A novel TRPA1 variant is associated with carbamazepine-responsive cramp-fasciculation syndrome.

Cramp-fasciculation syndrome (CFS) is a rare muscle hyperexcitability syndrome that presents with muscle cramps, fasciculations, and stiffness, as well as pain, fatigue, anxiety, hyperreflexia, and paresthesias. Although familial cases have been reported, a genetic etiology has not yet been identified. We performed whole-exome sequencing followed by validation and cosegregation analyses on a father-son pair with CFS. Both subjects manifested other hypersensitivity-hyperexcitability symptoms, including asthma, gastroesophageal reflux, migraine, restless legs syndrome, tremor, cold hyperalgesia, and cardiac conduction defects. Most symptoms improved with carbamazepine, consistent with an underlying cation channelopathy. We identified a variant in the transient receptor potential ankyrin A1 channel (TRPA1) gene that selectively cosegregated with CFS and the other hypersensitivity-hyperexcitability symptoms. This variant (c.2755C>T) resulted in a premature stop codon at amino acid 919 (p.Arg919*) in the outer pore of the channel. TRPA1 is a widely distributed, promiscuous plasmalemmal cation channel that is strongly implicated in the pathophysiology of the specific hypersensitivity-hyperexcitability symptoms observed in these subjects. Thus, we have identified a novel TRPA1 variant that is associated with CFS as part of a generalized hypersensitivity-hyperexcitability disorder. These findings clarify the diverse functional roles of TRPA1, and underscore the importance of this channel as a potential therapeutic target.

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.

Fine structure of RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA.

Nucleotide sequences of 50 RNA codons recognized by amphibian and mammalian liver transfer RNA preparations were determined and compared with those recognized by Escherichia coli transfer RNA. Almost identical translations were obtained with transfer RNA from guinea pig liver, Xenopus laevis liver (South African clawed toad), and E. coli. However, guinea pig and Xenopus transfer RNA differ markedly from E. coli transfer RNA in relative response to certain trinucleotides. Transfer RNA from mammalian liver, amphibian liver, and amphibian muscle respond similarly to trinucleotide codons. Thus the genetic code is essentially universal, but transfer RNA from one organism may differ from that of another in relative response to some codons.

Detection of alpha-transducin in retinal rods but not cones.

The distribution in chicken retina of the alpha subunit of transducin, the guanine nucleotide--binding protein that couples light-dependent activation of rhodopsin with activation of guanosine 3',5'-monophosphate phosphodiesterase, was determined with the aid of a specific antiserum. alpha-Transducin was found in rod photoreceptor cells but was not detected in cones. These results show that rods and cones differ with respect to alpha-transducin content and suggest that the processes of phototransduction may differ correspondingly in rods and cones.

Synapse formation between two clonal cell lines.

Clonal neuroblastoma x glioma hybrid cells frequently formed synapses with clonal mouse striated muscle cells. Clonal myotubes were similar to cultured mouse embryo myotubes with respect to acetylcholine sensitivity and other membrane properties examined. However, acetylcholine sensitivity measurements indicate that acetylcholine receptors of clonal myotubes are distributed more uniformly over the cell surface than the receptors of cultured mouse embryo myotubes.

Sequential translation of trinucleotide codons for the initiation and termination of protein synthesis.

Terminal events in protein synthesis were studied with trinucleotide codons. Initiator and terminator trinucleotides sequentially stimulate N-formyl-methionyl-tRNA binding to ribosomes and the release of free N-formyl-methionine from the ribosomal intermediate. The release factor discovered by Capecchi is also required. The trinucleotides UGA, UAA, and UAG were found to be terminator codons. This pattern of codon degeneracy has not been observed with other trinucleotides and transfer RNA.

Modulation of synapse formation by cyclic adenosine monophosphate.

Synapses between neuroblastoma-hybrid cells and myotubes exhibit a high degree of plasticity. Increase of cyclic adenosine monophosphate (AMP) levels of the hybrid cells for several days results in the appearance of functional voltage-sensitive Ca2+ channels, which are required for evoked secretion of acetylcholine. The results show that cyclic AMP regulates synaptogenesis by regulating the expression of voltage-sensitive Ca2+ channels, and suggest that cyclic AMP affects posttranslational modifications of some glycoproteins and cellular levels of certain proteins.

RNA codons and protein synthesis. Xi. Template activity of modified RNA codons.

Substituting 5'-, 2'-, 3'-terminal or 2'-internal ribose hydroxyls of oligonucleotides markedly affected their template activity in directing the binding of AA-sRNA to ribosomes. The relative template activity of oligo U preparations was as follows: p-5'-UpUpU > UpUpU > CH3O-p-5'-UpUpU > UpUpU-3'-p > UpUpU-3'-p-OCH3 > UpUpU-2',3'-cyclic phosphate. Trimers with (2'-5') phosphodiester linkages, (2'-5')-UpUpU and also (2'-5')-ApApA, did not serve as templates for phenylalanine- or lysine-sRNA, respectively. The relative template efficiency of oligo A preparations was as follows: p-5'-ApApA > ApApA > ApApA-3'-p > ApApA-2'-p. The hexamer, ApApApApApA was considerably more active as a template than the corresponding pentamer. These data indicate that two adjacent triplets are recognized by two AA-sRNA molecules bound to nearby ribosomal sites. A doublet with 5'-terminal phosphate, pUpC, served as a template for serine-sRNA, whereas a doublet without terminal phosphate, UpC, did not. Although the template efficiency of pUpC was lower than that of the triplet UpCpU the data show that serine-sRNA can recognize pUpC.

The three-dimensional structure of the vnd/NK-2 homeodomain-DNA complex by NMR spectroscopy.

The three-dimensional solution structure obtained by NMR of the complex formed between the uniformly singly15N and doubly13C/15N-labeled vnd/NK-2 homeodomain and its consensus 16 base-pair DNA binding sequence was determined. This work was carried out using the accepted repertoire of experiments augmented with a novel implementation of the water flipback technique to enhance signals from exchangeable amide protons. The results using this new technique confirm the existence of hydrogen bonding between the invariant Asn51 and the second adenine of the DNA binding sequence, as seen in crystal structures of other homeodomain-DNA complexes, but never before detected by NMR. Hydrogen bonding by Arg5 and Lys3 in the minor groove of the DNA appears to be responsible for two unusually upfield-shifted ribose H1' resonances. The DNA duplex is nearly straight and its structure is primarily that of B -DNA. A detailed comparison is presented for all available homeodomain-DNA structures including the vnd/NK-2 DNA complex, which demonstrates that homology is maintained in the protein structure, whereas for the orientation of the homeodomain relative to DNA, small but significant variations are observed. Interactions are described involving certain residues in specific positions of the homeodomain, namely Leu7, Thr41, and Gln50 of vnd/NK-2, where single amino acid residue mutations lead to dramatic developmental alterations. The availability of our previously determined three- dimensional structure of the vnd/NK-2 homeodomain in the absence of DNA allows us to assess structural changes in the homeodomain induced by DNA binding.

The three-dimensional solution structure of the NK-2 homeodomain from Drosophila.

We describe the NMR determination of the three-dimensional structure of a 77 amino acid residue protein, which consists of the 60 residue NK-2 homeodomain from Drosophila melanogaster and adjacent amino acid residues. The NK-2 homeodomain protein is part of a 723 amino acid residue protein which is expressed early in embryonic development in part of the central nervous system. NK-2 was characterized using both a natural abundance and a uniformly 15N enriched sample by two-dimensional and three-dimensional NMR experiments. The average root-mean-square deviation for 30 structures for residues 8 to 53 is 0.40 A for the backbone heavy-atoms and 0.72 A for the backbone and side-chain heavy-atoms. These structures were obtained from 986 NOE-derived upper and lower bound restraints. The three-dimensional structure contains three helices which consist of homeodomain amino acid residues 10 to 22, 28 to 38 and 42 to 52, as well as a turn between helix II and III, characteristic of homeodomains. Residues 53 to 60 of the DNA recognition helix are not fully ordered in the absence of DNA. In the free state this segment adopts a flexible but helix-like structure between residues 53 and 56 and is disordered from residues 57 to 60 although, as shown previously, the helix elongates by eight residues upon binding to DNA. The role of variable residues 52, 54 and 56 in determining the structure and flexibility of the recognition helix, as well as the stability of the NK-2 homeodomain as manifested by its thermal denaturation, are discussed.

Immunocytochemical localization of the renal neutral and basic amino acid transporter in rat adrenal gland, brainstem, and spinal cord.

A neutral and basic amino acid transporter (NBAT) cloned from rat kidney was recently localized to enteroendocrine cells and enteric neurons. We used an antibody directed against a synthetic peptide representing a putative extracellular domain of NBAT to determine whether this transporter was also present in other endocrine and neural tissues, including rat adrenal gland, brainstem, and spinal cord. Abundant, highly granular labeling for NBAT was observed in the cytoplasm of chromaffin and ganglion cells in the adrenal medulla. A small population of intensely labeled varicose processes was also seen in both the cortex and the medulla of the adrenal gland. More numerous, intensely labeled varicose processes were detected in brainstem and spinal cord nuclei, including the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, dorsal motor nucleus of the vagus, and intermediolateral cell column of the thoracic spinal cord. Significant perikaryal labeling for NBAT was only detected in brainstem and spinal cord following intraventricular colchicine treatment, which increased the number, distribution, and intensity of NBAT-immunolabeled cells. These NBAT-immunoreactive perikarya were most numerous in the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, and raphe nuclei. Ultrastructural examination of the nuclei of the solitary tract of normal rats showed that NBAT was localized predominantly to axon terminals. Within these labeled terminals, NBAT was associated with large dense core vesicles and discrete segments of plasma membrane. The observed localization of NBAT suggests that this renal specific amino acid transporter subserves a role as a vesicular or plasmalemmal transporter in monoamine-containing cells, including chromaffin cells and autonomic neurons.

Regional and subcellular distribution of a neutral and basic amino acid transporter in forebrain neurons containing nitric oxide synthase.

The neutral and basic amino acid transporter (NBAT) facilitates sodium-independent transport of L-amino acids in renal and intestinal epithelial cells and has been postulated to play a similar role in neurons. In previous studies, NBAT has been detected within enteric and brainstem autonomic neurons in a distribution similar to that of constitutive nitric oxide synthase (cNOS). Furthermore, L-arginine, the required precursor for nitric oxide synthesis, is an excellent NBAT substrate. Together, these findings suggest that NBAT may play a role in the regulation of nitric oxide synthesis, through the control of precursor availability. To gain insight into the potential physiological role of NBAT in central neurons, we used an antipeptide antiserum to examine the light and electron microscopic immunocytochemical localization of NBAT in the rat forebrain and to compare this distribution with that of cNOS. Immunolabeling for NBAT was detected within perikarya and dendrite-like processes that were most numerous in the frontal and cingulate cortex, the ventral striatum, the central amygdala, and the bed nucleus of the stria terminalis. Labeled varicose axonal processes were distributed most densely in the agranular insular cortex and the paraventricular nuclei of the thalamus and hypothalamus (PVH). Electron microscopy showed that immunogold labeling for NBAT was distributed along plasmalemmal and vacuolar membranes within somata, dendrites, and axonal profiles. Many of the NBAT-containing somata and dendrites contained detectable cNOS. Our results suggest that expression of NBAT may provide specific populations of cNOS-containing forebrain neurons with a unique mechanism for regulating somatodendritic synthesis of nitric oxide.

Control of opiate receptor-adenylate cyclase interactions by calcium ions and guanosine-5'-triphosphate.

Adenylate cyclase of homogenates of NG108-15 neuroblastoma x glioma hybrid cells is activated by low concentrations of Ca2+ ions and is inhibited by higher (greater than 0.1 mM) concentrations of Ca2+ ions. Activation of either opiate receptors by 10 microM morphine or alpha-adrenergic receptors by 10 microM norepinephrine inhibits adenylate cyclase by 55% in the absence of Ca2+ ions, and inhibits the Ca2+-dependent activation of adenylate cyclase by more than 90%. Concentrations of Ca2+ ions greater than 0.1 mM inhibit adenylate cyclase and also reduce the extent inhibition of adenylate cyclase by morphine but not by norepinephrine. Guanosine-5'-triphosphate (0.1-1 microM) is required for inhibition of adenylate cyclase by morphine. The results show that morphine inhibits adenylate cyclase by a guanosine-5-triphosphate-dependent process and that the extent of inhibition of adenylate cyclase by morphine or norepinephrine is a function of the Ca2+ ion concentration and the proportion of adenylate cyclase molecules that are activated or inhibited by Ca2+ ions.

Pharmacologic responses of cells of a neuroblastoma X glioma hybrid clone and modulation of synapses between hybrid cells and mouse myotubes.

Cells of the hybrid clone NG108-15 responded to 5-hydroxytryptamine (5-HT), dopamine or acetylcholine with graded depolarizations involving membrane conductance increases. Responses desensitized during continuous application of the neurotransmitters, and responses to 5-HT and dopamine cross-desensitized: a desensitizing application of one neurotransmitter also desensitized the hybrd cell to the other neurotransmitter. 5-HT and acetylcholine did not cross-desensitize. The hybrid cell 5-HT response was not attenuated by D-LSD, and was blocked by 10(-5) M morphine, although not via binding to naloxone-sensitive opiate receptors. 5-HT or the prostaglandin PGF2alpha caused the release of acetylcholine at the synapses of hybrid cells with mouse myotubes. Application of 5-HT or PGF2alpha also facilitated the synaptic release elicited by hybrid cell action potentials. Following treatment with the antimitotic agent cytosine arabinoside, co-cultures of hybrid cells and mouse myotubes exhibited plentiful synaptic connections only if maintained in medium containing 1 mM dibutyryl cAMP (dBcAMP). After X-irradiation, co-cultures were synaptically active even in the absence of dBcAMP. Thus, methods have been found to regulate both the short-term and long-term synaptic activity of NG108-15 hybrid cells.

Three cholinergic neuroblastoma hybrid cell lines that form few synapses on myotubes are deficient in acetylcholine receptor aggregation molecules and large dense core vesicles.

Three neuroblastoma X glioma hybrid cell lines that synthesize and release acetylcholine but that form few or no synapses with cultured skeletal muscle cells lack two characteristics of neuroblastoma or hybrid cell lines that do form many synapses with myotubes: large dense core vesicles and the ability to increase the number of nicotinic acetylcholine receptor clusters on co-cultured myotube membranes. Functional synapse formation on myotubes was increased by co-culturing myotubes and cells from one of the defective lines with neuroblastoma cells that induce myotube acetylcholine receptor aggregation but which synthesize little or no acetylcholine.

Formation of synapses between cells of a neuroblastoma X glioma hybrid clone and mouse myotubes.

Synapses form between cells of a neuroblastoma X glioma hybrid clone and cultured mouse skeletal myotubes. The synapses are cholinergic, and the acetylcholine release mechanism is dependent on calcium ions. The transmitter output of the synapses is low, with considerable variability in the latency and amplitude of the postsynaptic responses to presynaptic action potentials. The fine structure of physiologically identified functional junctions was examined electron microscopically. Small (50 nm) clear vesicles were seen presynaptically and there were areas with a wide (approx. 50 nm) gap containing basement membrane-like material between the pre- and postsynaptic cells. In addition, in some regions there was a densely staining material lining the muscle membrane and some suggestion of infolding of the muscle membrane. In none of the cases, however, have areas been found where small, clear vesicles cluster around pre- and postsynaptic membrane densities. Thus, functional synapses can occur in the absence of the highly organized synaptic structure seen at mature synapses.

Synapse selection based on differences in synapse turnover.

Rat retinal neurons formed transient synapses with rat muscle cells in culture only during a discrete period in development, from the 20th day of embryonic development to the 7th neonatal day. In contrast, chick embryo spinal cord neurons formed synapses at all developmental stages tested, from the 2nd to the 18th day of embryonic development. The percentage of cells from the spinal cord that formed synapses with muscle cells was maximum at 4 days of embryonic development and decreased thereafter. However, the number of synapses with muscle formed by cells from 8-day embryonic spinal cord did not decrease during 14 days of culture. Under identical conditions, all synapses formed between rat retinal neurons and muscle cells were terminated during 7 days of culture. These results show that differences in the rates of turnover of two populations of cholinergic synapses can result in the selective retention of one population of synapses and the loss of the other, and thereby alter the specificity of synaptic connections.

Forensic methods and the podiatric physician.

This is an introductory study of forensic podiatry. To elevate forensic podiatry to the level of forensic odontology and forensic anthropology, the podiatric medical profession must begin educational programs and research. A system for monitoring the activities of podiatrists involved in forensic medicine must be established to ensure that the high degree of integrity to which the profession is committed is maintained. By following these guidelines, the author believes that sometime in the future a podiatrist will be on the staff of every major police department in the country. At that point, the podiatric medical profession will have achieved unsurpassed status, recognition, and prestige.