C-Diazeniumdiolate Graminine in the Siderophore Gramibactin Is Photoreactive and Originates from Arginine.

Siderophores are synthesized by microbes to facilitate iron acquisition required for growth. Catecholate, hydroxamate, and α-hydroxycarboxylate groups comprise well-established ligands coordinating Fe(III) in siderophores. Recently, a C-type diazeniumdiolate ligand in the newly identified amino acid graminine (Gra) was found in the siderophore gramibactin (Gbt) produced by Paraburkholderia graminis DSM 17151. The N-N bond in the diazeniumdiolate is a distinguishing feature of Gra, yet the origin and reactivity of this C-type diazeniumdiolate group has remained elusive until now. Here, we identify l-arginine as the direct precursor to l-Gra through the isotopic labeling of l-Arg, l-ornithine, and l-citrulline. Furthermore, these isotopic labeling studies establish that the N-N bond in Gra must be formed between the Nδ and Nω of the guanidinium group in l-Arg. We also show the diazeniumdiolate groups in apo-Gbt are photoreactive, with loss of nitric oxide (NO) and H+ from each d-Gra yielding E/Z oxime isomers in the photoproduct. With the loss of Gbt's ability to chelate Fe(III) upon exposure to UV light, our results hint at this siderophore playing a larger ecological role. Not only are NO and oximes important in plant biology for communication and defense, but so too are NO-releasing compounds and oximes attractive in medicinal applications.

A suite of asymmetric citrate siderophores isolated from a marine Shewanella species.

Woodybactins A-D are a suite of new fatty acyl siderophores produced by the luminous marine bacterium Shewanella woodyi MS32. While this bacterium has a set of genes homologous to the biosynthetic gene cluster for aerobactin, aerobactin is not produced. The arrangement of these genes within the genome differs in S. woodyi MS32 when compared to E. coli and other species producing siderophores similar to aerobactin, and one synthetase gene which would append a second acyl-hydroxylysine to the terminal carboxylate of citrate is not functional. Within the suite of woodybactins A-D, which differ by the fatty acid appendage, one contains an unusual C9 (9:0) fatty acid and one contains a unique branched C9 iso (9:0 iso) fatty acid, as well as a C8 (8:0) and C10 (10:0) fatty acid.

Species-Recognition Program: A Computer-Assisted Approach to Recognizing Species.

Species recognition is a crucial component for many types of biological studies. To that end, broadly trained students must be able to accurately identify many different types of organisms. Courses that focus on learning the names of different species traditionally rely on preserved specimens viewed during class or laboratory time. Unfortunately, reliance on preserved specimens comes with many challenges in providing students with an optimal learning experience. The curriculum activity described here uses a modified PowerPoint file (species-recognition program-SRP) as a means of helping students learn to recognize and identify fishes based on subtle visual cues. Our results indicate that students were better able to identify fish species when using the SRP as a learning approach than when using preserved specimens. We suggest that the SRP approach to species recognition is an effective, viable alternative or supplement to preserved specimens that can be easily implemented in any course that emphasizes species identification. Information and materials are provided to enable instructors to create their own species-recognition programs. Journal of Microbiology & Biology Education.

Long-term retention of knowledge and critical thinking skills in developmental biology.

The primary goal of this project was to assess long-term retention of concepts and critical thinking skills in individuals who completed a Developmental Biology course. Undergraduates who had completed the course between 2006 and 2009 were recently contacted and asked to complete a professional goals survey and a multiple-choice developmental biology assessment test (DBAT) targeting four levels of learning. The DBAT was designed to assess students' retention of knowledge and skills related to factual recall, concept application, data analysis, and experimental design. Performance of the 2006-2009 cohorts was compared to that of students enrolled in 2010 who completed the DBAT at the beginning and the end of the semester. Participants from the 2010 course showed significant learning gains based on pre- and posttest scores overall and for each of the four levels of learning. No significant difference in overall performance was observed for students grouped by year from 2006-2010. Participants from the 2006-2009 cohorts scored slightly, but significantly, higher on average if they enrolled in graduate or professional training. However, performance on individual question categories revealed no significant differences between those participants with and without postundergraduate training. Scores on exams and a primary literature critique assignment were correlated with DBAT scores and thus represent predictors of long-term retention of developmental biology knowledge and skills.

Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca(2+) sparks in airway myocytes.

Short-lived, localized Ca(2+) events mediate Ca(2+) signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca(2+)-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca(2+) signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca(2+) signaling system composed of Ca(2+) sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca(2+)-activated K(+) (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca(2+) sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca(2+) spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca(2+)] resulting from Ca(2+) sparks, and the kinetic model of BK channels, we estimate that an average Ca(2+) spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca(2+) sparks using explicit knowledge of the spatial relationship between RYRs (the Ca(2+) source) and BK channels (the Ca(2+) target).

Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminals.

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+.

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx.

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

HETEROCHRONY AND DEVELOPMENTAL INNOVATION: EVOLUTION OF FEMALE GAMETOPHYTE ONTOGENY IN GNETUM, A HIGHLY APOMORPHIC SEED PLANT.

Seed plant female gametophytes are focal points for the evolutionary modification of development. From a structural perspective, the most divergent female gametophytes among all seed plants are found in Gnetum, a clade within Gnetales. Coenocytic organization at sexual maturity, absence of defined egg cells (free nuclei are fertilized), lack of centripetal cellularization, and postfertilization development of embryo-nourishing tissues are features of the female gametophytes of Gnetum unparalleled among seed plants. Although the female gametophyte of Gnetum retains the three basic phases of somatic development common to female gametophytes of plesiomorphic seed plants (free nuclear development, cellularization, cellular growth), the timing of fertilization has been accelerated relative to the rate of somatic development. As a consequence, the female gametophyte of Gnetum matures sexually (is fertilized) at a juvenile (compared with the ancestral somatic ontogeny) and free nuclear stage of somatic development, thereby precluding differentiation of egg cells. Unlike progenetic animals, where truncation of somatic ontogeny evolves in tandem with acceleration in the timing of sexual maturation, the female gametophyte of Gnetum completes the entire ancestral somatic ontogeny after precocious sexual maturation. This results in the evolution of postfertilization development of embryo-nourishing female gametophyte tissues, a phenomenon unique among seed plants. Nonheterochronic developmental innovations have also played important roles in the evolution of the female gametophyte of Gnetum. Centripetal cellularization, which is always associated with the phase change from coenocytic to cellular organization among plesiomorphic seed plant female gametophytes, is lacking in Gnetum. Instead, during early phases of development, apomorphic free nuclear organization is coupled with a highly anomalous pattern of cellularization. Stage-specific innovations during early development in the female gametophyte of Gnetum do not affect plesiomorphic aspects of later phases of development. Thus, a complex array of heterochronic and nonheterochronic developmental innovations have played critical roles in the ontogenetic evolution of the highly apomorphic female gametophyte of Gnetum.