General anesthetic action at an internal protein site involving the S4-S5 cytoplasmic loop of a neuronal K(+) channel

The structural bases of general anesthetic action on a neuronal K(+) channel were investigated using the series of homologous 1-alkanols, electrophysiology, and mutational analysis. Domain swapping between dShaw2 (alkanol-sensitive) and hKv3.4 (alkanol-resistant) and site-directed mutagenesis demonstrated that a 13-amino acid cytoplasmic loop (S4-S5) determines the selective inhibition of native dShaw2 channels by 1-alkanols. The S4-S5 loop may contribute to a receptor for both 1-alkanols and the inactivation particle, because the enhanced 1-alkanol sensitivity of hKv3.4 channels hosting S4-S5 mutations correlates directly with disrupted channel inactivation. Evidence of a discrete protein site was also obtained from the analysis of the relationship between potency and alkyl chain length, which begins to level off after 1-hexanol. Rapid application to the cytoplasmic side of inside-out membrane patches shows that the interaction between dShaw2 channels and 1-alkanols equilibrates in <200 ms. By contrast, the equilibration time is >1000-fold slower when the drug is applied externally to outside-out membrane patches. The data strongly favor a mechanism of inhibition involving a discrete internal site for 1-alkanols in dShaw2 K(+) channels. A new working hypothesis proposes that 1-alkanols lock dShaw2 channels in their closed conformation by a direct interaction at a crevice formed by the S4-S5 loop.     

Functionally active t1-t1 interfaces revealed by the accessibility of intracellular thiolate groups in kv4 channels

Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn(2+) coordination. Also, added Zn(2+) or a potent Zn(2+) chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is approximately 200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact alpha-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1--T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn(2+) binding.     

Comparative context analysis of codon pairs on an ORFeome scale

Codon context is an important feature of gene primary structure that modulates mRNA decoding accuracy. We have developed an analytical software package and a graphical interface for comparative codon context analysis of all the open reading frames in a genome (the ORFeome). Using the complete ORFeome sequences of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Escherichia coli, we show that this methodology permits large-scale codon context comparisons and provides new insight on the rules that govern the evolution of codon-pair context.     

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.     

Comparison of techniques for the measurement of skin temperature during exercise in a hot,humid environment

Exercising or working in a hot, humid environment can results in the onset of heat-related illness when an individual''s temperature is not carefully monitored. The purpose of the present study was to compare three techniques (data loggers, thermal imaging, and wired electrodes) for the measurement of peripheral (bicep) and central (abdominal) skin temperature. Young men and women (N = 30) were recruited to complete the present study. The three skin temperature measurements were made at 0 and every 10-min during 40-min (60% VO₂max) of cycling in a hot (39±2°C), humid (45±5% RH) environment. Data was statistically analyzed using the Bland-Altman method and correlation analysis. For abdominal skin temperature, the Bland-Altman limits of agreement indicated that data loggers (1.5) were a better index of wired than was thermal imaging (3.5), For the bicep skin temperature the limits of agreement was similar between data loggers (1.9) and thermal (1.9), suggesting the both were suitable measurements. We also found that when skin temperature exceeded 35°C, we observed progressively better prediction between data loggers, thermal imaging, and wired skin sensors. This report describes the potential for the use of data loggers and thermal imaging to be used as alternative measures of skin temperature in exercising, human subjects.     

A general method of protein purification for recombinant unstructured non-acidic proteins

Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.     

Coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease Xrn1

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells.     

Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel's closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment.