Tafamidis delays neurological progression comparably across Val30Met and non-Val30Met genotypes in transthyretin familial amyloid polyneuropathy.

BACKGROUND AND PURPOSE: To better characterize the effects of tafamidis in non-Val30Met patients with transthyretin familial amyloid polyneuropathy, this post hoc analysis compared the neurological results from a 12-month, open-label study of non-Val30Met versus Val30Met patients at month 12 from the 18-month, double-blind, placebo-controlled registration study. A baseline covariate adjusted analysis was used to control for differences in baseline neurological severity. METHODS: Neurological function was assessed using the Neuropathy Impairment Score - Lower Limbs (NIS-LL) in three cohorts: Val30Met tafamidis (n = 64), Val30Met placebo (n = 61) and non-Val30Met tafamidis (n = 21). The change in NIS-LL from baseline to month 12 for Val30Met and non-Val30Met tafamidis-treated patients was compared with the change from baseline at month 12 for Val30Met placebo-treated patients using a mixed-effects model for repeated measures (MMRM). RESULTS: The baseline adjusted mean (standard error) change in NIS-LL values at month 12 was similar for Val30Met [1.60 (0.78)] and non-Val30Met [1.62 (1.43)] tafamidis-treated patients and less than that observed in the Val30Met placebo-treated group [4.72 (0.77); P = 0.0055 for Val30Met and P = 0.0592 for non-Val30Met]. Based on the MMRM, the magnitude of change in both tafamidis-treated cohorts was similar across the range of observed baseline NIS-LL values, and was consistently less than that observed in the Val30Met placebo-treated group at month 12. CONCLUSIONS: This baseline-adjusted analysis demonstrated that tafamidis treatment delayed neurological progression comparably in Val30Met and non-Val30Met patients across a range of baseline NIS-LL values. Neurological progression in these two genotype groups may be more similar than previously considered.

Role of SNAREs and H+-ATPase in the targeting of proton pump-coated vesicles to collecting duct cell apical membrane.

Recycling of H(+)-ATPase to the apical plasma membrane, mediated by vesicular exocytosis and endocytosis, is an important mechanism for controlling H(+) secretion by the collecting duct. We hypothesized that SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins) may be involved in the targeting of H(+)-ATPase-coated vesicles. Using a tissue culture model of collecting duct H(+) secretory cells (inner medullary collecting duct (IMCD) cells), we demonstrated that they express the proteins required for SNARE-mediated exocytosis and form SNARE-fusion complexes upon stimulation of H(+)-ATPase exocytosis. Furthermore, exocytic amplification of apical H(+)-ATPase is sensitive to clostridial toxins that cleave SNAREs and thereby inhibit secretion. Thus, SNAREs are critical for H(+)-ATPase cycling to the plasma membrane. The process in IMCD cells has a feature distinct from that of neuronal cells: the SNARE complex includes and requires the vesicular cargo (H(+)-ATPase) for targeting. Using chimeras and truncations of syntaxin 1, we demonstrated that there is a specific cassette within the syntaxin 1 H3 domain that mediates binding of the SNAREs and a second distinct H3 region that binds H(+)-ATPase. Utilizing point mutations of the B1 subunit of the H(+)-ATPase, we document that this subunit contains specific targeting information for the H(+)-ATPase itself. In addition, we found that Munc-18-2, a regulator of exocytosis, plays a multifunctional role in this system: it regulates SNARE complex formation and the affinity of syntaxin 1 for H(+)-ATPase.

A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis.

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.

Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells.

The events that precipitate cell death and the stress proteins responsible for cytoprotection during ATP depletion remain elusive. We hypothesize that exposure to metabolic inhibitors damages mitochondria, allowing proapoptotic proteins to leak into the cytosol, and suggest that heat stress-induced hsp72 accumulation prevents mitochondrial membrane injury. To test these hypotheses, renal epithelial cells were transiently ATP depleted with sodium cyanide and 2-deoxy-D-glucose in the absence of medium dextrose. Recovery from ATP depletion was associated with the release into the cytosol of cytochrome c and apoptosis-inducing factor (AIF), proapoptotic proteins that localize to the intermitochondrial membrane space. Concomitant with mitochondrial cytochrome c leak, a seven- to eightfold increase in caspase 3 activity was observed. In controls, state III mitochondrial respiration was reduced by 30% after transient exposure to metabolic inhibitors. Prior heat stress preserved mitochondrial ATP production and significantly reduced both cytochrome c release and caspase 3 activation. Despite less cytochrome c release, prior heat stress increased binding between cytochrome c and hsp72. The present study demonstrates that mitochondrial injury accompanies exposure to metabolic inhibitors. By reducing outer mitochondrial membrane injury and by complexing with cytochrome c, hsp72 could inhibit caspase activation and subsequent apoptosis.

c-Src and HSP72 interact in ATP-depleted renal epithelial cells.

Disruption of cell contact sites during ischemia contributes to the loss of organ function in acute renal failure. Because prior heat stress protects cell contact sites in ATP-depleted renal epithelial cells in vitro, we hypothesized that heat shock protein 72 (HSP72), the major inducible cytoprotectant in mammalian cells, interacts with protein kinases that regulate cell-cell and cell-matrix interactions. ATP depletion increased the content of Tyr(416) Src, the activated form of this kinase. c-Src activation was associated with an increase in the tyrosine phosphorylation state of beta-catenin, paxillin, and vinculin, three c-Src substrate proteins that localize to and regulate cell contact sites. Prior heat stress inhibited c-Src activation and decreased the degree of tyrosine phosphorylation of all three Src substrates during ATP depletion and/or early recovery. HSP72 coimmunoprecipitated with c-Src only in cells subjected to heat stress. ATP depletion markedly increased the interaction between HSP72 and c-Src, supporting the hypothesis that HSP72 regulates Src kinase activity. These results suggest that alterations in the tyrosine phosphorylation state of proteins located at the cell-cell and cell-matrix interface mediate, at least in part, the functional state of these structures during ATP depletion and may be modulated by interactions between HSP72 and c-Src.

A pertussis toxin-sensitive 8-lipoxygenase pathway is activated by a nicotinic acetylcholine receptor in aplysia neurons.

Acetylcholine (ACh) activates two types of chloride conductances in Aplysia neurons that can be distinguished by their kinetics and pharmacology. One is a rapidly desensitizing current that is blocked by alpha-conotoxin-ImI and the other is a sustained current that is insensitive to the toxin. These currents are differentially expressed in Aplysia neurons. We report here that neurons that respond to ACh with a sustained chloride conductance also generate 8-lipoxygenase metabolites. The sustained chloride conductance and the activation of 8-lipoxygenase have similar pharmacological profiles. Both are stimulated by suberyldicholine and nicotine, and both are inhibited by alpha-bungarotoxin. Like the sustained chloride conductance, the activation of 8-lipoxygenase is not blocked by alpha-conotoxin-ImI. In spite of the similarities between the metabolic and electrophysiological responses, the generation of 8-lipoxygenase metabolites does not appear to depend on the ion current since an influx of chloride ions is neither necessary nor sufficient for the formation of the lipid metabolites. In addition, the application of pertussis toxin blocked the ACh-activated release of arachidonic acid and the subsequent production of 8-lipoxygenase metabolites, yet the ACh-induced activation of the chloride conductance is not dependent on a G protein. Our results are consistent with the idea that the nicotinic ACh receptor that activates the sustained chloride conductance can, independent of the chloride ion influx, initiate lipid messenger synthesis.

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells.

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.

Structure and expression of the Aplysia polyubiquitin gene.

The ubiquitin-proteasome pathway, which is up-regulated in response to sensitizing treatments with serotonin (5-HT), plays a critical role in inducing long-term facilitation (LTF) of sensory-to-motor synapses in Aplysia. We characterized the structure of the polyubiquitin gene of Aplysia and studied its expression. At least six ubiquitin coding units exist in tandem, one of which encodes a protein with an amino acid sequence identical to human ubiquitin. Although the synthesis of polyubiquitin is induced by strong stimuli in many organisms, we found that the expression of ubiquitin in Aplysia is not affected by protocols that produce LTF.

The human chin revisited: what is it and who has it?

Although the presence of a "chin" has long been recognized as unique to Homo sapiens among mammals, both the ontogeny and the morphological details of this structure have been largely overlooked. Here we point out the essential features of symphyseal morphology in H. sapiens, which are present and well-defined in the fetus at least as early as the fifth gestational month. Differences among adults in expression of these structures, particularly in the prominence of the mental tuberosity, are developmental epiphenomena and serve to emphasize the importance of studying this region in juveniles whenever possible. A survey of various middle to late Pleistocene fossil hominids for which juveniles are known reveals that these features are present in some late Pleistocene specimens assigned to H. sapiens, but not in all of the presumed anatomically modern H. sapiens (i.e., Qafzeh 8, 9, and 11). The adult specimens from Skhul, as well as the adult Qafzeh 7 specimen, are similarly distinctive in symphyseal morphology. Neanderthals are quite variable in their own right, and they as well as other middle to late Pleistocene fossils lack the symphyseal features of H. sapiens. Some of the latter are, however, seen in the Tighenif (Ternifine) mandibles.

Activation and degradation of the transcription factor C/EBP during long-term facilitation in Aplysia.

Long-term facilitation (LTF) of the sensory-to-motor synapses that mediate defensive reflexes in Aplysia requires induction of the transcription factor Aplysia CCAAT/enhancer binding protein (ApC/EBP) as an early response gene. We examined the time course of ApC/ EBP DNA binding during the induction of LTF: Binding activity was detected within 1 h of the sensitization treatment with serotonin, reached a maximum at 2 h, and decreased after 6 h. How are DNA binding and the turnover of ApC/EBP regulated? We find that phosphorylation of ApC/EBP by mitogen-activated protein (MAP) kinase is essential for binding. MAP kinase appears to be activated through protein kinase C. We also showed that ApC/EBP is degraded through the ubiquitin-proteasome pathway but that phosphorylation by MAP kinase renders it resistant to proteolysis. Thus, phosphorylation by MAP kinase is required for ApC/EBP to act as a transcription activator as well as to assure its stability early in the consolidation phase, when genes essential for the development of LTF begin to be expressed.

Purinergic modulation of Na(+),K(+),Cl(-) cotransport and MAP kinases is limited to C11-MDCK cells resembling intercalated cells from collecting ducts.

We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na(+),K(+), Cl(-) cotransport is inhibited up to 50% by ATP via its interaction with P(2Y) purinoceptors (Biochim. Biophys. Acta 1998. 1369:233-239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na(+),K(+),Cl(-) cotransport to purinergic regulation. We did not observe any effect of ATP on Na(+),K(+),Cl(-) cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and rat collecting ducts ATP decreased the carrier's activity by approximately 30%. ATP did not affect Na(+),K(+),Cl(-) cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to approximately 85% inhibition of this carrier in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP(3) and cAMP production and transient elevation of [Ca(2+)](i). In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in C11-MDCK only. Thus, our results reveal that regulation of renal Na(+),K(+),Cl(-) cotransport by P(2Y) receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in purinergic control of this ion carrier's activity.

ATP depletion increases tyrosine phosphorylation of beta-catenin and plakoglobin in renal tubular cells.

This study examines the hypothesis that the loss of integrity of the junctional complex induced by ATP depletion is related to alterations in tyrosine phosphorylation of the adherens junction proteins beta-catenin and plakoglobin. ATP depletion of cultured mouse proximal tubular (MPT) cells induces a marked increase in tyrosine phosphorylation of both beta-catenin and plakoglobin. The tyrosine phosphatase inhibitor vanadate has the same effect in ATP-replete (control) monolayers, whereas genistein, a tyrosine kinase inhibitor, reduces phosphorylation of both proteins in ATP-replete monolayers and prevents the hyperphosphorylation of these proteins with ATP depletion. This study also demonstrates that the fall in the transepithelial resistance of MPT monolayers induced by ATP depletion can be reproduced by treatment of ATP-replete monolayers with vanadate, whereas genistein substantially ameliorates the fall in transepithelial resistance induced by ATP depletion. Also, using immunofluorescence microscopy it was demonstrated that ATP depletion results in a marked diminution of E-cadherin staining in the basolateral membrane of MPT cells. Vanadate mimics this effect of ATP depletion, whereas genistein ameliorates the reduction in the intensity of E-cadherin staining induced by ATP depletion. Because it is has been well established that hyperphosphorylation of the catenins leads to dissociation of the adherens junction and to dysfunction of the junctional complex, it is proposed that the increase in tyrosine phosphorylation of catenins observed in MPT cells during ATP depletion contributes to the loss of function of the junctional complex associated with sublethal injury.

SNARE proteins regulate H(+)-ATPase redistribution to the apical membrane in rat renal inner medullary collecting duct cells.

The interaction of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins provides the necessary steps for vesicle docking fusion. In inner medullary collecting duct (IMCD) cells, acid secretion is regulated in part by exocytotic insertion and endocytotic retrieval of an H(+)-ATPase to and from the apical membrane. We previously suggested a role for SNARE proteins in exocytotic insertion of proton pumps in IMCD cells. The purpose of the present study was to determine whether SNARE proteins are associated with the 31-kDa subunit of H(+)-ATPase in IMCD cells during exocytosis and to determine the effects of clostridial toxins on SNARE-mediated trafficking of H(+)-ATPase. Cell acidification induced a marked increment of H(+)-ATPase in the apical membrane. However, pretreating cells with clostridial toxins blocked the cellular translocation of the 31-kDa subunit. Immunoprecipitation of IMCD cell homogenate, using antibodies against either the 31-kDa subunit of H(+)-ATPase or vesicle-associated membrane protein-2, co-immunoprecipitated N-ethylmaleimide-sensitive factor, alpha-soluble NSF attachment protein (alpha-SNAP), synaptosome-associated protein-23, syntaxin, and vesicle-associated membrane protein-2. Pretreatment with clostridial toxin resulted in reduced co-immunoprecipitation of H(+)-ATPase and syntaxin. These experiments document, for the first time, a putative docking fusion complex in IMCD cells and a physical association of the H(+)-ATPase with the complex. The sensitivity to the action of clostridial toxin indicates the docking-fusion complex is a part of the exocytotic mechanism of the proton pump.

Homeobox genes, fossils, and the origin of species.

Ever since Darwin there has been a history of debate on the tempo and mode of evolution. Is speciation a gradual process involving the accumulation of minute variations extant within a species, or is it rapid, the result of major organismal reorganization? Does one define a species on the basis of genes, morphology, or geographic or reproductive isolation? In this communication I present a model of evolutionary change that is based on the Mendelian inheritance of mutations in regulatory genes and the fact that most nonlethal mutations arise in the recessive state. Since the new recessive allele will spread through many generations without expression until there is a critical mass of heterozygotes capable of producing homozygotes for the mutation, the novel feature thus produced will appear abruptly in the population and in more than one individual. This picture of punctuation is consistent with the fossil record, which typically fails to provide evidence of smoothly transitional states of morphological change. Given that the first of their kind in the fossil record are organisms in which their novel characteristics are often more fully expressed or complex than in their descendants, it would seem that, after the mutation involving a regulatory gene is introduced, the general tendency is for its effects to become diminished. Among the implications for speciation is that this process does not depend on either reproductive isolation or genetic incompatibility. Rather, barring effects on reproductive organs or behavior, homozygotes for a novelty should be able to breed with heterozygotes and homozygotes for the wild state of the original population. This, in turn, suggests that the species barrier between individuals is probably a matter of mate recognition.