RAG-mediated V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice

Atm-deficient mice die of malignant thymic lymphomas characterized by translocations within the Tcr alpha/delta locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA (dsDNA) breaks that occur during RAG-dependent V(D)J recombination. We recently demonstrated that development of thymic lymphoma in Atm(-/-) mice was not prevented by loss of RAG-2. Thymic lymphomas that developed in Rag2(-/-) Atm(-/-) mice contained multiple chromosomal abnormalities, but none of these involved the Tcr alpha/delta locus. These findings indicated that tumorigenesis in Atm(-/-) mice is mediated by chromosomal translocations secondary to aberrant responses to dsDNA breaks and that V(D)J recombination is an important, but not essential, event in susceptibility. In contrast to these findings, it was recently reported that Rag1(-/-) Atm(-/-) mice do not develop thymic lymphomas, a finding that was interpreted as demonstrating a requirement for RAG-dependent recombination in the susceptibility to tumors in Atm-deficient mice. To test the possibility that RAG-1 and RAG-2 differ in their roles in tumorigenesis, we studied Rag1(-/-) Atm(-/-) mice in parallel to our previous Rag2(-/-) Atm(-/-) study. We found that thymic lymphomas occur at high frequency in Rag1(-/-) Atm(-/-) mice and resemble those that occur in Rag2(-/-) Atm(-/-) mice. These results indicate that both RAG-1 and RAG-2 are necessary for tumorigenesis involving translocation in the Tcr alpha/delta locus but that Atm deficiency leads to tumors through a broader RAG-independent predisposition to translocation, related to a generalized defect in dsDNA break repair.     

A structural basis for 14-3-3sigma functional specificity

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.     

Specific binding of vf14-3-3a isoform to the plasma membrane H+-ATPase in response to blue light and fusicoccin in guard cells of broad bean

The plasma membrane H(+)-ATPase is activated by blue light with concomitant binding of the 14-3-3 protein to the C terminus in guard cells. Because several isoforms of the 14-3-3 protein are expressed in plants, we determined which isoform(s) bound to the H(+)-ATPase in vivo. Four cDNA clones (vf14-3-3a, vf14-3-3b, vf14-3-3c, and vf14-3-3d) encoding 14-3-3 proteins were isolated from broad bean (Vicia faba) guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly in guard cells. The 14-3-3 protein that bound to the H(+)-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. The H(+)-ATPase immunoprecipitated from mesophyll cell protoplasts, which had been stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protein, although three 14-3-3 isoproteins were found in mesophyll cell protoplasts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with cyanogen bromide gave the identical migration profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H(+)-ATPase gave the predicted peptide masses of vf14-3-3a. Far western analysis revealed that the H(+)-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H(+)-ATPase in vivo is vf14-3-3a and that it may play a key role in the activation of H(+)-ATPase in guard cells.     

The 14-3-3tau phosphoserine-binding protein is required for cardiomyocyte survival

14-3-3 family members are intracellular dimeric phosphoserine-binding proteins that regulate signal transduction, cell cycle, apoptotic, and metabolic cascades. Previous work with global 14-3-3 protein inhibitors suggested that these proteins play a critical role in antagonizing apoptotic cell death in response to provocative stimuli. To determine the specific role of one family member in apoptosis, mice were generated with targeted disruption of the 14-3-3tau gene. 14-3-3tau(-/-) mice did not survive embryonic development, but haploinsufficient mice appeared normal at birth and were fertile. Cultured adult cardiomyocytes derived from 14-3-3tau(+/-) mice were sensitized to apoptosis in response to hydrogen peroxide or UV irradiation. 14-3-3tau(+/-) mice were intolerant of experimental myocardial infarction and developed pathological ventricular remodeling with increased cardiomyocyte apoptosis. ASK1, c-jun NH(2)-terminal kinase, and p38 mitogen-activated protein kinase (MAPK) activation was increased, but extracellular signal-regulated kinase MAPK activation was reduced, in 14-3-3tau(+/-) cardiac tissue. Inhibition of p38 MAPK increased survival in 14-3-3tau(+/-) mice subjected to myocardial infarction. These results demonstrate that 14-3-3tau plays a critical antiapoptotic function in cardiomyocytes and that therapeutic agents that increase 14-3-3tau activity may be beneficial to patients with myocardial infarction.     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

A switch in pathogenic mechanism in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in IFN-γ-inducible lysosomal thiol reductase-free mice

IFN-γ-inducible lysosomal thiol reductase (GILT) is an enzyme located in the Lamp-2-positive compartments of APC. GILT(-/-) mice are phenotypically normal, but their T cells exhibit reduced proliferation to several exogenously administered Ags that include cysteine residues and disulfide bonds. We undertook the present studies to determine if GILT(-/-) mice would process exogenously administered myelin oligodendrocyte glycoprotein (MOG), which contains disulfide bonds, to generate experimental autoimmune encephalomyelitis (EAE) to the endogenous protein. One possibility was that MOG(35-55) peptide would induce EAE, but that MOG protein would not. GILT(-/-) mice were relatively resistant to MOG(35-55)-induced EAE but slightly more susceptible to rat MOG protein-induced EAE than wild-type (WT) mice. Even though MOG(35-55) was immunogenic in GILT(-/-) mice, GILT APCs could not generate MOG(35-55) from MOG protein in vitro, suggesting that the endogenous MOG protein was not processed to the MOG(35-55) peptide in vivo. Immunization of GILT(-/-) mice with rat MOG protein resulted in a switch in pathogenic mechanism from that seen in WT mice; the CNS infiltrate included large numbers of plasma cells; and GILT(-/-) T cells proliferated to peptides other than MOG(35-55). In contrast to WT rat MOG-immunized mice, rat MOG-immunized GILT(-/-) mice generated Abs that transferred EAE to MOG(35-55)-primed GILT(-/-) mice, and these Abs bound to oligodendrocytes. These studies, demonstrating the key role of a processing enzyme in autoimmunity, indicate that subtle phenotypic changes have profound influences on pathogenic mechanisms and are directly applicable to the outbred human population.     

Developmental regulation of human gamma-globin genes in transgenic mice.

We report results showing that several gamma gene promoter elements participate in the developmental control of gamma-globin genes. Four gamma gene constructs with 5' truncated at -141, -201, -382, and -730 of the A gamma gene promoter linked to a micro locus control region (microLCR) cassette were used for production of transgenic mice and analysis of gamma gene expression during development. Mice carrying a microLCR -141 A gamma construct displayed downregulation of gamma gene expression in the adult stage of development, indicating that the proximal promoter contains elements participating in gamma gene silencing. Mice carrying a microLCR -201 A gamma or a microLCR -382 A gamma construct displayed high gamma gene expression in the fetal stage of development and complete loss of gamma gene downregulation in the adult stage, suggesting that the -141 to -201 gamma gene sequence contains elements which upregulate gamma gene expression and are dominant over the negative element 3' to -141. Extension of the promoter to -730 resulted in reappearance of gamma gene downregulation, suggesting that the -382 to -730 sequences contain an adult-stage-specific silencer. gamma gene expression in the microLCR -201 A gamma and the microLCR -382 A gamma transgenic mice was copy number dependent. All the microLCR -730 A gamma transgenic mice expressed gamma mRNA; however, gamma gene expression was copy number independent, indicating that levels of gamma gene expression were modulated by the surrounding chromatin. Our results suggest that multiple elements participate in gamma gene silencing. The findings in the microLCR-201 A gamma and microLCR -382 A gamma transgenic mice are interpreted to indicate that the LCR interacts not only with the minimal gamma gene promoter but also with sequences of the upstream promoter. We postulate that gamma gene downregulation is achieved when the interaction between LCR and the upstream promoter is disturbed by the silencer located in the -382 to -730 region. We propose that gamma gene silencing is achieved by the combined effect of negative elements located 3' to -141, the negative element located between -382 and -730, and the competition by the beta gene promoter during the adult stage of development.     

Immunoglobulin-secreting cells of maternal origin can be detected in B cell-deficient mice

It is well known that the transfer of immunoglobulins (Igs) from mother to young via milk contributes to the offspring's immune defense. The present study suggests that not only is IgG transmitted to progeny, but that functional maternal Ig-secreting cells (or B cells) can also be transferred to the neonate. We have used B cell-deficient (micro(-/-)) mice and found that a high proportion of them obtain long-lasting, partial reconstitution of their serum Ig levels if born to micro(+/-) mothers. In some of these serum IgG-positive micro(-/-) mice, Ig-secreting cells were detected in spleen and bone marrow. To ensure that cells of maternal origin were present in the progeny, micro(-/-) offspring born to micro(+/-) dams transgenic for green fluorescent protein (GFP) were used. In spleens and bone marrow from some of these micro(-/-)GFP(-/-) offspring, GFP-positive cells were detected, which demonstrated that cells of maternal origin could infiltrate the progeny. In addition, splenic Ig-secreting cells were detected in micro(-/-) mice that were born to micro(-/-) dams and transferred to a lactating micro(+/+) foster dam at birth. This indicates that maternal Ig-secreting cells can be transferred postnatally via milk.     

Overexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli and provides enhanced osmotic stress tolerance and ABA sensitivity in Arabidopsis thaliana

Previous studies have shown that the late embryogenesis abundant (LEA) group 3 proteins significantly respond to changes in environmental conditions. However, reports that demonstrate their biological role, especially in Arabidopsis, are notably limited. This study examines the functional roles of the Arabidopsis LEA group 3 proteins AtLEA3-3 and AtLEA3-4 in abiotic stress and ABA treatments. Expression of AtLEA3-3 and AtLEA3-4 is upregulated by ABA, high salinity, and osmotic stress. Results on the ectopic expression of AtLEA3-3 and AtLEA3-4 in E. coli suggest that both proteins play important roles in resistance to cold stress. Overexpression of AtLEA3-3 in Arabidopsis (AtLEA3-3-OE) confers salt and osmotic stress tolerance that is characterized during germination and early seedling establishment. However, AtLEA3-3-OE lines show sensitivity to ABA treatment during early seedling development. These results suggest that accumulation of AtLEA3-3 mRNA and/or proteins may help heterologous ABA re-initiate second dormancy during seedling establishment. Analysis of yellow fluorescent fusion proteins localization shows that AtLEA3-3 and AtLEA3-4 are mainly distributed in the ER and that AtLEA3-3 also localizes in the nucleus, and in response to salt, mannitol, cold, or BFA treatments, the localization of AtLEA3-3 and AtLEA3-4 is altered and becomes more condensed. Protein translocalization may be a positive and effective strategy for responding to abiotic stresses. Taken together, these results suggest that AtLEA3-3 has an important function during seed germination and seedling development of Arabidopsis under abiotic stress conditions.     

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose–phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.     

14-3-3 Facilitates Ras-Dependent Raf-1 Activation In Vitro and In Vivo

14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.     

Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase

The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity.     

DNA Damage and Replication Checkpoints in Fission Yeast Require Nuclear Exclusion of the Cdc25 Phosphatase via 14-3-3 Binding

In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins. Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant. These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks. Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus. Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus. In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant. These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA.     

油松毛虫雌蛾对油松松针两种手性化合物的触角电位反应

采用触角电位技术测定了油松毛虫雌蛾触角对油松挥发物的两对手性单萜的剂量反应,并在饱和剂量下测定了α-蒎烯、β-蒎烯手性化合物及其消旋体的触角电位值。剂量反应测试表明,油松毛虫雌蛾对(-)-α-蒎烯的饱和剂量为1 600 μL,对(+)-α-蒎烯、(+)-β-蒎烯、(-)-β-蒎烯的饱和剂量为800 μL。α-蒎烯右旋异构体的反应值高于(-)-α-蒎烯的反应值,说明(+)-α-蒎烯能够更有效的与感受器中的分子受体结合;β-蒎烯则相反,(-)-β-蒎烯的EAG反应值高于(+)-β-蒎烯,说明β-蒎烯的右旋异构体不能有效的与多数感受器中的分子受体结合。在饱和剂量下的测定结果表明油松毛虫雌蛾对α-蒎烯和β-蒎烯消旋体的反应均与对其手性异构体反应相当,说明油松毛虫雌蛾用同一个感受细胞来接受α-蒎烯的两个对映异构体,同样β-蒎烯的两个对映异构体也是被同一个受体细胞来接受。结果提示,手性化合物的比例可能在油松毛虫寄主识别中有重要作用。     

Hypotension and bradycardia during caloric restriction in mice are independent of salt balance and do not require ANP receptor

We hypothesized that caloric restriction (CR)-induced hypotension would correlate with increased sodium excretion through an atrial natriuretic peptide (ANP)-dependent mechanism. To test this hypothesis, the cardiovascular parameters of c57/Bl mice were measured with radiotelemetry while urine was collected. The 23-h mean blood pressure (BP) dropped from 108.6 +/- 1.8 to 92.7 +/- 2.4 mmHg, and 23-h heart rate dropped from 624 +/- 5 to 426 +/- 13 beats/min over 7 days of CR at 29 degrees C. Contrary to our hypothesis, urine sodium excretion decreased by 55% by day 7 of CR. Consistent with decreased sodium excretion was the drop in plasma ANP (from 82.4 +/- 4.3 to 68.0 +/- 5.8 pg/ml). To explore the possibility that CR lowers BP through an ANP receptor-dependent mechanism that is independent of its effect on sodium retention, we measured the cardiovascular parameters of mice deficient in the ANP receptor (NPR1(-/-)) or the ANP clearance receptor (NPR3(-/-)). Mean BP fell from 117.1 +/- 3.9 to 108.0 +/- 4.7 mmHg in the NPR1(-/-) mice and from 87.0 +/- 2.4 to 78.4 +/- 1.7 mmHg in the NPR3(-/-) mice during CR. These data indicate that the hypotension induced by CR does not depend on increased sodium excretion. Rather, it appears that the mouse responds to the low BP induced by CR with an increase in sodium reabsorption. Furthermore, circulating ANP levels and data from NPR1(-/-) and NPR3(-/-) mice suggest that the ANP pathway may not be involved in the cardiovascular response to CR.