Spider capture silk: performance implications of variation in an exceptional biomaterial

Spiders and their silk are an excellent system for connecting the properties of biological materials to organismal ecology. Orb-weaving spiders spin sticky capture threads that are moderately strong but exceptionally extensible, resulting in fibers that can absorb remarkable amounts of energy. These tough fibers are thought to be adapted for arresting flying insects. Using tensile testing, we ask whether patterns can be discerned in the evolution of silk material properties and the ecological uses of spider capture fibers. Here, we present a large comparative data set that allows examination of capture silk properties across orb-weaving spider species. We find that material properties vary greatly across species. Notably, extensibility, strength, and toughness all vary approximately sixfold across species. These material differences, along with variation in fiber size, dictate that the mechanical performance of capture threads, the energy and force required to break fibers, varies by more than an order of magnitude across species. Furthermore, some material and mechanical properties are evolutionarily correlated. For example, species that spin small diameter fibers tend to have tougher silk, suggesting compensation to maintain breaking energy. There is also a negative correlation between strength and extensibility across species, indicating a potential evolutionary trade-off. The different properties of these capture silks should lead to differences in the performance of orb webs during prey capture and help to define feeding niches in spiders.     

The structure-specific nicking of small heteroduplexes by the RAG complex: implications for lymphoid chromosomal translocations

During V(D)J recombination, the RAG complex binds at recombination signal sequences and creates double-strand breaks. In addition to this sequence-specific recognition of the RSS, the RAG complex has been shown to be a structure-specific nuclease, cleaving 3' overhangs and 3' flaps, and, more recently, 10 nucleotides (nt) bubble (heteroduplex) structures. Here, we assess the smallest size heteroduplex that core and full-length RAGs can cleave. We also test whether bubbles adjacent to a partial RSS are nicked any differently or any more efficiently than bubbles that are surrounded by random sequence. These points are important in considering what types and what size of non-B DNA structure that the RAG complex can nick, and this helps assess the role of the RAG complex in mediating lymphoid chromosomal translocations. We find that the smallest bubble nicked by the RAG complex is 3nt, and proximity to a partial or full RSS sequence does not affect the nicking by RAGs. RAG nicking efficiency increases with the size of the heteroduplex and is only about two-fold less efficient than an RSS when the bubble is 6nt. We consider these findings in the context of RAG nicking at non-B DNA structures in lymphoid chromosomal translocations.     

Within-winter flexibility in muscle and heart lipid transport and catabolism in passerine birds

Journal of Comparative Physiology B - Small birds in cold climates may show within-winter metabolic flexibility to match metabolic capacities to prevailing weather conditions. This flexibility may...     

The pH dependence of serpin-proteinase complex dissociation reveals a mechanism of complex stabilization involving inactive and active conformational states of the proteinase which are perturbable by calcium

Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k(diss, app)) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k(diss, app) for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of tosylarginine methyl ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10-100 mm Ca(2+) ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca(2+).     

Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication

Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.     

Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin

Michaelis complex, acylation, and conformational change steps were resolved in the reactions of the serpin, plasminogen activator inhibitor-1 (PAI-1), with tissue plasminogen activator (tPA) and trypsin by comparing the reactions of active and Ser 195-inactivated enzymes with site-specific fluorescent-labeled PAI-1 derivatives that report these events. Anhydrotrypsin or S195A tPA-induced fluorescence changes in P1'-Cys and P9-Cys PAI-1 variants labeled with the fluorophore, NBD, indicative of a substrate-like interaction of the serpin reactive loop with the proteinase active-site, with the P1' label but not the P9 label perturbing the interactions by 10-60-fold. Rapid kinetic analyses of the labeled PAI-1-inactive enzyme interactions were consistent with a single-step reversible binding process involving no conformational change. Blocking of PAI-1 reactive loop-beta-sheet A interactions through mutation of the P14 Thr --> Arg or annealing a reactive center loop peptide into sheet A did not weaken the binding of the inactive enzymes, suggesting that loop-sheet interactions were unlikely to be induced by the binding. Only active trypsin and tPA induced the characteristic fluorescence changes in the labeled PAI-1 variants previously shown to report acylation and reactive loop-sheet A interactions during the PAI-1-proteinase reaction. Rapid kinetic analyses showed saturation of the reaction rate constant and, in the case of the P1'-labeled PAI-1 reaction, biphasic changes in fluorescence indicative of an intermediate resembling the noncovalent complex on the path to the covalent complex. Indistinguishable K(M) and k(lim) values of approximately 20 microM and 80-90 s(-1) for reaction of the two labeled PAI-1s with trypsin suggested that a diffusion-limited association of PAI-1 and trypsin and rate-limiting acylation step, insensitive to the effects of labeling, controlled covalent complex formation. By contrast, differing values of K(M) of 1.7 and 0.1 microM and of k(lim) of 17 and 2.6 s(-1) for tPA reactions with P1' and P9-labeled PAI-1s, respectively, suggested that tPA-PAI-1 exosite interactions, sensitive to the effects of labeling, promoted a rapid association of PAI-1 and tPA and reversible formation of an acyl-enzyme complex but impeded a rate-limiting burial of the reactive loop leading to trapping of the acyl-enzyme complex. Together, the results suggest a kinetic pathway for formation of the covalent complex between PAI-1 and proteinases involving the initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational change steps that trap the proteinase in a covalent complex.     

Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.     

Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia

The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice approximately 100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-gamma and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-gamma and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-gamma drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.