Measurements of Antibacterial Activity of Seed Crude Extracts in Cultivated Rice and Wild Oryza Species.

Seeds are continuously exposed to a wide variety of microorganisms in the soil. In addition, seeds contain large amounts of carbon and nitrogen sources that support initial growth after germination. Thus, seeds in the soil can easily promote microbial growth, and seeds are susceptible to decay. Therefore, seed defense against microorganisms is important for plant survival. Seed-microbe interactions are also important issues from the perspective of food production, in seed quality and shelf life. However, seed-microbe interactions remain largely unexplored. In this study, we established a simple and rapid assay system for the antibacterial activity of rice seed crude extracts by colorimetric quantification methods by the reduction of tetrazolium compound. Using this experimental system, the diversity of effects of rice seed extracts on microbial growth was analyzed using Escherichia coli as a bacterial model. We used collections of cultivated rice, comprising 50 accessions of Japanese landraces, 52 accessions of world rice core collections, and of 30 wild Oryza accessions. Furthermore, we attempted to find genetic factors responsible for the diversity by genome-wide association analysis. Our results demonstrate that this experimental system can easily analyze the effects of seed extracts on bacterial growth. It also suggests that there are various compounds in rice seeds that affect microbial growth. Overall, this experimental system can be used to clarify the chemical entities and genetic control of seed-microbe interactions and will open the door for understanding the diverse seed-microbe interactions through metabolites.

Vero/BC-F: an efficient packaging cell line stably expressing F protein to generate single round-infectious human parainfluenza virus type 2 vector.

A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 10(8) median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.

Expression of ADAMTS4 in Ewing's sarcoma.

Ewing's sarcoma (EWS) is a malignant bone tumor that frequently occurs in teenagers. Genetic mutations which cause EWS have been investigated, and the most frequent one proved to be a fusion gene between EWS gene of chromosome 22 and the FLI1 gene of chromosome 11. However, a limited numbers of useful biological markers for diagnosis of EWS are available. In this study, we identified ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs) as a possible tumor marker for EWS using the retrovirus-mediated signal sequence trap method. ADAMTS4 is a secreted protein of 837 amino acids with a predicted molecular mass of 98-100 kDa. It is a member of metalloprotease family, is expressed mainly in cartilage and brain, and regulates the degradation of aggrecans. ADAMTS4 has been suggested to be involved in arthritic diseases and gliomas. Herein, we show that ADAMTS4 mRNA was expressed in all primary EWS samples and all EWS-derived cell lines examined, while its expression was detected only in small subpopulations of other solid tumors. Furthermore, ADAMTS4 expression was found to be regulated by EWS-FLI1 fusion gene-dependent manner. We also demonstrated that ADAMTS4 protein was highly expressed in tumor samples of the patients with EWS by using immunohistochemistry. These results suggest that ADAMTS4 is a novel tumor marker for EWS.

Mixed-lineage-leukemia (MLL) fusion protein collaborates with Ras to induce acute leukemia through aberrant Hox expression and Raf activation.

Mixed-lineage-leukemia (MLL) fusion oncogenes are closely involved in infant acute leukemia, which is frequently accompanied by mutations or overexpression of FMS-like receptor tyrosine kinase 3 (FLT3). Earlier studies have shown that MLL fusion proteins induced acute leukemia together with another mutation, such as an FLT3 mutant, in mouse models. However, little has hitherto been elucidated regarding the molecular mechanism of the cooperativity in leukemogenesis. Using murine model systems of the MLL-fusion-mediated leukemogenesis leading to oncogenic transformation in vitro and acute leukemia in vivo, this study characterized the molecular network in the cooperative leukemogenesis. This research revealed that MLL fusion proteins cooperated with activation of Ras in vivo, which was substitutable for Raf in vitro, synergistically, but not with activation of signal transducer and activator of transcription 5 (STAT5), to induce acute leukemia in vivo as well as oncogenic transformation in vitro. Furthermore, Hoxa9, one of the MLL-targeted critical molecules, and activation of Ras in vivo, which was replaceable with Raf in vitro, were identified as fundamental components sufficient for mimicking MLL-fusion-mediated leukemogenesis. These findings suggest that the molecular crosstalk between aberrant expression of Hox molecule(s) and activated Raf may have a key role in the MLL-fusion-mediated-leukemogenesis, and may thus help develop the novel molecularly targeted therapy against MLL-related leukemia.

C/EBPalpha and C/EBPvarepsilon induce the monocytic differentiation of myelomonocytic cells with the MLL-chimeric fusion gene.

CCAAT/enhancer binding proteins (C/EBPs) have an important function in granulocytic differentiation, and are also involved in the leukemogenesis of acute myeloid leukemia (AML). Their involvement in myelomonocytic leukemia, however, is still unclear. Therefore, the expression and function of C/EBPs in myelomonocytic cells with MLL-fusion genes were investigated. Retinoic acid (RA) induced monocytic differentiation in the myelomonocytic cell lines with MLL-fusion genes, THP-1, MOLM-14 and HF-6 cells, accompanied by monocytic differentiation with the upregulation of C/EBPalpha and C/EBPepsilon. Monocytic differentiation by RA treatment was confirmed in primary AML cells using a clonogenic assay. When the activity of C/EBPalpha or C/EBPepsilon was introduced into HF-6 cells, their cellular growth was arrested through differentiation into monocytes with the concomitant marked downregulation of Myc. Cebpe mRNA was upregulated by the induction of C/EBPalpha-ER, but not vice versa, thus suggesting that C/EBPepsilon may have an important function in the differentiation process. Introduction of Myc isoforms into HF-6 cells partially antagonized the C/EBPs effects. These findings suggest that the ectopic expression of C/EBPepsilon, as well as C/EBPalpha, can induce the monocytic differentiation of myelomonocytic leukemic cells with MLL-fusion gene through the downregulation of Myc, thus providing insight into the development of novel therapeutic approaches.

Voluntary running exercise attenuates the progression of endothelial dysfunction and arterial calcification in ovariectomized rats.

AIM: Loss of oestrogen synthesis capacity after menopause contributes to increases in arterial stiffness and calcification. Exercise training improves arterial stiffness and calcification. However, the mechanism of exercise training-induced improvement of arterial stiffness and calcification remains unclear. METHOD: We examined the mechanism by using aortas of sham-operated rats (sham control; SC), ovariectomized rats (OVX control; OC), OVX plus treatment with vitamin D(3) plus nicotine (VDN) rats (OV sedentary; OVSe), which is an animal model of endothelial dysfunction and arterial calcification, and voluntary running wheel exercise for 8 weeks plus OVX plus VDN rats (OV exercise; OVEx). RESULTS: The arterial tissue calcium and endothelin-1 (ET-1: a vasoconstrictor peptide and a potent regulator of arterial calcification) levels were significantly higher in OVSe rats compared with the SC and OC rats, whereas these levels in the OVEx rats were significantly lower than in the OVSe rats. Additionally, arterial expression of endothelial nitric oxide synthase (eNOS), which is an enzyme that produces nitric oxide (NO: a vasodilator substance), was reduced in OVSe rats. However, exercise training prevented the decrease in eNOS expression. Moreover, there was a significant positive correlation between arterial calcium level and arterial ET-1 level. CONCLUSION: These findings suggest that exercise training-induced improvement of ET-1 and NO prevents the impairment of endothelial function after menopause in females, and this improvement may result in less arterial calcification.

Identification of TSC-22 as a potential tumor suppressor that is upregulated by Flt3-D835V but not Flt3-ITD.

Transforming growth factor-beta (TGF-beta)-stimulated clone-22 (TSC-22) was originally isolated as a TGF-beta-inducible gene. In this study, we identified TSC-22 as a potential leukemia suppressor. Two types of FMS-like tyrosine kinase-3 (Flt3) mutations are frequently found in acute myeloid leukemia: Flt3-ITD harboring an internal tandem duplication in the juxtamembrane domain associated with poor prognosis and Flt3-TKD harboring a point mutation in the kinase domain. Comparison of gene expression profiles between Flt3-ITD- and Flt3-TKD-transduced Ba/F3 cells revealed that constitutive activation of Flt3 by Flt3-TKD, but not Flt3-ITD, upregulated the expression of TSC-22. Importantly, treatment with an Flt3 inhibitor PKC412 or an Flt3 small interfering RNA decreased the expression level of TSC-22 in Flt3-TKD-transduced cells. Forced expression of TSC-22 suppressed the growth and accelerated the differentiation of several leukemia cell lines into monocytes, in particular, in combination with differentiation-inducing reagents. On the other hand, a dominant-negative form of TSC-22 accelerated the growth of Flt3-TKD-transduced 32Dcl.3 cells. Collectively, these results suggest that TSC-22 is a possible target of leukemia therapy.

STAT5 induces macrophage differentiation of M1 leukemia cells through activation of IL-6 production mediated by NF-kappaB p65.

We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines

Molecular cloning of a human novel type I cytokine receptor related to delta1/TSLPR.

In a search for a human sequence related to a recently identified type I cytokine receptor delta1, which turned out to be a receptor subunit for a cytokine called TSLP, we have now identified a novel human type I cytokine receptor from a human T lymphocyte cDNA library. The deduced amino acid sequence of 371 residues has a typical signal sequence and a membrane-spanning region. The mature protein is predicted to have a molecular mass of 39,698 Da. The N-terminal extracellular region contains two fibronectin type III-like domains, four conserved cysteine residues, and a WSXWS box-like motif. The C-terminal intracellular region contains box 1 and box 2-like motifs. Thus, it has common characteristics of type I cytokine receptor family members, and we tentatively termed this protein CRLF2, which stands for cytokine receptor-like factor 2. Northern blot analysis revealed CRLF2 mRNA in liver, kidney, heart, and skeletal muscle. The fetal liver also expresses CRLF2 transcripts. The gene for CRLF2 was mapped to the pseudoautosomal region, Xp22.3 and Yp11.3 by FISH analysis, a region where genes encoding the IL-3 receptor alpha and the GM-CSF receptor alpha chains are also located. The biological function of this newly identified receptor is now under investigation.

A novel secreted form of immune suppressor factor with high homology to vacuolar ATPases identified by a forward genetic approach of functional screening based on cell proliferation.

In the search for stromal-derived growth factors, we have identified a novel secreted short form of immune suppressor factor (ISF) using a combination of a genetic approach and retrovirus-mediated functional screening. This protein, which we termed ShIF, was isolated based on its ability to support proliferation of a mutant clone S21, which was established from Ba/F3 cells that are usually interleukin-3-dependent but became dependent on a stroma cell line ST2 after chemical mutagenesis. ISF, a membrane protein harboring six transmembrane domains, was reported to have immunosuppressive functions. The coding region of ShIF started from the third transmembrane domain of ISF. Biochemical analysis demonstrated that ShIF was expressed in both the secreted and membrane-bound forms of 27-kDa protein, which was supposed to have an internal ATG present in the third transmembrane domain of ISF as a start codon. In addition to the full-length form of ISF, a major protein with a molecular size of 27 kDa was also expressed through the proteolytic process of ISF. ShIF resembles this naturally occurring short form of ISF (sISF). Deletion analysis of the major domains of ISF cDNA revealed that ShIF is an active functional domain of ISF with a capability to support proliferation of S21 cells. Enforced expression of ShIF in MS10 cells, bone marrow stroma cells that do not express endogenous ShIF or ISF, conferred on the cells an ability to support the growth of S21 cells as well as bone marrow cells. Interestingly, ShIF shows a high sequence homology to the C-terminal part of a 95-kDa yeast vacuolar H (+)-ATPase subunit, Vph1p (39%), and a 116-kDa proton pump (VPP1) (54%) of the rat and bovine synaptic vesicle. Therefore, it is possible that ShIF also acts as a proton pump and somehow prevents the cells from undergoing apoptosis.

MgcRacGAP is involved in cytokinesis through associating with mitotic spindle and midbody.

We have recently cloned a cDNA for a full-length form of MgcRacGAP. Here we show using anti-MgcRacGAP antibodies that, unlike other known GAPs for Rho family, MgcRacGAP localized to the nucleus in interphase, accumulated to the mitotic spindle in metaphase, and was condensed in the midbody during cytokinesis. Overexpression of an N-terminal deletion mutant resulted in the production of multinucleated cells in HeLa cells. This mutant lost the ability to localize in the mitotic spindle and midbody. MgcRacGAP was also found to bind alpha-, beta-, and gamma-tubulins through its N-terminal myosin-like domain. These results indicate that MgcRacGAP dynamically moves during cell cycle progression probably through binding to tubulins and plays critical roles in cytokinesis. Furthermore, using a GAP-inactive mutant, we have shown that the GAP activity of MgcRacGAP is required for cytokinesis, suggesting that inactivation of the Rho family of GTPases may be required for normal progression of cytokinesis.

Role of MgcRacGAP/Cyk4 as a regulator of the small GTPase Rho family in cytokinesis and cell differentiation.

To identify the key molecules that regulate differentiation of hematopoietic cells, we carried out retrovirus-mediated functional screening for cDNAs whose expression suppresses IL-6-induced differentiation of mouse myeloid leukemic M1 cells. From this screening, we obtained a full length cDNA encoding a mouse homologue of human MgcRacGAP. Overexpression of the anti-sense MgcRacGAP profoundly inhibited IL-6-induced macrophage-differentiation of M1 cells. On the other hand, overexpression of the full-length form of MgcRacGAP alone enhanced macrophage differentiation of M1 cells in response to IL-6, and induced macrophage differentiation of HL-60 leukemic cells. To determine how this protein regulates differentiation and proliferation, an antibody against MgcRacGAP was prepared. Immunohistochemical studies revealed that MgcRacGAP mainly localizes in the nucleus in interphase, accumulates on the mitotic spindle in metaphase, and is condensed in the midbody during cytokinesis. Overexpression of an N-terminal domain deletion mutant, which lacks the ability to localize to the midbody through association with tubulins, or a GAP-inactive mutant resulted in the formation of multinucleated cells in HeLa cells as well as in hemopoietic cells. Interestingly, MgcRacGAP in the midbody was phosphorylated probably on serine and threonine residues. These results indicate that MgcRacGAP regulates cytokinesis and cellular differentiation as a regulator of Rho family of GTPase and suggest that this process is controlled by some serine/threonine kinases.

A novel low-density lipoprotein receptor-related protein mediating cellular uptake of apolipoprotein E-enriched beta-VLDL in vitro.

We report here the identification of a novel member of the low-density lipoprotein receptor (the LDL receptor) family through signal sequence trap screening of a mouse lymphocyte cDNA library. The protein was termed LDL receptor-related protein 9 (LRP9). LRP9 is a type I membrane protein predicted to contain 696 amino acids with a calculated molecular mass of 74 764 Da. The NH(2)-terminal half of LRP9 contains two CUB domains separated by a single ligand-binding repeat. The second CUB domain is followed by a cluster of three additional ligand-binding repeats and a transmembrane domain. The COOH-terminal intracellular region contains a proline-rich region. LRP9 mRNA was expressed in the liver, kidney, lung, and heart at high levels, and in the spleen and brain at low levels. In situ hybridization analysis of mouse liver, kidney, and brain detected LRP9 transcripts in hepatocytes, sinusoidal lining cells, peritubular capillaries, choroid plexus, ependyma of the third ventricle, pia matter, and hippocampus. In particular, high levels of expression were observed in the vascular walls. Apolipoprotein E (apoE)-enriched beta-VLDL stimulated cellular cholesteryl ester formation in ldl-A7/LRP9. These results raise the possibility that this newly identified receptor, which is expressed in the liver, may play a physiological role in the uptake of apoE-containing lipoproteins.

Accuracy and repeatability of blood volume measurement by pulse dye densitometry compared to the conventional method using 51Cr-labeled red blood cells.

OBJECTIVE: To determine the accuracy and repeatability of pulse dye densitometry (PDD) in measuring blood volume (BV) by comparing it with the conventional method using 51Cr-labeled red blood cells (RI method) and by assessing sequential measurements. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENTS AND PARTICIPANTS: Eleven adult ICU patients who received cardiac surgery (1st ICU day). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: After injecting indocyanine green (10 or 20 mg) into the right atrium, its arterial concentration was continuously monitored at the nose and finger by PDD, and BV was calculated by back extrapolating the logarithmic dye concentration on the dye elimination curve between 2.5 and 5.5 min after mean transit time to each mean transit time with the least squares method. These measurements were repeated in eight patients and performed only once in the other three, and the BV was measured concurrently by the RI method one time. The Bland-Altman method was used for evaluating differences between methods and within methods. The (percentage) biases and standard deviations between the PDD and RI methods and between the successive measurements by PDD at the finger and nose were 0.26 +/- 0.491 (8.8 +/- 15.3%) and 0.004 +/- 0.251 (0.06 +/- 5.9%) with the probe on a nostril, and 0.16 +/- 0.561 (2.5 +/- 14.4%) and 0.19 +/- 0.311 (4.7 +/- 7.3%) using the finger probe. The bias between methods was less than 10%, and the repeatability of PDD was better. CONCLUSIONS: As PDD can measure BV with good repeatability and with a small bias compared to the RI method, serial changes in BV can be evaluated at the bedside of critically ill patients noninvasively and repeatedly.

MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells.

In a search for key molecules that prevent murine M1 leukemia cells from undergoing interleukin (IL)-6-induced differentiation into macrophages, we isolated an antisense complementary DNA (cDNA) that encodes full-length mouse MgcRac-GTPase-activating protein (GAP) through functional cloning. Forced expression of this antisense cDNA profoundly inhibited IL-6-induced differentiation of M1 cells into macrophage lineages. We also isolated a full-length human MgcRacGAP cDNA, which encodes an additional N-terminal polypeptide of 105 amino acid residues compared with the previously published human MgcRacGAP. In human HL-60 leukemic cells, overexpression of the full-length form of human MgcRacGAP alone induced growth suppression and macrophage differentiation associated with hypervacuolization and de novo expression of the myelomonocytic marker CD14. Analyses using a GAP-inactive mutant and 2 deletion mutants of MgcRacGAP indicated that the GAP activity was dispensable, but the myosin-like domain and the cysteine-rich domain were indispensable for growth suppression and macrophage differentiation. The present results indicated that MgcRacGAP plays key roles in controlling growth and differentiation of hematopoietic cells through mechanisms other than regulating Rac GTPase activity.

Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) in hematopoietic cells.

Hematopoietic cell growth and differentiation are controlled by a number of cytokines. Ligand stimulation induces rapid phosphorylation of the tyrosine residues of the cytokine receptor and a variety of cellular molecules. Among them, Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have recently been found to play a unique role in cytokine receptor-mediated intracellular signaling and hematopoietic cell development. Abnormal signaling of the JAK-STAT pathway results in hematopoietic disorders, including severe combined immunodeficiency and leukemia.

Molecular cloning and characterization of a mouse homolog of human TNFSF14, a member of the TNF superfamily.

A member of the tumor necrosis factor (TNF) superfamily, human TNFSF14 (hTNFSF14)/HVEM-L (herpes virus entry mediator ligand) was isolated as a cellular ligand for HVEM/TR2 and human lymphotoxin beta receptor (LTbetaR). TNFSF14 induces apoptosis and suppresses tumor formation. We have isolated a cDNA clone for a mouse homologue of hTNFSF14 by signal sequence trap (SST) screening which we recently developed. The deduced amino acid sequence of the mouse TNFSF14 (mTNFSF14) cDNA comprised 239 amino acid residues and was 77% identical to the hTNFSF14 protein. In Northern blot analysis, 2.1 kb and 4.2kb mTNFSF14 transcripts were detected in spleen and lung, and in heart, respectively. Fluorescence in situ hybridization analysis localized the mTNFSF14 gene Tnfsf14 to chromosome 17 which is tightly linked with Tnf, Lta, and Ltb.

Functional reconstitution of class II MHC-restricted T cell immunity mediated by retroviral transfer of the alpha beta TCR complex.

Transfer of the alphabeta TCR genes into T lymphocytes will provide a means to enhance Ag-specific immunity by increasing the frequency of tumor- or pathogen-specific T lymphocytes. We generated an efficient alphabeta TCR gene transfer system using two independent monocistronic retrovirus vectors harboring either of the class II MHC-restricted alpha or beta TCR genes specific for chicken OVA. The system enabled us to express the clonotypic TCR in 44% of the CD4+ T cells. The transduced cells showed a remarkable response to OVA323-339 peptide in the in vitro culture system, and the response to the Ag was comparable with those of the T lymphocytes derived from transgenic mice harboring OVA-specific TCR. Adoptive transfer of the TCR-transduced cells in mice induced the Ag-specific delayed-type hypersensitivity in response to OVA323-339 challenge. These results indicate that alphabeta TCR gene transfer into peripheral T lymphocytes can reconstitute Ag-specific immunity. We here propose that this method provides a basis for a new approach to manipulation of immune reactions and immunotherapy.

Constitutive activation of STAT5 by a point mutation in the SH2 domain.

We previously identified a constitutively active form of STAT (signal transducer and activator of transcription) 5A by polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening, which had two point mutations in the DNA-binding and transcriptional activation domains, and was designated STAT5A1*6. STAT5A1*6 showed markedly elevated DNA binding and transactivation activities with stable tyrosine phosphorylation and nuclear accumulation, and conferred autonomous cell growth on interleukin 3-dependent Ba/F3 cells. We now report another constitutively active mutant, STAT5A-N642H which has a single point mutation (N642H) in its SH2 domain, identified using the same strategy as that used to identify STAT5A1*6. STAT5A-N642H showed identical properties to those of STAT5A1*6 both biochemically and biologically. Interestingly the mutation in STAT5A-N642H resulted in restoration of the conserved critical histidine which is involved in the binding of phosphotyrosine in the majority of SH2-containing proteins. Introduction of an additional mutation (Y694F) to STAT5A-N642H, which disrupted critical tyrosine 694 required for dimerization of STAT5, abolished all the activities manifested by the mutant STAT5A-N642H, which indicates that dimerization is required for the activity of STAT5A-N642H as was the case for the wild-type STAT5A. The present findings also show that different mutations rendered STAT5A constitutively active, through a common mechanism, which is similar to that of physiological activation.