Etomoxir is an inhibitor of mitochondrial CPT1 (carnitine palmitoyltransferase 1) and thereby switches energy metabolism from fatty acids to glucose oxidation. Such a metabolic change may be beneficial in CHF (congestive heart failure). The ERGO (etomoxir for the recovery of glucose oxidation) study was designed in which etomoxir was tested at a dose of 80 and 40 mg compared with placebo for a period of 6 months in patients with CHF. As the principle measure of efficacy, a maximal exercise tolerance test and a submaximal 6-min corridor walk test were used. Secondary end points were echocardiographical dimensions and quality-of-life assessment scores. A total of 350 patients were planned to be screened, with the expectation that end point data would be available from approx. 260 patients. However, the study had to be stopped prematurely, because unacceptably high liver transaminase levels were detected in four patients taking etomoxir. At the termination of the study, 121 patients were randomized to placebo, 118 to 40 mg of etomoxir and 108 to 80 mg of etomoxir. At that time, 21 patients in the placebo group, 16 in the 40 mg of etomoxir group and 14 patients in the 80 mg of etomoxir group had completed the study. The mean increases in exercise time were 3.3, 10.2 and 19.4 s for the placebo, 40 mg of etomoxir and 80 mg of etomoxir groups respectively (P value was not significant). No changes were obvious in the 6-min corridor walk test or in echocardiographical parameters from baseline. The number of patients that completed the study was too small to demonstrate significant effects on exercise time, although there was a tendency towards an increase in exercise time. Therefore, before rejecting the hypothesis that inhibition of fatty acid oxidation might be beneficial in CHF, similar studies have to be performed using different inhibitors of fatty acid oxidation targeting CPT1 and other enzymes in this metabolic pathway.

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FhuD is the periplasmic binding protein of the ferric hydroxamate transport system of Escherichia coli. FhuD was isolated and purified as a His-tag-labeled derivative on a Ni-chelate resin. The dissociation constants for ferric hydroxamates were estimated from the concentration-dependent decrease in the intrinsic fluorescence intensity of His-tag-FhuD and were found to be 0.4 M for ferric aerobactin, 1.0 M for ferrichrome, 0.3 M for ferric coprogen, and 5.4 M for the antibiotic albomycin. Ferrichrome A, ferrioxamine B, and ferrioxamine E, which are poorly taken up via the Fhu system, displayed dissociation constants of 79, 36, and 42 M, respectively. These are the first estimated dissociation constants reported for a binding protein of a microbial iron transport system. Mutants impaired in the interaction of ferric hydroxamates with FhuD were isolated. One mutated FhuD, with a W-to-L mutation at position 68 [FhuD(W68L)], differed from wild-type FhuD in transport activity in that ferric coprogen supported promotion of growth of the mutant on iron-limited medium, while ferrichrome was nearly inactive. The dissociation constants of ferric hydroxamates were higher for FhuD(W68L) than for wild-type FhuD and lower for ferric coprogen (2.2 M) than for ferrichrome (156 M). Another mutated FhuD, FhuD(A150S, P175L), showed a weak response to ferrichrome and albomycin and exhibited dissociation constants two-to threefold higher than that of wild-type FhuD. Interaction of FhuD with the cytoplasmic membrane transport protein FhuB was studied by determining protection of FhuB degradation by trypsin and proteinase K and by cross-linking experiments. His-tag-FhuD and His-tag-FhuD loaded with aerobactin specifically prevented degradation of FhuB and were cross-linked to FhuB. FhuD loaded with substrate and also FhuD free of substrate were able to interact with FhuB.Transport of ferric hydroxamates into Escherichia coli requires outer membrane receptor proteins for certain ferric hydroxamates (6) and a periplasmic binding-protein-dependent system of transport across the cytoplasmic membrane that is common for all ferric hydroxamates (6, 24). The latter consists of the FhuD protein in the periplasm, the integral transport protein FhuB in the cytoplasmic membrane, and the FhuC protein, which is associated with the cytoplasmic membrane (7,8,26,27,29,42) and presumably energizes transport into the cytoplasm by ATP hydrolysis. Binding of substrates to the FhuD protein has been demonstrated by proteolysis experiments; only ferric hydroxamates that were transported, aerobactin, ferrichrome, and coprogen, prevented degradation of FhuD, while ferric hydroxamates that were not transported by the Fhu system of E. coli did not protect FhuD from being hydrolyzed (28). No data for the activity of periplasmic proteins for ferric siderophore transport systems other than those for FhuD exist. Studies of the interactions of ferric siderophores with their cognate periplasmic proteins are hampered by the very low concentrations of the binding protei...

Purpose: In patients with uveal melanoma, tumor cell dissemination and subsequent formation of metastases are confined mainly to the hematogenous route. Here, we sought to isolate circulating melanoma cells in peripheral blood of patients with primary uveal melanoma and clinically localized disease. Experimental Design: Blood samples from 52 patients with clinically localized uveal melanoma and from 20 control individuals were prospectively collected before therapy of the primary tumor. Tumor cells expressing the melanoma-associated chondroitin sulfate proteoglycan were enriched by immunomagnetic cell sorting and visualized by immunocytologic staining. Results were compared with clinical data at presentation. Uveal melanoma is the most common primary intraocular malignant tumor in adults and 98% of these patients present with clinically localized disease (1, 2). Despite successful local tumor control, one half of the patients with primary uveal melanoma will die from metastatic disease (3). The 10-year survival rates in patients with nonmetastatic tumors range from 81% (T 1 N 0 M 0 ) to 15% (T 4 N 0 M 0 ; ref. 4). Metastasis-related deaths occur as long as 35 years after diagnosis, indicating that disseminated tumor cells may stay quiescent for decades and change their biological behavior even after this time (5). As therapeutic options in metastatic disease are poor, with a mean survival of 12 to 14 months (6, 7), there is an urgent need for early identification of patients at increased risk for metastases. This will allow evaluation of adjuvant therapeutic strategies in high-risk patients.Established prognostic factors for clinically localized ocular disease include largest basal tumor diameter (LBD), ciliary body involvement, and extraocular growth (8 -17). A number of staging classifications use combinations of the abovementioned criteria (for review, see ref. 4). However, neither of these factors alone or in combination is adequate for predicting the occurrence of metastatic disease in an individual patient. Other prognostic factors such as histologic subtype (11 -14, 16, 18) as well as genomic changes (19 -21) of the primary tumor also correlate with clinical outcome. Yet, these factors are increasingly difficult to determine as patients are currently often treated with radiotherapy. Although a diagnostic biopsy may be done before treatment, in most cases tumor tissue is not available for analysis.Metastatic spread in uveal melanoma is confined to the hematogenous route as long as the conjunctiva is not infiltrated trans-sclerally (22 blood is an obligate (although not sufficient) event in the metastatic cascade. Detection of circulating tumor cells might therefore represent a unique tool to identify patients at increased risk for metastatic disease. A recent systematic metaanalysis (23) of the prognostic value of tumor cell detection in peripheral blood in melanoma patients identified and evaluated two major approaches, PCR-based detection of melanomaassociated mRNA (n = 52 studies) and our rece...

We conclude that fatty acid metabolism and in particular CPT-1 may be an excellent target for treatment of psoriasis.

Transport of iron (III) hydroxamates across the inner membrane of Escherichia coli depends on a binding protein-dependent transport system composed of the FhuB, C and D proteins. The FhuD protein, which is synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport in a fhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein. Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides which are normally exported across the inner membrane and can cause toxicity problems when overproduced.

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In conclusion, in this rabbit model of PVR, adjuvant decorin application during vitrectomy effectively reduces fibrosis and TRD development. In conjunction with no obvious histopathological toxicity signs, decorin represents a promising substance to inhibit PVR reactions.