Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Cyclic AMP metabolism in mouse parotid glands. Properties of adenylate cyclase, protein kinase and phosphodiesterase.

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands. Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity witha pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a Ka of 1.2 - 10(-7) M. Parotid cyclic AMP and cyclic GMP phosphodiesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least sixpeaks of enzyme activity in the pI range of 4-6. Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

Water, temperature and life

Cold is the fiercest and most widespread enemy of life on earth. Natural cold adaptation and survival are discussed in terms of physicochemical and biochemical water management mechanisms, relying on thermodynamic or kinetic stabilization. Distinctions are drawn between general effects of low temperature (chill) and specific effects of freezing. Freeze tolerance is a misnomer because tolerance does not extend to the cell fluids. Freezing is confined to the extracellular spaces where it acts as a means of protecting the cytoplasm against freezing injury. Freeze resistance depends on the phenomenon of undercooling, a survival mechanism that relies on the long-term maintenance of a thermodynamically highly unstable state. Correct water management involves many factors, among them the control of membrane composition and transmembrane osmotic equilibrium, the biosynthesis of compounds able to afford protection against injury through freeze desiccation and the availability (or inactivation) of biogenic ice nucleation catalysts.     

Determinants of the anesthetic sensitivity of two-pore domain acid-sensitive potassium channels: molecular cloning of an anesthetic-activated potassium channel from Lymnaea stagnalis

Certain two-pore domain K(+) channels are plausible targets for volatile general anesthetics, yet little is known at the molecular level about how these simple agents cause channel activation. The first anesthetic-activated K(+) current I(K(An)) that was characterized was discovered in the mollusk Lymnaea stagnalis and is remarkable for both its sensitivity to general anesthetics and its stereoselective responses to anesthetic enantiomers (Franks, N. P., and Lieb, W. R. (1988) Nature 333, 662-664 and Franks, N. P., and Lieb, W. R. (1991) Science 254, 427-430). Here we report the molecular cloning of a two-pore domain K(+) channel LyTASK from L. stagnalis and show that, when expressed in HEK-293 cells, it displays the same biophysical characteristics as the anesthetic-activated K(+) current I(K(An)). Sequence analysis and functional properties show it to be a member of the TASK family of channels with approximately 47% identity at the amino acid level when compared with human TASK-1 and TASK-3. By using chimeric channel constructs and site-directed mutagenesis we have identified the specific amino acid 159 to be a critical determinant of anesthetic sensitivity, which, when mutated to alanine, essentially eliminates anesthetic activation in the human channels and greatly reduces activation in LyTASK. The L159A mutation in LyTASK disrupts the stereoselective response to isoflurane while having no effect on the pH sensitivity of the channel, suggesting this critical amino acid may form part of an anesthetic binding site.     

Reconnaissance and latent learning in ants

We show that ants can reconnoitre their surroundings and in effect plan for the future. Temnothorax albipennis colonies use a sophisticated strategy to select a new nest when the need arises. Initially, we presented colonies with a new nest of lower quality than their current one that they could explore for one week without a need to emigrate. We then introduced a second identical low quality new nest and destroyed their old nest so that they had to emigrate. Colonies showed a highly significant preference for the (low quality) novel new nest over the identical but familiar one. In otherwise identical experiments, colonies showed no such discrimination when the choice was between a familiar and an unfamiliar high-quality nest. When, however, either all possible pheromone marks were removed, or landmarks were re-orientated, just before the emigration, the ants chose between identical low-quality new nests at random. These results demonstrate for the first time that ants are capable of assessing and retaining information about the quality of potential new nest sites, probably by using both pheromones and landmark cues, even though this information may only be of strategic value to the colony in the future. They seem capable, therefore, of latent learning and, more explicitly, learning what not to do.     

Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins

Chemical shift assignment is the first step in all established protocols for structure determination of uniformly labeled proteins by NMR. The explosive growth in recent years of magic-angle spinning (MAS) solid-state NMR (SSNMR) applications is largely attributable to improved methods for backbone and side-chain chemical shift correlation spectroscopy. However, the techniques developed so far have been applied primarily to proteins in the size range of 5–10 kDa, despite the fact that SSNMR has no inherent molecular weight limits. Rather, the degeneracy inherent to many 2D and 3D SSNMR spectra of larger proteins has prevented complete unambiguous chemical shift assignment. Here we demonstrate the implementation of 4D backbone chemical shift correlation experiments for assignment of solid proteins. The experiments greatly reduce spectral degeneracy at a modest cost in sensitivity, which is accurately described by theory. We consider several possible implementations and investigate the CANCOCX pulse sequence in detail. This experiment involves three cross polarization steps, from H to CA[i], CA[i] to N[i], and N[i] to C′[i−1], followed by a final homonuclear mixing period. With short homonuclear mixing times (<20 ms), backbone correlations are observed with high sensitivity; with longer mixing times (>200 ms), long-range correlations are revealed. For example, a single 4D experiment with 225 ms homonuclear mixing time reveals ∼200 uniquely resolved medium and long-range correlations in the 56-residue protein GB1. In addition to experimental demonstrations in the 56-residue protein GB1, we present a theoretical analysis of anticipated improvements in resolution for much larger proteins and compare these results in detail with the experiments, finding good agreement between experiment and theory under conditions of stable instrumental performance.     

Recurrent circuitry dynamically shapes the activation of piriform cortex

In the piriform cortex, individual odorants activate a unique ensemble of neurons that are distributed without discernable spatial order. Piriform neurons receive convergent excitatory inputs from random collections of olfactory bulb glomeruli. Pyramidal cells also make extensive recurrent connections with other excitatory and inhibitory neurons. We introduced channelrhodopsin into the piriform cortex to characterize these intrinsic circuits and to examine their contribution to activity driven by afferent bulbar inputs. We demonstrated that individual pyramidal cells are sparsely interconnected by thousands of excitatory synaptic connections that extend, largely undiminished, across the piriform cortex, forming a large excitatory network that can dominate the bulbar input. Pyramidal cells also activate inhibitory interneurons that mediate strong, local feedback inhibition that scales with excitation. This recurrent network can enhance or suppress bulbar input, depending on whether the input arrives before or after the cortex is activated. This circuitry may shape the ensembles of piriform cells that encode odorant identity.     

Land plants acquired active stomatal control early in their evolutionary history

Stomata are pores that regulate plant gas exchange [1]. They evolved more than 400 million years ago [2, 3], but the origin of their active physiological responses to endogenous and environmental cues is unclear [2-6]. Recent research suggests that the stomata of lycophytes and ferns lack pore closure responses to abscisic acid (ABA) and CO(2). This evidence led to the hypothesis that a fundamental transition from passive to active control of plant water balance occurred after the divergence of ferns 360 million years ago [7, 8]. Here we show that stomatal responses of the lycophyte Selaginella [9] to ABA and CO(2) are directly comparable to those of the flowering plant Arabidopsis [10]. Furthermore, we show that the underlying intracellular signaling pathways responsible for stomatal aperture control are similar in both basal and modern vascular plant lineages. Our evidence challenges the hypothesis that acquisition of active stomatal control of plant carbon and water balance represents a critical turning point in land plant evolution [7, 8]. Instead, we suggest that the critical evolutionary development is represented by the innovation of stomata themselves and that physiologically active stomatal control originated at least as far back as the emergence of the lycophytes (circa 420 million years ago) [11].     

Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.