Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.

Amperometry has been used for real-time electrochemical detection of the quantal release of catecholamines and indolamines from secretory granules in chromaffin and mast cells. Using improved-sensitivity carbon fiber electrodes, we now report the detection of quantal catecholamine release at the surface of somas of neonatal superior cervical ganglion neurons that are studded with axon varicosities containing synaptic vesicles. Local application of a bath solution containing high K+ or black widow spider venom, each of which greatly enhances spontaneous quantal release of transmitter at synapses, evoked barrages of small-amplitude (2-20 pA), short-duration (0.5-2 ms) amperometric quantal "spikes". The median spike charge was calculated as 11.3 fC. This figure corresponds to 3.5 x 10(4) catecholamine molecules per quantum of release, or approximately 1% that evoked by the discharge of the contents of a chromaffin granule.

In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts1

Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-14C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-14C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [33P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated α-glucans. Acid hydrolysis of the phosphorylated α-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.

Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice

Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor l-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug d-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.

Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix.

The cuticle of the silkworm Bombyx mori was demonstrated to contain pro-phenol oxidase [zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] and its activating cascade. The activating cascade contained at least one serine proteinase zymogen (latent form of pro-phenol oxidase activating enzyme). When the extracted cascade components were incubated with Ca2+, the latent form of pro-phenol oxidase activating enzyme was itself activated and, in turn, converted through a limited proteolysis of pro-phenol oxidase to phenol oxidase. Immuno-gold localization of prophenol oxidase in the cuticle using a cross-reactive hemolymph anti-pro-phenol oxidase antibody revealed a random distribution of this enzyme in the nonlamellate endocuticle and a specific orderly arrayed pattern along the basal border of the laminae in the lamellate endocuticle of the body wall. Furthermore, prophenol oxidase was randomly distributed in the taenidial cushion of the tracheal cuticle. At the time of pro-phenol oxidase accumulation in the body wall cuticle, no pro-phenol oxidase mRNA could be detected in the epidermal tissue, whereas free-circulating hemocytes contained numerous transcripts of pro-phenol oxidase. Our results suggest that the pro-phenol oxidase is synthesized in the hemocytes and actively transported into the cuticle via the epidermis.

Nucleotide sequence of the cDNA encoding the proenzyme of phenol oxidase A1 of Drosophila melanogaster.

Clones encoding pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] A1 were isolated from a lambda gt10 library that originated from Drosophila melanogaster strain Oregon-R male adults. The 2294 bp of the cDNA included a 13-bp 5'-noncoding region, a 2070-bp encoding open reading frame of 690 amino acids, and a 211-bp 3'-noncoding region. A hydrophobic NH2-terminal sequence for a signal peptide is absent in the protein. Furthermore, there are six potential N-glycosylation sites in the sequence, but no amino sugar was detected in the purified protein by amino acid analysis, indicating the lack of an N-linked sugar chain. The potential copper-binding sites, amino acids 200-248 and 359-414, are highly homologous to the corresponding sites of hemocyanin of the tarantula Eurypelma californicum, the horseshoe crab Limulus polyphemus, and the spiny lobster Panulirus interruptus. On the basis of the phylogenetic tree constructed by the neighbor-joining method, vertebrate tyrosinases and molluscan hemocyanins constitute one family, whereas pro-POs and arthropod hemocyanins group with another family. It seems, therefore, likely that pro-PO originates from a common ancestor with arthropod hemocyanins, independently to the vertebrate and microbial tyrosinases.

Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.