bluuebunny/crossref_metadata_2025 huggingface dataset:
Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics
Authors: de Tombe Pieter P. , Belus Alexandra , Piroddi Nicoletta , Scellini Beatrice , Walker John S. , Martin Anne F. , Tesi Chiara , Poggesi Corrado
Published: November 2006

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC50) and whether modulation of EC50 affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20°C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC50; ssTnI-chimera Tn exchange induced a further decrease in EC50 (in μM: endogenous Tn, 1.35 ± 0.08; cTnI, 1.04 ± 0.13; ssTnI-chimera Tn, 0.47 ± 0.03). EC50 was also decreased by application of 100 μM bepridil (control: 2.04 ± 0.03 μM; bepridil 1.35 ± 0.03 μM). Maximum tension was not different between any groups. Despite marked alterations in EC50, none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca2+ sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC50. This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.
Molecular modulation of calcium oxalate crystallization
Authors: De Yoreo James J. , Qiu S. Roger , Hoyer John R.
Published: November 2006

Calcium oxalate monohydrate (COM) is the primary constituent of the majority of renal stones. Osteopontin (OPN), an aspartic acid-rich urinary protein, and citrate, a much smaller molecule, are potent inhibitors of COM crystallization at levels present in normal urine. Current concepts of the role of site-specific interactions in crystallization derived from studies of biomineralization are reviewed to provide a context for understanding modulation of COM growth at a molecular level. Results from in situ atomic force microscopy (AFM) analyses of the effects of citrate and OPN on growth verified the critical role of site-specific interactions between these growth modulators and individual steps on COM crystal surfaces. Molecular modeling investigations of interactions of citrate with steps and faces on COM crystal surfaces provided links between the stereochemistry of interaction and the binding energy levels that underlie mechanisms of growth modification and changes in overall crystal morphology. The combination of in situ AFM and molecular modeling provides new knowledge that will aid rationale design of therapeutic agents for inhibition of stone formation.
Time course of ozone-induced changes in breathing pattern in healthy exercising humans
Authors: Schelegle Edward S. , Walby William F. , Adams William C.
Published: November 2006

We examined the time course of O3-induced changes in breathing pattern in 97 healthy human subjects (70 men and 27 women). One- to five-minute averages of breathing frequency (fB) and minute ventilation (V̇e) were used to generate plots of cumulative breaths and cumulative exposure volume vs. time and cumulative exposure volume vs. cumulative breaths. Analysis revealed a three-phase response; delay, no response detected; onset, fB began to increase; response, fB stabilized. Regression analysis was used to identify four parameters: time to onset, number of breaths at onset, cumulative inhaled dose of ozone at onset of O3-induced tachypnea, and the percent change in fB. The effect of altering O3 concentration, V̇e, atropine treatment, and indomethacin treatment were examined. We found that the lower the O3 concentration, the greater the number of breaths at onset of tachypnea at a fixed ventilation, whereas number of breaths at onset of tachypnea remains unchanged when V̇e is altered and O3 concentration is fixed. The cumulative inhaled dose of O3 at onset of tachypnea remained constant and showed no relationship with the magnitude of percent change in fB. Atropine did not affect any of the derived parameters, whereas indomethacin did not affect time to onset, number of breaths at onset, or cumulative inhaled dose of O3 at onset of tachypnea but did attenuate percent change in fB. The results are discussed in the context of dose response and intrinsic mechanisms of action.
Apparatus for measuring rat body volume: a methodological proposition
Authors: Hohl Rodrigo , de Oliveira Renato Buscariolli , Vaz de Macedo Denise , Brenzikofer René
Published: November 2006

We propose a communicating-vessels system to measure body volume in live rats through water level detection by hydrostatic weighing. The reproducibility, accuracy, linearity, and reliability of this apparatus were evaluated in two tests using previously weighed water or six aluminum cylinders of known volume after proper system calibration. The applicability of this apparatus to measurement of live animals (Wistar rats) was tested in a transversal experiment with five rats, anesthetized and nonanesthetized. We took 18 measurements of the volume under each condition (anesthetized and nonanesthetized), totaling 90 measurements. The addition of water volumes (50–700 ml) produced a regression equation with a slope of 1.0006 ± 0.0017, intercept of 0.75 ± 0.81 ( R2= 0.99999, standard error of estimate = 0.58 ml), and bias of ∼1 ml. The differences between cylinders of known volumes and volumes calculated by the system were <0.4 ml. Mean volume errors were 0.01–0.07%. Among the live models, the difference between the volumes obtained for anesthetized and nonanesthetized rats was 0.31 ± 2.34 (SD) ml ( n = 90). These data showed that animal movement does not interfere with the volume measured by the proposed apparatus, and neither anesthesia nor fur shaving is needed for this procedure. Nevertheless, some effort should be taken to eliminate air bubbles trapped in the apparatus or the fur. The proposed apparatus for measuring rat body volume is inexpensive and may be useful for a range of scientific purposes.
The production of reactive oxygen and nitrogen species by skeletal muscle
Authors: Jackson Malcolm J. , Pye Deborah , Palomero Jesus
Published: November 2006

Skeletal muscle has been recognized as a potential source for generation of reactive oxygen and nitrogen species for more than 20 years. Initial investigations concentrated on the potential role of mitochondria as a major source for generation of superoxide as a “by-product” of normal oxidative metabolism, but recent studies have identified multiple subcellular sites, where superoxide or nitric oxide are generated in regulated and controlled systems in response to cellular stimuli. Full evaluation of the factors regulating these processes and the functions of the reactive oxygen species generated are important in understanding the redox biology of skeletal muscle.
Sequential Positron Emission Tomography Using [18F]Fluorodeoxyglucose for Monitoring Response to Chemotherapy in Metastatic Breast Cancer
Authors: Couturier Olivier , Jerusalem Guy , N'Guyen Jean-Michel , Hustinx Roland
Published: November 2006

Purpose: To evaluate the clinical value of positron emission tomography (PET) for monitoring chemotherapy in metastatic breast cancer.

Experimental Design: Twenty patients with hormonorefractory or hormonoreceptor-negative multimetastatic breast cancer were prospectively included. PET studies were done at baseline, at day 21 after the first cycle and at day 21 after the third cycle of chemotherapy. Metabolic response was defined based on visual and various modes of standardized uptake value (SUV) analysis of sequential PET studies.

Results: After one cycle, PET indicated a partial response in 12 patients, stable disease in 7 patients, and progressive disease in 1 patient, according to the visual analysis. After three cycles, PET showed a complete response in 5 patients, partial response in 11 patients, stable disease in 3 patients, and progressive disease in 1 patient. Seventy-five percent of the patients showing a metabolic response on visual analysis effectively responded to the treatment. The average SUV decreased on both the second and the third PET study, but only changes measured after three cycles of chemotherapy predicted the clinical response to chemotherapy and the overall survival. All methods for calculating the SUV (normalized for body weight, body surface area, or lean body mass) provided similar results.

Conclusion: Semiquantitative analysis of [18F]fluorodeoxyglucose-PET studies done after three cycles of chemotherapy is useful for monitoring the response to chemotherapy in metastatic breast cancer.
Acceleration of Telomere Loss by Chemotherapy Is Greater in Older Patients with Locally Advanced Head and Neck Cancer
Authors: Unryn Brad M. , Hao Desiree , Glück Stefan , Riabowol Karl T.
Published: November 2006

Purpose: Chronic viral infection and combinations of chemotherapeutic drugs have been reported to accelerate telomere erosion. Here, we asked if chemoradiotherapy, using the single agent cisplatin, would accelerate telomere loss in head and neck cancer patients, and whether loss was linked to smoking status, age, gender, or stage of disease at diagnosis.

Experimental Design: Blood samples were collected from 20 patients with squamous cell cancer of the head and neck before, during, and after chemoradiotherapy. Following DNA isolation from peripheral blood mononuclear cells, telomere length was measured by terminal restriction fragment analysis.

Results: Chemoradiotherapy increased the rate of telomere erosion >100-fold. Telomere length before treatment in chemoradiotherapy patients was similar to age-matched controls. Although smokers began with significantly shorter telomeres, smoking status did not affect chemoradiotherapy-induced attrition, nor did gender or stage of disease. We also make the novel observation that a significantly greater telomere loss occurred in response to treatment in older patients, with those younger than 55 years losing an average of 400 bp of telomeric DNA compared with the 880 bp lost by those over 55 years.

Conclusions: The lack of telomere length difference before treatment suggests that shortened telomeres may not be a risk factor for development of head and neck cancer in the age range we examined. Chemoradiotherapy caused a severe telomere length reduction in all patients. The significant difference seen in the elderly (P = 0.018) suggests that chemoradiotherapy may have more severe effects on the replicative capacity of blood cells in older patients.
Loss of Krüppel-Like Factor 4 Expression Contributes to Sp1 Overexpression and Human Gastric Cancer Development and Progression
Authors: Kanai Masashi , Wei Daoyan , Li Qiang , Jia Zhiliang , Ajani Jaffer , Le Xiangdong , Yao James , Xie Keping
Published: November 2006

Purpose: Increasing evidence indicates that the transcription factor, Sp1, regulates the expression of multiple genes involved in tumor development and progression. We have recently reported that Sp1 overexpression is directly correlated with the angiogenic potential of and poor prognosis for human gastric cancer. However, the underlying mechanisms that result in Sp1 overexpression remain unclear.

Experimental Design: The expression of Sp1 and Krüppel-like factor 4 (KLF4), a potential tumor suppressor gene, in gastric cancer tissue was analyzed by immunohistochemistry and Western blot analysis. Alterations of Sp1 and KLF4 expression were achieved by gene transfer and verified by Northern and Western blot analyses. Furthermore, Sp1 promoter activity assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay were done to identify the KLF4 binding sites on the Sp1 promoter.

Results: Mutually exclusive expression of Sp1 and KLF4 was evident in gastric cancer and noncancerous tissue. Specifically, strong Sp1 expression but loss of KLF4 expression was found in cancer tissue, whereas the adjacent noncancerous tissue showed negative Sp1 expression but strong KLF4 expression. Enforced KLF4 expression repressed Sp1 expression at the promoter activity, mRNA, and protein levels. Moreover, a region within the proximal Sp1 promoter was identified to have overlapping KLF4- and Sp1-binding sites, to which KLF4 and Sp1 compete for binding. Sp1 positively regulated its own promoter, whereas KLF4 did the opposite.

Conclusions: Our data suggests that disruption of KLF4-mediated negative regulation contributes to the molecular events of Sp1 overexpression and to the development and progression of human gastric cancer.
Schedule-Dependent Synergy between the Heat Shock Protein 90 Inhibitor 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin and Doxorubicin Restores Apoptosis to p53-Mutant Lymphoma Cell Lines
Authors: Robles Ana I. , Wright Mollie H. , Gandhi Bheru , Feis Steven S. , Hanigan Christin L. , Wiestner Adrian , Varticovski Lyuba
Published: November 2006

Purpose: Loss of p53 function impairs apoptosis induced by DNA-damaging agents used for cancer therapy. Here, we examined the effect of the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) on doxorubicin-induced apoptosis in lymphoma. We aimed to establish the optimal schedule for administration of both drugs in combination and the molecular basis for their interaction.

Experimental Design: Isogenic lymphoblastoid and nonisogenic lymphoma cell lines differing in p53 status were exposed to each drug or combination. Drug effects were examined using Annexin V, active caspase-3, cell cycle, and cytotoxicity assays. Synergy was evaluated by median effect/combination index. Protein expression and kinase inhibition provided insight into the molecular mechanisms of drug interaction.

Results: Presence of mutant p53 conferred increased survival to single agents. Nevertheless, DMAG showed synergistic toxicity with doxorubicin independently of p53 status. Synergy required exposure to doxorubicin before DMAG. DMAG-mediated down-regulation of CHK1, a known HSP90 client, forced doxorubicin-treated cells into premature mitosis followed by apoptosis. A CHK1 inhibitor, SB-218078, reproduced the effect of DMAG. Administration of DMAG before doxorubicin resulted in G1-S arrest and protection from apoptosis, leading to additive or antagonistic interactions that were exacerbated by p53 mutation.

Conclusions: Administration of DMAG to doxorubicin-primed cells induced premature mitosis and had a synergistic effect on apoptosis regardless of p53 status. These observations provide a rationale for prospective clinical trials and stress the need to consider schedule of exposure as a critical determinant of the overall response when DMAG is combined with chemotherapeutic agents for the treatment of patients with relapsed/refractory disease.
Attractin Is Elevated in the Cerebrospinal Fluid of Patients with Malignant Astrocytoma and Mediates Glioma Cell Migration
Authors: Khwaja Fatima W. , Duke-Cohan Jonathan S. , Brat Daniel J. , Van Meir Erwin G.
Published: November 2006

Purpose: There are a limited number of noninvasive methods available for the monitoring of neoplastic disease in the central nervous system. The goal of our study was to find reliable markers that could be used for disease monitoring as well as to identify new targets for the therapeutic intervention for malignant astrocytoma (WHO grades 3 and 4).

Experimental Design: We employed proteomic techniques to identify secreted proteins in the cerebrospinal fluid that were specific to patients with malignant astrocytoma.

Results: Among 60 cerebrospinal fluid samples of patients with various central nervous system diseases, attractin was consistently found to be elevated in the samples of patients with malignant astrocytoma. To independently validate these results, we examined attractin expression in a new set of 108 normal and tumoral brain tissue specimens and found elevated expression in 97% of malignant astrocytomas, with the highest levels in grade 4 tumors. Using immunohistochemistry, we further showed that attractin is produced and secreted by the tumor cells. Finally, we showed that cerebrospinal fluid from brain tumor patients induces glioma cell migration and that attractin is largely responsible for this promigratory activity.

Conclusions: Our results find attractin to be a reliable secreted marker for high-grade gliomas. Additionally, our migration studies suggest that it may be an important mediator of tumor invasiveness, and thus, a potential target in future therapies.
Gastric and Intestinal Phenotypic Marker Expression in Early Differentiated-Type Tumors of the Stomach: Clinicopathologic Significance and Genetic Background
Authors: Tajima Yusuke , Yamazaki Kimiyasu , Makino Reiko , Nishino Nobukazu , Aoki Shigeo , Kato Masanori , Morohara Koji , Kaetsu Tsutomu , Kusano Mitsuo
Published: November 2006

Purpose: Gastric and intestinal phenotypic cell markers are expressed in gastric carcinomas, irrespective of their histologic type. In the present study, we determined the clinicopathologic significance of phenotypic marker expression in early-stage gastric differentiated-type tumors and the association between marker expression and genetic alterations.

Experimental Design: Phenotypic marker expression was determined by examining the expressions of human gastric mucin (HGM), MUC6, MUC2, and CD10 in 63 gastric adenomas, 133 early differentiated-type carcinomas, and 24 follow-up cases with gastric adenoma. Tumors were classified into gastric, gastric and intestinal mixed, or intestinal phenotypes according to the immunopositivity of the above markers. The presence of mutations in APC, K-ras, and p53 and the microsatellite instability status were also determined in all tumors.

Results: The expressions of HGM and MUC6, representing gastric or gastric and intestinal mixed phenotypes, were significantly associated with high-grade atypia in the 63 gastric adenomas. Among the 133 early differentiated-type carcinomas, HGM expression was significantly associated with mixed-type (with an undifferentiated-type component) tumors and lymph node metastasis. MUC2 expression was inversely associated with submucosal invasion. A multivariate analysis revealed that gastric adenomas were significantly associated with the intestinal phenotype and were inversely associated with p53 mutation compared with early differentiated-type carcinomas. Among all 196 tumors, APC mutation was significantly associated with CD10 expression and the intestinal phenotype and was inversely associated with the expressions of HGM and MUC6. The microsatellite instability status was significantly associated with MUC6 expression. Malignant transformation from gastric adenoma to carcinoma was shown in 5 of the 24 follow-up cases of gastric adenoma. The malignant transformation was significantly associated with the gastric and intestinal mixed phenotype and was inversely associated with APC mutation. No malignant transformation was found in intestinal phenotype gastric adenomas with APC mutation.

Conclusions: Our present findings show that phenotypic marker expression is associated with tumor aggressiveness during the early stage of gastric differentiated-type tumors. Differences in the biological behavior of tumors with different phenotypes may result from differences in the genetic backgrounds during the incipient phase of gastric tumorigenesis.
Long-term Events in Adult Patients with Clinical Stage IA-IIA Nonbulky Hodgkin's Lymphoma Treated with Four Cycles of Doxorubicin, Bleomycin, Vinblastine, and Dacarbazine and Adjuvant Radiotherapy: A Single-Institution 15-Year Follow-up
Authors: Brusamolino Ercole , Baio Ambrogia , Orlandi Ester , Arcaini Luca , Passamonti Francesco , Griva Vassiliki , Casagrande William , Pascutto Cristiana , Franchini Pietro , Lazzarino Mario
Published: November 2006

Purpose: To report on long-term events after short doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) chemotherapy and adjuvant radiotherapy in favorable early-stage Hodgkin's lymphoma.

Experimental Design: We monitored late events and causes of death over 15 years (median follow-up, 120 months) in 120 patients with nonbulky stage IA-IIA Hodgkin's lymphoma, treated with four cycles of ABVD and limited radiotherapy. Pulmonary and cardiac function tests were done throughout the follow-up. Outcome measures included cause-specific mortality, standardized mortality ratio, and standardized incidence ratio for secondary neoplasia.

Results: Projected 15-year event-free and overall survival were 78% and 86%, and tumor mortality was 3%. Standardized mortality ratio was significantly higher than 1 for both males (2.8; P = 0.029) and females (9.4; P = 0.003). The risk of cardiovascular events at 5 and 12 years was 5.5% and 14%, with a median latent time of 67 months (range: 23-179 months) from the end of radiotherapy. Pulmonary toxicity developed in 8% of patients; all had received mediastinal irradiation and the median time from radiotherapy to pulmonary sequelae was 76 weeks (range: 50-123 weeks). The risk of secondary neoplasia at 5 and 12 years was 4% and 8%, respectively, with no cases of leukemia. Fertility was preserved.

Conclusions: Long-term events were mostly related to radiotherapy; the role of short ABVD chemotherapy was very limited, as documented by fertility preservation and lack of secondary myelodysplasia/leukemia. A proportion of patients died from causes unrelated to disease progression and the excess mortality risk was mostly due to the occurrence of secondary neoplasms and cardiovascular diseases. A moderate dose reduction of radiotherapy from 40-44 Gy to 30-36 Gy did not decrease the risk of late complications; abolishing radiotherapy in nonbulky early-stage Hodgkin's lymphoma is being evaluated.
Variants of the hK2 Protein Gene (KLK2) Are Associated with Serum hK2 Levels and Predict the Presence of Prostate Cancer at Biopsy
Authors: Nam Robert K. , Zhang William W. , Klotz Laurence H. , Trachtenberg John , Jewett Michael A.S. , Sweet Joan , Toi Ants , Teahan Seamus , Venkateswaran Vasundara , Sugar Linda , Loblaw Andrew , Siminovitch Kathy , Narod Steven A.
Published: November 2006

Purpose: Increased levels of serum human kallikrein-2 (hK2) and an hK2 gene (KLK2) variant are positively associated for prostate cancer, but the relationships between them remain unclear. We examined five variants of the KLK2 gene to further define its relevance to prostate cancer susceptibility and hK2 levels.

Experimental Design: We genotyped 645 men with biopsy-proven prostate cancer (cases) and 606 males with biopsies negative for prostate cancer (controls) for five additional single nucleotide polymorphisms (SNP) across the KLK2 gene and also tested for serum hK2 levels. These SNPs were identified from sequencing the KLK2 gene among 20 patients with aggressive prostate cancer. Odds ratios (OR) for prostate cancer detection and haplotype analysis were done.

Results: Among the SNPs studied, the A allele of the KLK2-SNP1 (G > A, rs2664155) and the T allele of the KLK2-SNP5 (C > T, rs198977) polymorphisms showed positive associations with prostate cancer, adjusted ORs for KLK2-SNP1 AG and AA genotypes being 1.4 [95% confidence interval (95% CI), 1.2-1.8; P = 0.002] and for KLK2-SNP5 TT or CT genotypes being 1.3 (95% CI, 1.1-1.6; P = 0.05). Haplotype analyses also revealed a significant association between prostate cancer and the haplotype containing both risk alleles (ACCTT), OR being 5.1 (95% CI, 1.6-6.5; P = 0.005). Analysis of serum hK2 revealed hK2 levels to be significantly increased in association with KLK2-SNP1 AA and AG risk genotypes compared with the GG genotype (P = 0.001) and also in association with the ACCTT risk haplotype compared with the most common non-risk haplotype (P = 0.05).

Conclusions: These findings suggest a role for the KLK2 gene in prostate cancer susceptibility and imply that this role may be realized at least in part by the induction of increases in hK2 production.
Advances in Targeting Human Epidermal Growth Factor Receptor-2 Signaling for Cancer Therapy
Authors: Meric-Bernstam Funda , Hung Mien-Chie
Published: November 2006

Human epidermal growth factor receptor (HER)-2 is a member of the HER tyrosine kinase family, which regulates cell growth and proliferation. HER-2 is overexpressed in 20% to 30% of breast cancers and has been associated with an aggressive phenotype and a poorer prognosis, making it an appealing therapeutic target. Since 1998, the anti-HER-2 antibody trastuzumab has been used for the treatment of women with HER-2-positive metastatic breast cancer. Results from large trials have established a role for trastuzumab in the adjuvant setting for the treatment of high-risk primary breast cancer as well. Tyrosine kinase inhibitors that target HER-2 are also very promising therapies and are likely to be incorporated into clinical practice in the near future. HER-2-targeted therapies represent a major step forward in achieving our goal of delivering individualized targeted therapy for breast cancer. However, there are many unanswered questions about the optimal use of these agents. Ongoing research will better elucidate the best combination therapies to overcome resistance to HER-2-targeted agents and will help identify patients at high enough risk to warrant their toxicity.
Effects of Metoprolol on Sympathetic Remodeling and Electrical Remodeling at Infarcted Border Zone after Myocardial Infarction in Rabbits
Authors: Jiang Hong , Lu Zhibing , Yu Ying , Zhao Dongdong , Jian Xiaoli , Yang Bo , Huang Congxin
Published: November 2006

Background: The findings of sympathetic remodeling and its electrophysiological implications force us to rerecognize the drugs presently used. The aim of this study was toinvestigate the effects of metoprolol on sympathetic remodeling and electrical remodeling at the infarcted border zone (IBZ) after myocardial infarction (MI). Methods: Forty rabbits were randomly assigned into two groups: MI group (n = 20), ligation of the anterior descending coronary; Metoprolol group (n = 20), ligation of the anterior descending coronary and administration of oral metoprolol 5 mg/kg/day. Eight weeks after surgery, transmural dispersion of repolarization (TDR) at baseline, TDR and difference of TDR (ΔTDR) during sympathetic nerve stimulation were measured at the IBZ. The distribution and densities of growth associated protein 43 and tyrosine hydroxylase positive nerves at the IBZ were detected with immunohistochemical techniques. Results: The study was completed in the 36 surviving animals (18 rabbits in each group). The densities of growth associated protein 43 and tyrosine hydroxylase positive nerves in the Metoprolol group (2,550 ± 554 and 1,779 ± 458 µm2/mm2, respectively) were lower than in the MI group (3,217 ± 589 and 2,616 ± 528 µm2/mm2, respectively; both p < 0.01). TDR at baseline, TDR and ΔTDR during sympathetic nerve stimulation were shorter in the Metoprolol group than in the MI group (p < 0.01 for all). Conclusion: Metoprolol can inhibit sympathetic remodeling and electrical remodeling at the IBZ after MI. The association of metoprolol with improved electrical remodeling may be partly related to the inhibition of sympathetic remodeling.
The Prognostic Relevance of p16 Inactivation in Head and Neck Cancer
Authors: Koscielny Sven , Dahse Regine , Ernst Günther , von Eggeling Ferdinand
Published: November 2006

The inactivation of the tumor suppressor gene p16 plays an important role in the development of malignant tumors. p16 loss can result from point mutations, loss of heterozygosity (LOH) or methylation of the promoter region. A total of 67 samples of tumor tissue from squamous cell carcinoma of the oral cavity, the pharynx and the larynx were analyzed for an inactivation of p16. The results of the molecular-biological investigations were correlated with the known clinical prognostic parameters after a follow-up period of approximately 3 years. Methylation of the promoter region and LOH were the main mechanisms of p16 inactivation. Point mutations presented as rare events. An inactivation of p16 did not have any statistical influence on tumor prognosis. Patients with a p16 gene inactivated by promoter methylation appeared to have a slightly lower tendency for local and regional recurrences. The inactivation of the tumor suppressor gene p16 plays a role in the carcinogenesis of head and neck cancer.
MTHFR 677C→T and 1298A→C Polymorphisms: Evaluation of Maternal Genotypic Risk and Association with Level of Neural Tube Defect
Authors: Dalal Ashwin , Pradhan Mandakini , Tiwari Deepshikha , Behari Sanjay , Singh Uttam , Mallik G.K. , Das Vinita , Agarwal Sarita
Published: November 2006

Background: Neural tube defects (NTDs) are common birth defects (1 in 1,000) leading to significant morbidity and mortality. Periconceptional folic acid supplementation helps in prevention of 70% of NTDs. Recently, polymorphisms in genes encoding enzymes of the folate pathway have been implicated in causation of NTDs. Since the closure of neural tube occurs at multiple sites, the etiology of defect at different sites may be different – which explains the failure of folic acid supplementation to prevent all NTDs. Methods: Molecular analysis of methylenetetrahydrofolate reductase polymorphisms was carried out using polymerase chain reaction and restriction enzyme digestion. We studied the association of these polymorphisms in mothers with a previous child with NTD and further refined the risk by stratification based on level of defect. Results: The frequency of 677C→T homozygotes was higher in mothers with a previous child with NTD than the controls (OR = 1.6 (0.38–6.7), 95% CI, p = 0.72) but the difference was statistically insignificant. There was a significant difference in frequency of T alleles among mothers with a previous child with a ‘lower’ type of defect compared to controls (OR = 2.15 (1.13–4.1), 95% CI, p = 0.02). We did not find any significant association of 1298A→C polymorphism with the level of NTDs. Conclusions: We conclude that in the North Indian population, the 677C→T allele of the MTHFR gene may be associated with the occurrence of a lower type of NTD. This points towards the differential role of thermolabile MTHFR at different sites of neural tube closure.
Adrenarche in Prader-Willi Syndrome Appears Not Related to Insulin Sensitivity and Serum Adiponectin
Authors: Unanue Nancy , Bazaes Rodrigo , Iñiguez Germán , Cortés Fanny , Ávila Alejandra , Mericq Verónica
Published: November 2006

Prader-Willi syndrome (PWS) is a genetic disorder characterized by dysmorphic features, obesity, hypogonadism, hypotonia and mental retardation. Obesity has been linked to insulin resistance and the latter has also been associated with premature adrenarche. Since up to date a controlled study to investigate adrenarche and its hormonal regulation was lacking in PWS, our aim was to assess whether prepubertal PWS patients develop premature adrenarche and its relationship with markers of insulin sensitivity. Fourteen prepubertal children with PWS (6 M, 8 F) and 10 non-syndromal simple obese matched controls (5 M, 5 F) participated (mean age: 7.62 ± 1.84 years). A fasting blood sample was obtained for adrenal and ovarian androgens, sex hormone binding globulin, insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-1, leptin, adiponectin and a lipid profile. Thereafter an oral glucose tolerance test was performed. PWS patients were smaller at birth and a higher proportion displayed premature pubarche. No differences were found in testosterone, androstenedione, sex hormone binding globulin, free androgen index, homeostatic model assessment-IR, 2-hour insulin, leptin or adiponectin levels. 17-hydroxyprogesterone and DHEAS levels however, were significantly higher in PWS. IGF-I levels were significantly lower in PWS and correlated significantly with height SDS (p < 0.05). In conclusion, a higher proportion of premature adrenarche in our PW patients was observed, which was not explained by differences in insulin sensitivity or plasma levels of adipokines and IGF-I.
Nox4 Is Required for Maintenance of the Differentiated Vascular Smooth Muscle Cell Phenotype
Authors: Clempus Roza E. , Sorescu Dan , Dikalova Anna E. , Pounkova Lily , Jo Patricia , Sorescu George P. , Lassègue Bernard , Griendling Kathy K.
Published: November 2006

Objective—

The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have been implicated in VSMC differentiation, but the responsible sources are unknown. In this study, we investigated the role of Nox1 and Nox4-derived ROS in this process.

Methods and Results—

Primary VSMCs were used to study the relationship between Nox homologues and differentiation markers such as smooth muscle α-actin (SM α-actin), smooth muscle myosin heavy chain (SM-MHC), heavy caldesmon, and calponin. We found that Nox4 and differentiation marker genes were downregulated from passage 1 to passage 6 to 12, whereas Nox1 was gradually upregulated. Nox4 co-localized with SM α-actin–based stress fibers in differentiated VSMC, and moved into focal adhesions in de-differentiated cells. siRNA against nox4 reduced NADPH-driven superoxide production in serum-deprived VSMCs and downregulated SM-α actin, SM-MHC, and calponin, as well as SM-α actin stress fibers. Nox1 depletion did not decrease these parameters.

Conclusion—

Nox4-derived ROS are critical to the maintenance of the differentiated phenotype of VSMCs. These findings highlight the importance of identifying the specific source of ROS involved in particular cellular functions when designing therapeutic interventions.
Low Circulating Endogenous Secretory Receptor for AGEs Predicts Cardiovascular Mortality in Patients With End-Stage Renal Disease
Authors: Koyama Hidenori , Shoji Tetsuo , Fukumoto Shinya , Shinohara Kayo , Shoji Takuhito , Emoto Masanori , Mori Katsuhito , Tahara Hideki , Ishimura Eiji , Kakiya Ryusuke , Tabata Tsutomu , Yamamoto Hiroshi , Nishizawa Yoshiki
Published: November 2006

Objective—

Receptor for advanced glycation end-products (RAGE) is involved in diabetic vascular complications. We have recently shown that plasma endogenously secretory RAGE (esRAGE), an alternatively spliced form of RAGE, is closely associated with metabolic syndrome and atherosclerosis. Here, we evaluated if plasma esRAGE is a predictor of cardiovascular mortality in a cohort of 206 (171 nondiabetic) patients with end-stage renal diseases (ESRD).

Methods and Results—

The cohort was followed for a median of 111 months, and 74 deaths including 34 cardiovascular deaths were recorded. Plasma esRAGE was measured at baseline. Cumulative incidence of cardiovascular death by Kaplan-Meier estimation was significantly higher in subjects in the lowest tertile of plasma esRAGE than those in the middle or the highest tertile both in all and nondiabetic subjects alone. In all subjects, as compared with the lowest tertile of plasma esRAGE, the hazards ratios for the highest and middle tertile were 0.40 (95% CI, 0.18 to 0.89) and 0.26 (0.10 to 0.66), respectively. The higher risk for lower esRAGE was still significant even after adjusted either with body mass index, hypertension, dyslipidemia and vascular complications, but was confounded by age and diabetes.

Conclusions—

Low circulating esRAGE is a predictor for cardiovascular mortality in ESRD patients.
Biomarkers of Atherosclerotic Plaque Instability and Rupture
Authors: Koenig Wolfgang , Khuseyinova Natalie
Published: November 2006

Basic research over the last two decades has identified a large number of molecules pertinent to the atherosclerotic process, which have clearly improved our understanding of the underlying pathology. It is now well established that inflammation represents a major feature which is present in the vessel wall throughout all stages of the disease until the final pathophysiologic steps, representing plaque destabilization and eventually plaque rupture. Several cells typical for the atherosclerotic plaque, like monocyte-derived macrophages and T-lymphocytes are able to produce and secrete such mediator molecules, like cytokines, chemokines, growth-factors, enzymes, and disintegrins, which lead to activation of endothelial cells, proliferation of smooth muscle cells, lesion progression, and finally to the weakening of a vulnerable plaque by matrix degradation of its fibrous cap. Today, many of these molecules involved can be measured systemically by sensitive assays, and elevated concentrations in the circulation have been shown to be associated with future cardiovascular events. Determination of several of these molecules carries important prognostic information, independent of traditional risk factors, and may turn out to be useful in improving risk stratification. However, for most of these biomarkers the clinical utility has not yet been established.
Therapeutic Angiogenesis Using Novel Vascular Endothelial Growth Factor-E/Human Placental Growth Factor Chimera Genes
Authors: Inoue Natsuo , Kondo Takahisa , Kobayashi Koichi , Aoki Mika , Numaguchi Yasushi , Shibuya Masabumi , Murohara Toyoaki
Published: November 2006

Background—

Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis but causes adverse side effects such as edema or tissue inflammation. VEGF-E, found in the genome of the Orf virus, specifically binds to VEGF receptor-2 and shows mitotic activity on endothelial cells. Recently, we created two forms of VEGF-E and human placental growth factor (PlGF) chimera genes (VEGF-E chimera #9 and VEGF-E chimera #33), which are humanized genes with VEGF-E function but showing less antigenicity.

Methods and Results—

We examined potential proangiogenic activities of these chimera genes. Four types of expression plasmids (pCDNA3.1-LacZ, phVEGF-A, pVEGF-Echimera#9, and pVEGF-Echimera#33) were administered in a rat model of hindlimb ischemia. Either pVEGF-Echimera#9, pVEGF-Echimera#33, or phVEGF-A significantly increased the ratio of ischemic/normal hindlimb blood-flow compared with the control pCDNA3.1-LacZ treated group (by 1.5-fold, 1.5-fold, and 1.4-fold, respectively,
P
<0.05). Histochemical staining by alkaline phosphatase also revealed that either pVEGF-Echimera#9, pVEGF-Echimera#33, or phVEGF-A increased the capillary density compared with the pCDNA3.1-LacZ treated group (1.4-fold, 1.5-fold, and 1.5-fold, respectively,
P
<0.05). Furthermore, immunostaining for anti-ED1 revealed that fewer macrophages had infiltrated in both pVEGF-Echimera#9 and pVEGF-Echimera#33 groups compared with the phVEGF-A group (
P
<0.05).

Conclusions—

Novel VEGF-E/human PlGF chimera genes, pVEGF-Echimera#9, and pVEGF-Echimera#33 significantly stimulated angiogenesis in response to tissue ischemia to an almost identical extent to that induced by phVEGF-A with fewer tissue inflammation responses.
A Central Role for Nicotinic Cholinergic Regulation of Growth Factor–Induced Endothelial Cell Migration
Authors: Ng Martin K.C. , Wu Jenny , Chang Edwin , Wang Bing-yin , Katzenberg-Clark Regina , Ishii-Watabe Akiko , Cooke John P.
Published: November 2006

Objective—An endothelial nicotinic acetylcholine receptor (nAChR) participates in atherogenesis and tumorigenesis by promoting neovascularization. To date, the mechanisms of nAChR-mediated angiogenesis and their relationship to angiogenic factors, eg, VEGF and bFGF, are unknown.

Methods and Results—Nicotine induced dose-dependent human microvascular endothelial cell (HMVEC) migration, a key angiogenesis event, to an extent which was equivalent in magnitude to bFGF (10 ng/mL) but less than for VEGF (10 ng/mL). Unexpectedly, nAChR antagonism not only abolished nicotine-induced HMVEC migration but also abolished migration induced by bFGF and attenuated migration induced by VEGF. Transcriptional profiling identified gene expression programs which were concordantly regulated by all 3 angiogens (nicotine, VEGF, and bFGF), a notable feature of which includes corepression of thioredoxin-interacting protein (TXNIP), endogenous inhibitor of the redox regulator thioredoxin. Furthermore, TXNIP repression by all 3 angiogens induced thioredoxin activity. Silencing thioredoxin by small interference RNA abrogated all angiogen-induced migration while silencing TXNIP strongly induced HMVEC migration. Interestingly, nAChR antagonism abrogates growth factor (VEGF and bFGF)–mediated induction of thioredoxin activity.

Conclusions—Nicotine promotes angiogenesis via stimulation of nAChR-dependent endothelial cell migration. Furthermore, growth factor–induced HMVEC migration, a key angiogenesis event, requires nAChR activation—an effect mediated in part by nAChR-dependent regulation of thioredoxin activity.
The CX
3
C Chemokine Fractalkine Induces Vascular Dysfunction by Generation of Superoxide Anions
Authors: Schäfer Andreas , Schulz Christian , Fraccarollo Daniela , Tas Piet , Leutke Meike , Eigenthaler Martin , Seidl Stefan , Heider Peter , Ertl Georg , Massberg Steffen , Bauersachs Johann
Published: November 2006

Objective—

The chemokine fractalkine activates platelets and induces leukocyte adhesion to the endothelium. Expression of fractalkine and its receptor, CX
3
CR1, is elevated in coronary artery disease. We assessed the effects of fractalkine on vascular function in isolated rat aorta.

Methods and Results—

CX
3
CR1 expression was demonstrated in rat aortic endothelial and smooth muscle cells by immunohistochemistry, Western blot, and polymerase chain reaction (PCR). Fractalkine (up to 1 μg/mL) did not directly induce contractile or relaxant responses when applied to rat aortic rings in organ baths. Short-term incubation with fractalkine (1 μg/mL) for 5 minutes did not affect vascular reactivity. Pretreatment of isolated rat aortic rings with fractalkine for 2 hours impaired acetylcholine-induced nitric oxide (NO)-mediated relaxation after preconstriction with phenylephrine in a concentration-dependent manner. The concentration response to the NO donor DEA-NONOate was significantly shifted to the right. The radical scavenger tiron normalized the attenuated acetylcholine-induced relaxation after fractalkine incubation. Aortic superoxide formation was enhanced by fractalkine, which was inhibited by diphenyleneiodonium but not by inhibitors of xanthine oxidase or NO synthase.

Conclusion—

In addition to its role as a chemokine and adhesion molecule, fractalkine induces vascular dysfunction by stimulating vascular reactive oxygen species resulting in reduced NO bioavailability.
Altered Na+Channels Promote Pause-Induced Spontaneous Diastolic Activity in Long QT Syndrome Type 3 Myocytes
Authors: Fredj Sandra , Lindegger Nicolas , Sampson Kevin J. , Carmeliet Peter , Kass Robert S.
Published: November 2006

Long QT syndrome (LQTS) type 3 (LQT3), typified by the ΔKPQ mutation (LQT3 mutation in which amino acid residues 1505 to 1507 [KPQ] are deleted), is caused by increased sodium entry during the action potential plateau resulting from mutation-altered inactivation of the Nav1.5 channel. Although rare, LQT3 is the most lethal of common LQTS variants. Here we tested the hypothesis that cellular electrical dysfunction, caused not only by action potential prolongation but also by mutation-altered Na+entry, distinguishes LQT3 from other LQTS variants and may contribute to its distinct lethality. We compared cellular electrical activity in myocytes isolated from mice heterozygous for the ΔKPQ mutation (ΔKPQ) and myocytes from wild-type littermates. Current-clamp pause protocols induced rate-dependent spontaneous diastolic activity (delayed after depolarizations) in 6 of 7 ΔKPQ, but no wild-type, myocytes (n=11) tested. Voltage-clamp pause protocols that independently control depolarization duration and interpulse interval identified a distinct contribution of both depolarization duration and mutant Na+channel activity to the generation of Cai2+-dependent diastolic transient inward current. This was found at rates and depolarization durations relevant both to the mouse model and to LQT3 patients. Flecainide, which preferentially inhibits mutation-altered late Na+current and is used to treat LQT3 patients, suppresses transient inward current formation in voltage-clamped ΔKPQ myocytes. Our results demonstrate a marked contribution of mutation-altered Na+entry to the incidence of pause-dependent spontaneous diastolic activity in ΔKPQ myocytes and suggest that altered Na+entry may contribute to the elevated lethality of LQT3 versus other LQTS variants.
Haptoglobin Genotype Is a Regulator of Reverse Cholesterol Transport in Diabetes In Vitro and In Vivo
Authors: Asleh Rabea , Miller-Lotan Rachael , Aviram Michael , Hayek Tony , Yulish Michael , Levy Joanne E. , Miller Benjamin , Blum Shany , Milman Uzi , Shapira Chen , Levy Andrew P.
Published: November 2006

Two common alleles exist at the haptoglobin (Hp) locus, and the Hp2 allele is associated with an increased incidence of cardiovascular disease, specifically in diabetes mellitus (DM). Oxidative stress is increased in Hp2 mice and humans with DM. Oxidative modification of the apolipoprotein A-I inhibits reverse cholesterol transport. We sought to test the hypothesis that reverse cholesterol transport is impaired in Hp2 DM mice and humans. In vitro, using serum from non-DM and DM individuals, we measured cholesterol efflux from
3
H-cholesterol–labeled macrophages. In vivo, we injected
3
H-cholesterol–loaded macrophages intraperitoneally into non-DM and DM mice with the Hp1-1 or Hp2-2 genotype and monitored
3
H-tracer levels in plasma, liver, and feces. In vitro, in DM individuals only, we observed significantly decreased cholesterol efflux from macrophages incubated with serum from Hp2-1 or Hp2-2 as compared with Hp1-1 individuals (
P
<0.01). The interaction between Hp type and DM was recapitulated using purified Hp and glycated Hb. In vivo, DM mice loaded with
3
H-cholesterol–labeled macrophages had a 40% reduction in
3
H-cholesterol in plasma, liver, and feces as compared with non-DM mice (
P
<0.01). The reduction in reverse cholesterol transport associated with DM was significantly greater in Hp2-2 mice as compared with Hp1-1 mice (54% versus 25% in plasma; 52% versus 27% in liver; 57% versus 32% in feces;
P
<0.03). reverse cholesterol transport is decreased in Hp2-2 DM. This may explain in part the increased atherosclerotic burden found in Hp2-2 DM individuals.
Intra- and Interrater Reliability of Ischemic Lesion Volume Measurements on Diffusion-Weighted, Mean Transit Time and Fluid-Attenuated Inversion Recovery MRI
Authors: Luby Marie , Bykowski Julie L. , Schellinger Peter D. , Merino José G. , Warach Steven
Published: November 2006

Background and Purpose—

We investigated the intra- and interrater reliability of ischemic lesion volumes measurements assessed by different MRI sequences at various times from onset.

Methods—

Ischemic lesion volumes were measured for intrarater reliability using diffusion-weighted (DWI), mean transit time (MTT) perfusion and fluid-attenuated inversion recovery (FLAIR) MRI at chronic (>3 days from stroke onset) time points. A single intrarater reader, blind to clinical information and time point, repeated the volume measurements on two occasions separated by at least 1 week. Interrater reliability was also obtained in the second set of patients using acute DWI, MTT and chronic FLAIR MRI. Four blinded readers performed these volume measurements. Average deviations across repeat measurements per lesion and differences between sample means between the two measurements were calculated globally, ie, across all sequences and time points, and per reader type for each sequence at each time point.

Results—

There was good concordance of the mean sample volumes of the 2 intrarater readings (deviations were <4% and 2 mL globally, <2% and 2 mL for DWI, <6% and 7 mL for MTT, and <2% and 1 mL for FLAIR). There was also good concordance of the interrater readings (<5% and 2 mL globally).

Conclusions—

Repeat measurements of stroke lesion volumes show excellent intra- and interrater concordance for DWI, MTT and FLAIR at acute through chronic time points.