Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death:a mechanism for maintaining tissue integrity

Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme which cross-links proteins via e(g-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca2+ homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca2+ homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca2+, but it is independent of intracellular proteases and is near maximal after only 20min post-wounding. Using labelled primary amines as an indicator of tTG activity within these 'wounded cells', we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.

Pupil responses associated with coloured afterimages are mediated by the magno-cellular pathway

Sustained fixation of a bright coloured stimulus will, on extinction of the stimulus and continued steady fixation, induce an afterimage whose colour is complementary to that of the initial stimulus; an effect thought to be caused by fatigue of cones and/or of cone-opponent processes to different colours. However, to date, very little is known about the specific pathway that causes the coloured afterimage. Using isoluminant coloured stimuli recent studies have shown that pupil constriction is induced by onset and offset of the stimulus, the latter being attributed specifically to the subsequent emergence of the coloured afterimage. The aim of the study was to investigate how the offset pupillary constriction is generated in terms of input signals from discrete functional elements of the magno- and/or parvo-cellular pathways, which are known principally to convey, respectively, luminance and colour signals. Changes in pupil size were monitored continuously by digital analysis of an infra-red image of the pupil while observers viewed isoluminant green pulsed, ramped or luminance masked stimuli presented on a computer monitor. It was found that the amplitude of the offset pupillary constriction decreases when a pulsed stimulus is replaced by a temporally ramped stimulus and is eliminated by a luminance mask. These findings indicate for the first time that pupillary constriction associated with a coloured afterimage is mediated by the magno-cellular pathway. © 2003 Elsevier Science Ltd. All rights reserved.

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His7 and Ala8, can greatly improve resistance to this enzyme. Little research has assessed what effect Glu9-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu9 of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue (Lys9)GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys9)GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Self-organizing Map and Cellular Automata Combined Technique for Advanced Mesh Generation in Urban and Architectural Design

This paper presents a technique for building complex and adaptive meshes for urban and architectural design. The combination of a self-organizing map and cellular automata algorithms stands as a method for generating meshes otherwise static. This intends to be an auxiliary tool for the architect or the urban planner, improving control over large amounts of spatial information. The traditional grid employed as design aid is improved to become more general and flexible.

A comparison of spatial patterns of TDP-43 cellular inclusions in familial and sporadic frontotemporal lobar degeneration with TDP-43 proteinopathy

Abnormal protein aggregates of transactive response (TAR) DNA-binding protein (TDP-43) in the form of neuronal cytoplasmic inclusions (NCI), oligodendroglial inclusions (GI), neuronal internuclear inclusions (NII), and dystrophic neurites (DN) are the pathological hallmark of frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP). To investigate the role of phosphorylated TDP-43 (pTDP-43) in neurodegeneration in FTLD-TDP, the spatial patterns of the pTDP-43-immunoreactive NCI, GI, NII, and DN were studied in frontal and temporal cortex in three groups of cases: (1) familial FTLD-TDP caused by progranulin (GRN) mutation, (2) a miscellaneous group of familial cases containing cases caused by valosin-containing protein (VCP) mutation, ubiquitin associated protein 1 (UBAP1) mutation, and cases not associated with currently known genes, and (3) sporadic FTLD-TDP. In a significant number of brain regions, the pTDP-43-immunoreactive inclusions developed in clusters and the clusters were distributed regularly parallel to the tissue boundary. The spatial patterns of the inclusions were similar to those revealed by a phosphorylation-independent anti-TDP-43 antibody. The spatial patterns and cluster sizes of the pTDP-43-immunoreactive inclusions were similar in GRN mutation cases, remaining familial cases, and in sporadic FTLD-TDP. Hence, pathological changes initiated by different genetic factors in familial cases and by unknown causes in sporadic FTLD-TDP appear to follow a parallel course resulting in very similar patterns of degeneration of frontal and temporal lobes.

Cellular and molecular consequences of S100A4-induced motility in rat breast tumour Rama 37 cells

Since the first discovery of S100 members in 1965, their expressions have been affiliated with numerous biological functions in all cells of the body. However, in the recent years, S100A4, a member of this superfamily has emerged as the central target in generating new avenue for cancer therapy as its overexpression has been correlated with cancer patients’ mortality as well as established roles as motility and metastasis promoter. As it has no catalytic activity, S100A4 has to interact with its target proteins to regulate such effects. Up to date, more than 10 S100A4 target proteins have been identified but the mechanical process regulated by S100A4 to induce motility remains vague. In this work, we demonstrated that S100A4 overexpression resulted in actin filaments disorganisation, reduction in focal adhesions, instability of filopodia as well as exhibiting polarised morphology. However, such effects were not observed in truncated versions of S100A4 possibly highlighting the importance of C terminus of S100A4 target recognition. In order to assess some of the intracellular mechanisms that may be involved in promoting migrations, different strategies were used, including active pharmaceutical agents, inhibitors and knockdown experiments. Treatment of S100A4 overexpressing cells with blebbistatin and Y-27632, non muscle myosin IIA (NMMIIA) inhibitors, as well as knockdown of NMMIIA, resulted in motility enhancement and focal adhesions reduction proposing that NMMIIA assisted S100A4 in regulating cell motility but its presence is not essential. Further work done using Cos 7 cell lines, naturally lacking NMMIIA, further demonstrated that S100A4 is capable of regulating cell motility independent of NMMIIA, possibly through poor maturation of focal adhesion. Given that all these experiments highlighted the independency of NMMIIA towards migration, a protein that has been put at the forefront of S100A4-induced motility, we aimed to gather further understanding regarding the other molecular mechanisms that may be at play for motility. Using high throughput imaging (HCI), 3 compounds were identified to be capable of inhibiting S100A4-mediated migration. Although we have yet to investigate the underlying mechanism for their effects, these compounds have been shown to target membrane proteins and the externalisation of S100 proteins, for at least one of the compounds, leading us to speculate that preventing externalisation of S100A4 could potentially regulate cell motility.

Phytoestrogen-ind uced glucose uptake and cellular proliferation in L6 myotubesadipocyte function

Aims: Oestrogens are known to act on a number of tissues throughout the body via classical oestrogen receptors, alpha (ER-a) and beta (ER-beta). Previous research has shown that oestrogens can regulate skeletal muscle glucose uptake cellular proliferation. Thus, oestrogens and related molecules provide an interesting focus for research into possible therapies for the treatment of metabolic disorders and sarcopenia. Enterodiol and enterolactone are plant derived mammalian enterolignans which share a struc- tural similarity to the human oestrogen oestradiol. Methods: In the present study we incubated the differentiated rat skeletal muscle cell line L6 concentration ranges of both com- pounds in the presence/absence of oestrogen receptor antagonists and measured glucose uptake using the non-metabolised glucose analogue 2-NBDG. Cellular proliferation was also measured using a modified MTS assay. Results: Enterolactone was seen to cause a significant increase in cellular proliferation after 48h (a maximal 25% at 0.1nmol/l), in an ER-a dependent mechanism. Incubation with 10nmol/l and 100nmol/l enterodiol caused significant increases in 2-NBDG (5000% compared with control, p < 0.001) and 2h glucose depletion from media (15% increase compared with control, p < 0.05), also in an ER-a dependent way. These results suggest these dietary derived oestrogen-like molecules might be of potential use in targeting metabolic disorders or sarcopenia. Conclusion: We can report here that the phytoestrogen derived molecules enterodiol and enterolactone interact with ER-a in the myotubes to regulate glucose uptake and cellular proliferation respectively.

Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm – with or without anchoring them in the plasma membrane –, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised neither by adding a C-terminal stabilisation signal nor by anchoring the protein at the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

System design and resource management in the next-generation integrated WLAN and 3G cellular networks

Next-generation integrated wireless local area network (WLAN) and 3G cellular networks aim to take advantage of the roaming ability in a cellular network and the high data rate services of a WLAN. To ensure successful implementation of an integrated network, many issues must be carefully addressed, including network architecture design, resource management, quality-of-service (QoS), call admission control (CAC) and mobility management. ^ This dissertation focuses on QoS provisioning, CAC, and the network architecture design in the integration of WLANs and cellular networks. First, a new scheduling algorithm and a call admission control mechanism in IEEE 802.11 WLAN are presented to support multimedia services with QoS provisioning. The proposed scheduling algorithms make use of the idle system time to reduce the average packet loss of realtime (RT) services. The admission control mechanism provides long-term transmission quality for both RT and NRT services by ensuring the packet loss ratio for RT services and the throughput for non-real-time (NRT) services. ^ A joint CAC scheme is proposed to efficiently balance traffic load in the integrated environment. A channel searching and replacement algorithm (CSR) is developed to relieve traffic congestion in the cellular network by using idle channels in the WLAN. The CSR is optimized to minimize the system cost in terms of the blocking probability in the interworking environment. Specifically, it is proved that there exists an optimal admission probability for passive handoffs that minimizes the total system cost. Also, a method of searching the probability is designed based on linear-programming techniques. ^ Finally, a new integration architecture, Hybrid Coupling with Radio Access System (HCRAS), is proposed for lowering the average cost of intersystem communication (IC) and the vertical handoff latency. An analytical model is presented to evaluate the system performance of the HCRAS in terms of the intersystem communication cost function and the handoff cost function. Based on this model, an algorithm is designed to determine the optimal route for each intersystem communication. Additionally, a fast handoff algorithm is developed to reduce the vertical handoff latency.^

Foundational forensic techniques for cellular and ad hoc multi-hop networks

The Internet has become an integral part of our nation’s critical socio-economic infrastructure. With its heightened use and growing complexity however, organizations are at greater risk of cyber crimes. To aid in the investigation of crimes committed on or via the Internet, a network forensics analysis tool pulls together needed digital evidence. It provides a platform for performing deep network analysis by capturing, recording and analyzing network events to find out the source of a security attack or other information security incidents. Existing network forensics work has been mostly focused on the Internet and fixed networks. But the exponential growth and use of wireless technologies, coupled with their unprecedented characteristics, necessitates the development of new network forensic analysis tools. This dissertation fostered the emergence of a new research field in cellular and ad-hoc network forensics. It was one of the first works to identify this problem and offer fundamental techniques and tools that laid the groundwork for future research. In particular, it introduced novel methods to record network incidents and report logged incidents. For recording incidents, location is considered essential to documenting network incidents. However, in network topology spaces, location cannot be measured due to absence of a ‘distance metric’. Therefore, a novel solution was proposed to label locations of nodes within network topology spaces, and then to authenticate the identity of nodes in ad hoc environments. For reporting logged incidents, a novel technique based on Distributed Hash Tables (DHT) was adopted. Although the direct use of DHTs for reporting logged incidents would result in an uncontrollably recursive traffic, a new mechanism was introduced that overcome this recursive process. These logging and reporting techniques aided forensics over cellular and ad-hoc networks, which in turn increased their ability to track and trace attacks to their source. These techniques were a starting point for further research and development that would result in equipping future ad hoc networks with forensic components to complement existing security mechanisms.

Cancer therapy combining the modalities of hyperthermia and chemotherapy: In vitro cellular response after rapid heat accumulation in the cancer cell

Hyperthermia is usually used at a sub-lethal level in cancer treatment to potentiate the effects of chemotherapy. The purpose of this study is to investigate the role of heating rate in achieving synergistic cell killing by chemotherapy and hyperthermia. For this purpose, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted. The cytotoxicity, mode of cell death, induction of thermal tolerance and P-gp mediated MDR following the two different modes of heating were studied. Doxorubicin (DOX) was used as the chemotherapy drug. Indocyanine green (ICG), which absorbs near infrared light at 808nm (ideal for tissue penetration), was chosen for achieving rapid rate hyperthermia. A slow rate hyperthermia was provided by a cell culture incubator. The results show that the potentiating effect of hyperthermia to chemotherapy can be maximized by increasing the rate of heating as evident by the results from the cytotoxicity assay. When delivered at the same thermal dose, a rapid increase in temperature from 37°C to 43°C caused more cell membrane damage than gradually heating the cells from 37°C to 43°C and thus allowed for more intracellular accumulation of the chemotherapeutic agents. Different modes of cell death are observed by the two hyperthermia delivery methods. The rapid rate laser-ICG hyperthermia @ 43°C caused cell necrosis whereas the slow rate incubator hyperthermia @ 43°C induced very mild apoptosis. At 43°C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified. This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR.

Mitigating congestion by integrating time forecasting and realtime information aggregation in cellular networks

An iterative travel time forecasting scheme, named the Advanced Multilane Prediction based Real-time Fastest Path (AMPRFP) algorithm, is presented in this dissertation. This scheme is derived from the conventional kernel estimator based prediction model by the association of real-time nonlinear impacts that caused by neighboring arcs’ traffic patterns with the historical traffic behaviors. The AMPRFP algorithm is evaluated by prediction of the travel time of congested arcs in the urban area of Jacksonville City. Experiment results illustrate that the proposed scheme is able to significantly reduce both the relative mean error (RME) and the root-mean-squared error (RMSE) of the predicted travel time. To obtain high quality real-time traffic information, which is essential to the performance of the AMPRFP algorithm, a data clean scheme enhanced empirical learning (DCSEEL) algorithm is also introduced. This novel method investigates the correlation between distance and direction in the geometrical map, which is not considered in existing fingerprint localization methods. Specifically, empirical learning methods are applied to minimize the error that exists in the estimated distance. A direction filter is developed to clean joints that have negative influence to the localization accuracy. Synthetic experiments in urban, suburban and rural environments are designed to evaluate the performance of DCSEEL algorithm in determining the cellular probe’s position. The results show that the cellular probe’s localization accuracy can be notably improved by the DCSEEL algorithm. Additionally, a new fast correlation technique for overcoming the time efficiency problem of the existing correlation algorithm based floating car data (FCD) technique is developed. The matching process is transformed into a 1-dimensional (1-D) curve matching problem and the Fast Normalized Cross-Correlation (FNCC) algorithm is introduced to supersede the Pearson product Moment Correlation Co-efficient (PMCC) algorithm in order to achieve the real-time requirement of the FCD method. The fast correlation technique shows a significant improvement in reducing the computational cost without affecting the accuracy of the matching process.

Forecasting enrollment in differential assessment programs using cellular automata

Urban growth models have been used for decades to forecast urban development in metropolitan areas. Since the 1990s cellular automata, with simple computational rules and an explicitly spatial architecture, have been heavily utilized in this endeavor. One such cellular-automata-based model, SLEUTH, has been successfully applied around the world to better understand and forecast not only urban growth but also other forms of land-use and land-cover change, but like other models must be fed important information about which particular lands in the modeled area are available for development. Some of these lands are in categories for the purpose of excluding urban growth that are difficult to quantify since their function is dictated by policy. One such category includes voluntary differential assessment programs, whereby farmers agree not to develop their lands in exchange for significant tax breaks. Since they are voluntary, today’s excluded lands may be available for development at some point in the future. Mapping the shifting mosaic of parcels that are enrolled in such programs allows this information to be used in modeling and forecasting. In this study, we added information about California’s Williamson Act into SLEUTH’s excluded layer for Tulare County. Assumptions about the voluntary differential assessments were used to create a sophisticated excluded layer that was fed into SLEUTH’s urban growth forecasting routine. The results demonstrate not only a successful execution of this method but also yielded high goodness-of-fit metrics for both the calibration of enrollment termination as well as the urban growth modeling itself.