Effects of α2-Adrenergic Agonists and Antagonists on Photoreceptor Membrane Currents

Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presynaptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (greater than 100 microM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cell's resting potential as well as after a light step. Clonidine reduces voltage-dependent outward K+ currents (IA, an early current, ICa2+-K+, a late Ca2+-dependent current) that control the type B cell's excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the alpha 2-receptor antagonist, yohimbine (0.5 microM), but not the alpha 1-blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde-induced fluorescence or Falck-Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine-like synaptic effect on type B cells, therefore, could amplify conditioning-specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.     

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.     

Amino acid sequence and relative biological activity of eel atrial natriuretic peptide

A peptide exhibiting vasodepressor and natriuretic activities in rats was isolated from eel atria, and its primary structure was determined as H-Ser-Lys-Ser-Ser-Ser-Pro-Cys-Phe-Gly-Gly-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Tyr-Ser- Gly-Leu-Gly-Cys-Asn-Ser-Arg-Lys-OH. This peptide, termed eel atrial natriuretic peptide (ANP), has sequence homology of 59% to mammalian (human or rat) ANP, 52% to fowl ANP, and 46% to frog ANP. When the biological activity of synthetic eel ANP was compared with that of human ANP, the eel peptide was 110 times more potent for the vasodepressor activity in eels, nearly equipotent for the vasodepressor activity in quails, and 20 times less potent for the vasodepressor and natriuretic activity in rats.     

Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host

E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B.     

One-stage repair of blepharophimosis

Congenital blepharophimosis is a congenital anomaly characterized by abnormalities in the area of the eyes, including bilateral ptosis, shortening of the horizontal fissure of the lid, expansion of the intercanthal distance, and epicanthus inversus. The condition is subject to autosomal-dominant heredity and is said to occur more frequently in Orientals than in Occidentals. Over the past 9 years, we have surgically treated 11 cases of congenital blepharophimosis using a procedure in which levator resection and medial canthoplasty are performed in one stage. It has been commonly believed that when levator resection and medial canthoplasty are performed at the same time, tension in the eyelid becomes too strong to achieve favorable results; therefore, the standard procedure has been to divide the operation into two stages. In all 11 cases we experienced, however, it was possible to obtain good results with a single-stage operation.     

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derived peptide with vasoconstricting activity, was determined in an aqueous solution by means of a combination of NMR and distance geometry calculations. The resulting structure is characterized by an alpha-helical conformation in the sequence region, Lys9-Cys15. Furthermore, an extended structure and a turn structure exist in the Cys1-Ser4 and Ser5-Asp8 regions respectively, and no preferred conformation was found for the C-terminal part of the peptide which was not uniquely constrained by the NMR data. These structural elements, the alpha-helical structure in the sequence portion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys, are homologous to those found commonly in several neurotoxic peptides.     

Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.     

Calcium activates and inactivates a photoreceptor soma potassium current.

Light-induced currents were measured with a two-microelectrode voltage clamp of type B photoreceptor somata, which had been isolated by axotomy from all synaptic interactions as well as from all membranes capable of generating impulse activity. In artificial seawater (ASW), light elicited a transient early inward current, INa+, which depended on Na+o and had a linear current-voltage relation and an extrapolated reversal potential of 30-40 mV (absolute). In 0-Na+ ASW, light elicited a transient short-latency outward current that dependent on K+o, increased exponentially with more positive voltages (greater than or equal to -40 mV), and reversed at -70 to -75 mV. This outward current was not blocked by Ca++ channel blockers (e.g., Cd++, Co++) or substitution of Ba++o, for Ca++o, but was reduced by iontophoretic injection of EGTA. In both ASW and 0-Na+ ASW, light also elicited a delayed, apparently inward current, which was associated with a decreased conductance, depended on K+o, increased exponentially with more positive voltages (greater than or equal to -40 mV), reversed at the equilibrium potential for K+ flux in elevated K+o was eliminated by substitution of Ba++o for Ca++o, and was greatly reduced by Cd++o or Co++o. Thus, light elicited an early Ca++-dependent K+ current, IC, and a prolonged decrease of IC. Iontophoretic injection of Ca++ through a third microelectrode caused prolonged reduction of both IC and the light-induced decrease of IC, but did not alter ICa++ or the current-voltage relation of IC. Ruthenium red (1 microM) in the external medium caused a prolongation of the light-induced decrease of IC. Iontophoretic injection of EGTA often eliminated the light-induced IC decrease while decreasing peak IC (during depolarizing steps to -5 or 0 mV) by less than one-half. EGTA injection, on the average, did not affect steady state IC but reduced the light-induced decrease of steady state IC to approximately one-third of its original magnitude. The prolonged IC decrease, elicited by dim light in the absence of light-induced IC or INa+, was more completely eliminated by EGTA injection. It was concluded that light, in addition to inducing a transient inward Na+ current, causes both a transient increase and a prolonged decrease of IC via elevation of Ca++i.     

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.     

Human aldolase isozyme gene: the structure of multispecies aldolase B mRNAs.

A complete nucleotide sequence of human aldolase B mRNA was determined with a recombinant cDNA (pHABL120-3). The cDNA insert was composed of 1,652 bases excluding poly(A) tail and the sequence was consistent with the previous results reported by others. However, S1 nuclease mapping and subsequent genomic analysis allowed us to know that the clone possesses two more sites corresponding to 5'-termini in the 5'-noncoding region and another site of polyadenylation in the 3'-noncoding region. In fact, the major aldolase B mRNA species occupying 90% of the total mRNAs initiated at the predominant position corresponding to the position around -82 of the 5'-noncoding sequence in pHABL120-3 and terminated at the distal polyadenylation site. Second species accounting for 9% of the mRNAs initiated at the same site and terminated at the proximal polyadenylation site. The remainings have a longer 5'-noncoding sequence which starts from further upstream region of the major one and pHABL120-3 corresponds to one of these largest clones.