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.      

Normal vision can compensate for the loss of the circadian clock

Circadian clocks are thought to be essential for timing the daily activity of animals, and consequently increase fitness. This view was recently challenged for clock-less fruit flies and mice that exhibited astonishingly normal activity rhythms under outdoor conditions. Compensatory mechanisms appear to enable even clock mutants to live a normal life in nature. Here, we show that gradual daily increases/decreases of light in the laboratory suffice to provoke normally timed sharp morning (M) and evening (E) activity peaks in clock-less flies. We also show that the compound eyes, but not Cryptochrome (CRY), mediate the precise timing of M and E peaks under natural-like conditions, as CRY-less flies do and eyeless flies do not show these sharp peaks independently of a functional clock. Nevertheless, the circadian clock appears critical for anticipating dusk, as well as for inhibiting sharp activity peaks during midnight. Clock-less flies only increase E activity after dusk and not before the beginning of dusk, and respond strongly to twilight exposure in the middle of the night. Furthermore, the circadian clock responds to natural-like light cycles, by slightly broadening Timeless (TIM) abundance in the clock neurons, and this effect is mediated by CRY.     

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.     

Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.     

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.