A few selected applications of surface nonlinear optical spectroscopy.

As a second-order nonlinear optical process, sum-frequency generation is highly surface-specific and accordingly has been developed into a very powerful and versatile surface spectroscopic tool. It has found many unique applications in different disciplines and thus provided many exciting new research opportunities in surface and surface-related science. Selected examples are discussed here to illustrate the power of the technique.

Social transmission of reproductive behavior increases frequency of inherited disorders in a young-expanding population

The observation of high frequencies of certain inherited disorders in the population of Saguenay–Lac Saint Jean can be explained in terms of the variance and the correlation of effective family size (EFS) from one generation to the next. We have shown this effect by using the branching process approach with real demographic data. When variance of EFS is included in the model, despite its profound effect on mutant allele frequency, any mutant introduced in the population never reaches the known carrier frequencies (between 0.035 and 0.05). It is only when the EFS correlation between generations is introduced into the model that we can explain the rise of the mutant alleles. This correlation is described by a c parameter that reflects the dependency of children’s EFS on their parents’ EFS. The c parameter can be considered to reflect social transmission of demographic behavior. We show that such social transmission dramatically reduces the effective population size. This could explain particular distributions in allele frequencies and unusually high frequency of certain inherited disorders in some human populations.

A new mechanism of sound generation in songbirds

Our current understanding of the sound-generating mechanism in the songbird vocal organ, the syrinx, is based on indirect evidence and theoretical treatments. The classical avian model of sound production postulates that the medial tympaniform membranes (MTM) are the principal sound generators. We tested the role of the MTM in sound generation and studied the songbird syrinx more directly by filming it endoscopically. After we surgically incapacitated the MTM as a vibratory source, zebra finches and cardinals were not only able to vocalize, but sang nearly normal song. This result shows clearly that the MTM are not the principal sound source. The endoscopic images of the intact songbird syrinx during spontaneous and brain stimulation-induced vocalizations illustrate the dynamics of syringeal reconfiguration before phonation and suggest a different model for sound production. Phonation is initiated by rostrad movement and stretching of the syrinx. At the same time, the syrinx is closed through movement of two soft tissue masses, the medial and lateral labia, into the bronchial lumen. Sound production always is accompanied by vibratory motions of both labia, indicating that these vibrations may be the sound source. However, because of the low temporal resolution of the imaging system, the frequency and phase of labial vibrations could not be assessed in relation to that of the generated sound. Nevertheless, in contrast to the previous model, these observations show that both labia contribute to aperture control and strongly suggest that they play an important role as principal sound generators.

Rapid generation of incremental truncation libraries for protein engineering using α-phosphothioate nucleotides

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of α-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3′→5′ exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

Characterization of a high-voltage-activated IA current with a role in spike timing and locomotor pattern generation

Transient A-type K+ channels (IA) in neurons have been implicated in the delay of the spike onset and the decrease in the firing frequency. Here we have characterized biophysically and pharmacologically an IA current in lamprey locomotor network neurons that is activated by suprathreshold depolarization and is specifically blocked by catechol at 100 μM. The biophysical properties of this current are similar to the mammalian Kv3.4 channel. The role of the IA current both in single neuron firing and in locomotor pattern generation was analyzed. The IA current facilitates Na+ channel recovery from inactivation and thus sustains repetitive firing. The role of the IA current in motor pattern generation was examined by applying catechol during fictive locomotion induced by N-methyl-d-aspartate. Blockade of this current increased the locomotor burst frequency and decreased the firing of motoneurons. Although an alternating motor pattern could still be generated, the cycle duration was less regular, with ventral roots bursts failing on some cycles. Our results thus provide insights into the contribution of a high-voltage-activated IA current to the regulation of firing properties and motor coordination in the lamprey spinal cord.

P2Y1 purinergic receptors in sensory neurons: contribution to touch-induced impulse generation.

Somatic sensation requires the conversion of physical stimuli into the depolarization of distal nerve endings. A single cRNA derived from sensory neurons renders Xenopus laevis oocytes mechanosensitive and is found to encode a P2Y1 purinergic receptor. P2Y1 mRNA is concentrated in large-fiber dorsal root ganglion neurons. In contrast, P2X3 mRNA is localized to small-fiber sensory neurons and produces less mechanosensitivity in oocytes. The frequency of touch-induced action potentials from frog sensory nerve fibers is increased by the presence of P2 receptor agonists at the peripheral nerve ending and is decreased by the presence of P2 antagonists. P2X-selective agents do not have these effects. The release of ATP into the extracellular space and the activation of peripheral P2Y1 receptors appear to participate in the generation of sensory action potentials by light touch.