Time-Dependent Alterations of Gut Wall Integrity in Small Bowel Obstruction in Mice.

BACKGROUND: Small bowel obstruction (SBO) is one of the most common disorders in surgical emergency departments. Without resolution of the obstructed bowel segments, patients may develop multiorgan failure. The aim of this study was to investigate whether morphological damage of the intestinal wall during SBO may lead to molecular translocation and how this may impair intestinal motility. METHODS: C57Bl6 mice were laparotomized, and the small intestine was ligated 5 cm oral to the coecum for SBO. Controls received minilaparotomy only. Animals were sacrificed 3 h, 9 h, and 24 h after SBO. Morphological changes were evaluated on hematoxylin and eosin histology by a standardized score. Intestinal motility was determined by recording intraluminal pressure of the small intestine in vitro. Permeability was measured by fluorospectroscopy and ELISA of blood samples after oral gavage with fluorescein isothiocyanate (FITC)-dextrane and horse radish peroxidase. Data are mean ± SD. RESULTS: Three hours after SBO, FITC-dextrane uptake was increased to 187.6 ± 15.2 ng/mL compared to controls (P = 0.011). At 9 h, uptake of horse radish peroxidase (23.0 ± 8.6 ng/mL, 9.0 ± 6.3 ng/mL, P = 0.039) and FITC-dextrane (86.8 ± 17.8 ng/mL, 62.0 ± 1.6 ng/mL, P = 0.029) was higher compared to controls. Motility was increased to 162.2 ± 20.2 area under the curve (AUC) compared to 121.3 ± 20.3 AUC in controls, P = 0.009 and an increased histology score was observed at 9 h (3.2 ± 1.8 versus 0.6 ± 0.7, P = 0.003). Twenty-four hours after SBO, histology score was 3.8 ± 1.7, which was higher than 0.9 ± 0.7 in controls (P = 0.001). Intestinal motility was decreased 24 h after SBO compared to sham controls (146.0 ± 21.4 AUC versus 198.9 ± 21.2 AUC, P = 0.003). CONCLUSIONS: SBO entails a time dependent epithelial damage to the mucosa. In parallel, molecular changes in the gut mucosal barrier occur as early as 3 h after the onset of SBO with a subsequent increase in permeability. Initial intestinal hypermotility is followed by a decrease in motility.

Timing of surgery in ulcerative colitis in the biologic therapy era-the patient's perspective.

BACKGROUND: There is no general consensus regarding the ideal timing of surgery in patients with refractory ulcerative colitis (UC). Decision-making and timing of restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is influenced by treating physicians and patients themselves. The aim of this study was to determine whether or not patients would have preferred the operation to be performed earlier, at the same time, or at a later point of time and to determine the reasons for their preference. METHODS: Clinical data of 193 patients with UC who have undergone IPAA were documented in a prospective database at our institution between 2004 and 2015. From this database, 190 patients were identified and a standardized custom-made questionnaire was mailed for follow-up survey. Patients who did not respond were called by telephone and encouraged to complete the questionnaire. RESULTS: One hundred nine questionnaires were eligible for analysis (57.4%). Average time between diagnosis and surgery was 11.2 ± 10.8 years (mean ± SD). Indications for surgery were refractory disease (70.6%), colitis-associated colorectal cancer (11.0%), high-grade dysplasia or stenosis (11.9%), and septic complications of UC (6.4%); 39 of 77 patients (50.6%) with refractory UC reported to have preferred their operation to be carried out earlier as it was actually performed (16.8 ± 11.9 months). Refractory course of the disease was identified as a predictor for a retrospectively desired earlier surgical approach (p = 0.014). CONCLUSION: A substantial proportion of patients felt that they should have undergone surgery earlier than actually performed. It appears that timing of the decision to undergo surgery is suboptimal. This situation may be improved by earlier surgical consultation in the course of the disease.

Enteral Nutrition Support Reduces the Necessity of Total Parenteral Nutrition to Reach Patient-Specific Caloric Goals Postpancreaticoduodenectomy.

OBJECTIVES: Pancreaticoduodenectomy (PD) is associated with significant rates of postoperative complications. Although there is evidence that enteral nutrition support (ENS) may reduce postoperative sepsis, the true value of ENS in the abrogation of septic complications remains controversial. The aim of our study is to investigate the postoperative outcome of patients post-PD with and without ENS. METHODS: Using our prospective institutional database, we identified 202 patients from 2001 through 2009 who underwent PD. Of the 202 patients, 121 matched our inclusion criteria. In total, 67 of 121 (55.4%) patients received ENS, whereas 54 (44.6%) patients had no ENS and served as controls. Postoperative morbidity and mortality were recorded and analyzed. RESULTS: No significant differences were found in the postoperative morbidity of the patients. The anastomotic leak rate was 13% in both the ENS and control groups (P = 0.846). There was no difference in mortality within the two groups (4% vs 5%, P = 0.881). Significantly more patients in the control group received total parenteral nutrition (P = 0.033). CONCLUSIONS: ENS is not associated with lower rates of postoperative morbidity and mortality. It does, however, reduce the necessity of additional total parenteral nutrition to reach patient-specific caloric goals.

Hartmann's procedure or primary anastomosis?

Perforation following acute diverticulitis is a typical scenario during the first attack. Different classification systems exist to classify acute perforated diverticulitis. While the Hinchey classification, which is based on intraoperative findings, is internationally best known, the German Hansen-Stock classification which is based on CT scan is widely accepted within Germany. When surgery is necessary, sigmoid colectomy is the standard of care. An important question is whether patients should receive primary anastomosis or a Hartmann procedure subsequently. A priori there are several arguments for both procedures. Hartmann's operation is extremely safe and, therefore, represents the best option in severely ill patients and/or extensive peritonitis. However, this operation carries a high risk of stoma nonreversal, or, when reversal is attempted, a high risk in terms of morbidity and mortality. In contrast, primary anastomosis with or without loop ileostoma is a slightly more lengthy procedure as normally the splenic flexure needs to be mobilized and construction of the anastomosis may consume more time than the Hartmann operation. The big advantage of primary anastomosis, however, is that there is no need for the potentially risky stoma reversal operation. The most interesting question is when to do the Hartmann operation or primary anastomosis. Several comparative case series were published showing that primary anastomosis is feasible in many patients. However, no randomized trial is available to date. It is of note, that all non-randomized case series are biased, i.e. that patients in better condition received anastomosis and those with severe peritonitis underwent Hartmann's operation. This bias is undoubtedly likely to be present, even if not obvious, in the published papers! Our own data suggest that this decision should not be based on the extent of peritonitis but rather on patient condition and comorbidity. In conclusion, sigmoid colectomy and primary anastomosis is feasible and safe in many patients who need surgery for perforated diverticulitis, particularly when combined with loop ileostomy. Based on our own published analysis, however, we recommend performing Hartmann's operation in severely ill patients who carry substantial comorbidity, while the extent of peritonitis appears not to be of predominant importance.

The role of vagal innervation on the early development of postoperative ileus in mice.

BACKGROUND: Postoperative ileus (POI) involves an intestinal inflammatory response that is modulated by afferent and efferent vagal activation. We aimed to identify the potential influence of the vagus nerve on POI by tracking central vagal activation and its role for peripheral inflammatory changes during the early hours after surgery. METHODS: C57BL6 mice were vagotomized (V) 3-4 days prior to experiments, while control animals received sham vagotomy (SV). Subgroups underwent either laparotomy (sham operation; S-POI) or laparotomy followed by standardized small bowel manipulation to induce postoperative ileus (POI). Three hours and 9 h later, respectively, a jejunal segment was harvested and infiltration of inflammatory cells in intestinal muscularis was evaluated by fluorescein isothiocyanate (FITC) avidin and myeloperoxidase (MPO) staining. Moreover, the brain stem was harvested, and central nervous activation was investigated by Fos immunochemistry in both the nucleus of the solitary tract (NTS) and the area postrema (AP). Data are presented as mean ± SEM, and a p < 0.05 was considered statistically significant. KEY RESULTS: Three hour experiments revealed no significant differences between all experimental groups, except MPO staining: 3 h after abdominal surgery, there were significantly more MPO-positive cells in vagotomized S-POI animals compared to sham-vagotomized S-POI animals (26.7 ± 7.1 vs. 5.1 ± 2.4, p < 0.01). Nine hour postoperatively intramuscular mast cells (IMMC) were significantly decreased in the intestinal muscularis of V/POI animals compared to SV/POI animals (1.5 ± 0.3 vs. 5.9 ± 0.2, p < 0.05), while MPO-positive cells were increased in V/POI animals compared to SV/POI animals (713.2 ± 99.4 vs. 46.9 ± 5.8, p < 0.05). There were less Fos-positive cells in the NTS of V/POI animals compared to SV/POI animals (64.7 ± 7.8 vs. 132.8 ± 23.9, p < 0.05) and more Fos-positive cells in the AP of V/POI animals compared to SV/POI animals 9 h postoperatively (38.0 ± 2.0 vs. 13.7 ± 0.9, p < 0.001). CONCLUSIONS AND INTERFERENCES: Afferent nerve signaling to the central nervous system during the development of early POI seems to be mediated mainly via the vagus nerve and to a lesser degree via systemic circulation. During the early hours of POI, the intestinal immune response may be attenuated by vagal modulation, suggesting interactions between the central nervous system and the intestine.

The role of Cys108 in Trigonopsis variabilis d-amino acid oxidase examined through chemical oxidation studies and point mutations C108S and C108D.

Oxidative modification of Trigonopsis variabilis D-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12 mM), the two muteins were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change in D-amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O2-dependent reaction with D-Met which in comparison with the corresponding wild-type value was decreased two- and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50 degrees C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability of D-amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.

Co-morbidity and postsurgical outcome in patients with perforated sigmoid diverticulitis.

INTRODUCTION: It was previously reported that in patients with acute perforated diverticulitis with Hinchey categories I to III sigmoidectomy with primary anastomosis (PA) is superior to Hartmann's procedure (HP) as later closure of colostomy involves substantial morbidity. We evaluated our experience with PA for patients with perforated diverticulitis over a 10-year period and aimed to investigate whether Hinchey category or co-morbidity are more relevant for postoperative outcome. METHODS: Records of all patients treated at our institution for sigmoid diverticulitis between 1996 and 2006 were retrieved from an in-hospital database (N = 787, median age 66 years, range 30 to 94, female:male ratio 1.3:1); 73 patients who underwent immediate emergency surgery for perforated diverticulitis were included in this study. American Society of Anesthesiology (ASA) classification to gauge co-morbidity and Hinchey category for intraoperative extent of inflammation were evaluated as regards their relevance for postoperative mortality and major complications. RESULTS: 47 patients (64%) underwent sigmoid colectomy and PA, which was combined with loop ileostomy in 11 patients (15%). Sigmoid colectomy and HP was performed in 26 patients (36%). Major postoperative complications occurred in 26 patients (36%). In the PA group, 10 of 47 patients (21%) had anastomotic leakage. Three leakages occurred despite a loop ileostomy. Anastomotic leakage was independent of Hinchey category (Hinchey I: three patients, Hinchey II: four patients, Hinchey III: three patients, n.s.), but associated with co-morbidity (one patient ASA II, six patients ASA III, three patients ASA IV, P < 0.05). Total mortality was 12%. Seven patients died after HP and two patients after PA. No mortality was observed in PA patients with loop ileostomy. CONCLUSIONS: Emergency surgery for perforated sigmoid diverticulitis is associated with high morbidity and mortality rates. Anastomotic leakage was associated with patient co-morbidity rather than with intraoperative Hinchey category, suggesting that the decision to perform PA should better be based on patient's general condition rather than on intraoperative extent of inflammation.

Effect of bowel preparation on intestinal permeability and inflammatory response during postoperative ileus in mice.

BACKGROUND: Postoperative ileus entails pathophysiological changes in mucosal permeability and an intestinal inflammatory immune response. We hypothesized that preoperative selective decontamination of the digestive tract combined with preoperative mechanical bowel preparation might be advantageous to prevent or reduce permeability changes and immune response in postoperative ileus. METHODS: Postoperative ileus was induced in mice by standardized small bowel manipulation. Intervention groups received selective decontamination and/or intestinal lavage with normal saline simulating mechanical bowel preparation before postoperative ileus induction. At 1, 3, and 9 hours after surgery, ileum samples were harvested for measurements of fluorescein (332 Da) permeability, quantification of tumor necrosis factor α-mRNA level, and leukocyte infiltration of the intestinal wall. RESULTS: Mucosal fluorescein permeability increased at 1 hour (8.6 ± 1.1 vs 5.9 ± 0.9 10-6 cm/s; P < .01) and 3 hours (8.5 ± 0.6 vs 6.5 ± 0.2 10-6 cm/s; P < .05) after induction of postoperative ileus. This increase was prevented by mechanical bowel preparation and selective decontamination+mechanical bowel preparation interventions at both points in time. Expression of tumor necrosis factor α was more than 2-fold increased (P < .05) in the very early phase after induction of postoperative ileus but did not occur in mechanical bowel preparation-pretreated animals. Myeloperoxidase staining revealed that mechanical bowel preparation inhibited postoperative ileus-associated leukocyte infiltration of the intestinal muscularis at 3 and 9 hours after surgery, but not selective decontamination + mechanical bowel preparation treatment. The number of leukocytes after mechanical bowel preparation-only treatment remained at the level of sham-controls. CONCLUSION: Mechanical bowel preparation prevents permeability and leukocyte infiltration of the intestinal wall in the early phase of postoperative ileus in mice.

"Short-chain" alpha-1,4-glucan phosphorylase having a truncated N-terminal domain: functional expression and characterization of the enzyme from Sulfolobus solfataricus.

All known alpha-1,4-glucan phosphorylases (GPs) are active as homodimers and use their N-terminal domains for oligomerisation. Structure-based sequence comparison of a putative phosphorylase from the thermophilic crenarchaeon Sulfolobus solfataricus (SsGP) with the well characterized GP from Escherichia coli reveals that SsGP totally lacks the otherwise conserved regions for building the dimer interface. Because all efforts of producing functional SsGP in E. coli failed, we used heterologous gene expression in the hyperthermophilic archaeon Thermococcus kodakaraensis and isolated, in low amounts, SsGP harboring Strep-Tag II fused to the C-terminal Tyr-465 of the enzyme. The recombinant protein eluted in size exclusion chromatography with an apparent molecular mass of approximately 69 kDa, consistent with neither the mass expected for a monomer (55 kDa) nor that of a homodimer (110 kDa). The biochemical properties of SsGP were similar to those seen for other GPs containing the N-terminal elements for dimerisation, suggesting that the "short-chain" format of SsGP is fully appropriate for phosphorylase catalytic function and stability. However, the substrate specificity of SsGP differed from that reported for GPs from other thermophilic microorganisms.

Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme.

BACKGROUND: Orthophosphate recognition at allosteric binding sites is a key feature for the regulation of enzyme activity in mammalian glycogen phosphorylases. Protein residues co-ordinating orthophosphate in three binding sites distributed across the dimer interface of a non-regulated bacterial starch phosphorylase (from Corynebacterium callunae) were individually replaced by Ala to interrogate their unknown function for activity and stability of this enzyme. RESULTS: While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr28-->Ala, Arg141-->Ala) or decreased (Lys31-->Ala, Ser174-->Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45 degrees C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme. Loss of stability in the mutated phosphorylases appeared to be largely due to weakened affinity for orthophosphate binding. Binding of sulphate mimicking the crystallographically observed "non-covalent phosphorylation" of the phosphorylase at the dimer interface did not have an allosteric effect on the enzyme activity. CONCLUSIONS: The phosphate sites at the subunit-subunit interface of C. callunae starch phosphorylase appear to be cooperatively functional in conferring extra kinetic stability to the native dimer structure of the active enzyme. The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins. It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.

A survey of street children in northern Tanzania: how abuse or support factors may influence migration to the street.

In October 2006, a survey was undertaken of youth "on the streets" in the Arusha and Kilimanjaro regions of Tanzania (n = 1,923). The question of interest was if street children who live on streets full-time differ concerning reports of abuse and support, compared to reports of children who are only part-time on the streets, and to children who don't self-identify as "street children" at all. Results show full-time street children reporting significantly more abuse than part-time counterparts, or children who were not street children (mean difference = -1.44, P < .001). Concerning support scores, non-street children and part-time street children reported significantly more support from their family than full-time street children (mean difference = 1.70, P < .001). This information identifies possible reasons why vulnerable children migrate to live on the streets in the urban areas, and contributes to the limited literature and data on this subject.

Extrinsic afferent nerve sensitivity and enteric neurotransmission in murine jejunum in vitro.

Enteric and extrinsic sensory neurons respond to similar stimuli. Thus they may be activated in series or in parallel. Because signal transmission via synapses or mediator release would depend on calcium, we investigated its role for extrinsic afferent sensitivity to chemical and mechanical stimulation. Extracellular multiunit afferent recordings were made in vitro from paravascular nerve bundles supplying the mouse jejunum. Intraluminal pressure and afferent nerve responses were recorded under control conditions and under four conditions designed to interfere with enteric neurotransmission. We found that phasic intestinal contractions ceased after switching perfusion to Ca(2+)-free buffer with or without a purinergic P2 receptor antagonist, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or cadmium (blocking all Ca(2+)-channels) but not following omega-conotoxin GVIA (N-type Ca(2+)-channel blocker). Luminal HCl (pH 2) and 5-HT (500 microM) evoked peak firing of 17 +/- 4 impulses per second (imp/s) (n = 10) and 21 +/- 4 imp/s (n = 13) under control conditions. These responses were reduced to 4 +/- 2 imp/s and 5 +/- 2 imp/s by cadmium (n = 7, P < 0.05), to 7 +/- 2 imp/s and 6 +/- 1 imp/s by Ca(2+)-free perfusion (n = 6, P < 0.05), and to 3 +/- 1 imp/s and 4 +/- 1 imp/s by Ca(2+)-free perfusion with PPADS (n = 6, P < 0.05). Responses were unchanged by omega-conotoxin GVIA. Mechanical ramp distension of the intestinal segment to 60 cmH(2)O was not altered by any of the experimental conditions. We concluded that HCl and 5-HT activate extrinsic afferents via a calcium-dependent mechanism, which is unlikely to involve enteric neurons carrying N-type calcium channels. Extrinsic mechanosensitivity is independent of enteric neurotransmission. It appears that cross talk from the enteric to the extrinsic nervous system does not mediate extrinsic afferent sensitivity.

Prediction of caseness for mental pathology in Swiss conscripts: the Self-Screen Prodrome.

BACKGROUND: Basic military training (BMT) is an environment of higher stress levels than are encountered in civilian life. It may trigger mental disorders in predisposed individuals. To reduce BMT attrition because of mental problems a psychiatric assessment is part of the Swiss recruitment process. An initial screening survey that identifies vulnerable individuals will be useful to save both cost and effort when dealing with large populations, such as military draftees. Aims of this investigation are to verify the psychometric properties of the Self-Screen Prodrome (SPro), a newly developed, short screening tool for psychopathology, and to validate it against the Symptom Checklist-90-Revised (SCL-90-R), a well-established self-assessment instrument. METHOD: A sample of 12,380 male conscripts from the year 2003 were administered both the SPro and the SCL-90-R. Vulnerability was operationalized using the "caseness" definition of the SCL-90-R. RESULTS: Factor analysis demonstrated unidimensional scaling of the SPro, and this was supported by high internal reliability. Scores of nine or more symptoms on the SPro scale were found to successfully discriminate between SCL-90-R positive and negative cases. It is thus an adequate measure of general psychopathology (caseness). The association of p = 0.77 between the SPro and the SCL-90-R Global Severity Index (GSI) clearly supports concurrent validity. Our data also demonstrated that the SPro can distinguish individuals with self-reported mental health problems from those with no or few reported symptoms (cutoff > or = 9; sensitivity 89.3%; specificity 84.9%; AUC 0.942; CI 95% 0.935-0.948). CONCLUSION: Though replication and further research are needed, the SPro scale may currently be a useful screening tool for initial screening in a two-stage process of early detection of psychopathology.

Differential sensitization of afferent neuronal pathways during postoperative ileus in the mouse jejunum.

BACKGROUND: Postoperative ileus induces reflex inhibition of gastrointestinal motility and an intestinal inflammatory response. We aimed to determine whether afferent sensitivity is increased during postoperative ileus and whether alterations are cyclooxygenase-2 (COX-2)-dependent. METHODS: C57BL/6 mice underwent laparotomy followed by standardized small bowel manipulation to induce ileus or sham treatment. After 24 hours, extracellular multiunit mesenteric afferent nerve discharge was recorded in vitro from 2-cm segments of jejunum. Fos immunoreactivity was determined for neuronal activation in the vagal nucleus of the solitary tract (nTS) of the brain stem and leukocyte infiltration in the intestinal muscularis by myeloperoxidase stains. RESULTS: Serosal bradykinin (1 microM) was followed by an increase in afferent discharge to 65 +/- 5 imp x s(-1) in ileus segments compared with 37 +/- 6 imp x s(-1) in sham controls (P < 0.05). The response was attenuated to 31 +/- 7 imp x s(-1) after the selective COX-2 inhibitor 5,5-dimethyl-3-(flurorophenyl)-4-(4-methylsulfonyl) phenyl-2(5H)-furanone (DFU) in ileus segments. Afferent firing during ileus was augmented at luminal distension at 20 mm Hg but not at pressures up to 60 mm Hg. The number of Fos-positive neurons in the nTS was 110 +/- 45 during ileus compared with 7 +/- 4 in sham controls (-7.32 mm from bregma, P < 0.05) and did not differ after DFU. The intestinal muscularis contained more leukocytes during ileus compared with ileus segments after DFU and controls (both P < 0.05). CONCLUSION: This study provides direct evidence from afferent nerve recordings that sensitivity to bradykinin, which stimulates predominantly spinal afferents, is augmented during postoperative ileus involving a COX-2 pathway. Vagal afferents were also sensitized because low-threshold mechanosensitivity and neuronal activation in the nTS were increased.

Postoperative complications have little influence on long-term quality of life in Crohn's patients.

PURPOSE: The purpose of the study was to determine the influence of postoperative complications on long-term quality of life in patients after abdominal operations for Crohn's disease. MATERIALS AND METHODS: From 1996 to 2002, 305 Crohn's patients underwent abdominal surgery, and 66 patients developed postoperative complications. Quality of life was studied using a standardized questionnaire and four quality of life instruments. Sixty-six Crohn's patients with uneventful postoperative course matched for age, and follow-up time served as controls. RESULTS: Forty-eight patients (81%) in the complication group (32 major and 16 minor) and 43 patients (75%) in the control group answered the questionnaire. Postoperative follow-up time was 42 (10-94) and 41 months (13-94; median (range)). Quality of life was comparable between groups, except on the subscale "physical functioning" of the Short-form 36 on which patients with minor and major complications showed impaired quality of life compared to controls (67+/-6, 69+/-4, and 84+/-2%; mean+/-standard error of the mean; both p<0.05 vs controls). The incidence of Crohn's disease-related symptoms at follow-up was unaffected by complications (minor 63%, major 56% vs controls 70%; both not significant). CONCLUSION: Postoperative complications after abdominal operations for Crohn's disease do not impair long-term quality of life in general but may affect specific dimensions of quality of life like patients' physical function.

Mechanistic differences among retaining disaccharide phosphorylases: insights from kinetic analysis of active site mutants of sucrose phosphorylase and alpha,alpha-trehalose phosphorylase.

Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-d-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations.

Dissecting differential binding of fructose and phosphate as leaving group/nucleophile of glucosyl transfer catalyzed by sucrose phosphorylase.

Site-directed mutagenesis was used to examine the specificity of Leuconostoc mesenteroides sucrose phosphorylase for utilization of fructose and phosphate as leaving group/nucleophile of the reaction. The largest catalytic defect in Arg(137)-->Ala (approximately 60-fold) and Tyr(340)-->Ala (approximately 2500-fold) concerned phosphate dependent half-reactions whereas that in Asp(338)-->Asn (approximately 7000-fold) derived from disruption of steps where fructose departs or attacks. The relative efficiencies for enzyme glucosylation by sucrose compared with alpha-d-glucose-1-phosphate and enzyme deglucosylation by phosphate compared with fructose were 5.5 and 6.2 for wild-type, 19 and 2.0 for Arg(137)-->Ala, 950 and 0.17 for Tyr(340)-->Ala, and 0.05 and 180 for Asp(338)-->Asn, respectively. Asp(338) and Tyr(340) have a key role in differential binding of fructose and phosphate, respectively.

The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis.

Replacements of Asp-295 by Asn (D295N) and Glu (D295E) decreased the catalytic center activity of Leuconostoc mesenteroides sucrose phosphorylase to about 0.01% of the wild-type level (k(cat)=200s(-1)). Glucosylation and deglucosylation steps of D295N were affected uniformly, approximately 10(4.3)-fold, and independently of leaving group ability and nucleophilic reactivity of the substrate, respectively. pH dependences of the catalytic steps were similar for D295N and wild-type. The 10(5)-fold preference of the wild-type for glucosyl transfer compared with mannosyl transfer from phosphate to fructose was lost in D295N and D295E. Selective disruption of catalysis to glucosyl but not mannosyl transfer in the two mutants suggests that the side chain of Asp-295, through a strong hydrogen bond with the equatorial sugar 2-hydroxyl, stabilizes the transition states flanking the beta-glucosyl enzyme intermediate by > or = 23kJ/mol.

Enteral immunonutrition during sepsis prevents pulmonary dysfunction in a rat model.

BACKGROUND: Sepsis often results in severe pulmonary dysfunction. Via the thoracic duct, the lung is the first organ exposed to gut-derived inflammatory mediators released into mesenteric lymph during sepsis. AIM: To investigate whether an enteral immunonutrition during sepsis improves pulmonary function. METHODS: Mesenteric lymph was obtained from lymph fistula donor rats after intra peritoneal (i.p.) saline (control lymph) or lipopolysaccharide (sepsis lymph) injection. Sepsis lymph was also collected during enteral immunonutrition with omega-3 enriched, long-chain fatty acids (SMOF lipid). Control, sepsis, or sepsis-SMOF lymph was reinfused into the jugular vein of separate recipient rats. The lungs were then harvested, stained with hematoxylin-eosin, and analyzed for: (1) perpendicular parenchyma thickness of the alveolar wall; (2) myeloperoxidase-positive cells; and (3) terminal deoxynucleotidyl transferase Biotin-dUTP nick end labeling (TUNEL)-positive cells. RESULTS: Enteral immunonutrition during sepsis reduced the release of TNFalpha into mesenteric lymph by about 4.5-fold within the first 2 h. Infusion of sepsis lymph into recipient rats induced thickening of alveolar walls, inflammatory reaction, and apoptosis. Infusion of sepsis lymph obtained during enteral immunonutrition did not cause anatomical changes, induced only a mild inflammatory reaction, and prevented apoptosis in the lungs of recipient rats. CONCLUSIONS: Mediators in sepsis lymph induce pulmonary dysfunction such as an increased distance for oxygen transport, inflammatory reaction, and apoptosis. The lung may be protected by an enteral immunonutrition containing long-chain fatty acids.

Shielding design of an underground experimental area at point 5 of the CERN Super Proton Synchrotron (SPS).

Increasing projected values of the circulating beam intensity in the Super Proton Synchrotron (SPS) and decreasing limits to radiation exposure, taken with the increasing non-acceptance of unjustified and unoptimised radiation exposures, have led to the need to re-assess the shielding between the ECX and ECA5 underground experimental areas of the SPS. Twenty years ago, these experimental areas at SPS-Point 5 housed the UA1 experiment, where Carlo Rubbia and his team verified the existence of W and Z bosons. The study reported here describes such a re-assessment based on simulations using the multi-purpose FLUKA radiation transport code. This study concludes that while the main shield which is made of concrete blocks and is 4.8 m thick satisfactorily meets the current design limits even at the highest intensities presently planned for the SPS, dose rates calculated for liaison areas on both sides of the main shield significantly exceed the design limits. Possible ways of improving the shielding situation are discussed.