The ACh-evoked,Ca2+-activated Whole-cell K+ Current in Mouse Mandibular Secretory Cells. Whole-cell and Fluorescence Studies

In our previous studies on sheep parotid secretory cells, we showed that the K⁺ current evoked by acetylcholine (ACh) was not carried by the high-conductance voltage- and Ca²⁺-activated K⁺ (BK) channel which is so conspicuous in unstimulated cells, notwithstanding that the BK channel is activated by ACh. Since several studies from other laboratories had suggested that the BK channel did carry the ACh-evoked K⁺ current in the secretory cells of the mouse mandibular gland, and that the current could be blocked with tetraethylammonium (TEA), a known blocker of BK channels, we decided to investigate the ACh-evoked K⁺ current in mouse cells more closely. We studied whether the ACh-evoked K⁺ current in the mouse is inhibited by TEA and quinine. Using the whole-cell patch-clamp technique and microspectrofluorimetric measurement of intracellular Ca²⁺, we found that TEA and quinine do inhibit the ACh-evoked K⁺ current but that the effect is due to inhibition of the increase in intracellular Ca²⁺ evoked by ACh, not to blockade of a K⁺ conductance. Furthermore, we found that the K⁺ conductance activated when ionomycin is used to increase intracellular free Ca²⁺ was inhibited only by quinine and not by TEA. We conclude that the ACh-evoked K⁺ current in mouse mandibular cells does not have the blocker sensitivity pattern that would be expected if it were being carried by the high-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel. The properties of this current are, however, consistent with those of a 40 pS K⁺ channel that we have reported to be activated by ACh in these cells [16]. Received: 9 January 1996/Revised: 17 April 1996     

NAD+ is not utilized as a co-factor for DNA ligation by human DNA ligase IV

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD⁺ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD⁺, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD⁺ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD⁺ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD⁺ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD⁺ or ADP-ribose as adenylation donor for ligation.     

Activation of the Ano1 (TMEM16A) chloride channel by calcium is not mediated by calmodulin

The Ca²⁺-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca²⁺, but there is uncertainty whether Ca²⁺ binds directly to Ano1 or whether phosphorylation or additional Ca²⁺-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca²⁺ for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca²⁺-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca²⁺-activated K⁺ (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba²⁺, which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca²⁺ because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although Ano1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit Ano1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of Ano1 by Ca²⁺. Although CaM is not required for channel opening by Ca²⁺, work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel.     

Identification of p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130^Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130^Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130^Cas and its subsequent binding to several SH2 domains, which depended on both the p130^Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130^Cas further increased cell migration on FN and coexpression of the p130^Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130^Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130^Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.     

Separation of Gating Properties from Permeation and Block in mslo Large Conductance Ca-activated K+ Channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K⁺ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca²⁺ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca²⁺ block, and a slow voltage-dependent Ba²⁺ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca²⁺ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca²⁺ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca²⁺ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca²⁺ concentration range spanning three orders of magnitude indicating that over this range the internal Ca²⁺ concentration does not limit the ability of the channel to be activated by voltage.     

Muscimol Potentiation of Acidic Amino Acid Release from Cerebellar Synaptosomes Is Chloride Dependent

In previous studies we have shown that the depolarization-induced release of preaccumulated acidic amino acids and newly synthesized glutamate from cerebellar synaptosomal preparations is potentiated by γ-aminobutyric acid (GABA) agonists through a GABAergic presynaptic mechanism. Here we report a systematic analysis of the ionic requirements of the potentiating effect of muscimol on the high K⁺-evoked release of d-[³H]aspartate. Our studies show that: Ca²⁺, Na⁺, and Mg²⁺ are not required for muscimol to exert its effect; a depolarizing concentration of K⁺ is a necessary, but not sufficient, condition to observe the presynaptic effect in question; and a minimal Cl^- concentration (50–70 mM) is also required. A possible model based on these findings is proposed.     

Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca²⁺ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca²⁺ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca²⁺ release channel (RyR1). Here we report that SPC also displaces Ca²⁺-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca²⁺CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [³H]ryanodine binding assays. In the presence of Ca²⁺CaM, however, the addition of SPC did not reduce [³H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca²⁺CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca²⁺ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.     

Anti-H-2 hybridoma antibodies as immunoselective agents

Using immunoselection with an H-2K^k-specific monoclonal antibody following mutagenesis on an (H-2 ^k/H-2^d) F₁ cell line we have obtained variants that do not react with the selecting monoclonal antibody but continue to react with other monoclonal antibodies directed against the same gene product. The mutants fall into two classes based on their serological profile. This phenotype is suggestive of a structural mutation in the selected gene. If the genetic change involved is a point mutation (as opposed to a deletion), one should be able to obtain revertants. Using the fluorescence-activated cell sorter, we have been able to obtain from one of the monoclonal-antibody-nonseactive mutants cells that do bind the selecting antibody. In order to prove that the presumptive revertant is not a contaminant wild-type cell that inadvertantly got mixed into the resistant mutant, we first introduced an outside marker, resistance to the purine analogue 2-amino-6-mercaptopurine (6-thioguanine), into the monoclonal-antibody-resistant mutant. The revertants obtained using the cell sorter continue to express the nonselective phenotype of resistance to 6-thioguanine, showing that they are not wild-type cells. In addition, their serological characteristics are different from those of either the wild-type cells or the hybrid oma-resistant mutants from which they were derived. Based on the serological analyses, it would seem that we have isolated at least three variant forms of the H-2K^k-gene product.     

Exosomes Are Unlikely Involved in Intercellular Nef Transfer

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81^low/TSG101^low exosomes, but not in detergent-soluble AChE-/CD81^high/TSG101^high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81^low/TSG101^low exosomes and AChE-/CD81^high/TSG101^high exosomes.     

The involvement of Ca2+ in the Ca2+-effect on Photosystem-II oxygen evolution

A number of recent reports have concluded that Ca²⁺ is not released by treatments which are usually thought to induce the depletion of Ca²⁺. Consequently, it was proposed that the Ca²⁺ demand was not related to a specific rôle for Ca²⁺ in Photosystem-II oxygen evolution. In this letter, we scrutinize the data behind these conclusions and argue that, based on these data, it is premature to question the view that intrinsic Ca²⁺ is actually being released.     

AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca²⁺ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca²⁺ signaling can additionally induce autophagy independently of the Ca²⁺-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca²⁺ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca²⁺-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca²⁺ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca²⁺-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca²⁺ flux modifications. Importantly, using AMPK-α1^?/?α2^?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca²⁺-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.     

Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc

While elucidating the peculiar epitope of the α-PrP mAb IPC2, we found that PrP^Sc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the α-fold, suggesting oxidation may facilitate the conversion of PrP^C into PrP^Sc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs, whose epitopes comprise Met residues, readily detected PrP^C, but could not recognize most PrP^Sc bands unless they were vigorously reduced. Next, we showed that the α-Met pAbs did not recognize newly formed PrP^Sc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized. We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrP^C to PrP^Sc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.     

Cytochromes and gastric acid secretion A reevaluation of mucosal acidification experiments

Using an improved spectrophotometer, we have reinvestigated the report (Hersey, S.J. (1974) Biochim. Biophys. Acta 344, 157–203) that acidification of the mucosal surface of frog gastric mucosa produces a crossover point between flavoprotein and cytochrome $\text{b}$, thus identifying a site of energy coupling between the cytochrome and H⁺ transport systems. While we find spectrophotometric changes upon addition of HCl to the mucosal solution, we find similar changes upon addition of NaCl without pH change, but no changes when the pH is lowered by substitution of H⁺ for Na⁺ at constant osmolality. We show that osmolality changes, with consequent alteration in tissue light scattering, are responsible for these effects. Further, we can show that the pH changes used do not inhibit acid secretion, and that one cannot do so without osmolality increase. We conclude that the imputed crossover point is not demonstrated, and that models based on its existence must be revised.     

Tumor-initiating cells are not enriched in cisplatin-surviving BRCA1;p53-deficient mammary tumor cells in vivo

Although many breast cancers respond to chemotherapy or hormonal therapy, lack of tumor eradication is a central clinical problem preceding the development of drug resistant tumors. Using the K14cre;Brca1^F5-13/F5-13;p53^F2-10/F2-10mouse model for hereditary breast cancer, we have previously studied responses of mammary tumors generated in to clinically relevant anti-cancer drugs, including cisplatin. The BRCA1- and p53-deficient tumors generated in this model are hypersensitive to cisplatin and never become resistant to this agent due to the large, irreversible deletion in Brca1. We show here that even dose-dense treatment with a maximum tolerated dose of cisplatin does not result in complete tumor eradication. To explain this result we have addressed the hypothesis that the lack of eradication of drug-sensitive tumors is due to increased in vivo chemotherapy resistance of tumor-initiating cells (TICs). Using the CD24 and CD49f cell surface markers which detect normal mouse mammary stem cells, we have identified tumor-initiating cells in BRCA1- and p53-deficient tumors. In addition to the "OLE_LINK14">Lin^-/CD24⁺/CD49f⁺ subpopulation, we show that a larger population of Lin^-/CD24⁺/CD49f^- cells also has tumor-initiating capability in at least two serial orthotopic transplantations, suggesting that these are not more differentiated transit-amplifying cells. However, we did not find an enrichment of TICs in cisplatin-treated tumor remnants. We conclude that in this model the tolerance of the cisplatin-surviving cells cannot be attributed to special biochemical defense mechanisms of TICs.

     

Activity-dependent modulation of neuronal KV channels by retinoic acid enhances CaV channel activity

The metabolite of vitamin A, retinoic acid (RA), is known to affect synaptic plasticity in the nervous system and to play an important role in learning and memory. A ubiquitous mechanism by which neuronal plasticity develops in the nervous system is through modulation of voltage-gated Ca²⁺ (Ca_V) and voltage-gated K⁺ channels. However, how retinoids might regulate the activity of these channels has not been determined. Here, we show that RA modulates neuronal firing by inducing spike broadening and complex spiking in a dose-dependent manner in peptidergic and dopaminergic cell types. Using patch-clamp electrophysiology, we show that RA-induced complex spiking is activity dependent and involves enhanced inactivation of delayed rectifier voltage-gated K⁺ channels. The prolonged depolarizations observed during RA-modulated spiking lead to an increase in Ca²⁺ influx through Ca_V channels, though we also show an opposing effect of RA on the same neurons to inhibit Ca²⁺ influx. At physiological levels of Ca²⁺, this inhibition is specific to Ca_V2 (not Ca_V1) channels. Examining the interaction between the spike-modulating effects of RA and its inhibition of Ca_V channels, we found that inhibition of Ca_V2 channels limits the Ca²⁺ influx resulting from spike modulation. Our data thus provide novel evidence to suggest that retinoid signaling affects both delayed rectifier K⁺ channels and Ca_V channels to fine-tune Ca²⁺ influx through Ca_V2 channels. As these channels play important roles in synaptic function, we propose that these modulatory effects of retinoids likely contribute to synaptic plasticity in the nervous system.     

Are human faces and voices ornaments signaling common underlying cues to mate value?

In our daily lives, we constantly interact with people. We maintain relationships with families and friends. We collaborate with colleagues. We seek passion with our lovers and avoid conflicts with our enemies. How we divide the world into these and many other categories of people is initially guided by our first impressions of how they look and sound. Many times we are surprised when we hear someone on the phone whom we have not yet met face‐to face; they sound different from what we imagined. There are, however, many things that we are not surprised about in such situations. People are accurate at identifying sex, health, emotions, and age by both voices and faces.^3–12 There is good evidence that many seemingly disparate ornaments such as body and face,¹³ body and voice,^14–16 and face and odor¹⁹ may convey either backup signals of the same underlying quality^20,21 or convey signals of different underlying qualities that are used in conjunction to provide a more robust view of the organism's overall fitness.^22,23 Is this also true of face and voices? Until recently, little attention has been given to the idea that people's faces and voices might both signal the same underlying qualities related to hormone levels, and that we might use these hormonal fitness markers to provide a better picture of the signaler's overall mate value.^20,21,24 In this paper I first argue that aspects of voices and faces can be used as markers of hormonal status. Second, I argue that both vocal and facial features associated with hormonal status are used by people to assess mate quality.     

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

All mitochondrial tRNAs in Trypanosoma brucei derive from cytosolic tRNAs that are in part imported into mitochondria. Some trypanosomal tRNAs are thiolated in a compartment-specific manner. We have identified three proteins required for the thio modification of cytosolic tRNA^Gln, tRNA^Glu, and tRNA^Lys. RNA interference-mediated ablation of these proteins results in the cytosolic accumulation non-thio-modified tRNAs but does not increase their import. Moreover, in vitro import experiments showed that both thio-modified and non-thio-modified tRNA^Glu can efficiently be imported into mitochondria. These results indicate that unlike previously suggested the cytosol-specific thio modifications do not function as antideterminants for mitochondrial tRNA import. Consistent with these results we showed by using inducible expression of a tagged tRNA^Glu that it is mainly the thiolated form that is imported in vivo. Unexpectedly, the imported tRNA becomes dethiolated after import, which explains why the non-thiolated form is enriched in mitochondria. Finally, we have identified two genes required for thiolation of imported tRNA^Trp whose wobble nucleotide is subject to mitochondrial C to U editing. Interestingly, down-regulation of thiolation resulted in an increase of edited tRNA^Trp but did not affect growth.     

Prion formation,but not clearance,is supported by protein misfolding cyclic amplification

Prion diseases are fatal transmissible neurodegenerative disorders that affect animals including humans. The kinetics of prion infectivity and PrP^Sc accumulation can differ between prion strains and within a single strain in different tissues. The net accumulation of PrP^Sc in animals is controlled by the relationship between the rate of PrP^Sc formation and clearance. Protein misfolding cyclic amplification (PMCA) is a powerful technique that faithfully recapitulates PrP^Sc formation and prion infectivity in a cell-free system. PMCA has been used as a surrogate for animal bioassay and can model species barriers, host range, strain co-factors and strain interference. In this study we investigated if degradation of PrP^Sc and/or prion infectivity occurs during PMCA. To accomplish this we performed PMCA under conditions that do not support PrP^Sc formation and did not observe either a reduction in PrP^Sc abundance or an extension of prion incubation period, compared to untreated control samples. These results indicate that prion clearance does not occur during PMCA. These data have significant implications for the interpretation of PMCA based experiments such as prion amplification rate, adaptation to new species and strain interference where production and clearance of prions can affect the outcome.     

A comparison of Zn2+- and Ca2+-triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition

Intracellular Zn²⁺ toxicity is associated with mitochondrial dysfunction. Zn²⁺ depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn²⁺-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn²⁺-induced depolarization with the effects of Ca²⁺ in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca²⁺ caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg²⁺, ADP and cyclosporine A. Zn²⁺ also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn²⁺-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca²⁺ and Zn²⁺ in a calcein-retention assay. Consistent with the well-documented ability of Ca²⁺ to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn²⁺-treated mitochondria. Considered together, our results suggest that Ca²⁺ and Zn²⁺ depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn²⁺-induced depolarization, and that Zn²⁺ is not a particularly potent mitochondrial inhibitor.     

Analysis of putative miRNA function using a novel approach, GAPPS-miRTarGE

Deciphering the function of miRNA is one of the most important research subjects directed toward understanding the regulation of gene expression. Several experimental methodologies and bioinformatics programs have been developed, however, elucidating miRNA function has not been an easy task. Herein, we suggest a new method, GAPPS-miRTar^GE, which is a novel methodology for predicting miRNA function based on the proportion of mRNA targets expressed during embryonic developmental stages, the Theilers stages (TS), in mice. GAPPS-miRTar^GE is essentially a computational approach that groups miRNAs using shared expression patterns of their target genes during the 28 different TS. In this study, we present not only several examples derived from the GAPPS-miRTar^GE analyses that confirm previously known miRNA functions but also examples of function prediction for valid but functionally unknown miRNAs. Furthermore, we show that tissue-centered GAPPS-miRTar^GE, such as brain-centered or heart-centered, is useful for predicting miRNA function on a more detailed level.