Influence of stress resilience in adolescence on long-term risk of psoriasis and psoriatic arthritis among men: A prospective register-based cohort study in Sweden.

BACKGROUND: Although stress is considered to be a negative factor for psoriasis, no convincing scientific evidence of this association exists, largely because of difficulties in measuring stress. Stress resilience is the ability to cope with and adapt to stressful events. Stress resilience can be measured in a standardized way and used as a marker for chronic stress. OBJECTIVES: The objective of this study is to investigate whether low stress resilience in adolescence increases the risk for onset of psoriasis and psoriatic arthritis later in life. METHODS: A cohort of Swedish men (mean age 18.3 years), enrolled in compulsory military service between 1968 and 2005, was created using data from the Swedish Military Service Conscription Register (n = 1,669,422). Stress resilience at conscription was estimated using standardized semi-structured interviews, and was divided into three categories: low, medium and high. The men were followed from conscription until new-onset psoriasis or psoriatic arthritis, death or emigration or at the latest until 31 December 2019. Cox regression models adjusted for confounders at conscription were used to obtain hazard ratios (HRs) with 95% confidence intervals (CIs) for incident psoriasis and psoriatic arthritis. RESULTS: Men in the lowest stress resilience category had an increased risk of psoriasis and psoriatic arthritis (HR 1.31 (95% CI 1.26-1.36) and 1.23 (95% CI 1.15-1.32), respectively), compared with those in the highest stress resilience category. When including only hospitalized patients the HRs for psoriasis and psoriatic arthritis in the lowest stress resilience group were 1.79 (1.63-1.98) and 1.53 (1.32-1.77), respectively. CONCLUSIONS: This large, prospective register study suggests that low stress resilience in adolescence is associated with an increased risk of incident psoriasis among men. The results indicate that patients with psoriasis have an inherent psychological vulnerability, and highlight the importance of addressing psychological well-being in the management of psoriasis.

The Role of Water in Fast Plant Movements.

Plants moved onto land ∼450 million years ago and faced their biggest challenge: living in a dry environment. Over the millennia plants have become masters of regulating water flow and the toolkit they have developed has been co-opted to effect rapid movements. Since plants are rooted, these fast movements are used to disperse reproductive propagules including spores, pollen, and seeds. We compare five plants to demonstrate three ways, used alone or in combination, that water powers rapid movements: the direct capture of the kinetic energy of a falling raindrop propels gemmae from the splash cups of the liverwort, Marchantia; the loss of water powers the explosive dispersal of the spores of Sphagnum moss; the alternate loss and gain of water in the bilayer of the elaters of Equisetum drive the walk, jump, and glide of spores; the gain of water in the inner layer of the arils of Oxalis drive the eversion of the aril that jettisons seeds from the capsule; and the buildup of turgor pressure in the petals and stamens of bunchberry dogwood (Cornus canadensis) explosively propels pollen. Each method is accompanied by morphological features, which facilitate water movement as a power source. The urn shaped splash cups of Marchantia allow dispersal of gemmae by multiple splashes. The air gun design of Sphagnum capsules results in a symmetrical impulse creating a vortex ring of spores. The elaters of Equisetum can unfurl while they are dropping from the plant, so that they capture updrafts and glide to new sites. The arils of Oxalis are designed like miniature toy "poppers." Finally, in bunchberry, the softening of stamen filament tissue where it attaches to the anther allows them to function as miniature hinged catapults or trebuchets.

Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers.

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.

Positionally selective growth of embryonic spinal cord neurites on muscle membranes.

Motor neurons from distinct positions along the rostrocaudal axis generally innervate muscles or muscle fibers from corresponding axial levels. These topographic maps of connectivity are partially restored after denervation or transplantation under conditions in which factors of timing and proximity are eliminated. It is therefore likely that motor neurons and some intramuscular structures bear cues that bias synapse formation in favor of positionally matched partners. To localize these cues, we studied outgrowth of neurites from embryonic spinal cord explants on carpets of membranes isolated from perinatal rat muscles. Neurites from rostral (cervical) and caudal (lumbar) spinal cord slices exhibit distinct growth preferences. In many instances, rostrally derived neurites grew selectively on membranes from forelimb muscles or from a single thoracic muscle (the serratus anterior) when given a choice between these membranes and membranes from hindlimb muscles or laminin. Caudally derived neurites almost never exhibited such rostral preferences, but instead preferred membranes from hindlimb muscles or a single hindlimb muscle (the gluteus) to rostral muscles or laminin. Likewise, spinal neurites exhibited distinct position-related preferences for outgrowth on membranes of clonal myogenic cell lines derived from specific rostral and caudal muscles. Taken together these results suggest that the membranes of motor axons and myotubes bear complementary labels that vary with rostrocaudal position and regulate neuromuscular connectivity.

What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes?

Proteinases perform many beneficial functions that are essential to life, but they are also dangerous and must be controlled. Here we focus on one of the control mechanisms: the ubiquitous presence of protein proteinase inhibitors. We deal only with a subset of these: the standard mechanism, canonical protein inhibitors of serine proteinases. Each of the inhibitory domains of such inhibitors has one reactive site peptide bond, which serves all the cognate enzymes as a substrate. The reactive site peptide bond is in a combining loop which has an identical conformation in all inhibitors and in all enzyme-inhibitor complexes. There are at least 18 families of such inhibitors. They all share the conformation of the combining loops but each has its own global three-dimensional structure. Many three-dimensional structures of enzyme-inhibitor complexes were determined. They are frequently used to predict the conformation of substrates in very short-lived enzyme-substrate transition state complexes. Turkey ovomucoid third domain and eglin c have a Leu residue at P(1). In complexes with chymotrypsin, these P(1) Leu residues assume the same conformation. The relative free energies of binding of P(1) Leu (relative to either P(1) Gly or P(1) Ala) are within experimental error, the same for complexes of turkey ovomucoid third domain, eglin c, P(1) Leu variant of bovine pancreatic trypsin inhibitor and of a substrate with chymotrypsin. Therefore, the P(1) Leu conformation in transition state complexes is predictable. In contrast, the conformation of P(1) Lys(+) is strikingly different in the complexes of Lys(18) turkey ovomucoid third domain and of bovine pancreatic trypsin inhibitor with chymotrypsin. The relative free energies of binding are also quite different. Yet, the relative free energies of binding are nearly identical for Lys(+) in turkey ovomucoid third domain and in a substrate, thus allowing us to know the structure of the latter. Similar reasoning is applied to a few other systems.

Effect of single amino acid replacements on the thermodynamics of the reactive site peptide bond hydrolysis in ovomucoid third domain.

We have measured equilibrium constants, Khyd, at pN 6 for the hydrolysis of the reactive site peptide bond (bond between residues 18 and 19) in 42 sequenced variants (39 natural, 3 semisynthetic) of avian ovomucoid third domains. The values range from 0.4 to approximately 35. In 35 cases the effect of a single amino acid replacement on Khyd could be calculated, 13 are without effect and 22 range from a factor of 1.25 to 5.5. Several, but not all, of the effects can be rationalized in terms of residue-residue interactions that are affected by the reactive site hydrolysis. As the measurements are very precise it appears that additional measurements on designed rather than natural variants should allow for the precise measurement of side-chain--side-chain interaction energies.

Refined X-ray crystal structures of the reactive site modified ovomucoid inhibitor third domains from silver pheasant (OMSVP3*) and from Japanese quail (OMJPQ3*).

Tetragonal and triclinic crystals of two ovomucoid inhibitor third domains from silver pheasant and Japanese quail, modified at their reactive site bonds Met18-Glu19 (OMSVP3*) and Lys18-Asp19 (OMJPQ3*), respectively, were obtained. Their molecular and crystal structures were solved using X-ray data to 2.5 A and 1.55 A by means of Patterson search methods using truncated models of the intact (virgin) inhibitors as search models. Both structures were crystallographically refined to R-values of 0.185 and 0.192, respectively, applying an energy restraint reciprocal space refinement procedure. Both modified inhibitors show large deviations from the intact derivatives only in the proteinase binding loops (Pro14 to Arg21) and in the amino-terminal segments (Leu1 to Val6). In the modified inhibitors the residues immediately adjacent to the cleavage site (in particular P2, P1, P1') are mobile and able to adapt to varying crystal environments. The charged end-groups, i.e. Met18 COO- and Glu19 NH3+ in OMSVP3*, and Lys18 COO- and Asp19 NH3+ in OMJPQ3*, do not form ion pairs with one another. The hydrogen bond connecting the side-chains of Thr17 and Glu19 (i.e. residues on either side of the scissile peptide bond) in OMSVP3 is broken in the modified form, and the hydrogen-bond interactions observed in the intact molecules between the Asn33 side-chain and the carbonyl groups of loop residues P2 and P1' are absent or weak in the modified inhibitors. The reactive site cleavage, however, has little effect on specific interactions within the protein scaffold such as the side-chain hydrogen bond between Asp27 and Tyr31 or the side-chain stacking of Tyr20 and Pro22. The conformational differences in the amino-terminal segment Leu1 to Val6 are explained by their ability to move freely, either to associate with segments of symmetry-related molecules under formation of a four-stranded beta-barrel (OMSVP3* and OMJPQ3) or to bind to surrounding molecules. Together with the results given in the accompanying paper, these findings probably explain why Khyd of small protein inhibitors of serine proteinases is generally found to be so small.

Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution.

The molecular structure of the complex between bovine pancreatic alpha-chymotrypsin (EC 3.4.4.5) and the third domain of the Kazal-type ovomucoid from Turkey (OMTKY3) has been determined crystallographically by the molecular replacement method. Restrained-parameter least-squares refinement of the molecular model of the complex has led to a conventional agreement factor R of 0.168 for the 19,466 reflections in the 1.8 A (1 A = 0.1 nm) resolution shell [I greater than or equal to sigma (I)]. The reactive site loop of OMTKY3, from Lys13I to Arg21I (I indicates inhibitor), is highly complementary to the surface of alpha-chymotrypsin in the complex. A total of 13 residues on the inhibitor make 113 contacts of less than 4.0 A with 21 residues of the enzyme. A short contact (2.95 A) from O gamma of Ser195 to the carbonyl-carbon atom of the scissile bond between Leu18I and Glu19I is present; in spite of it, this peptide remains planar and undistorted. Analysis of the interactions of the inhibitor with chymotrypsin explains the enhanced specificity that chymotrypsin has for P'3 arginine residues. There is a water-mediated ion pair between the guanidinium group on this residue and the carboxylate of Asp64. Comparison of the structure of the alpha-chymotrypsin portion of this complex with the several structures of alpha and gamma-chymotrypsin in the uncomplexed form shows a high degree of structural equivalence (root-mean-square deviation of the 234 common alpha-carbon atoms averages 0.38 A). Significant differences occur mainly in two regions Lys36 to Phe39 and Ser75 to Lys79. Among the 21 residues that are in contact with the ovomucoid domain, only Phe39 and Tyr146 change their conformations significantly as a result of forming the complex. Comparison of the structure of the OMTKY3 domain in this complex to that of the same inhibitor bound to a serine proteinase from Streptomyces griseus (SGPB) shows a central core of 44 amino acids (the central alpha-helix and flanking small 3-stranded beta-sheet) that have alpha-carbon atoms fitting to within 1.0 A (root-mean-square deviation of 0.45 A) whereas the residues of the reactive-site loop differ in position by up to 1.9 A (C alpha of Leu18I). The ovomucoid domain has a built-in conformational flexibility that allows it to adapt to the active sites of different enzymes. A comparison of the SGPB and alpha-chymotrypsin molecules is made and the water molecules bound at the inhibitor-enzyme interface in both complexes are analysed for similarities and differences.

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I.

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.

Binding of amino acid side-chains to S1 cavities of serine proteinases.

The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (+/-2) degrees C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1, cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin A alpha (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 10(6) to 10(8). The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu- and Asp- are strongly unfavorable. The Pearson pro duct moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (-0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.

Strategies for Early Vaccination During Novel Influenza Outbreaks.

Ongoing research and technology developments hold the promise of rapid production and large-scale deployment of strain-specific or cross-protective vaccines for novel influenza viruses. We sought to investigate the impact of early vaccination on age-specific attack rates and evaluate the outcomes of different vaccination strategies that are influenced by the level of single or two-dose vaccine-induced protections. We developed and parameterized an agent-based model for two population demographics of urban and remote areas in Canada. Our results demonstrate that there is a time period before and after the onset of epidemic, during which the outcomes of vaccination strategies may differ significantly and are highly influenced by demographic characteristics. For the urban population, attack rates were lowest for children younger than 5 years of age in all vaccination strategies. However, for the remote population, the lowest attack rates were obtained for adults older than 50 years of age in most strategies. We found that the reduction of attack rates following the start of vaccination campaigns during the epidemic depends critically on the disease transmissibility, suggesting that for a sufficiently high transmissibility, vaccine delivery after the onset of epidemic has little or no effect, regardless of the population demographics.

Water molecules participate in proteinase-inhibitor interactions: crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B.

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water-mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol-1 A-2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site-directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein-complex stability.

Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues.

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. beta-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of gamma-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6-18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19-56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive-site peptide bond X18-Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive-site-intact inhibitor variants.

Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB.

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three beta-branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 A resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 A resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The beta-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type gamma-branched P1 residue for OMTKY3, Leu18I. Chi1 angles of approximately 40 degrees are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60 degrees, -60 degrees, or 180 degrees are more commonly observed but 40 degrees is not unfavorable for the beta-branched side chains. Thr18I Ogamma1 also forms a hydrogen bond with Ser195 Ogamma in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.

Imidazole myopathy. Production of the myopathy and its dependence on acetylcholine.

An acetylcholine-mediated myopathy has been produced in the soleus muscle of the rat by the daily injection of imidazole, a compound that accelerates the metabolism of adenosine 3':5' cyclic phosphate by activating the enzyme phosphodiesterase. The imidazole-treated muscles were found to have a lowered resting membrane potential. This study suggests that a decrease in resting membrane potential may make skeletal muscle more vulnerable to necrosis by acetylcholine released during normal activity.

Expression and characterization of elastase inhibitors from the ascarid nematodes Anisakis simplex and Ascaris suum.

Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex, have been isolated and characterized. Because these inhibitors are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679-689). To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that: (1) the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor (Ka = 4.5 x 10(9) and 6.4 x 10(9) M(-1), respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (Ka = 1.6 x 10(9) M(-1)); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (Ka = 1.2 x 10(6) M(-1)). In contrast to the Anisakis inhibitor, however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE (Ka = 3.5 x 10(10) M(-1)) and CHYM (Ka = 3.6 x 10(12) M(-1)). We also found that the determined reactive sites (P1-P'1) of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P'1 residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P1 and P'3 residues in the inhibitors. Elastase, which recognizes both alanine and leucine, canaccommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1.

Biochemical and ultrastructural changes in skeletal muscle induced by a creatine antagonist.

To evaluate the essentiality of creatine and phosphocreatine for the maintenance of the ultrastructure of skeletal muscle, chicks were fed a creatine antagonist, beta-guanidinobutyric acid (beta-GBA), as 2% of a Chow diet. Chicks fed beta-GBA exhibited growth retardation and weakness, and they accumulated large amounts of a monosubstituted guanidino compound, presumably beta-GBA, in their skeletal muscles. After 2 wk, there was a 74% decrease in the uptake of [14C]-1-creatine into pectoralis muscles of chicks fed beta-GBA. After 2 wk there as a significant decrease in phosphocreatine concentrations in pectoralis muscles from 20.1 +/- 2.8 mumoles per g wet weight (mean +/- S.D.) for 8 control chicks to 16.5 +/- 2.5 for 7 chicks fed beta-GBA. Selected fibers of the pectoralis and gastrocnemius muscles of chicks fed beta-GBA exhibited ultrastructural abnormalities including loss of thick and thin filaments, disruption of the Z band, dilated mitochondria, and dilated and displaced sarcoplasmic reticulum. The pectoralis muscles of chicks given 6% creatine in addition to 2% beta-GBA in the diet accumulated little beta-GBA, maintained normal phosphocreatine concentrations, and exhibited no significant ultrastructural abnormalities. These findings are the first experimental evidence that high concentrations of phosphocreatine are essential for the maintenance of the ultrastructural integrity of skeletal muscle.

Brain motilin-like peptides are localized within the cell nucleus.

The subcellular localization of motilin-like immunoreactive (MLIP) peptides in comparison to vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine amide-27-like peptide (PLP) was investigated in rat brain applying different subcellular fractionation techniques. Unlike VIP or PLP, motilin-like peptides were not located in synaptosomes, but in the cell nucleus. This is the first report of a non-vesicular localization of this neuropeptide and is suggestive of a possible non-neurotransmitter role of MLIP. Previous developmental studies point to a possible role for motilin-like peptides as trophic or developmental factors. These results open the possibility that brain motilin-like peptides may operate by binding to chromatin and regulating gene expression.

A morphological technique for exploring neuromuscular topography expressed in the mouse gluteus maximus muscle.

Motor neuron pools innervate muscle fibers forming an ordered topographic map. In the gluteus maximus (GM) muscle, as well as additional muscles, we and others have demonstrated electrophysiologically that there exists a rostrocaudal distribution of axon terminals on the muscle surface. The role of muscle fiber type in determining this topography is unknown. A morphological approach was designed to investigate this question directly. We combined three different methods in the same muscle preparation: (1) the uptake of activity-dependent dyes into selected axon terminals to define the spinal segmental origin of a peripheral nerve terminal; (2) the fluorescent labeling of nicotinic acetylcholine receptors to determine motor endplate size; (3) the immunocytochemical staining of skeletal muscle to determine fiber subtype. We applied these methods to the mouse GM muscle to determine the relationship between muscle fiber type and the topographic map of the inferior gluteal nerve (IGN). Results from this unique combination of techniques in the same preparation showed that axon terminals from more rostral spinal nerve segments of origin are larger on rostral muscle fibers expressing myosin heavy chain (MyHC) IIB epitope than caudal type IIB fibers. Because type IIB fibers dominate the GM, this suggests that for these rostral axons terminal size is independent of fiber type. How this axon terminal size is related to the topographic map is the next question to be answered.