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This article summarizes the major categories of ethical violations encountered during submission, review, and publication of scientific articles. We discuss data fabrication and falsification, plagiarism, redundant and duplicate publication, conflict of interest, authorship, animal and human welfare, and reviewer responsibility. In each section, pertinent historical background and citation of relevant regulations and statutes are provided. Furthermore, a specific case(s) derived from actual situations is (are) presented. These cases were chosen to highlight the complexities that investigators and journals must face when dealing with ethical issues. A series of discussion questions follow each case. It is our hope that by increasing education and awareness of ethical matters relevant to scientific investigation and publication, deviations from appropriate conduct will be reduced.

Human acid-sensing ion channel 1b (hASIC1b) is a [H.sup.+]-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment. two-electrode voltage clamp; Xenopus laevis oocytes; phorbol 12-myristate 13-acetate; chelerythrine; mutagenesis; phorbol 12,13-dibutyrate; protein kinase C

Despite intensive research, brain tumors remain among the most difficult type of malignancies to treat, due largely to their diffusely invasive nature and the associated difficulty of adequate surgical resection. To migrate through the brain parenchyma and to proliferate, glioma cells must be capable of significant changes in shape and volume. We have previously reported that glioma cells express an amiloride- and psalmotoxin-sensitive cation conductance that is not found in normal human astrocytes. In the present study, we investigated the potential role of this ion channel to mediate regulatory volume increase in glioma cells. We found that the ability of the cells to volume regulate subsequent to cell shrinkage by hyperosmolar solutions was abolished by both amiloride and psalmotoxin 1. This toxin is thought to be a specific peptide inhibitor of acid-sensing ion channel (ASIC1), a member of the Deg/ENaC superfamily of cation channels. We have previously shown this toxin to be an effective blocker of the glioma cation conductance. Our data suggest that one potential role for this conductance may be to restore cell volume during the cell's progression thorough the cell cycle and while the tumor cell migrates within the interstices of the brain. brain tumor; sodium; epithelial sodium channel; acid-sensing ion channel; psalmotoxin

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular [Ca.sup.2+] ([[Ca.sup.2+]].sub.i]) and intracellular pH ([pH.sub.i]) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) is a major regulator of [[Ca.sup.2+]].sub.i]. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [[Ca.sup.2+]].sub.i] and pHi in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [[Ca.sup.2+]].sub.i] was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [[Ca.sup.2+]].sub.i] increase than in normal astrocytes. This increase was prevented in nominally [Ca.sup.2+]-free buffer and by 2-APB, an inhibitor of storeoperated [Ca.sup.2+] channels. In addition, TG-activated [Ca.sup.2+] influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The p[H.sub.i] was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a [Na.sup.+]/[H.sup.+] exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular [Ca.sup.2+] homeostasis and pH regulation in tumor cells compared with normal human astrocytes. fura-2; BCECF; store-operated calcium channels

Cation selectivity and inhibition of malignant glioma [Na.sup.+] channels by Psalmotoxin 1. Am J Physiol Cell Physiol 287: C1282-C1291, 2004. First published July 14, 2004; doi:10.1152/ajpcell. 00077.2004.--Psalmotoxin 1 (a component of the venom of a West Indies tarantula) is a 40-amino acid peptide that inhibits cation currents mediated by acid-sensing ion channels (ASIC). In this study we performed electrophysiological experiments to test the hypothesis that Psalmotoxin 1 (PcTX1) inhibits [Na.sup.+] currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). In whole cell patch-clamped cultured GBM cells, the peptide toxin quickly and reversibly inhibited both inward and outward current with an I[C.sub.50] of 36 [+ or -] 2 pM. The same inhibition was observed in freshly resected GBM cells. However, when the same experiment was performed on normal human astrocytes, the toxin failed to inhibit the whole cell current. We also determined a cationic selectivity sequence for inward currents in three cultured GBM cell lines (SK-MG-1, U87-MG, and U251-MG). The selectivity sequence yielded a unique biophysical fingerprint with inward [K.sup.+] conductance approximately fourfold greater than that of [Na.sup.+], [Li.sup.+], and [Ca.sup.2+]. These observations suggest that PcTX1 may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas. patch clamp; amiloride; ion channels: acid-sensing ion channels

ENaC subunit-subunit interactions and inhibition by syntaxin 1A. Am J Physiol Renal Physiol 286:F1100-Fll06, 2004. First published March 2, 2004; 10.1152/ajprenal.00344.2003.--Amiloride-sensitive epithelial N[a.sup.+] channels (ENaCs) are subject to modulation by many factors. Recent data have also linked the Nethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery to this regulation of ENaC, but the molecular mechanisms that underlie this modulation are poorly understood. In this study, we demonstrate that syntaxin 1A physically interacts with ENaC and functionally regulates ENaC activity. Syntaxin 1A was able to coimmunoprecipitate in vitro-translated [gamma]-ENaC, but not [alpha]- or [beta]-ENaC. Also, using antibodies raised against [alpha]-, [beta]-, or [gamma] -ENaC, we detected syntaxin 1A in immunoprecipitates from Madin-Darby canine kidney cells stably transfected with [alpha][beta][gamma]-ENaC. In bilayers, syntaxin 1A inhibited ENaC, and this syntaxin 1A modulation of ENaC activity was eliminated by truncations of cytoplasmic domains of the ENaC subunits. Our findings provide evidence for a direct physical interaction between ENaC and syntaxin 1A and suggest involvement of ENaC's cytoplasmic domains in functional modulation of ENaC activity by syntaxin 1A. membrane; channels; bilayers; interaction

The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in [alpha]-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal [beta]- and [gamma]-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, [alpha]-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC. Liddle mutant; amiloride; inside-out patch; voltage clamp; post-M2 region

Human astrocytoma cells were examined using whole cell patch-clamp recording. An inward, amiloride-sensitive Na+ current was identified in four continuous cell lines originally derived from human glioblastoma cells and in three primary cultures of cells obtained from glioblastoma multiforme tumors. Also, cells freshly isolated from a resected medulloblastoma tumor exhibited this same characteristic inward current. Findings showed that the functional expression of amiloride-sensitive Na+ currents is a characteristic feature of malignant brain tumor cells and that this pathway may be a useful target for therapeutic intervention.

Research was conducted to examine the hypothesis that osmotic pressure regulates alpha-beta-gamma-rat epithelial Na+ channel (rENaC) expressed in Xenopus oocytes. The objectives of the study were to characterize the responses of a clone amiloride-sensitive Na+ channel, alpha-beta-gamma-rENaC heterologously expressed in Xenopus oocytes to changes in cell volume generated by altering external osmolarity. Results suggested that expressed rENaC may have a role in the regulatory volume increase of oocytes.

The hypothesis that alpha-1- and alpha-2-adrenergic receptors independently regulate lymphocyte amiloride-sensitive sodium channels (ASSC) by utilizing cAMP as a second messenger and by utilizing a GTP-binding protein was investigated. Direct measurements revealed that norepinephrine increased lymphocyte cAMP and activated ASSCs. Terazosin had no effect on the lymphocyte whole cell ASSC currents. This result implied the association between alpha-1-adrenergic receptors to lymphocyte ASSC regulation via GTP-binding proteins.

Research was conducted to examine the generation of new antibodies raised against the identified outwardly rectified Cl- channel (ORCC) candidate proteins and an associated purification procedure aimed at deriving a highly purified 115-kDa protein acting as an ORCC in bilayers. The influence of G-alpha-i protein precipitation on the rectification properties of ORCC was also studied. Results indicate that the loss of rectification of the ORCC was caused by the uncoupling of G-alpha-i proteins from the ORCC protein. The 115-kDa polypeptide was also identified as an ORCC.

A cell line derived from the Xenopus kidney, A6, was used to evaluate the effects of arginine vasotocin (AVT) on sodium and chloride transport in the presence of microtubular disarray. The AVT-dependent secretion of chloride was inhibited with the disruption of tubular structure but the amiloride-sensitive sodium flux was spared. Inhibition studies, reverse transcription polymerase chain reaction and immunocytochemistry revealed that chloride conductance in A6 is modulated by the cystic fibrosis transmembrane conductance regulator.

The molecular weight of the calcium activated chloride channel (CACC) isolated from the trachea of cows is 38 kDa while the predicted molecular weight of the protein transcribed from the postulated coding DNA of CACC is about 100 kDa. A truncated construct of the large protein, CACCX, was expressed in Xenopus eggs to determine if the 38 kDa channel could have been the end product of the posttranslational modification of the 100 kDa peptide. Results indicate that CACCX can function as a chloride channel.

Research shows that the beta- and gamma-subunits of an epithelial sodium channel work together to inhibit channel activity. Experiments designed to assess the effects of beta-subunit truncations on the open probability of channel activity using rat colon tissue are described. Immunofluorescence assays and cell current analysis techniques are reported.

Genetics research demonstrates that the cyclic adenosine monophosphate-gated chloride channel (CFTR) created on cystic fibrosis gene expression is controlled by the membrane protein syntaxin 1A. Syntaxin and the syntaxin-binding protein Mucn18 isoform n-Sec1 work together in CFTR regulation. Evidence suggests that epithelial syntaxin 1 suppresses CFTR currents. Further research in this area may be of use in cystic fibrosis studies.

Streaming potentials across cloned epithelial [Na.sup.+] channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient ([Delta][Pi]) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential ([Delta]P) on [Alpha][Beta][Gamma]-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a [Delta][Pi] negates channel activation by [Delta]P when the [Delta][Pi] was directed against [Delta]P. A streaming potential of 2.0 [+ or -] 0.7 mV was measured across [Alpha][Beta][Gamma]-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [[Na.sup.+]] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single [Na.sup.+] ion. A minimal effective pore diameter of 3 [Angstrom] that could accommodate two water molecules even in single file is in contrast with the 2-[Angstrom] diameter predicted from the selectivity properties of [Alpha][Beta][Gamma]-rENaC. The fact that activation of [Alpha][Beta][Gamma]-rENaC by [Delta]P can be reproduced by the imposition of [Delta][Pi] suggests that water movement through the channel is also an important determinant of channel activity.

The apical distribution of amiloride-sensitive sodium channels in A6 renal epithelial cell cultures was analyzed by high-resolution atomic force microscopy (AFM). NanoScope III and BioScope AFM imaging of antibody-labeled Na+ channels on the apical surface of the A6 epithelium indicated the localized distribution of amiloride-sensitive sodium channels in the apical microvilli. Furthermore, AFM techniques provided high-resolution imaging of cell surface topography and distribution of ion channels in Na+ transporting renal epithelial cells.

The biochemical aspects of protein kinase A (PKA)-induced phosphorylation in alveolar epithelial type II (ATII) Na+ channels isolated from adult rabbits were analyzed to determine the regulatory effects of G proteins. The sensitivity of ATII Na+ channels to amiloride and ethylisopropylamiloride was reduced by PKA-induced phosphorylation. Furthermore, the amiloride-sensitive Na+ channels isolated from rabbit ATII cells retained their ability to be regulated by PKA phosphorylation and exogenous Galphai-2 protein isoforms.

The interaction between bovine tracheal cystic fibrosis transmembrane conductance regulator and cytoskeletal proteins in the regulation of cloned epithelial Na2+ channels (ENaC) was analyzed in planar lipid bilayers. Coreconstitution of immunopurified bovine tracheal CFTR and alpha, beta, gamma-rat ENaC (rENaC) in planar lipid bilayers lowered single-channel open probability in the presence of actin. Furthermore, the short actin filaments produced their modulatory effects alpha-rENaC independent of the presence of beta- or gamma-subunits.

Knowledge on cellular physiology is vital in understanding the mechanisms are functions of different forms of cellular membrane transport. The principles of cellular physiology are essential in understanding the pathogenesis or progression of diseases that are caused by disruptions in normal membrane transport of metabolites. Furthermore, the basic principles of cellular physiology such as the mass balance principle, flow-force relationship, capacitance and thermodynamic equilibrium is important in understanding the mechanisms of cellular metabolism and life cycle.

Inositol phosphates are a family of water-soluble intracellular signaling molecules derived from membrane inositol phospholipids. They undergo a variety of complex interconversion pathways, and their levels are dynamically regulated within the cytosol in response to a variety of agonists. Relatively little is known about the biological function of most members of this family, with the exception of inositol 1,4,5-trisphosphate. Specifically, the biological functions of inositol tetrakisphosphates are largely obscure. In this paper, we report that D-myo-inositol 3,4,5,6-tetrakisphosphate (D-Ins(3,4,5,6)[P.sub.4]) has a direct biphasic (activation/inhibition) effect on an epithelial [Ca.sup.2+]-activated chloride channel. The effect of D-Ins(3,4,5,6)[P.sub.4] is not mimicked by other inositol tetrakisphosphate isomers, is dependent on the prevailing calcium concentration, and is influenced when channels are phosphorylated by calmodulin kinase II. The predominant effect of D-Ins(3,4,5,6)[P.sub.4] on phosphorylated channels is inhibitory at levels of intracellular calcium observed in stimulated cells. Our findings indicate the biological function of a molecule hitherto considered as an 'orphan' messenger. They suggest that the molecular target for D-Ins(3,4,5,6)[P.sub.4] is a plasma membrane [Ca.sup.2+]-activated chloride channel. Regulation of this channel by D-Ins(3,4,5,6)[P.sub.4] and [Ca.sup.2+] may have therapeutic implications for the disease states of both diabetic nephropathy and cystic fibrosis.

Sodium (Na+) transport plays an important role in fluid balance of the lung. To characterize the properties of Na+ transport, cultured mature fetal lung distal epithelial cells were investigated using biochemical techniques. Various types of lung Na+ channels were identified. Common to all the channels was the presence of an amiloride-binding subunit in the apical membranes. Protein kinase A and ATP increased the open probability of these channels whereas [N-ethyl-N-isopropyl]-2'-4'-amiloride and amiloride had the reverse effect. Sublethal hyperoxia increased the expression of Na+ channel mRNA.

Injections of pure protein kinase C (PKC) or the activation of PKC with phorbol-12-myristate-13-acetate (PMA) suppress the activity of amiloride-sensitive epithelial Na+ channels (alpha-beta-gamma-rENaC), cloned into Xenopus oocytes and planar lipid bilayers. Unlike other amiloride-sensitive channels, the alpha-beta-gamma-rENaC channels are unaffected by activation of protein kinase A (PKA). PKA is unable to activate the Na+ channels in oocytes pretreated with PMA. Results indicate that the alpha-beta-gamma-rENaC channels are the main Na+ channels in the epithelium.

Mechanical stretch causes a loss of cation selectivity and amiloride sensitivity of the amiloride-sensitive Na+ channels in immortalized human B lymphocytes. The inhibition of stretch-induced conductance by amiloride is less than that of hydrostatic pressure-induced conductance. Stretch increases membrane conductivity of both Na+ and K+. There is no stretch-induced current in the lymphocytes of individuals affected with Liddle's disease, as the patients have a mutant Na+ channel.

Liddle's disease is caused by abnormal activity of the peripheral blood lymphocytes' amiloride-sensitive Na+ channels due to a mutation in the beta-subunit of the channels. Na+ currents in individuals, affected with the genetic disease, are strongly activated and do not increase in the presence of 8-(-4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (cAMP). The cAMP-induced current is normalized by a peptide, consisting of deleted residues. In normal individuals the Na+ current is low. Amiloride inhibits the Na+ current in both affected and normal individuals.

A beta- or gamma-epithelial Na+ channel (ENaC) carboxy-terminal peptide blocks the strongly activated Na+ channels in lymphocytes of patients suffering from Liddle's disease. Analysis of a cloned ENaC confirms that elimination of carboxy-terminal region of the ENaC beta-subunit causes hypertensive genetic disease. The immunopurified Na+ channels from lymphocytes of patients are reconstituted in planar lipid bilayers.