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Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

Voltage-gated sodium (NaV) channels contain an α-subunit incorporating the channel’s pore and gating machinery composed of four homologous domains (DI–DIV), with a pore domain formed by the S5 and S6 segments and a voltage-sensor domain formed by the S1–S4 segments. During a membrane depolarization movement, the S4s in the voltage-sensor domains exert downstream effects on the S6 segments to control ionic conductance through the pore domain. We used lidocaine, a local anesthetic and antiarrhythmic drug, to probe the role of conserved Asn residues in the S6s of DIII and DIV in NaV1.5 and NaV1.4. Previous studies have shown that lidocaine binding to the pore domain causes a decrease in the maximum gating (Qmax) charge of ∼38%, and three-fourths of this decrease results from the complete stabilization of DIII-S4 (contributing a 30% reduction in Qmax) and one-fourth is due to partial stabilization of DIV-S4 (a reduction of 8–10%). Even though substitutions for the Asn in DIV-S6 in NaV1.5, N1764A and N1764C, produce little ionic current in transfected mammalian cells, they both express robust gating currents. Anthopleurin-A toxin, which inhibits movement of DIV-S4, still reduced Qmaxby nearly 30%, a value similar to that observed in wild-type channels, in both N1764A and N1764C. By applying lidocaine and measuring the gating currents, we demonstrated that Asn residues in the S6s of DIII and DIV are important for coupling their pore domains to their voltage-sensor domains, and that Ala and Cys substitutions for Asn in both S6s result in uncoupling of the pore domains from their voltage-sensor domains. Similar observations were made for NaV1.4, although substitutions for Asn in DIII-S6 showed somewhat less uncoupling.

A personal account of the author's experiences as a U.S. Civil War reenactor is presented, examining his early interest in the war growing up in Huntersville, West Virginia, his work as an historical interpreter at Point Park on Lookout Mountain, Tennessee, and his reenacting with Company A of the Confederate 36th Virginia Volunteer Infantry.

The XenopusCripto-1 protein is found only in cells comprising the animal hemisphere during embryogenesis because the Cripto-1 mRNA is translationally repressed in the vegetal hemisphere. This paper demonstrates that the RNA binding protein, Bicaudal-C, is responsible for the translational repression. Repression is cap-dependent but not poly(A)-dependent.

We show that microRNA-427 (miR-427) mediates the rapid deadenylation of maternal mRNAs after the midblastula transition (MBT) of Xenopus laevis embryogenesis. By MBT, the stage when the embryonic cell cycle is remodeled and zygotic transcription of mRNAs is initiated, each embryo has accumulated ∼109molecules of miR-427 processed from multimeric pri-miR-427 transcripts synthesized after fertilization. We demonstrate that the maternal mRNAs for cyclins A1 and B2 each contain a single miR-427 target sequence, spanning less than 30 nucleotides, that is both necessary and sufficient for deadenylation, and that inactivation of miR-427 leads to stabilization of the mRNAs. Although this deadenylation normally takes place after MBT, exogenous miRNAs produced prematurely in vivo can promote deadenylation prior to MBT, indicating that turnover of the maternal mRNAs is limited by the amount of accumulated miR-427. Injected transcripts comprised solely of the cyclin mRNA 3' untranslated regions or bearing a 5' ApppG cap undergo deadenylation, showing that translation of the targeted RNA is not required. miR-427 is not unique in promoting deadenylation, as an unrelated miRNA, let-7, can substitute for miR-427 if the reporter RNA contains an appropriate let-7 target site. We propose that miR-427, like the orthologous miR-430 of zebrafish, functions to down-regulate expression of maternal mRNAs early in development.

Sirolimus (rapamycin) is an immunosuppressant used in preventing allograft rejection and in drug-eluting stents to prevent restenosis after angioplasty. Zotarolimus, an analogue of sirolimus, was designed to have a shorter in vivo half-life. Zotarolimus was found to be mechanistically similar to sirolimus in having high-affinity binding to the immunophilin FKBP12 and comparable potency for inhibiting in vitro proliferation of both human and rat T cells. Rat pharmacokinetic studies with intravenous dosing demonstrated terminal elimination half-lives of 9.4 hours and 14.0 hours for zotarolimus and sirolimus, respectively. Given orally, T1/2values were 7.9 hours and 33.4 hours, respectively. Consistent with its shorter duration, zotarolimus showed a corresponding and statistically significant 4-fold reduction in potency for systemic immunosuppression in 3 rat disease models. Pharmacokinetic studies in cynomolgus monkey underpredicted the half-life difference between zotarolimus and sirolimus apparent from recent clinical data. In vitro inhibition of human coronary artery smooth muscle cell proliferation by zotarolimus was comparable to sirolimus. Drug-eluting stents for local delivery of zotarolimus to the vessel wall of coronary arteries are in clinical development. The pharmacological profile of zotarolimus suggests it may be advantageous for preventing restenosis with a reduced potential for causing systemic immunosuppression or other side effects.

Cyclin mRNAs are unstable in the adult cell cycle yet are stable during the first 12 cell divisions in Xenopus laevis. We recently reported that cyclin A1 and B2 maternal mRNAs are deadenylated upon completion of the 12th division (Audic et al. [2001] Mol. Cell Biol. 21:1662–1671). Deadenylation is mediated by the 3' untranslated region (UTR) of the mRNA and precedes the terminal disappearance of the cyclin proteins, with both processes requiring zygotic transcription. The purpose of the current study was (1) to ask whether deadenylation leads to translational repression and/or destabilization of endogenous cyclin A1 and B2 mRNAs, and (2) to further characterize the regulatory sequences required. We show that zygote-driven deadenylation leads to translational repression and mRNA destabilization. A 99-nucleotide region of the 3'UTR of the cyclin A1 mRNA mediates both deadenylation and destabilization. Surprisingly, two AU-rich consensus elements within this region are dispensable for this activity. These results suggest that zygote-dependent deadenylation, translational repression, and mRNA destabilization by means of novel 3'UTR elements contribute to the disappearance of maternal cyclins. They also suggest that translational control of cyclins may play a role in the transition to the adult cell cycle. These data concur with previous studies in Drosophila showing that zygote-mediated degradation of maternal cdc25 mRNA may be a general mechanism whereby transition to the adult cell cycle proceeds. © 2002 Wiley-Liss, Inc.

Recent revisions in the Xenopus laevis fate map led to the designation of the rostral/caudal axis and reassignment of the dorsal/ventral axis (Lane and Smith [1999] Development 126:423–434; Lane and Sheets [2000] Dev. Biol. 225:37–58). It is unprecedented to reassign primary embryonic axes after many years of research in a model system. In this review, we use insights about vertebrate development from anatomy and comparative embryology, as well as knowledge about gastrulation in frogs, to reexamine several traditional amphibian fate maps. We show that four extant maps contain information on the missing rostral/caudal axis. These maps support the revised map as well as the designation of the rostral/caudal axis and reassignment of the dorsal/ventral axes. To illustrate why it is important for researchers to use the revised map and nomenclature when thinking about frog and fish embryos, we present an example of alternative interpretations of “dorsalized” zebrafish mutations. © 2002 Wiley-Liss, Inc.

We investigated the contribution the four outermost basic residues (K1, R2, R3, R4) in segment 4 of domain III in the human cardiac Na channel (hH1a, NaV1.5) to the total gating charge (Qmax). Each of the four basic residues were mutated individually to a cysteine. In addition, R2 was also mutated to a glutamate. All mutant channels were transiently expressed with the α1 subunit in fused tsA201 cells. We used the relative reduction in Qmax caused by anthopleurin-A (ApA) toxin, a site-3 toxin known to inhibit the movement of gating charge associated with domain IV, to estimate the size of the contribution from each basic residue. Studies of the toxin’s ability to inhibit gating charge in mutant channels showed that R2 contributed 19–20% to the Qmax, R3 contributed 10%, and K1 and R4 made almost no contribution. In contrast to the outermost basic residue in the S4 of Shaker K channels and in the S4 of domain IV in hH1a, the outermost charge (K1) in domain III of Na channels is outside the voltage field.

Xenopus laevisembryogenesis is controlled by the inducing activities of Spemann's organizer. These inducing activities are separated into two distinct suborganizers: a trunk organizer and a head organizer. The trunk organizer induces the formation of posterior structures by emitting signals and directing morphogenesis. Here, we report that the fibroblast growth factor receptor (FGFR) signaling pathway, also known to regulate posterior development, performs critical functions within the cells of Spemann's organizer. Specifically, the FGFR pathway was required in the organizer cells in order for those cells to induce the formation of somitic muscle and the pronephros. Since the organizer influences the differentiation of these tissues by emitting signals that pattern the mesodermal germ layer, our data indicate that the FGFR regulates the production of these signals. In addition, the FGFR pathway was required for the expression of chordin, an organizer-specific protein required for the trunk-inducing activities of Spemann's organizer. Significantly, the FGFR pathway had a minimal effect on the function of the head organizer. We propose that the FGFR pathway is a defining molecular component that distinguishes the trunk organizer from the head organizer by controlling the expression of organizer-specific genes required to induce the formation of posterior structures and somitic muscle in neighboring cells. The implications of our findings for the evolutionarily conserved role of the FGFR pathway in the functions of Spemann's organizer and other vertebrate-signaling centers are discussed.

Activation of the Xenopus bone morphogenetic protein (BMP) pathway is coincident with the onset of zygotic transcription but requires maternal signaling proteins. The mechanisms controlling the translation of mRNAs that encode proteins of the BMP pathway were investigated by using polysome association as an assay for translational activity. Our results indicate that five different mRNAs encoding proteins of the BMP pathway were translationally regulated during Xenopus development. These mRNAs were either not associated or inefficiently associated with polysomes in oocytes, and each was recruited to polysomes at a different developmental stage. The Smad1 and ALK-2 mRNAs were recruited to polysomes during oocyte maturation, whereas the BMP-7 and XSTK9 mRNAs were recruited during the early stages of embryogenesis. The ALK-3 mRNA was not efficiently associated with polysomes during any maternal stage of development and was efficiently recruited to polysomes only after the onset of zygotic transcription. In general, for all stages except oocytes, polysome recruitment was associated with the presence of a 3′ poly(A) tail. However, there was not an obvious correlation between the absolute length of poly(A) and the efficiency of polysome recruitment, indicating that the relationship between poly(A) tail length and translation during early frog embryogenesis is complex. We further focused on the BMP-7 mRNA and demonstrated that sequence elements within the 3′UTR were necessary for recruitment of the BMP-7 mRNA to polysomes and sufficient to direct the addition of poly(A) and activate translation of a reporter during embryogenesis. Interestingly, the BMP-7 mRNA lacks the previously defined eCPE sequences proposed to direct poly(A) addition and translational activation during embryogenesis. The implications of our findings for translational regulation of maternal mRNAs during embryogenesis and for the activation of the BMP pathway are discussed.

A new fate map for mesodermal tissues in Xenopus laevispredicted that the prime meridian, which runs from the animal pole to the vegetal pole through the center of Spemann's organizer, is the embryo's anterior midline, not its dorsal midline (M. C. Lane and W. C. Smith, 1999, Development126, 423–434). In this report, we demonstrate by lineage labeling that the column 1 blastomeres at st. 6, which populate the prime meridian, give rise to the anterior end of the embryo. In addition, we surgically isolate and culture tissue centered on this meridian from early gastrulae. This tissue forms a patterned head with morphologically distinct ventral and dorsal structures. In situhybridization and immunostaining reveal that the cultured heads contain the anterior tissues of all three germ layers, correctly patterned. Regardless of how we dissect early gastrulae along meridians running from the animal to the vegetal pole, both the formation of head structures and the expression of anterior marker genes always segregate with the prime meridian passing through Spemann's organizer. The prime meridian also gives rise to dorsal, axial mesoderm, but not uniquely, as specification tests show that dorsal mesoderm arises in fragments of the embryo which exclude the prime meridian. These results support the hypothesis that the midline that bisects Spemann's organizer is the embryo's anterior midline.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Qmax) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of INa was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of INa were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Qmax by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Qmax to Gmax was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.

To assess the duration of immunosuppression in FK506-dosed pigs, an undiluted whole blood assay was established to measure reactivities of T cells in their physiological milieu. PMA and ionomycin were shown to induce IL-2 production in swine blood. The IC₅₀ of FK506 in inhibiting IL-2 production in whole blood and isolated PBMC stimulated with PMA and ionomycin measured 1.2 and 0.04 nM, respectively. These data underscore the influence of red blood cells and plasma proteins on drug potency. IL-2 levels were determined in blood drawn immediately before and 1, 24, 48, and 72 h after iv dosing. For pigs dosed with 0.05 mg/kg, 50% recovery of IL-2 production was observed at 16 h and 100% at 35 h after dosing. For pigs dosed with 0.15 mg/kg, 50% recovery was observed at 38 h and 100% at 72 h. Blood concentrations of FK506 at 50 and 100% recovery of IL-2 production measured 10.8 and 2.2 nM for pigs dosed with 0.05 mg/kg and 6.1 and 1.1 nM for pigs dosed with 0.15 mg/kg, respectively. These concentrations are severalfold higher than predicted from the IC₅₀ of FK506 for inhibiting IL-2 production in the whole blood assay. These data suggest that the true potency of FK506 in blood after dosing is influenced by additional factors, which could include plasma protein binding, the presence of active or interfering metabolites, serum interfering factors, and sequestration of drug in blood cells. Our results demonstrate the utility of an undiluted whole blood assay for assessing the duration of immunosuppression in drug-dosed animals and emphasize the importance of assessing drug potency in the whole blood environment ex vivo. Copyright 1999 Academic Press.

1Sodium channel ionic current (INa) and gating current (Ig) were compared for rat skeletal (rSkM1) and human heart Na+channels (hH1a) heterologously expressed in cultured mammalian cells at ∼13 °C before and after modification by site‐3 toxins (Anthopleurin A and Anthopleurin B).2For hH1a Na+channels there was a concordance between the half‐points (V½) of the peak conductance‐voltage (G–V) relationship and the gating charge‐voltage (Q–V) relationship with no significant difference in half‐points. In contrast, the half‐point of the Q–Vrelationship for rSkM1 Na+channels was shifted to more negative potentials compared with its G–Vrelationship with a significant difference in the half‐points of −8 mV.3Site‐3 toxins slowed the decay of INain response to step depolarizations for both rSkM1 and hH1a Na+channels. The half‐point of the G–Vrelationship in rSkM1 Na+channels was shifted by −8.0 mV while toxin modification of hH1a Na+channels produced a smaller hyperpolarizing shift of the V½by −3.7 mV.4Site‐3 toxins reduced maximal gating charge (Qmax) by 33% in rSkM1 and by 31% in hH1a, but produced only minor changes in the half‐points and slope factors of their Q–Vrelationships. In contrast to measurements in control solutions, after modification by site‐3 toxin the half‐points of the G–Vand the Q–Vrelationships for rSkM1 Na+channels demonstrated a concordance similar to that for hH1a.5Qmaxvs. Gmaxfor rSkM1 and hH1a Na+channels exhibited linear relationships with almost identical slopes, as would be expected if the number of electronic charges (e−) per channel was comparable.6We conclude that the faster kinetics in rSkM1 channels compared with hH1a channels may arise from inherently faster rate transitions in skeletal muscle Na+channels, and not from major differences in the voltage dependence of the channel transitions.

The relationship between leaf cuticle thickness and resistance to the powdery mildew Erysiphe cichoracearumwas investigated for nine feral Phloxtaxa. Cuticle thickness, the first potential host resistance feature, is variable within plants, although not correlated with leaf age, and both within and among taxa. There is also considerable variation within and among taxa in resistance response to artificial inoculations with a broadly compatible E. cichoracearumstrain. However, no significant correlations between cuticle thickness and resistance were observed. The implications of these results for anti‐mildew defenses in both feral and cultivated hosts are discussed.

It has long been known that cardiac glycosides can inhibit the membrane sodium-potassium (Na+-K+) pump, raising intracellular Na+. However, at clinical concentrations of cardiac glycosides, a change in intracellular Na+that correlates with a change in cardiac contraction has been very difficult to demonstrate. The recent use of Na+-sensitive microelectrodes in the experimental laboratory has made intracellular Na+measurements possible. A doubling of contraction strength in vitro is associated with a change of only approximately 1 mMintracellular Na+. Another membrane transport system, the Na+-Ca2+exchange system, exchanges extracellular Na+ for intracellular Ca2+. If this system is responsible for regulating intracellular Ca2+, then it would be very sensitive to the transmembrane Na+ concentration gradient. This influence of intracellular Na+ on Na+-Ca2+exchange is thought to be the cellular basis of the positive inotropic action of digitalis. However, a number of issues remain unresolved, such as the extent of Na+-K+pump inhibition by the level of cardiac glycoside achieved clinically.

The sodium (Na) channel is the fundamental unit of excitability in heart muscle. This channel has been very difficult to study in detail, because the major experimental tool, the voltage clamp, has been difficult to use in multicellular tissue. In the absence of more direct studies in the heart, it has been assumed that the sodium channel in the heart was the same as that in nerve tissue, where it could be studied quantitatively. However, the sodium channel is not likely to be the same as in nerve, because it responds differently to local anesthetics and to other drugs such as tetrodotoxin. It is essential to learn the details of the cardiac sodium channel, because it is the membrane process that underlies many lethal cardiac arrhythmias, and it is the molecular site of action of the most effective antiarrhythmic drugs.