Plumbagin suppresses tumor cell growth in oral squamous cell carcinoma cell lines.

OBJECTIVES: Plumbagin (PL), a naturally occurring quinoid, exerts antitumoral effects in diverse types of cancer cells. However, the effect of PL on tumor cell proliferation in oral squamous cell carcinoma (OSCC) remains poorly understood. In this study, we assessed the efficacy of PL, in human OSCC cells. METHODS: The effect of PL on the cell growth and apoptosis of OSCC cell lines was evaluated using MTT and Annexin V assays, respectively. The effect of PL on mitochondrial membrane potential loss and reactive oxygen species (ROS) generation was evaluated using flow cytometry analysis. RESULTS: MTT assay showed that PL dose-dependently suppressed OSCC cell growth, with IC50 values ranging from 3.87 to 14.6 µM. Flow cytometry analysis revealed that PL treatment resulted in a significant decrease in mitochondrial membrane potential and an increase in the number of apoptotic cells. Notably, ROS generation was significantly elevated after PL treatment. Furthermore, a ROS scavenger, N-acetylcysteine (NAC), clearly suppressed the decrease in mitochondrial membrane potential, increase of caspase-3/7 activity, and apoptosis after PL treatment. CONCLUSION: This study provides the considerable evidence of the tumor-suppressive effect of PL, thereby highlighting its therapeutic potential for OSCC treatment.

Usefulness of magnifying endoscopy with narrow-band imaging for determining the horizontal extent of early gastric cancer when there is an unclear margin by chromoendoscopy (with video).

BACKGROUND: Magnifying endoscopy (ME) with narrow-band imaging (NBI) may allow reliable delineation of the horizontal extent of early gastric cancers before endoscopic submucosal dissection (ESD). However, the advantages of ME with NBI over standard endoscopy with dye spraying (chromoendoscopy [CE]) have yet to be elucidated. OBJECTIVE: To investigate the usefulness and limitations of ME with NBI when CE is unsuccessful for determining the horizontal extent of early gastric cancer. DESIGN: Case series. SETTING: Single tertiary referral center. MATERIALS: Series of 350 consecutive early gastric cancers resected en bloc using ESD. INTERVENTION: ME with NBI for cancers with unclear margins by CE. MAIN OUTCOME MEASUREMENTS: The rate of successful delineation by ME with NBI for cancers that had demonstrated unclear margins using CE. RESULTS: The proportion of cancers showing unclear margins using CE was 18.9% (66/350). Of these, 62 of 66 cancers were examined using ME with NBI, with the entire margins successfully delineated in 72.6% (45/62) of the lesions that had shown unclear margins using CE. The success rate was 0% for undifferentiated cancers, significantly lower than that for differentiated lesions (P < .00001). LIMITATIONS: Even by using ME with NBI, endoscopic delineation remains difficult for undifferentiated lesions. CONCLUSIONS: ME with NBI is an excellent modality for identifying the entire margin of early gastric cancers, when the margins are unclear using CE.

Cross-city comparison of indoor air temperatures in air-conditioned spaces.

Field measurements were collected through physical measurements and observations in the cities of Seoul, Korea; Eugene and Portland, Oregon; and Yokohama, Japan, during the fall, winter, and summer seasons from 2005 to 2006. A total of 1733 data sets were collected (Seoul - 713; Oregon - 807; Yokohama - 213) in variety of multi-use buildings with the goal of examining operative temperatures and conditions encountered during everyday life. Of the four cities measured, winter and autumn indoor operative temperatures were highest in Seoul and lowest in Yokohama when normalized to outdoor conditions. In contrast, summer indoor operative temperatures were highest in Yokohama and lowest in Oregon. Clothing levels changed seasonally, and differences were observed between 'long-term occupants', 'residents', and 'transients.'

Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines.

Tyrosine hydroxylase (TH) catalyzes the conversion of L: -tyrosine to L: -dopa, which is the initial and rate-limiting step in the biosynthesis of catecholamines [CA; dopamine (DA), noradrenaline, and adrenaline], and plays a central role in the neurotransmission and hormonal actions of CA. Thus, TH is related to various neuro-psychiatric diseases such as TH deficiency, Parkinson's disease (PD), and schizophrenia. Four isoforms of human TH (hTH1-hTH4) are produced from a single gene by alternative mRNA splicing in the N-terminal region, whereas two isoforms exist in monkeys and only a single protein exist in all non-primate mammals. A catalytic domain is located within the C-terminal two-thirds of molecule, whereas the part of the enzyme controlling enzyme activity is assigned to the N-terminal end as the regulatory domain. The catalytic activity of TH is end product inhibited by CA, and the phosphorylation of Ser residues (Ser(19), Ser(31), and especially Ser(40) of hTH1) in the N-terminus relieves the CA-mediated inhibition. Ota and Nakashima et al. have investigated the role of the N-terminus of TH enzyme in the regulation of both the catalytic activity and the intracellular stability by producing various mutants of the N-terminus of hTH1. The expression of the following three enzymes, TH, GTP cyclohydrolase I, which synthesizes the tetrahydrobiopterin cofactor of TH, and aromatic-L: -amino acid decarboxylase, which produces DA from L: -dopa, were induced in the monkey striatum using harmless adeno-associated virus vectors, resulting in a remarkable improvement in the symptoms affecting PD model monkeys Muramatsu (Hum Gene Ther 13:345-354, 2002). Increased knowledge concerning the amino acid sequences of the N-terminus of TH that control enzyme activity and stability will extend the spectrum of the gene-therapy approach for PD.

Mouse sepiapterin reductase: an enzyme involved in the final step of tetrahydrobiopterin biosynthesis. Primary structure deduced from the cDNA sequence.

We carried out the cloning of a mouse cDNA encoding a sepiapterin reductase which is involved in the final step of tetrahydrobiopterin biosynthesis as a first step toward gene-targeting technique in mice. The sequence contained 1245 nucleotides consisting of an open reading frame of 783 nucleotides encoding a protein of 261 amino acid residues whose molecular weight was 27,851, a 5'-untranslated region of 21 nucleotides and a 3'-untranslated region of 441 nucleotides containing poly(A) tail. The amino acid sequence of mouse sepiapterin reductase revealed the identity of 88% with rat and 74% with human sequence.

Insulin-sensitive tyrosine kinase is increased in livers of adult obese Zucker rats: correction with prolonged fasting.

Adult obese Zucker rats (fa,fa) are hyperinsulinemic and insulin resistant. Specific insulin binding to crude membranes prepared from livers was 2.8% (per mg protein) in fatty animals compared with 7.9% in homozygous lean (Fa,Fa) and 9.0% in heterozygous lean (Fa,fa) animals. Insulin binding increased in liver membranes from fatty animals after a 72-h fast to 6.4%. The reduced insulin binding in livers from fatty rats was associated with elevated insulin-sensitive tyrosine kinase activity, which fell towards control values after the fast. The elevated tyrosine kinase activity was associated with an increased maximum velocity (Vmax) without a change in Michaelis-Menten constant (Km) for its substrates, ATP and poly(Glu,Tyr)4:1. These findings suggest that, in adult fatty rats, insulin-sensitive tyrosine kinase has increased intrinsic activity. Further, the effect of the prolonged fast on both insulin binding and kinase activity, suggest that in this model environmental factors, and not necessarily a genetic abnormality, may regulate liver insulin receptors and their kinase. Whether the inverse relationship of the kinase and insulin receptor number is the result of a compensatory mechanism remains to be elucidated.

Functional insulin-like growth factor I receptors are expressed by neural-derived continuous cell lines.

High affinity insulin-like growth factor I (IGF-I) receptors are expressed by two human neural derived cell lines, SK-N-SH and SK-N-MC. Specific [125I]IGF-I binding to crude membranes was 23.4% for SK-N-SH and 10.7% for SK-N-MC, with 50% inhibition of binding by unlabeled IGF-I between 0.6-0.7 nM. Scatchard analysis of crude membrane binding was linear, whereas Scatchard analysis after wheat germ agglutinin purification of the receptor became curvilinear. The IGF-I receptor alpha-subunits of SK-N-SH have an apparent Mr of 126K, whereas that for SK-N-MC is 132K. Despite these differences in alpha-subunit structure both cell lines demonstrate IGF-I-induced autophosphorylation of their own beta-subunits as well as specific IGF-I induced tyrosine kinase activity, suggesting normal coupling between the ligand-binding alpha-subunit and the tyrosine kinase-containing beta-subunit. Furthermore, IGF-I stimulated iododeoxyuridine uptake in both SK-N-SH and SK-N-MC in a dose-dependent manner, suggesting that these cells may be used to study the role of IGF-I action on neural tissues.

Positive charge intrinsic to Arg(37)-Arg(38) is critical for dopamine inhibition of the catalytic activity of human tyrosine hydroxylase type 1.

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-3, 4-dihydroxyphenylalanine, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by the feedback inhibition of the catecholamine products including dopamine. To rationalize the significant role of the N-terminal sequence Arg(37)-Arg(38) of human TH type 1 (hTH1) in determining the efficiency of feedback inhibition, we produced mutants of which the positively charged Arg(37)-Arg(38) site was replaced by electrically neutral Gly and/or negatively charged Glu and analyzed the degree of inhibition of these mutant enzymes by dopamine. The replacement of Arg by Gly reduced the inhibitory effect of dopamine on the catalytic activity measured in the basic pH range and the replacement of Arg by Glu was enough to abolish the inhibitory effect, although these mutations brought no significant changes to the circular dichroism spectrum. The prediction of the secondary structure of N-terminal residues 1-60 by computer software specified the location of the Arg(37)-Arg(38) sequence in the turn intervening between the two alpha-helices (residues 16-29 and residues 41-59). These results suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintains the characteristic of the turn and is responsible for the enzyme inhibition by dopamine.

Synthesis and Ca2+ antagonistic activity of 2-[2-[(aminoalkyl)oxy]-5-methoxyphenyl]-3,4-dihydro-4-methyl-3-oxo-2H- 1,4-benzothiazines.

As an extension of the previous investigation (J. Med. Chem. 1988, 31, 919), we synthesized a series of 2-[2-[(aminoalkyl)oxy]-5-methoxyphenyl]-3,4-dihydro-4-methyl-3-oxo-2H- 1,4-benzothiazines (3) and evaluated their Ca2+ antagonistic activities. Ca2+ antagonistic activity was measured with isolated depolarized guinea pig taenia cecum. On the basis of their potent Ca2+ antagonistic activity, six benzothiazines were selected and further evaluated for their vasocardioselectivity. Among these six compounds, the key compound 15 [3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[3,4- (methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo- 2H-1,4-benzothiazine hydrogen fumarate] was recognized as having the lowest cardioselectivity. Following optical resolution, the absolute configuration of the compound's optically active enantiomer was determined by means of X-ray crystallography of a synthetic precursor (+)-4a. The Ca2+ antagonistic activity of 15 was found to reside primarily in (+)-15 (which was about 7 times more potent than (-)-15). The in vitro study showed that (+)-15 had a low cardioselectivity compared to verapamil and diltiazem. This result suggests that (+)-15 would exhibit less adverse effects due to cardiac inhibition than diltiazem and verapamil in therapeutic use.

Peripheral administration of lipopolysaccharide enhances the expression of guanosine triphosphate cyclohydrolase I mRNA in murine locus coeruleus.

GTP cyclohydrolase I is the first and rate-limiting enzyme for the de novo biosynthesis of tetrahydrobiopterin, which is the cofactor for tyrosine hydroxylase. Lipopolysaccharide can modulate tetrahydrobiopterin production by upregulating GTP cyclohydrolase I protein expression in the locus coeruleus in the mouse brain. The increased supply of tetrahydrobiopterin in the locus coeruleus leads to increased tyrosine hydroxylase activity without affecting the level of tyrosine hydroxylase protein expression, resulting in an increase in norepinephrine turnover at the site. This study was performed to address whether the increase in GTP cyclohydrolase I protein is dependent on the de novo synthesis of GCH in the locus coeruleus. After i.p. administration of lipopolysaccharide, the mRNA expression of GTP cyclohydrolase I was examined. The expression level increased within 2 h, and reached to maximum level at 4 h after the lipopolysaccharide administration. However, the mRNA expression level of 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase, both of which are involved successively after GTP cyclohydrolase I in tetrahydrobiopterin biosynthesis, were not affected by the lipopolysaccharide administration. These results suggest that GTP cyclohydrolase I upregulation alone is enough to modulate tetrahydrobiopterin production in the locus coeruleus. In addition, the mRNA level of tyrosine hydroxylase was also not affected by the lipopolysaccharide administration. Taken together, the data indicate that GTP cyclohydrolase I plays a crucial role in regulating norepinephrine biosynthesis by a pathway the activity of which is triggered by lipopolysaccharide i.p. administration.

Characterization of cyclosporin A transport in cultured rabbit corneal epithelial cells: P-glycoprotein transport activity and binding to cyclophilin.

PURPOSE: The purpose of this study was to characterize cyclosporin A (CsA) uptake and transport in cultured rabbit corneal epithelial cells (RCECs). METHODS: CsA uptake was evaluated by measuring time-dependent 3H-CsA accumulation in confluent RCECs. Bidirectional 3H-CsA fluxes were measured across the RCEC layers grown on Transwell-COL culture plate inserts. The anti-P-gp monoclonal antibody C219 was used in western blot analysis to probe for the presence of P-gp in these cells. RESULTS: The accumulation of 3H-CsA was time and temperature dependent. Steady state was reached by 60 minutes. The initial uptake was saturable and was suppressed as a function of increases in preloading with unlabeled CsA. This uptake process was enhanced by metabolic inhibition with either 3-O-methylglucose, MG, or 10 mM NaN3 and 3-O-MG. The largest increase was obtained with 10 mM NaN3 in combination with 3-O-MG. In their presence, uptake increased by 40%. A multidrug-resistance (MDR)-reversing agent (i.e., 500 microM verapamil, 100 microM vincristine, 100 microM progesterone, 100 microM testosterone, 500 microM quinidine, or 100 microM chlorpromazine) significantly increased 3H-CsA accumulation. The largest increase was obtained with 500 microM quinidine (i.e., 36%). Conversely, verapamil and vincristine produced the largest inhibition of 3H-CsA efflux (i.e., 19% and 28%, respectively). However, in the presence of 10 microM unlabeled CsA, 3H-CsA efflux increased. 3H-CsA flux across RCEC layers showed marked directional asymmetry. The stromal (S) to tear (T) side transcellular 3H-CsA permeability coefficient (Ptrans) was approximately seven times higher than that in the T-to-S direction. The S-to-T Ptrans was reduced by an MDR-reversing agent by up to 40%. Western blot analysis of lysates revealed a 170-kDa membrane protein band. CONCLUSIONS: These results suggest that in RCEC the tear-side-facing membrane has a P-gp-mediated drug efflux pump. In addition, there is suggestive evidence for the presence of the cytosolic protein, cyclophilin. The presence of P-gp in these cells could help protect them from being damaged by the uptake of toxic substances.

A further prospective evaluation of an equation to predict daily lithium dose.

BACKGROUND: Recently, one prospective study confirmed the safety and accuracy of a lithium-dose prediction equation created by Zetin et al., but no definitive conclusion on the reliability of the equation has been established as yet. METHOD: The authors applied the Zetin et al. equation to 18 chronic male schizophrenic inpatients. Predicted doses to reach the serum lithium concentration of 0.4 mmol/L were calculated and prescribed in the form of lithium capsules. At Weeks 1 and 3 after treatment initiation, morning blood samples were collected about 12 hours after the last lithium dose for the measurement of serum lithium concentrations. RESULTS: None of the 18 patients achieved the desired concentration (0.4 mmol/L) exactly. The mean +/- SD of serum lithium concentrations at Week 1 was 1.01 +/- 0.29 mmol/L (range, 0.2-1.5) and at Week 3 was 0.94 +/- 0.35 mmol/L (range, 0.2-1.8). Lithium concentrations were lower than 0.4 mmol/L in only 1 patient and were higher than 0.4 mmol/L in the other 17 patients. The deviations from the unexpected value were significantly correlated with the renal function (blood urea nitrogen and serum creatinine levels) but not with the neuroleptic doses administered to the patients. Moreover, our patients were relatively older and weighed relatively less than the patients described in the previous prospective study. CONCLUSION: The Zetin et al. equation cannot always accurately predict a required lithium dose. Renal function data, even when they range within normal values, may be useful to improve the accuracy of the equation, particularly in patients who are older or weigh less than the norm.

TPA-induced neurite formation in a neuroblastoma cell line (SH-SY5Y) is associated with increased IGF-I receptor mRNA and binding.

The neuroblastoma cell line SH-SY5Y was cultured in the presence of TPA for three days. Increased neurite formation was noted as early as 24 hours after TPA was added. These changes were associated with an increase in IGF-I receptor binding as well as increased mRNA for the IGF-I receptor.

Inducibility of protein-reactive antibodies by peptide immunization: comparison of three epitope peptides of hen egg-white lysozyme.

Three epitope peptides of hen egg-white lysozyme (HEL) were tested for ability to induce antibodies reactive with native HEL. Each peptide was coupled to bovine gamma-globulin (B gamma G) and 4 rabbits were immunized with each peptide-B gamma G conjugate in complete Freund's adjuvant. The mean association constants (K0s) of HEL-reactive antibodies (HEL-R-Abs) from each immunizing group to [3H]acetyl HEL or to [3H]acetyl-peptide were measured in solution by a double antibody method. Only peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) induced high-affinity antibodies to HEL (K0 = 2.5 x 10(6)-2.3 x 10(7) M-1) among the three epitope peptides tested. The association constants of antipeptide loop I.II to [3H]acetyl peptide loop I.II were always one to two orders of magnitude higher than those to HEL. In addition, 50 to 80% of the anti-peptide loop I.II antibodies were reactive with native HEL. The specificity of anti-peptide loop I.II was directed to a conformational feature of the peptide rather than to native HEL and reactivity of the antibody to HEL was interpreted as a kind of cross-reaction. The HEL-R-Abs from anti-Ploop I.II antisera also manifested neutralizing activities against the enzymic activity of HEL when Micrococcus luteus was used as the substrate.

Characteristics of the epitope of protein-reactive anti-peptide antibodies.

The specificity of hen egg-white lysozyme (HEL)-reactive rabbit antibodies induced by the peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) of HEL was clarified by analyzing their cross-reactions with various avian lysozymes and their reaction with synthetic peptides (sequences 59-82) in which alanine was substituted for the amino acid at certain positions. The Arg-68 residue of HEL plays a dominant role in the binding, while Gly-71, Ser-72, Arg-73, and Pro-79 also contribute to the binding of two anti-Ploop I.II antibodies (rabbit number 125 and 126). These residues, although remote in sequence, are grouped together in the crystal structure of HEL and may form an area of contact with the antibody. Contributions by Trp-63, Ile-78, and Asn-77 to the binding of the two antibodies to HEL were excluded. These results support the idea that the anti-Ploop I.II antibodies recognize a conformational type of epitope which is similar to that of native HEL. The immunogenicity of the reduced and alkylated form of Ploop I.II was also tested, but it failed to induce an HEL-reactive antibody.

Insulin and IGF-I receptors in neuroblastoma cells: increases in mRNA and binding produced by glyburide.

Insulin and IGF-I binding to neuroblastoma cells (SK-N-MC) was increased by 13% and 7% respectively following a 24hr, incubation with the sulphonylurea glyburide. This increase in binding was associated with increased steady-state levels of insulin receptor and IGF-I receptor mRNA levels. Though insulin and IGF-I both stimulate glucose uptake into these cells, the increased binding following glyburide treatment was not associated with any change in glucose uptake.

Expression of GTP cyclohydrolase I in murine locus ceruleus is enhanced by peripheral administration of lipopolysaccharide.

Among the enzymes involved in the system for catecholamine biosynthesis, GTP cyclohydrolase I (GCH) contributes to the system as the first and rate-limiting enzyme for the de novo biosynthesis of tetrahydrobiopterin (BH4), which is the cofactor for tyrosine hydroxylase (TH). Therefore, we investigated whether the endotoxemia caused by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) can modulate BH4 production in the norepinephrine nuclei, i.e. the locus ceruleus (LC; A6) and central caudal pons (A5), in C3H/HeN mice and whether such a change in BH4, if any, can result in the modification of norepinephrine production in these nuclei. After a 5-microg i.p. injection of LPS, the protein expression of GCH and TH in both nuclei was examined by immunohistochemistry. The staining intensity of GCH-positive cells increased at 6 h, whereas no significant change in the staining intensity of TH-positive cells was detected. Next, we measured the contents of BH4, norepinephrine, and its metabolites 4-hydroxy-3-methoxyphenylglycol (MHPG) and DL-4-hydroxy-3-methoxymandelic acid (VMA) in these nuclei after LPS i.p. injection. The BH4 content increased to a statistically significant level at 2 and 4 h after the injection. The contents of MHPG and VMA also showed a time-course similar to that of BH4. These data can be rationalized to indicate that an increased supply of BH4 in the LC increased TH activity and resulted in an increase in norepinephrine production rate at the site. This is the first report that sheds light on BH4 as a molecule that intervenes during endotoxemia to increase norepinephrine production rate in the LC.

Characterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells.

Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [125I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC50 for [125I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the alpha subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B10) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the alpha subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the alpha subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosylation. Insulin induced autophosphorylation of the insulin receptor beta subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10(-9) to 10(-6) M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.

Effects of dopamine on N-terminus-deleted human tyrosine hydroxylase type 1 expressed in Escherichia coli.

N-Terminus-deleted mutants and wild-type human tyrosine hydroxylase type 1 were expressed in Escherichia coli (E. coli) and utilized to investigate the dopamine-induced decrease in the enzyme catalytic activity and also to identify the specific portion in the N-terminus that affects the efficiency of the inhibitory action of dopamine. Supernatants of bacterial lysates were used as enzyme samples. The pH profiles of the enzyme catalytic activity were affected according to the degree of the deletion. The deletion up to 39 amino acid residues was enough to abolish the inhibitory effect of dopamine in the basic pH range. These results suggest that the inhibition by dopamine of tyrosine hydroxylase activity is closely related to the amino acid sequence in the N-terminus of the enzyme.