NCCN practice guidelines for head and neck cancer. National Comprehensive Cancer Network.
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Head and Neck Neoplasms↗
Biomedical subjects
K W Hong
Publications and source records attributed to K W Hong.
Effect of rebamipide on liver damage and increased tumor necrosis factor in a rat model of endotoxin shock.
We investigated the effect of rebamipide, a novel antiinflammatory agent, on liver damage in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide, LPS). Endotoxemia for 6 hr resulted in a 5.9-fold rise in the serum levels of nitrite (P < 0.05) with a significant rise in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactic dehydrogenase (LDH), suggestive of liver dysfunction. The increased activities of serum ALT, AST, and LDH, but not serum nitrite were significantly inhibited by rebamipide (100 mg/kg, orally for five days). Myeloperoxidase activity in the liver was significantly elevated in the rats with endotoxemia by 2.4-fold (P < 0.05), which was also significantly inhibited by rebamipide. Upon LPS injection, serum TNF-alpha levels peaked at 1 hr after LPS (from 167.4 +/- 20.0 to 1570.0 +/- 100.0 pg/ml) and thereafter rapidly declined. The increased TNF-alpha level measured at 1 hr was significantly inhibited by pretreatment with rebamipide (100 mg/kg for five days). It is suggested that rebamipide exerts a strong protective effect on the LPS-induced liver damage through inhibition of activation of neutrophils and TNF-alpha production.
Alanine↗
Decreased CGRP level with increased sensitivity to CGRP in the pial arteries of spontaneously hypertensive rats.
It was aimed to investigate the importance of calcitonin gene-related peptide (CGRP) in maintenance of normal cerebral microcirculation. We examined both the functional (in vivo) and biochemical effects (in vitro) of CGRP on the pial arteries of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). When mock cerebrospinal fluid containing capsaicin (3 x 10(-7) M) was suffused over the cortical surface, the diameter of pial arteries of SHR was transiently increased and rapidly returned to the baseline level, while the capsaicin-induced increase in pial arterial diameters of WKY was large and sustained for a longer duration (> 10 min). Capsaicin-induced vasodilation was significantly attenuated by pretreatment with CGRP8-37, a CGRP1, receptor antagonist, in both WKY and SHR. On the other hand, cortical suffusion with CGRP (10(-9) approximately 10(-6) M) exerted a larger enhancement in the vasodilation of pial artery of SHR than WKY. The CGRP-induced vasodilation was significantly antagonized by CGRP8-37 in both WKY and SHR. The released level of CGRP-like immunoreactivity (CGRP-LI) from the pial artery was significantly lower in SHR (12.3 +/- 1.2 fmol/mm2/hr) than that in WKY (24.5 +/- 3.9 fmol/mm2/hr). CGRP (10(-6) M)-induced stimulation of cyclic AMP formation was rather larger in the pial arteries from SHR (50.2 +/- 5.8 fmol/mm2/30 min, p < 0.05) than those from WKY (34.5 +/- 3.8 fmol/mm2/30 min). These data suggest that, in the pial arteries of SHR, the transient vasodilation to capsaicin and enhanced vasodilation to CGRP are related to the decreased CGRP level in the cerebral microvascular beds, consequently leading to increased sensitivity of the CGRP receptors to CGRP.
Animals↗
Age-dependent changes in particulate and soluble guanylyl cyclase activities in urinary tract smooth muscle.
Regional and age specific differences are observed in the sodium nitroprusside induced relaxation responses in the urinary tract. To clarify these differences, guanylyl cyclase activity is assayed in particulate and soluble fractions from the ureter, bladder dome, and urethra of young (11-18 days), adult (90-100 days), and old adult (2-3 years) guinea pigs. The rank order of soluble guanylyl cyclase activities is urethra = ureter > bladder dome with the largest decreases with aging occurring in the bladder. Atrial natriuretic factor (10(7) M) increases particulate guanylyl cyclase activity in the three tissues at all ages tested, with the activity being highest in the ureter. ATP (0.5 mM) activates particulate guanylyl cyclase in the ureter, bladder and urethra of old adult guinea pigs, and enhances atrial natriuretic factor induced activation of particulate guanylyl cyclase in all tissues and at all ages tested. The higher levels of soluble guanylyl cyclase activity in the urethra and ureter compared to the bladder parallel sodium nitroprusside induced relaxation in these tissues.
Aging↗
A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene.
A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation.
Bacillus subtilis↗
Preventive effect of rebamipide on gastric mucosal cell damage evoked by activation of formyl-methionyl-leucyl-phenylalanine receptors of rabbit neutrophils.
We investigated the underlying mechanism by which rebamipide exerts a preventive effect on neutrophil-mediated gastric mucosal cell damage. The release of 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (an index of cytotoxicity) was significantly increased by 16.7% (P < .05) when 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein-acetomethyl ester (5 microM) loaded gastric mucosal cells were incubated with neutrophils (5 x 10(6) cells/well) that were activated by cytochalasin B (5 microM) and formyl-methionyl-leucyl-phenylalanine (fMLP) (1 nM). In the in vitro study, upon application of cytochalasin B and fMLP, formation of superoxide anion and release of myeloperoxidase increased with increased neutrophil aggregation. These parameters were attenuated by pretreatment with rebamipide (100-1000 microM) in a concentration-dependent manner. In the Scatchard analysis, the maximum binding of [3H]fMLP to neutrophils decreased from 0.57 to 0.44 pmol/2 x 10(6) cells (P < .05) by application of rebamipide (300 microM) with little change in K(D). Neutrophils isolated from rabbits orally treated with rebamipide (100 mg/kg for 3 days) also showed a decrease in the production of superoxide anion upon stimulation with fMLP and a decrease in the binding of [3H]fMLP to its receptors on the neutrophil plasma membrane (0.59-0.45 pmol/2 x 10(6) cells, P < .05). Taken together, it is suggested that the inhibitory effect of rebamipide on the neutrophil-mediated gastric mucosal cell injury is due, in part, to alterations in the neutrophil membrane that ultimately result in a decrease in the number of binding sites for fMLP to its receptors.
Alanine↗
tRNA-dependent amino acid transformations.
Aminoacyl-tRNAs are either synthesized directly by aminoacyl-tRNA synthetases or indirectly by tRNA-dependent transformation of mischarged tRNAs. The enzymes which participate in the indirect routes may be interesting targets in the development of novel therapeutic compounds. We have purified one such enzyme, Glu-tRNA(Gln) amidotransferase from Bacillus subtilis, to homogeneity and present the initial biochemical characterization data.
Bacillus subtilis↗
Genetic analysis of functional connectivity between substrate recognition domains of Escherichia coli glutaminyl-tRNA synthetase.
It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype on Escherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA(Gln): GlnRS: ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3' end of tRNA(Gln). The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischarge supF tRNA(Tyr) considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguish supE tRNA(CUA)(Gln) from tRNA2(Gln) considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K(m) for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.
Adenosine Triphosphate↗
Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.
Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.
Amino Acyl-tRNA Synthetases↗
Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.
An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.
Adenosine Triphosphate↗
Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging.
Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.
Amino Acyl-tRNA Synthetases↗
Glutaminyl-tRNA synthetase: from genetics to molecular recognition.
Accurately aminoacylated tRNAs are an a priori requirement for translation of the genetic code. They are synthesized by the aminoacyl-tRNA synthetases which select both the correct amino acid and tRNA from a total of more than 400 possible combinations. Genetic, biochemical and structural studies have begun to reveal the mechanisms by which this specificity is achieved by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). Sequence-specific interactions between GlnRS and tRNA(Gln) determine both the accuracy of tRNA selection and the efficiency of aminoacylation. Thus, amino acid recognition is tRNA-dependent. Consequently, while a noncognate tRNA may be recognized by GlnRS, the resulting tRNA-enzyme complex displays a considerably reduced affinity for glutamine compared to wild-type. This mechanism now provides a ready explanation as to why the majority of tRNA mischarging events, including those originally described over 25 years ago for GlnRS, impair cellular viability only to a limited degree.
Amino Acyl-tRNA Synthetases↗
Pharmacological coupling and functional role for CGRP receptors in the vasodilation of rat pial arterioles.
In this study, we investigated the signal transduction underlying the vasodilator action of calcitonin gene-related peptide (CGRP) in the rat pial arterioles. In an in vivo experiment, changes in pial arterial diameters (20.2 +/- 1.9 microns) were observed under suffusion with mock cerebrospinal fluid containing CGRP (10(-9)-10(-7) M) directly through a closed cranial window. Changes in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to CGRP and levcromakalim were measured in the pial arterioles in an in vitro experiment. CGRP-induced vasodilation and cAMP production were significantly inhibited by specific CGRP antibody serum and CGRP-(8-37) fragment, suggesting involvement of the CGRP1 receptor subtype. Vasodilation and increase in cAMP production evoked by CGRP were inhibited not only by glibenclamide (ATP-sensitive K+ channel blocker) but also by charybdotoxin (large-conductance Ca(2+)-activated K+ channel blocker), but this was not the case for the isoproterenol-induced vasodilation and cAMP production. These findings implicate the ATP-sensitive K+ channels and the large-conductance Ca(2+)-activated K+ channels in the CGRP receptor-coupled cAMP production for vasodilation. Further study is required to identify whether the cAMP-dependent K+ channel activation is related to CGRP-induced vasorelaxation of the rat pial arterioles.
Animals↗
Preventive effect of rebamipide on gastric lesions induced by ischemia-reperfusion in the rat.
Rebamipide (2-(4-chlorobenzoylamino)-3-[2-(1H)-quinolinon-4-yl] propionic acid), a novel antiulcer agent, has been reported to prevent various acute experimental gastric mucosal lesions and to accelerate the healing of chronic gastric ulcers. We investigated the effect of rebamipide on rat gastric mucosa damaged by exposure to 30 min of ischemia and 60 min of reperfusion (I/R) with continuous intragastric instillation of 0.1 N HCl (1 ml/100 g body weight) into the stomach. Rebamipide, at 30 and 100 mg/kg, i.p., reduced the mucosal damage score from 2.28 (I/R vehicle group) to 1.54 and 1.07, respectively. Pretreatment with rebamipide significantly reduced the activity of myeloperoxidase (an index of neutrophil infiltration) and preserved the activities of superoxide dismutase and nitric oxide synthase in the gastric mucosa with inhibition of malondialdehyde production. Thus, a negative correlation between the activities of nitric oxide synthase and myeloperoxidase (y = 4.35-9.45x, r = .67, P < .01) was observed. In an in vitro study, rebamipide inhibited N-formyl-met-leu-phe-induced chemotaxis of neutrophils and production of superoxide anion from opsonized zymosan-stimulated neutrophils. However, it did not affect the production of superoxide anion either by the xanthine-xanthine oxidase reaction or phorbol 12-myristate 13-acetate-stimulated neutrophils. Based on these results, it is suggested that rebamipide exerts a protective effect on the I/R-induced gastric mucosal damage through inhibition of mobilization and activation of neutrophils in association with an attenuation of the decreases in both superoxide dismutase and nitric oxide synthase activities, thereby preventing the gastric microcirculation from deterioration.
Alanine↗
Substrate selection by aminoacyl-tRNA synthetases.
The integration of genetic and biochemical approaches to study the crystal structure of the glutaminyl-tRNA synthetase (GlnRS):tRNA(Gln):ATP complex has elucidated the mechanism by which GlnRS selects its cognate tRNA for aminoacylation. Three principal types of interaction have been identified: interaction with specific bases in the cognate tRNA, rejection of non-cognate tRNAs, and activation of the active site upon cognate tRNA binding. The recent solving of the crystal structure of tryptophanyl-tRNA synthetase (TrpRS) has allowed comparable studies to be initiated in an aminoacyl-tRNA synthetase which, unlike GlnRS, does not require tRNA binding prior to amino acid activation.
Amino Acyl-tRNA Synthetases↗
Pharmacological evidence that calcitonin gene-related peptide is implicated in cerebral autoregulation.
In anesthetized rats, we examined the possibility that calcitonin gene-related peptide (CGRP, a neuropeptide) released in response to transient hypotension may contribute to the reflex autoregulation of cerebral blood flow. Changes in pial arterial diameter (mean 33.0 +/- 1.1 microns) with changes in systemic arterial blood pressure (mean 101.9 +/- 2.7 mmHg) were observed directly through a closed cranial window. In capsaicin-treated rats (depletor of CGRP and substance P, 50 nmol capsaicin injected intracisternally 24 h before experiment), vasodilatation, which was evoked on transient hypotension, and vasoconstriction on reverse of hypotension were markedly attenuated or almost abolished. When changes in pial arterial diameter were plotted as a function of changes in blood pressure, the slopes of regression lines for vasodilatation and vasoconstriction were markedly reduced after capsaicin treatment. Similar reductions were evidenced under suffusion of CGRP antibody serum (1:1,000) and after CGRP receptor desensitization but not after substance P receptor desensitization. Pretreatment with glibenclamide, a K(+)-channel antagonist, also caused severe alterations in the autoregulatory vasomotor responses to hypotension and its reverse. Suffusion with mock cerebrospinal fluid, containing either CGRP or cromakalim, a K(+)-channel opener, dilated the pial artery in a concentration-dependent manner, and their effects were antagonized by glibenclamide. Substance P produced a vasodilatation, which was unaffected by glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Characterization of PD 121981- and CGP 42112-induced unmasking of low concentration effects of angiotensin II in rabbit abdominal aorta.
The unmasking of the low concentration effect of angiotensin II (AII) was identified within the concentration ranges of 10(-13) to 10(-11) M of AII by PD 121981 (5-diphenylacetyl-1-(4-methoxy-3-methylbenzyl)- 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-pyridine-6-carboxylic acid) and 10(-12) to 3 x 10(-10) M of AII by CGP 42112 (nicotinic acid-Tyr-(N alpha-benzyl-oxycarbonyl-Arg)Lys-His-Pro-IIe-OH), AT2 antagonists, in association with the ordinary contraction curve, i.e., high concentration effect (at 3 x 10(-10)-10(-6) M of AII), in the rabbit abdominal aorta. Thus, they showed clear biphasic features of AII-induced contraction curves. However, this was not the case for angiotensin I and angiotensin III. This PD 121981-evoked low concentration effect of AII was selectively inhibited by DuP 753 (0.01-1 nM), dithiothreitol (10 and 100 microM), pertussis toxin (50 and 300 ng/ml, for 2 hr), nifedipine (1 and 10 microM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (1 and 3 microM), which suggests the receptors were the AT1 subtype. However, the high concentration effect of AII was not affected by these drugs within the concentration ranges used in the present studies. These myographic results were almost consistent with the features of the intracellular Ca++ changes. Thus, it was concluded that the receptors that mediate the low concentration effect of AII belong to the AT1 subtype. However, the current study did not determine the mechanism by which PD 121981 and CGP 42112 evoked the up-regulation of the AT1 receptors.
Angiotensin II↗
Effect of ouabain on relaxation induced by cromakalim in human and canine mesenteric arteries.
We investigated the effect of cromakalim, a K+ channel opener, that activates indirectly the Na(+)-K+ pump, in association with increased K+ conductance in the mesenteric arteries. In 65% of human mesenteric arteries tested, the concentration-dependent relaxation curves for cromakalim were biphasic: the low concentration (< 10(-7) M) effect was preferentially inhibited by ouabain, whereas the higher concentration effect was significantly inhibited by glibenclamide. In branches of canine mesenteric artery, the cromakalim-induced relaxation was inhibited by pretreatment with ouabain (1 microM) as well as by glibenclamide (1 microM). The reduction in contraction of human and canine mesenteric arterial strips caused by cromakalim was totally reversed by pretreatment with ouabain (1 microM) or glibenclamide (1 microM). On the other hand, in canine mesenteric artery, cromakalim caused a significant stimulation of 22Na+ influx and ouabain-sensitive 86Rb+ uptake in association with increased 86Rb+ efflux, all of which were inhibited by glibenclamide (1 microM). Thus, it is suggested that cromakalim possesses the additional property to stimulate the Na(+)-K+ pump through an elevation in intracellular Na+, resulting in strong relaxation of blood vessels.
Adult↗