Localization of the Functional Domains of Human Tissue Inhibitor of Metalloproteinases-3 and the Effects of a Sorsby's Fundus Dystrophy Mutation

A transient COS-7 cell expression system was used to investigate the functional domain arrangement of tissue inhibitor of metalloproteinases-3 (TIMP-3), specifically to assess the contribution of the amino- and carboxylterminal domains of the molecule to its matrix metalloproteinase (MMP) inhibitory and extracellular matrix (ECM) binding properties. Wild type TIMP-3 was entirely localized to the ECM in both its glycosylated (27 kDa) and unglycosylated (24 kDa) forms. A COOH-terminally truncated TIMP-3 molecule was found to be a non- ECM bound MMP inhibitor, whereas a chimeric TIMP molecule, consisting of the NH2-terminal domain of TIMP-2 fused to the COOH-terminal domain of TIMP-3, displayed ECM binding, albeit with a lower affinity than the wild type TIMP-3 molecule. Thus the functional domain arrangement of TIMP-3 is analogous to that seen in TIMP-1 and -2, namely that the NH2-terminal domain is responsible for MMP inhibition whereas the COOH-terminal domain is most important in mediating the specific functions of the molecule. A mutant TIMP-3 in which serine 181 was changed to a cysteine, found in Sorsby’s fundus dystrophy, a hereditary macular degenerative disease, was also expressed in COS-7 cells. This gave rise to an additional 48-kDa species (possibly a TIMP-3 dimer) that retained its ability to inhibit MMPs and localize to the ECM. These data favor the hypothesis that the TIMP-3 mutations seen in Sorsby’s fundus dystrophy contribute to disease progression by accumulation of mutant protein rather than by the loss of functional TIMP-3

Clinical features of a novel TIMP-3 mutation causing Sorsby's fundus dystrophy: implications for disease mechanism

AIMS: To describe the phenotype in three family members affected by a novel mutation in the gene coding for the enzyme tissue inhibitor of metalloproteinase-3 (TIMP-3). METHODS: Three members of the same family were seen with a history of nyctalopia and visual loss due to maculopathy. Clinical features were consistent with Sorsby's fundus dystrophy. Exon 5 of the gene coding for TIMP-3 was amplified by the polymerase chain reaction, single strand conformation polymorphism analysis undertaken and exon 5 amplicons were directly sequenced. RESULTS: Onset of symptoms was in the third to fourth decade. Five of six eyes had geographic macular atrophy rather than neovascularisation as a cause for central visual loss. Peripheral retinal pigmentary disturbances were present. Scotopic ERGs were abnormal in all three. Mutation analysis showed a GT transversion in all three resulting in a premature termination codon, E139X, deleting most of the carboxy terminal domain of TIMP-3. CONCLUSIONS: The patients described had a form of Sorsby's fundus dystrophy which fell at the severe end of the spectrum of this disease. Postulated disease mechanisms include deposition of dimerised TIMP-3 protein

A novel tissue inhibitor of metalloproteinases-3 mutation reveals a common molecular phenotype in sorsby's fundus dystrophy

Sorsby’s fundus dystrophy (SFD) is a dominantly inherited degenerative disease of the retina that leads to loss of vision in middle age. It has been shown to be caused by mutations in the gene for tissue inhibitor of metalloproteinases-3 (TIMP-3). Five different mutations have previously been identified, all introducing an extra cysteine residue into exon 5 (which forms part of the C-terminal domain) of the TIMP-3 molecule; however, the significance of these mutations to the disease phenotype was unknown. In this report, we describe the expression of several of these mutated genes, together with a previously unreported novel TIMP-3 mutation from a family with SFD that results in truncation of most of the C-terminal domain of the molecule. Despite these differences, all of these molecules are expressed and exhibit characteristics of the normal protein, including inhibition of metalloproteinases and binding to the extracellular matrix. However, unlike wild-type TIMP-3, they all form dimers. These observations, together with the recent finding that expression of TIMP-3 is increased, rather than decreased, in eyes from patients with SFD, provides compelling evidence that dimerized TIMP-3 plays an active role in the disease process by accumulating in the eye. Increased expression of TIMP-3 is also observed in other degenerative retinal diseases, including the more severe forms of agerelated macular degeneration, the most common cause of blindness in the elderly in developed countries. We hypothesize that overexpression of TIMP-3 may prove to be a critical step in the progression of a variety of degenerative retinopathies

Investigating possible retinal biomarkers of head trauma in Olympic boxers using optical coherence tomography (OCT)

Purpose: Changes to retina have been reported after a number of neurodegenerative conditions. The purpose of this study was to investigate retinal structures in Olympic boxers exposed to frequent head blows. Methods:Retinal imaging offers potential as a non-invasive biomarkers of neuropathology. Macula and retinal nerve fibre layer (RNFL) thickness was measured using optical coherence tomography (OCT) in UK Olympic boxers attending two mandatory eye screening programs, 18 months apart. Data from the two eye-screenings provide longitudinal data of retinal change over time. Sedentary healthy subjects (controls) without past or present history of concussion were also screened at the time of the second boxer screening to provide comparison cross-sectional data. Results: Sixteen Olympic boxers aged 20-33 years and 20 sedentary healthy controls, aged 24-45 years were recruited. Significant macula thickening was observed over time (18 months) in 75% of right and 50% of left eye sectors. For RNFL, left eye quadrants thickened. For right eye RNFL quadrants, thickening and thinning of this layer was observed. Cross-sectional results showed thinner macula sectors and RNFL quadrants in Olympic boxers compared to controls. Conclusion: Significant change to macula and RNFL densities, occurring over an 18 month interval is an unexpected finding in otherwise heathy elite sportsmen. In addition, macula and RNFL were thinner than healthy sedentary controls. OCT may prove clinically useful as a candidate retinal biomarker of neuropathological change after mild traumatic brain injury and/or repeat head blows

Neural correlates of the behavioral-autonomic interaction response to potentially threatening stimuli

Subjective assessment of emotional valence is typically associated with both brain activity and autonomic arousal. Accurately assessing emotional salience is particularly important when perceiving threat. We sought to characterize the neural correlates of the interaction between behavioral and autonomic responses to potentially threatening visual and auditory stimuli. Twenty-five healthy male subjects underwent fMRI scanning whilst skin conductance responses (SCR) were recorded. One hundred and eighty pictures, sentences, and sounds were assessed as “harmless” or “threatening.” Individuals’ stimulus-locked, phasic SCRs and trial-by-trial behavioral assessments were entered as regressors into a flexible factorial design to establish their separate autonomic and behavioral neural correlates, and convolved to examine psycho-autonomic interaction (PAI) effects. Across all stimuli, “threatening,” compared with “harmless” behavioral assessments were associated with mainly frontal and precuneus activation with specific within-modality activations including bilateral parahippocampal gyri (pictures), bilateral anterior cingulate cortex (ACC) and frontal pole (sentences), and right Heschl’s gyrus and bilateral temporal gyri (sounds). Across stimulus modalities SCRs were associated with activation of parieto-occipito-thalamic regions, an activation pattern which was largely replicated within-modality. In contrast, PAI analyses revealed modality-specific activations including right fusiform/parahippocampal gyrus (pictures), right insula (sentences), and mid-cingulate gyrus (sounds). Phasic SCR activity was positively correlated with an individual’s propensity to assess stimuli as “threatening.” SCRs may modulate cognitive assessments on a “harmless–threatening” dimension, thereby modulating affective tone and hence behavior

Towards the architecture of an instructional multimedia database

The applicability of multimedia databases in education may be extended if they can serve multiple target groups, leading to affordable costs per unit for the user. In this contribution, an approach is described to build generic multimedia databases to serve that purpose. This approach is elaborated within the ODB Project ('Instructional Design of an Optical DataBase'); the term optical refers to the use of optical storage media to hold the audiovisual components. The project aims at developing a database in which a hypermedia encyclopedia is combined with instructional multimedia applications for different target groups at different educational levels. The architecture of the Optical Database will allow for switching between application types while working (for instance from tutorial instruction via the encyclopedia to a simulation and back). For instruction, the content of the database is thereby organized around so-called standard instruction routes: one route per target group. In the project, the teacher is regarded as the manager of instruction.\ud \ud From that perspective, the database is primarily organized as a teaching facility. Central to the research is the condition that the architecture of the Optical Database has to enable teachers to select and tailor instruction routes to their needs in a way that is perceived as logical and easy to use

Specific targeting of PEGylated liposomal doxorubicin (Doxil®) to tumour cells using a novel TIMP3 peptide

Doxorubicin is a cytotoxic anthracycline derivative that has been used as a chemotherapeutic in many different forms of human cancer with some success. However, doxorubicin treatment has several side-effects, the most serious of which is cardiomyopathy, that can be fatal. Doxorubicin encapsulation in PEGylated liposomes (Doxil®) has been shown to increase tumour localisation and decrease cardiotoxicity. Conversely, the stability of such liposomes also leads to increased circulation times and accumulation in the skin, resulting in palmar planter erythrodysesthesia, while also limiting release of the drug at the tumour site. Specific targeting of such liposomes to tumour cells has been attempted using various receptor-specific peptides and antibodies. However, targeting a single epitope limits the likely number of tumour targets and increases the risk of tumour resistance through mutation. In this report, Doxil® was coupled to peptide sequence p700 derived from tissue inhibitor of metalloproteinase 3. This Doxil® -P700 complex results in an approximately 100-fold increase in drug uptake, relative to Doxil® alone, by both mouse and human breast cancer cells and immortalised vascular cells resulting in an increase in cytotoxicity. Using p700 to target liposomes in this way may enable specific delivery of doxorubicin or other drugs to a broad range of cancers

Lentiviral shRNA knock-down of ADAMTS-5 and-9 restores matrix deposition in 3D chondrocyte culture

Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS‐1, ‐4, ‐5, ‐8, ‐9 and ‐15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell‐lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock‐down of ADAMTS‐5 and ‐9 expression in these cells and, by culturing them on three‐dimensional (3D) scaffolds, show that reduction in expression of ADAMTS‐5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue‐engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS‐5 may be a therapeutic target in the treatment of arthritic disease

Exploring interacting chiral spin chains in terms of black hole physics

In this paper we explore the properties of a one-dimensional spin chain in the presence of chiral interactions, focusing on the system's transition to distinct chiral phases for various values of the chiral coupling. By employing the mean-field theory approximation we establish a connection between this chiral system and a Dirac particle in the curved spacetime of a black hole. Surprisingly, the black-hole horizon coincides with the interface between distinct chiral phases. We examine the chiral properties of the system for homogeneous couplings and in scenarios involving position-dependent couplings that correspond to black-hole geometries. To determine the significance of interactions in the chiral chain we employ bosonization techniques and derive the corresponding Luttinger liquid model. Furthermore, we investigate the classical version of the model to understand the impact of the chiral operator on the spins and gain insight into the observed chirality. Our findings shed light on the behavior of the spin chain under the influence of the chiral operator, elucidating the implications of chirality in various contexts, including black-hole physics