Influence of adrenal demedullation on stress-related behaviour in Wistar rats

The influence of adrenal demedullation on stress-related behaviour (chronic immobilization) was investigated in respect to the following parameters and physiological processes: number of animals surviving the exposure, blood pressure behaviour, endogenous opioid system, pain sensitivity, endogenous level of substance P (SP) in pituitary and hypothalamus. The results showed that chronic immobilization stress and adrenal demedullation induce the same direction of alterations of physiological processes and parameters. This fact suggests the involvement of common regulatory mechanisms both in demedullation and chronic stress. An important process in this respect may be the induction of functional insufficiency of the adrenal medulla especially concerning the adrenal peptidergic mechanisms. Contrary to the sham operated rats the demedullated animals showed differences in their stress-related behaviour of parameters of adaptive regulatory processes. In rats with demedullation either no additional stress-related alterations of parameters in relation to the general adaptation processes were induced or the demedullation-induced alterations were antagonized partially or completely by chronic immobilization.     

Post-feeding hyperammonaemia in patients with transjugular intrahepatic portosystemic shunt and liver cirrhosis: role of small intestinal ammonia release and route of nutrient administration

BACKGROUND: Hyperammonaemia is a pathogenetic factor for hepatic encephalopathy that may be augmented after a transjugular intrahepatic portosystemic shunt (TIPS). Experimental data suggest that hyperammonaemia may be caused to a large extent by metabolism of small intestinal enterocytes rather than colonic bacteria. AIMS: To evaluate if ammonia release and glutamine metabolism by small intestinal mucosa contribute to hyperammonaemia in vivo in patients with liver cirrhosis. METHODS: Using TIPS to examine mesenteric venous blood, we measured mesenteric venous-arterial concentration differences in ammonia and glutamine in patients with liver cirrhosis before, during, and after enteral (n = 8) or parenteral (n = 8) isonitrogenous infusion of a glutamine containing amino acid solution. RESULTS: During enteral nutrient infusion, ammonia release increased rapidly compared with the post-absorptive state (65 (58-73) v. 107 (95-119) micromol/l after 15 min; mean (95% confidence interval)) in contrast with parenteral infusion (50 (41-59) v. 62 (47-77) micromol/l). This resulted in a higher portal ammonia load (29 (21-36) v. 14 (8-21) mmol/l/240 minutes) and a higher degree of systemic hyperammonaemia (14 (11-17) v. 9 (6-12) mmol/l/240 minutes) during enteral than parenteral infusion. The mesenteric venous-arterial concentration difference in glutamine changed from net uptake to release at the end of the enteral infusion period (-100 (-58 to -141) v. 31 (-47-110) micromol/l) with no change during parenteral nutrition. CONCLUSIONS: These data suggest that small intestinal metabolism contributes to post-feeding hyperammonaemia in patients with cirrhosis. When artificial nutrition is required, parenteral nutrition may be superior to enteral nutrition in patients with portosystemic shunting because of the lower degree of systemic hyperammonaemia.     

Bidirectional binding of invariant chain peptides to an MHC class II molecule

T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.     

Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor (Kd=120 nM,MW=734 Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

Metastasis is a complex multistep process (1, 2) employing, among others, mechanisms governing actin cytoskeleton dynamics involving integrin signaling and actin regulatory proteins (3–5). So far, all approved antimetastatic drugs antagonize integrins (6) or inhibit downstream kinases (7, 8) (SI Appendix, Fig. S1). In the metastatic setting however, these drugs appear to have only limited success (9–13) and 5-y survival is not increasing satisfactorily (14, 15), making new approaches in antimetastatic drug development essential to meet this urgent medical need.The enabled/vasodilator stimulated phosphoprotein protein family (Ena/VASP) acts as a crucial hub in cell migration by linking actin filaments to invadopodia and focal adhesions (16–22). Due to their role in the transformation of benign lesions into invasive and metastatic cancer, Ena/VASP proteins are discussed as part of the invasive signature and as a marker of breast carcinogenesis (23–25). At the advanced tumor stage, the protein family is overexpressed (26–28), which has been shown to increase migration speed in vivo (29) and to potentiate invasiveness (30). Yet, no sufficiently potent probes to interfere with Ena/VASP in vivo have been reported.The three vertebrate Ena/VASP family members, enabled homolog (ENAH), VASP, and Ena-VASP-like (EVL), share a tripartite structural organization in which two Ena/VASP homology domains (EVH1 and EVH2) are separated by a more divergent proline-rich central part. Interactions of the EVH2 domain are involved in the elongation and protection of barbed-end actin filaments from capping proteins and tetramerization (31, 32). EVH1 folds into a structured globular domain that interacts with proteins at focal adhesions (33), the leading edge (34, 35), and invadopodia (36, 37) by recognizing the motif [F/W/L/Y]PxϕP (35, 38) (ϕ hydrophobic, x any; SI Appendix, Fig. S3) in poly-L-proline type II helix (PPII) conformation.In the course of our research into small molecules as potential inhibitors of protein–protein interactions (39) we recently in silico designed and stereo-selectively synthesized scaffolds, coined ProMs, which mimic pairs of prolines in PPII conformation (40). The modular combination of different ProMs thereby allowed us to generate nonpeptidic secondary-structure mimetics that fulfill the steric requirements of the addressed proline-rich motif-recognizing domain (41–47). For the EVH1 domain, our proof-of-concept study yielded a canonically binding, nontoxic, cell-membrane-permeable, 706-Da inhibitor 1 (Fig. 1A) composed of two different ProM scaffolds and 2-chloro-(L)-phenylalanine (2-Cl-Phe) (40). While the synthetic inhibitor 1 represents the compound with the highest reported affinity toward Ena/VASP EVH1 domains, a further improvement was required for in vivo experiments. Here we report successful structure-based optimization of inhibitor 1 based on 22 high-resolution crystal structures of ENAH EVH1 in complex with different inhibitors (SI Appendix, Tables S1–S6), including the well-resolved C-terminal binding epitope TEDEL of ActA from Listeria monocytogenes (48). Newly identified interaction sites adjacent to the C terminus of 1 were addressed by in silico designed and stereo-selectively synthesized modifications of the ProM-1 scaffold (Fig. 1A). While drastically increasing the affinity against a rather flat protein surface we conserved structural simplicity, low molecular weight, nontoxicity, and cell-membrane permeability. Potent compounds against Ena/VASP were shown to also act in vivo, i.e., by inhibiting cancer cell extravasation in zebrafish at only 1 μM, thereby paving the way for future preclinical studies.Open in a separate windowFig. 1.(A) Structure of the first-generation Ena/VASP EVH1 inhibitor 1. All compositions share the N-acetylated 2-chloro-phenylalanine unit (blue) attached to a central ProM-2 scaffold (red). Esterification of the C terminus renders the inhibitors cell-membrane permeable (40). (B) General (modular) architecture of nonpeptidic, conformationally preorganized inhibitors used in this study. Structural variation (pink) was achieved by replacing the C-terminal ProM-1 unit (green) by ProM-9, ProM-13, ProM-12, ProM-15, or ProM-17 (Table 1).     

Saliva control issues in the neurologically challenged. A 30 year experience in team management

OBJECTIVE: The objective of this paper is to report the experience of a multidisciplinary team (otolaryngologist, speech-language pathologists, pediatric dentist, and social worker) functioning at a pediatric rehabilitation center have had in the management of 1487 neurologically challenged individuals with saliva control issues assessed from 1976 to the end of 2004. METHOD: The role of each team member is outlined. Management decisions have consisted of no treatment, utilization of oral-motor training program, elimination of contributing situational factors, utilization of medication, surgery and Botox injections of the major salivary glands. RESULTS: Oral-motor programs to improve oral-motor function is essential initially if there is patient compliance. Surgery was recommended in the majority of patients. Submandibular duct relocation with simultaneous sublingual gland excision remains the procedure of first choice for persistent significant drooling. Aspiration is much less common than drooling and is more problematic to treat. Simultaneous ligation of the submandibular and parotid ducts (aka 4-duct ligation) is promising. A recent initiative to inject the major salivary glands with Botox is being evaluated. CONCLUSIONS: Team evaluation with consensus decision making has worked well in this patient population with complex medical conditions.     

Endogenous opioid dependence--a basic pathophysiological phenomenon of stress-induced and genetically fixed disturbances in adaptation--influence of substance P.

Disturbances in adaptive processes can be induced by chronic exposition to stress or can result from a genetical predisposition. Experimental data of chronically stressed Wistar rats and of spontaneously hypertensive rats (SHR) demonstrate a relation between a decreased level of substance P (SP) in adrenals, the existence of a dependence on endogenous opioid peptides and an increased regulatory level of blood pressure. The endogenous level of SP was determined by using a RIA. The dependence (physical) on endogenous opioid peptides was detected by using the method of "gut dependence". SP injection i.p. once a day for 4 d antagonized the dependence on endogenous opioid peptides and normalized the increased level of blood pressure in both animal models. Investigations on SHR had shown that the adaptive effect of SP on blood pressure and endogenous opioid dependence is bound to the premise of an acute stimulated endogenous opioid system at the moment of SP-application. Experimental findings suggest that different systems of opioid peptides take part in the etiopathogenesis of genetically predisposed hypertension of SHR and in stress-induced increase of blood pressure level of Wistar rats. The effect of SP on blood pressure and endogenous opioid dependence will be discussed as a result of the modulatory influence on the cholinergic-opioid-peptidergic interaction.     

Effect of substance P on blood pressure in rats, with and without hypokinetic stress

The effect of substance P (SP) on blood pressure was studied in normotensive rats and in rats with hypokinesia-induced hypertension. To characterize the SP effect, the influence on the blood pressure value, intra-individual variation and interindividual changes was investigated. Whereas SP corrected the hypokinesia-induced changes (increased blood pressure, increase in the intra-individual blood pressure variations) in the stressed animals, no SP effect was observed in the normotensive control rats. These findings are discussed with a view to the assumed ability of SP to act as a regulatory peptide (regulide).     

Weight gain in long-term survivors of kidney or liver transplantation—Another paradigm of sarcopenic obesity?

ObjectiveObesity in transplant recipients is a frequent phenomenon but data from body composition analyses in long-term survivors are limited. Body composition and energy metabolism were studied in patients after liver (LTX) and kidney (KTX) transplantation and patients with liver cirrhosis (LCI) or on chronic hemodialysis (HD) and compared to healthy controls.MethodsIn 42 patients 50.0 mo (median; range 17.1–100.6) after LTX and 30 patients 93.0 mo (31.2–180.1) after KTX as wells as in LCI (n = 39) or HD (n = 10) patients mid-arm muscle and fat area, body cell mass, and phase angle (bioimpedance analysis), and resting energy expenditure (indirect calorimetry, REE_CALO) were measured.ResultsObesity was more prevalent in LTX (17%) than LCI (3%) and in KTX (27%) than in HD (10%). In LTX and KTX, phase angle was higher than in end-stage disease (LTX 5.6° [4.1–7.2] versus LCI 4.4° [2.9–7.3], P < 0.001; KTX 5.9° [4.4–8.7] versus HD 4.3° [2.9–6.8]) but was lower in all patient groups than in controls (7.1°; 4.6–8.9; P < 0.001). In LCI and HD REE_CALO was higher than predicted, while in LTX and KTX REE_CALO was not different from predicted REE.ConclusionsDespite excellent graft function, many long-term LTX or KTX survivors exhibit a phenotype of sarcopenic obesity with increased fat but low muscle mass. This abnormal body composition is observed despite normalization of the hypermetabolism found in chronic disease and cannot be explained by overeating. The role of appropriate nutrition and physiotherapy after transplantation merits further investigation.     

Über den Einfluß der Blutentziehung und der Bluttransfusion auf den Stickstoffstoffwechsel

Zusammenfassung Kleinere, mittlere und auch größere Blutentziehungen beeinflussen die Stickstoffbilanz, und zwar im positiven Sinne. Besonders bemerkenswert ist die längere Zeit andauernde Nachwirkung des ausgeführten Eingriffes.Inhaber eines Stipendiums der Rockefeller-Stiftung.