The assessment game in sand fiddler crab contests for breeding burrows

The single enlarged claw of male sand fiddler crabs, Uca pugilator, is used in contests for control of breeding burrows. The larger of the two contestants has the larger claw and usually wins. Males use one or more of 10 agonistic elements that vary in intensity from a no-contact extension of the claw to the flip of an opponent. We used the sequence of elements employed and the duration of unstaged, naturally occurring contests in a South Carolina salt marsh to evaluate three models of extended contests: (1) energetic war of attrition, (2) cumulative assessment and (3) sequential assessment. Contests usually began with elements of low action intensity and often proceeded to elements of high intensity. Elements of higher intensity were correlated with both contest duration and the number of contest elements. Contest duration increased as opponents became more evenly matched in size, a result consistent with both cumulative and sequential assessment models. Variation in duration increased as the relative sizes of opponents increased, also in accordance with sequential assessment. The absolute size of the smaller contestant had no effect on contest duration, in contrast to predictions based on cumulative assessment or energetic war of attrition models. Contestants that lost a fight were more likely to engage immediately in another fight without loss of contest intensity, if their previous fight had been long and intense. This result is inconsistent with contests of endurance, such as the energetic war of attrition or the cumulative assessment game, but it is consistent with the ritualized display of strength and fighting skill. Thus, sequential assessment appears to best explain ritualized fiddler crab contests. Cumulative assessment, however, may be the appropriate model for extended, nonritualized, all-out fights. Cumulative assessment may also explain the tenure of individuals on breeding grounds where multiple engagements are likely to test endurance and tolerance to damage over a period of days. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

Different secretory pathways of renin from mouse cells transfected with the human renin gene

Mammalian cells in culture, transfected with human renin gene, can provide a useful tool for studying renin biosynthesis and secretion. We transfected fibroblast cells (mouse L929 and Chinese hamster ovary cells) and pituitary tumor cells (mouse AtT-20) with the human renin gene and a selectable plasmid (pSV2Neo). Transfected fibroblasts synthesize prorenin only. Prorenin is secreted by fibroblasts constitutively and the secretion is not influenced by 8-bromo-cAMP. On the other hand, transfected AtT-20 cells synthesized both prorenin and mature active renin. Transfected AtT-20 cells release prorenin by constitutive secretion but mature renin is secreted by a regulated mechanism since the secretion of the former is not influenced by 8-bromo-cAMP but the release of the latter is significantly stimulated. Our studies demonstrate that human renin may be secreted by at least two cellular pathways: prorenin by a constitutive pathway and mature renin by a regulated pathway. These transfected cells may provide useful models for studies of human renin synthesis, processing, and secretion.     

Distribution and innervation of short, interdigitated muscle fibers in parallel-fibered muscles of the cat hindlimb

The cat hindlimb contains several long, biarticular strap muscles composed of parallel muscle fascicles that attach to short tendons. Three of these muscles--sartorius, tenuissimus, and semitendinosus--were studied by dissecting individual gold-stained fibers and determining the surface distribution of acetylcholinesterase-stained end-plate zones. In each muscle, fascicles were composed of muscle fibers that ran only part of the fascicle length and tapered to end as fine strands that interdigitated with other tapering fibers within the muscle mass. Most muscle fibers measured 2-3 cm in length. Fascicles of muscle fibers were crossed by short transverse bands of endplates (1 mm wide by 1-5 mm long) that were spaced at fairly regular intervals from the origin to the insertion of the muscle. The endplate pattern suggested that the fiber fascicles were organized into multiple longitudinal strips. In the sartorius, the temporospatial distribution of electromyographic (EMG) activity evoked by stimulating fine, longitudinal branches of the parent nerve confirmed that each strip was selectively innervated by a small subset of the motor axons. These axons appeared to distribute their endings throughout the entire length of the fascicles, providing for synchronous activation of their in-series fibers.     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

Purification of two high molecular weight proteases from rabbit reticulocyte lysate

We have purified two high molecular weight proteases approximately 400-fold from rabbit reticulocyte lysate. Both enzymes hydrolyze 125I-alpha-casein and 4-methylcoumaryl-7-amide peptides with tyrosine, phenylalanine, or arginine at the P1 position. Both are inhibited by hemin, thiol reagents, chymostatin, and leupeptin. They differ, however, by other criteria. Degradation of 125I-lysozyme-ubiquitin conjugates and succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide by the larger 26 S protease is stimulated by ATP. Based on sedimentation, gel filtration, and nondenaturing polyacrylamide gel electrophoresis, the ATP-dependent protease has a molecular weight of 1,000,000 +/- 100,000 and is a multisubunit complex. The smaller 20 S protease has a molecular weight of 700,000 +/- 20,000 and is composed of 8-10 separate subunits with Mr values between 21,000 and 32,000. It does not require nucleotides for degradation of protein or peptide substrates. This smaller enzyme is similar, if not identical, to the "multicatalytic proteinase complex" first described by Wilk and Orlowski (Wilk, S., and Orlowski, M. (1983) J. Neurochem. 40, 842-849).     

Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels

The hepatic vitamin A-storing Ito cell has been implicated as a causative cell in hepatic fibrogenesis. Using a modification of a recent method (Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8681-8685), rat Ito cells were isolated and passaged in vitro on collagen-coated plastic dishes through cell generation 40-50. The collagen synthetic phenotype for Ito cells grown on various extracellular matrices was demonstrated by immunofluorescence and quantitated by competition enzyme-linked immunosorbent assays. When grown on a type I collagen matrix, Ito cells produced type IV greater than type III greater than type I collagen. When grown on a type IV collagen matrix, the cells produced relatively equal amounts of types I and III collagen. The absolute amounts of type I collagen produced were greater when cells were grown on type IV versus type I matrix. When 10(-5) M retinol was added to cell cultures, there was a uniform increase in type III collagen regardless of matrix type but a decrease in type I collagen when cells were grown on a type IV matrix and a large increase in type I collagen when cells were grown on a type I collagen matrix. The levels of cellular retinol binding protein, a key cytosolic retinol transport protein, were quantitated by high performance liquid chromatography and compared for cells grown on type I versus type IV collagen matrices. It was found that cells on a type I matrix contain 4.96 +/- 2.8 times more cellular retinol binding protein than do cells grown on a type IV matrix. In conclusion, Ito cell collagen synthesis may be altered by underlying extracellular matrix and exogenous retinol. This in vitro culture system should allow the study of regulatory factors and possible therapeutic anti-fibrogenic mediators.     

Structure-function relationships in heparin cofactor II: spectral analysis of aromatic residues and absence of a role for sulfhydryl groups in thrombin inhibition

This study characterizes the structural and functional significance of sulfhydryl residues in human plasma heparin cofactor II (HCII). For quantification of sulfhydryl groups, the extinction coefficient of HCII was redetermined and found to be 0.593 ml mg-1 cm-1 using second-derivative spectroscopy and multicomponent analysis assuming 4, 10, and 2 residues of tryptophan, tyrosine, and tyrosine-O-sulfate per mole of protein, respectively. The results show that tyrosine-O-sulfate residues in HCII and in cholecystokinin peptide fragments (as model compounds) do not significantly contribute to the absorbance spectrum from 280 to 300 nm. A total of three sulfhydryl groups per mole of HCII was detected by Ellman's reagent titration, with or without treatment with dithioerythritol, indicating the absence of intramolecular disulfide bonds. Incubation of HCII with 0.1-10 mM dithioerythritol did not diminish its heparin-enhanced thrombin inhibition activity. Treatment with various sulfhydryl-specific reagents, including p-mercuribenzoate, HgCl2, and N-substituted maleimide derivatives, inactivated HCII. Titration with Ellman's reagent after these reactions identified the modification site as a cysteinyl residue(s). However, complete methanethio derivatization of the sulfhydryl groups of HCII using methyl methanethiosulfonate did not alter heparin-catalyzed thrombin inhibition. These results indicate that the sulfhydryl groups of HCII are not essential for thrombin inhibition. HCII differs from antithrombin III, which contains an essential disulfide bond for heparin-dependent thrombin inhibition (Longas, M. O., et al. (1980) J. Biol. Chem. 255, 3436). Furthermore, within the "serpin" (serine proteinase inhibitor) superfamily, HCII resembles chicken ovalbumin in occurrence of sulfhydryl residues and reactivity with various sulfhydryl group-directed compounds.