changes in heart and skeletal muscles of wild-type and Plgf^–/– mice following prolonged and sustained physical training. Sedentary Plgf^–/– mice showed a reduced capillary density in both heart and skeletal muscles as compared to wild-type mice (P < 0.05). However, after a 6-week training period, heart/body weight ratio, citrate synthase activity, vessel density and capillary/myocyte ratio were significantly increased in both wild-type and Plgf^–/– mice (all P < 0.05). At the same time intercapillary distance was significantly reduced. Finally, acute exercise was not associated with any change in PlGF protein level in the skeletal muscle. Our results demonstrate that PlGF is not necessary for exercise-training-induced angiogenesis. We thus suggest that the role of PlGF is confined to the selective regulation of angiogenesis only under pathological conditions.     

Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development

Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.The auxin binding protein 1 (ABP1) was first isolated from maize plants based on its ability to bind auxin (1). The crystal structure of ABP1 demonstrated clearly that ABP1 has an auxin-binding pocket and, indeed, binds auxin (2). However, the elucidation of the physiological functions of ABP1 has been challenging because the first reported abp1 T-DNA insertion mutant in Arabidopsis was not viable (3). Nevertheless, ABP1 has been recognized as an essential gene for plant development and as a key component in auxin signaling (4–9). Because viable abp1 null mutants in Arabidopsis were previously unavailable, alternative approaches have been used to disrupt ABP1 function in Arabidopsis to determine the physiological roles of the protein. Cellular immunization approaches were used to generate ABP1 knockdown plants (10, 11). Inducible overexpression of the single chain fragment variable regions (scFv12) of the anti-ABP1 monoclonal antibody mAb12 both in cell lines and in Arabidopsis plants presumably neutralizes the endogenous ABP1 activities (10, 11). Two such antibody lines, SS12S and SS12K, have been widely used in many ABP1-related studies (4, 6, 9–11). The results obtained from the characterization of the antibody lines suggest that ABP1 regulates cell division, cell expansion, meristem activities, and root development (4, 6, 10, 12, 13). Transgenic plants that overexpress ABP1 antisense RNA were also used to elucidate the physiological functions of ABP1 (4, 10). Moreover, missense point mutation alleles of abp1 have also been generated through the Arabidopsis TILLING project. One such TILLING mutant, named abp1-5, harbors a mutation (His94 >Tyr) in the auxin-binding pocket and has been widely used in many ABP1-related studies (4, 8, 9). Previous studies based on the antisense lines, antibody lines, and Arabidopsis mutant alleles have led to the conclusion that ABP1 is essential for embryogenesis, root development, and many other developmental processes. However, the interpretation of results generated by using the ABP1 antisense and antibody lines are not straightforward and off-target effects have not been completely ruled out. We believe that characterization of abp1 null plants is urgently needed to unambiguously define the roles of ABP1 in auxin signaling and in plant development.In the past several years, studies of the presumed ABP1-mediated auxin signal transduction pathway were carried out in several laboratories. It has been hypothesized that ABP1 is an auxin receptor mediating fast, nongenomic effects of auxin (4–6, 8, 9), whereas the TIR1 family of F-box protein/auxin receptors are responsible for auxin-mediated gene regulation (14, 15). One of the proposed functions of ABP1 is to regulate subcellular distribution of PIN auxin efflux carriers (6, 9, 13). Furthermore, a recent report suggests that a cell surface complex consisting of ABP1 and transmembrane receptor-like kinases functions as an auxin receptor at the plasma membrane by activating the Rho-like guanosine triphosphatases (GTPases) (ROPs) in an auxin-dependent manner (8). ROPs have been reported to play a role in regulating cytoskeleton organization and PIN protein endocytosis (5, 6). However, it is important to unequivocally determine the biological processes that require ABP1 before extensive efforts are directed toward elucidating any ABP1-mediated signaling pathways.In this paper, we generate and characterize new abp1 null mutants in Arabidopsis. We are interested in elucidating the molecular mechanisms by which auxin regulates flower development because our previously identified auxin biosynthetic mutants display dramatic floral defects (16–18). Because ABP1 was reported as an essential gene and ABP1 binds auxin (2, 3), we decided to determine whether ABP1 plays a role in flower development. We used our recently developed ribozyme-based CRISPR gene editing technology (19) to specifically inactivate ABP1 during flower development. Unexpectedly, we recovered a viable abp1 mutant (abp1-c1, c stands for alleles generated by using CRISPR) that contains a 5-bp deletion in the first exon of ABP1. We also isolated a T-DNA abp1 allele (abp1-TD1) that harbors a T-DNA insertion in the first exon of ABP1. We show that both abp1-c1 and abp1-TD1 are null mutants. Surprisingly, the mutants were indistinguishable from wild-type (WT) plants at all of the developmental stages we analyzed. Our data clearly demonstrate that ABP1 is not an essential gene and that ABP1 does not play a major role in auxin signaling and Arabidopsis development under normal growth conditions.     

Identification and characterization of polymorphic variations of the ataxia telangiectasia mutated (ATM) gene in childhood Hodgkin disease

There are conflicting reports about the involvement of single nucleotide polymorphisms (SNPs) of the ataxia telangiectasia mutated (ATM) gene with cancer, and the consequences of these SNPs for ATM function remain unclear. We therefore sought to identify SNPs of the ATM gene in pediatric Hodgkin disease (HD) and to analyze ATM function in cells from patients with these SNPs. We have identified SNPs of the ATM gene in 5 of 14 children (S1455R, n = 1; H1380Y, n = 1; N1650S, n = 2; and I709I, n = 1). One patient had nonsense-associated altered splicing of the ATM gene. Lymphoblastoid cell lines expressing the S1455R and N1650S exhibited defective ATM-mediated p53 phosphorylation and Chk2 activation; cells expressing the H1380Y exhibited defective c-Abl activation after X-irradiation. Expression of the N1650S in ATM-null fibroblasts conferred only partial hyperradiosensitivity. Furthermore, the introduction of N1650S ATM into U2OS cells, which express wild-type ATM, showed reduced p53-Ser15 phosphorylation, suggesting a dominant-negative effect of the N1650S over the wild-type ATM protein. We conclude that the rare polymorphic variants of the ATM gene that we identified in children with HD encode functionally abnormal proteins, and we discuss the possible genetic risk factors for childhood HD.     

AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress

Mammalian brain connectivity requires the coordinated production and migration of billions of neurons and the formation of axons and dendrites. The LKB1/Par4 kinase is required for axon formation during cortical development in vivo partially through its ability to activate SAD-A/B kinases. LKB1 is a master kinase phosphorylating and activating at least 11 other serine/threonine kinases including the metabolic sensor AMP-activated protein kinase (AMPK), which defines this branch of the kinome. A recent study using a gene-trap allele of the β1 regulatory subunit of AMPK suggested that AMPK catalytic activity is required for proper brain development including neurogenesis and neuronal survival. We used a genetic loss-of-function approach producing AMPKα1/α2-null cortical neurons to demonstrate that AMPK catalytic activity is not required for cortical neurogenesis, neuronal migration, polarization, or survival. However, we found that application of metformin or AICAR, potent AMPK activators, inhibit axogenesis and axon growth in an AMPK-dependent manner. We show that inhibition of axon growth mediated by AMPK overactivation requires TSC1/2-mediated inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. Our results demonstrate that AMPK catalytic activity is not required for early neural development in vivo but its overactivation during metabolic stress impairs neuronal polarization in a mTOR-dependent manner.     

Oxytocin is required for nursing but is not essential for parturition or reproductive behavior.

Oxytocin, a neurohypophyseal hormone, has been traditionally considered essential for mammalian reproduction. In addition to uterine contractions during labor and milk ejection during nursing, oxytocin has been implicated in anterior pituitary function, paracrine effects in the testis and ovary and the neural control of maternal and sexual behaviors. To determine the essential role(s) of oxytocin in mammalian reproductive function, mice deficient in oxytocin have been generated using embryonic stem cell technology. A deletion of exon 1 encoding the oxytocin peptide was generated in embryonic stem cells at a high frequency and was successfully transmitted in the germ line. Southern blot analysis of genomic DNA from homozygote offspring and in situ hybridization with an exonic probe 3' of the deletion failed to detect any oxytocin or neurophysin sequences, respectively, confirming that the mutation was a null mutation. Mice lacking oxytocin are both viable and fertile. Males do not have any reproductive behavioral or functional defects in the absence of oxytocin. Similarly, females lacking oxytocin have no obvious deficits in fertility or reproduction, including gestation and parturition. However, although oxytocin-deficient females demonstrate normal maternal behavior, all offspring die shortly after birth because of the dam's inability to nurse. Postpartum injections of oxytocin to the oxytocin deficient mothers restore milk ejection and rescue the offspring. Thus, despite the multiple reproductive activities that have been attributed to oxytocin, oxytocin plays an essential role only in milk ejection in the mouse.     

Raf-1 is not required for megakaryocytopoiesis or TPO-induced ERK phosphorylation

Thrombopoietin stimulates extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in megakaryocytes, and the classic mitogen-activated protein (MAP) kinase (Raf/mitogen-induced extracellular kinase [MEK]/ERK) pathway has been implicated directly and indirectly to play a critical role in megakaryocytopoiesis. However, the involvement of specific Raf family members in megakaryocytopoiesis is unknown. raf-1(-/-) mice were therefore used to directly determine the role of Raf-1 in megakaryocytopoiesis. Surprisingly, raf-1(-/-) mice have a modestly higher platelet count than their raf-1(+/+) littermates. Nonetheless, the absence of Raf-1 does not alter thrombopoietin-induced expansion of primary megakaryocyte-lineage cells, the development of apoptotic megakaryocytes in the presence or absence of thrombopoietin, or the development of megakaryocyte DNA ploidy distribution. Moreover, raf-1(-/-) megakaryocytes do not have a compensatory increase in A-Raf or B-Raf expression, and thrombopoietin-induced ERK1/2 phosphorylation is similar in raf-1(-/-) and raf-1(+/+) megakaryocytes. These unexpected findings demonstrate that Raf-1 is dispensable for megakaryocytopoiesis, and for thrombopoietin-induced ERK1/2 activation in primary megakaryocyte-lineage cells.     

Glycosylation site-binding protein is not required for N-linked glycoprotein synthesis.

In prior studies we identified a 57-kDa protein in the lumen of the endoplasmic reticulum that, in addition to having both protein disulfide isomerase and thyroid hormone-binding protein activities, bound a photoaffinity probe containing the N-glycosylation-site sequence Asn-Xaa-Ser/Thr. It was hypothesized that this multifunctional protein, called glycosylation site-binding protein (GSBP), participated in the process of N-glycosylation of proteins. To test this hypothesis we have employed various conditions to deplete the lumen of GSBP and then assess the level of N-glycosylation catalyzed by oligosaccharyltransferase (OTase). Although most conditions leading to depletion resulted in partial loss of OTase activity, this loss was independent of the extent of GSBP depletion. Indeed, virtually complete loss (greater than 99%) of GSBP with partial retention of OTase activity was frequently observed. Moreover, repletion of the microsomal lumen with GSBP did not restore OTase activity to control levels. Thus, no correlation between GSBP content and OTase activity before or after reconstitution was found. These results suggest that this multifunctional 57-kDa protein is not an essential component of the enzymatic reaction in which oligosaccharide chains are transferred from dolichyl-P-P-GlcNAc2Man9Glc3 to nascent polypeptides or to synthetic tripeptide acceptors.