Oxidants act as chemorepellents in Paramecium by stimulating an electrogenic plasma membrane reductase activity

Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptation and cellular sensory integration. While millimolar amounts of many attractants hyperpolarize and cause faster forward swimming, oxidants are repellents that depolarize and cause backward swimming at micromolar concentrations. The non-permeant oxidants cytochrome c, nitro blue tetrazolium and ferricyanide are repellents with half maximal concentrations of 0.4 M, 2.2 M and 100 M respectively. In vivo reductase activities follow the same order of potencies. The concentration dependence of the cytochrome c reductase activity is well correlated with cytochrome c-induced depolarizations. This suggests that plasma membrane reduction of external cytochrome c is electrogenic, causing membrane depolarization and chemorepulsion. The reductase activity also appears to be voltage dependent. Depolarization by either K+, Na+, Ca+ or Mg+ correlates with inhibition of both in vivo reductase activities and cytochrome c-induced membrane potential changes. These responses were also seen in deciliated cells, showing that the body plasma membrane is sufficient for the response. Both chloroquine and diphenyleneiodonium inhibited reductase activities but only at unusually high concentrations. This activity showed no pH dependence in the physiological range. We propose that a plasma membrane bound NADPH-dependent reductase controls oxidant-induced depolarizations and consequent chemorepulsion.Abbreviations bmv Body plasma membrane vesicles - BPS Bathophenanthroline disulfonate - cAMP Cyclic adenosine monophosphate - cmv Ciliary membrane vesicles - cyt c Cytochrome c - DPI Diphenyleneiodonium - EC ₅₀ Concentration for 50% effectiveness - FeCN Ferricyanide [Fe(CN)₆–3] - FeEDTA Ethylenediaminetetracetic acid (ferric-sodium salt) - GTP Guanosine 5-triphosphate - KCN Potassium cyanide - mM Millimolar - MOPS 3-(N-morpholino) propanesulfonic acid - mV Millivolts - NADH Nicotinamide adenine dinucleotide (reduced form) - NADPH Nicotinamide adenine dinucleotide phosphate (reduced form) - NBT Nitro blue tetrazolium - nm Nanometer - pCMB p-Chloromercuribenzoate - PMA Phorbol 12-myristate 13-acetate - s.d. Standard deviation - SOD Superoxide dismutase - Tris Tris(hydroxymethyl)aminomethane - M Micromolar     

Extracellular GTP Causes Membrane-Potential Oscillations through the Parallel Activation of Mg2+ and Na+ Currents in Paramecium tetraurelia

Paramecium tetraurelia responds to extracellular GTP (≥ 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either Mg²⁺ or Na⁺ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar to that of the membrane-potential and behavioral responses. These currents were also Mg²⁺- and Na⁺-dependent, suggesting that GTP acts through Mg²⁺-specific (I _Mg) and Na⁺-specific (I _Na) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg²⁺ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks I _Mg (``eccentric'), while Na<sup>+</sup> failed to support GTP responses in ``fast-2,' a mutant that specifically lacks I _Na. Both mutants responded normally to GTP if the alternative cation was provided. As I _Mg and I _Na are both Ca²⁺-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca²⁺ concentration. Indeed, applying GTP to cells in the absence of either Mg²⁺ or Na⁺ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known voltage-dependent Ca²⁺ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor–coupled Ca²⁺ conductance. Received: 28 October 1996/Revised: 24 December 1996     

Increase in percutaneous muscle biopsy yield with a suction-enhancement technique

Hennessey, James V., Joseph A. Chromiak, ShirleyDellaVentura, Jennifer Guertin, and David B. MacLean. Increasein percutaneous muscle biopsy yield with a suction-enhancementtechnique. J. Appl. Physiol. 82(6):1739-1742, 1997.The percutaneous muscle biopsy technique is usedin clinical practice and biomedical research. We developed a newenhanced-suction technique [suction-enhancing nipples(SEN)] and compared it with techniques currently in practice byassessing biopsy yields on anesthetized pigs. We applied the enhanced-suction technique to human subjects participating in aclinical trial. In the pig, there was a mean 91% (1.9-fold) increasein the size of the samples obtained with the 4-mm needle when SEN wasused and a mean 507% (fivefold) increase in sample size when the SENwas applied to the 6-mm needles. Nine passes of the 6-mm needle withSEN obtained from five consecutive human subjects yielded a meanindividual sample size of 109.4 mg or 219.4 mg per needle pass whenusing the double-sample technique. Adequate tissue samples forhistomorphometric and other analyses were obtained in all samplesobtained. The percutaneous muscle biopsy performed with enhancedsuction using inexpensive, readily available nipples enhances tissueyield two- to fivefold.

     

Setal patterns of the wings ofAphelinus,Mesidia, andMesidiopsis [hym.: aphelinidae], their values as taxonomic characters

Comparisons of fore wings from 27 separate populations of 13 species ofAphelinus, Mesidia, andMesidiopsis revealed that the portion of the wing posterior to the major veins is divided into 8 setal regions (the subcostal cell constitutes a 9th). Each species has a characteristic number and/or placement of setae within each region. In some species the interpopulation variability is quite small, butEphelinus asychis Walker shows large variations associated with the species of host aphid attacted; individuals reared fromTherioaphis trijolii (Monell) are hairier in several regions than those reared fromMyzus persicae (Sulzer). A nomenclature for setae in the various wing regions is proposed.     

Direct evidence for the cytoplasmic location of the NH2- and COOH-terminal ends of the Neurospora crassa plasma membrane H+-ATPase

Reconstituted proteoliposomes containing Neurospora plasma membrane H+-ATPase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the NH2 and COOH termini of the H+-ATPase relative to the lipid bilayer. Treatment of the proteoliposomes with trypsin in the presence of the H+-ATPase ligands Mg2+, ATP, and vanadate produces approximately 97-, 95-, and 88-kDa truncated forms of the H+-ATPase similar to those already known to result from cleavage at Lys24, Lys36, and Arg73 at the NH2-terminal end of the molecule. These results establish that the NH2-terminal end of the H+-ATPase polypeptide chain is located on the cytoplasmic side of the membrane. Treatment of the same proteoliposome preparation with trypsin in the absence of ligands releases approximately 50 water-soluble peptides from the proteoliposomes. Separation of the released peptides by high performance liquid chromatography and spectral analysis of the purified peptides identified only a few peptides with the properties expected of a COOH-terminal, tryptic undecapeptide with the sequence SLEDFVVSLQR, and NH2-terminal amino acid sequence analysis identified this peptide among the possible candidates. Quantitative considerations indicate that this peptide must have come from H+-ATPase molecules oriented with their cytoplasmic portion facing outward, and could not have originated from a minor population of H+-ATPase molecules of reverse orientation. These results directly establish that the COOH-terminal end of the H+-ATPase is also located on the cytoplasmic side of the membrane. These findings are important for elucidating the topography of the membrane-bound H+-ATPase and are possibly relevant to the topography of other aspartyl-phosphoryl-enzyme intermediate ATPases as well.     

Covalent binding of lipids to cytoskeletal proteins of mouse mammary epithelial cells.

Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. Primary cultures of MMEC were incubated in the presence of 3H-palmitate for 4 h. A cytoskeletal (CS) fraction was prepared by treatment of the cells with 1.5M KCl and 1% Triton X-100. The residual material, consisting primarily of keratin and actin filaments was exhaustively (10-20 rounds, including sonications) extracted with chloroform/methanol to remove non-covalently bound labeled lipids. The CS protein was then acid-hydrolyzed and the chloroform-soluble products subjected to thin layer chromatography (TLC). Two-thirds of the covalently bound radiolabel appeared as a very hydrophobic peak on a TLC system optimized for separation of neutral lipids. Ten percent separated into 4-5 peaks on a polar lipid TLC system. A small amount of label was traced to fatty acid-like components. Autoradiography of two-dimensional gels indicated that all the CS proteins resolvable by Coomassie blue staining were also radiolabeled. The results are discussed in terms of CS-lipid-membrane interactions.     

Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.     

Confidence limits and sample size for determining nonhost status of fruits and vegetables to tephritid fruit flies as a quarantine measure

Quarantine measures including treatments are applied to exported fruit and vegetable commodities to control regulatory fruit fly pests and to reduce the likelihood of their introduction into new areas. Nonhost status can be an effective measure used to achieve quarantine security. As with quarantine treatments, nonhost status can stand alone as a measure if there is high efficacy and statistical confidence. The numbers of insects or fruit tested during investigation of nonhost status will determine the level of statistical confidence. If the level of confidence of nonhost status is not high, then additional measures may be required to achieve quarantine security as part of a systems approach. Certain countries require that either 99.99 or 99.9968% mortality, as a measure of efficacy, at the 95% confidence level, be achieved by a quarantine treatment to meet quarantine security. This article outlines how the level of confidence in nonhost status can be quantified so that its equivalency to traditional quarantine treatments may be demonstrated. Incorporating sample size and confidence levels into host status testing protocols along with efficacy will lead to greater consistency by regulatory decision-makers in interpreting results and, therefore, to more technically sound decisions on host status.     

Chemoattraction to lysophosphatidic acid does not require a change in membrane potential in Tetrahymena thermophila

LPA (lysophosphatidic acid), a known chemoattractant for many types of eukaryotic cells, is also a reliable chemoattractant for Tetrahymena. Since LPA receptors are GPCRs (G-protein coupled receptors) in many cell types and several putative GPCR sequences can be found in the Tetrahymena Genome Database, we are interested to determine whether similar GPCR pathways can be used for chemosensory transduction in Tetrahymena. To confirm our procedures, we tested the known chemoattractant proteose peptone (at 1.0 mg/ml), which caused hyperpolarization and increased forward swimming speed in Tetrahymena, consistent with the current model for ciliate chemoattraction. Although 10 μM LPA did not produce these same responses, it was still an effective chemoattractant. PTX (pertussis toxin) blocked attraction to both of these compounds, suggesting a possible G-protein involvement in chemoattraction. Both of these chemoattractants also decreased the basal percent of cells showing direction changes [PDC (percent directional change)] and the duration of backward swimming in 0.5 mM Ba2+ (a general excitability assay). LPA probably causes chemoattraction in Tetrahymena by decreasing the basal PDC without changing either membrane potential or swim speed. Since a pertussis-sensitive G-protein might modulate the ciliate voltage-dependent Ca2+ channels, we propose that LPA acts through an uncharacterized GPCR to lower the PDC by decreasing cellular excitability. These combined behavioural and electrophysiological analyses support the novel hypothesis that chemoattraction to some attractants, like LPA, can occur without hyperpolarization and increased swim speed in Tetrahymena.