Cyclic presence and absence of conspecifics alters circadian clock phase but does not entrain the locomotor activity rhythm of the fruit fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms.     

The Opisthokonta and the Ecdysozoa may not be clades: stronger support for the grouping of plant and animal than for animal and fungi and stronger support for the Coelomata than Ecdysozoa

In considering the best possible solutions for answering phylogenetic questions from genomic sequences, we have chosen a strategy that we suggest is superior to others that have gone previously. We have ignored multigene families and instead have used single-gene families. This minimizes the inadvertent analysis of paralogs. We have employed strict data controls and have reasoned that if a protein is not capable of recovering the uncontroversial parts of a phylogenetic tree, then why should we use it for the more controversial parts? We have sliced and diced the data in as many ways as possible in order to uncover the signals in that data. Using this strategy, we have tested two controversial hypotheses concerning eukaryotic phylogenetic relationships: the placement of arthropoda and nematodes and the relationships of animals, plants, and fungi. We have constructed phylogenetic trees from 780 single-gene families from 10 completed genomes and amalgamated these into a single supertree. We have also carried out a total evidence analysis on the only universally distributed protein families that can accurately reconstruct the uncontroversial parts of the phylogenetic tree: a total of five families. In doing so, we ignore the majority of single-gene families that are universally distributed as they do not have the appropriate signals to recover the uncontroversial parts of the tree. We have also ignored every protein that has ever been used previously to address this issue, simply because none of them meet our strict criteria. Using these data controls, site stripping, and multiple analyses, 24 out of 26 analyses strongly support the grouping of vertebrates with arthropods (Coelomata hypothesis) and plants with animals. In the other two analyses, the data were ambivalent. The latter finding overturns an 11-year theory of Eukaryotic evolution; the first confirms what has already been said by others. In the light of this new tree, we re-analyze the evolution of intron gain and loss in the rpL14 gene and find that it is much more compatible with the hypothesis presented here than with the Opisthokonta hypothesis.     

psbG is not a photosystem two gene but may be an ndh gene

A gene of the chloroplast genome has been designated the psbG gene on the basis that in maize the gene product is a 24-kDa polypeptide of photosystem two (PS2) (Steinmetz, A. A., Castroviejo, M., Sayre, R. T., and Bogorad, L. (1986) J. Biol. Chem. 261, 2485-2488). We have located and sequenced the equivalent gene in wheat (Triticum aestivum) and have raised specific antibodies to the gene product following its expression in Escherichia coli as a beta-galactosidase fusion protein. Using these antibodies, we have investigated the location of the gene product in various thylakoid membrane fractions of pea (Pisum sativum). The gene product of apparent molecular mass 27-28 kDa is severely depleted in PS2-enriched membrane preparations and its distribution between stromal and granal regions of the membrane is distinct to that of the psbC gene product which is known to be a core polypeptide of PS2. We therefore conclude that psbG does not code for a component of PS2 but instead suggest that it is present in a novel protein complex of the thylakoid membrane. On the basis of 1) the conserved overlap between psbG and ndhC, a chloroplast gene which shows significant homology to a mitochondrial gene that codes for a subunit of the NADH-ubiquinone oxidoreductase of mitochondria, and 2) sequence similarity between the psbG gene product and the ndh gene product of E. coli, which codes for a respiratory NADH dehydrogenase, we propose that this ill-defined complex functions as a NADH or NADPH-plastoquinone oxidoreductase.     

Gripping during climbing of arboreal snakes may be safe but not economical

On the steep surfaces that are common in arboreal environments, many types of animals without claws or adhesive structures must use muscular force to generate sufficient normal force to prevent slipping and climb successfully. Unlike many limbed arboreal animals that have discrete gripping regions on the feet, the elongate bodies of snakes allow for considerable modulation of both the size and orientation of the gripping region. We quantified the gripping forces of snakes climbing a vertical cylinder to determine the extent to which their force production favoured economy or safety. Our sample included four boid species and one colubrid. Nearly all of the gripping forces that we observed for each snake exceeded our estimate of the minimum required, and snakes commonly produced more than three times the normal force required to support their body weight. This suggests that a large safety factor to avoid slipping and falling is more important than locomotor economy.     

Atrial gland cells synthesize a family of peptides that can induce egg laying inAplysia

Endogenous peptides induce egg laying in the marine mollusc Aplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying. Atrial glands were incubated in a mixture of 3H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE. Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparent Mr 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparent Mr 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm. Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule. Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pI's in peak D.(ABSTRACT TRUNCATED AT 400 WORDS)     

Entrainment of eclosion rhythm in Drosophila melanogaster populations reared for more than 700 generations in constant light environment

In this paper, we report the results of our extensive study on eclosion rhythm of four independent populations of Drosophila melanogaster that were reared in constant light (LL) environment of the laboratory for more than 700 generations. The eclosion rhythm of these flies was assayed under LL, constant darkness (DD) and three periodic light-dark (LD) cycles (T20, T24, and T28). The percentage of vials from each population that exhibited circadian rhythm of eclosion in DD and in LL (intensity of approximately 100 lux) was about 90% and 18%, respectively. The mean free-running period (τ) of eclosion rhythm in DD was 22.85 ± 0.87 h (mean ± SD). Eclosion rhythm of these flies entrained to all the three periodic LD cycles, and the phase relationship (ψ) of the peak of eclosion with respect to “lights-on” of the LD cycle was significantly different in the three periodic light regimes (T20, T24, and T28). The results thus clearly demonstrate that these flies have preserved the ability to exhibit circadian rhythm of eclosion and the ability to entrain to a wide range of periodic LD cycles even after being in an aperiodic environment for several hundred generations. This suggests that circadian clocks may have intrinsic adaptive value accrued perhaps from coordinating internal metabolic cycles in constant conditions, and that the entrainment mechanisms of circadian clocks are possibly an integral part of the clockwork.     

Pseudosubstrate sequence may not be critical for autoinhibition of smooth muscle myosin light chain kinase.

It has been hypothesized that basic residues in the autoinhibitory region of myosin light chain (MLC) kinase, which resemble the substrate sequence, interact with the catalytic core via charge interaction and thus inhibit the kinase activity (pseudosubstrate inhibitory hypothesis). In the present study, we produced seven MLC kinase mutants in which the residues in the autoinhibitory region are deleted to various extents, and determined the residues crucial for the autoinhibition of the kinase activity. The activities of MT799 (1-799) and MT796 (1-796) were completely inhibited, whereas MT793 (1-793), MT791 (1-791), MT787 (1-787) and MT783 (1-783) were constitutively active. The tryptic proteolysis of MT799 and MT796 activated the kinase activity, presumably due to the removal of the residues essential for autoinhibition. The mutants which showed the constitutively active kinase activity were not further activated by tryptic proteolysis, suggesting that the residues crucial for autoinhibition were already deleted. On the other hand, MT795 (1-795) was partially constitutively active (33% of maximum activity) and the tryptic proteolysis further activated the enzyme activity, suggesting that MT795 loses part of the residues essential for autoinhibition. The substitution of the residues Tyr794-Met795 but not Lys793 of untruncated MLC kinase significantly increased the Ca2+/calmodulin-independent kinase activity. These results clearly show that the region Tyr794-Met795-Ala796 is critical for autoinhibition. This study shows that the pseudosubstrate sequence is not critical for the autoinhibition mechanism of MLC kinase.     

Coupled, but not uncoupled, fluxes in a neuronal glutamate transporter can be activated by lithium ions

In the central nervous system a family of related (Na(+)-K(+))-coupled glutamate transporters remove the transmitter from the cleft and prevent its neurotoxic actions. In addition to this coupled uptake, these transporters also mediate a sodium- and glutamate-dependent uncoupled anion conductance. Most models assume that the initial steps for both processes are the same, leading to the anticipation that both may exhibit a similar requirement for cations. In this study we have tested this idea in the neuronal glutamate transporter EAAC-1. We report that in this transporter lithium can replace sodium in the coupled uptake. Strikingly, the glutamate-dependent gating of the uncoupled conductance mediated by EAAC-1 has a strict requirement for sodium; lithium cannot substitute for it. Moreover, we describe two mutants, T370S and G410S, in which the cation selectivity of the two processes is affected differently. In both mutants sodium, but not lithium, can support coupled transport. On the other hand, the sodium selectivity of the gated anion conductance in oocytes expressing the mutant transporters is not affected. Our observations indicate that although both the coupled and the uncoupled fluxes are sodium-dependent, the conformation gating the anion conductance is different from that during substrate translocation.     

Aseptic cure of pulmonary paracoccidioidomycosis can be achieved after a previous subcutaneous immunization of susceptible but not resistant mice

The murine model of paracoccidioidomycosis, the most important South American endemic mycosis, mimics the human disease: resistance is associated with preserved cellular immunity while T-cell anergy is related with susceptibility. In the present study we asked whether a previous s.c. infection which induces strong cellular immunity would protect mice against a lethal pulmonary challenge. It was found that susceptible but not resistant mice developed immunoprotection and aseptic cure of infection. Immunoprotection led to reversal of DTH anergy, increased levels of antibodies and pulmonary IL-12, IL-2 and IL-4 indicating a balanced type 1/type 2 response. On the contrary, no marked differences in A/Sn infection and immunity were observed. Depletion experiments showed that immunoprotection required the cooperative action of CD4(+) and CD8(+) T cells in association with IFN-gamma and IL-12. Altogether, these observations demonstrated that susceptible hosts can develop sterilizing immunity and defined the main immunological requirements to control secondary paracoccidioidomycosis.     

Autophagy can promote but is not required for epithelial cell extrusion in the amnioserosa of the Drosophila embryo

During Drosophila embryogenesis the majority of the extra-embryonic epithelium known as the amnioserosa (AS) undergoes programmed cell death (PCD) following the completion of the morphogenetic process of dorsal closure. Approximately ten percent of AS cells, however, are eliminated during dorsal closure by extrusion from the epithelium. Using biosensors that report autophagy and caspase activity in vivo, we demonstrate that AS cell extrusion occurs in the context of elevated autophagy and caspase activation. Furthermore, we evaluate AS extrusion rates, autophagy, and caspase activation in embryos in which caspase activity or autophagy are altered by genetic manipulation. This includes using the GAL4/UAS system to drive expression of p35, reaper, dINR (ACT) and Atg1 in the AS; we also analyze embryos lacking both maternal and zygotic expression of Atg1. Based on our results we suggest that autophagy can promote, but is not required for, epithelial extrusion and caspase activation in the amnioserosa.     

Evidence that substance P may be a modulator rather than a transmitter of noxious mechanical stimulation

Substance P (SP), injected intrathecally, produced a dose-related increase in responsiveness (hyperalgesia) in a pressure test for nociceptive thresholds. Pretreatment with two doses of SP produced complete desensitization to this response but did not alter base-line responsiveness. The hyperalgesic response to SP and the lack of change in base line following desensitization to SP suggest a modulator rather than a transmitter role for SP in the transmission of noxious mechanical stimulation.     

Too much of a good thing: light can be bad for photosynthesis.

Even though light is the ultimate substrate for photosynthetic energy conversion, it can also harm plants. This toxicity is targeted to the water-splitting photosystem II and leads to damage and degradation of the reaction centre D1-polypeptide. The degradation of this very important protein appears to be a direct consequence of photosystem II chemistry involving highly oxidizing radicals and toxic oxygen species. The frequency of this damage is relatively low under normal conditions but becomes a significant problem for the plant with increasing light intensity, especially when combined with other environmental stress factors. However, the plant survives this photoinhibition through an efficient repair system which involves an autoproteolytic activity of the photosystem II complex, D1-polypeptide synthesis and reassembly of active complexes.     

This is not the end of limnology (or of science): the world may well be a lot simpler than we think

1. Reynolds (1998) recently wrote a short piece in this journal lamenting the state of the art of freshwater ecology. Others have recently foreshadowed the end of science altogether. It is my argument here that the end of science is not nigh and that there are fundamental advances to be made in understanding ecosystem function. Despite changes to the funding base of freshwater ecology over the years, the discipline can continue to make fundamental contributions to ecology. We have an excellent base of raw material to work with, however, collected.
2. As a rebuttal R eynolds (1997) I present evidence that ecosystems (and freshwater ecosystems in particular) may well be a lot simpler than we think. Buried deep within a very complex world there are some general modes of behaviour, determined by fundamental principles, which impart certain kinds of high level order and predictability.
3. By means of six propositions I argue the case for the existence of these fundamental principles and present empirical evidence for each.
4. In conclusion it is clear that there is a need for fundamental information about the role of biodiversity in ecosystem function. There is also a need to understand the interplay between environmental perturbations, biodiversity and functional groups which together determine the cycling of energy and materials within freshwater and estuarine systems. While we have considerable information about northern hemisphere aquatic ecosystems less is known about southern hemisphere systems.     

Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.     

Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins

Recent evidence suggests the presence in resting platelets of centrally located compartments of glycoprotein (GP) IIb-IIIa. We have employed an experimental procedure which dissociates and antigenically denatures the surface compartment of GP IIb-IIIa and allows internal compartments of GP IIb-IIIa to be studied immunochemically and functionally in intact platelets. When gel-filtered platelets are incubated with 0.25 mM EGTA at 37 degrees C for 30 min, and then supplemented for 30 min with 5 mM calcium, they lose their ability to bind GP IIb-IIIa complex-specific monoclonal antibody Fab fragments. However, when such platelets are subsequently stimulated with thrombin, GP IIb-IIIa-specific Fabs are again able to bind in large amounts to the platelet surface, in concert with the appearance of substantial amounts of receptors for fibrinogen and fibronectin. In immunoprecipitation experiments, we have found that this thrombin-displayed pool of GP IIb-IIIa originates from a pool that is not labeled by lactoperoxidase-catalyzed radioiodination of intact resting platelets. In immunofluorescence experiments, we have found that EGTA-incubated platelets contain a large sequestered internal pool of GP IIb-IIIa which upon thrombin stimulation is translocated to the platelet surface. Additional experiments suggest that this centrally located compartment may be surface connected in resting platelets and that it is accessible to some extracellular proteins, but not others.     

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone

A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.