MRI features of extraocular muscle metastases compared to those of other extraocular muscle diseases of non-thyroid origin.

AIM: To identify the magnetic resonance imaging (MRI) features of metastases to the extraocular muscles (EOM metastases). MATERIAL AND METHODS: The MRI features of 19 patients with EOM metastases were compared with those of 24 patients with EOM diseases of non-thyroid origin. MRI was used to assess the number of tumours, morphology, signal intensity on T2-weighted images, enhancement patterns, and apparent diffusion coefficient (ADC) values. RESULTS: Single muscular involvement was observed in 10 patients, and multiple muscular involvement was observed in nine patients. The morphology was focally discrete in nine patients, and diffuse infiltrative in 10 patients; all the nine patients with focal discrete morphology presented with single muscular lesions. On T2-weighted images, the signal intensities were intermediate or low in 15 patients and a mixture of high and intermediate in four patients. In 14 patients for whom contrast-enhanced images were available, ring enhancement (n=5), heterogeneous diffuse enhancement (n=5), and homogeneous enhancement (n=4) were seen. The mean ADC value was 0.98 × 10-3 mm2/s. Compared to other EOM diseases of non-thyroid origin, single muscular presentation, focal discrete morphology, the presence of hyperintensity on T2-weighted images, and ring or heterogeneous enhancement were significantly more frequent in EOM metastases. CONCLUSION: The MRI features of EOM metastases showed two main patterns: a single discrete mass and multiple infiltrative masses. In addition to the presentation as a single discrete mass, the presence of hyperintensity on T2-weighted images and ring or heterogeneous enhancement can aid in the differentiation of EOM metastases from other EOM diseases.

An anatomical hypothesis: a "concentric-structured model" for the theoretical understanding of the surgical anatomy in the upper mediastinum required for esophagectomy with radical mediastinal lymph node dissection.

Understanding the surgical anatomy is the key to reducing surgical invasiveness especially in the upper mediastinal dissection for esophageal cancer, which is supposed to have a significant impact on curability and morbidity. However, there is no theoretical recognition regarding the surgical anatomy required for esophagectomy, although the surgical anatomy in abdominal digestive surgery has been developed on the basis of embryological findings of intestinal rotation and fusion fascia. Therefore, we developed a hypothesis of a 'concentric-structured model' of the surgical anatomy in the upper mediastinum based on human embryonic development. This model was characterized by three factors: (1) a concentric and symmetric three-layer structure, (2) bilateral vascular distribution, and (3) an 'inter-layer potential space' composed of loose connective tissue. The concentric three-layer structure consists of the 'visceral layer', the 'vascular layer', and the 'parietal layer': the visceral layer containing the esophagus, trachea, and recurrent laryngeal nerves as the central core, the vascular layer of major blood vessels surrounding the visceral core to maintain the circulation, and the parietal layer as the outer frame of the body. The bilateral vascular distribution consists of the inferior thyroid arteries and bronchial arteries originating from the bilateral dorsal aortae in an embryo. This bilateral vascular distribution may be related to the formation of the proper mesentery of the esophagus and frequent lymph node metastasis observed in the visceral layer around recurrent laryngeal nerves. The three concentric layers are bordered by loose connective tissue called the 'inter-layer potential space'. This inter-layer potential space is the fundamental factor of our concentric-structured model as the appropriate surgical plane of dissection. The peripheral blood vessels, nerves, and lymphatics transition between each layer, thereby penetrating this loose connective tissue forming the inter-layer potential space. Recurrent laryngeal nerves also transition from the vascular layer after branching off from the vagal nerves and then ascend consistently in the visceral layer. We investigated the validity of this concentric-structured model, confirming the intraoperative images and the surgical outcomes of thoracoscopic esophagectomy in a prone position (TSEP) before and after the introduction of this hypothetical anatomy model. A total of 226 patients with esophageal cancer underwent TSEP from January 2015 to December 2016. After the introduction of this model, the surgical outcomes in 105 patients clearly improved for the operation time of the thoracoscopic procedure (160 min vs. 182 min, P = 0.01) and the incidence of recurrent laryngeal nerve palsy (19.0% vs. 36.4%, P = 0.004). Moreover, we were able to identify the concentric and symmetric layer structure through surgical dissection along the inter-layer potential space between the visceral and vascular layers ('viscero-vascular space') in all 105 cases after introduction of the hypothetical model. The concentric-structured model based on embryonic development is clinically beneficial for achieving less-invasive esophagectomy by ensuring a theoretical understanding of the surgical anatomy in the upper mediastinum.

Anti-allodynic action of the disease-modifying anti-rheumatic drug iguratimod in a rat model of neuropathic pain.

OBJECTIVE: Patients with rheumatoid arthritis experience nociceptive as well as neuropathic pain. The effect of iguratimod (IGU), a disease-modifying anti-rheumatic drug, on neuropathic pain in a rat model of chronic constriction injury (CCI) was examined in this study. METHODS: CCI was induced by making four ligations on the left sciatic nerve. Rats with stable signs of static allodynia were selected 2 weeks after the surgery and drug treatments were started (day 0). The test drugs were orally administered once daily for 15 days. The threshold of mechanical pain response in the hind paw was evaluated by the von Frey hair test in a blinded manner. To observe histological changes in the spinal cord, the L4 region was subjected to immunohistochemical analysis for the detection of microglial cells. RESULTS: IGU showed an anti-allodynic effect on CCI-induced neuropathic pain at days 6 and 14, but not at 90 min after the first administration of IGU. This effect of IGU was observed until day 21. Furthermore, IGU decreased the number of Iba-1-positive cells, which had been increased at the ipsilateral side of the dorsal horn by CCI. CONCLUSIONS: These results suggest that IGU suppresses neuropathic pain via a different mechanism from that of current therapeutics.

Adsorption efficiency of natural materials for low-concentration cesium in solution.

In this study, several natural materials were investigated in order to clarify their potential use as cesium (Cs) adsorbents in situ. Four materials--carbonized rice hull, beech sawdust, oak sawdust, and charcoal (Japanese cedar)--which were previously shown to have Cs adsorption capabilities, were examined. Cs adsorption experiments were conducted using different initial Cs and adsorbent concentrations. The physical properties, adsorption isotherms, and adsorption processes were then examined, so as to exploit the Cs adsorption characteristics in the field. Based on these findings, carbonized rice hull and beech sawdust were selected as effective Cs adsorbents. It was found that these materials show continuous and stable Cs adsorption rates for different initial Cs concentrations. The adsorption efficiency of these two adsorption materials in combination was considered, and it was shown that the adsorption isotherms for carbonized rice hull and beech sawdust follow the Freundlich model. Furthermore, the beech sawdust adsorption process exhibited better agreement with the calculated values obtained via the adsorption rate model and the adsorption kinetics model than did the carbonized rice hull adsorption.

Kinetic Control of the Li0.9Mn1.6Ni0.4O4 Spinel Structure with Enhanced Electrochemical Performance.

The development of more sustainable societies has become an urgent goal worldwide. Electrical batteries are currently seen as one of the most important energy storage technologies for the development of decarbonized societies. However, many lithium-ion battery manufacturers currently utilize cobalt, a toxic and hazardous mineral, in their batteries. Lithium-deficient manganese nickel oxide spinels are considered promising candidates owing to their high potential and environmental friendliness. Their electrochemical performance highly depends on their average and local structures, such as phase purities, lattice parameters, and cation sites. Thus, a synthesis protocol should be designed to control these structural parameters to improve their electrochemical performance. In this study, we controlled the average and local structures of Li0.9Mn1.6Ni0.4O4 spinels obtained by co-precipitation by optimizing their cooling rates. High-resolution techniques, including transmission electron microscopy, synchrotron X-ray diffraction, and Auger-composition analysis combined with density functional theory calculations, X-ray absorption spectroscopy, and electrochemical analysis, were used to understand the average and local structural variations and their effects on the electrochemical properties. As a result, the control of oxygen diffusion at different cooling rates can promote the rearrangement of the structure, resulting in a cation-disordered spinel with minimal variations in lattice parameters and composition. Excellent electrochemical properties were noted in the cation-disordered spinel with high crystallinity and a slightly oxygen-rich surface produced via optimized cooling rates.

AICAR and metformin, but not exercise, increase muscle glucose transport through AMPK-, ERK-, and PDK1-dependent activation of atypical PKC.

Activators of 5'-AMP-activated protein kinase (AMPK) 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), metformin, and exercise activate atypical protein kinase C (aPKC) and ERK and stimulate glucose transport in muscle by uncertain mechanisms. Here, in cultured L6 myotubes: AICAR- and metformin-induced activation of AMPK was required for activation of aPKC and ERK; aPKC activation involved and required phosphoinositide-dependent kinase 1 (PDK1) phosphorylation of Thr410-PKC-zeta; aPKC Thr410 phosphorylation and activation also required MEK1-dependent ERK; and glucose transport effects of AICAR and metformin were inhibited by expression of dominant-negative AMPK, kinase-inactive PDK1, MEK1 inhibitors, kinase-inactive PKC-zeta, and RNA interference (RNAi)-mediated knockdown of PKC-zeta. In mice, muscle-specific aPKC (PKC-lambda) depletion by conditional gene targeting impaired AICAR-stimulated glucose disposal and stimulatory effects of both AICAR and metformin on 2-deoxyglucose/glucose uptake in muscle in vivo and AICAR stimulation of 2-[(3)H]deoxyglucose uptake in isolated extensor digitorum longus muscle; however, AMPK activation was unimpaired. In marked contrast to AICAR and metformin, treadmill exercise-induced stimulation of 2-deoxyglucose/glucose uptake was not inhibited in aPKC-knockout mice. Finally, in intact rodents, AICAR and metformin activated aPKC in muscle, but not in liver, despite activating AMPK in both tissues. The findings demonstrate that in muscle AICAR and metformin activate aPKC via sequential activation of AMPK, ERK, and PDK1 and the AMPK/ERK/PDK1/aPKC pathway is required for metformin- and AICAR-stimulated increases in glucose transport. On the other hand, although aPKC is activated by treadmill exercise, this activation is not required for exercise-induced increases in glucose transport, and therefore may be a redundant mechanism.

Doxorubicin, cisplatin, and fluorouracil combination therapy for metastatic esophageal squamous cell carcinoma.

The chemotherapy regimen currently used for treating esophageal and gastric carcinoma has been either epirubicin, cisplatin, and fluorouracil (5-FU) or docetaxel, cisplatin, and 5-FU. Here, we report the efficacy and toxicity of doxorubicin, cisplatin, and 5-FU for only esophageal squamous cell carcinoma (ESCC). Between January 2000 and October 2008, a total of 41 ESCC patients with a distant metastasis were enrolled. The most common sites of metastasis were liver (26, 63.4%), lung (9, 22.0%), and bone (8, 19.5%). Doxorubicin was administered on day 1 at 30 mg/m(2) , cisplatin on days 1-5 at 14 mg/m(2)/day, and 5-FU on days 1-5 at 700 mg/m(2)/day. The median number of cycles was 2.0 (range 1-8). The dose intensities of doxorubicin, cisplatin, and 5-FU were 92.9, 92.4, and 92.5%, respectively. The overall response rate was 43.9%; one showed complete response, 17 showed partial response, 13 showed a stable disease, and 10 showed progressive disease (PD). The median survival time was 306 days (95% CI = 74-935) and the 1-year survival rate was 37.6%. Grade 3 neutropenia was seen in seven patients and grade 4 in one patient. Grade 3 fatigue, anorexia, mucositis, and diarrhea were observed in three, two, two, and one patient, respectively. This regimen is effective as a first-line therapy for ESCC with distant metastasis.

Role of atypical protein kinase C in activation of sterol regulatory element binding protein-1c and nuclear factor kappa B (NFkappaB) in liver of rodents used as a model of diabetes, and relationships to hyperlipidaemia and insulin resistance.

AIMS/HYPOTHESIS: Previous findings in rodents used as a model of diabetes suggest that insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but, unexpectedly, conserved in liver, despite impaired hepatic protein kinase B (PKB/Akt) activation. Moreover, aPKC at least partly regulates two major transactivators: (1) hepatic sterol receptor binding protein-1c (SREBP-1c), which controls lipid synthesis; and (2) nuclear factor kappa B (NFkappaB), which promotes inflammation and systemic insulin resistance. METHODS: In Goto-Kakizaki rats used as a model of type 2 diabetes, we examined: (1) whether differences in hepatic aPKC and PKB activation reflect differences in activation of IRS-1- and IRS-2-dependent phosphatidylinositol 3-kinase (PI3K); (2) whether hepatic SREBP-1c and NFkappaB are excessively activated by aPKC; and (3) metabolic consequences of excessive activation of hepatic aPKC, SREBP-1c and NFkappaB. RESULTS: In liver, as well as in muscle, IRS-2/PI3K activation by insulin was intact, whereas IRS-1/PI3K activation by insulin was impaired. Moreover, hepatic IRS-2 is known to control hepatic aPKC during insulin activation. Against this background, selective inhibition of hepatic aPKC by adenoviral-mediated expression of mRNA encoding kinase-inactive aPKC or short hairpin RNA targeting Irs2 mRNA and partially depleting hepatic IRS-2 diminished hepatic SREBP-1c production and NFkappaB activities, concomitantly improving serum lipids and insulin signalling in muscle and liver. Similar improvements in SREBP-1c, NFkappaB and insulin signalling were seen in ob/ob mice following inhibition of hepatic aPKC. CONCLUSIONS/INTERPRETATION: In diabetic rodent liver, diminished PKB activation may largely reflect impaired IRS-1/PI3K activation, while conserved aPKC activation reflects retained IRS-2/PI3K activity. Hepatic aPKC may also contribute importantly to excessive SREPB-1c and NFkappaB activities. Excessive hepatic aPKC-dependent activation of SREBP-1c and NFkappaB may contribute importantly to hyperlipidaemia and systemic insulin resistance.

Effect of Te substitution on crystal structure and transport properties of AgBiSe2 thermoelectric material.

Silver bismuth diselenide (AgBiSe2) has attracted much attention as an efficient thermoelectric material, owing to its intrinsically low lattice thermal conductivity. While samples synthesized using a solid-state reaction showed n-type conductivity and their dimensionless figure of merit (ZT) reached ∼1 by electron doping, theoretical calculations predicted that a remarkably high thermoelectric performance can be achieved in p-type AgBiSe2. In this paper, we present the effect of Te substitution on the crystal structure and thermoelectric properties of AgBiSe2, expecting p-type conductivity due to the shallowing of the energy potential of the valence band. We found that all AgBiSe2-xTex (x = 0-0.8) prepared using a solid-state reaction exhibits n-type conductivity from 300 to 750 K. The room-temperature lattice thermal conductivity decreased to as low as 0.3 W m-1 K-1 by Te substitution, which was qualitatively described using the point defect scattering model for the solid solution. We show that ZT reaches ∼0.6 for x = 0.8 at a broad range of temperatures, from 550 to 750 K, due to the increased power factor, although the carrier concentration has not been optimized yet.

Minerals and Trace Elements in Milk, Milk Products, Infant Formula, and Adult/Pediatric Nutritional Formula, ICP-MS Method: Collaborative Study, AOAC Final Action 2015.06, ISO/DIS 21424, IDF 243.

AOAC Final Action Official MethodSM 2015.06 "Minerals and Trace Elements in Milk, Milk Products, Infant Formula and Adult/Pediatric Nutritional Formula, ICP-MS Method" was collaboratively studied. Note that "milk, milk products" has now been added to the title of the Final Action method because whole milk and several dairy ingredients were successfully incorporated into the collaborative study for the purpose of developing an International Organization for Standardization/International Dairy Federation standard (ISO/DIS 21424; in progress). The method determines sodium, magnesium, phosphorus, potassium, calcium, iron, manganese, zinc, copper, chromium, molybdenum, and selenium by inductively coupled plasma (ICP)-MS after microwave digestion. Ten laboratories participated in the study, and data from five different model ICP-MS units were represented. Thirteen products, five placebo products, and six dairy samples were tested as blind duplicates in this study, along with a standard reference material, for a total 50 samples. The overall repeatability and reproducibility for all samples met Standard Method Performance Requirements put forth by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals, with a few exceptions. Comparisons are made to ICP-atomic emission data from a collaborative study of AOAC Official Method 2011.14 carried out concurrently on these same samples.

Surface discharge of heavy metals from low farmland.

Runoff heavy metals from farmland were examined using the field data for the summer of 2005. The observation farmland is located on lowland where the irrigation water was contaminated with the drained water from the upstream farmlands. The area of the farmland is 11.2 ha, of which 6.0 ha and 4.5 ha have been used for rice paddy fields and soybean cultivation, respectively. During the observation, heavy metal concentrations at the downstream end were usually found to be higher than those in the irrigation water. That is, the heavy metal concentrations increased due to the passage of the water through the farmland. This increase in the heavy metal concentrations is not equal to the discharge of the heavy metal because the evaporation on the surface of the paddy field and the absorption by plants makes the surface water volume small. The discharged load from the farmland generally indicates the gross surface load from the farmland. When the effects of circulation irrigation on the heavy metal concentrations are estimated, the discharged load from the farmland should be calculated as the net surface load. When the runoff heavy metals from the circulation irrigation farmland are estimated, it is important to consider the inflowing heavy metals with irrigation water. All the heavy metal types observed in this study were discharged from the farmland. The net surface loads of Cr, Fe, Cd, and Pb were 371 microg m(-2) day(-1), 14.9 mg m(-2) day(-1), 0.26 microg m(-2) day(-1), and 3.3 microm( -2) day(-1), respectively.

Application of the Dental Hygiene Human Needs Conceptual Model and the Oral Health-Related Quality of Life Model to the dental hygiene curriculum in Japan.

This paper reports the incorporation of the Dental Hygiene Human Needs Conceptual Model (DHHN) and the Oral Health-Related Quality of Life Model (OHRQL) into a dental hygiene curriculum in Japan. A simulated patient practice was offered to 67 dental hygiene students. In the practice activity, all students were introduced to the use of an OHRQL assessment tool. A DHHN assessment tool was utilized additionally only by the experimental student group. The statistical analysis of the post-practice survey showed that the OHRQL instrument was more helpful in assessment and problem identification than the DHHN instrument. By contrast, text-based analysis of dental hygiene diagnostic statements showed that the experimental group identified more domains of patients' human needs deficits than the control group. This suggested the possibility that the DHHN model helped them to see patients from broader perspectives. However, it was difficult for students to design care plans attending to the domains of the models. Also, in considerations to the cultural issues, the validity and equivalence of the Japanese versions of both models should be further investigated. Within the limitation of the present study, the results suggested that incorporation of the combination of the DHHN and OHRQL models can be useful in a dental hygiene curriculum, as each tool helps students expand the perspective from which they view client. Further improvements in learning strategies should facilitate the effective utilization of these models.

Effect of tumor necrosis factor-alpha on insulin signal transduction in rat adipocytes: relation to PKCbeta and zeta translocation.

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.

Differential effect of the antidiabetic thiazolidinediones troglitazone and pioglitazone on human platelet aggregation mechanism.

Troglitazone and pioglitazone, antidiabetic thiazolidinediones, are known to improve insulin resistance. However, the effect of these drugs on platelet aggregation remains unclear. The chemical structure of troglitazone contains vitamin E. Accordingly, we studied the effect of troglitazone, pioglitazone, and vitamin E on thrombin-induced platelet aggregation, metabolism of phosphoinositide, protein phosphorylation, protein kinase C (PKC)-alpha and -beta, and phosphatidylinositol (PI) 3-kinase activation in vitro in human platelets. Maximum platelet aggregation by ADP, collagen, and thrombin decreased in the presence of 0.1-1 micromol/l troglitazone and 500 nmol/l vitamin E for 60 min compared with controls. However, pioglitazone did not inhibit ADP-, collagen-, or thrombin-induced platelet aggregation. Pretreatment with troglitazone and vitamin E, but not with pioglitazone, resulted in decreases in thrombin-induced phosphatidic acid production, hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, and 47-kDa protein phosphorylation. Thrombin-induced PKC-alpha and -beta activation in membrane fraction was suppressed by pretreatment with troglitazone and vitamin E, but not with pioglitazone. Separately, troglitazone and pioglitazone stimulated PI 3-kinase activity, but thrombin-induced PI 3-kinase activation was suppressed by pretreatment with troglitazone and pioglitazone for 60 min. These results suggest that troglitazone and vitamin E, but not pioglitazone, have a potent inhibitory effect on platelet aggregation via suppression of the thrombin-induced activation of phosphoinositide signaling in human platelets. Finally, the chemical structure of vitamin E may contribute to the inhibitory effect of troglitazone on platelet aggregation in human platelets.

Increased expression of ribosomal genes during inhibition of ribosome assembly in Escherichia coli.

We have examined a prediction made from the ribosome feedback regulation model of ribosomal RNA and transfer RNA synthesis in Escherichia coli. This model proposes that non-translating "free" ribosomes act directly or indirectly as negative feedback inhibitors to regulate the transcription of rRNA and tRNA operons. One prediction of this model is that preferential inhibition of the assembly of ribosomes (without inhibiting macromolecular synthesis) should lead to a deficiency of free ribosomes which should, in turn, cause a stimulation of rRNA (and tRNA) synthesis. We have examined this prediction in vivo by causing the preferential inhibition of synthesis of certain ribosomal proteins by an overproduction of the translational repressor ribosomal protein S4. In agreement with our model, we have observed a preferential stimulation of ribosomal protein messenger RNA and rRNA synthesis under these conditions. These results suggest that ribosomes in the cellular pool, rather than incomplete ribosomal particles or free rRNA, are monitored by cells to regulate the rate of rRNA synthesis, and give further support to this proposed model.

Glucocorticoid-induced insulin resistance associates with activation of protein kinase C isoforms.

We studied glucocorticoid-induced insulin resistance and possible role of protein kinase C (PKC). Pretreatment with dexamethasone, prednisolone and corticosterone for 60 min decreased insulin-induced [3H] 2-deoxyglucose (DOG) uptake in isolated rat adipocytes. Preincubation with Go6976, LY379196 or myristoylated PKC pseudosubstrate, conventional PKC inhibitor, but not cycloheximide or RU38486, recovered dexamethasone-induced insulin resistance. Dexamethasone activated immunoprecipitates with anti-PKC alpha, beta, and zeta antibodies. PKC zeta activity in adipocytes increased to 163%, and 264% from basal level (100%) with dexamethasone and insulin treatment, respectively. Dexamethasone provoked redistribution of both PKC beta and zeta from the cytosol to the membrane. These results indicate that dexamethasone activates both conventional and atypical PKC. However, conventional PKC is more important in glucocorticoid-induced insulin resistance.

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles.

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.

Regulation of BCL-6 gene expression in human myeloid/monocytoid leukemic cells.

We demonstrated in the present study that the BCL-6 transcripts were detectable not only in B cells, but also in circulating granulocytes and monocytes from normal individuals, and in human acute nonlymphocytic leukemia cells of certain subtypes (M3, M4, M5). Then, with an assumption that the BCL-6 gene expression may be related to the differentiation of myeloid cells, we analyzed the inducibility of BCL-6 gene expression along monocytic lineage differentiation in HL-60 and U-937 cells by treating them with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although the expression of BCL-6 transcripts was very low or undetectable in untreated HL-60 or U-937 cells, treatment of these cells with TPA to induce monocytic differentiation resulted in an apparent increase of BCL-6 mRNA, suggesting that BCL-6 gene expression is not limited to B cells and it is closely associated with monocytic lineage differentiation. The BCL-6 transcripts in TPA-treated U-937 cells were superinduced by the treatment with cycloheximide (CHX) and the half-life of the BCL-6 mRNA was apparently prolonged when TPA-treated U-937 cells were exposed to CHX in the presence of actinomycin D (ACD). Furthermore, the nuclear run-on assay revealed that the BCL-6 transcription signals were enhanced by TPA treatment. These results suggest that the increase of BCL-6 mRNA in U-937 cells stimulated with TPA to induce monocytic lineage differentiation is mediated by both transcriptional and post-transcriptional regulation.

Cell-permeable ceramide inhibits the growth of B lymphoma Raji cells lacking TNF-alpha-receptors by inducing G0/G1 arrest but not apoptosis: a new model for dissecting cell-cycle arrest and apoptosis.

We examined the effects of a cell-permeable ceramide analog, C2-ceramide, on the growth of TNF-alpha-resistant B lymphoma Raji cells lacking TNF-alpha-receptors (TNF-R). C2-ceramide inhibited the clonal growth of not only TNF-alpha-sensitive myeloid leukemia cells (HL60 and U937) but also Raji cells. Following stimulation with C2-ceramide, HL60 and U937 cells showed apoptotic cell death, whereas Raji cells did not show a detectable level of apoptosis. However, a cell-cycle arrest in G0/G1 phase was observed in Raji cells after the treatment with C2-ceramide, which was accompanied by the dephosphorylation of retinoblastoma (RB) gene products and decreased expression of p53 proteins. Failure of C2-ceramide to induce apoptosis in Raji cells might be explained by the lack or low expression of apoptosis-inducing proteins by two lines of evidence: (1) Raji cells were resistant to apoptosis induced by ceramide even in the presence of transcription/translation inhibitors; (2) Bax protein expression was not detectable in Raji cells, although Bcl-2 protein expression in Raji cells was even less than that in HL60 and U937 cells. Moreover, protein kinase C (PKC), whose activation has been described to inhibit ceramide-induced apoptosis, inhibitor H-7 did not induce apoptotic cell death in Raji cells, suggesting that an imbalance between PKC and ceramide pathways is not the reason for the resistance of Raji cells against ceramide-induced apoptosis. Finally, ceramide-induced activation of nuclear factor kappaB (NF-kappaB) was observed in Raji cells as well as HL60 cells, indicating that activation of this molecule may not be specific for apoptosis. By using the present model, one can dissect cell-cycle arrest and apoptosis induced by ceramide.