Effects of Cold Stimulation on Mitochondrial Activity and VEGF Expression in vitro.

We aimed to clarify the effects of cold stimulation at various temperatures on mitochondrial activity and vascular endothelial growth factor (VEGF) expression in vitro. Human fibroblast, human mesenchymal stem cell, and rat skeletal muscle myoblast cell lines were used. For each cell type, cells were divided into 4 groups and stimulated in various cold temperatures (0, 4, 17 and 25°C) 3 times for 15 min each by placement on crushed ice or floating on cold water set at each temperature. Control cells were subjected to warm water at 37°C. Factors related to mitochondrial activity, mitochondrial DNA copy numbers, and VEGF expression were analyzed 24 h after the last cold stimulation. In all cell types, significant increases of factors related to mitochondrial activity and mitochondrial DNA copy numbers were seen in the 4°C and 17°C-stimulated cells compared with control cells. In rat skeletal muscle cells stimulated at 4°C, VEGF expression significantly increased compared to the control cells. Our data suggest that cold stimulation at certain temperatures promotes mitochondrial activity, biogenesis and VEGF expression.

[Metastatic sternal tumor from thyroid papillary carcinoma; report of a case].

A 68-year-old male was referred to our department for treatment of a metastatic sternal tumor in the manubrium sterni. Primary lesion was papillary carcinoma of the left lobe of the thyroid gland. Total thyroidectomy, cervical lymph node dissection, resection of manubrium sterni with concomitant resection of bilateral clavicles, 1st ribs and 2nd ribs, and chest wall reconstruction using Marlex Mesh were performed on January 29, 2001. Considering relatively good prognosis and good response to multimodality therapy, surgical resection of sternal metastatic lesion from differentiated thyroid cancer seems to be a choice of therapy as a part of multimodality approach, including surgery, radioiodine and external radiation therapy, to thyroid cancer with systemic spread.

The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily.

SM-11044 is the only beta-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [(125)I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

Analysis of treatment results of hypopharyngeal cancer.

Sixty-three patients with squamous cell carcinoma of the hypopharynx were treated at the University of Tokyo between 1985 and 1993. Twelve patients were treated with surgery alone, 16 with preoperative irradiation, 26 with postoperative irradiation, and nine with irradiation alone. A tumor dose of 50 Gy (25 fractions/5 weeks) was used in postoperative irradiation, 40-60 Gy in preoperative irradiation, and 70 Gy in irradiation alone. When "number of indications," including, for example, partial pharyngectomy, positive or close (< or = 5 mm) margin, and extracapsular extension, was used for multivariate analysis in patients who underwent surgery, it proved to be the only significant prognostic factor for gross survival. Six of seven patients with a positive surgical margin or close margin who received postoperative irradiation had local recurrence. A tumor dose of 50 Gy (25 fractions/5 weeks) in postoperative irradiation is not enough. It is difficult to cure by irradiation metastases to the parapharyngeal lymph nodes large enough to be detected with CT. It is necessary to irradiate parapharyngeal lymph nodes prophylactically, but 50 Gy in postoperative irradiation may not be enough from our results. The same may be true for metastases to the paratracheal nodes, which lie close to the lower poles of the thyroid gland or within the superior mediastinum and are difficult to access through surgery. In the patients who were operated on, N stage and the number of lymph node metastases were significant for gross survival. When "number of indications" was used for analysis, "number of indications" proved to be the most significant prognostic factor for gross survival.

Characterization of a novel iodocyanopindolol and SM-11044 binding protein, which may mediate relaxation of depolarized rat colon tonus.

Studies under blockade of alpha-, beta1-, and beta2-adrenoreceptors revealed a good correlation between the responses of rat colon relaxation of depolarized tonus and of rat adipocyte lipolysis elicited by catecholamines or BRL-37344, a selective beta3-adrenoreceptor agonist, suggesting beta3-adrenoreceptor stimulation. In contrast, SM-11044, a nonselective beta-adrenoreceptor agonist, stimulated colon relaxation more efficiently than lipolysis; its effects were differently antagonized by cyanopindolol with pA2 values of 8.31 in colon and of 7.32 in adipocytes. Binding studies in rat colon smooth muscle membranes using [125I]iodocyanopindolol under blockade of adrenaline and serotonin receptors revealed the existence of a single class of sites (Kd = 11.0 nM, Bmax = 716.7 fmol/mg protein). The specific binding was saturable and reversible and was displaced by SM-11044 but not by BRL-37344, isoproterenol, noradrenaline, adrenaline, serotonin, nor dopamine. This binding site was photoaffinity labeled using [125I]iodocyanopindolol-diazirine. The labeling was prevented by SM-11044 but not by BRL-37344. The amino-terminal amino acid sequences of the high performance liquid chromatography-purified peptides generated by enzymatic and chemical cleavages of the affinity labeled 34-kDa protein confirmed that the novel iodocyanopindolol or SM-11044 binding protein of rat colon smooth muscle membranes is different from known adrenaline, serotonin, or dopamine receptors. Its functional role might include the relaxation of depolarized colon.

Preservation of the larynx after resection of a carcinoma of the posterior wall of the hypopharynx: versatility of a free flap patch graft.

BACKGROUND: Preservation of the larynx after resection of a pharyngeal tumor remains a challenging problem for the head and neck surgeon. METHODS: Nine patients with T1 or T2 carcinoma of the posterior wall of the hypopharynx (UICC 1987), who were treated surgically between 1984 and 1994, were studied. All patients underwent surgical resection of the tumor with laryngeal preservation and immediate reconstruction with free flap transfer. A free forearm flap was transferred in four patients and a free jejunal patch graft, in five patients. RESULTS: There was one flap loss, due to venous thrombosis. Successful larynx preservation was achieved in the remaining eight patients (89%). Although there was one local control failure, three patients remained free of disease for more than 5 years. CONCLUSIONS: Laryngeal preservation surgery using a free flap patch graft has proven very beneficial in selected cases with a carcinoma of the posterior wall of the hypopharynx.

Significance of aminopyrine breath test as a parameter of hepatic functional reserve in 40% partial hepatectomy of rats with CCl4-induced liver injury.

Rats with CCl4-induced liver injury underwent partial (40%) hepatectomy. The [14C]aminopyrine breath test (ABT) values in rats with CCl4-induced liver injury were reduced by 34% compared with those in rats with normal liver. Preoperative ABT values clearly discriminated between survivors and those that died following 40% partial hepatectomy in rats CCl4-induced liver injury (P < 0.05). Hepatic protein synthesis was remarkably enhanced in CCl4-induced liver injury compared with normal liver (P < 0.001), and this was inversely correlated with ABT values (P < 0.001). These data show that the enhanced hepatic protein synthesis could induce a decrease of hepatic functional reserve. ABT seems to be a useful preoperative test for predicting surgical mortality following hepatectomy.

Effect of BRL-35135 on LTB4-induced guinea pig eosinophil chemotaxis.

The effect of an atypical beta-adrenoceptor agonist, BRL-35135 on leukotriene B4-induced-guinea pig eosinophil chemotaxis was studied. BRL-35135 and SC-41930 (leukotriene B4-antagonist) inhibited the chemotaxis in a concentration-dependent manner (IC50 = 9.0 x 10(-6) and 2.6 x 10(-7) M, respectively). However, isoproterenol, fenoterol and another atypical beta-agonist, BRL-37344 had no effects. The inhibitory effect of BRL-35135 was not affected by (+/-)-propranolol (10(-4) M). In contrast, the nonselective beta-adrenoceptor antagonist, (-)-alprenolol (10(-4) M) dextrally shifted the inhibitory curve of BRL-35135. The response to BRL-35135 was antagonized in a competitive manner by (-)-alprenolol, with the slope of the Schild plot close to unity, and a pA2 value of 5.62. These findings suggest that guinea pig eosinophils possess an "atypical receptor", which differs from either beta 1-, beta 2- or atypical beta-adrenoceptor on guinea pig ileum, and through which eosinophil chemotaxis can be modulated by BRL-35135.

In vitro study of a novel atypical beta-adrenoceptor agonist, SM-11044.

SM-11044 (L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl) propyl] serine pyrrolidine amide hydrobromide) stimulated the relaxation of guinea pig ileum (EC50: 3.0 x 10(-8) M), trachea (EC50: 1.3 x 10(-7) M), lung parenchyma (EC50: 2.1 x 10(-6) M) and the increase in right atrial rate (EC50: 6.9 x 10(-6) M). It also induced lipolysis of rat white adipocytes (EC50: 1.2 x 10(-6) M). Both the relaxant response of ileum and the lipolytic response of adipocytes to SM-11044 were resistant to inhibition by propranolol (10(-6) M), but were antagonized by cyanopindolol (10(-6) M). In contrast, the responses to SM-11044 in trachea, lung parenchyma and right atrium were almost completely abolished by propranolol (10(-6) M). Furthermore, the selectivity of SM-11044 relative to isoproterenol was ileum greater than adipocytes greater than trachea (beta 2) greater than lung (beta 2) greater than atrium (beta 1). These results suggest that SM-11044 is a selective agonist of atypical beta-adrenoceptors that are resistant to antagonism by propranolol but sensitive to cyanopindolol. The receptor binding and adenylate cyclase stimulating activity of SM-11044 were also examined on transfected Chinese hamster ovary cells expressing human beta 1-, beta 2- or beta 3-adrenoceptors. SM-11044 inhibited the binding of [125I]iodocyanopindolol to the three types of receptors in a concentration-dependent manner. The selectivity in terms of Ki values was beta 3 (Ki: 1.3 x 10(-6) M) greater than beta 2 (Ki: 4.1 x 10(-6) M) greater than beta 1 (Ki: 18.1 x 10(-6) M)-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the selective PAF antagonist SM-10661 on an asthmatic model. 1. Effect on passive anaphylactic bronchoconstriction in guinea pigs.

The effect of SM-10661, a selective antagonist of platelet-activating factor (PAF), on passive anaphylactic bronchoconstriction was examined in guinea pigs. A challenge of ovalbumin to passively sensitized guinea pigs induced bronchoconstriction, which peaked at 4 min. When SM-10661 was administered intravenously 2 min before ovalbumin challenge, bronchoconstriction was inhibited dose-dependently with an ID50 of 68 mg/kg. In guinea pigs pretreated with 15 micrograms/kg mepyramine which is a suboptimal dose, antigen-induced bronchoconstriction peaked at 4-6 min, but was inhibited by SM-10661 with an ID50 of 21 mg/kg. When guinea pigs were pretreated intravenously with 2.5 mg/kg mepyramine, 1 mg/kg indomethacin and 0.01 mg/kg propranolol, the antigen-induced bronchoconstriction peaked at 6 min. SM-10661 inhibited the response with an ID50 of 45 mg/kg. Histamine- and leukotriene D4-induced bronchoconstrictions were unaffected by up to 100 mg/kg SM-10661. Ovalbumin challenge of minced lungs from passively sensitized guinea pigs triggered the release of leukotrienes and histamine. SM-10661 had no effect on the antigen-induced release of peptide leukotrienes or histamine up to 10(-4) M. These results indicate that SM-10661 may be a useful tool to investigate the role of PAF in antigen-induced anaphylactic bronchoconstriction.

Effect of the selective PAF antagonist SM-10661 on an asthmatic model. 2. Effect on antigen-induced dual asthmatic response and infiltration of leukocytes into airways in actively sensitized conscious guinea pigs.

The effect of a selective platelet-activating factor (PAF) receptor antagonist, SM-10661, on antigen-induced dual asthmatic response and leukocyte infiltration into the airways of actively sensitized conscious guinea pigs was investigated. The animals were pretreated with mepyramine maleate (10 mg/kg i.p.) and then challenged with ovalbumin. The inhalation of ovalbumin caused an immediate decline in specific airway conductance (sGaw) which peaked at 5 min after the challenge. sGaw gradually returned to the baseline 6 hr after the challenge. After the early asthmatic response (EAR), a second phase change, a late asthmatic response (LAR) in sGaw peaking at 17-20 hr was observed. When 1% w/v disodium cromoglycate was inhaled for 2 min on two occasions, EAR was not affected, but LAR was significantly inhibited. Oral administration of 50 mg/kg fenoterol (30 min before challenge) significantly inhibited EAR, but had no significant effect on LAR. SM-10661 administered orally 1 hr before the challenge inhibited EAR dose-dependently (50% inhibitory dose (ID50); 59 mg/kg, but was even more effective against the LAR (ID50); 13-16 mg/kg). When 30 mg/kg of SM-10661 was administered orally 6 hr after ovalbumin challenge, LAR was completely inhibited. The number of total cells, macrophages and eosinophils in bronchoalveolar lavage fluids rose significantly 17 hr after antigen challenge compared to that observed after saline challenge. The number of neutrophils and lymphocytes also increased in response to the ovalbumin challenge, but not significantly. SM-10661 (30 mg/kg) administered orally 1 hr before the challenge significantly inhibited the increase in total cells, macrophages and eosinophils.(ABSTRACT TRUNCATED AT 250 WORDS)

[Clinical observations of acute low-tone sensorineural hearing loss considered as cochlear hydrops].

Acute low-tone sensorineural hearing loss (ALHL) has the following three criteria; obscure origin, acute onset and sensorineural hearing loss limited to low frequencies. Sixteen cases of ALHL which were considered as cochlear hydrops using glycerol test and electrocochleogram were studied. All patients visited our department within two weeks after onset and were followed up for one year or more after initial examination. The subjective symptoms, the character of vestibular and hearing impairment and prognosis of 16 cases with ALHL were also investigated. The results were as follows. 1. Patients complained of ear fullness rather than hearing impairment. Four patients were unaware of hearing loss. 2. Recruitment phenomenon was found in all of 15 cases examined. Vestibular findings were mostly normal, except that spontaneous nystagmus was found in two cases and head-shaking nystagmus in one. 3. Recurrence and fluctuation of hearing impairment occurred in 14 cases. Three cases had an attack of rotatory vertigo once and two has diagnosed as Meniere's disease later. 4. During three months prior to last examination, hearing was stabilized in nine cases and continued to fluctuate in seven cases. In the former, hearing was improved in four cases, unchanged in three, and worsened in two. 5. Two patients underwent an endolymphatic sac operation and have had a good prognosis. 6. The authors suggest that most of ALHL should be considered as transient cochlear hydrops because the subjective symptom and audiological and vestibular findings of our cases are similar to those of cases which were reported as ALHL by other investigators. According to the findings of glycerol test and electrocochleogram, endolymphatic hydrops in ALHL is considered to exist in all turns of cochlea.

1-Azacycloalkyl-1,4-benzodiazepin-2-ones with antianxiety-antidepressant actions.

A series of 1-azacycloalkyl-1,4-benzodiazepin-2-ones were synthesized from 1-azacycloalkyl-2-benzoylanilines and corresponding imines and then evaluated for their central nervous system activities. Pharmacological data showed that some of these compounds have potent antidepressant properties, as assessed by their antagonism of tetrabenzine (TBZ) induced ptosis and their inhibition of [3H]norepinephrine uptake into rat brain synaptosomes, as well as their moderate antianxiety properties of preventing of pentylenetetrazol (PTZ) convulsion, suppressing conflict behavior, and displacing potential for [3H]diazepam binding. Introduction of a halogen substituent at position 7 of the 1,4-benzodiazepine ring lengthened the anti-PTZ effects, although the peak effect was slightly reduced and clearly enhanced the anti-PTZ and anticonflict properties. Introduction of Cl to the ortho position of the phenyl ring at position 5 greatly reduced the antidepressant properties. The secondary amine function of the azacyclic ring at position 1 was essential for the production of the antidepressant properties. Of these new series, 7-fluoro-5-(2-fluorophenyl)-1,3-dihydro-1-(4-piperidinyl)-2H-1,4-benzodi azepin-2 -one has the potential to become a useful antidepressant drug with a moderate antianxiety property.