Modulation of Na+-channels by neurotoxins produces different effects on [3H]ACh release with mobilization of distinct Ca2+-channels

1. Voltage-gated Na⁺ channels are responsible for initiation and conduction of action potentials. The arrival of an action potential at nerve terminal increases intracellular Na⁺ and Ca²⁺ concentrations. Calcium entry into neurons through voltage-dependent calcium channels is associated with a variety of intracellular processes. Scorpion neurotoxins have been used as tools to investigate mechanisms involved in neurotransmitter release. Tityustoxin (TsTX) is an -type toxin that delays Na⁺-channel inactivation. Toxin- (TiTX-) is a -type toxin that induces Na⁺-channel activation at resting potentials.2. In the present work, we describe the effects of both toxins on [³H]acetylcholine ([³H]ACh) release from rat cerebrocortical synaptosomes, in the presence or absence of the calcium channels blockers: -conotoxin-GVIA (-CgTx), 1 M; -agatoxin-IVA (-Aga), 30 nM; -conotoxin-MVIIC (-MVIIC), 1 M; or verapamil, 1M.3. TsTX evokes [³H]ACh release in a concentration-dependent manner with a gradual increase up to saturation at concentrations of 500 nM. However, release of ACh evoked by TiTX- was not linear regarding the toxin concentration. The [³H]-ACh release evoked by TsTX or TiTX- was partially inhibited by -CgTx or -Aga, and blocked with -MVIIC. Verapamil (1 M) had no effect. Tetrodotoxin blocked [³H]ACh release evoked by both toxins.4. These results show that different actions on Na⁺-channels produce different effects on [³H]ACh release with involvement of distinct presynaptic Ca²⁺-channels, which supports the idea that sodium channels may modulate neurotransmitter release.     

Differences of the electrical and nicotinic receptor stimulation-evoked liberation of norepinephrine from chicken sympathetic neurons in culture: Possible involvement of different pools of the transmitter

We studied the release of [³H]norepinephrine from chicken sympathetic neurons in culture evoked by nicotinic and electrical stimulation with an intention to establish functional identity or nonidentity of the two stimuli in investigations of neurotransmitter release. Nicotinic stimulation evoked extracellular calcium dependent release of [³H]norepinephrine and the rise of intracellular calcium concentration. The release was completely blocked by nicotinic antagonists hexamethonium (100 mol/l) and mecamylamine (10 mol/l), and decreased by tetrodotoxin (0.3 mol/l) and -conotoxin (0.1 mol/l) to 17% and 27%, resp. The intracellular calcium response was decreased by nicotinic antagonists and tetrodotoxin, but not changed by -conotoxin. The electrical stimulation-evoked release was blocked by both tetrodotoxin and -conotoxin, and decreased by previous electrical, but not nicotinic, stimulation. The differential sensitivity to -conotoxin and tetrodotoxin, and the inability of nicotinic stimulation to decrease the liberation by following electrical stimulation may suggest the mobilization of different pools of the transmitter.     

A new metabolic pathway from n-dodecane to α, ω-dodecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-dodecanedioic acid via ,-dodecanediol from n-dodecane using a mutant S76 of Candida tropicalis was studied.It was found that resting cells of S76 produce ,-dodecanediol from n-dodecane. This intermediate was identified by different analytical methods. With n-dodecanol as substrate the quantitative changes in the concentrations of ,-dodecanediol as well as other intermediates, e.g. monoacid, -hydroxy acid and ,-dioic acid produced by resting cells of S76 for different periods of time were determined. With ,-dodecanediol as the sole carbon source, quantitative changes of -hydroxy acid and ,-dioic acid produced by S76 were also recorded.The results confirm the existence of a new metabolic pathway via ,-diol in the course of ,-dioic acid formation from n-alkane in the mutant S76 of C. tropicalis.     

Degradation pathways from n-tridecane to α, ω-tridecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-tridecanedioic acid via n-tridecanoic acid and via ,-tridecanediol from n-tridecane in the mutant S 76 of Candida tropicalis was studied. It was found that resting cells of S 76 produced ,-tridecanediol from n-tridecane.With n-tridecanol as substrate, the ,-diol could also be detected. The mutant S 76 was able to produce ,-tridecanedioic acid using either n-tridecanol or n-tridecanoic acid as the sole carbon source. Quantitative changes in the concentration of -hydroxy tridecanoic acid and other intermediates were recorded during the formation of ,-dioic acid.The results confirm the existence of two metabolic pathways mentioned above in the course of ,-dioic acid formation from odd n-alkane in the mutant S 76 of C. tropicalis.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

A frequency-time domain 3D COSY-J technique for measurement of1H−31P coupling constants in oligonucleotides

Summary A frequency-time domain 3D NMR technique has been developed for measurement of heteronuclear coupling constants in oligonucleotides employing a combination of COSY andJ-resolved techniques. The method employs frequency-selective excitation to generate the ₁ axis and 2D FT to generate the ₂ and ₃ axes. The procedure yields high resolution, especially along the ₁ axis. The technique is demonstrated on a dinucleotide.     

Inserted sequence in the mitochondrial 23S ribosomal RNA gene of the yeast Saccharomyces cerevisiae.

Summary The sequence organization of the yeast mit-DNA region carrying the large ribosomal RNA gene and the polar locus was examined. Hybridization studies using rho^- deletion mutants and electron microscopy of the heteroduplexes formed between 23S rRNA and the appropriate restriction fragments, lead to the conclusion that the 23S rRNA¹ gene of the ⁺ strains is split by an insertion sequence of 1,000–1,100 bp. In contrast, no detactable insertion was found in the 23S rRNA gene of the ^- strains. The size and the location of the insert found in the 23S rRNA gene of the ⁺ strains appear to be identical to those of the sequence which had previously been found to characterize the difference (at the locus) between the mitDNA of the wild type strains carrying the ⁺ or ^- alleles (Jacq et al., 1977).     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

Characteristics of [125I]ω-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain

Characteristic of [¹²⁵I]-conotoxin (-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]: DSP) was systematically investigated in crude membranes from chick whole brain. [¹²⁵I]-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [¹²⁵I]-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [¹²⁵I]-CgTX labeling of the 216 kDa band and specific [¹²⁵I]-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [¹²⁵I]-CgTX using DSP involves the specific binding sites of [¹²⁵I]-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.     

Chronic administration of a nitric oxide synthase inhibitor,Nω-nitro-l-arginine,and drug-induced increase in cerebellar cyclic GMP in vivo

N-nitro-l-arginine (N^G-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 g/10 l/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in N-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.Part of this work was presented at the Experimental Biology 93 FASEB Meeting at New Orleans, March 1993.     

Restriction fragment analysis of ‘null’ forms at the Gli-1 loci of bread and durum wheats

Summary Wheat accessions lacking some of the - and -gliadin components encoded by the Gli-1 loci on the short arm of chromosome 1D in bread wheat and chromosome 1A in durum wheat were studied by two-dimensional polyacrylamide gel electrophoresis and restriction fragment analysis. Digested genomic DNAs of normal and null forms were probed with a cDNA clone related to -/-gliadins and with a genomic clone encoding an LMW subunit of glutenin. The hybridisation patterns with the -/-gliadin probe were similar to those of cvs Chinese Spring and Langdon used as standards for bread and durum wheats, respectively, but several restriction fragments located on the 1D chromosome of bread wheat and the 1A chromosome of durum wheat were absent in the null forms. In addition, specific LMW glutenin fragments encoded by the same chromosomes were also absent in the null forms, suggesting that simultaneous deletions of blocks of genes for both -/-gliadins and LMW glutenins had occurred. Comparisons of the protein and RFLP patterns enabled some proteins to be mapped to specific restriction fragments.     

Antigen selectivity characteristic of polyclonal antibodies against omega-conotoxin GVIA and N-type voltage-dependent calcium channels

The antibodies against omega-conotoxin GVIA (-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B₁Nt (N-terminal segment [residues 1–13] of BI ₁ subunits of VDCCs) were prepared, and the selectivity for each antigen -CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti–-CTX GVIA antibody against -CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A–Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM -CTX GVIA, but not by -CTX SVIB (N-type VDCC blocker), -CTX MVIIC (N- and P-type VDCC blocker), or -Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC₅₀ values were about 1.2 × 1028 and 1.3 × 1028 M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 M B1Nt. These results suggest that the antibodies prepared against -CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against -CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti -CTX GVIA antibody must also be useful for the radioimmunoassay of -CTX GVIA.     

Metabolic formation of dodecanedioic acid from n-dodecane by a mutant of Candida tropicalis

Summary These experiments studied the metabolic formation of a,-dodecanedioic acid using the mutant S 76 developed from the wild strain Candida tropicalis 1230 (capable of producing large amounts of a,-dodecanedioic acid).Our results show for the first time that 12-hydroxydodecanoic acid was excreted into the medium as a free acid.n-Dodecanol and n-dodecanoic acid were also detected in the n-dodecane medium. The mutant S 76 was able to produce a,-dodecanedioic acid using either n-dodecanol or dodecanoic acid as the sole carbon source. Quantitative cahnges in the concentrations of 12-hydroxy-dodecanoic acid and other intermediates were recorded during the formation of a,-dodecanedioic acid. S 76 was rapidly able to convert large amounts of 12-hydroxy-dodecanoic acid to a,-dodecanedioic acid.The formation of a,-dodecanedioic acid from n-dodecane via the sequence n-dodecanoln-dodecanoic acid 12-hydroxy-dodecanoic acid was confirmed.     

Cultivation of the agarophyte Gelidium pristoides in Algoa Bay,South Africa

Biofilms were allowed to develop on wooden slides of the River Red Gum (Eucalyptus camaldulensis Dehnh., Myrtaceae) submerged in two billabongs of south-eastern Australia. The slides were placed in the photic zone and the aphotic zone, and the biofilms sampled after eight week's growth over the summer of 1989–1990 and winter of 1990. Bacterial numbers, estimated with epifluorescence microscopy, ranged from 4–78 × 10⁶ cells cm^–2. Bacteria were more abundant in the photic zone than the aphotic zone, and more abundant in summer than winter. Fewer than 0.5% of the bacteria could be cultivated on nutrient agar plates. Concentrations of phospholipids ranged from 8–79 ng cm^–2, which corresponded to bacterial abundances of 2–17 × 10⁶ cells cm^–2. Fifty five phospholipid fatty acids (PLFA) were identified, of which 16:0 (13–29% of total PFLA) was the most common. Other abundant PFLA included 16:17c (6–28%), 18:26 (3–16%), 18:33 (4–12%), 18:19c (3–5%), 18:l7c (5–11%) and 18:0 (2–8%). Minor PLFA included 14:0, i and a 15:0, 15:0, 16:l5c, 16:113c, 18:36, 18:43, 20:46 and 20:53. The PLFA profiles of the biofilms were quite different from those of the sediments and plankton. There was a clear distinction between the PLFA profiles of summer and winter biofilms, but less evidence for unequivocal site or light-regime effects.     

Occurrence of Curtobacterium sp. possessing ω-cyclohexyl fatty acids in soil with zinc added

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed -cyclohexyl fatty acids. -Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of -cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.The bacteria grew almost normally in zinc-enriched medium, and -cyclohexyl undecanoic acid increased with zinc concentration. Zinc added to the medium was not abundant in the cell fraction, and the ratio of increase of zinc in the cells was not so high as in the culture medium. These results suggested that -cyclohexyl fatty acids are related to the zinc tolerance of the isolated strain, and that this tolerance depends on low permeability of the membrane to zinc.     

Similarities of omega gliadins from<Emphasis Type="Italic"> Triticum urartu</Emphasis> to those encoded on chromosome 1A of hexaploid wheat and evidence for their post-translational processing

The -gliadins encoded on chromosome 1 of the A genome were purified from Triticum aestivum L. (2n=6x=42, AABBDD) cv. Butte86, nullisomic 1D-tetrasomic 1A of cv. Chinese Spring (CS N1DT1A), and the diploid T. urartu (2n=2x=14, AA). Reverse-phase high-performance liquid chromatography combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gliadin extracts from CS nullisomic-tetrasomic (NT) lines confirmed the assignment to chromosome 1A. The purified -gliadins were characterized by mass spectrometry and N-terminal sequencing. The 1A-encoded -gliadins were smaller than 1B- or 1D-encoded -gliadins. The N-terminal amino acid sequences for 1A -gliadin mature peptides were nearly identical to those for the T. urartu -gliadins and were more similar to 1D -gliadin sequences than to sequences for T. monococum -gliadins, barley C-hordeins, or rye -secalins. They diverged greatly from the N-terminal sequences for the 1B -gliadins. The data suggest that T. urartu is the A-genome donor, and that post-translational cleavage by an asparaginyl endoprotease produces those -gliadins with N-terminal sequences beginning with KEL.Communicated by J. Dvorak     

Evaluation of errors of interproton distances and correlation time determined from NMR cross-relaxation rates

Summary We have analyzed a combined use of the two-dimensional nuclear Overhauser effect in the laboratory frame (NOESY) and in the rotating frame (ROESY) to determine interproton distances and correlation time in medium-sized rigid molecules (Davis, 1987). This method can be applied in the intermediate motional regime, 0.2 < _oc, < 5, (_c, correlation time, (_o resonance frequency). Error limits depend on the motional regime and are smallest near _oc=1.14.The method was tested on six geminal proton pairs in the bicyclic octapeptide (S-deoxo--[R]-OH-Ile³ amaninamide, Mw =870) for which at 297 K in DMSO, a correlation time of 1.0 ns, with a standard deviation of 0.12 ns, and an interproton distance of 1.87 Å, with standard deviation of 0.04 Å, are obtained.     

Linkage of the β-Like ω-Globin Gene to α-Like Globin Genes in an Australian Marsupial Supports the Chromosome Duplication Model for Separation of Globin Gene Clusters

The structure, function, and evolutionary history of globin genes have been the subject of extensive investigation over a period of more than 40 years, yet new globin genes with highly specialized functions are still being discovered and much remains uncertain about their evolutionary history. Here we investigate the molecular evolution of the -globin gene family in a marsupial species, the tammar wallaby, Macropus eugenii. We report the complete DNA sequences of two -like globin genes and show by phylogenetic analyses that one of these genes is orthologous to embryonically expressed -globin genes of marsupials and eutherians and the other is orthologous to adult expressed -globin genes of marsupials and eutherians. We show that the tammar wallaby contains a third functional -like globin gene, -globin, which forms part of the -globin gene cluster. The position of -globin on the 3 side of the -globin cluster and its ancient phylogenetic history fit the criteria, originally proposed by Jeffreys et al. (1980), of a fossil -globin gene and suggest that an ancient chromosome or genome duplication preceded the evolution of unlinked clusters of - and -globin genes in mammals and avians. In eutherian mammals, such as humans and mice, -globin has been silenced or translocated away from the -globin locus, while in marsupials -globin is coordinately expressed with the adult -globin gene just prior to birth to produce a functional hemoglobin (_{2 2}).     

Isolation and characterization of an ω3-desaturation-defective mutant of an arachidonic acid-producing fungus,Mortierella alpina 1S-4

A mutant considered to be defective in the conversion of n-6 to n-3 fatty acids (3-desaturation) was derived from a 5-desaturation-defective mutant (Mut44) of Mortierella alpina 1S-4, after treating its spores with N-methyl-N-nitro-N-nitrosoguanidine. This mutant cannot produce 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid or any other n-3 fatty acids, of which about 10% was found in its parental strain upon cultivation at 12°C. The mutant's growth rate was comparable to that of the parental strain when grown at 28°C, but it became much slower when the mutant grew at 12°C, at which the lag phase for Mut44 was about 2 d but 5 d for the mutant.Abbreviations 18:33 9(Z),12(Z),15(Z)-octadecatrienoic acid - 18:43 6(Z),9(Z),12(Z),15(Z)-octadecatetraenoic acid - 20:43 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid - AA arachidonic acid - DHGA dihomo--linolenic acid - EPA 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid - GLC gas-liquid chromatography - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PC phosphatidylcholine     

Characteristics of the Inhibitory Effect of Calmodulin on Specific [125I]Omega-Conotoxin GVIA Binding to Crude Membranes from Chick Brain

The characteristics of the inhibitory effect of calcium ion (Ca²⁺)/calmodulin (CaM) on specific [¹²⁵I]-omega-conotoxin GVIA (¹²⁵I--CTX) binding and on the labeling of ¹²⁵I--CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca²⁺/CaM depended on the concentrations of free Ca²⁺ and CaM. The IC₅₀ values for free Ca²⁺ and CaM were about 2.0 × 10^–8 M and 3.0 g protein/ml, respectively. The inhibitory effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca²⁺/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca²⁺ channel ₁ subunits) with ¹²⁵I--CTX using a cross-linker. These results suggest that Ca²⁺/CaM affects specific ¹²⁵I--CTX binding sites, probably N-type Ca²⁺ channel ₁ subunits, in crude membranes from chick whole brain.