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Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.

Excessive nitric oxide (NO) production has been implicated to be responsible for the development of septic shock. To determine whether plasma nitrite/nitrate (NOx) levels are related to the severity of systemic inflammatory response syndrome (SIRS) and the degree of multiple organ dysfunction, we studied plasma NOx levels in 70 patients with SIRS consisting of noninfectious SIRS (n = 32), sepsis (n = 23), and septic shock (n = 15). Infection is a microbial phenomenon characterized by an inflammatory response to the presence of microorganism. Positive culture for microorganism is regarded as infectious SIRS (sepsis and septic shock) and negative culture is regarded as noninfectious SIRS. Plasma samples collected from each patient within 24 h from admission to the intensive care unit were subjected for measurement of NOx levels, the stable end products of NO, by the high performance liquid chromatography-Greiss system. Mean plasma NOx levels in patients with SIRS were 52.8 +/- 44 microM/L, ranging from 8.1 to 186.2 microM/L. Plasma NOx levels were positively correlated with Acute Physiology, Age, and Chronic Health Evaluation (APACHE) III score (r = 0.414, P < 0.01) and sequential organ failure assessment (SOFA) score (r = 0.433, P < 0.01). Plasma NOx levels in patients with sepsis (51.0 +/- 38.5 microM/L) and septic shock (94.5 +/- 53.7 microM/L) were significantly (P < 0.01) higher than those in patients with noninfectious SIRS (25.8 +/- 16.9 microM/L) and healthy subjects (29.6 +/- 8.9 microM/L). Our study shows that plasma NOx levels are increased in patients with infectious, but not noninfectious SIRS, which increase as the severity of SIRS and the development of multiple organ dysfunction syndrome, suggesting its possible pathogenic role in SIRS.

Recipients for liver transplantation often have portosystemic shunts due to portal hypertension. It is an important problem whether such shunts should be ligated during operations. Ligating the shunts seems of benefit for increasing portal blood flow to the liver, but it is sometimes difficult technically, and it is invasive to the patient. We experienced a recipient with huge portosystemic shunts and no esophageal varices before living-related liver transplantation. Some shunts were ligated during operation to increase portal blood flow to the graft. Unfortunately, the patient suffered severe bleeding from esophagogastric varices after he underwent retransplantation owing to accidental liver failure. Based on our experience, extreme care should be exercised to avoid varicose bleeding after ligating the portosystemic shunts of liver transplantation patients.

A 48-year-old male patient was admitted to our institution with left hypochondralgia. Preoperative serum CEA was 504 ng/ml. Under a radiological diagnosis of simultaneous double cancer of the pancreas and lung, resection was performed. Pathological study revealed primary pancreatic cancer with lung metastasis. After the operation, serum CEA fell to 55 ng/ml. Seventeen months later, bowel obstruction occurred and serum CEA was 140 ng/ml. One course of systemic chemotherapy, using the combination of CDDP, 5-FU and Leucovorin, was successful. After another three months, however, the same clinical symptoms occurred. With no response to the prior chemotherapy regimen, surgery was undertaken and a mass at the mesentery was removed. Fifty-nine months have passed since the initial operation and the patient is free of disease, though his serum CEA is around 50 ng/ml. In conclusion, there are cases of pancreatic cancer in which tumor dormancy can be achieved by postoperative 5-FU based chemotherapy.

Objectives: To test the hypotheses (1) that nitric oxide (NO) production is stimulated after cardiovascular surgery and is related to the hyperdynamic state and (2) that NO production is more prominent in patients with cardiopulmonary bypass., Design: Prospective, clinical study., Setting: Intensive care unit in a university hospital., Patients: One hundred patients after cardiovascular surgery: coronary artery bypass graft with (n=53) and without (n=17) cardiopulmonary bypass, valve surgery with cardiopulmonary bypass (n=23) and thoracic aortic replacement with cardiopulmonary bypass (n=7)., Interventions: None., Measurements and Results: Urinary nitrite/nitrate (NOx) excretion was measured by the high-performance liquid chromatography-Griess system as an index of endogenous NO production during the first 2 postoperative days. Hemodynamic variables, hematologic variables and serum C-reactive protein concentrations were measured after the operation. Urinary NOx concentrations were 146+/-70 and 190+/-93 micro mol/l, and the amounts of NOx excreted in the urine were 23+/-10 and 18+/-8 micro mol/h on the 1st and 2nd days, respectively. Urinary NOx excretions were positively correlated with the cardiac index (P<0.01), but inversely correlated with the systemic vascular resistance index (P<0.01). Urinary NOx concentrations were positively correlated with serum C-reactive protein concentrations (P<0.01), but inversely correlated with the cardiopulmonary bypass time (P<0.01). The urinary NOx concentration was highest in patients undergoing coronary artery bypass graft without cardiopulmonary bypass., Conclusion: These findings suggest, firstly, that NO production is stimulated by a surgical inflammatory response and, secondly, that the endogenous NO contributes to the increase in cardiac output that accompanies the reduced systemic vascular resistance after cardiovascular surgery.

Objective: BCR-ABL is a unique oncoprotein of which sole expression can cause cancer. A number of signaling molecules were shown to be activated by BCR-ABL. One of the important molecules that contributes to BCR-ABL-mediated cell proliferation is signal transducer and activator of transcription (STAT) 5. To elucidate the mechanism of BCR-ABL-mediated leukemogenesis, a role of pim-1, one of the important target genes of STAT5, was investigated., Materials and Methods: A temperature-sensitive mutant of p210(BCR-ABL) was introduced in interleukin-3-dependent murine hematopoietic cell line Ba/F3 cells, and downstream signaling after activation of BCR-ABL was investigated. Effects of the expression of a dominant-negative (dn) Pim-1 and a dn STAT5A in BCR-ABL-driven cell proliferation also were studied in Ba/F3 cells., Results: We found that pim-1 was markedly up-regulated following activation of BCR-ABL tyrosine kinase with activation of STAT5. Overexpression of pim-1 alone induced cytokine-independent cell growth of Ba/F3 cells in a dose-dependent manner. However, expression of the dn Pim-1 did not affect growth of Ba/F3 cells transformed by BCR-ABL, whereas that of the dn STAT5A did suppress it., Conclusion: Pim-1 is one of the redundant molecules that contributes to induction of autonomous cell growth and is dispensable for leukemogenesis by BCR-ABL.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.

Objective: To verify the hypothesis that the gastric intraluminal PCO2 (PgCO2) changes independently of the change in cardiac output (CO) during and after cardiovascular surgery using cardiopulmonary bypass (CPB), and that the elevation of PgCO2 affects the patients' morbidity., Design: Prospective, noninterventional study., Setting: Medical/surgical intensive care unit and operating theater of a university hospital., Patients: Sixteen adults patients receiving elective cardiovascular surgery using CPB., Interventions: None., Measurements and Main Results: After induction of anesthesia, the patients were fitted with a gastric tube equipped at the tip with a CO2 sensor (ion-selective field effect transistor) that can continuously measure real-time PgCO2, and a pulmonary artery catheter capable of monitoring continuous CO (CCO) and end-tidal CO2. Data from the devices was uploaded to a personal computer every 2 mins until the catheter was pulled off based on clinical judgment (PgCO2 values were blinded to everyone except the investigator). One patient expired as a result of multiple organ failure subsequent to sepsis, and postoperative morbidity assessed by the peak SOFA (sequential organ failure assessment) score (mean +/- SD 6.9 +/- 3.5; range, 2-13) was correlated with the peak PgCO2 during intensive care unit stay (mean +/- SD 74.1 +/- 30.7 mm Hg; range, 45-169 mm Hg) (p < .01, by regression analysis). The peak PgCO2 during surgery (mean +/- SD 71.1 +/- 18.1 mm Hg; range, 44-115 mm Hg) had no correlation with the postoperative morbidity. From analysis of CCO before, during, and after returning from the above 60 mm Hg of PgCO2, PgCO2 changed independently of CCO., Conclusions: PgCO2 changed independently of CCO, and its postoperative elevation was related to morbidity, even in the group of patients with a good outcome. Continuous monitoring of PgCO2 is useful for the detection of morbidity and can be expected to help elucidate the pathophysiology of change of PgCO2.

2-(2'-hydroxy-5'-methylphenyl)-benzotriazole (HMB) is nominated as the restricted ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for HMB was investigated by HPLC. After dissolving HMB in cream or oil with tetrahydrofuran and making the constant volume with methanol, the test solution was prepared. Twenty micro-liter of the test solution was determined by HPLC by using CAPCELL PAK C18 column, the mixture of methanol and water (17:3) as mobile phase, the detection wavelength at 306 nm. The concentrations of 10 to 80 micrograms/ml HMB gave the linear relationship between the concentrations and the peak areas. In creams and oil 0.1% and 1% HMB were sufficiently determined by the established method.

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.

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

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.

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.

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., (Copyright 2001 S. Karger AG, Basel)

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.

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.

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.

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.

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.

In a complementary DNA (cDNA) screening of murine Th2-skewed lymphocytes with our recently developed signal sequence trap method termed SST-REX, a novel type 1 cytokine receptor, Delta1 (delta1), was identified. Although delta1 is ubiquitously expressed in multiple tissues, the expression level is higher in Th2-skewed lymphocytes than in Th1-skewed ones. The delta1 cDNA encodes a 359-amino acid type 1 membrane protein. The extracellular domain of 206 amino acids showed 24% identity with the murine common gamma receptor that is shared among the receptors for interleukin(IL)-2, IL-4, IL-7, IL-9, and IL-15. The membrane-proximal region of delta1 includes a box1 motif, which is important for association with Janus kinases (JAKs), and showed a significant homology with that of the mouse erythropoietin receptor (EPOR). A box2 motif was also found in close proximity to the box1 region. Dimerization of the cytoplasmic region of delta1 alone did not transduce proliferative signals in IL-3-dependent cell lines. However, the membrane-proximal region of delta1 could substitute for that of human EPOR in transmitting proliferative signals and activating JAK2. These results suggest that delta1 is a subunit of cytokine receptor that may be involved in multiple receptor systems and play a regulatory role in the immune system and hematopoiesis.

We previously established a high-efficiency, retrovirus-mediated expression cloning method. Using this system, we now have developed an expression cloning method (FL-REX; fluorescence localization-based retrovirus-mediated expression cloning) in which cDNAs can be isolated based on the subcellular localization of their protein products. Complementary DNAs generated from mRNA using random hexamers were fused to the cDNA of green fluorescent protein (GFP) in the pMX retrovirus vector. The resulting cDNA-GFP fusion library was transfected into retrovirus-packaging cells, and the derived retroviruses were used to infect NIH 3T3 cells. Infected cells then were screened to identify cDNAs of interest through the subcellular localization of the GFP-fusion products. Using FL-REX, we have identified 25 cDNAs, most of which showed reasonable subcellular localization as GFP-fusion proteins, indicating that FL-REX is useful for identification of proteins that show specific intracellular localization.

We recently identified several constitutively active forms of signal transducers and activators of transcription 5 (STAT5) using polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening. All constitutively active STAT5 showed constitutive phosphorylation on their tyrosine residues and induced factor-independent growth in a mouse interleukin-3-dependent cell line, Ba/F3. Sequence analysis of these active STAT5 revealed two important mutations: S710F and N642H. The N642H mutation localized in the SH2 domain was able to induce autonomous growth of Ba/F3 cells by itself, whereas S710F in the effector domain was able to induce autonomous growth of Ba/F3 cells in concert with a second mutation including H298R and E150G. Recently, constitutive activation of STAT5 has been reported in patients' leukemic cells and is implicated in leukemogenesis. We attempted to clarify whether leukemic cells harbored activating mutations primarily in STAT5 proteins, and analyzed the sequence of STAT5 derived from 49 leukemic patients. No mutations were found, however, in the regions surrounding S710 and N642 of STAT5A and corresponding residues of STAT5B. We also cloned full-length cDNAs for STAT5s from three patients whose leukemic cells exhibited constitutive tyrosine phosphorylation of the STAT5 protein and expressed the derived STAT5 proteins in Ba/F3 cells. However, none of these clones exhibited constitutive tyrosine phosphorylation or gave rise to FI proliferation of Ba/F3 cells. These results indicate that constitutive activation of STAT5 is a secondary event in most leukemias.

Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through gamma(c)-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7-responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3(-/-) mice. Newborn Jak3(-/-) mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4(+), CD8(+), and B220(+)/IgM(+) splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3-treated Jak3(-/-) mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3-treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7Ralpha(+)/IL-3Ralpha(+) cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R(+) lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-beta1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.

We recently experienced a high frequency of biliary complications after orthotopic liver transplantation in rats (22 of 25 cases (88%): biloma, 20 cases; biliary peritonitis, 2 cases). These complications seemed to be rare in general, but some researchers reported such cases and addressed them mainly through rearterialization. The biliary complications we encountered were found to be associated with necrosis of the donor bile duct and an opportunistic infection of Enterobacteriaceae. After administering appropriate antibiotics, the complications significantly diminished (2 of 25 cases (8%), P = 0.0001). The nonarterialized bile duct, which becomes ischemic soon after liver transplantation, appears to be susceptible to infections. Such opportunistic infections may prevent the development of arterial collaterals, causing bile duct necrosis and the subsequent leakage of bile juice. When biliary complications frequently occur after nonarterialized liver transplantation in rats, the possibility of an opportunistic infection should thus be considered.

STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.

Cytokines manifest their function through regulation of gene expression. We searched for immediate-early cytokine responsive genes by the mRNA differential display technique using interleukin-3 (IL-3)-dependent OTT-1 cells, and have isolated a novel cDNA which encodes 210 amino acids and shows 87% amino acid identity to human SNAP-23 (synaptosomal-associated protein of 23 kD). The message for this protein (mouse SNAP-23) was induced in OTT-1 cells by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5. The experiment using C-terminal deletion mutants of the common beta subunit (betac) of IL-3/GM-CSF/IL-5 receptors showed that expression of SNAP-23 was associated with the Ras-Raf-MAPK pathway, but not with the JAK-STAT pathway. Moreover, SNAP-23 was induced in response to a wide variety of cytokines, including IL-2, IL-3, IL-5, IL-10, stem cell factor, G-CSF, GM-CSF, leukemia inhibitory factor, and erythropoietin. Constitutive expression of SNAP-23 was seen in various tissues, including heart, lung, kidney, liver, spleen, and small intestine. Possible involvement of SNAP-23 in cytokine signal transduction is discussed.

The tyrosine kinase Jak3 plays a key role in transducing signals from the IL-2, -4, -7, -9, and -15 receptors. Mice lacking Jak3 exhibit a profound, early block in both B and T cell development. To examine the mechanisms whereby Jak3 influences T cell function, we have reconstituted thymic development in Jak3-/- animals by introducing a Jak3 transgene in which expression was driven by the lck proximal promoter. Thymic reconstitution required Jak3 kinase activity, as catalytically inactive Jak3 did not restore early thymic development. Furthermore, the thymus-restricted expression pattern of the transgene allowed us to assess the requirement for Jak3 in peripheral T cells. In these mice, loss of Jak3 expression was associated with a failure to proliferate in response to antigen receptor crosslinking, the accumulation of T cells manifesting an activated cell surface phenotype, and an increased CD4/CD8 ratio among peripheral T cells, all of which are characteristics that were observed in Jak3-/- animals. Finally, we present data which suggest that peripheral T cells proliferate more rapidly in vivo and also undergo apoptosis more rapidly, upon loss of Jak3. Hence Jak3 exerts effects on mature peripheral T lymphocytes, as well as on thymocytes, resulting in the proper maintenance of circulating, quiescent cells.

The characteristic features of cytokines are functional pleiotropy and redundancy. Each cytokine is produced by a variety of cell types and acts on a wide range of target cells and tissues. Many cytokines have overlapping biological activities in the same cells. It was originally thought that each cytokine has a specific receptor and a unique signal transduction system. However, extensive studies on cytokines and their receptors revealed that many cytokines share receptor subunits and signal transduction system, and that biological functions of a single cytokine can vary depending on the status of the cells. Therefore, it is important to know the structure and function of cytokine receptors to understand the pleiotropy and redundancy as well as specificity of cytokines. Among signal transduction pathways, recently identified Jak/STAT pathway, which connects activation of the receptor complexes and transcription of various genes directly, would give us further insights in the mechanisms of cytokine action.

The transcription of human T-cell leukemia virus type I (HTLV-I) provirus starts from a promoter located in the 5' long terminal repeat (LTR). We have identified a second promoter at the 3' end of the pol gene. This internal promoter expresses the Tax transactivator protein, but does not require Tax for its activity. Furthermore, we have found the novel enhancer motif AGTTCTGCCC, which are located near the initiation site. We have named the sequence HIRE (HTLV-I internal regulatory element). The HIRE binding protein is a ubiquitous protein. We purified this protein from the HTLV-I producing cell line MT-2 cells by DNA affinity chromatography. SDS-PAGE analysis revealed four major bands (70, 85, 115 and more than 200 kDa) and some minor bands on the gel. We renatured each major protein and showed the 70 and 115 kDa proteins bind to DNA, although the 115 kDa protein seemed to bind nonspecifically. We have designated these components as HIRF (HTLV-I internal regulatory factor).

Hematopoiesis is regulated through the binding of cytokines to receptors of the cytokine receptor superfamily. Although lacking catalytic domains, members of the cytokine receptor superfamily mediate ligand-dependent activation of protein tyrosine phosphorylation through their association and activation of members of the Janus kinase (Jak) family of protein tyrosine kinases. The activated Jaks phosphorylate the receptors which creates docking sites for SH2-containing signaling proteins which are tyrosine phosphorylated following their association with the complex. Among the substrates of tyrosine phosphorylation are members of the signal transducers and activators of the transcription family of proteins (Stats). Various cytokines induce the tyrosine phosphorylation and activation of one or more of the seven family members. The pattern of Stat activation provides a level of cytokine individuality that is not observed in the activation of other signaling pathways. The role of various Stats in the biological responses to cytokines has been assessed through the analysis of receptor mutations which disrupt Stat activation and more recently by disruption of the genes in mice. Our results have demonstrated that the activation of Stat5a and Stat5b by erythropoietin is critical for the activation of a number of immediate early genes but is not required for a mitogenic response. Mice in which the genes for Stat4 and Stat6 are disrupted are viable but lack functions that are mediated by interleukin 12 (IL-12) or IL-4, respectively, suggesting that these Stats perform very specific functions in immune responses.

The chemical shifts of 31P and 1H in thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) (TmDOTP5-) are approximately two orders of magnitude more sensitive to temperature than are water proton and 19F shifts. In the physiologically relevant pH range, the 31P and 1H chemical shifts of TmDOTP5- are linear functions of temperature between 25 and 47 degrees C. The results indicate that using TmDOTP5- can provide measurements of temperature in vivo that are significantly more accurate than methods based on water and fluorocarbon chemical shifts.

Between 1980 and 1995, 10 patients underwent thoracotomy for pulmonary aspergillosis. In six patients, hemoptysis and bloody sputum were the chief complaints. The other complaints were nonspecific. Six patients had a history of pulmonary tuberculosis, and two of those patients underwent upper lobectomy. Aspergillosis had developed in the residual space. A fungus ball was observed on the preoperative chest X-ray and CT scan films in seven patients. Lobectomy was done in three patients, segmentectomy in two, and partial pulmonary resection in four. The patients with lesions that had grown in the residual space underwent curettage with muscle plombage. Three patients underwent thoracoplasty. An additional operation was done in two patients because of poor residual lung expansion. No patient had recurrence. We conclude that surgical treatment should be based on symptoms and on pathological findings.

Traumatic diaphragmatic hernia requires urgent surgical treatment. A 51-year-old female was injured in a speeding car. She had dyspnea, and resistance on the abdominal wall on physical examination. Intra-thoracic and abdominal visceral injury was suspected. A chest roentogenogram and CT scan revealed an obvious diaphragmatic herniation on the left which necessitated emergency operation. On the right semi-lateral position, left thoracoabdominal spiral incision was made through the 7th intercostal space. Good exposure of the chest and abdomen was easily obtained. Herniated organs and the abdominal cavity were thoroughly examined with ease. The diaphragm was repaired directly. She had an uneventful postoperative recovery, and was discharged in 17 days. Thoracoabdominal spiral incision offered an excellent operative exposure for the patient with a possible combined thoracic and abdominal visceral injury.

Signal transducers and activators of transcription (Stats) are activated by tyrosine phosphorylation in response to cytokines, and are thought to mediate many of their functional responses. Stat6 is activated in response to interleukin (IL)-4 and may contribute to various functions including mitogenesis, T-helper cell differentiation and immunoglobulin isotype switching. To evaluate the role of Stat6, we generated Stat6-null mice (Stat6 -/-) by gene disruption in embryonic stem cells. The mice were viable, indicating the lack of a non-redundant function in normal development. Although naive lymphoid cell development was normal, Stat6 -/- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE. In contrast, IL-4-mediated proliferation was only partly affected.

Expression of major histocompatibility complex of lung cancer was examined to study the malignant potential of the tumor using immunochemical staining (anti-HLA class I monoclonal antibody; w6/32). Nuclear DNA contents and the labeling index of proliferating cell nuclear antigen (PCNA) were measured as well. Forty three advanced (p-stage IIIa) lung cancers resected by lobectomy (33 cases), pneumonectomy (9 cases), and segmentectomy (1 case) entered the study. Mediastinal lymph node dissection was performed in every case during the operation. Of the 43 cases, expression of HLA class I was positive in 17 and negative in 26. The patients with positive HLA expression showed significantly better prognosis than those with negative HLA expression in terms of 5 year survival (p < 0.01). The patients with aneuploid pattern and positive HLA class I expression had the best prognosis on the survival curve (p < 0.01). When the PCNA positive rate was high, the prognosis was poor. And the patients with negative PCNA and positive HLA class I expression had significantly better prognosis than the others (p < 0.01). These findings suggest that the expression of HLA class I on the tumor cells is an important prognostic factor in the patients with NSCLC.

The Janus tyrosine kinases (Jaks) play a central role in signaling through cytokine receptors. Although Jak1, Jak2, and Tyk2 are widely expressed, Jak3 is predominantly expressed in hematopoietic cells and is known to associate only with the common gamma (gamma c) chain of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors. Homozygous mutant mice in which the Jak3 gene had been disrupted were generated by gene targeting. Jak3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, Jak3 plays a critical role in gamma c signaling and lymphoid development.

Four patients underwent resection of mediastinal malignant tumors with invasion to the superior vena cava. Two patients had invasive thymoma, one seminoma, and one metastatic mediastinal lymph nodes of unknown origin. Prior to resection of the tumor, an ePTFE graft was anastomosed between the innominate vein and the right atrium to maintain the venous drainage from the brain and the upper extremities. In two patients, the superior vena cava was reconstructed by patch angioplasty after the tumor with a part of the vena cava was safely resected. One patient died of acute respiratory failure, but the other three are alive and well without any evidence of graft obstruction. This safe and useful method in order to prevent cerebral congestion during and after resection of the tumor.

Human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, including two essential trans-activators for viral replication, Rev and Tat. Both Rev and Tat have a nucleolar targeting signal and are actually located predominantly in the nucleoli. Within the nucleoli, Rev is localized to the combined regions of the dense fibrillar (DFC) and the granular (GC) components. Tat does not colocalize precisely with any nucleolar component tested, but partly overlaps regions of the DFC and the GC. Regions of both Rev and Tat are overlapped by the distribution of the major nucleolar protein B23. Overexpression of Rev causes nucleolar ballooning and general structural deformity with aberrant accumulation of rRNAs, whereas Tat does not have that effect. B23 is markedly accumulated in those nucleoli deformed by Rev. Components of the nucleolar DFC, GC, and fibrillar center domains are not accumulated but dispersed in few small spots or larger patches within the enlarged nucleoli. Cytophotometric DNA determinations revealed that transient expression of Rev results in accumulation of G2, prophase, and mitotic cells which have failed cytokinesis, suggesting that Rev is capable of preventing or slowing the progression through mitosis. Tat, in contrast, does not affect the cell cycle. We speculate, based on these results, that Rev represses cell growth by inhibiting the transport of ribosomal proteins and preribosomal particles across the nuclear envelope and affecting the cell cycle, both of which may be related to proposed functions of B23.

The rex-encoded protein (Rex) of human T-cell leukemia virus type I (HTLV-I) is responsible for the cytoplasmic accumulation of incompletely spliced mRNAs that encode the virion structural proteins. Rex is known to be located predominantly in the cell nucleoli in transient transfections or in the isolated nuclei of HTLV-I-infected cells. However, precise location of Rex under physiological conditions has not been determined unequivocally. Here we report that Rex is primarily located as intranucleolar speckles in HTLV-I-infected cells, except for a few nucleoplasmic speckles. This is in contrast to the more diffuse nucleolar distribution of the rev-encoded protein (Rev) of human immunodeficiency virus type 1 (HIV-1), the functional homologue to Rex, in HIV-1-infected cells. Accumulation of Rev is associated with disruption of nucleolar structure and cell death, whereas Rex does not have these effects. The difference in distribution of Rex and Rev within the nucleoli may reflect the difference of toxicity toward the host cells. Involvement of the nucleolus in processing of certain mRNAs is also discussed.

Hepatic cells receive dual blood supply of the arterial and portal systems, but hepatoma has been thought to be supplied completely by hepatic artery. However, transcatheter hepatic artery embolization (TAE) has not been able to damage hepatoma entirely. For the study on the relationship between hepatoma and portal vein, I gave intraportal infusions of bromodeoxyuridine. (BrdU), an analogue of thymidine, to 10 patients with hepatoma at the time of surgery, and counted BrdU-positive nuclei immunohistochemically using the anti-BrdU monoclonal antibody. The labeling index, or percentage of BrdU-labelled cells, was 2.0 +/- 1.1% (mean +/- SD) in the cases without TAE and 11.9 +/- 4.2% in the other after TAE. On the other hand, examining the specimens with Microfil injected into the portal branch showed the distribution of portal branches in the hepatoma by radiograph or microscope. It is concluded that hepatoma does not receive arterial blood supply alone but the portal vein participates in vascular distribution of hepatoma, and the participation gets larger after TAE. It is suggested that this finding could be of great importance in planing treatment of patients with hepatoma.

The transcription initiation signals in retroviruses lie within the long terminal repeat (LTR) sequences. We have found a new transcriptional promoter in a central portion of the genome of human T-cell leukaemia virus type I (HTLV-I). The transcription start site is located just upstream to the ATG codon of the transcriptional trans-activator molecule, tax protein (Tax). The internal promoter may provide a new insight into gene expression of HTLV-I. The mechanism of leukaemogenesis by the defective HTLV-I is also discussed. Furthermore, we have identified two repeats of a novel enhancer sequence AGTTCT, which are located around the initiation site. We call the sequence HIRE (HTLV-I Internal Regulatory Element).

The rev protein (Rev) of the human immunodeficiency virus type 1 (HIV-1) is known as a post-transcriptional regulator of viral gene expression. It is located in the cell nucleolus. Transiently expressed Rev caused nucleolar ballooning and deformity with aberrant accumulation of rRNAs, and de novo synthesis of rRNAs decreased dramatically in these cells. However, similarly expressed rex protein (Rex) of the human T-cell leukemia virus type I, which is a functional homologue to Rev, did not affect nucleolar structure and function. Rev expression resulted in cell death with nucleolar destruction in an inducible cell line. Analysis of Rev mutants revealed that both the nucleolar targeting signal of Rev and the multimerization domain are prerequisites to the nucleolar disintegration by Rev. Human T-cells acutely infected with HIV-1 contained nucleoli which were deformed and filled with Rev, but chronically infected cells had intact nucleoli. Involvement of Rev in cytopathic effects in HIV-1 infection is discussed.

One of the salient pathological features of rheumatoid arthritis is synovial cell proliferation with bone erosion. Despite extensive investigation, the factors essential for synovial cell proliferation remain to be identified. Recent studies suggest that human T cell leukemia virus type I (HTLV-I) may play an important role in synovial overgrowth observed in patients with one type of chronic inflammatory synovitis. In order to confirm and extend these observations, we have established synovial cell clones (SCCs) from three HTLV-I carriers who demonstrated synovial overgrowth but were otherwise asymptomatic. HTLV-I proviral DNA randomly integrated into the cellular genome was present in 20-30% of SCCs. The SCCs carrying HTLV-I proviral DNA and expressing the tax gene exhibited high levels of proliferative potential. HTLV-I was found to function as a transcriptional trans-activator in these SCCs. Moreover, transfection of the tax expression plasmid into SCCs resulted in the same phenotype of increased proliferation and cytokine expression as exhibited by HTLV-I provirus-carrying and tax-expressing SCCs. These data suggest that tax plays a critical role not only in leukemogenesis but also in synovial overgrowth in humans.

A novel biphenotypic cell line carrying t(9;11)(p22;q23), TA-1, was established from the peripheral blood of a patient with acute undifferentiated leukemia. The TA-1 cells simultaneously expressed lymphoid (CD19, CD20) and myeloid characteristics (CD13, CD33, myeloperoxidase) on the same cells. When the cells were treated with tetraphorbol acetate, cytoplasmic mu chain was induced and the fluorescence intensity of CD13 was increased. These findings suggested that TA-1 cells have a bidirectional maturation capacity, as well as biphenotypic features. Molecular analysis disclosed differences in the rearranged bands, corresponding to one allele of the immunoglobulin heavy chain gene (IgH) and the T cell antigen receptor gamma gene (TCR gamma), between the non-cultured cells and the cell line, while showing identical rearranged patterns of another allele of both these genes and the TCR beta gene. These results suggest that the non-cultured cells and the established cell line have the same clonal origin and that the latter is a clonal descendant of the former.

According to differences in mobility on SDS-polyacrylamide gel electrophoresis, calpastatins (inhibitor proteins of the calcium-dependent proteinase calpain) are classified into the tissue type (100-120 kDa) and the erythrocyte type (70 kDa), which lacks the amino-terminal domains (domains L and 1). We investigated the molecular diversity of calpastatin in human hematopoietic cells by Western-blot analysis and by the reverse-transcription-polymerase-chain reaction method. While the mononuclear and polymorphonuclear cells in peripheral blood showed the tissue type (110 and 114 kDa), a cell line of erythroid cells (JK-1) showed both the tissue type (110 kDa) and the erythrocyte type (70 kDa) at approximately equal ratios. When the lysate of JK-1 cells was incubated in the presence of ATP, the 110-kDa form was degraded much faster than the 70-kDa form. In human erythrocytes, the 110-kDa form was identified as the tissue type by an antibody recognizing domain L, and this form was also present in addition to the predominant 70-kDA form. JK-1 cells, as well as nucleated cells in peripheral blood, contained calpastatin mRNA with exon-3-deleted. Glioblastoma and fibroblast cell lines expressed the nondeleted calpastatin mRNA in addition to the deletion type, and they showed bands corresponding to 117 kDa as well as 110 and 114 kDa. The 117-kDa band was detectable by an anti-exon 3 peptide antibody. These results suggest that diversity among the tissue type calpastatins is caused by both alternative splicing and post-translational processing whereas the apparent conversion from the tissue type to the erythrocyte type is caused by proteolytic processing.

To evaluate the effects of exercise on aortic wall elasticity and elastic components, young male rats underwent various exercise regimes for 16 weeks. In the exercised rats, the aortic incremental elastic modulus decreased significantly when under physiological strain. The aortic content of elastin increased significantly and the calcium content of elastin decreased significantly in the exercised group. The accumulated data from the exercised and sedentary groups revealed that the elastin calcium content was related positively to the incremental elastic modulus. We concluded that physical exercise from an early age decreases the calcium deposit in aortic wall elastin and that this effect probably produced in the exercised rats a distensible aorta.

Rex protein, the posttranscriptional regulator of human T-cell leukemia virus type I (HTLV-I), is required for the control of viral structural protein expression and virus replication. Rex is a phosphoprotein found predominantly in the cell nucleolus, whose function is thought to be regulated by its nucleolar localization and phosphorylation. Therefore, we investigated the in vivo phosphorylation of Rex protein in more detail. Phosphorylation of Rex occurred in all HTLV-I-infected cell lines examined in vivo, primarily at serine residues and to a very small extent at threonine residues. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) led to significant but transient enhancement of the incorporation of [32P]orthophosphate into Rex protein. N-terminal truncation of Rex protein abolished TPA-dependent phosphorylation. Chymotryptic digestion of phosphorylated Rex yielded two phosphopeptides. In vivo phosphorylation sites were identified as serine residues 70 and 177 and threonine residue 174. Serine 70 was a TPA-dependent phosphorylation site within a regulatory domain. We have already shown that the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) specifically blocked accumulation of viral unspliced gag-pol mRNA. Therefore, the phosphorylation at serine 70 may be involved in the regulation of Rex function in response to extracellular stimuli.