Formation of disulphide bonds within the mammalian endoplasmic reticulum (ER) requires the combined activities of Ero1α and protein disulphide isomerase (PDI). As Ero1α produces hydrogen peroxide during oxidation, regulation of its activity is critical in preventing ER-generated oxidative stress. Here, we have expressed and purified recombinant human Ero1α and shown that it has activity towards thioredoxin and PDI. The activity towards PDI required the inclusion of glutathione to ensure sustained oxidation. By carrying out site-directed mutagenesis of cysteine residues, we show that Ero1α is regulated by non-catalytic disulphides. The midpoint reduction potential (E°′) of the regulatory disulphides was calculated to be approximately −275 mV making them stable in the redox conditions prevalent in the ER. The stable regulatory disulphides were only partially reduced by PDI (E°′∼−180 mV), suggesting either that this is a mechanism for preventing excessive Ero1α activity and oxidation of PDI or that additional factors are required for Ero1α activation within the mammalian ER.

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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 NH 2 -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 NH 2 -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.The matrix metalloproteinases (MMPs) 1 are a family of zincdependent endopeptidases that exist in both secreted and membrane bound forms. The enzymes are initially expressed as inactive pro-enzymes becoming activated by proteolytic cleavage of their amino termini. The activity of MMPs is tightly regulated by the tissue inhibitors of metalloproteinases (TIMPs), a family of secreted proteins currently comprising four members (TIMP-1-TIMP-4) (1-4). The balance between MMPs and TIMPs regulates the integrity of the proteinacious extracellular matrix (ECM) and thus plays a key role in a wide range of physiological processes that include embryonic development, connective tissue remodeling, wound healing, glandular morphogenesis, and angiogenesis. An imbalance in MMP/ TIMP expression has been implicated in various diseases such as erosive joint disease, cardiovascular disease, and cancer (reviewed in Refs. 5-7).The TIMPs form high affinity 1:1 complexes with the active forms of most MMPs (reviewed in Ref. 8) but show varying specificity for different pro-MMPs allowing TIMPs to control the activation of specific MMPs (9 -12). Activities have also been ascribed to the TIMPs that are independent of their ability to inhibit MMPs; for example, anti-angiogenic and erythroid-potentiating activities have been described for TIMP-1 that are independent of MMP inhibition (13,14). Likewise TIMP-2 shows MMP independent inhibition of endothelial tube formation (15). These differences in TIMP ...

Aim: To investigate the use of intravitreal triamcinolone acetonide (IVTA) for the treatment of diabetic macular oedema (DMO) unresponsive to previous laser photocoagulation. Method: A retrospective, interventional, non-comparative case series. There were 30 eyes of 22 consecutive patients with refractory DMO. An intravitreal injection of triamcinolone acetonide at the dose of 4 mg in 0.1 ml was administered. Best corrected visual acuity was measured at each examination. In addition the central macular thickness was quantitatively measured by optical coherence tomography (OCT) examination at each visit. The amount of hard exudates deposition in the macula was subjectively evaluated using colour fundus photographs. Results: 30 eyes of 22 patients completed 6 months or more of follow up and were included in the study. Mean (SD) visual acuity improved from 0.17 (0.12) at baseline to 0.34 (0.18), 0.36 (0.16), and 0.31 (0.17) at the 1, 3, and 6 month follow up respectively. Mean (SD) OCT macular thickness decreased from 476 (98.32) mm at baseline to 277.46 (96.77) mm, 255.33 (95.73) mm, and 331.25 (146.76) mm at the 1, 3, and 6 month follow up period respectively. 18 and seven eyes completed 12 months and 18 months of follow up, respectively. Mean (SD) visual acuity was 0.36 (0.15) and 0.35 (0.16) at the 12 and 18 month follow up period respectively. 12 eyes received two, seven eyes received three, and two eyes received four IVTA injections. The mean (SD) interval between the first and second IVTA injection was 5.7 (2.67) months and between the second and third was 5.7 (3.25) months. Hard exudates were present in the macula at baseline in all eyes. Progressive reduction in the number and size of the hard exudates was noted after IVTA in all cases. Intraocular pressure was raised above 21 mm Hg in 12 (40%) of 30 eyes. Two eyes developed posterior subcapsular cataract and two developed vitreous haemorrhage. Conclusions: IVTA is a promising treatment for patients with DMO refractory to laser treatment. IVTA is effective in improving vision, reducing macular thickness, and inducing reabsorption of hard exudates. Further investigation is warranted to assess the safety of IVTA for the treatment of DMO. D iabetic retinopathy is the leading cause of blindness in patients aged 20-74 years in the United States.

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