Cycles Of Repair Research Articles

Determinants of initiation and progression of idiopathic pulmonary fibrosis

IPF is a devastating disease with few therapeutic options. The precise aetiology of IPF remains elusive. However, our understanding of the pathologic processes involved in the initiation and progression of this disease is improving. Data on the mechanisms underlying IPF have been generated from epidemiologic investigations as well as cellular and molecular studies of human tissues. Although no perfect animal model of human IPF exists, pre-clinical animal studies have helped define pathways which are likely important in human disease. Epithelial injury, fibroblast activation and repetitive cycles of injury and abnormal repair are almost certainly key events. Factors which have been associated with initiation and/or progression of IPF include viral infections, abnormal cytokine, chemokine and growth factor production, oxidant stress, autoimmunity, inhalational of toxicants and gastro-oesophageal reflux disease. Furthermore, recent evidence identifies a role for a variety of genetic and epigenetic abnormalities ranging from mutations in surfactant protein C to abnormalities in telomere length and telomerase activity. The challenge remains to identify additional inciting agents and key dysregulated pathways that lead to disease progression so that we can develop targeted therapies to treat or prevent this serious disease.

Stem Cell Populations and Regenerative Potential in Chronic Inflammatory Lung Diseases

Several acute and chronic inflammatory pathologies of the lung are accompanied by structural modifications of airway mucosa that vary depending on the severity, duration and type of the disease. These morphological changes, that determine organ dysfunction, are not always reversible. Indeed, the cycle of injury and repair, influencing airway wall re- generation, may sometimes break off and an exacerbation of the pathology may occur. The mechanisms at the base of airway remodelling during inflammation have been widely studied and numerous evidences indicate that the molecular dialogue among the cells of the mucosa has an essential role in orchestrating cell differentiation and tissue repair. In this review, we revise old notions on pulmonary morphology at the light of some of the most recent discoveries concerning stem cell differentiation, tissue homeostasis and organ regeneration of the lung.

Influence of menstrual cycle on circulating endothelial progenitor cells

Endothelial progenitor cells (EPCs) are circulating mononuclear cells that participate in angiogenesis. The aim of this study was to determine the influence of the menstrual cycle on the number and function of EPCs, and to investigate their relationship with circulating concentrations of sex steroids and inflammatory mediators. Ten healthy nulliparous, premenopausal, non-smoking women with regular menses were studied over a single menstrual cycle. Venepuncture was performed in the menstrual, follicular, peri-ovulatory and luteal phases. EPCs were quantified by flow cytometry (CD133(+)CD34(+)KDR(+) phenotype) and the colony-forming unit (CFU-EPC) functional assay. Circulating concentrations of estradiol, progesterone and inflammatory mediators (TNF-alpha, IL-6, sICAM-1 and VEGF) were measured by immunoassays. The numbers of CD133(+)CD34(+)KDR(+) cells were higher in the follicular phase (0.99 +/- 0.3 x 10(6) cells/l) compared with the peri-ovulatory phase (0.29 +/- 0.1 x 10(6) cells/l; P < 0.05). In contrast, the numbers of CFU-EPCs did not vary over the menstrual cycle. There were no correlations between EPCs and concentrations of either circulating sex steroids or inflammatory mediators. CD133(+)CD34(+)KDR(+) cells but not CFU-EPCs vary during the menstrual cycle. Our findings suggest a potential role for circulating EPCs in the normal cycle of physiological angiogenesis and repair of the uterine endometrium that is independent of circulating sex steroids or inflammatory mediators.

High-Intensity Focused Ultrasound Ablation of Ex Vivo Bovine Achilles Tendon

Small tears in tendons are a common occurrence in athletes and others involved in strenuous physical activity. Natural healing in damaged tendons can result in disordered regrowth of the underlying collagen matrix of the tendon. These disordered regions are weaker than surrounding ordered regions of normal tendon and are prone to re-injury. Multiple cycles of injury and repair can lead to chronic tendinosis. Current treatment options either are invasive or are relatively ineffective in tendinosis without calcifications. High-intensity focused ultrasound (HIFU) has the potential to treat tendinosis noninvasively. HIFU ablation of tendons is based on a currently-used surgical analog, viz., needle tenotomy. This study tested the ability of HIFU beams to ablate bovine tendons ex vivo. Two ex vivo animal models were employed: a bare bovine Achilles tendon (deep digital flexor) on an acoustically absorbent rubber pad, and a layered model (chicken breast proximal, bovine Achilles tendon central and a glass plate distal to the transducer). The bare-tendon model enables examination of lesion formation under simple, ideal conditions; the layered model enables detection of possible damage to intervening soft tissue and consideration of the possibly confounding effects of distal bone. In both models, the tissues were degassed in normal phosphate-buffered saline. The bare tendon was brought to 23°C or 37°C before insonification; the layered model was brought to 37°C before insonification. The annular array therapy transducer had an outer diameter of 33 mm, a focal length of 35 mm and a 14-mm diameter central hole to admit a confocal diagnostic transducer. The therapy transducer was excited with a continuous sinusoidal wave at 5.25 MHz to produce nominal in situ intensities from 0.23–2.6 kW/cm 2. Insonification times varied from 2–10 s. The focus was set over the range from the proximal tendon surface to 7 mm deep. The angle of incidence ranged from 0° (normal to the tissue surface) to 15°. After insonification, tendons were dissected and photographed, and the dimensions of the lesions were measured. Transmission electron micrographs were obtained from treated and untreated tissue regions. Insonification produced lesions that mimicked the shape of the focal region. When lesions were produced below the proximal tendon surface, no apparent damage to overlying soft tissue was apparent. The low intensities and short durations required for consistent lesion formation, and the relative insensitivity of ablation to small variations in the angle of incidence, highlight the potential of HIFU as a noninvasive treatment option for chronic tendinosis. (E-mail: muratore@rrinyc.org)

The Psb27 Protein Facilitates Manganese Cluster Assembly in Photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

Copper acclimation in juvenile fathead minnows: Is a cycle of branchial damage and repair necessary?

Fathead minnows exposed to sublethal Cu concentrations may undergo branchial damage followed by repair, and may also develop enhanced Cu tolerance. The primary objective of this study was to determine whether the cycle of damage and repair was necessary for the development of enhanced Cu tolerance. Inferences regarding damage and repair were made from changes in the whole body Na + of juvenile (0.5 g) fathead minnows ( Pimephales promelas). Two experiments were conducted in which juvenile minnows were exposed to various sublethal Cu concentrations for 16 d. In the first experiment, fish were exposed to one of four different Cu concentrations (0, 73, 118, 189 μg/L Cu) then challenged with an 8-d exposure at 302 μg Cu/L. Only the fish exposed to the highest Cu dose experienced enhanced Cu tolerance relative to the other three doses. In the second experiment, whole body Na + was monitored in fish exposed to one of five different Cu exposures (0, 70, 127, 202, 289 μg/L Cu). Fish exposed to 70 μg/L Cu did not experience a significant decline in whole body Na + at any point during the 16-d exposure period. The reduction in whole body Na + was short lived and moderate (17%) in the fish exposed to 127 μg/L Cu, but more severe (>30%) and longer lasting in the fish exposed to 202 μg/L Cu. At 289 μg/L Cu, the fish experienced irreversible reductions in whole body Na + and ultimately died. When taken together, results from these two experiments suggest that enhanced tolerance will only develop in fathead minnows that have experienced a pronounced, relatively long-term cycle of branchial damage and repair.

Activation of Satellite Cells in the Intercostal Muscles of Patients With Chronic Obstructive Pulmonary Disease

The respiratory muscles of patients with chronic obstructive pulmonary disease (COPD) display evidence of structural damage in parallel with signs of adaptation. We hypothesized that this can only be explained by the simultaneous activation of satellite cells. The aim of this study was to analyze the number and activation of those cells along with the expression of markers of microstructural damage that are frequently associated with regeneration. The study included 8 patients with severe COPD (mean [SD] forced expiratory volume in 1 second, 33% [9%] of predicted) and 7 control subjects in whom biopsies were performed of the external intercostal muscle. The samples were analyzed by light microscopy to assess muscle fiber phenotype, electron microscopy to identify satellite cells, and real-time polymerase chain reaction to analyze the expression of the following markers: insulin-like growth factor 1, mechano growth factor, and embryonic and perinatal myosin heavy chains (MHC) as markers of microstructural damage; Pax-7 and m-cadherin as markers of the presence and activation of satellite cells, respectively; and MHC-I, IIa, and IIx as determinants of muscle fiber phenotype. The patients had larger fibers than healthy subjects (54 [6] vs 42 [4] microm(2); P< .01) with a similar or slightly increased proportion of satellite cells, as measured by ultrastructural analysis (4.3% [1%] vs 3.7% [3.5%]; P>.05) or expression of Pax-7 (5.5 [4.1] vs 1.6 [0.8] arbitrary units [AU]; P< .05). In addition, there was greater activation of satellite cells in the patients, as indicated by increased expression of m-cadherin (3.8 [2.1] vs 1.0 [1.2] AU; P=.05). This was associated with increased expression of markers of microstructural damage: insulin-like growth factor 1, 0.35 (0.34) vs 0.09 (0.08) AU (P< .05); mechano growth factor, 0.45 (0.55) vs 0.13 (0.17) AU (P=.05). The intercostal muscles of patients with severe COPD show indirect signs of microstructural damage accompanied by satellite cell activation. This suggests the presence of ongoing cycles of lesion and repair that could partially explain the maintenance of the structural properties of the muscle.

Airway Epithelial Stem Cells and the Pathophysiology of Chronic Obstructive Pulmonary Disease

Characteristic pathologic changes in chronic obstructive pulmonary disease (COPD) include an increased fractional volume of bronchiolar epithelial cells, fibrous thickening of the airway wall, and luminal inflammatory mucus exudates, which are positively correlated with airflow limitation and disease severity. The mechanisms driving general epithelial expansion, mucous secretory cell hyperplasia, and mucus accumulation must relate to the effects of initial toxic exposures on patterns of epithelial stem and progenitor cell proliferation and differentiation, eventually resulting in a self-perpetuating, and difficult to reverse, cycle of injury and repair. In this review, current concepts in stem cell biology and progenitor-progeny relationships related to COPD are discussed, focusing on the factors, pathways, and mechanisms leading to mucous secretory cell hyperplasia and mucus accumulation in the airways. A better understanding of alterations in airway epithelial phenotype in COPD will provide a logical basis for novel therapeutic approaches.

Cancer surveillance in inflammatory bowel disease: new molecular approaches

Patients with chronic inflammatory bowel disease, such as ulcerative colitis and Crohn's disease, have an increased risk of colorectal cancer. Life-long colonoscopy surveillance is performed to detect the presence of dysplasia, but this approach is expensive and time-consuming. Thus, there is intensive research to identify molecular factors with prognostic value. This review summarizes recent research, with a special emphasis on the mechanisms underlying these molecular alterations. The role of chromosomal instability in the progression to inflammatory bowel disease-associated colorectal cancer is clear and likely relates to chronic cycles of injury, inflammation, repair and telomere shortening. The role of microsatellite instability has been a subject of discussion, and data suggest that microsatellite instability in inflammatory bowel disease might be different from microsatellite instability in sporadic colorectal cancer. Methylation, as a mechanism of gene silencing, also plays a role in ulcerative colitis tumorigenesis. Chronic inflammation has been linked to p53 activation and oxidative stress, contributing to the extensive genomic DNA damage observed in ulcerative colitis. Improved understanding of the molecular biology of cancer progression in inflammatory bowel disease will hopefully lead to the identification of useful prognostic biomarkers. Efforts are needed to prove the clinical utility of the most promising markers now identified.

Simulation for product life cycle management

PurposeEffective product life cycle management will save costs and resources and improve customer service. Seeks to present a simulation model of the complete life cycle of a batch of products undergoing breakdown and repair and to show the ability of the model to predict future waste arisings and cost savings.Design/methodology/approachARENA system simulation software is used for a novel application – a full life cycle of manufacture, use, repair and ultimate disposal. Two batches of products are compared: products with and without features which improve reliability. The number of replications for the Monte Carlo process can be calculated from the statistics of the model data.FindingsThe model demonstrates the predicted flows of products through their life cycle. The software has in‐built probability distributions that are not fully suitable for the problem modelled, requiring some artificial treatment, especially when using the delay function. The number of replications should be increased, requiring additional computer time.Practical implicationsThe model is potentially valuable for producers wishing to predict the effect on future costs or the risk of modifying designs. The method can also be used to assist waste management using the output graphs of disposed components or products; hence the economics of component remanufacture or reuse can be modelled.Originality/valueThe paper presents the first known application of manufacturing system simulation software for modelling product life cycles.

Cyclic exposure to ozone alters distal airway development in infant rhesus monkeys

Inner city children exposed to high levels of ozone suffer from an increased prevalence of respiratory diseases. Lung development in children is a long-term process, and there is a significant period of time during development when children growing up in urban areas are exposed to oxidant air pollution. This study was designed to test whether repeating cycles of injury and repair caused by episodes of ozone exposure lead to chronic airway disease and decreased lung function by altering normal lung maturation. We evaluated postnatal lung morphogenesis and function of infant monkeys after 5 mo of episodic exposure of 0.5 parts per million ozone beginning at 1 mo of age. Nonhuman primates were chosen because their airway structure and postnatal lung development is similar to those of humans. Airway morphology and structure were evaluated at the end of the 5-mo exposure period. Compared with control infants, ozone-exposed animals had four fewer nonalveolarized airway generations, hyperplastic bronchiolar epithelium, and altered smooth muscle bundle orientation in terminal and respiratory bronchioles. These results suggest that episodic exposure to environmental ozone compromises postnatal morphogenesis of tracheobronchial airways.

Carpal tunnel syndrome—The role of the subsynovial connective tissue

textabstractCarpal tunnel syndrome (CTS) is the most common compression neuropathy, yet the cause of the compression is in most cases idiopathic. The most common clinical finding associated with the nerve compression is non-inflammatory fibrosis of the subsynovial connective tissue (SSCT) which surrounds the flexor tendons in the carpal canal. The normal SSCT has a multi-layer gliding mechanism, reminiscent of a series of sleeves around the tendon, with each successive sleeve being connected to its neighbors with fine collagenous fibers. We have conducted a series of experiments which have outlined the pathology, kinematics and material properties of the SSCT in normal individuals and in individuals with CTS, and have identified specific features within the SSCT of individuals with CTS that suggest that injury to the SSCT may play a role in the etiology of CTS. Specifically, we have identified evidence of a shearing injury of the SSCT, with fibrosis, and obliteration o! f the gliding, sleeve-like mechanism, being greatest close to the tendon, and with a tendency of greater degrees of fibrosis being observed in more severe cases of CTS. This fibrosis may be associated with tethering of the SSCT to the underlying tendon, which may increase the work of finger movement in patients with CTS, and impair differential finger movement. Based on these observations, we propose a vicious cycle of SSCT injury and repair as a working hypothesis of the etiology of CTS, and have developed a rabbit model to test this hypothesis in vivo.

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients

BackroundCigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR's). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers.MethodsThe study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative.ResultsThe expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients.ConclusionOur data suggest a smoke related change in the phenotype of AM's and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract.

How to Make a Barrett Esophagus: Pathophysiology of Columnar Metaplasia of the Esophagus

Barrett esophagus is defined as a specialized intestinal replacing the squamous epithelium of the esophageal mucosa in response to gastroesophageal reflux. Barrett metaplasia is a healing process that develops to protect the esophagus from further damage. Although mechanisms by which Barrett metaplasia evolves toward dysplasia and adenocarcinoma have been extensively studied, the process by which squamous epithelium is replaced by specialized intestinal metaplasia is poorly understood. Barrett esophagus develops when defense mechanisms in the esophageal mucosa (luminal secretion of mucus, bicarbonate, growth factors, etc.) are overwhelmed by an ongoing cycle of mucosal injury and repair. Hydrogen ion, pepsin, trypsin, and bile acids are considered harmful agents that synergistically invade the esophageal mucosa. Areas of destroyed squamous epithelium are then progressively reepithelized by a columnar epithelium that may originate from multipotent stem cells located within the basal layer of the normal esophageal mucosa or in the ducts of submucosal glands.

Chaco Canyon reburial programme

Chaco Culture National Historical Park in northwestern New Mexico contains a wealth of archaeological resources, including 150 large earth and masonry structures under active management and preservation. In response to loss of original fabric from exposure over the last 100 years and more, as well as from continuous cycles of maintenance and repair, an extensive and long-term reburial programme was embarked upon in the late 1980s. The overall context of the site and the decision to undertake reburial as a principal conservation strategy is described in Part I of this paper. Part II provides a summary of the results of partial reburial at Chetro Ked, one of the ‘great houses’ of the canyon, in which protection of original timber was the main objective. Most of the wood at Chetro Ked could be covered only by a shallow overburden of soil, necessitating a specialized reburial design and materials to exclude moisture. Recent evaluation of the efficacy of the wood reburial was undertaken. Problems and shortcomings that were identified have led to re-design of part of the reburial and more careful attention to quality control during the intervention, as well as to selection of more appropriate geosynthetic materials. Additional monitoring techniques have been developed to allow direct withdrawal of samples of wood for assessment of deterioration.

Postnatal remodeling of the neural components of the epithelial-mesenchymal trophic unit in the proximal airways of infant rhesus monkeys exposed to ozone and allergen

Nerves and neuroendocrine cells located within the airway epithelium are ideally situated to sample a changing airway environment, to transmit that information to the central nervous system, and to promote trophic interactions between epithelial and mesenchymal cellular and acellular components. We tested the hypothesis that the environmental stresses of ozone (O 3) and house dust mite allergen (HDMA) in atopic infant rhesus monkeys alter the distribution of airway nerves. Midlevel bronchi and bronchioles from 6-month-old infant monkeys that inhaled filtered air (FA), house dust mite allergen HDMA, O 3, or HDMA + O 3 for 11 episodes (5 days each, 0.5 ppm O 3, 8 h/day followed by 9 days recovery) were examined using immunohistochemistry for the presence of Protein gene product 9.5 (PGP 9.5), a nonspecific neural indicator, and calcitonin gene-related peptide (CGRP). Along the axial path between the sixth and the seventh intrapulmonary airway generations, there were small significant ( P < 0.05) decrements in the density of epithelial nerves in monkeys exposed to HDMA or O 3, while in monkeys exposed to HDMA + O 3 there was a greater significant ( P < 0.05) reduction in epithelial innervation. In animals exposed to O 3 or HDMA + O 3 there was a significant increase in the number of PGP 9.5 positive/CGRP negative cells that were anchored to the basal lamina and emitted projections in primarily the lateral plain and often intertwined with projections and cell bodies of other similar cells. We conclude that repeated cycles of acute injury and repair associated with the episodic pattern of ozone and allergen exposure alter the normal development of neural innervation of the epithelial compartment and the appearance of a new population of undefined PGP 9.5 positive cells within the epithelium.

Role of carbohydrates in repair of human respiratory epithelium using an in vitro model.

The epithelial layer in the conducting airway provides a primary protective barrier. Repair of this barrier normally occurs rapidly after damage, but is compromised in diseases such as asthma. We have developed a human in vitro model system to test our hypothesis that cell surface glycoconjugate-based interactions are required for the normal repair of damaged epithelium. Lectins having narrow carbohydrate specificities were used to identify and block specific carbohydrate moieties on human airway-derived epithelial cells in culture. The lectin wheat germ agglutinin bound to N-acetyl glucosamine and inhibited the repair of epithelial damage while having little effect on cell viability. In contrast, other N-acetyl glucosamine binding lectins had no effect even when bound to the cell surface. The involvement of glycoconjugates was confirmed by pre-incubating the lectin with its specific sugar, preventing the inhibition of repair. These results indicate that lectin-binding sites are involved in epithelial repair and may be important in the repetitive cycles of injury and repair seen in asthma. This model system provides an insight into the role of glycoconjugates and will help to determine the function of specific carbohydrate groups in epithelial repair. These may present a target for therapeutic intervention in respiratory and other diseases.

In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of Cyclooxygenase-2 enzyme: mechanistic considerations

Purpose : Selective cyclooxygenase-2 inhibitors have been reported to enhance the tumor response to radiation in vivo, but the cellular mechanisms underlying the radiosensitizing effect are not understood. In the present study, we investigated several possible mechanisms using a murine sarcoma cell culture system. Methods and Materials : Cells derived from a murine sarcoma, designated NFSA, were cultured in vitro and exposed to different (either single or split) doses of radiation with and without a pretreatment of SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-l-yl] benzene sulfonamide), a selective cyclooxygenase-2 (COX-2) inhibitor. The cells were assayed for clonogenic survival to determine the radiosensitizing effect of SC-236. In addition, MTT assay and TUNEL assay were performed to determine the effects of SC-236 and radiation on the cell survival and cell cycle distribution. RNase protection assay was performed on the total RNA extract using probes that encoded for selected cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases. To monitor the extent of COX-2 activity and its role in radiosensitization, the cellular content of prostaglandin E2, a major metabolite of COX-2 activity on arachidonic acid, was also determined. Results : The cell clonogenic survival assay showed that SC-236 significantly enhanced tumor cell radiosensitivity: 50 μM SC-236 increased it by a factor of 1.51 at the 0.1 cell survival level. Treatment with SC-236 (50 μM, 3 days) removed the “shoulder” region on the radiation survival curve, suggesting that the drug inhibited repair of sublethal radiation damage. The inhibition was confirmed by split-dose experiments where two doses (3 Gy each) of radiation were given 4 h apart. The cells exposed to radiation only repaired the damage by a factor of 1.44, whereas those treated with SC-236 plus radiation repaired it by a factor of 1.1 only. Whereas SC-236 induced apoptosis in these NFSA cells, radiation did not. No further increase in apoptosis was observed when the cells were exposed to both SC-236 and radiation, suggesting that SC-236 did not render tumor cells more susceptible to radiation-induced apoptosis. The RNase protection assay showed that SC-236 (50 μM, 3 days) inhibited the expression of cyclins A and B, as well as cyclin-dependent kinase-1. Inhibition of these cell cycle regulatory elements by SC-236 was associated with the arrest of cells in the radiosensitive G2-M phase (67%), determined by flow cytometry. Conclusions : SC-236 significantly enhanced radiosensitivity of tumor cells; the magnitude of sensitivity was dependent on the drug’s concentration. The likely mechanisms involve accumulation of cells in the radiosensitive G2-M phase of the cell cycle and inhibition of repair from sublethal radiation damage.

The role of DNA adduct structure and conformation in cisplatin resistance

Cisplatin belongs to the group of platinum anti-tumour compounds widely used in the treatment of testicular and ovarian cancers and a variety of other solid tumours. The target for cisplatin is DNA, with which it binds efficiently to form a variety of monoadducts and cross-links, either between adjacent bases on the same strand of DNA or on opposing strands. These DNA lesions contribute to the cytotoxicity of cisplatin because they block DNA replication and promote cell death. In addition, several cellular proteins bind preferentially to cisplatin-damaged DNA, which could stimulate transduction pathways and ultimately signal apoptosis.One such protein is MutS, a component of the mismatch repair (MMR) machinery that removes aberrantly inserted bases from newly synthesized DNA following replication. Binding of MutS to cisplatin adducts is thought to result in a continuous, futile cycle of repair on the opposing DNA strand, ultimately leading to cell death. Loss of MMR in tumour cells confers cisplatin resistance, and there has been considerable investigation into the development of platinum compounds for use in cisplatin-resistant disease, and whose cytotoxicity is not circumvented by loss of MMR. Drugs that contain the bulky, non-polar diaminocyclohexane (DACH) ligand in place of the amine ligands of cisplatin appear to be promising in this respect. The DACH ligand, present in drugs such as oxaliplatin, is thought to have a different structural conformation in DNA than the corresponding cisplatin adduct, and perhaps provides a less recognizable structure for MutS.Zdraveski et al. [1xMutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA. Zdraveski, Z.Z. et al. J. Biol. Chem. 2002; 277: 1255–1260Crossref | PubMed | Scopus (75)See all References][1] have investigated this hypothesis by examining the binding of MutS to DNA-containing adducts formed by cisplatin and a DACH analogue. In vitro MutS binding assays revealed that purified MutS bound to cisplatin-modified DNA with twofold greater affinity than DNA modified with the DACH analogue. Sensitivity assays using Escherichia coli mutants corroborate these results; E. coli deficient in methylation, a process essential to MMR that distinguishes between newly synthesized and template DNA strands following replication, were twofold more sensitive to cell death by cisplatin than to DACH analogues. DACH-containing platinum drugs might therefore be less dependent on MMR-mediated cytotoxicity, and exert their cell killing effects by another mechanism, such as replication blockage. Indeed, the equal sensitivity of recombination-deficient mutants to both drugs suggest that the adducts formed by both cisplatin and DACH analogues block replication with equal efficiency.This study adds to the existing evidence that drugs that contain DACH ligands, such as oxaliplatin, might overcome the cisplatin resistance of MMR-deficient tumours. Furthermore, this study demonstrates the importance of structural differences in the structural conformation of DNA adducts formed by cytotoxic drugs, and supports the development of platinum drugs based on their coordination chemistry to combat drug resistance in human tumours.

The effect of two episodes of denervation and reinnervation on skeletal muscle contractile function.

Sensory or motor "baby-sitting" has been proposed as a clinical strategy to preserve muscle integrity if motion-specific axons must regenerate over a long distance to reach denervated target muscles. Denervated muscles are innervated temporarily by using axons from nearby sensory or motor nerves. After motion specific motor axons have reached the target, the baby-sitter nerve is severed and motion-specific axons are directed to the target. Although this strategy minimizes denervation time, the requisite second episode of denervation and reinnervation might be deleterious to muscle contractile function. This study was designed to test the hypothesis that two sequential episodes of skeletal muscle denervation and reinnervation result in greater force and power deficits than a single peripheral nerve injury and repair. Adult Lewis rats underwent either transection and epineurial repair or sham exposure of the left peroneal nerve. After a 4-month recovery period, the contractile properties of the extensor digitorum longus muscle of the sham exposure group (control, n = 9) and one of the nerve division and repair groups (repair group 1, n = 9) were evaluated with measurements of the maximum tetanic isometric force, peak power, and maximal sustained power. A third group of rats underwent a second cycle of nerve division and repair (repair group 2, n = 9) at this same time point. Four months postoperatively, contractile properties of the extensor digitorum longus muscles were evaluated. Maximum tetanic isometric force and peak power were significantly reduced in repair group 2 rats as compared with repair group 1 and control rats. Maximal sustained power was not significantly different between the groups. These data support our working hypothesis that skeletal muscle contractile function is adversely affected by two cycles of denervation and reinnervation as compared with a single episode of nerve division and repair.