Gating properties conferred on BK channels by the beta3b auxiliary subunit in the absence of its NH(2)- and COOH termini

Both beta1 and beta2 auxiliary subunits of the BK-type K(+) channel family profoundly regulate the apparent Ca(2)+ sensitivity of BK-type Ca(2)+-activated K(+) channels. Each produces a pronounced leftward shift in the voltage of half-activation (V(0.5)) at a given Ca(2)+ concentration, particularly at Ca(2)+ above 1 microM. In contrast, the rapidly inactivating beta3b auxiliary produces a leftward shift in activation at Ca(2)+ below 1 microM. In the companion work (Lingle, C.J., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. J. Gen. Physiol. 117:583-605, this issue), we have shown that some of the apparent beta3b-mediated shift in activation at low Ca(2)+ arises from rapid unblocking of inactivated channels, unlike the actions of the beta1 and beta2 subunits. Here, we compare effects of the beta3b subunit that arise from inactivation, per se, versus those that may arise from other functional effects of the subunit. In particular, we examine gating properties of the beta3b subunit and compare it to beta3b constructs lacking either the NH(2)- or COOH terminus or both. The results demonstrate that, although the NH(2) terminus appears to be the primary determinant of the beta3b-mediated shift in V(0.5) at low Ca(2)+, removal of the NH(2) terminus reveals two other interesting aspects of the action of the beta3b subunit. First, the conductance-voltage curves for activation of channels containing the beta3b subunit are best described by a double Boltzmann shape, which is proposed to arise from two independent voltage-dependent activation steps. Second, the presence of the beta3b subunit results in channels that exhibit an anomalous instantaneous outward current rectification that is correlated with a voltage dependence in the time-averaged single-channel current. The two effects appear to be unrelated, but indicative of the variety of ways that interactions between beta and alpha subunits can affect BK channel function. The COOH terminus of the beta3b subunit produces no discernible functional effects.     

Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues

An auxiliary beta2 subunit, when coexpressed with Slo alpha subunits, produces inactivation of the resulting large-conductance, Ca(2+) and voltage-dependent K(+) (BK-type) channels. Inactivation is mediated by the cytosolic NH(2) terminus of the beta2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH(2) terminus in the inactivation process. The beta2 NH(2) terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2-4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH(2)-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.     

Anti-Predator Strategies and Grouping Patterns in White-Tailed Deer and Mule Deer

White-tailed deer ( Odocoileus virginianus ) and mule deer ( O. hemionus ) are closely related species of similar size that differ in their anti-predator behavior. White-tails flee from coyotes ( Canis latrans ), whereas mule deer typically stand their ground and attack this predator. I used observations of coyotes hunting deer to identify: (i) changes in group structure made in response to coyotes; and (ii) the relationship between group structure and the risk of predation for each species.
In response to coyotes, groups of mule deer merged with other groups and individuals bunched together. Predation attempts were more likely to escalate when groups split and individuals failed to bunch. Coyotes typically attacked mule deer that were in outlying positions, and these deer had to move to central positions to end attacks. Due to the high frequency of attacks on small groups as well as to the level of dilution of risk, individuals in small mule deer groups were at high risk of being attacked compared with those in larger groups. In contrast to mule deer, white-tails made no consistent changes in group size or formation, and coyotes attacked individuals in central as well as in outlying positions. Variation in aspects of group cohesion was not related to the vulnerability of white-tails, and there was no obvious difference in the risk of attack facing individuals in groups of different size. These results suggest that coyote predation selects for relatively large, cohesive groups in mule deer, apparently because this type of group improves their ability to deter coyotes. Coyote predation does not have similar effects on groups formed by white-tails, which use flight rather than deterrence to avoid predation. The benefits of responding cohesively, occupying certain positions within groups, and forming groups of a certain size can vary widely depending on the anti-predator strategies used by an animal.     

Lab-Scale Biodegradation Study of BTEX under the Unsaturated Condition and Its Effect on Soil Matric Potential

Benzene, toluene, ethylbenzene, and xylene are collectively known as BTEX which contributes to volatile environmental contaminants. This present study investigates the microbial degradation of BTEX in batch and continuous soil column experiments and its effects on soil matric potential. Batch degradation experiments were performed with different initial concentrations of BTEX using the BTEX tolerant culture isolated from petroleum-contaminated soil. In batch study, the degradation pattern for single substrate showed that xylene was degraded much faster than other compounds followed by ethylbenzene, toluene, and benzene with the highest μ_max = 0.140 h^?1 during initial substrate concentration of 100 mg L^?1. Continuous degradation experiments were performed in a soil column with an inlet concentration of BTEX of about 2000 mg L^?1 under unsaturated flow in anaerobic condition. BTEX degradation pattern was studied with time and the matric potential of the soil at different parts along the length of the column were determined at the end of the experiment. In continuous degradation study, BTEX compounds were degraded with different degradation pattern and an increase in soil matric potential was observed with an increase in depth from top to bottom in the column with applied suction head. It was found that column biodegradation contributed to 69.5% of BTEX reduction and the bacterial growth increased the soil matric potential of about 34% on an average along the column height. Therefore, this study proves that it is significant to consider soil matric potential in modeling fate and transport of BTEX in unsaturated soils.     

Sucrose metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Centrosomes and cancer.

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.