Systems analysis of cancer cell heterogeneity in caspase-dependent apoptosis subsequent to mitochondrial outer membrane permeabilization.

Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac(-/-), similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAP(Adv)) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP.

Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype.

BACKGROUND: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. METHODS: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. RESULTS: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. CONCLUSION: We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.

Cell survival or apoptosis: rooperol's role as anticancer agent.

Nontoxic hypoxoside, isolated from Hypoxis, is converted to cytotoxic rooperol in the presence of beta-glucosidase. In this study, we investigated rooperol's mechanism of action. IC50 values of hypoxoside and rooperol were determined against the HeLa, HT-29, and MCF-7 cancer cell lines, and peripheral blood mononuclear cells. DNA cell cycle arrest occurred in late G1 and/or early S phases, associated with increased p21(Waf1/Cip1) levels. Apoptosis was shown by caspase-3 and/or caspase-7 activation, phosphatidylserine translocation, DNA fragmentation, cell blebbing, and apoptotic body formation. Increased phospho-Akt, phospho-Bcl-2, and p21(Waf1/Cip1) proteins, and cell size correspond to cell survival strategies (associated with endoreduplication).

Outcomes of surgical treatment of intrathoracic stomach.

The purpose of this study is to assess the long-term outcomes after surgical repair of intrathoracic stomach. Prospectively collected data was retrospectively reviewed. Patients underwent a phone questionnaire 1 year postoperatively to assess gastroesophageal reflux disease-related symptoms and surgical satisfaction. In addition, objective evaluation for integrity of hiatal hernia repair was undertaken either by esophagram or endoscopy. Any recurrence was considered a failure. Forty-one patients underwent surgical repair of a large paraesophageal hernia with intrathoracic stomach during the study period. Thirty-four patients underwent a laparoscopic repair, and seven patients underwent a transthoracic repair. An antireflux procedure was performed on 28 patients, and 13 patients had only hernia reduction and hiatal closure. In the laparoscopic group, two patients required conversion to open laparotomy, as one was unable to tolerate the pneumoperitoneum, and the other had mediastinal bleeding. Thirty-eight (93%) were available for 1-year follow-up. There were three (7.8%) recurrences, one requiring emergency transabdominal repair, and the other two being asymptomatic 1-cm recurrences. All patients report a high degree of satisfaction with surgery. There is a high incidence of short esophagus in patients with intrathoracic stomach. The surgical repair is safe and durable, with high patient satisfaction at 1-year follow-up.

Is it possible to differentiate tuberculous and cryptococcal meningitis in HIV-infected patients using only clinical and basic cerebrospinal fluid characteristics?

BACKGROUND: Tuberculous and cryptococcal meningitis (TBM and CM) are the most common causes of opportunistic meningitis in HIVinfected patients from resource-limited settings, and the differential diagnosis is challenging. OBJECTIVE: To compare clinical and basic cerebrospinal fluid (CSF) characteristics between TBM and CM in HIV-infected patients. METHODS: A retrospective analysis was conducted of clinical, radiological and laboratory records of 108 and 98 HIV-infected patients with culture-proven diagnosis of TBM and CM, respectively. The patients were admitted at a tertiary centre in São Paulo, Brazil. A logistic regression model was used to distinguish TBM from CM and derive a diagnostic index based on the adjusted odds ratio (OR) to differentiate these two diseases. RESULTS: In multivariate analysis, TBM was independently associated with: CSF with neutrophil predominance (odds ratio (OR) 35.81, 95% confidence interval (CI) 3.80 - 341.30, p=0.002), CSF pleocytosis (OR 9.43, 95% CI 1.30 - 68.70, p=0.027), CSF protein >1.0 g/L (OR 5.13, 95% CI 1.38 - 19.04, p=0.032) and Glasgow Coma Scale <15 (OR 3.10, 95% CI 1.03 - 9.34, p=0.044). Nausea and vomiting (OR 0.27, 95% CI 0.08 - 0.90, p=0.033) were associated with CM. Algorithm-related area under the receiver operating characteristics curve was 0.815 (95% CI 0.758 - 0.873, p<0.0001), but an accurate cut-off was not derived. CONCLUSION: Although some clinical and basic CSF characteristics appear useful in the differential diagnosis of TBM and CM in HIVinfected patients, an accurate algorithm was not identified. Optimised access to rapid, sensitive and specific laboratory tests is essential.

The apoptotic and autophagic properties of two natural occurring prodrugs, hyperoside and hypoxoside, against pancreatic cancer cell lines.

Pancreatic cancer is only the 12th most common cancer, but the fourth leading cause of cancer-related deaths in the world. This is due to late prognosis, poor response to chemotherapy and early metastases. Natural prodrugs may play an important role in the treatment of pancreatic cancer. The main aim of this study was to determine the cytotoxicity of five natural prodrugs, namely harpagoside, hyperoside, hypoxoside, oleuropein and polydatin, by investigating apoptosis and autophagy as possible mechanism/s of action. Hypoxoside and hyperoside have shown selective cytotoxicity at IC50 values of ∼25 and 50µM against INS-1 and MIA PaCa-2 pancreatic cancer cells, respectively. Hypoxoside and hyperoside induced G2/M phase arrest and caspase-3 activation in INS-1 and MIA PaCa-2 cells, respectively. Hoechst/phalloidin staining confirmed morphological changes, including condensed chromatin multinucleation, membrane blebbing and loss of cytoskeletal arrangement in INS-1 and MIA PaCa-2 cells. Acridine orange staining was absent in INS-1 (hypoxoside) and MIA PaCa-2 (hyperoside) treated cells, whereas LC3B expression was not significantly increased. INS-1 and MIA PaCa-2 treated cells favour the cell death pathway, apoptosis, over the cell survival pathway, autophagy.

Superinduction of phenylalanine ammonia-lyase in gherkin hypocotyls caused by the inhibitor, L-alpha-aminooxy-beta-phenylpropionic acid.

The extractable activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) and the concentration of sugar esters of p-coumaric and ferulic acids in the hypocotyls of etiolated gherkin seedlings increase upon irradiation with white light. Treatment of intact seedlings with the phenylalanine ammonia-lyase inhibitors alpha-aminooxyacetic acid and L-alpha-aminooxy-beta-phenylpropionic acid during illumination causes enhanced formation of the lyase and reduces the accumulation of hydroxycinnamic acids. Enzyme activity in excised hypocotyl segments floating on buffer increases in the dark as well as in the light, while hydroxycinnamic acids accumulate only in the light. Phenylalanine ammonia-lyase formation in the segments is inhibited by cinnamic acid and, to a lesser extent, p-coumaric acid, while it is slightly enhanced by caffeic acid and is not affected by ferulic acid. Aminooxyphenylpropionate dramatically promotes phenylalanine ammonia-lyase formation in the segments in darkness and light prevents the accumulation of hydroxycinnamic acids in the light. Aminooxyphenylpropionate does not, however, affect the time course of apparent lyase formation and decay. Cinnamic acid, the product of the lyase reaction, antagonizes the effect of aminooxyphenylpropionate. It is proposed that the reaction product(s) are involved to some extent in the regulation of the pool of active lyase in the hypocotyl tissue.

A new concept for implementation of a required general surgery clerkship.

BACKGROUND: Two years ago our institution abbreviated the junior internal medicine and general surgery clerkships to accommodate a 4-week family practice clerkship and a 4-week elective clerkship. As a consequence, 1-month mandatory internal medicine and general surgery clerkships were placed in the senior year. METHODS: The surgical disorders most commonly encountered by the generalist are discussed. The senior students spend 4 weeks with a community-based surgeon. All lectures are presented by full-time faculty and adhere to the student manual, which is designed to coincide with examination material. Three histories and physicals are reviewed by the course director to determine utilization of critical thinking skills. The development of healthy interpersonal and professional relationships is addressed by a 2-hour module on the essentials of integrity, compassion, humility, and self-knowledge. A faculty development seminar provides an awareness of course objectives and logistics. Student grades are determined by the preceptor's evaluation (50%), an in-house written examination (50%), and submission of adequate history and physicals. RESULTS: Subjective reviews by students (n = 115) reveal that although only 27% of the students care to pursue a surgical practice, 85% feel that their time was effectively spent and 83% feel that the clerkship should be offered to future fourth year medical students. Seventy percent of submitted history and physicals (n = 420) exhibit appropriate critical thinking skills. CONCLUSIONS: We are currently in the midst of our third year of implementation. The students are receiving insight into a surgical approach to common disease processes. History and physical examination skills and healthy interpersonal relationships are reinforced. Although change is often difficult to accomplish and accept, the positive response to the newly formatted senior curriculum has exceeded expectations.

Are long hours and hard work detrimental to end-clerkship examination scores?

BACKGROUND: Third-year medical students' complaints focus on the number of hours worked and subsequent lack of study time among three general surgery blocks. We hypothesize that this difference between the surgical blocks does not adversely influence student examination scores. METHODS: Student scores for the academic years 1996-97 to 1997-98 for the National Board of Medical Examiners (NBME) surgery subtests were compiled. A comparison of two "slow" general surgery blocks (B/C) with one "busy" block (A) was made using a two-tailed t test. A multiple regression analysis was also employed. Finally, United States Medical Licensing Examination (USMLE) part I scores were used to determine equivalency of groups. RESULTS: No significant difference existed between block A and blocks B/C in USMLE part I and NBME (P = 0.35 and 0.16 respectively). However, USMLE and rotation sequence influenced NBME scores (P < 0.001). CONCLUSION: The data suggest that no difference exists in examination scores between students assigned to a busy general surgery block versus those students assigned to slow blocks.

CO2 laser endoscopic posterior partial transverse cordotomy for bilateral paralysis of the vocal fold.

OBJECTIVE/HYPOTHESIS: A clinical evaluation of CO2 laser endoscopic posterior partial transverse cordotomy (EPPTC) in patients with severely compromised airway due to bilateral paralysis of the vocal fold. STUDY DESIGN: An inception cohort of 25 patients over a 10-year period. METHODS: The CO2 laser EPPTC was unilateral in 15 patients and bilateral in 10. Variables were tested for potential statistical relation to successful rehabilitation of the airway. RESULTS: The use of the CO2 laser never resulted in adverse side effects. Complications were not encountered. A one-step, successful restoration of the airway was achieved in 68% (17/25) of patients. In univariate analysis, the CO2 laser EPPTC was statistically more likely to be successful if bilateral EPPTC was performed (P = .018). None of the following variables--age, sex, cause of bilateral paralysis, prior treatment, laser parameters, and duration of postoperative antibiotherapy and oral steroids--was statistically related to a successful restoration of the airway. Revision CO2 laser EPPTC, performed in six patients, resulted in an overall 92% (23/25) rate for a successful restoration of the airway. The overall tracheotomy rate was 8% (2/25). CONCLUSION: The authors' data confirm the safety, ease of performance, and efficiency of the CO2 laser EPPTC in patients with bilateral vocal fold paralysis. This report also suggests that the completion of bilateral CO2 laser EPPTC statistically increases the likelihood of restoring the airway in a one-step surgical procedure.

Laparoscopic reoperative antireflux surgery.

BACKGROUND: Antireflux operations for gastroesophageal reflux disease whether performed open or laparoscopically can fail and may require reoperation to control new, recurrent symptoms or operation-related complications. We report our experience with the laparoscopic reoperation for failed antireflux procedures. METHODS: Between 1995 and 2000, 37 patients underwent laparoscopic reoperative antireflux procedures. The mean age and weight were 52 years and 181.5 pounds. The main presenting symptoms were heartburn (n = 18), respiratory reflux (n = 4), chest pain (n = 3), regurgitation (n = 1), and dysphagia (n = 10). The mean duration between the first operation and recurrence of symptoms was 18 months, and the duration between the two procedures was 25 months. The operation was completed laparoscopically in 32 patients (86.5%): Nissen fundoplication (n = 27) and Toupet fundoplication (n = 9). RESULTS: Intraoperative and postoperative complications occurred in 6 and 14 patients, respectively. Fundoplication disruption was the most common cause of primary surgery failure. The mean hospital stay was 4 days. At a mean follow-up of 26.5 months, results were excellent to good (65%), fair (21.5%), and poor (13.5%). CONCLUSION: Laparoscopic reoperative antireflux procedures are technically feasible with acceptable preliminary results.

Goniometric techniques for range-of-motion assessment.

The authors hope that this article promotes greater understanding of the methodology available for measuring and recording joint ROM and fosters movement towards the eventual adoption of a single integrated goniometric system for the evaluation and recording of musculoskeletal impairment and disability. Towards this end, the authors have focused on applications of the Neutral-Zero Measuring Method and SFTR documentation and Recording System, and have highlighted the advantages of this system when compared to more conventional approaches. Examination and recording procedures have been described briefly, and examples of appropriate instruments and their applications have been illustrated. The reader is referred to more extensive discussions elsewhere of the measurement techniques themselves.

Psychosocial adaptation to amputation: the role of sociodemographic variables, disability-related factors and coping strategies.

This study examined the roles of sociodemographic variables, disability-related factors, and coping strategies as predictors of the psychosocial adaptation of 61 persons with amputations. Psychosocial adaptation was conceptualized as a multifaceted outcome criterion and was measured by seven scales from the Reactions to Impairment and Disability Inventory (RIDI) and the Acceptance of Disability (AD) scale. A series of multiple regression analyses indicated that both a set of sociodemographic variables and disability-related factors (age, duration of amputation, type of amputation) and a set of coping strategies (action problem-solving, emotion-focusing, behavioral/problem disengagement, cognitive disengagement) accounted, albeit differentially, for significant portions of the variance in the outcome measures of psychosocial adaptation to amputation. Of the various coping strategies, active problem-solving was negatively associated with the psychosocial reactions of depression and internalized anger (RIDI) but it was positively associated to adjustment (RIDI) and acceptance of disability (AD). Emotion-focusing and cognitive disengagement were positively associated with anxiety, depression, and externalized hostility (RIDI) and negatively associated with acceptance of disability (AD). Measurement and theoretical implications are briefly outlined.

Systems analysis of BCL2 protein family interactions establishes a model to predict responses to chemotherapy.

Apoptotic desensitization is a hallmark of cancer cells, but present knowledge of molecular systems controlling apoptosis has yet to provide significant prognostic insights. Here, we report findings from a systems study of the intrinsic pathway of apoptosis by BCL2 family proteins and clinical translation of its findings into a model with applications in colorectal cancer (CRC). By determining absolute protein quantifications in CRC cells and patient tumor samples, we found that BAK and BAX were expressed more highly than their antiapoptotic inhibitors. This counterintuitive finding suggested that sole inhibition of effector BAX and BAK could not be sufficient for systems stability in nonstressed cells. Assuming a model of direct effector activation by BH3-only proteins, we calculated that the amount of stress-induced BH3-only proteins required to activate mitochondrial apoptosis could predict individual death responses of CRC cells to 5-fluorouracil/oxaliplatin. Applying this model predictor to protein profiles in tumor and matched normal tissue samples from 26 patients with CRCs, we found that differences in protein quantities were sufficient to model the increased tumor sensitivity to chemotherapy compared with normal tissue. In addition, these differences were sufficient to differentiate clinical responders from nonresponders with high confidence. Applications of our model, termed DR_MOMP, were used to assess the impact of apoptosis-sensitizing drugs in lowering the necessary dose of state-of-the-art chemotherapy in individual patients. Together, our findings offer a ready clinical tool with the potential to tailor chemotherapy to individual patients.

Rooperol as an antioxidant and its role in the innate immune system: an in vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Biologically active rooperol is formed when the glucose subunits of the nontoxic glycoside, hypoxoside, are cleaved by ß-glucosidase. Hypoxoside is isolated from Hypoxis, a medicinal plant genus frequently used by the indigenous people of South Africa as an immune system booster. The aim of this study was to investigate rooperol's antioxidant and anti-inflammatory properties using the ferric reducing ability of plasma (FRAP) assay, NO and ROS production, and phagocytosis. MATERIALS AND METHODS: Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages was induced using 10-100 nM 1,25(OH)(2)D(3) and PMA over 72 h. Differentiation was confirmed by light microscopy and flow cytometry. Undifferentiated and/or differentiated cells were treated with DMSO (0.25 v/v%, vehicle control), hypoxoside (50 µg/mL), rooperol (20 µg/mL) or PMA (10/20 nM, positive control). ROS production was measured in undifferentiated and differentiated monocyte-macrophages using DCFH-DA and flow cytometry. Phagocytosis of pHrodo™ Escherichia coli BioParticles(®) was measured using pre-treated monocyte-macrophage differentiated U937 cells. NO production was measured in monocyte-macrophage differentiated U937 cells using DAF-2 DA and flow cytometry. RESULTS: Rooperol was shown to have similar or greater antioxidant potential than ascorbic acid. Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages were confirmed morphologically (cell attachment, clump- and pseudopodia-formation) and biochemically (CD11b and CD14 cell surface marker expression). Rooperol significantly increased ROS and NO production, and phagocytosis in undifferentiated and/or differentiated human promonocytic U937 leukemia cells. Hypoxoside had no or very little effect on ROS and NO production, and phagocytosis. CONCLUSION: This study confirms previous reports that hypoxoside has to be converted to rooperol to be biologically active. The FRAP assay confirms the antioxidant capacity of rooperol seen in previous studies, whereas rooperol's induction of ROS and NO production, and phagocytosis constitute novel findings. Possible mode(s) of action for the in vitro anti-inflammatory activities of rooperol may be explained by ROS and NO production, and phagocytosis.

Clinical measurements of joint motion and position in the neutral-zero method and SFTR recording: basic principles.

The SFTR method of recording joint motion and position in the internationally accepted neutral-zero method provides a truly international system avoiding confusion of language and terminology. It allows one to record and read data in one way only, thus facilitating communication. It is short, precise, easy to learn and suitable for computer use. It also allows recording of strength of agonist and antagonist muscles.

Development of an improved automated gas-chromatographic chiral analysis system: application to non-natural amino acids and natural protein hydrolysates.

In the use of combinatorial chemistry as a novel strategy for drug discovery, the chirality assessment of building blocks used for library construction is particularly important in the evaluation of biological actions of generated libraries. The procedure for chiral analysis of lead compounds in screening may be of a high priority, particularly in the case of the protection of intellectual rights. Previously, an automated amino acid analysis system using enantiomer labeling was developed. The system incorporates a reactor, which allows automated esterification and acylation of amino acids, and is connected to an on-line gas chromatographic system. A capillary column coated with a chiral phase is employed for the separation of the enantiomers. This particular system is improved with a newly constructed "high-throughput auto-derivatizer" in combination with a new GC-system. The resulting data can be processed by newly constructed software. The analyses of amino acid derivatives or hydrolysates of proteins and peptides are carried out routinely within ca. 45 min, including derivatization. Using this system several non-natural amino acids were tested with respect to the stereoisomeric configuration. In addition, acid hydrolysates of food proteins and tissues obtained by autopsy were analyzed as an application to proteome research.

Quality control of peptide drugs. Chiral amino acid analysis versus standard for icatibant acetate.

A method of chiral amino acid analysis versus standard is presented. By treating the peptide sample and a chiral standard whose intrinsic chiral composition is known in the same way, i.e. simultaneous hydrolysation and analysis, exact corrections can be made for the sequence-related and many hydrolysation-related racemisations. The accuracy obtained in this way permits use of the results for quality control of the chiral purity of peptide drugs. Using the bradykinin antagonist Icatibant acetate (INNM) the method was validated with respect to its precision, accuracy, specificity, limit of detection, and robustness.