Signal polymorphism in the web-decorating spider Argiope argentata is correlated with reduced survivorship and the presence of stingless bees, its primary prey.

Many spiders, and in particular those in the genus Argiope, spin highly visible web decorations whose function and significance are the subject of spirited debate. In this work, we present data to address two of the competing hypotheses that fuel this controversy. In particular, we examine the relationship between the presence of web decorations and spider survivorship (predator-protection hypothesis) and the relationship between the presence of prey and spider decorating behavior (the prey-attraction hypothesis). Our laboratory studies reveal that the decorating behavior of the spider A. argentata has a genetic component but that the expression of decorating behavior tends to be elicited only when a spider is well fed. Furthermore, our field studies show that in the presence of abundant stingless bees, spider decorating behavior is induced. Nevertheless, our field surveys also suggest that spiders that decorate their webs show reduced survivorship. We propose that the high correlation between web decorating in the presence of stingless bees supports the hypothesis that A. argentata engage in decorating behavior when attracting or targeting specific prey types. However, we also propose that web decorations attract the predators of A. argentata because high-frequency decorators suffer lower survivorship than spiders that decorate moderately or rarely. These findings suggest that spider web decorating behavior is affected by conflicting selection pressures: the positive effect of prey attraction versus the negative effect of predator attraction. Due to the heritable component of decorating behavior, web decorating among A. argentata is likely to be particularly sensitive to the spider's local ecology as well as local patterns of gene flow.

Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection.

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Ordered intracellular RecA-DNA assemblies: a potential site of in vivo RecA-mediated activities.

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli. We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.

Doublecortin mutations cluster in evolutionarily conserved functional domains.

Mutations in the X-linked gene doublecortin ( DCX ) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development.

Doublecortin, a stabilizer of microtubules.

X-linked lissencephaly is a severe brain malformation affecting males. Recently it has been demonstrated that the doublecortin gene is implicated in this disorder. In order to study the function of Doublecortin, we analyzed the protein upon transfection of COS cells. Doublecortin was found to bind to the microtubule cytoskeleton. In vitro assays (using biochemical methods, DIC microscopy and electron microscopy) demonstrate that Doublecortin binds microtubules directly, stabilizes them and causes bundling. In vivo assays also show that Doublecortin stabilizes microtubules and causes bundling. Doublecortin is a basic protein with an iso-electric point of 10, typical of microtubule-binding proteins. However, its sequence contains no known microtubule-binding domain(s). The results obtained in this study with Doublecortin and our previous work on another lissencephaly gene ( LIS1 ) emphasize the central role of regulation of microtubule dynamics and stability during neuronal morphogenesis.

DNA protection by stress-induced biocrystallization.

The crystalline state is considered to be incompatible with life. However, in living systems exposed to severe environmental assaults, the sequestration of vital macromolecules in intracellular crystalline assemblies may provide an efficient means for protection. Here we report a generic defence strategy found in Escherichia coli, involving co-crystallization of its DNA with the stress-induced protein Dps. We show that when purified Dps and DNA interact, extremely stable crystals form almost instantaneously, within which DNA is sequestered and effectively protected against varied assaults. Crystalline structures with similar lattice spacings are formed in E. coli in which Dps is slightly over expressed, as well as in starved wild-type bacteria. Hence, DNA-Dps co-crystallization is proposed to represent a binding mode that provides wide-range protection of DNA by sequestration. The rapid induction and large-scale production of Dps in response to stress, as well as the presence of Dps homologues in many distantly related bacteria, indicate that DNA protection by biocrystallization may be crucial and widespread in prokaryotes.

Brain mechanisms in fatal cardiac arrhythmia.

Intravenous injection of histamine to rabbits was used as a prototype in an investigation of the mechanism of sudden death due to anaphylaxis and other causes. The "dive" reflex, bradycardia due to activation of the ophthalmic branch of the fifth cranial nerve, was induced in thirty-seven of the animals while they inhaled a very small amount of cigarette smoke. Associated with the resulting bradycardia were lowered blood pH and increased serum content of lactic acid and potassium and increased peripheral arterial constriction with elevation of diastolic blood pressure. Intravenous injection of 1 ml of histamine in the presence of the dive reflex induced potentially fatal ventricular arrhythmia, but no cardiac disturbance when administered while the dive reflex was inactive, thereby strongly suggesting that sudden death in anaphylaxis may involve an overzealous response to a normally protective neural reflex.

Structure of the alpha beta tubulin dimer by electron crystallography.

The alphabeta tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes. Each tubulin monomer binds a guanine nucleotide, which is nonexchangeable when it is bound in the alpha subunit, or N site, and exchangeable when bound in the beta subunit, or E site. The alpha- and beta-tubulins share 40% amino-acid sequence identity, both exist in several isotype forms, and both undergo a variety of posttranslational modifications. Limited sequence homology has been found with the proteins FtsZ and Misato, which are involved in cell division in bacteria and Drosophila, respectively. Here we present an atomic model of the alphabeta tubulin dimer fitted to a 3.7-A density map obtained by electron crystallography of zinc-induced tubulin sheets. The structures of alpha- and beta-tubulin are basically identical: each monomer is formed by a core of two beta-sheets surrounded by alpha-helices. The monomer structure is very compact, but can be divided into three functional domains: the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins.

Visualizing the secondary structure of tubulin: three-dimensional map at 4 A.

We are in the process of determining the structure of tubulin using electron crystallography of zinc-induced, crystalline sheets. We have now extended the resolution to 4 A, and there are many features in the map that appear to show details of the secondary structure. X-ray crystallographers are well aware of the problems of interpreting maps with such limited resolution, and the additional problem of the missing cone of data inherent in electron crystallography may make interpretation even more difficult. To investigate how reliably these maps can be interpreted, we have calculated density maps of a known structure, actin, under conditions similar to those of the tubulin map. Results of these simulations support the limited interpretations we made previously in the 6.5-A maps and the more extensive interpretations we make here in the 4-A map. Most of the secondary structure of the tubulin dimer can now be identified.

Interpreting a medium-resolution model of tubulin: comparison of zinc-sheet and microtubule structure.

We previously used electron crystallography of zinc-induced two-dimensional crystalline sheets of tubulin to construct a medium-resolution three dimensional (3-D) reconstruction (at 6.5 A) of this protein. Here we present an improved model, and extend the interpretation to correlate it to microtubule structure. Secondary sequence predictions and projection density maps of subtilisin-cleaved tubulin provide information on the location of the C-terminal portion, which has been suggested to be involved in the binding of microtubule-associated proteins. The zinc-sheet tubulin model is compared to microtubules in two ways; comparison of electron diffraction from the zinc-sheets to electron diffraction from microtubules, and by docking the zinc-sheet protofilament 3-D model into a helical reconstruction from ice-embedded microtubules. By correlating the zinc-sheet protofilament to a reconstruction of axonemal protofilaments, we assigned polarity to the protofilament in our model. The polarity assignment together with our model for dimer boundaries and the assignment of alpha- and beta-monomers in our reconstruction, provides a microtubule model where the alpha-monomer crowns the plus- (or fast-growing) end of the microtubule and contact is made in the centrosome with gamma-tubulin via the beta-monomer.

Application of chaos theory to a model biological system: evidence of self-organization in the intrinsic cardiac nervous system.

The neutral organization that determines the specific beat-to-beat pattern of cardiac behavior is expected to be demonstrated in the independent regulation of the RR intervals (chronotropy) and the corresponding QT subintervals (inotropy), as the former defines the rate of contraction and the latter has a linear negative correlation with the peak pressure inside the contracting ventricular muscles. The neurons of the isolated cardiac nervous system, many of which are located in the fat-pads of the heart, exhibit the same types of mechanical and chemical receptors and the same types of cholinergic and noradrenergic effectors as those found in the neural superstructure. In the surgically isolated and perfused rabbit heart we studied the responses of the QT and RR intervals evoked by block of coronary blood flow. We found that if we separated each RR cycle into QT and RR-QT components, then the dynamics of variation for each subinterval series often had the same fractional number of degrees of freedom (i.e., chaotic dimensions), a finding which suggests they are both regulated by the same underlying system. The ischemia/anoxia evoked transient dimensional increases and separations between the two subinterval series that, after the temporary divergence, reconverged to having the same lower value. The dimensional fluctuations occurred repeatedly and preceded or coincided with alterations in the magnitude and sign of the slope of QT vs RR-QT. We interpret the dimensional fluctuations of the two subinterval series as correlates of adaptation-dependent self-organization and reorganization in the underlying intrinsic cardiac nervous system during accumulating ischemia/anoxia. Such attempts at functional reorganization in this simple neurocardiac system may explain the transient dimensional changes in the RR intervals that precedes by 24 hrs the occurrences of fatal ventricular fibrillation in high-risk cardiac patients.

Preservation of 2-D crystals of tubulin for electron crystallography.

Zinc-induced sheets of tubulin are two-dimensional crystalline polymers that constitute an ideal sample for high resolution studies of tubulin by electron crystallography. We show that these 2-dimensional tubulin crystals can be stabilized by taxol against low-temperature depolymerization and degradation with time, easing the way for the preparation of electron microscopy samples. The preservation of the crystals to high resolution has been tested with different embedding media. While glucose-embedded samples diffract poorly, samples embedded in tannin consistently diffract to a resolution of at least 3.5 A. Even better results are obtained by embedding with a combination of tannin and glucose, which improves the flatness of the crystals and allows the collection of isotropic high-resolution data from tilted specimens.

Structure of tubulin at 6.5 A and location of the taxol-binding site.

Tubulin, the major component of microtubules, is a heterodimer of two chains, alpha and beta, both of relative molecular mass 50,000 (Mr50K) and with 40-50% identity. The isotypic variety and conformational flexibility of tubulin have so far made it impossible to obtain crystals for X-ray work. Structural knowledge of tubulin has been limited to about 20 A from X-ray diffraction of oriented microtubules, and from electron microscopy of microtubules and zinc-induced crystalline sheets in negative stain. The sheets consist of protofilaments similar to those in microtubules but associated in an antiparallel arrangement, and their two-dimensional character is ideal for high-resolution electron microscopy. Here we present a three-dimensional reconstruction of tubulin to 6.5 A resolution, obtained by electron crystallography of zinc-induced two-dimensional crystals of the protein. The alpha- and beta-subunits appear topologically similar, in agreement with their sequence homology. Several features can be defined in terms of secondary structure. An apparent alpha-helical portion, adjacent to both interdimer and inter-protofilament contacts, is tentatively attributed to a segment near the carboxy terminus of the protein. We can assign the alpha- and beta-subunits on the basis of projection studies of the binding of taxol, which show one taxol site per tubulin heterodimer, in agreement with the known stoichiometry of taxol in microtubules. These studies indicate that taxol affects the interaction between protofilaments; to our knowledge, this is the first time that a ligand-binding site has been visualized in the tubulin molecule.

Shortening of the QT interval of the EKG is associated primarily with increased ventricular contractility rather than heart rate.

The objective of this study was to gather direct evidence on whether the duration of the QT interval relates primarily to heart rate or to ventricular contractility. The electrocardiographic and cardiodynamic consequences of electrical stimulation (15 V, 5 ms, 10Hz) of various intrathoracic sympathetic efferent neuronal structures were studied in 10 anesthetized mongrel dogs. Stimulation of efferent sympathetic axons in the right intraganglionic nerve, which innervates the sinoatrial node, induced tachycardia (110 +/- 5 - 133 +/- 6 bpm; p < 0.01) without significantly altering right or left ventricular intramyocardial ventricular chamber pressures. The QT interval, as determined by leads I, II and III of the EKG and a transthoracic lead, was not affected by this intervention 310 +/- 8 - 302 +/- ms). Increasing heart rate to a similar degree (111 +/- 3 - 131 +/- 3 bpm) by right atrial pacing did not induce changes in the QT interval. When right (23 +/- 3 - 49 +/- 8 mm Hg; p < 0.01) and left (81 +/- 10 - 127 +/- 19 mm Hg; p < 0.01) ventricular forces were augmented without concomitant increases in heart rate by stimulating efferent sympathetic axons in the left caudal pole cardiopulmonary nerve the QT interval shortened (322 +/- 11 - 290 +/- 12 ms; p < 0.01). Only when an efferent sympathetic nerve, that contains fibers destined for both the sinoatrial node and the ventricles was stimulated did both heart rate and ventricular contractility augment and QT shorten (318 +/- 10 - 290 +/- 11 ms; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinesin does not support the motility of zinc-macrotubes.

Moving along a microtubule, kinesin follows a course parallel to the protofilaments; but it is not known whether kinesin binds exclusively on a single protofilament. The presence of zinc during tubulin polymerization induces sheets where neighboring protofilaments are antiparallel. If kinesin could support the motility of these zinc-sheets, then the binding site for a kinesin molecule would be limited to a single protofilament. Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] reported that kinesin moves along zinc-sheets. We found that zinc-sheets grown under their conditions often had a microtubule-like structure along one edge. We confirmed the possibility that the motility observed by Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] is attributed to the microtubule-like structure rather than the zinc-sheet. To resolve the question of whether kinesin can recognize an antiparallel protofilament lattice, we investigated the kinesin-mediated motility of zinc-macrotubes. At higher free zinc concentrations, zinc-sheets roll up as macrotubes, free of edges. In the presence of 10 microM taxol and 100 nM free Zn2+ at pH 6.8, the samples were shown by electron microscopy to contain only macrotubes. Under these buffer conditions, kinesin could bind strongly to axonemal doublets in the presence of AMP-PNP, and generate motility in the presence of ATP, but kinesin did not bind to nor move the macrotubes. This shows that kinesin cannot bind efficiently to nor move on the anti-parallel lattice; it is possible (though not necessary) that the groove between two parallel protofilaments is required for kinesin's motility.

Specimen flatness of glucose-embedded biological materials for electron crystallography is affected significantly by the choice of carbon evaporation stock.

Imperfect specimen flatness can be a significant limitation in the application of electron crystallography to high-resolution structure analysis of biological macromolecules. We now report that the choice of solid carbon stock that is used to make evaporated carbon films can have a very great effect on the preparation of flat specimens of glucose-embedded purple membrane. The degree of purity of the carbon does not seem to be the controlling factor, and other likely factors such as the type of mica used as a substrate, the evaporation apparatus used (and its limiting vacuum), and the use of a continuous versus an interrupted evaporation protocol do not have a discernible influence. The physical or chemical basis for the observed differences in specimen flatness is still unknown; however, the important conclusion that we can communicate at this point is that the choice of evaporating material does have a major effect on the flatness of purple membrane, the specimen used here. The implication is that different sources of carbon stock should be tried whenever difficulty is encountered in the preparation of suitably flat specimens of biological macromolecules.

Ventricular arrhythmias induced by chemically modified intrinsic cardiac neurones.

OBJECTIVE: The aim was to investigate whether intrinsic cardiac neurones can be involved in the genesis of ventricular arrhythmias. METHODS: Nicotinic, muscarinic, beta adrenergic, peptidergic, and amino acidergic agonists, as well as purinergic compounds, were individually administered in microliter quantities adjacent to spontaneously active in situ right atrial neurones in 57 anaesthetised dogs before and after acute decentralisation. RESULTS: Ventricular arrhythmias were induced in one third of the dogs following neurochemical administration. Ventricular arrhythmias are induced much less frequently when intrathoracic extracardiac neurones are modified chemically. Salvos of ventricular premature contractions or ventricular tachycardias were elicited when intrinsic cardiac neurones were modified locally applied nicotine, bethanechol, isoprenaline, angiotensin II, bradykinin, substance P, vasoactive intestinal polypeptide, glutamate, or adenosine. In 60% of those instances in which intrinsic cardiac neuronal activity was modified by a neurochemical, ventricular arrhythmias were elicited. When arrhythmias were induced, activity generated by chemically modified intrinsic cardiac neurones increased from 0.7(SD 0.2) to 2.2(0.4) impulses.s-1 (p < 0.05). Following decentralisation of the intrinsic cardiac nervous system, repeat administration of the same neurochemicals into the same loci elicited ventricular arrhythmias in 42% of those dogs in which ventricular arrhythmias had been elicited previously. Neuronal activity increased [0.8(0.5) to 2.1(0.6) impulses.s-1; p < 0.05] in 86% of these instances. CONCLUSIONS: Intrinsic cardiac neurones can be involved in the genesis of ventricular arrhythmias.

Diaphragmatic spasm: a neglected cause of dyspnoea and chest pain.

The study included 17 patients, 12 women and 5 men, with a recurrent symptom complex involving chest pain and dyspnoea characterized by inability to get a full breath. Some attacks had subsided spontaneously. Others had lasted hours or days. When examined by fluoroscopy during an attack, each subject was found to have a nearly maximally contracted (flat) diaphragm. In some of them the attack was promptly interrupted by a small intravenous injection of sodium amytal. In others it could be aborted by a conscious effort at full expiration. The syndrome associated with diaphragmatic spasm is discussed in comparison with other noncardiac sources of chest pain and dyspnoea.

Tubulin conformation in zinc-induced sheets and macrotubes.

The protein tubulin is the main constituent of microtubules. Previous studies have shown that zinc ions induce the formation of crystalline sheets and macrotubes of tubulin. Both crystal types are suitable for structural studies by electron crystallography. However, crystallographic structural analysis of tubulin has been hampered by limited crystal size and quality and the inability to control crystal polymorphism. We can obtain well-ordered crystals which are grown upon prolonged incubations (up to 24 hr). The presence of NaCl delays the degradation of the crystals, and addition of the protease inhibitor pepstatin improves crystal quality. The crystal form (sheet or macrotube) can be controlled with incubation conditions. The size of the crystals can reach up to 2 microns in width for the sheets and up to 0.5 microns in diameter for the macrotubes. Both crystal types can reach several micrometers in length. Comparison of the projection maps of the two crystal structures shows that adjacent protofilaments in the macrotubes are shifted by about 6 A relative to their positions in the sheets. Observable changes of monomer shape appear to allow close interprotofilament contacts to be maintained in both crystal forms. Images of glucose-embedded specimens obtained under these conditions give structural information beyond 4 A resolution. Merging of high- and low-resolution data allows for unambiguous assignment of monomer boundaries to high-resolution features.