A calcium-dependent potassium current is increased by a single-gene mutation inParamecium

Summary The membrane currents of wild typeParamecium tetraurelia and the behavioral mutantteaA were analyzed under voltage clamp. TheteaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs⁺ and external TEA⁺. This, along with previous work (Satow, Y., Kung, C., 1976,J. Exp. Biol. 65:51–63) identified this as a K⁺ current. It was further found to be a calcium-activated K⁺ current since this increased outward K⁺ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutationpwB, which blocks the inward calcium current, also blocks this increased outward K⁺ current inteaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K⁺ current are the same for mutant and wild type, theteaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of theteaA mutant. TheteaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of theteaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K⁺ current which prematurely repolarizes the action potential.     

MicroRNA-related cofilin abnormality in Alzheimer's disease

Rod-like structures composed of actin and the actin-binding protein cofilin are found in Alzheimer's disease (AD) patients. However, the mechanisms underlying formation of these structures and their pathological consequences are still largely unknown. We found that microRNAs 103 and 107 repress translation of cofilin, and that reduced levels of miR-103 or miR-107 are associated with elevated cofilin protein levels and formation of rod-like structures in a transgenic mouse model of AD. These results suggest that microRNAs may play an important role in cytoskeletal pathology in AD.     

An Intragenic Suppressor of a Calmodulin Mutation in Paramecium: Genetic and Biochemical Characterization

We describe a suppressor of the calmodulin mutant cam1 in Paramecium tetraurelia. The cam1 mutant, which has a SER----PHE change at residue 101 of the third calcium-binding domain, inhibits the activity of the Ca(2+)-dependent K+ current and causes exaggerated behavioral responses to most stimuli. An enrichment scheme, based on an increased sensitivity to Ba2+ in cam1 cells, was used to isolate suppressors. One such suppressor, designated cam101, restores both the activity of the Ca(2+)-dependent K+ current and behavioral responses of the cells. We show that the cam101 mutant is an intragenic suppressor of cam1, based on genetic and microinjection data. The cam101 calmodulin is shown to be similar to wild-type calmodulin in terms of its ability to stimulate calmodulin-dependent phosphodiesterase at low concentrations of free calcium. However, the cam101 calmodulin has a reduced affinity for a monoclonal antibody to wild-type Paramecium calmodulin, as does the parental cam1 calmodulin, and a different mobility on acid-urea gels relative to both wild-type and cam1 calmodulin. We have been able to demonstrate that the isolation of intragenic suppressors of a calmodulin mutation is possible, which allows for the further genetic analysis of structure-function relationships in the calmodulin molecule.     

Promoter methylation in head and neck squamous cell carcinoma cell lines is significantly different than methylation in primary tumors and xenografts

Studies designed to identify novel methylation events related to cancer often employ cancer cell lines in the discovery phase of the experiments and have a relatively low rate of discovery of cancer-related methylation events. An alternative algorithm for discovery of novel methylation in cancer uses primary tumor-derived xenografts instead of cell lines as the primary source of nucleic acid for evaluation. We evaluated DNA extracted from primary head and neck squamous cell carcinomas (HNSCC), xenografts grown from these primary tumors in nude mice, HNSCC-derived cell lines, normal oral mucosal samples, and minimally transformed oral keratinocyte-derived cell lines using Illumina Infinum Humanmethylation 27 genome-wide methylation microarrays. We found >2,200 statistically significant methylation differences between cancer cell lines and primary tumors and when comparing normal oral mucosa to keratinocyte cell lines. We found no statistically significant promoter methylation differences between primary tumor xenografts and primary tumors. This study demonstrates that tumor-derived xenografts are highly accurate representations of promoter methylation in primary tumors and that cancer derived cell lines have significant drawbacks for discovery of promoter methylation alterations in primary tumors. These findings also support use of primary tumor xenografts for the study of methylation in cancer, drug discovery, and the development of personalized cancer treatments.     

Intermolt development reduces oxygen delivery capacity and jumping performance in the American locust (<Emphasis Type="Italic">Schistocerca americana</Emphasis>)

Among animals, insects have the highest mass-specific metabolic rates; yet, during intermolt development the tracheal respiratory system cannot meet the increased oxygen demand of older stage insects. Using locomotory performance indices, whole body respirometry, and X-ray imaging to visualize the respiratory system, we tested the hypothesis that due to the rigid exoskeleton, an increase in body mass during the intermolt period compresses the air-filled tracheal system, thereby, reducing oxygen delivery capacity in late stage insects. Specifically, we measured air sac ventilation frequency, size, and compressibility in both the abdomen and femur of early, middle, and late stage sixth instar Schistocerca americana grasshoppers. Our results show that late stage grasshoppers have a reduced air sac ventilation frequency in the femur and decreased convective capacities in the abdomen and femur. We also used X-ray images of the abdomen and femur to calculate the total proportion of tissue dedicated to respiratory structure during the intermolt period. We found that late stage grasshoppers had a lower proportion of their body dedicated to respiratory structures, especially air sacs, which convectively ventilate the tracheal system. These intermolt changes make oxygen delivery more challenging to the tissues, especially critical ones such as the jumping muscle. Indeed, late stage grasshoppers showed reduced jump frequencies compared to early stage grasshoppers, as well as decreased mass-specific CO₂ emission rates at 3 kPa PO₂. Our findings provide a mechanism to explain how body mass changes during the intermolt period reduce oxygen delivery capacity and alter an insect’s life history.     

Governance Profiles and the Management of the Uses of Large Marine Ecosystems

Interest in the management of the environment and its resources on an ecosystem basis has been increasing in both terrestrial and marine contexts. The emergence of the concept of large marine ecosystems (LMEs) is one important example of this development. LMEs have been examined through five linked modules: (1) productivity of the ecosystem; (2) fish and fisheries; (3) pollution and ecosystem health; (4) socioeconomic conditions; and (5) governance. The first three focus on natural systems, while the last two concentrate on human interactions with those systems. To date the first three have received the greatest attention but as attention has turned to development and implementation of management strategies, greater consideration has being given to the human dimension of LMEs represented by the latter two modules. This article focuses on governance, a matter that is of fundamental importance because it shapes the pattern of human use of the natural environment. Efforts to promote ecosystem-based management occur within different governance frameworks; these frameworks and their associated dynamics must be understood in the same fashion that the structure and interplay of the elements of the natural ecosystem need to be comprehended. Just as natural science employs baseline studies to gauge change over time, this paper asserts the need for similar studies relevant to governance aspects of ecosystem use. After identifying and describing the roles of three major and generic governance institutions, we suggest the development in each LME of a governance profile that outlines and analyzes the existing governance framework. Moreover, we propose to consider governance change over time to assess whether such shifts represent movement in the direction of greater ecosystem focus.     

Identification of the major cytoplasmic regions of the Neurospora crassa plasma membrane H(+)-ATPase using protein chemical techniques

The transmembrane topography of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using purified, reconstituted components and direct protein chemical techniques. Reconstituted proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin to liberate peptides present on the cytoplasmic surface of the H(+)-ATPase as recently described (Hennessey, J.P., Jr., and Scarborough, G. (1990) J. Biol. Chem. 265, 532-537. The released peptides were then separated from the proteoliposomes by gel filtration chromatography and further purified by high performance liquid chromatography. Fourteen such peptides were identified by NH2-terminal amino acid sequence analysis, directly defining these parts of the molecule as present on the cytoplasmic surface of the membrane. Moreover, this information identified several additional flanking stretches as likely to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return. These results and the results of other recent experiments establish 417 residues of the 919 present in the ATPase molecule, at positions 2-100, 186-256, 441-663, and 897-920, as cytoplasmically located. Taken together with the results of our preliminary investigations of the membrane embedded sectors of the ATPase, this information allows the formulation of a reasonably detailed model for the transmembrane topography of the ATPase polypeptide chain.     

Human parainfluenza virus type 1 C proteins are nonessential proteins that inhibit the host interferon and apoptotic responses and are required for efficient replication in nonhuman primates

Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C', C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C-) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C-) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C-) and rHPIV1-C(F170S), a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C-) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C-), whereas only the anti-IFN activity is disabled in rHPIV1-C(F170S). In African green monkeys (AGMs), rHPIV1-P(C-) was considerably more attenuated than rHPIV1-C(F170S), suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C-) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.