Structural analysis of bovine pancreatic thread protein

Pancreatic thread protein (PTP) forms double helical threads in the neutralpH range after purification, undergoing freely reversible,pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Grosset al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing >99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH₂-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca²⁺-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.     

Effects of boron and calcium nutrition on the establishment of the Rhizobium leguminosarum–pea (Pisum sativum) symbiosis and nodule development under salt stress

The effects of modifying boron (B) and calcium (Ca²⁺) concentrations on the establishment and development of rhizobial symbiosis in Pisum sativum plants grown under salt stress were investigated. Salinity almost completely inhibited the nodulation of pea plants by Rhizobium leguminosarum bv. viciae 3841. This effect was prevented by addition of Ca²⁺ during plant growth. The capacity of root exudates derived from salt‐treated plants to induce Rhizobium nod genes was not significantly decreased. However, bacterial adsorption to roots was highly inhibited in plants grown with 75 mM NaCl. Moreover, R. leguminosarum 3841 did not grow in minimal media containing such salt concentration. High Ca²⁺ levels enhanced both rhizobial growth and adsorption to roots, and increased nodule number in the presence of high salt. Nevertheless, the nodules developed were not functional unless the B concentration was also increased. Because B has a strong effect on infection and cell invasion, these processes were investigated by fluorescence microscopy in pea nodules harbouring a R. leguminosarum strain that expresses green fluorescent protein. Salt‐stressed plants had empty nodules and only those treated with high B and high Ca²⁺ developed infection threads and exhibited enhanced cell and tissue invasion by Rhizobium. Overall, the results indicate that Ca²⁺ promotes nodulation and B nodule development leading to an increase of salt tolerance of nodulated legumes.     

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume–Rhizobium symbiosis

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus , cerberus , which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti . Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.     

Distinct glycoconjugate cell surface structures make the pelagic diatom Thalassiosira rotula an attractive habitat for bacteria

Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser scanning microscopy (CLSM) after staining with fluorescently labeled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co-cultivated with a natural bacterial community and in two- or three-partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose-containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co-cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono- and co-cultures with an abundant diatom of costal seas.     

Evolution of adhesive mechanisms in cribellar spider prey capture thread: evidence for van der Waals and hygroscopic forces

Sticky prey capture threads are produced by many members of the spider infraorder Araneomorphae. Cribellar threads are plesiomorphic for this clade, and viscous threads are apomorphic. The outer surface of cribellar thread is formed of thousands of fine, looped fibrils. Basal araneomorphs produce non-noded cribellar fibrils, whereas more derived members produce noded fibrils. Cribellar fibrils snag and hold rough surfaces, but other forces are required to explain their adherence to smooth surfaces. Threads of Hypochilus pococki (Hypochilidae) formed of non-noded fibrils held to a smooth plastic surface with the same force under low and high humidities. In contrast, threads of Hyptiotes cavatus and Uloborus glomosus (Uloboridae) formed of noded fibrils held with greater force to the same surface at intermediate and high humidities. This supports the hypothesis that van der Waals forces allow non-noded cribellar fibrils to adhere to smooth surfaces, whereas noded fibrils, owing to the hydrophilic properties of their nodes, add hygroscopic forces at intermediate and high humidities. Thus, there appear to have been two major events in the evolution of adhesive mechanisms in spider prey capture thread: the addition of hydrophilic nodes to the fibrils of cribellar threads and the replacement of cribellar fibrils by viscous material and glycoprotein glue.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77 , 1–8.     

Quantitative assessment of tear production: A review of methods and utility in dry eye drug discovery

The successful development of a therapeutic agent targeting treatment of dry eye syndrome necessitates the demonstration of drug efficacy for both sign and symptom endpoints. As numerous therapeutic strategies incorporate a secretagogue function into their overall mechanism of action, the quantitative assessment of tear production serves as a logical endpoint to anchor “sign” efficacy. Although several methods including the Schirmer, the phenol red thread and tear clearance tests exist, their utility in clinical evaluations of novel therapeutics is unclear. The purpose of this review is to summarize findings and conclusions describing the performance of each of these tests so as to gain insight into which, if any, is most applicable for use in discovering new dry eye therapeutics.     

Hanging by a thread: Post-attack defense of caterpillars

Caterpillars use a diverse range of anti-predator defenses, including camouflage, making and hiding in shelters, mimicry, regurgitating, rolling, and biting. Some caterpillars also drop to the ground and hang themselves by a silk thread. This hanging behavior has been described for a long time but has surprisingly been overlooked in entomological research. In this study, we aimed to identify (1) the taxonomic distribution of the species showing the hanging behavior and (2) the type of sensory stimulus that induces the hanging behavior. We first located caterpillars in the forest and stimulated each caterpillar with three different types of sensory stimuli sequentially: visual approaching, wind-blowing, and poking. For those who responded to none of the stimuli, we further delivered harder tactile stimuli (pinching and detaching from the plants by grabbing) and observed whether they performed the hanging behavior. Among the surveyed 88 species, 46 species (comprising 11 different families) were confirmed to perform the hanging behavior. Most species responded to neither visual nor wind stimulus, but about half of the tested individuals responded to one of the tactile stimuli. Our results suggest that hanging by a silk thread is widespread across a range of lepidopteran groups, and they use this behavior as a post-attack defense.     

Transcellular progression of infection threads in Medicago truncatula roots is associated with locally confined cell wall modifications

1. Download : Download high-res image (88KB)
2. Download : Download full-size image

     

Webs of theridiid spiders: construction, structure and evolution

Understanding the web construction behaviour of theridiid (comb-footed) spiders is fundamental to formulating specific evolutionary hypotheses and predictions regarding the reduction of orb-webs. We describe for the first time in detail the web construction behaviour of Achaearanea tepidariorum , Latrodectus geometricus , Theridion sisyphium and T. varians as well as webs of a range of other theridiids. In our survey we distinguish four major web types. Among webs with gumfooted lines, we distinguish between webs with a central retreat ( Achaearanea -type) and those with a peripheral retreat ( Latrodectus -type). Among webs without gumfooted lines, we distinguish between those which contain viscid silk ( Theridion -type) and those with a sheet-like structure, which do not ( Coleosoma -type). Theridiid gumfoot-webs consist of frame lines that anchor them to surroundings and support threads which possess viscid silk. Building of gumfooted lines constitutes a unique stereotyped behaviour and is most probably homologous for Nesticidae and Theridiidae. Webs remained in place for extended periods and were expanded and repaired, but no regular pattern of replacement was observed. We suggest that the cost of producing and maintaining viscid silk might have led to web reduction, at least in theridiids.  © 2003 The Linnean Society of London. Biological Journal of the Linnean Society , 2003, 78 , 293−305.