Transfer of habituation between stimulation sites of the siphon withdrawal reflex in Aplysia californica

Habituation of the siphon withdrawal reflex (SWR) can be evoked by iterative tactile stimuli presented to one of several sites, including the siphon and gill. The SWR evoked at an arbitrary "test" site did not habituate when stimuli were presented at 20-min intervals. However, there was a large decrease in the reflex evoked at the test site when the trial was preceded by 10 repetitive stimuli (interstimuli interval = 30 s) presented to the opposite "habituation" site. Transfer of habituation occurred from gill to siphon stimulation sites, and vice versa. There was a concomitant decrease in the excitatory input evoked in the central siphon motor neurons LDS1 and LDS3. Moreover, transfer of habituation occurred after the abdominal ganglion (central nervous system) was removed. There was little change in the magnitude of the control responses or transfer of habituation after deganglionation. Since transfer of habituation between stimulation sites of the SWR was similar to that reported previously for the gill withdrawal reflex, it was suggested that a common mechanism may underlie the two behaviors.     

Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin.

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

Effect of Cetylpyridinium Chloride on Microbial Adhesion to Hexadecane and Polystyrene

Microbial adhesion at the oil-water interface is a subject of both basic interest (e.g., as a technique for the measurement of hydrophobicity) and applied interest (e.g., for use in two-phase oil-water mouthwashes for the desorption of oral microorganisms). In general, surfactants inhibit microbial adhesion to oils and other hydrophobic surfaces. In the present study, we demonstrated that the cationic surfactant cetylpyridinium chloride (CPC) significantly enhanced microbial adhesion to hexadecane and various oils, as well as to the solid hydrophobic surface polystyrene. CPC increased adhesion to hexadecane of Escherichia coli, Candida albicans and Acinetobacter calcoaceticus MR-481 and of expectorated oral bacteria from near 0% to over 90%. The CPC concentration required for optimal enhancement of adhesion was a function of the initial cell density. This phenomenon was inhibited by high salt concentrations and, in the case of E. coli, by a low pH. CPC-pretreated cells were able to bind to hexadecane, but CPC-pretreated hexadecane was unable to bind untreated cells. Another cationic, surface-active antimicrobial agent, chlorhexidine gluconate, was similarly able to promote microbial adhesion to hexadecane. The results suggest that (i) CPC enhances microbial adhesion to hexadecane by binding via electrostatic interactions at the cell surface, thus diminishing surface charge and increasing cell surface hydrophobicity, and (ii) this phenomenon may have applications in oral formulations and in the use of hydrocarbon droplets as a support for cell immobilization.     

Structural, bioinformatic, and in vivo analyses of two Treponema pallidum lipoproteins reveal a unique TRAP transporter

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of "tetratricopeptide repeat" (TPR) motifs. The crystal structure of recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).     

Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes

Traditional cultivation-based methods to quantify microbial abundance are not suitable for analyses of microbial communities in environmental or medical samples, which consist mainly of uncultured microorganisms. Recently, different cultivation-independent quantification approaches have been developed to overcome this problem. Some of these techniques use specific fluorescence markers, for example ribosomal ribonucleic acid targeted oligonucleotide probes, to label the respective target organisms. Subsequently, the detected cells are visualized by fluorescence microscopy and are quantified by direct visual cell counting or by digital image analysis. This article provides an overview of these methods and some of their applications with emphasis on (semi-)automated image analysis solutions.     

Improving evolutionary models of protein interaction networks

MOTIVATION: Theoretical models of biological networks are valuable tools in evolutionary inference. Theoretical models based on gene duplication and divergence provide biologically plausible evolutionary mechanics. Similarities found between empirical networks and their theoretically generated counterpart are considered evidence of the role modeled mechanics play in biological evolution. However, the method by which these models are parameterized can lead to questions about the validity of the inferences. Selecting parameter values in order to produce a particular topological value obfuscates the possibility that the model may produce a similar topology for a large range of parameter values. Alternately, a model may produce a large range of topologies, allowing (incorrect) parameter values to produce a valid topology from an otherwise flawed model. In order to lend biological credence to the modeled evolutionary mechanics, parameter values should be derived from the empirical data. Furthermore, recent work indicates that the timing and fate of gene duplications are critical to proper derivation of these parameters. RESULTS: We present a methodology for deriving evolutionary rates from empirical data that is used to parameterize duplication and divergence models of protein interaction network evolution. Our method avoids shortcomings of previous methods, which failed to consider the effect of subsequent duplications. From our parameter values, we find that concurrent and existing existing duplication and divergence models are insufficient for modeling protein interaction network evolution. We introduce a model enhancement based on heritable interaction sites on the surface of a protein and find that it more closely reflects the high clustering found in the empirical network.     

Inducible overexpression of the FUM1 gene in Saccharomyces cerevisiae: localization of fumarase and efficient fumaric acid bioconversion to L-malic acid

Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.     

Effect of hypoproteinemia on lung fluid balance in awake newborn lambs

To study the influence of plasma protein concentration on fluid balance in the newborn lung, we measured pulmonary arterial and left atrial pressures, lung lymph flow, and concentrations of protein in lymph and plasma of eight lambs, 2-3 wk old, before and after we reduced their plasma protein concentration from 5.8 +/- 0.3 to 3.6 +/- 0.6 g/dl. Each lamb underwent two studies, interrupted by a 3-day period in which we drained protein-rich systemic lymph through a thoracic duct fistula and replaced fluid losses with feedings of a protein-free solution of electrolytes and glucose. Each study consisted of a 2-h control period followed by 4 h of increased lung microvascular pressure produced by inflation of a balloon in the left atrium. Body weight and vascular pressures did not differ significantly during the two studies, but lung lymph flow increased from 2.6 +/- 0.1 ml/h during normoproteinemia to 4.1 +/- 0.1 ml/h during hypoproteinemia. During development of hypoproteinemia, the average difference in protein osmotic pressure between plasma and lymph decreased by 1.6 +/- 2 Torr at normal left atrial pressure and by 4.9 +/- 2.2 Torr at elevated left atrial pressure. When applied to the Starling equation governing microvascular fluid balance, these changes in liquid driving pressure were sufficient to account for the observed increases in lung fluid filtration; reduction of plasma protein concentration did not cause a statistically significant change in calculated filtration coefficient. Protein loss did not influence net protein clearance from the lungs nor did it accentuate the increase in lymph flow associated with left atrial pressure elevation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anandamide administration alone and after inhibition of fatty acid amide hydrolase (FAAH) increases dopamine levels in the nucleus accumbens shell in rats

Although endogenous cannabinoid systems have been implicated in the modulation of the rewarding effects of abused drugs and food, little is known about the direct effects of endogenous ligands for cannabinoid receptors on brain reward processes. Here we show for the first time that the intravenous administration of anandamide, an endogenous ligand for cannabinoid receptors, and its longer-lasting synthetic analog methanandamide, increase the extracellular dopamine levels in the nucleus accumbens shell of awake, freely moving rats, an effect characteristic of most drugs abused by humans. Anandamide produced two distinctly different effects on dopamine levels: (1) a rapid, transient increase that was blocked by the cannabinoid CB1 receptor antagonist rimonabant, but not by the vanilloid VR1 receptor antagonist capsazepine, and was magnified and prolonged by the fatty acid amide hydrolase (FAAH) enzyme inhibitor, URB597; (2) a smaller delayed and long-lasting increase, not sensitive to CB1, VR1 or FAAH blockade. Both effects were blocked by infusing either tetrodotoxin (TTX, 1 microm) or calcium-free Ringer's solution through the microdialysis probe, demonstrating that they were dependent on the physiologic activation of dopaminergic neurotransmission. Thus, these results indicate that anandamide, through the activation of the mesolimbic dopaminergic system, participates in the signaling of brain reward processes.