Protection Against Chemically-Induced Oxidative Gastrointestinal Tissue Injury in Rats by Bismuth Salts

Oxygen free radicals (OFR) are implicated in thepathogenesis of stress, chemically induced gastriclesions, and gastrointestinal injury. Theconcentration-dependent scavenging abilities of bismuthsubsalicylate (SBS), colloidal bismuth subcitrate (CBS), andselected OFR scavengers, including superoxide dismutase(SOD), catalase, mannitol, and allopurinol were examinedagainst biochemically or chemically generated superoxide anion, hydroxyl radical, andhypochlorite radical plus hypochlorous acid based on achemiluminescence assay. Furthermore, both gastric (GM)and intestinal mucosa (IM) were individually exposed in vitro to these free radical generatingsystems, and the concentration-dependent protectiveabilities of SBS and CBS against lipid peroxidation (LP)were compared with selected OFR scavengers. In addition, 24-hr fasted rats were orally treated with thenecrotizing agents 0.6 M HCl, 0.2 M NaOH, 80% ethanol,and aspirin (200 mg/kg). The extent of tissue injury inthe GM and IM was determined by assessing LP, DNA fragmentation, and membrane microviscosity.Dose- and time-dependent in vivo protective abilities ofCBS (100 mg/kg) and SBS (15 mg/kg) were also assessed.Following incubations with superoxide anion and hydroxyl radical generating systems in thepresence of 125 mg SBS/liter, approximately 47% and 61%inhibitions were observed in the chemiluminescenceresponse, respectively, while 48% and 46% inhibitions were observed with 125 mg CBS/liter. SBS andCBS exerted similar abilities towards hypochloriteradical plus hypochlorous acid. Approx. 3.1- and3.7-fold increases in LP were observed in the GM and IMof rats following oral administration of 0.6 MHCl. Pretreatment of the rats with SBS and CBS decreased0.6 M HCl-induced LP in the GM by approx. 39% and 27%,respectively, with similar decreases in LP in the IM. SBS exhibited better protectiveabilities towards 0.6 M HCl and 0.2 m NaOH-induced GMand IM injury as compared to CBS. SBS and CBS providedsimilar protection towards 80% ethanol-induced gastric injury, while CBS exerted a superior protectiveability towards aspirin-induced gastric injury. Theresults demonstrate that both SBS and CBS can scavengereactive oxygen species and prevent tissue damage produced by OFR.     

Synthesis,Physicochemical Properties,and Cytotoxicity of a Series of 5′-Ester Prodrugs of 5-Iodo-2′-deoxyuridine

Five aliphatic 5-esters of 5-iodo-2deoxyuridine (IDU) were synthesized via an acid chloride alcoholysis reaction. The solubility in pH 7.4 phosphate buffer, lipophilicity as determined by partition experiments in octanol/pH 7.4 buffer, and cytotoxicity of these potential prodrugs were evaluated. The esters showed a 43- to 250-fold increase in lipophilicity and a 1.6- to 14-fold decrease in aqueous solubility relative to IDU. At a concentration of 50 µM, all esters showed reduced cytotoxicity toward uninfected Vero cells relative to IDU.     

Prodrugs of 5-Iodo-2′-Deoxyuridine for Enhanced Ocular Transport

Problems associated with the use of 5-iodo-2-deoxyundine (IDU) in the treatment of herpes simplex keratitis can be attributed largely to the polar nature of IDU resulting in its poor permeability across the lipoidal epithelial layer of the corneal membrane. Five aliphatic 5-esters of IDU were synthesized and evaluated as prodrugs for potential use in the treatment of deep ocular infections such as stromal keratitis, iritis, and even retinitis. A parabolic relationship between in vitro corneal membrane permeability and carbon chain length of prodrugs is evident. For a given prodrug, enzymatic hydrolysis proceeded most readily in iris–ciliary body, followed by cornea and aqueous humor. An increase in carbon chain length made the prodrugs more enzymatically labile but more resistant to chemical hydrolysis at pH 7.4 and 34°C. The 5-butyryl ester of IDU exhibited an approximately fourfold increase in aqueous humor IDU concentration relative to IDU at 25 min following instillation of 25-µl 5 mM solutions.     

Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferative factors.

Normal human epidermal keratinocytes (KC) grown under conditions that maintain the undifferentiated state are highly motile. Migration of these cells as measured in two different assays (migration out of an agarose drop explant, and into micropore filters in a modified Boyden chamber), is stimulated by fibronectin (FN) and to a lesser extent by thrombospondin (TSP). In contrast, laminin (LN) inhibits KC migration. Cultivation of the cells for 1 day under conditions that induce differentiation (ie, in the presence of 1.4 mM Ca2+) suppresses KC motility. A number of soluble growth modulating polypeptide factors also influence KC migration. Transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) stimulate KC motility. These factors simultaneously induce KC production of FN and a significant portion of the stimulated motility can be inhibited with antibodies to FN. EGF and somatomedin-C (SM-C), but not TGF-beta, also stimulate TSP production while EGF and SM-C (but not TGF-beta) induce KC proliferation. In contrast to these factors, interferon-gamma (INF-gamma) inhibits KC production of both FN and TSP and concomitantly inhibits both motility and proliferation. These data suggest that KC properties essential for normal wound healing (ie, motility and proliferation) are regulated by both extracellular matrix molecules and soluble peptide factors. Finally, these effects of various growth promoting and antiproliferative factors on KCs may, in part, be mediated through alteration in the endogenous production of extracellular matrix molecules by KCs.     

Enhanced 3D PET OSEM reconstruction using inter-update Metz filtering

We present an enhancement of the OSEM (ordered set expectation maximization) algorithm for 3D PET reconstruction, which we call the inter-update Metz filtered OSEM (IMF-OSEM). The IMF-OSEM algorithm incorporates filtering action into the image updating process in order to improve the quality of the reconstruction. With this technique, the multiplicative correction image--ordinarily used to update image estimates in plain OSEM--is applied to a Metz-filtered version of the image estimate at certain intervals. In addition, we present a software implementation that employs several high-speed features to accelerate reconstruction. These features include, firstly, forward and back projection functions which make full use of symmetry as well as a fast incremental computation technique. Secondly, the software has the capability of running in parallel mode on several processors. The parallelization approach employed yields a significant speed-up, which is nearly independent of the amount of data. Together, these features lead to reasonable reconstruction times even when using large image arrays and non-axially compressed projection data. The performance of IMF-OSEM was tested on phantom data acquired on the GE Advance scanner. Our results demonstrate that an appropriate choice of Metz filter parameters can improve the contrast-noise balance of certain regions of interest relative to both plain and post-filtered OSEM, and to the GE commercial reprojection algorithm software.     

Cell membrane-associated MT1-MMP-dependent activation of pro-MMP-2 in A375 melanoma cells.

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375. RESULTS: Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay. CONCLUSION: Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.