Protective role of quercetin against cisplatin-induced hair cell damage in zebrafish embryos.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the protective effects of quercetin on cisplatin-induced hair cell damage in transgenic zebrafish embryos. MATERIALS AND METHODS: Five days postfertilization zebrafish embryos were exposed to 1 mM cisplatin and quercetin at 10, 50, 100, or 200 µM for 4 h. Hair cells within neuromasts of the supraorbital, otic, and occipital lateral lines were analyzed by fluorescent microscopy (n = 10). Survival of hair cells was calculated as the average number of hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy. RESULTS: Hair cell damage in neuromasts was decreased by co-treatment of quercetin and cisplatin (quercetin 100 µM: 8.6 ± 1.1 cells; 1 mM cisplatin only: 5.0 ± 0.5 cells; n = 10, p < 0.05); apoptosis of hair cells examined by special stain was also decreased by quercetin. The ultrastructure of hair cells within neuromasts was preserved in zebrafish by the combination of quercetin (100 µM) and cisplatin (1 mM). CONCLUSION: In conclusion, quercetin showed protective effects against cisplatin-induced toxicity in a zebrafish model. The results of this study suggest the possibility of a protective role of quercetin against cisplatin-induced apoptotic cell death in zebrafish.

Adjuvant hepatic intra-arterial iodine-131-lipiodol following curative resection of hepatocellular carcinoma: a prospective randomized trial.

BACKGROUND: The purpose of the present study was to determine whether intrahepatic injection of (131)I-lipiodol (Lipiodol) is effective against recurrence of surgically resected hepatocellular carcinoma (HCC). METHODS: From June 2001 through March 2007, this nationwide multi-center prospective randomized controlled trial enrolled 103 patients 4-6 weeks after curative resection of HCC with complete recovery (52: Lipiodol, 51: Control). Follow-up was every 3 months for 1 year, then every 6 months. Primary and secondary endpoints were recurrence-free survival (RFS) and overall survival (OS), respectively, both of which were evaluated by the Kaplan-Meier technique and summarized by the hazard ratio (HR). The design was based on information obtained from a similar trial that had been conducted in Hong Kong. RESULTS: The Lipiodol group showed a small, and nonsignificant, improvement over control in RFS (HR = 0.75; 95 % confidence interval [95 % CI] 0.46-1.23; p = 0.25) and OS (HR = 0.88; 95 % CI 0.51-1.51; p = 0.64). Only two serious adverse events were reported, both with hypothyroidism caused by (131)I-lipiodol and hepatic artery dissection during angiography. CONCLUSIONS: The randomized trial provides insufficient evidence to recommend the routine use of (131)I-lipiodol in these patients.

Low incidence of penetrating trauma in a high-volume tertiary center: 10-year mortality review.

BACKGROUND: Trauma morbidity and mortality outcome is better in high-volume trauma centers. However, there are few publications investigating the experience of high-volume centers with high non-trauma emergency load but seeing a relatively low incidence of trauma. The objective of this study is to review the presentation and outcomes for the low volume of patients presenting with penetrating injuries in a high-volume hospital. METHODS: Data were extracted from the Singapore General Hospital database between 1998 and 2007. There were 1,233 patients who sustained penetrating injuries and were brought to the hospital during the 10-year period. Of these, only 78 patients had injury severity score (ISS) values of 16 or more. In the same period, there were 1,270 patients with ISS > 15 who were admitted with blunt injury. SPSS 10.1 was used to conduct univariate and multivariate analyses to elucidate risk factors for mortality. RESULTS: Age, ISS, and trauma injury severity score (TRISS) were significant predictors of mortality. Gender and type of injury were not predictive of mortality. Mortality outcomes were independently predicted by age, TRISS, and ISS. The most common site of injury was the chest, followed closely by the head and neck. The abdomen/pelvis was the third most common site of injury. There was no significant difference in anatomical site injury pattern between the survivors and non-survivors. For both groups, chest injuries and head and neck injuries dominated, with maximal abdominal/pelvic injuries a distant third. CONCLUSION: With a trauma system in place, high-volume centers with a low volume of penetrating injury patients can still manage uncommon injuries without jeopardizing patient care.

Comparing the efficacy of sunitinib with sorafenib in xenograft models of human hepatocellular carcinoma: mechanistic explanation.

Hepatocellular carcinoma (HCC) is the fifth most common and third deadliest malignancy. Sorafenib has demonstrated 44% survival advantage over placebo and has emerged as a standard of care in advanced HCC. The therapeutic effects of sorafenib are however transient and hence additional treatment options are warranted. In this study, we aimed to compare the efficacy of sunitinib relative to sorafenib, two potent inhibitors of protein tyrosine kinases involved in tumor growth, metastasis, or angiogenesis. We reported that sorafenib and sunitinib suppressed tumor growth, angiogenesis, cell proliferation, and induced apoptosis in both orthotopic and ectopic models of HCC. However, the antitumor effect of 50 mg/kg sorafenib was greater than that of 40 mg/kg sunitinib. Sorafenib inhibited p-eIF4E Ser209, p-p38 Thr180/Tyr182 and reduced survivin expression. This was not seen with sunitinib. In addition, the antitumor and apoptotic effects of sorafenib, which are associated with upregulation of fast migrating Bim and ASK1 and downregulation of survivin, were greater than that of sunitinib. These observations explained in part the apparent superior anti-tumor activity of sorafenib compared to sunitinib. In conclusion, sunitinib demonstrated an inferior anti-tumor activity compared to sorafenib in ectopic and orthotopic models of human HCC. It remains to be seen whether such observations would be recapitulated in humans.

Sunitinib (SUTENT, SU11248) suppresses tumor growth and induces apoptosis in xenograft models of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common and third deadliest primary neoplasm. Since HCC is a particularly vascular solid tumor, we determined the antitumor and antiangiogenic activities of sunitinib malate, a potent inhibitor of two receptors involved in angiogenesis - vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). In the present study, we reported that treatment of HepG2 and SK-Hep-1 cells with sunitinib led to growth inhibition and apoptosis in a dose-dependent fashion. Sunitinib inhibited phosphorylation of VEGFR-2 at Tyr951 and PDGFR-beta at Tyr1021 both in vitro and in vivo. Sunitinib also suppressed tumor growth of five patient-derived xenografts. Sunitinib-induced tumor growth inhibition was associated with increased apoptosis, reduced microvessel density and inhibition of cell proliferation. This study provides a strong rationale for further clinical investigation of sunitinib in patients with hepatocellular carcinoma.

Computed tomographic appearance of colorectal hepatic metastases.

INTRODUCTION: This article reviews the various computed tomography (CT) appearances of hepatic metastases from colorectal primaries and assesses the frequency of occurrence of the various patterns. MATERIALS AND METHODS: This is a retrospective study of the CT appearances of histologically proven colorectal hepatic metastases in a group of 52 patients who had undergone surgical hepatic resection between January 1994 and December 2001. A total of 74 hepatic metastatic lesions were reviewed. All lesions were examined in the portal venous phase. RESULTS: A discernible rim was seen in 54 lesions (73%). Thick rim was present in 36 lesions (48.6%) and thin rim in 18 lesions (24.3%). Enhancement of the rim was present in 62 cases (83.8%). Increased central attenuation was seen in 38 lesions (51.4%). Of these, the centre was heterogeneous in 76.3% and scar-like in 23.7%. A non-enhancing rim was seen in 12 lesions (16.2%) which appeared as lesions with "bevelled edge". Thick enhancing rim with non-enhancing centre was the most common combination in 15 lesions (20.3%). CONCLUSION: An enhancing rim could be seen in 83.8% of lesions. Increased central attenuation was present in 51.4% of the lesions. Familiarity with the various CT appearances may facilitate identification and diagnosis of colorectal liver metastases.

Prevalence of Helicobacter pylori in gastric cancer in a South-East Asian population by 14C-urea breath test.

BACKGROUND: Helicobacter pylori is believed to play an important role in the aetiology of gastric cancer. There is a great variability in seropositivity and histological frequency of H. pylori in gastric cancer. The present prospective study investigates the prevalence of H. pylori infection in gastric cancer patients using 14C-urea breath testing. METHODS: Patients with endoscopic biopsy-proven gastric cancer were fasted for 6 h prior to ingesting 18.5 x 104 Bq of 14C-urea cocktail orally. Breath samples were collected after 20 min by asking them to blow into a hyamine solution and measurements were read in a scintillation counter. RESULTS: Fifty out of 51 patients (98%) with gastric cancer were positive on the 14C-urea breath test compared to 29 patients (61%) who were positive on histology. There was no association between sex, age or tumour site, stage, differentiation, Lauren type and H. pylori status. The test was negative in one patient with cardial tumour in which histology of the resected specimen was also negative for the bacteria. CONCLUSIONS: Active H. pylori infection is highly prevalent in gastric cancer in a South-East Asian population. The 14C-urea breath test is a highly sensitive method for detecting the presence of H. pylori even in gastric adenocarcinoma irrespective of the stage.

The diversity of beetle assemblages in different habitat types in Sabah, Malaysia.

The diversity of beetle assemblages in different habitat types (primary forest, logged forest, acacia plantation and oil palm plantation) in Sabah, Malaysia was investigated using three different methods based on habitat levels (Winkler sampling, flight-interception-trapping and mist-blowing). The overall diversity was extremely high, with 1711 species recorded from only 8028 individuals and 81 families (115 family and subfamily groups). Different degrees of environmental changes had varying effects on the beetle species richness and abundance, with oil palm plantation assemblage being most severely affected, followed by acacia plantation and then logged forest. A few species became numerically dominant in the oil palm plantation. In terms of beetle species composition, the acacia fauna showed much similarity with the logged forest fauna, and the oil palm fauna was very different from the rest. The effects of environmental variables (number of plant species, sapling and tree densities, amount of leaf litter, ground cover, canopy cover, soil pH and compaction) on the beetle assemblage were also investigated. Leaf litter correlated with species richness, abundance and composition of subterranean beetles. Plant species richness, tree and sapling densities correlated with species richness, abundance and composition of understorey beetles while ground cover correlated only with the species richness and abundance of these beetles. Canopy cover correlated only with arboreal beetles. In trophic structure, predators represented more than 40% of the species and individuals. Environmental changes affected the trophic structure with proportionally more herbivores (abundance) but fewer predators (species richness and abundance) in the oil palm plantation. Biodiversity, conservation and practical aspects of pest management were also highlighted in this study.

Controlled localized buckling responses of orthodontic arch wires.

The orthodontic arch wire is often activated locally, in transverse bending and/or longitudinal torsion, to engage an individual malaligned tooth. Arch wires with substantial flexibilities and elastic ranges in bending are available. Several clinical reports of distal displacements of molars with appliances activated by locally buckling the arch wire have appeared in the recent published literature. This article contains an explanation of buckling or "column" action and the postbuckling response of a wire, and a report of the results of a controlled, in-vitro study of a sample of 256 wire segments subjected to activation-deactivation, buckling-postbuckling-unbuckling cycles. Continuous force-displacement diagrams were obtained from mechanical tests run at oral temperature. Four orthodontics-relevant, mechanical characteristics were quantified from each diagram, and each specimen was subjected to posttest evaluation for inelastic behavior. Although the deformation of the buckled wire is, in fact, bending, the force-displacement diagrams obtained differed substantially from their familiar counterparts generated in transverse bending. Judging from the force magnitudes induced as the deactivation half-cycles commenced as well as the deactivation rates, not all of the 8 wires seem to be clinically suitable for activation initiated by buckling. Magnitudes of springback were substantial from activations as large as 6 mm, and only 2 of the 8 wires exhibited full deactivations less than 80% of their activating displacements. This relatively new mode of arch wire activation that enables delivery to the dentition of mesiodistal pushing forces has substantial potential for clinical application from several biomechanical standpoints.

The modified dipeptide, enalapril, an angiotensin-converting enzyme inhibitor, is transported by the rat liver organic anion transport protein.

Oatp1, the organic anion transport polypeptide, is an integral membrane protein cloned from rat liver that mediates the uptake of various organic anions such as bromosulfophthalein (BSP) and taurocholate (TCA). Recent studies by others revealed that the thrombin inhibitor, CRC 220, a modified dipeptide, was transported by oatp1. The present study was designed to examine whether another modified peptide, enalapril, an angiotensin-converting enzyme inhibitor, was also a substrate. Transport was studied with enalapril (1 to 800 micromol/L, with [3H]enalapril) in a HeLa cell line stably transfected with oatp1-cDNA under the regulation of a Zn2+-inducible promoter. Noninduced transfected cells (without zinc) that did not express oatp1 failed to take up enalapril. In contrast, cells expressing oatp1 transported enalapril, estrone sulfate (E1S), taurolithocholic acid sulfate (TLCAS), and the glutathione conjugate of BSP (BSPGSH). Uptake of enalapril by oatp1 at 37 degreesC was substantially higher than that at 4 degreesC. The rate at 37 degreesC (uptake rates for induced - noninduced, transfected cells) was linear over 5 minutes and was concentration-dependent, characterized by a Km of 214 +/- 67 micromol/L and a Vmax of 0.51 +/- 0.15 nmol/min/mg protein. Enalapril uptake was inhibited competitively by BSP (at 1, 5, 10, and 50 micromol/L) and TCA (at 5, 25, and 100 micromol/L) with inhibition constants (Ki) of 2 and 32 micromol/L, respectively. The metabolite enalaprilat was, however, not transported by oatp1. That oatp1 is not a general transporter of anionic compounds was further shown by the lack of transport of harmol sulfate, benzoate, and hippurate. These observations attest to the role of oatp1 as a specific transporter for at least two classes of pharmacologically important peptides.

Estimation of rate constants for reactions of pulmonary microvascular angiotensin converting enzyme with an inhibitor and a substrate in vivo.

We developed a method of measuring the mole quantity of pulmonary angiotensin-converting enzyme (ACE) bound by a partially saturating dose of an ACE inhibitor injected i.v. For each test animal (11 guinea pigs), tracer (nonsaturating) doses of the ACE substrate [14C]benzoyl-Ala-Gly-Pro (14C-BAGP) and the ACE inhibitor 3H-RAC-X-65 were coinjected at timed intervals for a total of four studies per animal. The injectate used for the second study contained, in addition, a partially saturating dose of unlabeled RAC-X-65. With indicator-dilution techniques supplemented with measurements of fractional hydrolysis of 14CBAGP and uptake of 3H-RAC-X-65 during a single transit through the pulmonary vascular bed, the following parameters were computed: plasma flow (Qp), (kcat/Km)[E] vector c, k1[E] vector c and Eb, where [E] is the concentration of active ACE, Eb is the mole quantity of ACE bound by inhibitor, kcat/Km is the second-order rate constant for substrate hydrolysis, k1 is the inhibitor-ACE association rate constant and vector c is capillary mean transit time. As shown elsewhere (Catravas et al., 1990; Catravas and White, 1984), the product of Qp (in liters per second) multiplied by (kcat/Km)[E] vector c is (kcat/Km)E, and the product of Qp multiplied by k1[E] vector c is k1E, where E is the mole quantity of ACE. Values of (kcat/Km)Eb and k1Eb were computed and divided by Eb to obtain kcat/Km and k1. The fractional degree of inhibition conferred by a partially saturating dose of an ACE inhibitor can be understood to be the ratio Eb/ET, where ET is total ACE. With Eb in moles and the ratio Eb/ET, we computed the mole quantity of ET. By measuring the rate of recovery of ACE activity following partial inhibition of ACE, an apparent dissociation rate constant, k(dissoc), was computed. With k(dissoc) and K1, an apparent Ki was computed. The following computations were obtaine: ET of 0.90 +/- 0.20 (S.E.M.) nmol; kcat/Km, 5.16 +/- 0.89E + 06 M-1.sec-1; k1, 1.26 +/- 0.21E + 06 M-1.sec-1; k(dissoc), 6.47 +/- 0.63E - 04 sec-1 and Ki, 5.13E - 10 M. Although we focused on the characterization of ACE, the methods developed are general and may be applicable to studies of other vascular surface proteins, including other enzymes and hormone receptors.

Characterization of rat pulmonary vascular aminopeptidase P in vivo: role in the inactivation of bradykinin.

The nonapeptide bradykinin (BK) is hydrolyzed at multiple sites during a single passage through the rat pulmonary vascular bed. Hydrolysis of one bond, Arg1-Pro2, appears to be catalyzed by an aminoacylproline hydrolase called aminopeptidase P (AmP). To help clarify its role in BK degradation, we have characterized rat pulmonary AmP in vivo in terms of its ability to react with intravascular substrates, its saturability and its contributions to the inactivation of circulating BK. By using indicator dilution methodology, hydrolysis of tracer doses of the AmP substrate Arg-Pro-Pro-[3H]benzylamide ([3H]APPB) during a single transit through the pulmonary vascular bed was measured. Transpulmonary hydrolysis of [3H]APPB obeyed first-order enzyme kinetics and was inhibited by carrier substrate (APPB) and two alternative AmP substrates, BK and des-Arg9-BK. APPB, des-Arg9-BK and des-Arg1-BK, all capable of binding to AmP in vitro, potentiated hypotensive effects of BK injected i.v. A saturating dose of APPB, 2 mumol/kg, in coinjections with BK, potentiated effects of i.v. BK by about 4-fold when pulmonary angiotensin converting enzyme (ACE) was active or inhibited completely. Complete inhibition of ACE potentiated blood pressure effects of i.v. BK by 40- to 120-fold. When both AmP and ACE were inhibited, the effects of i.v. BK were potentiated by up to 800-fold, and the hypotensive effects of BK injected i.v. on systemic mean arterial blood pressure were equivalent to effects of BK injected into the ascending aorta (i.a.); the BK i.v. and i.a. log dose-response curves were virtually superimposable.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiation-induced early pulmonary endothelial ectoenzyme dysfunction in vivo: effect of indomethacin.

We investigated the early effects of radiation on pulmonary endothelial function in vivo 7-8 hr after exposure of rabbits to a single dose of 30 Gy to the chest. Utilizing multiple indicator-dilution techniques, we measured rates and kinetics of hydrolysis of the synthetic substrates [3H]benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP) by endothelial-bound angiotensin-converting enzyme (ACE) and of 5'[14C]-AMP by endothelial-bound 5'-nucleotidase (NCT) and binding of the synthetic ACE inhibitor [3H]RAC-X-65 during a single transpulmonary passage in anesthetized, artificially ventilated, open-chest rabbits in which both systemic and pulmonary circulations were fully supported by an extracorporeal pump. We have shown that these techniques and the use of the aforementioned probes provide reliable information on pulmonary endothelial function in vivo. Radiation to the chest produced endothelial ectoenzyme dysfunction, as reflected in altered available perfused capillary surface area and altered enzyme kinetics of all probes (decreases in substrate hydrolysis, inhibitor binding, first- and second-order kinetic constants) over a wide range of pulmonary blood flow values (reflecting approximately 60-200% of normal cardiac output). Indomethacin prevented most of these alterations in partially as well as fully recruited lungs. We conclude that impairment of endothelial ectoenzyme activity is an early event in the pathogenesis of radiation-induced lung damage, which occurs independently of hemodynamic influences and may involve synthesis of arachidonic acid metabolites.

Assay of pulmonary microvascular endothelial angiotensin-converting enzyme in vivo: comparison of three probes.

We monitored the activity of pulmonary microvascular endothelial-bound angiotensin-converting enzyme (ACE) in vivo by means of multiple indicator-dilution-type techniques, utilizing three different probes: the hydrolysis of two substrates, [3H]-benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP), and the binding of the inhibitor [3H]RAC-X-65 (RAC), all measured during a single transpulmonary passage in anesthetized rabbits, placed on total heart bypass, so that both systemic and pulmonary circulations were fully supported by means of a two-channel extracorporeal pump. Experiments were performed at pulmonary blood flows (Qb) of 250, 400, 560, and 800 ml/min in control or indomethacin-pretreated rabbits. ACE activity was also compared to that of pulmonary microvascular endothelial-bound 5'-nucleotidase, by measuring the dephosphorylation of its natural substrate 5'-[14C]AMP. We calculated substrate utilization, mean lung transit time (t), and volume of distribution (i.e., central blood volume) of all substrates, as well as inhibitor binding. We also calculated Amax/Km and Bmax products of enzyme mass and kinetic constants for substrates and inhibitor, respectively. As Qb increased, Amax/Km values for all three substrates and Bmax increased linearly, indicating microvascular recruitment. In experiments in which either BPAP and 5'-AMP metabolism or BAGP metabolism and RAC binding were studied concomitantly, a linear relationship was observed between Qb-induced changes in Amax/Km values of BPAP vs 5'-AMP as well as in Amax/Km of BAGP vs Bmax of RAC. Similarly, increasing Qb increased central blood volume and decreased t. Indomethacin had no effect on most of the hemodynamic or enzyme parameters measured. We conclude that in vivo assays of ACE proceed as predicted by Michaelis-Menten kinetics and offer insights into pulmonary endothelial pathophysiology.

N-glycosylation of forms of angiotensin converting enzyme from four mammalian species.

To help clarify bases for the molecular weight and surface charge heterogeneities of forms of somatic angiotensin converting enzyme (ACE), we examined for differences in N-glycosylation. ACE preparations purified from human, guinea pig, rat and rabbit tissues were found to be heterogeneous in terms of numbers of N-glycosylated sites (7-8 sites per molecule of ACE) and in types of structures of oligosaccharides used for glycosylation (complex versus high mannose oligosaccharide contents). Our findings, taken with reports of potential N-glycosylation sites and amino acid sequencing data, indicate that ACE forms can differ in terms of degrees of glycosylation, sites of glycosylation and structures of attached oligosaccharide units.

A comparison of guinea pig serum angiotensin converting enzyme with forms of angiotensin converting enzyme from human, rat and rabbit tissues.

Guinea pig serum angiotensin converting enzyme (ACE) activities exceed ACE activities of other mammalian sera by as much as two magnitudes. To examine the possibility that guinea pig ACE has a superior catalytic efficiency, we purified it to apparent homogeneity and compared it to highly purified forms of ACE from human seminal plasma, rat lungs and rabbit lungs. The first 24 amino acid residues of guinea pig and rat ACE forms were 96% identical with the sequence of human ACE. Second order rate constants (kcat/Km) for guinea pig, human and rabbit forms of ACE on reaction with benzoyl-Phe-Ala-Pro were identical (1.6E-09 M-1 min-1). Their dissociation constants on reaction with the ACE inhibitor RAC-X-65 were within a narrow range (10-16 pM). Thus, the high ACE activity of guinea pig serum is owing to high enzyme concentration and not to superior catalytic efficiency.

A radiochemical assay for aminopeptidase N.

We developed an assay for aminopeptidase N (AmN) in which substrate, Arg-Phe-[3H]anilide (24.9 Ci/mmol), can be used at concentrations (1-200 nM) well below Km (12 microM) and at or below enzyme concentration ([E]). Such reaction conditions simulate those in vivo where peptide hormones in picomolar concentrations (<< Km) are degraded by nano- or micromolar concentrations of enzyme. The Arg-Phe-[3H]anilide:AmN reaction obeyed first-order enzyme kinetics when human serum, human seminal plasma, guinea pig serum, or homogeneous porcine kidney AmN was used as enzyme source and substrate was within the concentration range of 1-200 nM. For porcine AmN, kcat/Km was 1.47 x 10(9) M-1 min-1, kcat 17,640 min-1. Human serum AmN was in a concentration (about 4.6 nM) in great excess over those reported for substrates such as angiotensin III. Several advantages accrue under conditions of first-order enzyme kinetics: (1) Vmax/Km is measured directly. (2) When kcat/Km is known, [E] can be computed in mol/liter. (3) IC50 values for alternative substrates can be taken as Km values. (4) IC50 values for inhibitors are Ki values when Ki >> [E]. Arg-Phe-[3H]anilide can be used to measure AmN activity in the presence of chromophores and fluorophores that interfere with photometric and fluorometric assays. We have confirmed that alleged substrates such as angiotensin III and Met-Lys- and Lys-bradykinin are bound by AmN with high affinities (Km values, 5.7, 9.1, and 14.3 microM). Bovine pulmonary artery endothelial cell cultures were found to possess AmN-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)