Identification and Enzymatic Analysis of an Archaeal ATP-Dependent Serine Kinase from the Hyperthermophilic Archaeon Staphylothermus marinus.

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O-phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk-SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here, we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk-SerK from Euryarchaeota (42 to 45% identical). Tk-serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli. All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity for l-Ser but not other compounds, including d-Ser, l-threonine, and l-homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as phosphate donors. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5'-triphosphates over triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis (Tk-SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk-SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases.

Reactivity of autoantibodies against not only erythrocytes but also hepatocytes in sera of mice with malaria.

In order to further examine the reactivity of autoantibodies, mice were infected with a non-lethal strain of Plasmodium yoelii. Parasitemia appeared between days 10 and 21. During this period, hyperglycemia and hypothermia were serially obeserved and this phenomenon resembled stress-associated responses. In parallel with parasitemia, autoantibodies appeared against nucleus and double-stranded DNA in the sera. To examine further the reactivity of autoantibodies against tissues, immunohistochemical staining using sera from mice with or without malaria was conducted. Autoantibodies contained reactivity to erythrocytes in the spleen, bone marrow and peripheral blood, especially against tissues obtained from mice with malaria. In the liver and intestine, autoantibodies reacted with hepatocytes and intestinal epithelial cells, respectively. These results suggested that the reactivity of autoantibodies against erythrocytes and hepatocytes might be associated with the modulation of the disease course in malaria.

Subcutaneous Panniculitis-like T Cell Lymphoma Diagnosed with a Slowly Progressive Course: A Case Report.

Panniculitis is an inflammation that occurs in subcutaneous adipose tissue. Panniculitis includes physical panniculitis (e.g., traumatic) and infectious panniculitis (e.g., bacterial, fungal, subcutaneous panniculitis-like T cell lymphoma [SPCTL], etc.). Accurate diagnosis is crucial due to similar clinical presentation of all types of panniculitis. Here, we report a case of SPCTL which was initially diagnosed with traumatic panniculitis. A 15-year-old male patient was admitted to a previous hospital due to a progressively enlarged right flank and inguinal mass after an abdominal bruise. He was initially diagnosed with traumatic panniculitis, but the mass expanded throughout the chest and abdomen accompanied by a fever of over 11 months. Computed tomography (CT) revealed a subcutaneous mass in the anterior chest and abdominal wall. Fludeoxyglucose F18 (FDG) uptake was observed at those lesions using FDG-positron emission tomography (PET). A biopsy of the mass lesion was performed, during which SPCTL was diagnosed based on pathological examination. He was initially treated with prednisolone and cyclosporine A for two weeks. His fever went down, but subcutaneous mass in the chest and abdominal wall persisted. Therefore, he received a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen. After 6 courses of CHOP, CT revealed no disease evidence. He remained in complete remission at 30 months of therapy.

Spontaneously Resolved Suspected Hemophagocytic Lymphohistiocytosis or Macrophage Activating Syndrome Associated with Neonatal Lupus Erythematosus: A Case Report.

OBJECTIVES: To present a rare case of neonatal lupus erythematosus (NLE) associated with suspected hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS). CASE PRESENTATION: A female infant weighing 2,995 g was born to a mother without medical history of any disease. At birth, the patient had erythematous papules on her face and trunk. She was admitted at 1 day of age with elevated C-reactive protein levels. The patient was diagnosed with NLE based on the presence of anti-Ro/SSA and anti-La/SSB antibodies. Thereafter, it became clear that the antibody levels in her mother were also elevated. At 20 days of age, the infant showed elevated transaminases, ferritin, triglyceride, and soluble interleukin-2 receptor levels. Although HLH or MAS was suspected, she did not fulfill the diagnostic criteria. Thereafter, these abnormal values spontaneously improved, and the skin rash improved with the use of topical steroids. The patient was discharged at 39 days of age. At 1 year of age, the patient's growth and development were normal. CONCLUSION: NLE should be considered in infants with an unexplained skin rash at birth. When a diagnosis is made, close observation of the infant's clinical features is needed to determine whether they will develop HLH or MAS.

Human T cell leukemia virus type 2 (HTLV-2) Tax2 has a dominant activity over HTLV-1 Tax1 to immortalize human CD4+ T cells.

While human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia, a close relative, HTLV-2, is not associated with any leukemia. HTLV-1 and HTLV-2 encode the Tax1 and Tax2 proteins, respectively, which are essential for the immortalization of human T cells by the respective viruses, thereby causing persistent infection. In this study, we compared Tax1 and Tax2 with respect to their immortalization activity in human T cells. Lentivirus-mediated transduction of the tax2 gene into human peripheral blood mononuclear cells stimulated with phytohemagglutinin and interleukin-2 in 96-well plates induced outgrowing T cells in most wells, but the cells infected with the control viruses died within 3 weeks. Surprisingly, the number of outgrowing cells induced by Tax2 was much higher than that induced by Tax1, and the appearance of outgrowing cells by Tax2 was earlier than that induced by Tax1. Nevertheless, both Tax2 and Tax1 preferentially immortalized CD4(+) T cells, but not CD8(+) T cells. Our study showed that HTLV-2 Tax2 can immortalize human CD4(+) T cells, and the activity is much higher than that of Tax1. The distinct T cell immortalization activities of Tax2 and Tax1 might therefore play a role in the different pathogeneses observed for these two viruses.

Extracellular ATP-stimulated macrophages produce macrophage inflammatory protein-2 which is important for neutrophil migration.

Macrophages are the major source of the chemokines macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), which play a major role in neutrophil migration to sites of inflammation. Although extracellular ATP from inflammatory tissues induces several immune responses in macrophages, it is unclear whether ATP-stimulated macrophages affect neutrophil migration. Therefore, the aim of the present study was to investigate the role of ATP-induced MIP-2 production by macrophages. When ATP was injected intraperitoneally into mice, the number of neutrophils within the peritoneal cavity markedly increased, along with the levels of MIP-2 and KC in the peritoneal lavage fluid. Consistent with this, ATP induced MIP-2 production, but not that of KC, by peritoneal exudate macrophages (PEMs) in vitro. This occurred via interactions with the P2X(7) receptor and P2Y(2) receptor. Furthermore, treatment of PEMs with ATP led to the production of reactive oxygen species. The ATP-induced MIP-2 production was inhibited by treatment with the antioxidant N-acetyl-l-cysteine. Also, MIP-2 production was inhibited by pre-incubating PEMs with inhibitors of extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase. The MIP-2 neutralization reduced the increase in neutrophil numbers observed in ATP-treated mice. Taken together, these results suggest that increased production of reactive oxygen species by ATP-stimulated macrophages activates the signalling pathways that promote MIP-2 production which, in turn, induces neutrophil migration.

Natural killer T cells suppress zymosan A-mediated granuloma formation in the liver by modulating interferon-γ and interleukin-10.

Wild-type (WT) and CD1d(-/-) [without natural killer (NK) T cells] mice were treated with zymosan A to induce granuloma formation in the liver. Increased granuloma formation was seen in NKT-less mice on days 7 and 14 after administration. WT mice showed limited granuloma formation, and zymosan A eventually induced NKT cell accumulation as identified by their surface marker (e.g. CD1d-tetramer). Zymosan A augmented the expression of Toll-like receptor 2 on the cell surface of both macrophages and NKT cells. One possible reason for accelerated granuloma formation in NKT-less mice was increased production of interferon- γ (IFN-γ); a theory that was confirmed using IFN-γ(-/-) mice. Also, zymosan A increased interleukin-10 production in WT mice, which suppresses IFN-γ production. Taken together, these results suggest that NKT cells in the liver have the potential to suppress zymosan A-mediated granuloma formation.

Coincidence of autoantibody production with the activation of natural killer T cells in α-galactosylceramide-mediated hepatic injury.

Natural killer T (NKT) cells are known to be specifically activated by α-galactosylceramide (α-GalCer) via their interaction with CD1d. At that time, NKT cells mediate autoreactivity and eventually induce hepatic injury. As these immune responses resemble acute autoimmune hepatitis, it was examined whether autoantibody production and the activation of autoantibody-producing B-1 cells were accompanied by this phenomenon. Autoantibodies against Hep-2 cells and double-stranded DNA were detected in sera as early as day 3 (showing a peak at day 14) when mice were treated with α-GalCer. On day 3, B220(low) cells appeared in the liver. These B220(low) cells were CD5(-) (i.e. B-1b cells) and CD69(+) (an activation marker). Primarily, such B220(low) cells were present in the peritoneal cavity, but the proportion of B220(low) cells increased with the administration of α-GalCer even at this site. In parallel with the appearance of B220(low) cells in the liver, hepatic lymphocytes acquired the potential to produce autoantibodies in in vitro cell culture in the presence of lipopolysaccharide. These results suggested that hepatic injury induced by α-GalCer administration resembled acute autoimmune hepatitis and that the major effector lymphocytes were NKT cells with autoreactivity and autoantibody-producing B-1 cells.

Identification and characterization of autoantibody-producing B220(low) B (B-1) cells appearing in malarial infection.

Mice with malaria showed unique immunological responses, including the expansion of NK1.1(-)TCR(int) cells (extrathymic T cells). Since TCR(int) cells with autoreactivity and autoantibody-producing B cells (B-1 cells) are often simultaneously activated under autoimmune conditions, it was examined whether B-1 cells were activated in the course of malarial infection. From days 14 after infection, B220(low) B-1 cells appeared in the liver and spleen. The number of B220(low) B cells was highest at day 14, but the ratio was highest at days 28-35. In parallel with the appearance of B220(low) cells, autoantibodies against HEp-2 cells and double-stranded DNA were detected in sera. These B220(low) cells had phenotypes of CD44(high), CD23(-) and CD62L(-). In sharp contrast, conventional B220(high) B cells (B-2 cells) were CD44(low), CD23(+) and CD62L(+). These results suggested that malaria immune responses were not mediated by conventional T and B cells but resembled the responses during autoimmune diseases.

Co-appearance of autoantibody-producing B220(low) B cells with NKT cells in the course of hepatic injury.

Severe hepatic injury is induced by Concanavalin A (Con A) administration in mice, the major effector cells being CD4(+) T cells, NKT cells and macrophages. Since autologous lymphocyte subsets are associated with tissue damage, Con A-induced hepatic injury is considered to be autoimmune hepatitis. However, it has remained to be investigated how autoantibodies and B-1 cells are responsible for this phenomenon. In this study, it was demonstrated that autoantibodies which were detected using Hep-2 cells in immunofluorescence tests and using double-strand (ds) DNA in the ELISA method, appeared after Con A administration (a peak at day 14). Moreover, autoantibody-producing B220(low) cells (i.e., B-1 cells) also appeared at this time. Purified B220(low) cells were found to have a potential to produce autoantibodies. These results suggest that Con A-induced hepatic injury indeed includes the mechanism of autoimmune hepatitis.

A cyclooxygenase-2 inhibitor ameliorates behavioral impairments induced by striatal administration of epidermal growth factor.

Consistent with the hypothesis that neuroinflammatory processes contribute to the neuropathology of schizophrenia, the protein levels of epidermal growth factor (EGF) and its receptor ErbB1 are abnormal in patients with schizophrenia. To evaluate neuropathological significance of this abnormality, we established an animal model for behavioral deficits by administering EGF into the striatum and evaluated the effects of cyclooxygenase-2 (Cox-2) inhibitor celecoxib. Intracranial infusion of EGF into the striatum of adult male rats activated ErbB1 and induced neurobehavioral impairments observed in several schizophrenia models. Unilateral EGF infusion to the striatum lowered prepulse inhibition (PPI) in a dose-dependent manner and impaired latent learning of active shock avoidance without affecting basal learning ability. Bilateral EGF infusion similarly affected PPI. In contrast, EGF infusion to the nucleus accumbens did not induce a behavioral deficit. Intrastriatal EGF infusion also increased Cox-2 expression, elevated tyrosine hydroxylase activity, and upregulated the levels of dopamine and its metabolites. Subchronic administration of celecoxib (10 mg/kg, p.o.) ameliorated the abnormalities in PPI and latent learning as well as normalized dopamine metabolism. We conclude that this EGF-triggered neuroinflammatory process is mediated in part by Cox-2 activity and perturbs dopamine metabolism to generate neurobehavioral abnormalities.

Relationship between diseases accompanied by tissue destruction and granulocytes with surface adrenergic receptors.

It is well-known that physiological phenomena and certain diseases, including neonatal granulocytosis, age-associated granulocytosis, periodontitis, pancreatitis, Crohn's disease, ulcerative colitis, hemorrhoids, endometriosis, and NSADs-enteritis, are accompanied by tissue destruction and granulocytosis. We investigated what is a key factor connecting tissue destruction and granulocytosis, attention being focused on adrenergic receptors on granulocytes and stress-induced sympathetic nerve stimulation. If we introduce the concept that "granulocytosis and subsequent tissue destruction are induced by sympathetic nerve stimulation," the mechanisms underlying many physiological phenomena and the etiology of several uncurable diseases in humans can be clearly understood.

Structural Study on the Reaction Mechanism of a Free Serine Kinase Involved in Cysteine Biosynthesis.

A free serine kinase (SerK) is involved in l-cysteine biosynthesis in the hyperthermophilic archaeon Thermococcus kodakarensis. The enzyme converts ADP and l-serine (Ser) into AMP and O-phospho-l-serine (Sep), which is a precursor of l-cysteine. SerK is the first identified enzyme that phosphorylates free serine, while serine/threonine protein kinases have been well studied. SerK displays low sequence similarities to known kinases, suggesting that its reaction mechanism is different from those of known kinases. Here, we determined the crystal structures of SerK from T. kodakarensis (Tk-SerK). The overall structure is divided into two domains. A large cleft is found between the two domains in the AMP complex and in the ADP complex. The cleft is closed in the ternary product complex (Sep, AMP, and Tk-SerK) and may also be in the ternary substrate complex (Ser, ADP, and Tk-SerK). The closure may reorient the carboxyl group of E30 near to the Oγ atom of Ser. The Oγ atom is considered to be deprotonated by E30 and to attack the ß-phosphate of ADP to form Sep. The substantial decrease in the activity of the E30A mutant is consistent with this mechanism. Our structures also revealed the residues that contribute to the ligand binding. The conservation of these residues in uncharacterized proteins from bacteria may raise the possibility of the presence of free Ser kinases not only in archaea but also in bacteria.

Protection against malaria due to innate immunity enhanced by low-protein diet.

Mice were fed ad libitum with a normal diet (25% protein) or low-protein diets (0-12.5% protein) for a wk and then infected with a nonlethal or lethal strain of Plasmodium yoelii, that is, blood stage infection. The same diet was continued until recovery. Mice fed with a normal diet showed severe parasitemia during nonlethal infection, but survived the infection. They died within 2 wk in the case of lethal infection. However, all mice fed with low-protein diets survived without apparent parasitemia (there were small peaks of parasitemia) in cases of both nonlethal and lethal strains. These surviving mice were found to have acquired potent innate immunity, showing the expansion of NK1.1 -TCRint cells and the production of autoantibodies during malarial infection. Severe combined immunodeficiency (scid) mice, which lack TCRint cells as well as TCRhigh cells, did not survive after malarial infection of lethal strain of P. yoelii, even when low-protein diets were given. These results suggest that low-protein diets enhanced innate immunity and inversely decreased conventional immunity, and that these immunological deviations rendered mice resistant against malaria. The present outcome also reminds us of our experience in the field study of malaria, in which some inhabitants eventually avoided contracting malaria even after apparent malarial infection.

An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism.

Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization.

USP10 Is an Essential Deubiquitinase for Hematopoiesis and Inhibits Apoptosis of Long-Term Hematopoietic Stem Cells.

Self-renewal, replication, and differentiation of hematopoietic stem cells (HSCs) are regulated by cytokines produced by niche cells in fetal liver and bone marrow. HSCs must overcome stresses induced by cytokine deprivation during normal development. In this study, we found that ubiquitin-specific peptidase 10 (USP10) is a crucial deubiquitinase for mouse hematopoiesis. All USP10 knockout (KO) mice died within 1 year because of bone marrow failure with pancytopenia. Bone marrow failure in these USP10-KO mice was associated with remarkable reductions of long-term HSCs (LT-HSCs) in bone marrow and fetal liver. Such USP10-KO fetal liver exhibited enhanced apoptosis of hematopoietic stem/progenitor cells (HSPCs) including LT-HSCs but not of lineage-committed progenitor cells. Transplantation of USP10-competent bone marrow cells into USP10-KO mice reconstituted multilineage hematopoiesis. These results suggest that USP10 is an essential deubiquitinase in hematopoiesis and functions by inhibiting apoptosis of HSPCs including LT-HSCs.

Association of CD8+ natural killer T cells in the liver with neonatal tolerance phenomenon.

BACKGROUND: Neonatal tolerance is a very interesting phenomenon, because even allogeneic skin grafts are not rejected in these mice at the adult stage. However, the underlying mechanism remains unclear. METHODS: In this study we prepared such tolerant C57BL/6 (B6) mice (H-2b) by the injection of allogeneic lymphocytes of BALB/c origin (H-2d) at the neonatal stage. RESULTS: The total number of liver lymphocytes in these tolerant mice was found to increase when it was examined at the adult stage. Nevertheless, the retention of allogeneic lymphocytes that were injected at the neonatal stage was highest in the spleen. It is speculated that these allogeneic lymphocytes stimulate the hepatic immune system via the portal vein and that such stimulation maintains the tolerance phenomenon. Indeed, these tolerant mice showed elevated levels of IL-2R beta+ CD3int cells (i.e., extrathymic T cells) and NK1.1+ CD3int cells (i.e., NKT cells) in the liver. Even more interestingly, the number and proportion of CD8+ NKT cells, which are usually a minor population in normal mice, increased among NKT cells in the liver of tolerant mice. This became much more prominent when tolerant mice were grafted with allogeneic (H-2d) skin. CONCLUSION: In conjunction with additional data from a cell-transfer experiment and a splenectomy experiment, our results suggest that CD8+ NKT cells in the liver of tolerant mice might be intimately associated with the neonatal tolerance phenomenon.

Unique immunomodulation by electro-acupuncture in humans possibly via stimulation of the autonomic nervous system.

Cumulative evidence suggests that immunologic responses are under the regulation of the autonomic nervous system. Since acupuncture has recently been reported to modulate the autonomic nervous system, we investigated the possibility that acupuncture eventually modulates the immune system. In the present study, electro-acupuncture was applied in young volunteer subjects. As for the proportions of granulocytes and lymphocytes in the blood, there were three groups: (1) granulocytosis and lymphocytopenia; (2) granulocytopenia and lymphocytosis; and (3) normal pattern. Interestingly, with the administration of acupuncture, the status of subjects with relatively low levels of granulocytes and high levels of lymphocytes shifted to Group 1, whereas that of subjects with high levels of granulocytes and low levels of lymphocytes shifted to Group 2. In other words, acupuncture tended to normalize the pattern of leukocytes. We confirmed that acupuncture induced parasympathetic nerve stimulation, resulting in a decrease in the heart rate. These results suggest possible mechanisms underlying how acupuncture ameliorates the condition of patients with many chronic diseases.