bluuebunny/crossref_metadata_2025 huggingface dataset:
Developing the Rural Dimension of Business-Support Policy
Authors: Anderson Duncan , Tyler Peter , McCallion Trevor
Published: August 2005

The development of rural business-support policy depends on the proper identification of those factors that have a differential impact on the success of rural as opposed to urban businesses. A number of studies have pointed to differences between urban and rural businesses in the business constraints and opportunities they face, and how they respond to them. This issue is investigated by use of a multivariate econometric model to analyse data generated by a survey of almost 800 businesses in all areas of Northern Ireland. The locations of the businesses in the study were mapped carefully according to an urban, accessible-rural, and remote-rural spatial typology. There is particular interest in the shape and form that rural business support policy might take in order to assist businesses in rural areas where there will be a reduction in traditional EU Common Agricultural Policy (CAP) assistance as well as a phased withdrawal of Objective 1 Structural Fund support. This is currently the case in Northern Ireland. The research should be seen as a pilot study designed to demonstrate how further work might be undertaken in an area where the search for appropriate interventions is likely to be a continuing theme in policy debate. Further research might usefully be directed towards examining the factors that are responsible for causing some of the differences observed. The research suggests that the business areas that will have the greatest impact on rural business competitiveness, relative to their more urban counterparts, are training, business-service provision, transport and communication infrastructure, costs of production, and the environment. This complex set of factors cuts across traditional departmental and national boundaries and highlights the importance of ensuring customised rural business support responses and the effective ‘rural proofing’ of strategically important public policies in general.
Multifocal Acquired Sensory and Motor Neuropathy
Authors: Magda Paul , Latov Norman , Brannagan Thomas H , Goldfarb Adina , Chin Russell L , Sander Howard W
Published: August 2005

The electrodiagnostic studies of 13 consecutive patients with multifocal sensory and motor neuropathy of unknown etiology were reviewed to determine whether they exhibit features of demyelination or axonal degeneration. The type and frequency of demyelinating features, fulfillment of electrodiagnostic criteria for chronic inflammatory demyelinating polyneuropathy (CIDP), and response to immunotherapy were noted. Of 13 patients, 11 had at least one electrodiagnostic feature of demyelination at presentation and 2 had none. Seventeen percent to 77% of the patients fulfilled at least one of the published electrodiagnostic CIDP criteria, depending on the criteria used, but the number of demyelinating features per patient was less than reported for unselected patients with CIDP. Patients with multifocal sensory and motor neuropathy had a similar percentage of nerves with partial conduction block or F-wave prolongation as reported for unselected CIDP, but a smaller percentage of nerves exhibiting prolonged distal compound muscle action potential duration, distal latency prolongation or slowed conduction velocities. All treated patients, including 2 who did not meet any CIDP criteria, had at least a moderate response to immunotherapy. The results indicate that a large majority of, but not all, patients with idiopathic multifocal sensory and motor neuropathies exhibit electrodiagnostic features of demyelination, although fewer than seen in classic CIDP.
Polyneuropathy in Osteosclerotic Myeloma Coexisting With Hyaline Vascular Castleman's Disease
Authors: Sahota Preeti , Das Chandi P , Prabhakar Sudesh , Rajwanshi A , Singh Paramjeet , Khurana Dheeraj
Published: August 2005

Peripheral neuropathy is usually the presenting feature of POEMS syndrome. Approximately 50% of patients with POEMS syndrome are associated with osteosclerotic myeloma, a rare variant of multiple myeloma, and some with Castleman's disease, an unusual lymphoproliferative disorder. The multicentric plasma cell variant of Castleman's disease is usually associated with systemic disorders rather than its localized form of mediastinal lymphoid hyperplasia characterized by hyalinization of follicles and interfollicular vascular proliferation. We report a 48-year-old woman who presented with progressive sensorimotor demyelinating polyradiculoneuropathy, bilateral optic disc edema, hepatosplenomegaly, generalized lymphadenopathy, and skin changes. There was associated thrombocytosis, hypothyroidism, hypoparathyroidism, mixed osteolytic and osteosclerotic bone lesions, monoclonal gammopathy of IgG lambda type, and hyaline vascular type of Castleman's disease. This combination of POEMS syndrome, osteosclerotic myeloma with a hyaline vascular type of Castleman's disease is uncommon.
Percutaneous Conchotome Biopsy of the Deltoid and Quadricep Muscles in the Diagnosis of Neuromuscular Disorders
Authors: Poulsen Mette Buch , Bojsen-Moller Marie , Jakobsen Johannes , Andersen Henning
Published: August 2005

Clinical usefulness and safety of percutaneous conchotome biopsy of the deltoid muscle and of the quadriceps muscle was compared in patients suspected to have a neuromuscular disorder. In 121 samples from the quadriceps muscle and in 39 samples from the deltoid muscle, the quantity of muscle fibers was equal and the fraction of biopsies resulting in a specific diagnosis was similar (50% and 56%, respectively). Also, biopsy from the 2 locations resulted in the same distribution of neurogenic, inflammatory, and myopathic disorders. After quadriceps muscle biopsy, 2 patients developed vasovagal syncopes and one patient a local hematoma, whereas no complications occurred in the patients subjected to deltoid muscle biopsy. We conclude that biopsy from the deltoid muscle is clinically as useful and safe as biopsy from the quadriceps muscle.
Dendritic Cell Adhesion Is Enhanced on Endothelial Cells Preexposed to Calcineurin Inhibitors
Authors: Schlichting Christoph L , Schareck Wolfgang D , Weis Michael
Published: August 2005

Chronic rejection remains a major complication in solid organ transplantation. Host alloreactive T cells (TC) can be activated by donor dendritic cells (DCs; direct allorecognition) or by recipient DCs (indirect allorecognition). A fundamental aspect of DC function is vascular invasion to present donor antigens to recipient naive TC in secondary lymphoid organs. We investigated the impact of calcineurin inhibitors on DC binding and transmigration to allogeneic human microvascular endothelial cells (ECs) with and without blocking of specific adhesion molecules. Recipient immature DCs were generated by culturing CD14+ human peripheral blood monocytes with GM-CSF and IL-4. DC adhesion and transmigration were investigated on allogeneic ECs preincubated with increasing concentrations of cyclosporine and tacrolimus. Experiments were repeated in the presence of blocking antibodies against LFA-1, PECAM-1, VCAM-1, and ICAM-1. Endothelial stimulation with cyclosporine A (100 and 300 ng/mL) and tacrolimus (15 ng/mL) significantly enhanced DC-EC adhesion and transmigration (P < 0.01). LFA-1 blockade on DCs significantly reduced cyclosporine- and tacrolimus-induced DC adhesion (P < 0.001). VCAM-1 blockade on ECs partially reversed cyclosporine-induced DC adhesion (P < 0.001), whereas DC adhesion under tacrolimus exposure was significantly decreased by ICAM-1 (P < 0.01) and PECAM-1 (P < 0.001) blockade. DC binding and transmigration on allogeneic ECs exposed to calcineurin inhibitors is concentration-dependently increased. Different adhesion molecule patterns on ECs are responsible for enhanced DC invasion under cyclosporine and tacrolimus exposure. We speculate that long-term immunosuppression mediates enhanced invasion of recipient DCs to the donor organ and therefore may aggravate chronic rejection.
Improved Mitochondrial Bioenergetics by Anesthetic Preconditioning During and After 2 Hours of 27°C Ischemia in Isolated Hearts
Authors: An Jianzhong , Camara Amadou K. S , Riess Matthias L , Rhodes Samhita S , Varadarajan Srinivasan G , Stowe David F
Published: August 2005

We examined if sevoflurane given before cold ischemia of intact hearts (anesthetic preconditioning, APC) affords additional protection by further improving mitochondrial energy balance and if this is abolished by a mitochondrial KATP blocker. NADH and FAD fluorescence was measured within the left ventricular wall of 5 groups of isolated guinea pig hearts: (1) hypothermia alone; (2) hypothermia + ischemia; (3) APC (4.1% sevoflurane) + cold ischemia; (4) 5-HD + cold ischemia, and (5) APC + 5-HD + cold ischemia. Hearts were exposed to sevoflurane for 15 minutes followed by 15 minutes of washout at 37°C before cooling, 2 hours of 27°C ischemia, and 2 hours of 37°C reperfusion. The KATP channel inhibitor 5-HD was perfused before and after sevoflurane. Ischemia caused a rapid increase in NADH and a decrease in FAD that waned over 2 hours. Warm reperfusion led to a decrease in NADH and an increase in FAD. APC attenuated the changes in NADH and FAD and further improved postischemic function and reduced infarct size. 5-HD blocked the cardioprotective effects of APC but not APC-induced alterations of NADH and FAD. Thus, APC improves redox balance and has additive cardioprotective effects with mild hypothermic ischemia. 5-HD blocks APC-induced cardioprotective effects but not improvements in mitochondrial bioenergetics. This suggests that mediation of protection by KATP channel opening during cold ischemia and reperfusion is downstream from the APC-induced improvement in redox state or that these changes in redox state are not attenuated by KATP channel antagonism.
Vasorelaxant and Antioxidant Activity of Flavonols and Flavones: Structure-Activity Relationships
Authors: Woodman Owen L , Meeker William F , Boujaoude Mirna
Published: August 2005

We investigated the structure-activity relationships regarding vascular and antioxidant activity of a range of synthetic flavonols and flavones with 3 or fewer hydroxyl (OH) or methoxyl substitutions. The relaxant responses and ability of the flavones/flavonols to inhibit phenylephrine (PE)- and Ca2+-induced contraction was determined in rat isolated thoracic aorta. The ability of these compounds to reduce the level of superoxide and preserve endothelium-dependent relaxation in the presence of oxidative stress was also examined. Four compounds impaired contraction to PE or Ca2+, in the potency order 3′-hydroxyflavonol > 3′,4′-dihydroxyflavonol > 7,4′-dihydroxyflavonol > 3′,4′-dihydroxyflavone. Flavonol, 3′,4′-dimethoxyflavonol, and flavone were significantly less active. The flavonoids caused concentration-dependent reductions in superoxide produced by rat aorta in the presence of NADPH. The most active compounds, 3′,4′-dihydroxyflavonol and 7,4′-dihydroxyflavonol, preserved endothelium-dependent relaxation in the presence of oxidative stress caused by pyrogallol or xanthine/xanthine oxidase. The results indicate that the catechol group is not critical for vascular relaxant or antioxidant activity, but rather, the important determinants for higher vascular and antioxidant activity of these compounds are the presence of a C3 OH group and the total number of OH substituents, respectively. These results have allowed the identification of the structural characteristics that promote vascular and antioxidant activity of flavonols, which may lead to the development of agents useful in treatment of cardiovascular disease.
Lowering of Elevated Ambulatory Blood Pressure by HMG-CoA Reductase Inhibitors
Authors: Terzoli Laura , Mircoli Luca , Raco Roberta , Ferrari Alberto U
Published: August 2005

Controversial results were reported as to a possible blood pressure-lowering effect of statins. This may relate to methodological limitations (blood pressure measuring techniques) or to putative different effects of statins in different biologic conditions (cholesterol or blood pressure levels, age, etc). Patients with cholesterol >200 mg/dL and no previous statin treatment underwent 24-hour ambulatory blood pressure (ABP) monitoring and were classified as normotensives or hypertensives according to their ABP. They were randomized to statin (n = 51, simvastatin or pravastatin, 10-20 mg/d; atorvastatin, 5-10 mg/d) or control treatment (n = 23, soy lecithin, 20 g/d) for 2 months, after which ABP assessment was repeated. No consistent treatment-related reduction in ABP was observed in lecithin-treated patients (either hypertensives or normotensives) or in statin-treated normotensive patients (−0.7 ± 5.1/−1.0 ± 4.6 mm Hg, both P = ns). In contrast, statin-treated hypertensive patients showed lower systolic and diastolic blood pressure (−5.7 ± 5.8/−3.5 ± 3.9 mm Hg, both P < 0.001), the effect was entirely accounted for by reduced daytime values with no change in nighttime values, and it was unrelated to the concomitant statin-induced cholesterol reduction. Statins moderately but significantly lower blood pressure in patients with high (but not with normal) ABP; the effect is confined to the daytime period and is unrelated to the extent of the cholesterol lowering.
Hemodynamic Effect of Intracoronary Administration of Levosimendan in the Anesthetized Pig
Authors: Grossini E , Caimmi P P , Molinari C , Teodori G , Vacca G
Published: August 2005

In this study the hemodynamic effects of intracoronary injection of levosimendan in anesthetized pigs and the mechanisms involved were examined. In 12 anesthetized pigs instrumented for measurement of heart rate (HR), aortic blood pressure (ABP), central venous pressure (CVP), left ventricular end-diastolic blood pressure, left ventricular contractility and relaxation, and mean coronary blood flow (CBF), levosimendan has been injected into the left anterior descending coronary artery at doses corresponding to the ones commonly used in clinics as bolus administration but adapted to the measured CBF. In a further 9 pigs levosimendan has been administered after the blockade of α and β adrenoceptors, muscarinic receptors, and coronary nitric oxide synthase (NOS) to investigate the action mechanism of the drug. The intracoronary bolus administration of doses of levosimendan corresponding to 12 and 24 μg/kg in 10 minutes exerted, respectively, CBF increases of 26.3% and 41.3% of the control values in the absence of changes in the other hemodynamic variables. The blockade of the autonomic nervous system did not prevent the coronary vasodilation, which was, however, abolished by the NOS inhibition. The intracoronary administration of levosimendan exerts positive effects on myocardial blood supply without changes in ABP, HR, CVP, or in myocardial kinetics. The coronary effects of levosimendan are related to NO production.
Mechanisms of the Dilator Action of Danshen (Salvia miltiorrhiza) on Rat Isolated Femoral Artery
Authors: Lam Francis Fu Yuen , Yeung John Hok Keung , Cheung Jessica Ho Yan
Published: August 2005

This study investigates the actions of Danshen crude extract (Salvia miltiorrhiza) on rat isolated femoral artery rings precontracted with phenylephrine. Low concentrations of Danshen (10 to 30 μg/mL) enhanced the phenylephrine-precontracted tone by a maximum of 31.20 ± 2.71%. At concentrations 100 μg/mL or above, Danshen relaxed the precontracted tone, with full relaxation obtained at 1 mg/mL. Involvement of endothelium-dependant mechanisms in the dilator effect of Danshen was investigated by pretreatment of the artery rings with a cyclooxygenase inhibitor flurbiprofen (10 μM), a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 μM), a muscarinic receptor antagonist atropine (100 nM), and by mechanical removal of the endothelium; none of these procedures produced a significant change on the Danshen-induced effect. Involvement of endothelium-independent mechanisms was investigated in endothelium-denuded artery rings pretreated with a histamine H2 receptor antagonist cimetidine (10 μM), a β-adrenoceptor antagonist propranolol (100 nM), an adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 μM), a guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM), and a potassium channel inhibitor tetraethylammonium (TEA, 10 and 100 mM); only TEA was effective in partially inhibiting the Danshen-induced effect. These findings suggest the dilator action of Danshen on rat femoral artery was mediated in part by the opening of TEA-sensitive K+ channels in the smooth muscle cells. Muscarinic receptors, histamine receptors, β-adrenoceptors, endothelium-derived relaxant factors, adenylyl cyclase, and guanylyl cyclase-dependent pathways did not play a role in its vasodilatory effect.
Two Inotropes With Different Mechanisms of Action: Contractile, PDE-Inhibitory and Direct Myofibrillar Effects of Levosimendan and Enoximone
Authors: Szilágyi Szabolcs , Pollesello Piero , Levijoki Jouko , Haikala Heimo , Bak István , Tósaki Árpád , Borbély Attila , Édes István , Papp Zoltán
Published: August 2005

We characterized the Ca2+-sensitizing and phosphodiesterase (PDE)-inhibitory potentials of levosimendan and enoximone to assess their contributions to the positive inotropic effects of these drugs. In guinea pig hearts perfused in the working-heart mode, the maximal increase in cardiac output (55%, P < 0.05) was attained at 50 nM levosimendan. The corresponding value for enoximone (36%) was significantly smaller (P < 0.05) and was observed at a higher concentration (500 nM). In permeabilized myocyte-sized preparations levosimendan evoked a maximal increase of 55.8 ± 8% (mean ± SEM) in isometric force production via Ca2+ sensitization (pCa 6.2, EC50 8.4 nM). Enoximone up to a concentration of 10 μM failed to influence the isometric force. The PDE-inhibitory effects were probed on the PDE III and PDE IV isoforms. Levosimendan proved to be a 1300-fold more potent and a 90-fold more selective PDE III inhibitor (IC50 for PDE III 1.4 nM, and IC50 for PDE IV 11 μM, selectivity factor ∼8000) than enoximone (IC50 for PDE III 1.8 μM, and IC50 for PDE IV 160 μM, selectivity factor ∼90). Hence, our data support the hypothesis that levosimendan exerts positive inotropy via a Ca2+-sensitizing mechanism, whereas enoximone does so via PDE inhibition with a limited PDE III versus PDE IV selectivity.
Endothelial Nitric Oxide Synthase Inhibition Does Not Alter Endothelial Progenitor Cell Colony Forming Capacity or Migratory Activity
Authors: Hoetzer Greta L , Irmiger Heather M , Keith Rebecca S , Westbrook Kimber M , DeSouza Christopher A
Published: August 2005

Endothelial nitric oxide synthase (eNOS) activity has been shown to play a pivotal role in the mobilization of endothelial progenitor cells (EPCs) into the circulation from bone marrow. Indeed, in eNOS-deficient mice, exercise-induced EPC mobilization is severely diminished. We determined ex vivo whether circulating EPC colony-forming capacity and migratory activity are influenced by eNOS activity. Peripheral-blood mononuclear cells were isolated from 20 healthy adults and preplated for 2 days, and nonadherent cells were further cultured for 7 days in the presence and absence of NG-nitro-L-arginine methyl ester (L-NAME, 300 μM, an eNOS antagonist) to determine EPC colony-forming units. Migratory activity of EPCs, cultured with and without L-NAME (300 μM) was determined by utilizing a modified Boyden chamber. The number of EPC colony-forming units was not significantly different when cultured in the absence or presence of L-NAME (21 ± 5 vs 18 ± 5). Moreover, eNOS inhibition did not alter EPC migratory activity; mean fluorescence was similar in samples cultured with (983 ± 126 RFUs) and without (962 ± 105 RFUs) L-NAME. These in vitro results suggest that, in contrast to EPC mobilization from the bone marrow, eNOS does not exert a modulatory influence on the functional capacity of circulating EPCs to either form colonies or migrate.
Lack of Evidence of Association of p21WAF1/CIP1 Polymorphism with Lung Cancer Susceptibility and Prognosis in Taiwan
Authors: Shih Chuen‐Ming , Lin Pey‐Tzy , Wang Hui‐Chun , Huang Wei‐Chi , Wang Yi‐Ching
Published: August 2005

An association between the Arg allele of the p21WAF1/CIP1 codon 31 polymorphism and lung cancer has been reported. However, the genotype distribution of the p21 codon 31 polymorphism, as well as the association of this polymorphism with lung cancer risk and prognosis, remain undefined in the Taiwanese population. Therefore, we investigated the genotype distribution of the p21 codon 31 polymorphism in 155 lung cancer patients and 189 non‐cancer controls. The genotype frequencies in the Taiwanese non‐cancer controls were 0.51 (Ser) and 0.49 (Arg). χ2 analysis indicated significant differences in Taiwanese genotype distribution of p21 from those reported for Swedes (P=0.001), Caucasians (P=0.001), Indians (P=0.001), and African‐Americans (P=0.001). However, our data did not demonstrate an association of the Arg allele of the p21 polymorphism with lung cancer risk in Taiwan. Lung cancer patients with Ser/Arg and Arg/Arg genotypes were at a nonsignificant 1.15‐fold increased risk of lung cancer when compared to individuals with the Ser/Ser genotype (95%CI, 0.70–1.86). In addition, although p21 is a downstream target of p53, we found no significant correlation of the p21 polymorphism with the p53 polymorphism and p53 gene mutation in lung cancer patients. We further investigated the association of the p21 polymorphism with prognosis in 154 lung cancer patients. Patients with the Ser/Ser genotype tended to have a poorer prognosis than those with the Ser/Arg and Arg/Arg genotypes (P=0.097, by the log rank test). Our data suggest that the p21 codon 31 polymorphism may not play a significant role in cancer susceptibility and the prognosis of lung cancer patients in Taiwan.
Low Susceptibility of Long‐Evans Cinnamon Rats to N‐Butyl‐N‐(4‐hydroxybutyl)‐nitrosamine‐induced Urinary Bladder Carcinogenesis and Inhibitory Effect of Urinary Copper
Authors: Chone Yoshifumi , Kinouchi Takemi , Yamada Takamasa , Suzuki Yasuo , Kitaura Keisuke , Jiao Zhongxian , Minami Takanori , Bando Yoshimi , Uehara Hisanori , Mochizuki Masataka , Ohnishi Yoshinari , Izumi Keisuke
Published: August 2005

We studied the susceptibilities to N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐induced urinary bladder carcinogenesis of male Long‐Evans Cinnamon (LEC), F344 and Long‐Evans Agouti (LEA) rats. Male rats (n=21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN‐treated LEC and F344 rats were 12% and 76%, respectively (P < 0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively (P < 0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N‐butyl‐N‐(3‐carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats (P < 0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28‐ and 50‐week‐old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two‐stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN induced bladder carcinogenesis. In a two‐stage carcinogenesis study using LEC rats, oral administration of D‐penicillamine decreased urinary copper excretion, and increased BBN‐induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.
Expression of Drug Resistance‐related Genes in Head and Neck Squamous Cell Carcinoma and Normal Mucosa
Authors: Hirata Shitau , Katoh Osamu , Oguri Tetsuya , Watanabe Hiromitsu , Yajin Koji
Published: August 2005

We examined the expression levels of mRNA for multidrug resistance 1 (MDR1), multidrug resistance‐associated protein (MRP), human canalicular multispecific organic anion transporter (cMOAT), lung resistance‐related protein (LRP), topoisomerase IIα, β(Topo IIα, β) and topoisomerase I (Topo I) genes in human head and neck squamous cell carcinoma (HNSCC) specimens and mucosa (HNM) specimens, to elucidate their roles in relation to the biological characteristics and drug resistance in vivo. Fifty‐eight samples (45 head and neck carcinomas and 13 head and neck mucosa) obtained during surgical resection or biopsy from 38 patients were analyzed using the quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) method. MDR1, MRP, LRP, Topo IIα, Topo IIβ, and Topo I gene transcripts were detected in all the samples tested, but cMOAT mRNA was not detected in them. Comparisons of the expression levels in HNSCC with those in HNM showed that the Topo IIα gene expression level was higher in HNSCC than in HNM (P=0.0298). Moreover, the Topo IIα mRNA level was significantly higher in metastatic lymph node samples of HNSCC than in HNM samples (P=0.0205). There were no significant differences in the six genes' expression levels between samples exposed to platinum drugs and those not exposed to platinum drugs. These results suggest that it may be effective in anticancer therapy to use topoisomerase‐targetting drugs against HNSCC, especially metastatic neck tumors, and that the expression of these genes in HNSCC is not associated with platinum drug exposure.
HeLa Cell Transformants Overproducing Mouse Metallothionein Show in vivo Resistance to cis‐Platinum in Nude Mice
Authors: Toyoda Haruka , Mizushima Tomoe , Satoh Masahiko , Iizuka Narushi , Nomoto Akio , Chiba Harumi , Mita Masaharu , Naganuma Akira , Himeno Seiichiro , Imura Nobumasa
Published: August 2005

Plasmid pSV2MT‐I encoding mouse metallothionein‐I (MT‐I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi‐copies of mouse MT‐I cDNA in their genomes were isolated. These transformants produced 4 to 20‐fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis‐platinum (cis‐DDP) in vitro. To study the role of MT in resistance to cis‐DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3‐fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 μmol/kg of cis‐DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis‐DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis‐DDP resistance may underestimate its importance in in vivo situations.
Dihydropyrimidine Dehydrogenase and Messenger RNA Levels in Gastric Cancer: Possible Predictor for Sensitivity to 5‐Fluorouracil
Authors: Ishikawa Yoichiro , Kubota Tetsuro , Otani Yoshihide , Watanabe Masahiko , Teramoto Tatsuo , Kumai Koichiro , Takechi Teiji , Okabe Hiroyuki , Fukushima Masakazu , Kitajima Masaki
Published: August 2005

We investigated the correlation between tumor sensitivity to 5‐fluorouracil (5‐FU) and enzymatic activities of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in human gastric cancer specimens. Forty‐one patients with advanced gastric cancer gave informed consent and were enrolled in the study. Biopsy specimens of gastric cancer were obtained preoperatively through gastrofiberscopy and used to determine TS and DPD messenger RNA (mRNA) levels. TS and DPD enzyme activity and mRNA levels were also measured in resected tumor tissue samples obtained after surgical resection. TS and DPD activity were measured using the TS‐binding assay and a radioenzymatic assay, respectively, while mRNA levels were measured by semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), with co‐amplification of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an internal standard. 5‐FU sensitivity of resected tumor specimens was measured by the tetrazolium‐based colorimetric assay (MTT assay). Both TS and DPD mRNA levels correlated well between biopsied and resected tumor specimens. A statistically significant correlation was also observed between mRNA levels in biopsied specimens and enzymatic activities in resected specimens. DPD levels significantly correlated with 5‐FU sensitivity, such that high DPD activity and high DPD mRNA levels resulted in low sensitivity to 5‐FU. In contrast, no correlation was observed between TS activity or TS mRNA levels and 5‐FU sensitivity. We conclude that tumor DPD mRNA level, as assessed from biopsy specimens obtained by gastrofiberscopy, may be a useful indicator in predicting tumor sensitivity to 5‐FU in patients with gastric cancer.
Increased Expression of Cyclin D1, Cyclin E and p21Cip1 Associated with Decreased Expression of p27Kip1 in Chemically Induced Rat Mammary Carcinogenesis
Authors: Jang Tae Jung , Kang Myung Soo , Kim Heesoo , Kim Dong Hoon , Lee Jong Im , Kim Jung Ran
Published: August 2005

We induced rat mammary tumors in 7‐week‐old female Sprague‐Dawley rats by intragastric administration of 7,12‐dimethylbenz(a)anthracene (DMBA), and analyzed by immunohistochemistry the expression of cyclin D1, cyclin E, p21Cip1, and p27Kip1 in carcinomas, atypical tumors, and benign tumors as well as normal mammary glands from the control group. Proliferation status was assessed by immunohistochemistry using bromodeoxyuridine (BrdU). A sequential increase in cyclin D1‐, cyclin E‐, and p21Cip1‐positive epithelial cells was observed from normal mammary glands, to atypical tumors, to carcinomas. In contrast, carcinomas showed a significantly lower number of epithelial cells immunoreactive to p27Kip1 when compared with atypical tumors, benign tumors and normal mammary glands. The immunoreactivities of BrdU, cyclin D1, cyclin E, and p21Cip1 were positively correlated, whereas that of p27Kip1 appeared inversely correlated to those of the others. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and western blot analysis were also performed to determine the mRNA and protein levels of cyclins and cyclin‐dependent kinase inhibitors in tumors and normal mammary glands. The protein levels for cyclin D1, cyclin E and p21Cip1 in carcinomas and atypical tumors were significantly higher than those in benign tumors, while normal mammary glands showed negligible expression. On RT‐PCR, tumors showed higher mRNA levels of cyclin D1 and cyclin E than those of normal mammary glands. Our results suggest that rat mammary carcinogenesis involves increased expression of cyclin D1, cyclin E, and p21Cip1, associated with decreased expression of p27Kip1
The Characteristics of Epstein‐Barr Virus (EBV)‐positive Diffuse Large B‐Cell Lymphoma: Comparison between EBV+ and EBV‐ Cases in Japanese Population
Authors: Kuze Tetuo , Nakamura Naoya , Hashimoto Yuko , Sasaki Yoshikazu , Abe Masafumi
Published: August 2005

We have investigated 114 cases with diffuse large B‐cell lymphoma (DLBCL) to clarify the characteristics of DLBCL with Epstein‐Barr virus (EBV) infection. Thirteen cases (11.4%) showed EBVencoded RNA 1 (EBER1) signals by RNA in situ hybridization. EBV‐encoded latent membrane protein 1 (LMP1) and EBV‐encoded nuclear antigen 2 (EBNA2) were expressed in 11 and 4 cases, respectively. Expression of CD30, Bcl‐6 and immunoglobulin (Ig) was found in 92%, 31% and 23% with EBV+ DLBCL, and in 15%, 79% and 82% with EBV‐ DLBCL, respectively. The sequence of rearranged Ig heavy chain (IgH) variable (V) region gene was analyzed in 5 cases with EBV+ DLBCL and 61 cases with EBV‐ DLBCL. Somatic mutation was found in all cases except one with EBV‐ DLBCL. Average mutation frequency was 9.6% in EBV+ DLBCL vs. 11.5% in EBV‐ DLBCL. The rates of replacement mutation vs. silent mutation (R/S values) in complementarity determining region II and framework region III were 2.7 and 1.5 in EBV+ DLBCL, 2.6 and 1.4 in EBV‐ DLBCL. Crippling mutation generating a stop codon was found in 2 of 5 cases (40%) with EBV+ DLBCL, but none of 61 cases (0%) with EBV‐ DLBCL. These findings suggest that EBV+ DLBCL and EBV‐ DLBCL were both derived from germinal center (GC) or post‐GCB cells, and EBV+ DLBCL frequently have a non‐functional IgH gene owing to crippling mutation.
Allelic Loss of 14q and 22q, NF2 Mutation, and Genetic Instability Occur Independently of c‐kit Mutation in Gastrointestinal Stromal Tumor
Authors: Fukasawa Tomoki , Chong Ja‐Mun , Sakurai Shinji , Koshiishi Naoki , Ikeno Rie , Tanaka Akira , Matsumoto Yoshiro , Hayashi Yukiko , Koike Morio , Fukayama Masashi
Published: August 2005

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Since c‐kit mutation occurs only in one‐third of GIST, there might be other molecular mechanisms. Loss of heterozygosity (LOH), microsatellite instability (MSI) and NF2 gene mutation were investigated in 22 GISTs (9 low‐risk and 13 high‐risk tumors). LOH and MSI were evaluated using 41 markers on 21 chromosomal arms, and NF2 gene mutation was examined by PCR‐SSCP. High frequency of LOH was observed on 14q (9/19, 47%), and 22q (17/22, 77%). The frequencies were similar in low‐risk and high‐risk tumors, and were unrelated with gastric or intestinal origin. Two other abnormalities, additional LOH on other chromosomes and MSI at more than two loci, were characteristic of the high‐risk tumors (P < 0.05). NF2 gene mutation was identified in two cases showing 22q‐LOH (8 bp deletion on the splice donor site of exon 7, and 1 bp insertion at position 432 of exon 4, which resulted in nonsense mutation). There was no significant correlation between these results and c‐kit gene mutation, which was observed in 8 of 22 tumors. Suppressor genes on 14q and 22q may be involved, independently of c‐kit gene mutation, in the development of GIST. NF2 contributes as a tumor suppressor in a small subset of GIST. These abnormalities are presumably followed by increased genetic instability.
In vivo and in vitro Interactions between Human Colon Carcinoma Cells and Hepatic Stellate Cells
Authors: Shimizu Sadatoshi , Yamada Nobuya , Sawada Tetsuji , Ikeda Kazuo , Kawada Norifumi , Seki Syuichi , Kaneda Kenji , Hirakawa Kosei
Published: August 2005

Stromal reaction is important for the growth of cancer both in primary and metastatic sites. To demonstrate this reaction during the hepatic metastasis of human colon carcinoma, we histologically investigated alterations to the distribution and phenotype of hepatic stellate cells (HSCs), the only mesenchymal cells in the liver parenchyma, using a nude mouse model. Intrasplenically injected colon carcinoma LM‐H3 cells migrated into the space of Disse and underwent proliferation, in close association with hepatocytes and HSCs, at 2 days. At 14 days, HSCs were accumulated around the tumor mass and expressed α‐smooth muscle actin, a marker for HSC activation. We next investigated in vitro the growth factors involved in the interactions between LM‐H3 cells and HSCs. Conditioned medium of rat HSCs which underwent culture‐induced activation contained platelet‐derived growth factor (PDGF)‐AB, hepatocyte growth factor (HGF) and transforming growth factor (TGF)β, and could augment LM‐H3‐cell proliferation and migration. Neutralizing antibodies against PDGF‐AA and PDGF‐BB and those against PDGF‐BB and HGF inhibited proliferation and migration, respectively, of LM‐H3 cells, whereas antibody against TGF‐β had no effect. LM‐H3 cells expressed PDGF receptors‐α and ‐β and c‐met. Conditioned medium of LM‐H3 cells contained PDGF‐AB, and could enhance HSC proliferation and migration. This augmenting effect was suppressed by treatment with anti‐PDGF‐AB antibody. The present study has demonstrated that bidirectional interactions involving PDGF and HGF take place in vitro between colon carcinoma cells and HSCs, raising the possibility that similar interactions might be involved in the stromal reaction during hepatic metastasis.
Synergistic Suppressive Effect of Double Transfection of Tumor Necrosis Factor‐α and Interleukin 12 Genes on Tumorigenicity of Meth‐A Cells
Authors: Fujiwara Hitoshi , Yamauchi Naofumi , Sato Yasushi , Sasaki Katsunori , Takahashi Minoru , Okamoto Tetsuro , Sato Tsutomu , Iyama Satoshi , Koshita Yoshikazu , Hirayama Michiaki , Yamagishi Hisakazu , Niitsu Yoshiro
Published: August 2005

Tumor necrosis factor‐α(TNF‐α) and interleukin 12 (IL‐12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer/lymphocyteactivated killer (NK/LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1‐type cellular immunity and enhances the activities of natural killer T (NKT), NK/LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK/LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor‐specific immunity was confirmed by rechallenge with parental Meth‐A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF‐αand IL‐12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.
A Computational Model for Quantitative Analysis of Cell Cycle Arrest and Its Contribution to Overall Growth Inhibition by Anticancer Agents
Authors: Kuh Hyo‐Jeong , Nakagawa Shin‐ichi , Usuda Jitsuo , Yamaoka Katsunori , Saijo Nagahiro , Nishio Kazuto
Published: August 2005

Most anticancer agents induce cell cycle arrest (cytostatic effect) and cell death (cytotoxic effect), resulting in the inhibition of population growth of cancer cells. When asynchronous cells are to be examined, the currently used flow cytometric method can not provide checkpoint‐specific and quantitative information on the drug‐induced cell cycle arrest. Hence, despite its significance, no good method to analyze in detail the mechanism of cell cycle arrest and its contribution to overall growth inhibition induced by an anticancer agent has yet been established. We describe in this study the development of a discrete time (Markov model)‐based computational model for cell cycle progression/arrest with transition probability (TPi) as a model parameter. TPi was calculated using model equations that include easily measurable parameters such as the fraction of cells in each cell cycle phase and population doubling time. The TPi was then used to analyze checkpoint‐specific and quantitative changes in cell cycle progression. We also used TPi in a Monte‐Carlo simulation to predict growth inhibition caused by cell cycle arrest only. Human SCLC cells (SBC‐3) exposed to UCN‐01 were used to validate the model. The model‐predicted growth curves agreed with the observed data for SBC‐3 cells not treated or treated at a cytostatic concentration (0.2μM) of UCN‐ 01, indicating validity of the present model. The changes in TPi indicated that UCN‐01 reduced the G1‐to‐S transition rate and increased the S‐to‐G2/M and G2/M‐to‐G1 transition rates of SBC‐3 cells in a concentration‐ and time‐dependent manner. When the model‐predicted growth curves were compared with the observed data for cells treated at a cytotoxic concentration (2 μM, they suggested that 22% out of 65% and 32% out of 73% of the growth inhibition could be attributed to the cell cycle arrest effect after 48 h and 72 h exposure, respectively. In conclusion, we report here the establishment of a novel method of analysis that can provide checkpoint‐specific and quantitative information about cell cycle arrest induced by an anticancer agent and that can be used to assess the contribution of cell cycle arrest effect to the overall growth inhibition.
Effective Cancer Targeting Using an Anti‐tumor Tissue Vascular Endotheliumspecific Monoclonal Antibody (TES‐23)
Authors: Wakai Yukiko , Matsui Junji , Koizumi Keiichi , Tsunoda Shin‐ichi , Makimoto Hiroo , Ohizumi Iwao , Taniguchi Kenji , Kaiho Shin‐ichi , Saito Hiroyuki , Utoguchi Naoki , Tsutsumi Yasuo , Nakagawa Shinsaku , Ohsugi Yoshiyuki , Mayumi Tadanori
Published: August 2005

Immunoconjugate targeting of solid tumors has not been routinely successful because the endothelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an antitumor tissue endothelium‐specific monoclonal antibody (TES‐23). TES‐23, an IgG1 monoclonal antibody raised against rat KMT‐17 fibrosarcoma‐derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES‐23‐NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti‐tumor effects against rat KMT‐17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of 125I‐labeled TES‐23 into BALB/c mice bearing Meth‐A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES‐23 was greater than that of the control IgG. In the present study, we report the anti‐tumor effects of this monoclonal antibody in mice bearing Meth‐A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES‐23 might recognize these antigens.
Cytotoxicity of cis‐[((1R,2R)‐1,2‐Cyclohexanediamine‐N,N')bis(myristato)]‐platinum (II) Suspended in Lipiodol in a Newly Established Cisplatinresistant Rat Hepatoma Cell Line
Authors: Kishimoto Shuichi , Miyazawa Kenji , Terakawa Yoko , Ashikari Hiromi , Ohtani Aya , Fukushima Shoji , Takeuchi Yoshikazu
Published: August 2005

The cytotoxic activity of cis‐[((1R,2R)‐1,2‐cyclohexanediamine‐N,N')bis(myristato)]platinum (II) (SM‐11355) was evaluated in a cisplatin (CDDP)‐resistant tumor cell line, and compared with that of CDDP. H4‐II‐E/CDDP with acquired resistance to CDDP was established by continuous exposure of a rat hepatic tumor line, H4‐II‐E, to increasing concentrations of CDDP over 12 months. Compared with the parental cell line, this cell line exhibited an 8.8‐fold increase in resistance to CDDP and was not cross‐resistant to 1,2‐diaminocyclohexane platinum (II) dichloride (DPC). There were no differences in sensitivity to six non‐platinum antitumor drugs between H4‐II‐E and H4‐II‐E/CDDP, which suggests that H4‐II‐E/CDDP is not multidrug‐resistant. Intracellular platinum accumulation and the formation of a platinum‐DNA adduct following CDDP exposure were significantly reduced in H4‐II‐E/CDDP compared to the parental cell line. The acquired CDDP resistance in H4‐II‐E/CDDP appeared to be predominantly due to reduced CDDP uptake. H4‐II‐E/CDDP was also resistant to CDDP suspended in Lipiodol (CDDP/Lipiodol), but was not crossresistant to SM‐11355 suspended in Lipiodol (SM‐11355/Lipiodol). Also, there were no differences in intracellular platinum accumulation or the formation of platinum‐DNA adducts after SM‐11355/Lipiodol exposure between H4‐II‐E and H4‐II‐E/CDDP. These results suggest that acquired CDDP resistance in H4‐II‐E/CDDP does not influence the cytotoxic activity of SM‐11355/Lipiodol.
Improved in vivo Antitumor Efficacy and Reduced Systemic Toxicity of Carboxymethylpullulan‐peptide‐doxorubicin Conjugates
Authors: Nogusa Hideo , Hamana Hiroshi , Uchida Naomi , Maekawa Ryuji , Yoshioka Takayuki
Published: August 2005

The antitumor efficacy of the conjugate of doxorubicin (DXR) and carboxymethylpullulan (CMPul) with Phe‐Gly spacer (CMPul‐FG‐DXR) was evaluated using murine tumor models and compared with that of DXR. The conjugate exhibited higher antitumor efficacy against Lewis lung carcinoma than DXR. Complete tumor regression followed by long‐term tumor‐free survival was frequently observed when CMPul‐FG‐DXR was administered i.v. three times at a dose equivalent to 10 mg/kg of DXR. The superior survival as well as anti‐metastatic effect of CMPul‐FG‐DXR in comparison with DXR was also demonstrated with the M5076 murine reticulosarcoma model. Body weight loss in mice treated with the conjugate was less than that in the DXR‐treated group, indicating lower systemic toxicity of CMPul‐FG‐DXR. Simply mixing CMPul with DXR did not enhance the antitumor activity of DXR, showing that the conjugation of DXR with CMPul is necessary for improved antitumor activity. However, no enhanced antitumor efficacy of the conjugates was observed against a non‐solid tumor model such as P388 leukemia. In summary, improved antitumor efficacy with reduced systemic toxicity of CMPul‐FG‐DXR was demonstrated in the present study. CMPul‐FG‐DXR may be useful as a cancer chemotherapy agent against solid tumors and metastases.
Suppression of N‐Nitrosomethylbenzylamine‐induced Rat Esophageal Tumorigenesis by Dietary Feeding of 1′‐Acetoxychavicol Acetate
Authors: Kawabata Kunihiro , Tanaka Takuji , Yamamoto Tomohiro , Ushida Jun , Hara Akira , Murakami Akira , Koshimizu Kouichi , Ohigashi Hajime , Stoner Gray D. , Mori Hideki
Published: August 2005

The modifying effects of 1′‐acetoxychavicol acetate (ACA) on N‐nitrosomethylbenzylamine (NMBA)‐induced esophageal tumorigenesis were investigated in male F344 rats. At 5 weeks of age, all test animals, except those given the test chemical alone, and the control rats received s.c. injections of NMBA (0.5 mg/kg body weight/injection, three times per week) for 5 weeks. At the termination of the study (20 weeks), 75% of rats treated with NMBA alone had esophageal neoplasms (papillomas). However, the groups given a dose of 500 ppm ACA during the initiation phase developed a significantly reduced incidence of tumors (29%; P < 0.01). Exposure to ACA (500 ppm) during the post‐initiation phase also decreased the frequency of the tumors (38%; P < 0.05). A reduction of the incidence of preneoplastic lesions (hyperplasia or dysplasia) was obtained when ACA was administered in the initiation phase (P < 0.01). Cell proliferation in the esophageal epithelium, determined by assay of proliferating cell nuclear antigen (PCNA), was lowered by ACA (P < 0.05). Blood polyamine contents in rats given NMBA and the test compound were also smaller than those of rats given the carcinogen (P < 0.05). These findings suggest that dietary ACA is effective in inhibiting the development of esophageal tumors by NMBA when given during the initiation or post‐initiation phase, and such inhibition is related to suppression of cell proliferation in the esophageal epithelium.