The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor-induced proliferation in hematopoietic cells.

The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

Antagonistic control of cell fates by JNK and p38-MAPK signaling.

During the development and organogenesis of all multicellular organisms, cell fate decisions determine whether cells undergo proliferation, differentiation, or aging. Two independent stress kinase signaling pathways, p38-MAPK, and JNKs, have evolved that relay developmental and environmental cues to determine cell responses. Although multiple stimuli can activate these two stress kinase pathways, the functional interactions and molecular cross-talks between these common second signaling cascades are poorly elucidated. Here we report that JNK and p38-MAPK pathways antagonistically control cellular senescence, oncogenic transformation, and proliferation in primary mouse embryonic fibroblasts (MEFs). Similarly, genetic inactivation of the JNK pathway results in impaired proliferation of fetal hepatoblasts in vitro and defective adult liver regeneration in vivo, which is rescued by inhibition of the p38-MAPK pathway. Thus, the balance between the two stress-signaling pathways, MKK7-JNK and MKK3/6-p38-MAPK, determines cell fate and links environmental and developmental stress to cell cycle arrest, senescence, oncogenic transformation, and adult tissue regeneration.

G proteins activate ATP-sensitive K+ channels by antagonizing ATP-dependent gating.

To determine whether G proteins activate cardiac ATP-sensitive K+ (KATP) channels by regulating intracellular ATP (ATPi)-dependent gating, currents were measured in inside-out patches. When ATPi closed KATP channels, activators of endogenous G proteins, GTP (plus adenosine or acetylcholine), GTP gamma S, or AlF-4 stimulated channels, an effect prevented by GDP beta S. In the absence of ATPi, G protein activators were ineffective. Intracellular nucleoside diphosphates restored KATP channel openings after the "rundown" of spontaneous activity. Only when ATPi suppressed nucleoside diphosphate-induced openings, GTP gamma S or AlF-4 enhanced KATP channel activity. Active forms of exogenous G protein subunits (G alpha i-1, G alpha i-2, or G alpha o) activated only KATP channels closed by ATPi. G proteins stimulate cardiac KATP channels apparently by antagonizing ATPi-dependent inhibitory gating. Regulation of ligand-dependent gating represents a distinct type of G protein modulation of ion channels.

Relationship between expression of 5-fluorouracil metabolic enzymes and 5-fluorouracil sensitivity in esophageal carcinoma cell lines.

5-Fluorouracil (5-FU) is a key drug in the treatment of esophageal squamous cell carcinoma (ESCC). Gene expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), has recently been investigated in order to predict the 5-FU sensitivity of several cancers. We examined the relationship between such gene expression and 5-FU sensitivity in 25 ESCC cell lines. TS, DPD, TP and OPRT mRNA levels were assessed by real-time polymerase chain reaction. The 50% inhibitory concentrations (IC50) of 5-FU in 25 ESCC cell lines were determined by cell proliferation assay. IC50 values for 5-FU ranged from 1.00 to 39.81 micromol/L. There were significant positive correlations between IC50 and TS mRNA expression (R(2) = 0.5781, P < 0.0001) and DPD mRNA expression (R(2) = 0.3573, P = 0.0016). There were no correlations between IC50 and TP or OPRT mRNA expression. TS and DPD mRNA expression levels may be useful indicators in predicting the anti-tumor activity of 5-FU in ESCC.

Gene expression profiling of the response of esophageal carcinoma cells to cisplatin.

Cisplatin is the most common chemotherapeutic agent used in esophageal cancer. However, sensitivity to cisplatin varies greatly between patients. It is important to identify the gene(s) that are related to the sensitivity to cisplatin in esophageal cancer patients. The IC50 for cisplatin was measured for 15 esophageal cancer cell lines (TE1-5, TE8-15, KYSE140, and KYSE150). RNA was extracted from each of these cell lines and a normal esophageal epithelial cell line, namely, Het1A, and gene expression profiles were analyzed using an oligonucleotide microarray consisting of 34 594 genes. TE4 was highly resistant and TE12, 14, and 15 were sensitive to cisplatin. Thirty-seven genes were differentially expressed in the cisplatin-resistant esophageal cancer cell line. Our investigation provides a list of candidate genes that may be associated with resistance to cisplatin in esophageal cancer cells, which may serve as a basis for additional functional studies.

Identification of candidate genes involved in the radiosensitivity of esophageal cancer cells by microarray analysis.

Radiotherapy plays a key role in the control of tumor growth in esophageal cancer patients. To identify the patients who will benefit most from radiation therapy, it is important to know the genes that are involved in the radiosensitivity of esophageal cancer cells. Hence, we examined the global gene expression in radiosensitive and radioresistant esophageal squamous cell carcinoma cell lines. Radiosensitivities of 13 esophageal cancer cell lines were measured. RNA was extracted from each esophageal cancer cell line and a normal esophageal epithelial cell line, and the global gene expression profiles were analyzed using a 34 594-spot oligonucleotide microarray. In the clonogenic assay, one cell line (TE-11) was identified to be highly sensitive to radiation, while the other cell lines were found to be relatively radioresistant. We identified 71 candidate genes that were differentially expressed in TE-11 by microarray analysis. The up-regulated genes included CABPR, FABP5, DSC2, GPX2, NME, CBR3, DOCK8, and ABCC5, while the down-regulated genes included RPA1, LDOC1, NDN, and SKP1A. Our investigation provided comprehensive information on genes related to radiosensitivity of esophageal cancer cells; this information can serve as a basis for further functional studies.

Long-term Follow-up of Laparoscope-Assisted Living Donor Hepatectomy.

BACKGROUND: We have introduced and performed laparoscope-assisted surgery in living donor hepatectomy. The objective of this study was to investigate the long-term results of laparoscope-assisted living donor hepatectomy. METHODS: From 2006 to 2016, laparoscope-assisted living donor hepatectomy was performed in 11 patients (laparoscopic group), and conventional open living donor hepatectomy was performed in 40 patients (conventional group). Intraoperative and postoperative complications were evaluated according to the Clavien-Dindo classification and analyzed in the laparoscopic group for comparison with the conventional group. RESULTS: The median postoperative follow-up period was 88 months (range, 58-120 months) in the laparoscopic group. One donor in the conventional group died from a motor vehicle crash 16 months after surgery. All others were alive and returned to their preoperative activity level. Regarding intraoperative and early (≤90 days after surgery) postoperative complications, 1 patient (1/11, 9%) showed biliary fistula (Grade IIIa) in the laparoscopic group. In the conventional group, 6 patients (6/40, 15%) showed surgical complications of Grade I in 2 patients and Grade II in 4 patients. Regarding late (>90 days after surgery) postoperative complications, biliary stricture was observed in 1 patient of the laparoscopic group; this patient developed hepatolithiasis 6 years after surgery, and endoscopic lithotomy and extracorporeal shockwave lithotripsy were performed, resulting in successful treatment. Late complications were not observed in the conventional group. CONCLUSION: One donor in the laparoscopic group showed Grade IIIa late complications. The introduction of laparoscopic surgery to living donor hepatectomy should be performed carefully.

Induction of starfish oocyte maturation by the beta gamma subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm.

beta gamma subunits of G proteins were purified from starfish oocytes, and their role in the induction of oocyte maturation by 1-methyladenine was investigated. When injected into starfish oocytes, the purified beta gamma subunit of the starfish G protein induced germinal vesicle breakdown (GVBD) faster than that of bovine brain G protein. Injection of the starfish beta gamma into cytoplasm near the germinal vesicle (GV) induced GVBD earlier than when injected into the GV or the cytoplasm near the plasma membrane. Fluorescent-labeled beta gamma was retained in the injected area even after GVBD. Injected beta gamma also induced the formation of maturation-promoting factor as well as an increase of histone H1 kinase activity. These results suggest that beta gamma dissociates from alpha-subunit by the stimulation of 1-methyladenine and interacts with a cytoplasmic effector, which results in formation of active cdc2 kinase.

Accelerated turnover of blood glucose in pertussis-sensitized rats due to combined actions of endogenous insulin and adrenergic beta-stimulation.

1. Epinephrine-induced hyperglycemia was attenuated by the treatment of rats with pertussis vaccine, but this attenuation was abolished when endogenous insulin was suppressed by streptozotocin or anti-insulin serum. It was concluded that epinephrine-induced hyperglycemia was counterbalanced by the hypoglycemic action of insulin, the secretion of which was markedly potentiated in pertussis-sensitized rats. 2. Without epinephrine, no hypoglycemia developed in pertussis-sensitized rats despite the higher blood level of insulin. Tracer experiments with [14C,3H] glucose or [14C]bicarbaonate showed that, in pertussis-sensitized rats, more glucose was liberated into the blood from hepatic gluconeogenesis at the expense of hepatic glycogenesis, thereby accelerating the turnover of blood glucose. 3. Since this activation of hepatic glucose production was reduced by propranolol, a beta-adrenergic blocking agent, it is very likely that adrenergic beta-stimulation is, at least partly, responsible for the metabolic alterations observed in pertussis-sensitized rats.

Isolation of a cDNA encoding mouse DNA topoisomerase III which is highly expressed at the mRNA level in the testis.

A cDNA (mTOP3) encoding mouse DNA topoisomerase III (topo III) was cloned using the human TOP3 cDNA as a probe. The deduced amino acid sequence of mouse topo III showed 86.8% identity with that of human topo III. Mouse TOP3 mRNA was highly expressed in the testis in comparison with other tissues. The TOP3 mRNA level in the testis increased slightly 14 days after birth and showed a marked increase in 17 days, times when the cells in pachytene phase begin to appear and increase in number.

Involvement of the beta gamma subunits of inhibitory GTP-binding protein in chemoattractant receptor-mediated potentiation of cyclic AMP formation in guinea pig neutrophils.

Cellular cyclic AMP formation in response to prostaglandin (PG) E1 was markedly potentiated by the chemoattractant formyl-Met-Leu-Phe (fMLP) in guinea pig neutrophils. This potentiation by fMLP was abolished by prior treatment of the cells with pertussis toxin, but not by the prevention of an fMLP-induced intracellular Ca2+ increase in the cells, indicating the direct involvement of the inhibitory GTP-binding protein (Gj), but not Ca2+, in the fMLP-induced potentiation of cyclic AMP formation. Cyclic AMP formation in the neutrophils was also unique in response to forskolin; the diterpene inhibited cyclic AMP formation stimulated by PGE1 plus fMLP at low concentrations, but it slightly stimulated the basal and fMLP-induced cyclic AMP formation at high concentrations. Such a forskolin-induced inhibition was also observed in the adenylyl cyclase of the cell membranes and detergent extract therefrom only when the cyclase was activated by GTP or its nonhydrolyzable analogue (GTP gamma S). The forskolin-inhibitable activity could be affinity-purified from the GTP gamma S-treated cell membranes with a forskolin-agarose column. The cyclase appeared to be purified as a complex with the GTP gamma S-bound alpha subunit of the stimulatory GTP-binding protein (Gs alpha), but not with the beta gamma subunits, as judged from immunoblot analysis with specific antisera. The GTP gamma S-bound Gs alpha-stimulated cyclase activity was further enhanced by beta gamma, and this enhancement was again inhibited by forskolin. These results suggest that the GTP-bound Gs alpha produced by PGE1 receptor stimulation and the beta gamma subunits released from Gj by fMLP receptor stimulation were acting synergistically in the cyclic AMP formation of intact neutrophils.

cDNA cloning of mouse BLM gene, the homologue to human Bloom's syndrome gene, which is highly expressed in the testis at the mRNA level.

We cloned a cDNA encoding the mouse homologue to human Bloom's syndrome gene (BLM). The deduced amino acid sequence of mouse Blm showed 76% identity to the human sequence with very high homology in seven consecutive domains characteristic of DNA and RNA helicases. The expression of mBLM mRNA was examined in various tissues. Extremely high expression was observed in the testis as compared with other tissues. The mBLM mRNA level in the testis began to increase 12-14 days after birth, corresponding to the appearance of cells in the pachytene phase.

Cloning of two isoforms of mouse DNA helicase Q1/RecQL cDNA; alpha form is expressed ubiquitously and beta form specifically in the testis.

We cloned cDNAs encoding mouse homologues for the human DNA helicase Q1/RecQL (human helicase Q1) which has homology with the Escherichia coli RecQ protein and found that they encode two isoforms. The two isoforms are identical over the entire sequence except for the carboxyl terminal sequence spanning less than 30 amino acids. One of the two isoforms, alpha, contains a sequence, KKRK, in the carboxyl terminus, which is also contained in human helicase Q1 and was confirmed to function as the nuclear localization signal. The other form, beta, does not contain such a sequence. Expression of mouse helicase Q1 mRNA is extremely and relatively high in the testis and the thymus, respectively. RT-PCR analysis revealed that helicase Q1alpha was expressed in all tissues tested and the beta form was expressed only in the testis. A survey of expression of Q1alpha and Q1beta mRNA in the testis after birth revealed that Q1alpha mRNA is expressed in all testes of mice aged from 7 days to 8 weeks, and the expression of Q1beta mRNA begins 14 days after birth, corresponding to the appearance of cells in the pachytene stage.

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells.

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.

On the mechanism of G protein beta gamma subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel.

The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

Selective coupling of purified alpha-subunits of pertussis toxin-substrate GTP-binding proteins to endogenous receptors in rat brain membranes treated with N-ethylmaleimide.

The membrane fraction prepared from rat brain was incubated with 0.5 mM N-ethylmaleimide (NEM) for 10 min. 3H-labelled agonist binding to muscarinic, A1-adenosine, opiate and alpha 2-adrenergic receptors was markedly inhibited by this NEM treatment of membranes, which interfered with the subsequent ADP-ribosylation of endogenous G-proteins by pertussis toxin. This indicated that the toxin target cysteine residues of the G-protein were modified by NEM. The NEM-induced inhibition of agonist bindings was mostly reversed by reconstitution of the alpha-subunits of purified Gi or Go into the membranes. The NEM-induced inhibition, together with the reversal by the G alpha reconstitution, was due to changes in the relative number of high- to low-affinity receptors solely without change in the total (high- plus low-affinity) receptor number. Thus, in NEM-treated membranes endogenous G-proteins become uncoupled from receptors, which were coupled to either Gi alpha or Go alpha. Reconstitution of NEM pre-treated membranes showed that Go acted in preference to Gi in interaction with muscarinic receptors and vice versa in interaction with three other types of receptor. The possible involvement of Go in mediating phospholipase C activation and Gi in mediating adenylate cyclase inhibition is discussed.

Immunochemical comparison of pertussis toxin substrates in brain and peripheral tissues.

The tissue distribution of pertussis toxin-sensitive GTP-binding proteins was examined using specific antibodies raised against the purified alpha-subunit of G0 from bovine brain or against synthetic peptides predicted from cDNAs for distinct Gi subtypes. GTP-binding proteins were partially purified from membrane fractions prepared from rabbit tissues including brain, heart, liver, lung, erythrocytes and neutrophils. Brain contained both G0 and Gi1. Gi1 was also found to be abundant in heart. All peripheral tissues contained readily detectable amounts of Gi2, whereas only barely detectable amounts of Gi2 were found in brain. Gi3 was found to be prominent in erythrocytes and exists as a minor component of G proteins in neutrophils and liver. Thus, Gi2 appears to be widely disseminated in peripheral rabbit tissues, while other pertussis toxin substrates are more limited in their distribution.

Perfusion of the pancreas isolated from pertussis-sensitized rats: potentiation of insulin secretory responses due to beta-adrenergic stimulation.

In order to study the mechanism by which pertussis-sensitized rats showed enhanced insulin secretory responses to various secretagogues (Sumi, T., and M. Ui, Endocrinology 97: 352, 1975), pancreases of rats receiving a single injection of Bordetella pertussis cells 3 days before were perfused with Krebs-Ringer solution, and release of insulin therefrom was compared with that from the pancreases of normal rats. Much more insulin was released from the pancreas of the pertussis-sensitized rat than from the pancreas of the normal rat in response to glucose, arginine, glibenclamide and 3-isobuty-l-methylxanthine. The inhibition of insulin secretion caused by epinephrine, norepinephrine or phenylephrine via alpha-adrenergic receptors in the pancreas of normal rats was no longer observable with the pancreas from pertussis-sensitized rats. Instead, the addition of epinephrine with or without phentolamine gave rise to a marked secretion of insulin from the pancreas of pertussis-sensitized rats which was prevented by propranolol. It is concluded that a single injection of B. pertussis into rats results in a sustained modification of insulin secretory processes in the pancreatic beta-cells in such a manner as to favor insulin secretory responses to beta-adrenergic stimulation and other secretagogues.