Effects of peripheral administration of a Neuromedin U receptor 2-selective agonist on food intake and body weight in obese mice.

BACKGROUND: Neuromedin U (NMU) is a neuropeptide with various physiological functions, including regulation of smooth-muscle contraction, blood pressure, stress responses and feeding behaviors. NMU activates two distinct receptors, NMUR1 and NMUR2, which are predominantly expressed in peripheral tissues and the central nervous system (CNS), respectively. It is reported that the NMU signaling system regulates food intake (FI) and body weight (BW) via NMUR2, suggesting that an NMUR2 agonist exhibiting anorectic effects would be a potential therapy for obesity. METHODS: Antiobesity effects of NMUR2 activation were assessed using a recently developed, novel NMUR2-selective agonist, NMU-7005 (a polyethylene glycolated octapeptide). Here we assessed cumulative FI and BW loss after peripheral administration of NMU-7005 in NMUR2 knockout and diet-induced obese mice. To gain mechanistic insights, we performed immunohistochemical analysis of c-Fos-like protein expression in the brain. RESULTS: We found that NMU-7005 was a NMUR2-selective agonist with little activity toward NMUR1. The anorectic effect of NMU-7005 was completely abrogated in NMUR2 knockout mice. Repeated subcutaneous administration of NMU-7005 showed a potent antiobesity effect with FI inhibition (P<0.025) in diet-induced obese mice. NMU-7005 in combination with the glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide showed an additive antiobesity effect, suggesting that NMUR2-mediated anorectic action is different from that of GLP-1R agonists. NMU-7005 also elicited a minimal conditioned taste-aversive effect, while the effect of liraglutide was significant. As c-Fos expression was upregulated in the hypothalamus and the medulla oblongata in NMU-7005-administered mice, the pharmacological effects of NMU-7005 appeared to be mediated via activation of the CNS. CONCLUSION: Our results demonstrated that a novel NMUR2-selective agonist, NMU-7005, is a beneficial tool for the elucidation of NMUR2-mediated physiological functions, which is a promising therapeutic strategy for treating obesity.

Final overall survival analysis from the phase III J-ALEX study of alectinib versus crizotinib in ALK inhibitor-naïve Japanese patients with ALK-positive non-small-cell lung cancer.

BACKGROUND: Mature progression-free survival (PFS) data from the phase III J-ALEX study showed superiority for alectinib versus crizotinib [hazard ratio (HR) 0.37, 95% confidence interval (CI) 0.26-0.52; median PFS 34.1 versus 10.2 months, respectively] in advanced ALK (anaplastic lymphoma kinase)-positive non-small-cell lung cancer (NSCLC). Overall survival (OS) data were immature (HR 0.80, 99.8799% CI 0.35-1.82) at the time of data cut-off (30 June 2018). We report final OS data after ≥5 years of follow-up. PATIENTS AND METHODS: ALK inhibitor naive Japanese patients who were chemotherapy naive or had received one prior chemotherapy regimen were enrolled. Patients were randomized to receive alectinib 300 mg (n = 103) or crizotinib 250 mg (n = 104) twice daily until progressive disease, unacceptable toxicity, death, or withdrawal. The primary endpoint was independent review facility-assessed PFS, with OS (not fully powered) as a secondary endpoint. RESULTS: Median duration of OS follow-up was 68.6 months with alectinib and 68.0 months with crizotinib. Treatment with alectinib did not prolong OS relative to crizotinib (HR 1.03, 95.0405% CI 0.67-1.58; P = 0.9105). Five-year OS rates were 60.9% (95% CI 51.4-70.3) with alectinib and 64.1% (95% CI 54.9-73.4) with crizotinib. In total, 91.3% (n = 95/104) of crizotinib-treated patients and 46.6% (n = 48/103) of alectinib-treated patients received at least one subsequent anticancer therapy. After study drug discontinuation, 78.8% of patients in the crizotinib arm switched to alectinib, while 10.7% of patients in the alectinib arm switched to crizotinib as a first subsequent anticancer therapy. Patients randomized to crizotinib tended to switch treatment earlier than those randomized to alectinib. CONCLUSION: Final OS analysis from J-ALEX did not show superiority of alectinib to crizotinib; this result was most likely confounded by treatment crossover. Alectinib remains a standard of care for the treatment of patients with advanced ALK-positive NSCLC.

Chemosensitivity of patients with recurrent esophageal cancer receiving perioperative chemotherapy.

Perioperative chemotherapy (CT) and chemoradiotherapy are widely used for advanced esophageal cancer. We evaluated the chemosensitivity of patients displaying recurrent esophageal cancer after esophagectomy with perioperative CT. From the database at National Cancer Center Hospital in Tokyo, we extracted recurrent esophageal cancer cases after perioperative CT and evaluated the effectiveness of the first CT against the recurrent disease according to the duration between termination of the original perioperative CT and recurrence with treatment-free intervals (TFIs) 6 months. Systemic CT for their recurrent disease was performed for 30 esophageal cancer patients after perioperative CT. All patients received 5-fluorouracil and cisplatin as perioperative CT, with relapses occurring at TFIs 6 months in 19 patients (all received platinum-containing regimens). The response rate of patients experiencing a recurrence at TFIs 6 months was 0 and 37% (P = 0.029), the median progression-free survival was 2.8 and 4.8 months (log-rank P = 0.001) and the median overall survival was 6.1 and 10.2 months (log-rank P = 0.012), respectively. Recurrence at the TFI

ADP-ribosylation of myelin basic protein by cholera toxin.

Cholera toxin ADP-ribosylates four types of myelin basic proteins (MBPs) of Mr 14,000, 17,500, 19,000 and 22,000 in rat brain myelin. On an analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MBP underwent mono- and multi-(ADP-ribosyl)ation by cholera toxin and thus modified MBP migrated on the gel as several discrete protein bands, the molecular masses of which were apparently larger by 500-2000 daltons than that of the corresponding untreated MBP. On average, 1.1 mol of ADP-ribosyl residue was incorporated into 1 mol of MBP. Four types of purified MBPs were also ADP-ribosylated by cholera toxin dependent on GTP and the protein factor for the ADP-ribosylation. The results show evidence that MBP is one of major and specific substrates of cholera toxin in brain membranes.

Non-invasive assessment of the age related changes in stiffness of major branches of the human arteries.

The static mechanical properties of major branches of the human arteries (common carotid artery, abdominal aorta, femoral artery, and brachial artery) were studied in 39 subjects, aged 6-81 years, using an ultrasonic phase locked echo tracking system that allows continuous transcutaneous measurement of the diameter of the artery. The stiffness indices were calculated from the relation between systemic blood pressure and arterial diameter. With advancing age there was a significant increase in the diameter of all arteries with a reduction in percentage change in diameter. The stiffness index increased with age in all arteries; however, in the brachial and femoral arteries there was considerable variation in the individual values for a given age. The age associated increase in stiffness was statistically significant only in the common carotid artery and the abdominal aorta. Although the mechanical properties of the peripheral arteries were significantly influenced by the measuring environment, the calculated stiffness indices were less vulnerable to these stimuli in the central arteries. These results indicate that the stiffness indices of the peripheral muscular arteries are modified appreciably by vasoactive stimuli and that the mechanical properties of the deeper elastic arteries provide sufficiently reliable information about changes caused by aging and arteriosclerosis. The new ultrasonic method used appears to be suitable for this analysis.

GTPase activity associates with the inhibitory GTP-binding regulatory component of adenylate cyclase purified from rat brain.

The inhibitory regulatory component of adenylate cyclase (Ni) was highly purified from rat brain synaptic membranes. A low Km GTPase activity was always associated with Ni through the purification, and the recovery of GTPase activity correlated well with that of Ni. Purified Ni was hardly ADP-ribosylated by islet-activating protein (IAP). A heat-labile factor in the fraction of the stimulative regulatory component (Ns) restored ADP-ribosylation and also activated the GTPase about 2-fold. NaF which was reported to interact with Ni markedly reduced GTPase activity. The purified Ni fraction inhibited adenylate cyclase only in the presence of a heat-stable factor found in the partially purified regulatory component. GTPase and inhibitory activities were weak in myelin which contained only a small amount of Ni. These findings support the view that GTPase activity is an intrinsic activity of Ni and some factors are necessary for the function of Ni.

Inhibition of catalytic unit of adenylate cyclase and activation of GTPase of Ni protein by beta gamma-subunits of GTP-binding proteins.

A protein factor which inhibited adenylate cyclase was purified to apparent homogeneity from rat brain and identified as the beta gamma-subunits of the GTP-binding regulatory proteins of adenylate cyclase. (i) The beta gamma-subunits (protein factor) inhibited the partially purified catalytic unit of adenylate cyclase in the presence of an activator, forskolin or the stimulative regulatory protein (Ns), to 60 and 40% of the control, respectively; inhibition of the catalytic unit in the presence of forskolin required no guanine nucleotides. (ii) The subunits enhanced the GTPase activity of the purified alpha-subunit of the inhibitory regulatory protein (Ni alpha) 3.8-fold. (iii) The subunits stimulated ADP-ribosylation of Ni alpha catalyzed by islet-activating protein (pertussis toxin). ADP-ribosylation had no effect on the GTPase activity of Ni alpha in the presence of the beta gamma-subunits. The results suggest that direct inhibition of the catalytic unit by the beta gamma-subunits liberated from Ni is essential for the receptor-mediated inhibition of adenylate cyclase.

Preparation of subfragment-1 from abalone smooth muscle myosin.

Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.

Inhibitory effect of MgATP on the release of regulatory light chain from scallop myosin and light chain composition of scallop myosin hybridized with abalone light chain 2 at 30 degrees C.

The experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30 degrees C were studied. Substantially all RLC was released from myosin by incubation for 5 min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+, while the effect of Mg2+ was about 10,000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP. The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30 degrees C for 5 min produced a hybrid myosin. In the presence of 5 mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.

Two isoforms of regulatory light chain of abalone smooth muscle myosin.

Abalone myosin contains two kinds of light chain, regulatory light chain (LC2) and essential light chain (LC1) according to SDS-PAGE. Three distinct light chain bands were observed on polyacrylamide gel electrophoresis of purified abalone myosin in the presence of urea (urea-PAGE). The slower two components showed had mobility on SDS-PAGE and they also showed regulatory activity as the regulatory light chain. They were termed LC2-a and LC2-b in order of increasing mobility on urea-PAGE and isolated by DE-32 ion exchange column chromatography in the presence 8 M urea. The ratio of LC2-a and LC2-b in the central portion of adductor muscle of abalone (LC2-a: LC2-b = 7:3) was different from that (1:1) in the peripheral portion. These results suggest that the two light chains are isoforms of the regulatory light chain. The amino acid compositions of LC2-a and LC2-b were very similar to each other except for the Cys content. The UV absorption spectra were also quite similar, as were the UV difference absorption spectra induced by Ca2+. Phosphorylation was not detectable with the myosin light chain kinase of chicken gizzard. The Ca2+ concentration dependencies of Mg-ATPase activity of LC2-a or LC2-b hybridized abalone myosin (a-myosin, b-myosin) were similar to each other in the absence of rabbit F-actin, but differed in the presence of actin. The b-myosin had a higher maximum value of actomyosin ATPase activity and a lower apparent binding constant of actin and myosin than a-myosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Difference UV-absorption spectrum of scallop adductor myosin induced by ATP.

A difference UV-absorption spectrum induced by ATP was observed with scallop myosins purified from both striated and smooth adductor muscles. The difference spectra showed a positive peak at 289 nm with a shoulder around 295 nm, and two small negative troughs around 280 nm. Tryptophanyl movement similar to that in rabbit skeletal myosin is indicated. Some tyrosyl movements in scallop myosins, however, may be in the opposite direction.

Light chains of abalone myosin. UV absorption difference spectrum and resensitization of desensitized scallop myosin.

1. Abalone myosin consisted of one kind of heavy chain and two kinds of light chain according to SDS gel electrophoresis. The molecular weights of the light chains were estimated as 16,600 daltons (Light Chain-1: LC-1) and 14,500 daltons (Light Chain-2: LC-2). 2. The amino acid composition of LC-2 was not appreciably different from that of LC-1 except that the valine residue was 1 mol per mol of LC-2. 3. both LC-1 and LC-2 showed a calcium-induced UV absorption difference spectrum through the apparent binding constants for Ca2+ were low (2.5 x 10(-3) M for LC-1 and 3.2 x 10(-4) M for LC-2). 4. The modification of carboxyl groups of LC-2 by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide caused the disappearance of the calcium-induced UV absorption difference spectrum. 5. Manganese ion could also induce a UV absorption difference spectrum with these light chains although the concentration of Mn2+ required to produce the difference spectrum was very high. 6. Abalone LC-2 bound to desensitized scallop myosin and restored the calcium sensitivity of the myosin but LC-1 did not.

Activation of rat brain adenylate cyclase by proteases: involvement of distinct protein components in the activation by ?-chymotrypsin and trypsin.

Adenylate cyclase in synaptic plasma membranes from rat brain is activated by ?-chymotrypsin or trypsin. These proteases also activate adenylate cyclase reconstituted from the catalytic subunit of adenylate cyclase and the partially purified fraction of the GTP-binding proteins containing both the stimulatory and inhibitory GTP-binding proteins. Properties of the activation of reconstituted adenylate cyclase by the proteases are as follows. (1) The proteases do not directly activate the catalytic subunit. However, the pre-treatment of the partially purified GTP-binding proteins with ?-chymotrypsin (100 ?g/ml) increases the subsequently reconstituted cyclase activity at least 3-fold. Trypsin (10-30 ?g/ml) much more weakly enhances the cyclase activity. (2) ?-Chymotrypsin and trypsin synergistically activate the cyclase. (3) Trypsin but not ?-chymotrypsin no longer activates the cyclase when the purified stimulatory GTP-binding protein (Gs) replaces the partially purified GTP-binding proteins. (4) The stimulatory effects of ?-chymotrypsin and trypsin on the cyclase activity are little or slight unless 5?-guanylylimidodiphosphate (Gpp(NH)p) is present in the reconstitution. (5) The purified ??-subunits of the GTP-binding proteins markedly inhibit adenylate cyclase. This inhibition is nearly completely attenuated by treating the ??-subunits with ?-chymotrypsin (> 10 ?g/ml). (6) Trypsin (1-10 ?g/ml) inactivates the GTPase of the ?-subunit of the inhibitory GTP-binding protein (Gi). This inactivation of the GTPase seems to correlate with the activation of the reconstituted adenylate cyclase by trypsin. We conclude that two distinct protein components are involved in the activation of adenylate cyclase by ?-chymotrypsin and trypsin. One component sensitive to ?-chymotrypsin is probably the ??-subunits of the GTP-binding proteins. The other component sensitive to trypsin may be the ?-subunit of Gi.

A method for culturing and transplanting biliary epithelial cell from syrian golden hamster.

The present paper describes the establishment of a method for simultaneous culturing of biliary epithelial cells (BECs) from the gall bladder (GB), extrahepatic bile duct (EBD) and intrahepatic bile duct (IBD) of the hamster. GB, EBD and IBD were cut from the biliary tree after collagenase perfusion of the liver. These biliary segments were minced into fragments. The fragments were embedded in collagen gel and cultured in Dulbecco's modified Eagle medium/HamF12 medium containing 10% fetal bovine serum. The various cells subsequently spread from the fragments and formed cellular sheets. After the fragments and flattened cells were removed with the aid of a Pasteur pipette under phase-contrast microscopy, the sheets remaining were found to be composed of cuboidal cells. These cuboidal cells were shown to express gamma glutamyl transpeptidase and cytokeratin 7, which are known to be specific markers of BECs. Ultrastructurally, a large number of microvilli were observed on the luminal surface and junctional complex and interdigitation was identifiable on the lateral surfaces. BEC cultures were subcultured by digestion with collagenase and dispase and then dissociated by subsequent digestion in trypsin and ethylenediaminetetraacetic acid and then maintained on collagen gel for up to 8 weeks. After several passages, the BECs in culture eventually increased in size and showed vacuoles in the cytoplasm. They demonstrated irreversible growth arrest at 9 weeks. The BECs tended to form cystic structures when the BECs with collagen gel were transplanted into the interscapular fat pads of syngeneic hamsters. We established a method for culturing and transplanting biliary cells from syrian golden hamsters. This method may help to clarify the mechanism of hepatobiliary diseases.

Is extensive lymphadenectomy necessary for surgical treatment of intramucosal carcinoma of the stomach?

This study was undertaken to elucidate those histological and gross features associated with gastric carcinoma that can be adequately treated by gastrectomy with less aggressive lymphadenectomy. The frequency of metastasis to the lymph nodes was analyzed in 514 cases of resected, solitary, gastric carcinomas. The frequency of metastasis to the lymph nodes increased in proportion to the increase in the extent of penetration by the cancer into the gastric wall. Lymph nodes were not involved in cases of intramucosal carcinoma of the intestinal type, by Laurén's histological classification. By contrast, metastasis to the lymph nodes was observed in cases of intramucosal carcinoma of the diffuse type, by Laurén's classification. We conclude that extensive lymphadenectomy is not mandatory for patients with intramucosal carcinoma of the stomach of the protruded type, since the lymph nodes do not become involved in this type of gastric carcinoma.

One-pot aerosol synthesis of ordered hierarchical mesoporous core-shell silica nanoparticles.

A mixed surfactant approach has been successfully employed in an aerosol-based synthesis of spherical silica particles exhibiting a new core-shell structure where the shell and the core exhibit different ordered mesoporosity and pore sizes.

Primary hepatic carcinoid tumor.

A primary hepatic carcinoid tumor arising in a 49-year-old woman is reported. The patient was admitted with multiple hepatic tumors and treated by a left lobectomy and cholecystectomy. Cut sections of the specimen revealed a solid and necrotic mass, measuring 10 x 12 x 13 cm, with multiple small satellite nodules. Histologically, the tumor cells had small oval-shaped nuclei and presented with a trabecular arrangement and rosette-like formation. Both Grimelius and Fontana-Mason stainings were positive. The tumor cells were positive for chromogranin A and negative for other antigens. Ultrastructural studies of the tumor cells revealed duct-like formation with microvilli and a cluster of dense small immature neurosecretory granules in the cytoplasm. These findings were consistent with those of carcinoid tumors. Postoperatively, the patient was treated with repeated transcatheter arterial chemoembolization for any remnant tumors. However, she died of the disease 5 years after the initial surgery. The autopsy findings suggested the primary site to be the liver.

Observation of triply charged metal ion clusters by electrospray and laser spray

Studies of the gas phase ion chemistry of triply charged metal ions, M(3+) = Sc(3+), Y(3+), La(3+), Ce(3+), and Yb(3+), were made by electrospray and laser spray. Triply charged ion ligand complexes, M(3+)(ligand)(n) were produced in the gas phase by electrospray and laser spray for the following ligands; glucose; sucrose; raffinose; cyclodextrin; ginsenoside Rb(1); dimethyl sulfoxide (DMSO) and hexamethylphosphoramide (HMPA). The ion evaporation mechanism must be invoked to explain the transfer of more surface active ions (e.g., NH(4)(+)(H(2)O)(n)) in solution to the gas phase, while the transfer of low surface active ions (e.g., La(3+)(sucrose)(n)) may be explained by the charged residue model. In general, the laser spray gives stronger ion signals than electrospray for aqueous and water/methanol solutions. The laser spray is found to be more suitable for the observation of ions with larger solvation energies (e.g., Sc(3+)(DMSO)(n)). These results may be due to the enrichment of the sample concentration by the selective vaporization of the volatile solvent on the tip of the stainless steel capillary and also to the finer droplet formation caused by the laser irradiation. Copyright 1999 John Wiley & Sons, Ltd.

Influences of an infusion of lipid emulsion on phagocytotic activity of cultured Kupffer's cells in septic rats.

An experimental study was undertaken to study the influences of an infusion of lipid emulsion on phagocytosis of Kupffer's cells in septic rats. Sepsis was induced in 13 rats by ligating the cecum. Five of them received glucose as the sole nonprotein calorie (septic-glucose group), four of the rats received 25% of the nonprotein calorie with lipid emulsion, Intralipid (septic-lipid group), and the remaining four rats did not receive any intravenous solution and were allowed access to water (septic-fasted group). Another four rats which received neither intravenous solution nor ligation of the cecum served as the control group. The intravenous infusion was carried out for 72 hr. The phagocytotic activity of Kupffer's cells was determined by the ability to engulf latex particles with a size of 1.09 micron, in vitro. The phagocytotic activity was enhanced by the presence of sepsis but it was inhibited by starvation. The difference in the phagocytotic activity between the septic-glucose group and the septic-lipid group was not significant. These results suggest that, insofar as an in vitro study is concerned, a 72-hr infusion of lipid emulsion at a rate of 25% of the total nonprotein calorie does not influence the phagocytotic activity of cultured Kupffer's cell obtained from septic rats.

Effects of total parenteral nutrition on the development of intestinal endotoxemia in rats: a comparison between glucose and lipid.

The effects of parenteral nutrition (PN) and of the difference in the PN regimens between glucose and lipid emulsion on the development of endogenous endotoxemia were studied in 40 Wister rats. Endotoxemia was induced by occluding the superior mesenteric vein (SMV) for 30 min. The plasma endotoxin in the portal blood at the time of the release of the SMV occlusion and that in the arterial blood 10 min after the release were quantified. Twenty of the 40 rats had received PN for 48 hr prior to the SMV occlusion. Ten of these 20 rats received the total nonprotein calorie (TNPC) solely with glucose, and the other 10 rats received 25% of the TNPC with lipid emulsion. Ten rats had been allowed free access to lab food until the SMV occlusion. The remaining 10 rats underwent neither the SMV occlusion nor PN, and served as the control group. Both the portal and the arterial endotoxin increased after the release of the SMV occlusion, however the portal endotoxin was higher than that of the arterial one. Both the portal and the arterial endotoxin of the rats supported by PN were significantly lower than those of the rats nourished by lab food, while they were higher than the control values. The difference in the PN regimens did not cause any alteration in the endotoxin levels. These results indicate that the development of intestinal endotoxemia was not influenced by the difference in the PN regimens, but it was rather influenced by a presence of intestinal content.