Design and characterization of a hybrid miniprotein that specifically inhibits porcine pancreatic elastase

Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.     

Thermal acclimation, growth, and burst swimming of threespine stickleback: enzymatic correlates and influence of photoperiod

Threespine sticklebacks (Gasterosteus aculeatus) that had been reared in the laboratory under natural photoperiods were acclimated to 23 degrees and 8 degrees C in late spring under increasing day lengths and again in late fall under decreasing day lengths. The parents of these fish were from the anadromous Isle Verte population. In the spring, cold- and warm-acclimated fish grew at the same rates and attained similar condition factors (mass L(-3)), although food intake was considerably higher at 23 degrees C. As both groups had similar increases in mass and condition, the higher axial muscle activities of citrate synthase and phosphofructokinase (measured at 20 degrees C) after cold acclimation were likely a direct response to temperature. Multiple regression analysis showed that axial muscle levels of cytochrome C oxidase and citrate synthase were correlated with the burst swimming speeds of the spring sticklebacks, while growth rates were positively correlated with lactate dehydrogenase levels in pectoral and axial muscles and creatine kinase levels in the axial muscle. In the fall, the fish in both acclimation groups grew little, although they fed at similar rates as in the spring experiment. Overall, the sticklebacks showed lower burst swimming speeds in the fall. In both spring and fall, the burst speeds of cold- and warm-acclimated sticklebacks only differed at warm temperatures. In the spring experiment, the cold-acclimated fish swam faster, whereas in the fall experiment the warm-acclimated fish swam faster despite their lower percentage of axial muscle. Swimming speeds were measured both at a fish's acclimation temperature and after 12 h at the other temperature. Cold-acclimated sticklebacks seem to have more facility in rapidly adjusting to warm temperatures when they have experienced increasing rather than decreasing day lengths, perhaps as a result of the requirements of the spring migration to the intertidal breeding grounds.     

High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction (SNiPTag)

Single nucleotide polymorphisms (SNPs) are becoming widely recognized as the new currency for gene mapping as increasing numbers are discovered. Here we outline a method for their rapid analysis based on an allele-specific polymerase chain reaction (PCR) which employs a competitive approach, whereby both allele-specific primers are present in the same reaction and carry different fluorescent labels. This procedure is simple and amenable to high-throughput genotyping using conventional automated sequencing equipment, and no post-PCR modifications are required. Verification of the procedure was carried out by comparison of results derived by this method with those from restriction enzyme digestion of the ALDH2 exon 12 functional polymorphism (Glu-487-Lys) in 109 individuals. Additionally, we have examined all combinations of nucleotide substitutions and shown them to be differentiated by this method. As proof of concept, several assays were combined and loaded on a single gel lane/capillary to substantially improve throughput. This was made possible by designing the PCR products to be of different lengths and no interference was observed between products differing in size by only six nucleotides. We outline a number of test assays for well-characterized SNPs in human candidate genes for behavioral disorders.     

Alterations in metabolism and gene expression in brain regions during cuprizone-induced demyelination and remyelination

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down-regulated during demyelination and up-regulated during remyelination. Genes related to microglia/macrophages appeared in a time-course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.     

Hormonal and behavioral responses of starlings during a confrontation with males or females at nest boxes during the reproductive season

We investigated in an aviary experiment the behavioral and hormonal responses of European starlings (Sturnus vulgaris) that were moved from a same sex group to an aviary containing either a nest box alone, a nest box and another male, or a nest box and a female. Luteinizing hormone (LH) and testosterone (T) levels increased significantly and independently of the situation, suggesting that nest boxes were the most important stimulus affecting the levels of these hormones. Some birds occupied more than two boxes (winners), and others a single or no box (losers). Levels of T increased less in males that did not acquire a nest box. However, the increase in LH was similar in all males after the test. Singing was positively correlated with T levels. Winners started singing earlier and sang more during a contest than losers. In the presence of females LH increased more in winners than in losers, while the increase in T was similar in both groups. In females, there was no increase in T but LH increased in the presence of males. Levels were higher in females paired with winners than in females paired with losers. Finally, winners advertised their nest boxes more frequently than losers. These results indicate that within a relatively short time frame levels of LH and T increase following the transfer from a flock to a territorial situation and can react independently from each other depending on reproductive circumstances. For males, the possession of a nest box and, for females, the qualities of the male seemed to be the most important factors stimulating reproduction.     

TGF-beta 1 regulates adhesion of mucosal mast cell homologues to laminin-1 through expression of integrin alpha 7

Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-beta1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-beta1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 +/- 3.6% (mean +/- SEM) in the absence of TGF-beta1 to 58.7 +/- 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-beta1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcepsilonRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin alpha(7) by MMC homologues grown in the presence of TGF-beta1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the alpha(7)-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1.     

Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones

The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones.     

Effect of reproduction on escape responses and muscle metabolic capacities in the scallop Chlamys islandica Müller 1776

In scallops, gametogenesis leads to mobilization of glycogen and proteins from the adductor muscle towards the gonad. This mobilization is likely to diminish the metabolic capacities of the adductor muscle and thereby the scallops' escape response. We examined the escape response in terms of number of valve claps until exhaustion, rate of clapping and the recovery during and after valve closure in adult scallops, Chlamys islandica, sampled at different stages in the reproductive cycle (immature, mature, before and after spawning). In parallel, we measured muscle glycogen, protein and phosphoarginine contents, the oxidative capacity of mitochondria isolated from the adductor muscle and levels of muscle enzymes which are active during exercise and recovery. The number of claps (24-26), rate of clapping ( approximately 13 clapsmin(-1)) and phosphoarginine and arginine kinase levels were similar during the different reproductive stages. All immature scallops responded to restimulation immediately after opening their valves, while only 62% of mature, 82% of prespawned and 38% of spawned scallops responded. Immature animals completely recovered their initial swimming capacity within 4 h of opening their valves, but mature, prespawned and spawned scallops needed 18, 12 and 18 h, respectively. Overall phasic adductor muscle from mature, prespawned and spawned animals showed decreased glycogen phosphorylase, phosphofructokinase, pyruvate kinase (except for prespawned), octopine dehydrogenase and citrate synthase levels, a deterioration of the oxidative capacity of mitochondria and a marked decrease in glycogen content compared to immature scallops. Therefore, during gonadal maturation and spawning, C. islandica did not change its clapping capacity, but slowed its recuperation from exhausting burst exercise, both during and after valve closure, likely due to the decreased metabolic capacity of the adductor muscle.     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

PCR Detection of Coxiella burnetii from Different Clinical Specimens, Especially Bovine Milk, on the Basis of DNA Preparation with a Silica Matrix

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.