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Biomedical subjects

S R Yeh

Publications and source records attributed to S R Yeh.

At least 19 recordsLinked to original sources
PubMed2001

Dual and opposing modulatory effects of serotonin on crayfish lateral giant escape command neurons.

Serotonin modulates afferent synaptic transmission to the lateral giant neurons of crayfish, which are command neurons for escape behavior. Low concentrations, or high concentrations reached gradually, are facilitatory, whereas high concentrations reached rapidly are inhibitory. The modulatory effects rapidly reverse after brief periods of application, whereas longer periods of application are followed by facilitation that persists for hours. These effects of serotonin can be reproduced by models that involve multiple interacting intracellular signaling systems that are each stimulated by serotonin. The dependence of the neuromodulatory effect on dose, rate, and duration of modulator application may be relevant to understanding the effects of natural neuromodulation on behavior and cognition and to the design of drug therapies.

Animals↗
PubMed2001

Hierarchical folding of intestinal fatty acid binding protein.

Intestinal fatty acid binding protein (IFABP) is a member of the lipid binding protein family, members of which have a clam shell type of motif formed by two five-stranded beta-sheets. Understanding the folding mechanism of these proteins has been hindered by the presence of an unresolved burst phase. By initiating the reaction with a sub-millisecond mixer and following its progression by Trp fluorescence, we discovered three distinct phases in the folding reaction of the W6Y mutant of IFABP from which we postulate the following sequence of events. The first phase (k(1) > 10 000 s(-1)) involves collapse of the polypeptide chain around a hydrophobic core. During the second phase (k(2) approximately 1500 s(-1)), beta-strands B-G, mostly located on the top half of the clam shell structure, propagate from this hydrophobic core. It is followed by the final phase (k(3) approximately 5 s(-1)) involving the formation of the last three beta-strands on the bottom half of the clam shell and the establishment of the native hydrogen bonding network throughout the protein molecule.

Amino Acid Substitution↗
PubMed2000

Flavohemoglobin, a globin with a peroxidase-like catalytic site.

Biochemical studies of flavohemoglobin (Hmp) from Escherichia coli suggest that instead of aerobic oxygen delivery, a dioxygenase converts NO to NO3(-) and anaerobically, an NO reductase converts NO to N(2)O. To investigate the structural features underlying the chemical reactivity of Hmp, we have measured the resonance Raman spectra of the ligand-free ferric and ferrous protein and the CO derivatives of the ferrous protein. At neutral pH, the ferric protein has a five-coordinate high-spin heme, similar to peroxidases. In the ferrous protein, a strong iron-histidine stretching mode is present at 244 cm(-1). This frequency is much higher than that of any other globin discovered to date, although it is comparable to those of peroxidases, suggesting that the proximal histidine has imidazolate character. In the CO derivative, an open and a closed conformation were detected. The distal environment of the closed conformation is very polar, where the heme-bound CO strongly interacts with the B10 Tyr and/or the E7 Gln. These data demonstrate that the active site structure of Hmp is very similar to that of peroxidases and is tailored to perform oxygen chemistry.

Bacterial Proteins↗
PubMed2000

A cooperative oxygen binding hemoglobin from Mycobacterium tuberculosis. Stabilization of heme ligands by a distal tyrosine residue.

The homodimeric hemoglobin (HbN) from Mycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O(2), CO, and OH(-) derivatives of the wild type protein and the B10 Tyr --> Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.

Carbon Monoxide↗
PubMed1999

A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis.

Two putative hemoglobin genes, glbN and glbO, were recently discovered in the complete genome sequence of Mycobacterium tuberculosis H37Rv. Here, we show that the glbN gene encodes a dimeric hemoglobin (HbN) that binds oxygen cooperatively with very high affinity (P(50) = 0.013 mmHg at 20 degrees C) because of a fast combination (25 microM(-1).s(-1)) and a slow dissociation (0.2 s(-1)) rate. Resonance Raman spectroscopy and ligand association/dissociation kinetic measurements, along with mutagenesis studies, reveal that the stabilization of the bound oxygen is achieved through a tyrosine at the B10 position in the distal pocket of the heme with a conformation that is unique among the globins. Physiological studies performed with Mycobacterium bovis bacillus Calmette-Guérin demonstrate that the expression of HbN is greatly enhanced during the stationary phase in aerobic cultures but not under conditions of limited oxygen availability. The results suggest that, physiologically, the primary role of HbN may be to protect the bacilli against reactive nitrogen species produced by the host macrophage.

Amino Acid Sequence↗
PubMed1999

Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate.

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

Animals↗
PubMed1999

Ligand exchange during unfolding of cytochrome c.

The productive folding pathway of cytochrome c passes through an obligatory HW intermediate in which the heme is coordinated by a solvent water molecule and a native ligand, His-18, prior to the formation of the folded HM state with both the native His-18 and Met-80 heme coordination. Two off pathway intermediates, a five-coordinated state (5C) and a bis-histidine state (HH), were also identified during the folding reaction. In the present work, the thermodynamics and the kinetics of the unfolding reaction of cytochrome c were investigated with resonance Raman scattering, tryptophan fluorescence spectroscopy, and circular dichroism. The objective of these experiments was to determine if the protein opens up and diverges into the differing heme ligation states through a many pathway mechanism or if it passes through intermediate states analogous to those observed during the folding reaction. Equilibrium unfolding results indicate that, in contrast to 5C, the stability of HH with respect to HW decreases as the concentration of GdnHCl increases. The difference in their response to the denaturant indicates that the polypeptide structure of 5C is relatively loose as compared with HH in which the polypeptide is misfolded. Time-resolved resonance Raman measurements show that strikingly similar ligand exchange reactions occur during unfolding as were observed during folding. Combined with fluorescence data, a kinetic model is proposed in which local structural rearrangements controlled by heme ligand exchange reactions appear prior to the global relaxation of the polypeptide chain.

Animals↗
PubMed1998

Neuronal coincidence detection by voltage-sensitive electrical synapses.

Coincidence detection is important for functions as diverse as Hebbian learning, binaural localization, and visual attention. We show here that extremely precise coincidence detection is a natural consequence of the normal function of rectifying electrical synapses. Such synapses open to bidirectional current flow when presynaptic cells depolarize relative to their postsynaptic targets and remain open until well after completion of presynaptic spikes. When multiple input neurons fire simultaneously, the synaptic currents sum effectively and produce a large excitatory postsynaptic potential. However, when some inputs are delayed relative to the rest, their contributions are reduced because the early excitatory postsynaptic potential retards the opening of additional voltage-sensitive synapses, and the late synaptic currents are shunted by already opened junctions. These mechanisms account for the ability of the lateral giant neurons of crayfish to sum synchronous inputs, but not inputs separated by only 100 microsec. This coincidence detection enables crayfish to produce reflex escape responses only to very abrupt mechanical stimuli. In light of recent evidence that electrical synapses are common in the mammalian central nervous system, the mechanisms of coincidence detection described here may be widely used in many systems.

Animals↗
PubMed1998

Folding intermediates in cytochrome c.

Folding of cytochrome c from its low pH guanidine hydrochloride (Gdn-HCl) denatured state revealed a new intermediate, a five-coordinate high spin species with a water molecule coordinated to the heme. Incorporation of this five-coordinated intermediate into the previously reported ligand exchange model can quantitatively account for the observed folding kinetics. In this new model, unfolded cytochrome c is converted to its native structure through an obligatory folding intermediate, the histidine-water coordination state, whereas the five-coordinate state and a bis-histidine state are off-pathway intermediates. When the concentration of Gdn-HCl in the refolding solution was increased, an acceleration of the conversion from the bis-histidine coordinated state to the histidine-water coordinated state was observed, demonstrating that the reaction requires unfolding of the mis-organized polypeptide structure associated with the bis-histidine state.

Amino Acid Sequence↗
PubMed1997

Neuronal adaptations to changes in the social dominance status of crayfish.

The effect of superfused serotonin (5-HT; 50 microns) on the synaptic responses of the lateral giant (LG) interneuron in crayfish was found to depend on the social status of the animal. In socially isolated animals. 5-HT persistently increased the response of LG to sensory nerve shock. After social isolates were paired in a small cage, they fought and determined their dominant and subordinate status. After 12 d of pairing, 5-HT reversibly inhibited the response of LG in the social subordinate and reversibly increased the response of LG in the social dominant crayfish. The effect of 5-HT changed approximately linearly from response enhancement to inhibition in the new subordinate over the 12 d of pairing. If, after 12 d pairing, the subordinate was reisolated for 8 d, the response enhancement was restored. If the subordinate, instead, was paired with another subordinate and became dominant in this new pair, the inhibitory effect of 5-HT changed to an enhancing effect over the next 12 d of pairing. If, however, two dominant crayfish were paired and one became subordinate, the enhancing effect of 5-HT persisted in the new subordinate even after 38 d pairing. These different effects of serotonin result from the action of two or more molecular receptors for serotonin. A vertebrate 5-HT, agonist had no effect on social isolates but reversibly inhibited the response of LG in both dominant and subordinate crayfish. The inhibitory effects of the agonist developed approximately linearly over the first 12 d of pairing. A vertebrate 5-HT2 agonist persistently increased the response of LG in isolate crayfish and reversibly increased the response of the cell in dominant and subordinate crayfish. Finally, although neurons that might mediate these effects of superfused 5-HT are unknown, one pair of 5-HT-immunoreactive neurons appears to contact the LG axon and initial axon segment in each abdominal ganglion in its projection caudally from the thorax.

Adaptation, Physiological↗
PubMed1997

Folding of cytochrome c initiated by submillisecond mixing.

Cytochrome c folding was initiated using a new solution mixer that provides a time window which covers over 90% of the burst phase unresolved by conventional stop-flow measurements. Folding was followed by resonance Raman scattering. Kinetic analysis of the high frequency Raman data indicates that a nascent phase occurs within the mixing dead time of 100 microseconds. A significant fraction of the protein was found to be trapped in a misfolded bis-histidine form during the nascent phase at pH 4.5, thereby preventing the protein from folding rapidly and homogeneously. The nascent phase was followed by a haem-ligand exchange phase that populates the native histidine-methionine coordinated form through a thermodynamically controlled equilibrium.

Animals↗
PubMed1997

Ligand exchange during cytochrome c folding.

Submillisecond folding of cytochrome c reveals that a nascent phase appears within the mixing dead time of 100 microseconds, followed by a ligand exchange reaction during which His 26/33, water and Met 80 are inter-exchanged as haem ligands through a thermodynamically controlled equilibrium. In the ligand exchange phase, the rate of formation of a misfolded histidine-histidine coordinated state (HH) decreases by two orders of magnitude as the pH is reduced from 5.9 to 4.5 due to the protonation of the misligated His 26/33. The activation energy barriers for the transitions from the histidine-water coordinated form (HW) to the histidine-methionine coordinated form and the HH form are 18 and 4 kcal mol-1 respectively, at pH 4.8. The activation energy barrier for protein to escape from the misligated HH to the HW form was measured to be 12 kcal mol-1, demonstrating the kinetic trapping effect of the misligated bis-histidine form. The development of the polypeptide tertiary structure near the haem is concomitant with the coordination of the native haem axial ligand.

Animals↗
PubMed1996

The effect of social experience on serotonergic modulation of the escape circuit of crayfish.

The neuromodulator serotonin has widespread effects in the nervous systems of many animals, often influencing aggression and dominance status. In crayfish, the effect of serotonin on the neural circuit for tailflip escape behavior was found to depend on the animal's social experience. Serotonin reversibly enhanced the response to sensory stimuli of the lateral giant (LG) tailflip command neuron in socially dominant crayfish, reversibly inhibited it in subordinate animals, and persistently enhanced it in socially isolated crayfish. Serotonin receptor agonists had opposing effects: A vertebrate serotonin type 1 receptor agonist inhibited the LG neurons in dominant and subordinate crayfish and had no effect in isolates, whereas a vertebrate serotonin type 2 receptor agonist enhanced the LG neurons' responses in all three types of crayfish. The LG neurons appear to have at least two populations of serotonin receptors that differ in efficacy in dominant, subordinate, and socially isolate crayfish.

Animals↗
PubMed1996

Model studies of DNA photorepair: enthalpy of cleavage of a pyrimidine dimer measured by photothermal beam deflection calorimetry.

The enzyme DNA photolyase mediates the repair of pyrimidine dimers. This repair step, a net retro [2 + 2] reaction, proceeds through either the cation or anion radical of the pyrimidine dimer. In order to understand how electron transfer makes the repair process possible, its energetics have been examined by photothermal beam deflection calorimetry, fluorescence quenching and quantum yield studies. The enthalpy for the cleavage reaction of cis-syn 1,3-dimethylthymine dimer itself was found to be -19 kcal/mol. In addition, from the redox potentials, the enthalpies for the cleavage reactions of the dimer cation radical and the anion radical were determined to be -19 kcal/mol and -28 kcal/mol, respectively.

Calorimetry↗
PubMed1994

The onset of response habituation during the growth of the lateral giant neuron of crayfish.

1. The postembryonic development of the crayfish LG tailflip command neuron's response to mechanosensory input was studied with standard electrophysiological techniques in animals between 1 and 12 cm long. 2. LG neurons are present in each abdominal hemisegment where they receive direct and indirect excitatory input from mechanosensory afferents. In both small and large crayfish, electrical stimulation of an abdominal ganglionic nerve containing those afferents evoked a compound excitatory postsynaptic potential (EPSP) with an early, reliable alpha component and a later, depression-prone beta wave. It is known that the alpha and beta components are produced by inputs from primary mechanosensory afferents and interneurons, respectively. 3. In crayfish < 2 cm long, LG was excited by the alpha component. When superthreshold, the alpha component triggered a single spike; additional excitation provided by the later beta wave presumably was preempted by refractoriness following the alpha spike and by recurrent inhibition of LG excited by the spike. LG was excited reliably by the alpha component in response to repeated superthreshold stimulation. 4. In crayfish between 2 and 3 cm, LG was excited more readily by the beta wave than by the alpha component. LG's beta spike response habituated to repeated stimulation at 1 Hz, and the beta EPSP depressed whereas the alpha component was largely unchanged. The appearance of the cellular substrates of habituation correlates with the reported onset of behavioral habituation of the tailflip response. Higher stimulus levels brought the alpha EPSP to threshold. Repetitive stimulation at these levels reliably evoked LG spikes from the alpha EPSP.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗
PubMed1994

Changes in synaptic integration during the growth of the lateral giant neuron of crayfish.

1. The effect of growth on the electrotonic structure and synaptic integrative properties of the lateral giant (LG) interneuron was assessed from anatomic and electrophysiological measurements of LGs in small (1-2.4 cm) and large (9-11.2 cm) crayfish and from calculated responses of mathematical models of these neurons. Postsynaptic responses of small and large LGs were compared with model responses to determine whether the differences in the neurons' responses result from growth-related changes in their physical characteristics. 2. LG neurons in the terminal abdominal ganglia of small and large crayfish are similar in shape but differ in size according to an approximately isometric pattern of growth. The soma diameter of the large LG is 2.2 times larger than the small LG, the major ipsilateral dendrite is 2.8 times longer and 3.6 times greater in diameter, and the axon is 7.6 times longer and 4.5 times greater in diameter. The projected area of the major ipsilateral dendrite of LG in the horizontal plane of the terminal abdominal ganglion is 27 times larger in the large than in the small crayfish. 3. LG's input resistance was nearly 80% smaller in the large (167 K omega) than in the small (742 K omega) crayfish when measured at or near the initial axon segment. The cell's membrane time constant displayed an opposite relationship, with the value in the large crayfish (20.9 ms) nearly two-and-a-half times larger than the value in the small crayfish (8.6 ms). 4. Simultaneous recordings were made from the distal portion of the ipsilateral dendrite and the initial axon segment of small and large LGs to determine how excitatory postsynaptic potentials (EPSPs) are attenuated or filtered by the electrotonic properties of the different sized cells. In the small LG, the fast alpha and the slower beta components of compound EPSPs evoked by sensory nerve stimulation were similarly attenuated. In the large LG, the alpha component of the compound EPSP was much more attenuated and smoothed than the slower beta component. 5. Multicompartment models of small and large LGs were constructed and used to test whether differences in the two neurons' physical properties could account for the differences in their passive response properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗
PubMed1992

Characterization of 3H-serotonin (5-HT) binding and effects on the phosphoinositides (PI) turnover in cultured C6 glioma and N2 neuroblastoma cells from rodents.

3H-5-HT (serotonin) binding and its displacement by various specific subtype ligands and effects on the phosphoinositides (PI) turnover were studied in cultured C6 glioma and N2 neuroblastoma cells from rodents. Saturation analysis of 3H-5-HT binding to C6 cells revealed that its Kd and Bmax were 3.0 nM and 18.0 pmole/mg protein respectively. DOI.HCl (5-HT2 agonist) and ketanserin (5-HT2 antagonist) had the highest affinities in the drug-displacement of 3H-5-HT binding to C6 cells studied. The IC50 values for DOI-HCl and ketanserin were 7.5 x 10(-7) and 3.5 x 10(-8) M respectively. 5-HT also induced 3H-PI breakdown and generated 3H-IP. The EC50 values for 5-HT for this event were in the dose range between 0.5 to 1.5 microM, and this 5-HT-induced response could be blocked by 5-HT2 antagonist ketanserin more effectively than the 5-HT1 antagonist or 5-HT3 antagonist studied. 3H-5-HT binding to N2 cells revealed that its Kd and Bmax were 4.0 nM and 80 pmole/mg protein respectively in the saturation analysis study. The drug-displacement of this binding revealed that MDL 72222 (5-HT3 antagonist) had a higher affinity than ketanserin. The IC50 values for MDL 72222 and ketanserin were 10 nM and 10 microM respectively, when 3 nM of 3H-5-HT was used. Our results indicate that the predominant receptor subtype of 5-HT in C6 and N2 cells are 5-HT2, and 5-HT3 respectively, and that the PI turnover is linked to 5-HT2, but not 5-HT3 receptor activation.

Animals↗