Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain

In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.     

Somatostatin inhibits dendritic cell responsiveness to Helicobacter pylori

Somatostatin is a regulatory peptide found in abundance in the stomach. We have previously shown that somatostatin is required for IL-4-mediated resolution of Helicobacter pylori gastritis. In the current study, we hypothesize that somatostatin acts directly on antigen-presenting cells in the stomach to lessen the severity of gastritis. To test this hypothesis, we first show that CD11c+ dendritic cells are present in the infected tissue of mice with H. pylori-induced gastritis. Pretreatment of bone marrow-derived dendritic cells with somatostatin results in decreased IL-12 production, and lower splenocyte proliferation induced by H. pylori-stimulated dendritic cells. Furthermore, octreotide, a somatostatin analogue, is more potent than somatostatin in suppressing IL-12 release by H. pylori-stimulated dendritic cells through an NF-kappaB-independent pathway. In addition, IL-4 stimulates somatostatin secretion from dendritic cells. In conclusion, somatostatin inhibits dendritic cell activation by H. pylori; a possible mechanism by which IL-4 mediates resolution of gastritis. We suggest that octreotide may be effective in treating immune-mediated diseases of the stomach.     

Optimization of the dilute maleic acid pretreatment of wheat straw

Background  

In this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620-derived photoprobe

On the basis of the structure of IRL-1620, a specific agonist of the endothelin-B receptor subtype (ET(B)), a few photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing the photoreactive amino acid, p-benzoyl-l-phenylalanine in position 5 showed, as assessed with endothelin-A (ET(A)) and ET(B) receptor paradigms, pharmacological properties very similar to those of IRL-1620. The binding capacity of the probe was also evaluated on transfected Chinese hamster ovary (CHO) cells overexpressing the human ET(B) receptor. Data showed that binding of the radiolabeled peptide was inhibited by ET-1 and IRL-1620. Therefore, this photolabile probe was used to label the ET(B) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 49 kDa. An excess of ET-1 or IRL-1620 completely abolished the formation of the complex, showing the selectivity of the photoprobe. Digestions of the [Bpa(5),Tyr((125)I)(6)]IRL-1620-ET(B) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacts. Results showed that Endo Lys-C digestion gave a 3.8-kDa fragment corresponding to the Asp(274)-Lys(303) segment, whereas migration after V8 digestion revealed a fragment of 4.6 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp(275)-Asp(313) stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.6 kDa, corresponding to Gln(267)-Met(296). Thus, the combined cleavage data strongly suggested that the agonist binding domain of ET(B) includes a portion of the fifth transmembrane domain, between residues Trp(275) and Met(296).     

Monohydroxylated poly(3-hydroxyoctanoate) oligomers and its functionalized derivatives used as macroinitiators in the synthesis of degradable diblock copolyesters

The presence of a hydroxyl group at the end of poly(3-hydroxyoctanoate) oligomers, noted PHO oligomers, is required to prepare diblock copolymers with improved properties by ring-opening polymerization of cyclic monomer as epsilon-caprolactone. Several chemical methods such as basic hydrolysis, acid-catalyzed reaction with APTS, and methanolysis were used to prepare well-defined low molar masses PHO oligomers. The methanolysis reaction was allowed to proceed for 10-60 min to produce PHO oligomers with Mn values ranging from 20,000 to 800 g mol-1 with low polydispersity index. Detailed analysis of the MALDI-TOF mass spectra of the obtained oligomers has revealed the presence of linear structures bearing methyl ester on one side and hydroxyl end group on the other side. The same procedure was applied to poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate), PHOU, a poly(3-hydroxyalkanoate) containing unsaturated units in its side chains. These oligomers were further used to initiate the polymerization of epsilon-caprolactone by varying the PHO (or PHOU) and PCL lengths. By copolymerization with epsilon-caprolactone, the properties of PHO or PHOU have been improved. The crystallinity of the obtained copolymers was modified by controlling the length of the two different blocks. The unsaturations in the side chains of the PHOU block were oxidized in acid carboxylic functions to obtain a novel artificial biopolyester. Moreover, degradation was followed to study the influence of carboxylic groups on the hydrolysis of the copolymers.     

Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.