Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase.

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the "PEST" sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.

Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase.

Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (BCR downstream signaling 1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling.

Distribution of fibroblast growth factor-5 in rat hypothalamus, and its possible role as a regulator of feeding behaviour.

We previously reported that a transcript of fibroblast growth factor-5 (FGF-5) was more abundant in the brain of postnatal and adult mice than in the embryonic brain. This suggested that FGF-5 plays some role in the mature brain. Here, we have investigated the spatiotemporal expression and function of FGF-5 in the adult rat hypothalamus with the emphasis on feeding behaviour. In situ hybridization experiments demonstrated that, in both adequately fed and fasted (20 h) rats, FGF-5 transcripts were present within several nuclei in the hypothalamus (viz. the magnocellular part of the paraventricular nucleus, supraoptic nucleus, arcuate nucleus, median eminence, and ventromedial hypothalamic nucleus), but not in the lateral hypothalamic area. Quantitative detection of FGF-5 mRNA in the hypothalamus (especially in the paraventricular nucleus) indicated that food deprivation (20 h) reduced the expression of this gene to almost one-half of that seen in the control (fed) rats. The expression recovered to the control level after 1 h re-feeding, and this recovery persisted for several hours. Furthermore, FGF-5, when infused into the third ventricle, consistently reduced food intake, water intake and body weight gain, all in a dose-dependent manner. These results suggest that FGF-5 in the hypothalamus acts as a physiological regulator of feeding behaviour, and that its decreased expression during food deprivation may be important in stimulating appetite.

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase.

BACKGROUND: Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening. RESULTS: One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter. CONCLUSION: Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.

Identification of PU.1 and Sp1 as essential transcriptional factors for the promoter activity of mouse tec gene.

Tec is a cytoplasmic protein-tyrosine kinase abundantly expressed in hematopoietic precursor cells. To investigate the mechanism regulating the expression of Tec molecule, we cloned and analysed 5' flanking region of mouse tec gene up to -2kb from the transcriptional initiation site. Luciferase assays using successive deletion mutants demonstrated that regions from -364 to -323 and from -122 to -63, which contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively, are important for the transcriptional activity. Gel-shift and supershift assays revealed that PU.1 and Sp1 bind to the these regions through their consensus binding motifs. In addition, introduction of mutations into these motifs resulted in marked decrease in the promoter activity. These results indicate that PU.1 and Sp1 are essential for the transcriptional activity of the tec promoter and suggest that the cooperation of PU.1 and Sp1 plays a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineages.

Enhancement of TNF-alpha synthesis by overexpression of G alpha z in a mast cell line.

Ag stimulation of mast cells via the IgE receptor (Fc epsilon RI) elicits production and release of numerous cytokines. This activation of Fc epsilon RI initiates various tyrosine kinase-dependent signaling cascades, which ultimately result in the de novo synthesis of cytokines. To date, no heterotrimeric G proteins have been implicated in this process. Here we report that the alpha subunit of the heterotrimeric G protein, Gz, can regulate production of the cytokine, TNF-alpha. The alpha subunit was overexpressed in a cultured mast cell line (RBL-2H3) known to contain G alpha z. In stimulated cells, overexpression of G alpha z significantly enhanced the production of TNF-alpha. This effect of G alpha z appeared to be restricted in that constitutive synthesis of the cytokine, TGF-beta, and Ag-stimulation of the phosphoinositide-dependent secretory pathway were not significantly affected. Thus, G alpha z, a heterotrimeric G protein, appeared to modulate the stimulatory pathways for induction of TNF-alpha synthesis in RBL-2H3 cells.

Glucose and energy metabolism in rat liver after ischemic damage assessed by 13 C and 31 P NMR spectroscopy.

Glucose and energy metabolism in rat liver after ischemic damage was investigated by in vivo 31P NMR spectroscopy, 1H-detected 13C NMR spectroscopy, and in vitro 13C NMR spectroscopy using [1-13C]glucose as a tracer. Arterial ketone body ratio (AKBR; acetoacetate/beta-hydroxybutylate) and oxygen consumption of isolated mitochondria were also examined to evaluate hepatic function. The rats were divided into three groups: (A) without ischemia, (B) 10-min ischemia, and (C) 30-min ischemia. ATP was almost depleted at 10 min of ischemia and recovered after reperfusion, but the recovery was not complete. The recovery after 30-min ischemia was smaller than that after 10-min ischemia. [13C]Glucose was infused immediately after the reperfusion, and in vivo 1H-detected 13C NMR demonstrated sequential glucose incorporation into the liver. However, the incorporation depended on the blood sugar levels and did not reflect hepatic function. In vitro 13C NMR disclosed the glycogen C-1 signal in the nonischemic group and alanine C-3 and lactate C-3 signals in the ischemic groups. The intensity of glycogen was correlated positively (r = 0.648, P = 0.002) and those of alanine and lactate were correlated negatively (r = -0.831, P < 0.005 and r = -0.710, P = 0.005, respectively) to the ATP levels as measured by in vivo 31P NMR. These results suggested that ATP level participates in glycogenesis and gluconeogenesis in the liver. The AKBR and oxygen consumption of isolated mitochondria were the highest in the 10-min ischemia group, which might reflect mitochondrial compensatory response to the decreased ATP level.

Antiallergic dimeric prenylbenzoquinones from Ehretia microphylla.

The ethyl acetate-soluble portion of the MeOH extract of Ehretia microphylla showed inhibitory activity on exocytosis in antigen-stimulated rat basophils. The bioassay-guided separation of this fraction afforded five biologically active compounds. By means of chemical and spectroscopic methods, the structures of these compounds, which include microphyllone, a unique dimeric prenylbenzoquinone, and its congeners, were elucidated.

HRX gene rearrangement in secondary acute lymphoblastic leukemia.

The human tri-thorax gene (HRX) also called ALL-1 (Acute Lymphocytic Leukemia-1) as well as MLL (Myeloid-lymphoid or Mixed-lineage Leukemia) gene, is disrupted in the majority of leukemias with chromosomal abnormalities involving 11q23. The alteration of the gene is related to leukemogenesis of various types such as acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), and acute mixed lineage leukemia. The gene is also rearranged in cases of secondary AML developing after exposure to chemotherapeutic agents, especially topoisomerase II inhibitors. In at least one report, genomic analysis of this recombination site showed the breakpoint to be a topoisomerase II binding site and that exposure to the inhibitor could induce the rearrangement. If exposure induces the rearrangement of the gene, secondary ALL as well as secondary AML could occur after exposure to these agents, because the type of leukemias with rearranged HRX gene is not limited to AML. We present here such a case of secondary ALL with this gene rearrangement which occurred during adjuvant chemotherapy for breast cancer. Although less cases of secondary ALL are reported in comparison with those of secondary AML, such case reports have been accumulating. The incidence of this type of leukemia should be clarified in the future.

Studies with transfected and permeabilized RBL-2H3 cells reveal unique inhibitory properties of protein kinase C gamma.

To characterize protein kinase C (PKC) gamma, an isozyme found exclusively in brain and spinal cord, its cDNA was introduced into basophilic RBL-2H3 cells that lack this isozyme. The expression of PKC gamma significantly attenuated antigen-induced responses including hydrolysis of inositol phospholipids, increase in cytosolic calcium, and secretion of granules but enhanced antigen-induced release of arachidonic acid. Instead of a sustained increase in cytosolic calcium, antigen now induced calcium oscillations; possibly as a consequence of suppression of the phospholipase C activity and incomplete emptying of internal calcium stores. In addition, PKC gamma appeared to inhibit activation of other PKC isozymes because phorbol 12-myristate 13-acetate failed to act synergistically with the Ca(2+)-ionophore on secretion. This was confirmed in other studies where PKC gamma was shown to suppress the transduction of stimulatory signals by other isozymes of PKC on provision of these isozymes to PKC-depleted permeabilized cells. The studies in total indicated that only PKC gamma was capable of inhibiting both early and distal signals for secretion including those signals transduced by endogenous isozymes of PKC.

Medical consultation rate of allergic rhinitis and pollinosis surveillance in Aichi, Japan.

The medical consultation rate of allergic rhinitis (AR) was analysed using Japan National Health Insurance records of Aichi Prefecture for May 1989. Data collected from 88 cities, towns and villages were tabulated and divided into five-year age groups. The standardized medical consultation rate (SMCR) of AR in each municipality was then calculated. It was found that SMCR of AR did not correlate well with the pollen count for Japanese cedars, Japanese cypresses or gramineae, respectively, but a weak correlation with the mean yearly levels of nitrogen dioxide was suggested by the data. There was a significant positive correlation between SMCR of AR and the mean yearly levels of suspended particulate matter, the major element of which is diesel exhaust particulate.

Presacral epidermal cyst found in an adult male with a high CEA content: report of an unusual case.

A 63-year-old Japanese man presented with constipation, having noticed flat stools for several years. Digital examination of the rectum, followed by barium enema, colono-fiberscopy, computed tomography (CT), and magnetic resonance imaging (MRI) revealed an oval mass located between the retrorectal and presacral space without any mucosal lesion. This mass had narrowed the rectal lumen by compressing the rectum anteriorly. Although the plasma levels of the tumor markers were within the normal range, those of the tumor contents were elevated with a carcinoembryonic antigen (CEA) of 118 ng/mL, while the alpha-fetoprotein (AFP) value was 1 ng/mL. The tumor was completely extirpated through an abdominal incision, and there has been no evidence of recurrence thus far. Histological examination showed that the tumor wall was made of keratinized stratified squamous epithelium without any cutaneous adnexal structure, and hence it was diagnosed as an epidermal cyst. CEA was identified in these benign epithelial cells by immunoperoxidase staining using a monoclonal antibody. To the best of our knowledge, there have been only four other cases with a presacral epidermal cyst documented in the Japanese literature, all of whom were female. Our patient is the first reported case of an adult male with a presacral epidermal cyst.

Energy metabolism of the heart and the liver in brain-dead dogs as assessed by 31P NMR spectroscopy.

The energy metabolism of the heart and the liver was assessed in brain-dead dogs by 31P nuclear magnetic resonance spectroscopy. The mean blood pressure and pulse rate changed respectively from 136.1 +/- 9.3 mm Hg (mean +/- SEM) and 126.7 +/- 4.4/min in the control period, to 212.9 +/- 15.8 mm Hg and 146.7 +/- 13.3/min during Cushing phenomenon (CU period), and to 60.0 +/- 6.4 mm Hg and 60.0 +/- 2.9/min after completion of brain death (BD period). The ratio of creatine phosphate to inorganic phosphate (PCr/Pi) of the heart decreased from 5.00 +/- 1.08 to 3.24 +/- 0.80 in the CU period and increased to the levels of the control values in the early BD period. The intracellular pH of the heart decreased from 7.20 +/- 0.07 to 6.91 +/- 0.02 in the CU period and increased to 7.12 +/- 0.01 in the BD period. The positive relationship between the Pi/PCr and the rate-pressure product (BP x PR) indicates that the regulation of the oxidative metabolism by free ADP was well maintained except at some points in the CU period (Pi/PCr = 8.77 x 10(-6) BP x PR + 0.164, r = 0.704). This suggested that the remarkable hypotension and bradycardia in the BD period is not associated with cardiac energy failure. Although the energy states of both the heart and the liver in brain-dead dogs were adequately maintained in the BD period, the changes were different.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma amino acid profile in relation to arterial ketone body ratio in surgical patients.

A total of 357 plasma amino acid profiles from 93 surgical patients were statistically analyzed in relation to the changes in arterial ketone body ratio, which reflects the hepatic mitochondrial redox state. When the arterial ketone body ratio was above 0.7, all plasma amino acid levels were within the normal range. When it was between 0.7 and 0.4, plasma levels of aspartate, glutamate, valine, isoleucine, leucine, ornithine, and arginine decreased, and plasma levels of tyrosine, phenylalanine, proline, and methionine increased. Furthermore, when it was below 0.4, almost all plasma amino acids markedly increased. These results indicate that arterial ketone body ratio accurately reflects the alterations in plasma amino acid profile, and can serve as an indicator for providing nutritional support by amino acid supplement in surgical patients.

Morbidity of allergic rhinitis based on the National Health Insurance records of Japan.

The morbidity of allergic rhinitis (AR) based on Japan National Health Insurance records was calculated from 1981 to 1990. The data shows a three-fold increase of the rate for AR patients in those 10 years. Additionally, Aichi Prefecture's morbidity of AR was analysed for May 1991. When cross-sectional methods were used for May 1991, it was found that the morbidity of AR showed little correlation with the pollen count for Japanese cedars and Japanese cypresses. However, the results suggested possible correlations between the morbidity of AR and the mean yearly levels of the pollutant components SPM and NO2.

Molecular cloning and chromosomal localization of the key subunit of the human N-methyl-D-aspartate receptor.

A complementary DNA encoding the key subunit of the human N-methyl-D-aspartate (NMDA) receptor (NMDAR1) has been cloned using a probe derived from the rat NMDAR1 cDNA. The cDNA encodes a 938-amino acid protein, which shows 99% amino acid homology with the rat counterpart. Of the 7 of 938 amino acids which are different, three occur in the region of the signal peptide and the others in the extracellular amino-terminal domain preceding the 4 putative transmembrane segments. Expression in Xenopus oocytes demonstrated that the single protein encoded by the cloned cDNA possesses the electrophysiological and pharmacological properties characteristic of the NMDA receptor, including Ca2+ permeability, voltage-dependent Mg2+ block, and inhibition by selective antagonists such as Zn2+ and channel blockers. The high evolutionary conservation in the structure and properties of NMDAR1 argues strongly for the importance of this receptor in functions of glutamate neurotransmission. RNA blot analysis showed abundant expression of mRNA whose size is about 4.5 and 4.8 kilonucleotides. The human gene encoding the NMDAR1 subunit has been mapped to chromosome 9q34.3 by the analyses of blot hybridization of a DNA panel of human/hamster somatic cell hybrids and fluorescence in situ hybridization of human chromosomes.

[Effect of XKJ-001, a crude drug preparation, on body water distribution and water excretion in mice].

The effect of XKJ-001, a crude drug preparation based on Seisho-ekki-to, was investigated on the hematocrit, plasma volume, extracellular and interstitial fluid volumes as well as water excretion in mice. Mice were housed in an animal room maintained at 34 degrees C for 3 d with water and food freely available. While the hematocrit, extracellular and interstitial fluid volumes increased, the plasma volume decreased. These results suggest that the distribution of body water in mice housed at high environmental temperature exhibit the state of water metabolism disorders (Suitai) described in Kampo medicine. After the administration of XKJ-001 (3 g/kg, once a day) for 5 d, mice were housed in an animal room maintained at 34 degrees C for 3 d. The administration of XKJ-001 was allowed to continue on the day 0, day 1 and day 2. XKJ-001 inhibited the increase in hematocrit and the changes in body water distribution of mice induced by high environmental temperature. An effect of XKJ-001 on water excretion in mice was investigated in comparison with hydrochlorothiazide (HTZ). Distilled water (D.W., 100 ml/kg) or bicarbonate saline (B.S., 100 ml/kg) was intraperitoneally injected immediately after the oral administration of XKJ-001 (1.5 g/kg) or HTZ (15 mg/kg). The water excretion was enhanced after 3 h for XKJ-001 and after 6 h for HTZ after the intraperitoneal injection of D.W. As for the intraperitoneal injection of BS, HTZ enhanced the water excretion, however, XKJ-001 exhibited no effect. These results suggest that XKJ-001 has activities on water maldistribution and facilitates the water excretion.