Impact of parenteral n-3 fatty acids on experimental acute colitis.

The present study was undertaken to investigate the effects of parenteral lipid emulsions (LE) enriched with n-3 fatty acids (n-3 FA) in experimental acute colitis. Seventy-four adult male Wistar rats were randomized into six groups, five of which had acetic acid-induced colitis. The animals received a fat-free diet and water ad libitum in individual metabolic cages. By a central venous catheter, saline was infused (0.5 ml/h) into the control groups CS (without colitis) and CC (with colitis), while the test groups received specific LE for 7 days. The n-3/n-6 FA ratio and the lipidic compositions regarding long chain (LCT) and medium chain (MCT) triglycerides were: group L--1:7.7 (LCT, n = 12), M--1:7.0 (MCT and LCT, n = 12), LW-3--1:4.5 (LCT plus n-3 FA, n = 12) and MW-3--1:3.0 (MCT and LCT plus n-3 FA, n = 13). The frequency of diarrhea, oral intake/body weight ratio, intestinal alterations, macrophage cellularity were evaluated and colonic concentrations of leukotrienes (LTB4, LTC4), prostaglandins (PGE2) and thromboxanes (TXB2) were measured. Groups M, MW-3 and LW-3 had less diarrhea than the CC group (P<0.05). Average oral intake/body weight ratio in MW-3 animals was comparable to the CS and better than the CC group. n-3 FA treated rats (LW-3 and MW-3) presented less intestinal inflammatory alterations than CC rats. Mucosal ulcer formation in MW-3 group did not differ from CS rats. M and MW-3 rats had less macrophages in the colon than the CC group. Compared with CC group, lower concentrations of LTB4 in the CS, LW-3 and MW-3 groups; of PGE2 in the CS, M and MW-3 groups; and of TXB2 in the CS and MW-3 groups were found. Mean concentrations of LTC4 did not differ among the groups. Thus, a LCT-containing LE with a low n-3-n-6 ratio does not modify inflammatory colitis manifestations; LE with a high n-3-n-6 ratio reduces diarrhea, preserves oral intake-weight ratio, attenuates morphological consequences and decreases colonic concentrations of inflammatory mediators; MCT/LCT-containing LE with 1:3 n-3-n-6 ratio exerts the most profound beneficial impact on the inflammatory response.

A new structural class of selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome.

We describe the identification and in vitro characterization of a series of 2-aminobenzylstatine derivatives that inhibit non-covalently the chymotrypsin-like activity of the 20S proteasome. Our initial SAR data demonstrate that the 2-aminobenzylstatine core structure can effectively serve as the basis for designing potent, selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome.

Modeling of the binding mode of a non-covalent inhibitor of the 20S proteasome. Application to structure-based analogue design.

The 2-aminobenzvlstatine derivative I is a 20S proteasome inhibitor of a novel chemical type identified by high throughput screening. The compound specifically inhibits the chymotrypsin-like catalytic activity of the human proteasome with an IC50 value in the micromolar range. Using the crystal structure of the yeast proteasome, we modeled the structure of the human proteasome in complex with 1. As one of the first applications of the model in our oncology programme targeting the proteasome, we designed an analogue of the inhibitor having enhanced stacking/hydrophobic interactions with the enzyme. One order of magnitude in inhibitory potency was gained.

Proteasome- and p38-dependent regulation of ERK3 expression.

Proteasome inhibition leads to accumulation of transcription factors, heat shock proteins, cyclins, and other proteasome substrate proteins by blocking their proteolytic degradation. An increase in gene transcription upon proteasome inhibition was found for a group of proteins, including p21(WAF1/CIP1), ubiquitin, and transcription factors. In this study, we have demonstrated selective up-regulation of extracellular signal-regulated kinase 3 (ERK3) mRNA and protein expression upon treatment with peptide-based proteasome inhibitors or lactacystin. ERK3 is a family member of the mitogen-activated protein kinases (also called ERK) that are key mediators of signal transduction from the cell surface to the nucleus. ERK3 up-regulation is independent of the p53, Bcl2, and caspase 3 status of cells. p38 pathway kinase inhibitors prevent proteasome-dependent ERK3 induction and enhance the antiproliferative effect of proteasome inhibitors. MCF-7 cells expressing ERK3 ectopically show increased resistance toward proteasome inhibition. The results indicate that ERK3 expression is a consequence of p38 pathway activation and most probably represents an intracellular defense or rescue mechanism against cell stress and damage induced by proteasome inhibition.

Cysteinyl-leukotriene generation as a biomarker for survival in the critically ill.

OBJECTIVE: To evaluate the capacity of cysteinyl-leukotriene generation in the progression of critical illness compared with that in healthy volunteers and to clarify interrelationships between the rate of leukotriene generation, severity of the disease, and clinical outcome. DESIGN: Prospective, observational study. SETTING: Surgical intensive care unit (ICU) in a German university hospital. PATIENTS: We studied 14 ICU patients (nine men, five women; aged 42-82 yrs) suffering from systemic inflammatory response syndrome, sepsis, or sepsis syndrome, with a calculated sepsis severity score of 17.7+/-4.2 and a Simplified Acute Physiology score of 17.6+/-3.0. In addition, five healthy volunteers (three men, two women; aged 34-38 yrs) were included in the study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood samples were obtained every second day from septic patients until discharge from the ICU or death. Leukotriene C4 (LTC4) synthesizing capacity was assessed in isolated and stimulated leukocytes (Ca-ionophore) by using combined reversed-phase, high-pressure liquid chromatography and radioimmunoassay methods. Initially, all patients synthesized less LTC4 than the healthy subjects. In patients who did not survive, the low LTC4 generation persisted throughout the observation period, whereas in surviving patients, its formation was normalized during convalescence. In surviving patients, LTC4 concentrations correlated with sepsis severity score. CONCLUSIONS: LTC4 generation is impaired in sepsis and may serve as a biomarker for survival in the critical ill.

A novel efficient method to identify beta-glucuronidase activity in rat small intestine.

BACKGROUND: Recent epidemiologic studies promote the notion that high intake of food rich in phytochemicals protects against degenerative diseases such as coronary heart diseases and cancer. Phytochemicals are detoxified in mammalian tissues by conjugation with glucuronic acid yielding less active glucuronide conjugates. However, in several tissues glucuronide conjugates are reactivated by the cleaving enzyme beta-glucuronidase. The aim of the present study was to develop a routinely manageable, rapid technique to localize the beta-glucuronidase activity in the small intestinal tissue. METHODS: Histologic slices of rat duodenum, jejunum, and ileum were incubated with a specific chromogenic beta-glucuronidase substrate, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GlcU). After enzymatic cleavage, X-GlcU yields 5-bromo-4-chloro-3-indol, a dark blue crystalline precipitate easily monitored by light microscopic technique. RESULTS: The number and intensity of the crystals were highest in the jejunum and lowest in the ileum. In all three sections of the small intestine, the highest activity was observed at the villar tip and in the tela submucosa and only moderate activity in other layers of the intestinal tissue. CONCLUSIONS: By using the X-GlcU-technique, it could be demonstrated convincingly that beta-glucuronidase exists in all three segments of the rat small intestine. The proposed method is an efficient, simple, and convenient method to visualize beta-glucuronidase activity.

Assessment of resveratrol bioavailability in the perfused small intestine of the rat.

Resveratrol, which is present in grapes, wine and peanuts, is believed to possess chemoprotective properties such as anticarcinogenic effects and to provide protection against cardiovascular diseases. Little is known, however, about its intestinal absorption. We investigated the absorption and metabolism of resveratrol by using an isolated preparation of luminally and vascularly perfused rat small intestine. A synthetic perfusate free from blood components was used as vascular medium with a perfluorocarbon as oxygen carrier. Luminal media consisted of a bicarbonate buffered sodium chloride solution spiked with resveratrol in physiological, nutritionally relevant concentrations (28, 34 and 57 micromol/l, respectively). Viability was maintained during the entire perfusion and no significant differences between resveratrol and control perfusions for oxygen consumption, arterial pressure, lactate-pyruvate ratio and acid-base homeostasis were observed. Vascular uptake of luminally administered resveratrol was 20.5%. The majority of the absorbed resveratrol was conjugated to yield resveratrol glucuronide (16.8%), which was also the main luminal metabolite (11.2%). Lesser amounts of resveratrol sulfate, 3.0% and 0.3%, were found on the luminal and vascular side, respectively, while only minute amounts of resveratrol and resveratrol conjugates (1.9%) were found in the intestinal tissue. The structures of the resveratrol conjugates were verified by liquid chromatography coupled with mass spectometry (LC-MS). The results demonstrate an ample uptake and metabolic conversion of resveratrol. The proposed perfusion model serves as a tool to evaluate intestinal absorption and metabolic handling of phytochemicals, a pertinent input to the ongoing discussion about their health benefits.

Proteasome inhibitor induced gene expression profiles reveal overexpression of transcriptional regulators ATF3, GADD153 and MAD1.

The ubiquitin/proteasome pathway has been implicated in a wide variety of cellular processes and the number of substrates degraded by the proteasome is impressive. Most prominently, the stability of a large number of transcription factors is regulated by ubiquitination. To elucidate pathways regulated by the proteasome, gene expression profiles were generated, comparing changes of mRNA expression of 7900 genes from the UniGene collection upon exposure of cells to the proteasome inhibitors Lactacystin, Lactacystin-beta-lactone or MG132 by means of microarray based cDNA hybridization. The three profiles were very similar, but differed significantly from a gene expression profile generated with the histone deacetylase inhibitor Trapoxin A, indicating that the observed alterations were indeed due to proteasome inhibition. Two of the most prominently induced genes encoded the growth arrest and DNA damage inducible protein Gadd153 and the activating transcription factor ATF3, both transcription factors of the CCAAT/enhancer binding protein (C/EBP) family. A third gene encoded for the transcriptional repressor and c-Myc antagonist Mad1. Our results suggest that proteasome inhibition leads to upregulation of specific members of transcription factor families controlling cellular stress response and proliferation. Oncogene (2000).

Cost containment through L-alanyl-L-glutamine supplemented total parenteral nutrition after major abdominal surgery: a prospective randomized double-blind controlled study.

BACKGROUND & AIMS: Glutamine is recognized as a conditionally essential amino acid. Recent studies indicate that glutamine-containing total parenteral nutrition improves nitrogen economy, enhances gastrointestinal and immune functions and shortens hospital stay. METHODS: Thirty-seven patients (19 w and 18 m; age 61. 4+/-10.4 years; BMI 23.7+/-2.8 kg/m(2)) following major abdominal surgery receiving an isonitrogenous isoenergetic TPN with or without alanyl-glutamine supplementation (0.5 g/kg BW/day), were evaluated in a double-blind, randomized, controlled trial over a five-day period by measuring nitrogen balance, selected biochemical parameters and length of hospital stay. RESULTS: Supplemental alanyl-glutamine improved the overall mean (-3.5+/-1.6 vs. -5.5+/-1. 4 g N;P<0.05) and cumulative nitrogen balance (-14.1+/-9.1 vs. -21.7+/-11.4 g N;P<0.05) compared with the isonitrogenous, isoenergetic standard regimen. Alanyl-glutamine normalized plasma glutamine concentration and reduced the length of hospital stay (12.8+/-2.6 vs. 17.5+/-6.4 days;P<0.05). CONCLUSIONS: The results of the study confirm that supplementation with synthetic alanyl-glutamine dipeptide is associated with cost containment due to shortened hospitalization and improved nitrogen economy.

Protein isoaspartyl methyltransferase protects from Bax-induced apoptosis.

Protein L-isoaspartyl methyltransferase (Pimt) is a highly conserved enzyme utilising S-adenosylmethionine (AdoMet) to methylate aspartate residues of proteins damaged by age-related isomerisation and deamidation. We have been particularly interested in this enzyme since addition of the compound CGP3466 to primary rat astroglia cell cultures resulted in an upregulation of Pimt at the mRNA level, as shown here by semi-quantitative RT-PCR. CGP3466 is a compound related to the anti-Parkinson's drug R-(-)-deprenyl, which has been shown to protect from neural apoptosis induced by trophic factor withdrawal [Tatton et al., 1994. J. Neurochem. 63, 1572]. The pro-apoptotic gene Bax is required in the cascade of events following withdrawal [Deckwerth et al., 1996. Neuron 17, 401]. We therefore investigated whether Pimt overexpression was able to affect Bax-induced apoptosis in primary mouse cortical neurons. Our results show that Pimt is indeed able to protect from Bax-induced apoptosis. Furthermore, this activity is not restricted to brain-specific cell types, since the same effect is also demonstrated in COS1 cells. In addition, mutational analysis suggests that the protective effect is dependent on the adenosine methionine-binding motif, which is well conserved in protein methyltransferases, and that a mutation destroying this motif crucially affects cytoskeletal structures of the cell.

Determination of glutamine in muscle protein facilitates accurate assessment of proteolysis and de novo synthesis-derived endogenous glutamine production.

BACKGROUND: Results of tracer studies indicate that skeletal muscle contributes to approximately 70% of overall glutamine production in healthy adults; the contribution of de novo synthesis being estimated at approximately 60%. However, measurement of the de novo synthesis rate in muscle tissue requires knowledge of the appearance rate of glutamine in plasma and the quantity of glutamine derived from intracellular proteolysis. Thus, the content of glutamine in muscle protein is a prerequisite for an accurate calculation. OBJECTIVE: The objective of the study was to measure glutamine in muscle protein. DESIGN: Muscle specimens (open biopsies) were obtained from humans (10 men and 4 women), rats (n = 4), cows (n = 4), and pigs (n = 4). Glutamine was assessed via prehydrolysis derivatization, rapid microwave-enhanced acid hydrolysis, and 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) reversed-phase HPLC, and expressed per mg alkali-soluble protein (ASP) and DNA. RESULTS: Glutamine concentrations in muscle cell protein of various species ranged from 41 to 49 microg/mg ASP; the differences were not species related. The combined means (+/-SDs) for the 4 species were 43.6 +/- 4.9 microg/mg ASP and 11.9 +/- 2.0 mg/mg DNA, respectively. In humans, there was no apparent influence of age, sex, or BMI. CONCLUSIONS: Direct and specific measurements of glutamine in intact muscle protein were 50% lower than assumed previously. We used data compiled from earlier studies to recalculate the contributions of proteolysis and de novo synthesis to the endogenous production of glutamine in selected age groups of healthy humans; these contributions remained remarkably constant at approximately 13% and approximately 87%, respectively.

Glyceraldehyde-3-phosphate dehydrogenase, the putative target of the antiapoptotic compounds CGP 3466 and R-(-)-deprenyl.

R-(-)-Deprenyl (Selegiline) represents one of the drugs currently used for the treatment of Parkinson's disease. This compound was shown to protect neurons or glias from programmed cell death in a variety of models. The mechanism of action of neuroprotection as well as inhibition of apoptosis remains elusive. CGP 3466 is a structurally related analog of R-(-)-deprenyl that exhibits virtually no monoamine oxidase type B inhibiting activity but is neuroprotective in the picomolar concentration range. We showed specific binding of CGP 3466 to glyceraldehyde-3-phosphate dehydrogenase by affinity binding, by affinity labeling, and by means of BIAcore(R) technology. Apoptosis assays based on the human neuroblastoma cell line PAJU established the importance of this interaction for mediating drug-induced inhibition of programmed cell death.