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.

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.

Despite having a common P1 Leu, eglin C inhibits alpha-lytic proteinase a million-fold more strongly than does turkey ovomucoid third domain.

Results of the inhibition of alpha-lytic proteinase by two standard mechanism serine proteinase inhibitors, turkey ovomucoid third domain (OMTKY3) and eglin C, and many of their variants are presented. Despite similarities, including an identical P1 residue (Leu) in their primary contact regions, OMTKY3 and eglin C have vastly different association equilibrium constants toward alpha-lytic proteinase, with Ka values of 1.8 x 10(3) and 1.2 x 10(9) M(-1), respectively. Although 12 of the 13 serine proteinases tested in our laboratory for inhibition by OMTKY3 and eglin C are more strongly inhibited by the latter, the million-fold difference observed here with alpha-lytic proteinase is the largest we have seen. The million-fold stronger inhibition by eglin C is retained when the Ka values of the P1 Gly, Ala, Ser, and Ile variants of OMTKY3 and eglin C are compared. Despite the small size of the S1 pocket in alpha-lytic proteinase, interscaffolding additivity for OMTKY3 and eglin C holds well for the four P1 residues tested here. To better understand this difference, we measured Ka values for other OMTKY3 variants, including some that had residues elsewhere in their contact region that corresponded to those of eglin C. Assuming intrascaffolding additivity and using the Ka values obtained for OMTKY3 variants, we designed an OMTKY3-based inhibitor of alpha-lytic proteinase that was predicted to inhibit 10,000-fold more strongly than wild-type OMTKY3. This variant (K13A/P14E/L18A/R21T/N36D OMTKY3) was prepared, and its Ka value was measured against alpha-lytic proteinase. The measured Ka value was in excellent agreement with the predicted one (1.1 x 10(7) and 2.0 x 10(7) M(-1), respectively). Computational protein docking results are consistent with the view that the backbone conformation of eglin C is not significantly altered in the complex with alpha-lytic proteinase. They also show that the strong binding for eglin C correlates well with more favorable atomic contact energy and desolvation energy contributions as compared to OMTKY3.

Morphometric analysis of neuromuscular topography in the serratus anterior muscle.

Groups of neurons form ordered topographic maps on their targets, and defining the mechanisms that develop such maps, and re-connect them after disruption, has biological as well as clinical importance. The neuromuscular system is an accessible and well-studied model for defining the principles that guide map formation, both during its development and its reformation after motor nerve damage. We present evidence for the expression of this map at the level of nerve terminal morphology and muscle fiber type in the serratus anterior muscle. Morphometric analyses indicate, first, a rostrocaudal difference in nerve terminal size depending on the ventral root of origin of the axons. Second, motor endplates are larger on type IIB than type IIA muscle fibers. Third, whereas IIB muscle fibers are distributed rather evenly along the rostrocaudal axis of the muscle, the more rostral type IIB fibers are preferentially innervated by anteriorly derived (C6) motor neurons, and more caudal IIB fibers are preferentially innervated by posteriorly derived (C7) motor neurons. This inference is supported by analysis of the size of nerve terminals formed in each muscle sector by rostral and caudal roots, and by evidence that the larger terminals are on IIB fibers. These results demonstrate a subcellular expression of neuromuscular topography in the serratus anterior muscle (SA) muscle in the form of differences in nerve terminal size. These results provide deeper insights into the organization of a neuromuscular system. They also offer a rationale for a topographic map, that is, to allow spinal motor centers to activate selectively different compartments within a muscle.

Ephrin-A5 overexpression degrades topographic specificity in the mouse gluteus maximus muscle.

Motor neurons project onto specific muscles with a distinct positional bias. We have previously shown using electrophysiological techniques that overexpression of ephrin-A5 degrades this topographic map. Here, we show that positional differences in axon terminal areas, an entirely different parameter of neuromuscular topography, are also eliminated with ephrin-A5 overexpression. Therefore, we now have both morphological and electrophysiological approaches to explore the mechanisms of neuromuscular topography.

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.

Denervation and age modify neuromuscular positional selectivity.

The rostrocaudal position of neurons within the spinal motor pool maps systematically onto the surface of several muscles in mammals. In an effort to understand the mechanisms that generate such maps, we have been studying choices made by embryonic spinal cord neurons on muscle membrane substrates in the in vitro stripe assay. In this report we explore the effects of postnatal age of the muscle on neurite choice, and how prior denervation modifies this choice. Our results further differentiate rostral from caudal motor neurons in preferring one substrate to another. First, caudal neurites prefer to grow on P6 neonatal caudal over rostral membranes, but lose this ability to distinguish axial position of origin in older muscles. Rostral neurites prefer growth on rostral membranes, but this preference also diminishes with age. Second, when adult muscles have been denervated, both rostral and caudal neurites regain their positional growth selectivity. Third, caudal neurites are particularly sensitive to substrate choice. When growing on a preferred substrate (gluteus) caudal neurites prefer neonatal over adult membranes. These results support the concept of fundamental differences in the growth preferences of rostral and caudal spinal neurites. These differences will assist in the identification of molecular guidance cues that determine the formation of neuromuscular positional maps.

Development of inhibition by ephrin-A5 on outgrowth of embryonic spinal motor neurites.

The spinal motor pool maps systematically onto the surface of muscles. This map is detectable in rat embryonic muscles, and is partially restored after reinnervation. Recent evidence shows that either overexpression or deletion of the ephrin-A5 gene significantly disrupts the map, suggesting that ephrin-A5 plays a critical role in the formation of this topography. Several studies have demonstrated that ephrin-A5 is a repulsive molecule in the nervous system, including the neuromuscular system. To examine the development of sensitivity of ventral spinal axons to this inhibitory ligand, slices of E11 to E15 embryonic rat spinal cords were cocultured with membranes derived from ephrin-A5-expressing cell lines. We detected a progressive expression of inhibition by ephrin-A5 between E11 and E15. By E15, rostral and caudal spinal neurites showed clear differences in responsiveness to the ephrin-A5 ligand. Further, we found that at this age caudal neurites are more sensitive to changes of ephrin-A5 concentration along a gradient. In addition, growth cones of caudal, more than rostral, neurites tended to assume a collapsed shape in the presence of the ligand. These results demonstrate a progressive development of sensitivity to ephrin-A5, and suggest a divergence in this sensitivity between rostral and caudal spinal cord neurites. These results provide further insight into how subtle rostrocaudal differences in the development of sensitivity to ephrin-A5 may explain, in part, neuromuscular topography.

Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases.

An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

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.

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.

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.

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.

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.

Thermodynamic criterion for the conformation of P1 residues of substrates and of inhibitors in complexes with serine proteinases.

Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the +/-400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aalpha with eglin c () and with turkey ovomucoid third domain (). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) ( and ) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeply inserted into the enzyme's specificity pocket as it is in the (Lys+)18 turkey ovomucoid third domain complex with chymotrypsin.

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.

Synaptic competition during the reformation of a neuromuscular map.

We have been studying the mechanisms whereby pools of motor neurons establish a rostrocaudal bias in the position of their synapses in some skeletal muscles. The serratus anterior (SA) muscle of the rat displays a rostrocaudal topographic map before birth, and the topography is re-established after denervation. In this report, we explore the potential role of synaptic competition between innervating axons as a means of generating topographic specificity. We followed the progress of the reformation of this map in neonatal animals under conditions that enhanced the likelihood of observing synaptic competition. This was accomplished by forcing caudal axons to regenerate ahead of rostral axons onto a surgically reduced SA muscle. In this way, caudal (C7) motor neurons had unopposed access to vacated synaptic sites on the remaining rostral half of the SA before the return of the rostral (C6) axons. Intracellular recording revealed that 2 d after the second denervation, most of the reinnervated end plates contained only axons from the C7 branch; the remaining reinnervated end plates received input from C6 only or were multiply innervated by C6 and C7 axons. After 6 d, the pattern was reversed, with most end plates innervated exclusively by C6. After 17 d, axons from C6 were the sole input to reinnervated end plates. During the transition from C7- to C6-dominated input, at end plates coinnervated by C6 and C7 axons, the average quantal content from C6 was the same as that from C7; after 7 d, the quantal content of C6 was greater than that of C7. We have thus developed an experimental situation in which the outcome of synaptic competition is predictable and can be influenced by the positional labels associated with axons from different levels in the spinal cord.

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.

Interscaffolding additivity. Association of P1 variants of eglin c and of turkey ovomucoid third domain with serine proteinases.

Standard mechanism protein inhibitors of serine proteinases share a common mechanism of interaction with their cognate enzymes. The P1 residue of the inhibitor interacts with the enzyme in a substrate-like manner. Its side chain becomes imbedded in the S1 cavity of the enzyme. The nature of P1, the primary specificity residue, greatly affects the strength and specificity of the enzyme inhibitor association. In canonical inhibitors, residues P4-P2'(P3'), where P1-P1' is the reactive site, share a common main chain conformation that does not change on complex formation. The remainder of the inhibitor's structure, the scaffolding, is not always common. Instead, there are at least 20 inhibitor families, each with a different scaffolding. In this paper, we ask whether the differences in standard free energy of association of enzyme-inhibitor complexes upon P1 mutations are independent of the nature of the scaffolding. We have already reported on 25 P1 variants of turkey ovomucoid third domain, a member of the Kazal inhibitor family, interacting with six different serine proteinases. Here, we report on seven different P1 variants of eglin c, a potato I family member, interacting with the same six serine proteinases under the same conditions. The differences in standard free energy on P1 mutations in the eglin c system agree very well, when P1 Pro is omitted. Complete agreement indicates that these P1 residues are interscaffolding additive. This is consistent with the superimposition of the high-resolution structures of eglin c and of turkey ovomucoid third domain with chymotrypsin. In both cases, the P1 Leu side chain is similarly oriented in almost indistinguishable specificity pockets of the enzyme.