Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k(cat)/K-m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K-s. Values of k(cat/)K(m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k(cat)/K-m for the CySB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 X resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S-1 subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity

Influence of Portosystemic Shunt on Liver Regeneration after Hepatic Resection in Pigs

Objective. The minimal amount of liver mass necessary for regeneration is still a matter of debate. The aim of the study was to analyze liver regeneration factors after extended resection with or without portosystemic shunt. Methods. An extended left hemihepatectomy was performed in 25 domestic pigs, in 15 cases after a portosystemic H-shunt. The expression of Ki-67, VEGF, TGF-α, FGF, and CK-7 was analyzed in paraffin-embedded tissue sections. Results. The volume of the remnant liver increased about 2.5-fold at the end of the first week after resection. With 19 cells/10 Glisson fields versus 4/10, Ki-67-expression was significantly higher in the H-shunt group. VEGF- and CK-7-expressions were significantly higher in the control group. No significant change was found in FGF-expression. The expression of TGF-α was higher, but not significantly, in the control group. Conclusions. The expression of Ki-67, and therefore hepatocyte regeneration, was increased in the shunt group. The expression of CK-7 on biliary epithelium and the expression of VEGF, however, were stronger in the control group

Impact of response evaluation for resectable esophageal adenocarcinoma – A retrospective cohort study

AbstractIntroduction: The standard treatment concept in patients with locally advanced adenocarcinoma of the esophagogastric junction is neoadjuvant chemotherapy, followed by tumor resection in curative intent. Response evaluation of neoadjuvant chemotherapy using histopathological tumor regression grade (TRG) has been shown to be a prognostic factor in patients with esophageal cancer. Methods: We assessed the impact of the various methods of response control and their value in correlation to established prognostic factors in a cohort of patients with adenocarcinoma at the gastroesophageal junction treated by neoadjuvant chemotherapy. Results: After neoadjuvant chemotherapy, in 56 consecutive patients with locally advanced (T2/3/4 and/or N0/N1) esophageal adenocarcinoma an oncologic tumor resection for curative intent was performed. Median follow-up was 44 months. Histopathological tumor stages were stage 0 in 10.7%, stage I in 17.9%, stage II in 21.4%, stage III in 41.1% and stage IV 8.9%. The 3-year overall survival (OS) rate was 30.3%. In univariate analysis, ypN-status, histopathological tumor stage and tumor regression grade correlated significantly with overall survival (p = 0.022, p = 0.001, p = 0.035 respectively). Clinical response evaluation could not predict response and overall survival (p = 0.556, p = 0.254 respectively). Conclusion: After preoperative chemotherapy, outcomes of esophageal carcinoma are best predicted utilizing pathological tumor stage and histologic tumor regression. Clinical response assessments were not useful for guidance of treatment

Sequencing of folding events in Go-like proteins

We have studied folding mechanisms of three small globular proteins: crambin (CRN), chymotrypsin inhibitor 2 (CI2) and the fyn Src Homology 3 domain (SH3) which are modelled by a Go-like Hamiltonian with the Lennard-Jones interactions. It is shown that folding is dominated by a well-defined sequencing of events as determined by establishment of particular contacts. The order of events depends primarily on the geometry of the native state. Variations in temperature, coupling strengths and viscosity affect the sequencing scenarios to a rather small extent. The sequencing is strongly correlated with the distance of the contacting aminoacids along the sequence. Thus $\alpha$-helices get established first. Crambin is found to behave like a single-route folder, whereas in CI2 and SH3 the folding trajectories are more diversified. The folding scenarios for CI2 and SH3 are consistent with experimental studies of their transition states.Comment: REVTeX, 12 pages, 11 EPS figures, J. Chem. Phys (in press

Natural history, clinical pattern, and surgical considerations of pneumatosis intestinalis

<p>Abstract</p> <p>Objective</p> <p>Pneumatosis intestinalis has been increasingly detected in recent years with the more frequent use of computed tomography for abdominal imaging of the intestine. The underlying causes of the gas found during radiographic studies of the bowel wall can vary widely and different hypotheses regarding its pathophysiology have been postulated. Pneumatosis intestinalis often represents a benign condition and should not be considered an argument for surgery. However, it can also require life-threatening surgery in some cases, and this can be a difficult decision in some patients.</p> <p>Methods</p> <p>The spectrum of pneumatosis intestinalis is discussed here based on various computed tomographic and surgical findings in patients who presented at our University Medical Centre in 2003-2008. We have also systematically reviewed the literature to establish the current understanding of its aetiology and pathophysiology, and the possible clinical conditions associated with pneumatosis intestinalis and their management.</p> <p>Results</p> <p>Pneumatosis intestinalis is a primary radiographic finding. After its diagnosis, its specific pathogenesis should be ascertained because the appropriate therapy is related to the underlying cause of pneumatosis intestinalis, and this is sometimes difficult to define. Surgical treatment should be considered urgent in symptomatic patients presenting with an acute abdomen, signs of ischemia, or bowel obstruction. In asymptomatic patients with otherwise inconspicuous findings, the underlying disease should be treated first, rather than urgent exploratory surgery considered. Extensive and comprehensive information on the pathophysiology and clinical findings of pneumatosis intestinalis is provided here and is incorporated into a treatment algorithm.</p> <p>Conclusions</p> <p>The information presented here allows a better understanding of the radiographic diagnosis and underlying aetiology of pneumatosis intestinalis, and may facilitate the decision-making process in this context, thus providing fast and adequate therapy to particular patients.</p

The regeneration capacity of the flatworm Macrostomum lignano—on repeated regeneration, rejuvenation, and the minimal size needed for regeneration

The lion’s share of studies on regeneration in Plathelminthes (flatworms) has been so far carried out on a derived taxon of rhabditophorans, the freshwater planarians (Tricladida), and has shown this group’s outstanding regeneration capabilities in detail. Sharing a likely totipotent stem cell system, many other flatworm taxa are capable of regeneration as well. In this paper, we present the regeneration capacity of Macrostomum lignano, a representative of the Macrostomorpha, the basal-most taxon of rhabditophoran flatworms and one of the most basal extant bilaterian protostomes. Amputated or incised transversally, obliquely, and longitudinally at various cutting levels, M. lignano is able to regenerate the anterior-most body part (the rostrum) and any part posterior of the pharynx, but cannot regenerate a head. Repeated regeneration was observed for 29 successive amputations over a period of almost 12 months. Besides adults, also first-day hatchlings and older juveniles were shown to regenerate after transversal cutting. The minimum number of cells required for regeneration in adults (with a total of 25,000 cells) is 4,000, including 160 neoblasts. In hatchlings only 1,500 cells, including 50 neoblasts, are needed for regeneration. The life span of untreated M. lignano was determined to be about 10 months