Michael Keese

Inhibition of glutathione reductase by dinitrosyl-iron-dithiolate complex.

The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-l-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 μm, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a K i of 14 μm, and ak 3 of 1.3 × 10−3s−1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/orS-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specificallyN-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein.

Dinitrosyl-Dithiol-Iron Complexes, Nitric Oxide (NO) Carriers In Vivo, as Potent Inhibitors of Human Glutathione Reductase and Glutathione-S-Transferase

Human glutathione reductase (GR) and rat liver glutathione-S-transferases (GSTs) had been shown to be inhibited by the nitric oxide (NO) carrier S-nitroso-glutathione (GSNO). We have now extended these studies by measuring the effects of dinitrosyl-iron complexed thiols (DNIC-[RSH]2) on human GR, GST and glutathione peroxidase. DNIC-[RSH]2 represent important transport forms of NO but also of iron ions and glutathione in vivo. Human GR was found to be inhibited by dinitrosyl-iron-di-glutathione (DNIC-[GSH]2) and dinitrosyl-iron-di-L-cysteine (DNIC-Cys2) in two ways: both compounds were competitive with glutathione disulfide (GSSG), the inhibition constant (Ki) for reversible competition of DNIC-[GSH]2 with GSSG being approximately 5 microM; preincubating GR for 10 min with 4 microM DNIC-[GSH]2 and 40 microM DNIC-Cys2, respectively, led to 50% irreversible enzyme inactivation. More than 95% GR inactivation was achieved by incubation with 36 microM DNIC-[GSH]2 for 30 min. This inhibition depended on the presence of NADPH. Absorption spectra of inhibited GR showed that the charge-transfer interaction between the isoalloxazine moiety of the prosthetic group flavin adenine dinucleotide (FAD) and the active site thiol Cys63 is disturbed by the modification. Cys2 and FAD could be ruled out as sites of the modification. Isolated human placenta glutathione-S-transferase and GST activity measured in hemolysates were also inhibited by DNIC-[GSH]2. This inhibition, however, was reversible and competitive with reduced glutathione, the Ki being 20 nM. The inhibition of GST induced by GSNO was competitive with reduced glutathione (GSH) (Ki = 180 microM) and with the second substrate of the reaction, 1-chloro-2,4,-dinitrobenzene (Ki = 170 microM). An inhibition of human glutathione peroxidase by GSNO or DNIC-[RSH]2 was not detectable. Inactivation of GR by DNIC-[GSH]2 is by two orders of magnitude more effective than modification by GSNO; this result and the very efficient inhibition of GST point to a role of DNIC-[RSH]2 in glutathione metabolism.

Microscopy of bacterial translocation during small bowel obstruction and ischemia in vivo--a new animal model.

BackgroundExisting animal models provide only indirect information about the pathogenesis of infections caused by indigenous gastrointestinal microflora and the kinetics of bacterial translocation. The aim of this study was to develop a novel animal model to assess bacterial translocation and intestinal barrier function in vivo.MethodsIn anaesthetized male Wistar rats, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli was administered by intraluminal injection in a model of small bowel obstruction. Animals were randomly subjected to non-ischemic or ischemic bowel obstruction. Ischemia was induced by selective clamping of the terminal mesenteric vessels feeding the obstructed bowel loop. Time intervals necessary for translocation of E. coli into the submucosal stroma and the muscularis propria was assessed using intravital microscopy.ResultsBacterial translocation into the submucosa and muscularis propria took a mean of 36 ± 8 min and 80 ± 10 min, respectively, in small bowel obstruction. Intestinal ischemia significantly accelerated bacterial translocation into the submucosa (11 ± 5 min, p < 0.0001) and muscularis (66 ± 7 min; p = 0.004). Green fluorescent protein-transfected E. coli were visible in frozen sections of small bowel, mesentery, liver and spleen taken two hours after E. coli administration.ConclusionsIntravital microscopy of fluorescent bacteria is a novel approach to study bacterial translocation in vivo. We have applied this technique to define minimal bacterial transit time as a functional parameter of intestinal barrier function.

Influence of TNFA on the formation of liver metastases in a syngenic mouse model

The level of TNFα expression is increased after partial hepatectomy, and experimental evidence exists that TNFα plays a key role in liver regeneration. Contradictory results are reported about the influence of TNFα on tumor growth: on the one hand, stimulation of tumor growth in various animal models and, on the other hand, intraperitoneally administered TNFα leads to reduced metastasis formation. TNFα may be one responsible factor for increased metastasis formation after surgical trauma. The objective of our study was to clarify the influence of TNFα on the formation of liver metastases in a syngenic mouse model in vivo. We used a novel marker system, EGFP transfected C26 tumor cells for in vivo observation of metastasis formation by intravital microscopy. We analyzed the effect of intraperitoneal TNFα‐injection on tumor cell adhesion, extravasation and tumor development. The expression of ICAM‐1, VCAM‐1 and E‐Selectin was measured by Western blot and immunohistochemical staining. Tumor load was assessed by determining EGFP in Western blots. GdCl3 was employed 24 and 48 hr before tumor cell injection to selectively deplete the liver of functioning Kupffer cells. We observed significantly more extravasated tumor cells in the TNFα‐pre‐treated animals at early time points with increased expression of adhesion molecules. Measurement of the EGFP levels showed fewer liver metastases in the TNFα‐pretreated animals at day 8. After GdCl3 pretreatment even lower levels of EGFP, i.e., fewer metastases and also lower expression levels of ICAM‐1, VCAM‐1 and E‐Selectin could be observed. TNFα, acts in a bidirectional manner: whereas TNFα facilitates tumor cell adhesion and extravasation of C26 tumor cells by inducing the expression of adhesion molecules, at later time points, TNFα seems to hinder the formation of liver metastases.

The Gut Origin of Bacterial Pancreatic Infection during Acute Experimental Pancreatitis in Rats

Background: Infections are frequent complications and determine clinical course and outcome in severe pancreatitis. A novel animal model was used to assess minimal transit time of bacterial translocation (BT) across the gut mucosa in vivo using green fluorescent protein-transfected Escherichia coli and intravital video microscopy. Methods: Three hours after induction of acute pancreatitis by i.p. injection of 40 µg/kg cerulein, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli were injected into the lumen of a small bowel reservoir formed by ligature in anesthetized Wistar rats. Translocation of E. coli was assessed by intravital microscopy. Animals were sacrificed 5 h after induction of pancreatitis. Results: BT across the mucosa and into the muscularis propria took a mean ± SD of 36.4 ± 8 min and 80.9 ± 9.5 min, respectively, in sham animals. Pancreatitis resulted in a significantly shorter minimal transit time across the mucosa (16.4 ± 4.9 min, p = 0.007) and into the muscularis propria (47.7 ± 2.5 min, p = 0.001). E. coli were detected on frozen cross-sections and on bacteriological examination of pancreatic tissue in animals with acute pancreatitis but not in controls. Discussion: Intravital microscopy of fluorescent bacteria is a new approach towards studying BT in vivo. Minimal transit time of BT serves as a novel functional aspect of mucosal barrier function during acute pancreatitis. The observation of fluorescent bacteria translocating from the small bowel lumen into the pancreas provides substantial experimental proof for the gut-origin-hypothesis of infectious complications in pancreatitis.

Doxorubicin and mitoxantrone drug eluting beads for the treatment of experimental peritoneal carcinomatosis in colorectal cancer.

We investigated the therapeutic efficiency of sulfonate‐modified polyvinyl alcohol beads loaded with doxorubicin, irinotecan or mitoxantrone in vitro and in vivo in a model of experimental peritoneal carcinomatosis (PC). In vitro, cell proliferation was efficiently impaired by doxorubicin drug eluting bead (DEB) treatment while mitoxantrone DEBs were less effective than. Irinotecan showed little effect for both DEBs and free drug. Apoptosis was not different between free mitoxantrone and the DEB form while more apoptosis induction was observed in cells incubated with free doxorubicin and irinotecan. Experimental PC was produced in mice. The therapeutic efficiency of either mitoxantrone and doxorubicin DEB or free drugs were compared. Mice were treated either once on day 12 or by 3 repetitive applications on days 7, 10 and 12. Mice treated by DEBs showed less weight loss and mortality. Therapeutic effect was determined by measuring tumor volume and tumor load on the day 15 after tumor inoculation. For the single application on the day 12, an advantage could be observed for the free drugs. After 3 repeated injections of both free and mitoxantrone DEB no difference in tumor load or tumor volume could be observed. Least tumor load and tumor volume was observed in mice that received 3 repeated injections of doxorubicin DEB. No animal survived 3 injections of free doxorubicin. We conclude that bead encapsulation of chemotherapeutic drugs may show the advantage of less toxicity in peritoneal spread of colorectal cancer.

Liver regeneration in FGF‐2‐deficient mice: VEGF acts as potential functional substitute for FGF‐2

Background/Aims: The angiogenic properties, its role in mesoderm differentiation and cell culture studies implicate an important role of fibroblast growth factor (FGF‐2) in liver regeneration. The aim of the study was to evaluate this role in a FGF‐2 knockout mouse model.

Fluorescence lifetime imaging microscopy of chemotherapy-induced apoptosis resistance in a syngenic mouse tumor model

During cancer therapy with DNA‐damaging drug‐agents, the development of secondary resistance to apoptosis can be observed. In the search for novel therapeutic approaches that can be used in these cases, we monitored chemotherapy‐induced apoptosis resistance in a syngenic mouse tumor model. For this, syngenic murine colorectal carcinoma cells, which stably expressed a FRET‐based caspase‐3 activity sensor, were introduced into animals to induce peritoneal carcinomatosis or disseminated hepatic metastases. This syngenic system allowed in vitro, in vivo and ex vivo analysis of chemotherapy induced apoptosis induction by optically monitoring the caspase‐3 sensor state in the tumor cells. Tumor tissue analysis of 5‐FU treated mice showed the selection of 5‐FU‐induced apoptosis resistant tumor cells. These and chemo‐naive fluorescent tumor cells could be re‐isolated from treated and untreated mice and propagated in cell culture. Re‐exposure to 5‐FU and second line treatment modalities in this ex‐vivo setting showed that 5‐FU induced apoptosis resistance could be alleviated by imatinib mesylate (Gleevec). We thus show that syngenic mouse systems that stably express a FRET‐based caspase‐3 sensor can be employed to analyse the therapeutic efficiency of apoptosis inducing chemotherapy.

Near-infrared fiber optic spectroscopy as a novel diagnostic tool for the detection of pancreatic cancer

We have investigated the application of near-infrared (NIR) fiber-optic spectroscopy for the diagnosis of pancreatic cancer. Cluster analysis of the Fourier transformed near-infrared (FTNIR) fiber-optic spectra of surgically resected pancreatic tumor tissues allowed discrimination of tumor from normal tissue with high sensitivity and specificity. The sensitivity of the method using spectral information of the CH stretching first overtone region (5951-5608 cm(-1)) was 83.3% with a specificity of 83.3%. Based on the CH stretching second overtone region (8605-7938 cm(-1)) we could achieve a sensitivity of 88.9% and specificity of 72.2%. These findings suggest that NIR spectroscopy offers the potential for minimally invasive in-vivo diagnosis of pancreatic cancer.

Microinjected glutathione reductase crystals as indicators of the redox status in living cells.

The flavoenzyme glutathione reductase catalyses electron transfer reactions between two major intracellular redox buffers, namely the NADPH/NADP+ couple and the 2 glutathione/glutathione disulfide couple. On this account, microcrystals of the enzyme were tested as redox probes of intracellular compartments. For introducing protein crystals into human fibroblasts, different methods (microinjection, particle bombardment and optical tweezers) were explored and compared. When glutathione reductase crystals are present in a cytosolic environment, the transition of the yellow Eox form to the orange‐red 2‐electron reduced charge transfer form, EH2, is observed. Taking into account the midpoint potential of the Eox/EH2 couple, the redox potential of the cytosol was found to be <−270 mV at pH 7.4 and 37°C. As a general conclusion, competent proteins in crystalline – that is signal‐amplifying – form are promising probes for studying intracellular events.