The reaction of nitrogen monoxide with the haemocyanins of the crayfish Astacus leptodactylus and the snail Helix pomatia.

The rate of the reaction of Astacus leptodactylus methaemocyanin with NO follows the Henderson-Hasselbalch equation with a pKa of 5.85, suggesting that one imidazole ligand of Cu was exchanged for NO. The reaction is blocked by F- as a bridging ligand. The same imidazole residue might be responsible for the decomposition of nitrosylhaemocyanin, [Cu1NO+CuII], with an unlocated binding site for NO, into methaemocyanin and NO, as the rate increase with pH. NO could react preferentially with CuA of Helix pomatia methaemocyanin, CuA'IICuBII, as it possibly has only two histidine ligands instead of the three of CuA in Astacus haemocyanin. This difference might explain the higher formation rate and the much greater stability of Helix nitrosylhaemocyanin. The fast reaction is governed by a pKa of 6.80, probably of a bridging mu-aquo ligand. With F- or a mu-hydroxo bridging ligand a low reaction rate was still observed, in contrast with Astacus methaemocyanin. Helix nitrosylhaemocyanin was transformed by N3- into methaemocyanin with the liberation of N2 and N2O. This methaemocyanin could almost quantitatively be regenerated with H2O2. Helix nitrosylhaemocyanin was only partially regenerated by a direct treatment with H2O2 and almost quantitatively by HONH2 in a similar fairly fast reaction, followed by a much slower one.     

The reaction of nitrite with the haemocyanin of the Roman snail (Helix pomatia).

The reaction of nitrite at pH 5.0-7.0 with the deoxyhaemocyanin of a mollusc, the Roman snail (Helix pomatia), yielded nitrosylhaemocyanin (CuIA.NO+ CuIIB), in contrast with the formation of methaemocyanin with the deoxyhaemocyanin of the crustacean Astacus leptodactylus (mud crayfish). With Helix haemocyanin 1 NO was thereby liberated per active site, as shown by m.s., as against 2 NO with Astacus haemocyanin. Helix nitrosylhaemocyanin was characterized in c.d. by the negative extremum at 336 nm (CuIA.NO+) and by the mononuclear e.p.r. signal at g = 2 (CuIIB). Binuclear e.p.r. signals have been observed after the addition of nitrite to methaemocyanins. With Astacus methaemocyanin, no further reaction occurred, whereas with Helix methaemocyanin the mononuclear e.p.r. signal, characteristic for nitrosylhaemocyanin gradually appeared. This formation of Helix nitrosylhaemocyanin implicates the binding, most likely on CuIIA, of a second nitrite besides a bridging nitrite, so that a dismutation into NO and NO2 can occur there. A further dismutation of NO2 yields nitrite and nitrate. The formation of the latter was demonstrated by Raman spectrometry. The reaction rate of Helix methaemocyanin with nitrite decreased with increasing pH according to the Henderson-Hasselbalch equation with a pKa value of 6.77, attributed to a mu-aquo bridging ligand, which can be exchanged for nitrite, in equilibrium with a mu-hydroxo ligand which cannot. These data also favour the formulation of the final reaction product as nitrosylhaemocyanin instead of semi-methaemocyanin, with or without bound nitrite.     

The reaction of nitrite with the haemocyanin of Astacus leptodactylus.

The reaction of nitrite at pH 5.7 with deoxyhaemocyanin of Astacus leptodactylus yielded methaemocyanin in two one-electron steps, as nitrite was reduced to NO. This methaemocyanin could be almost fully regenerated by an anaerobic treatment with HONH2, in contrast with the methaemocyanin prepared with H2O2. A destruction of active sites on treating oxyhaemocyanin with HONH2 explains the partial regeneration of methaemocyanin under air, as traces of H2O2 are formed in the autoxidation of HONH2. The reaction rate of nitrite with deoxyhaemocyanin is almost 15 times that with oxyhaemocyanin. The slope of -1.0 for the logarithm of the pseudo-first-order rate constants plotted against pH indicates that HNO2 is the reacting species. Methaemocyanin was e.p.r.-undetectable, but a binuclear signal was observed at g = 2 on binding nitrite to methaemocyanin. This signal disappeared with a pKa of 6.50, suggesting that a mu-aquo bridging ligand, which can be replaced by nitrite, is deprotonated to a mu-hydroxo bridging ligand, which resists substitution by nitrite. The intensity of this triplet e.p.r. signal allowed the determination of the association constant of nitrite to the active site of Astacus methaemocyanin and yielded a value of 237 M-1 at pH 5.7. The interpretation by some authors of nitrosylhaemocyanin as a nitrite derivative of semimethaemocyanin is contradicted by this rapid reaction of nitrite with copper(I) in deoxyhaemocyanin and in semi-methaemocyanin and by the low binding constant of nitrite to the active site of methaemocyanin.     

The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.     

Chemotaxonomical investigations on the occurrence of sialic acids in protostomia and deuterostomia

Several tissues of different protostome species were tested in respect to the occurence of sialic acid. There is no sialic acid in the turbellarian Euplanaria gonocephala and the mollusc Helix pomatia. In contrast to other tissues the digestive gland of the decapod crustaceans Astacus leptodactylus and Uca tangeri contains 60–80 μg of sialic acid per gram wet weight. Radioactive tracer experiments showed that Astacus leptodactylus cannot synthesize sialic acid by using the specific precursor N-acetyl-mannosamine but obviously resorbs exogenous sialic acid taken up with the food and stores it in the digestive gland. It is suggested that protostomes in general are devoid of endogenous sialic acid whereas it is abundant in deuterostomes. Contradictory to existing results low amounts of a resorcinol positive substance, probably a sialic acid, were detected in the deuterostome tunicate Phallusia mammilata.     

Iron nitrosyl hemoglobin formation from the reactions of hemoglobin and hydroxyurea

Hydroxyurea represents an approved treatment for sickle cell anemia and acts as a nitric oxide donor under oxidative conditions in vitro. Electron paramagnetic resonance spectroscopy shows that hydroxyurea reacts with oxy-, deoxy-, and methemoglobin to produce 2-6% of iron nitrosyl hemoglobin. No S-nitrosohemoglobin forms during these reactions. Cyanide and carbon monoxide trapping studies reveal that hydroxyurea oxidizes deoxyhemoglobin to methemoglobin and reduces methemoglobin to deoxyhemoglobin. Similar experiments reveal that iron nitrosyl hemoglobin formation specifically occurs during the reaction of hydroxyurea and methemoglobin. Experiments with hydroxyurea analogues indicate that nitric oxide transfer requires an unsubstituted acylhydroxylamine group and that the reactions of hydroxyurea and deoxy- and methemoglobin likely proceed by inner-sphere mechanisms. The formation of nitrate during the reaction of hydroxyurea and oxyhemoglobin and the lack of nitrous oxide production in these reactions suggest the intermediacy of nitric oxide as opposed to its redox form nitroxyl. A mechanistic model that includes a redox cycle between deoxyhemoglobin and methemoglobin has been forwarded to explain these results that define the reactivity of hydroxyurea and hemoglobin. These direct nitric oxide producing reactions of hydroxyurea and hemoglobin may contribute to the overall pathophysiological properties of this drug.     

EPR stopped-flow studies of the reaction of the tyrosyl radical of protein R2 from ribonucleotide reductase with hydroxyurea.

The reaction of the functional tyrosyl radical in protein R2 of ribonucleotide reductase from E. coli and mouse with the enzyme inhibitor hydroxyurea has been studied by EPR stopped-flow techniques at room temperature. The rate of disappearance of the tyrosyl radical in E. coli protein R2 is k2 = 0.43 M-1 s-1 at 25 degrees C. The reaction follows pseudo-first-order kinetics up to 450 mM hydroxyurea indicating that no saturation by hydroxyurea takes place even at this high concentration. Transient nitroxide-like radicals from hydroxyurea have been detected for the first time in the reaction of hydroxyurea with protein R2 from E. coli and mouse, indicating that 1-electron transfer from hydroxyurea to the tyrosyl radical is the dominating mechanism in the inhibitor reaction. The hydroxyurea radicals appear in low steady-state concentrations during 2-3 half-decay times of the tyrosyl radical and disappear thereafter.     

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.     

Neuropeptide-like substances in the retina of intact and tentaculectomized snails

1. Immunocytochemical investigations in normal and regenerated eyes of Helix aspersa Müller revealed a positive immunoreactivity toward antibodies directed against four biologically active peptides.2. Eighty per cent of tentaculectomized animals regenerated all tentacular structures and the immunoreactivity was found to be identical in normal and regenerated retinas.3. Type I photosensory cells are the only αMSH-positive structures, whereas the reactivity to the three other antibodies is localized in one (rarely two) retinal ganglion cell(s).4. Using an elution technique, the colocalization of the three substances has been demonstrated in a unique ganglion cell.5. These results seem to indicate that the α MSH-like substance(s) play(s) an important role in visual mechanisms, particularly in photoreception.6. The colocalization of the three other substances in a unique ganglion cell suggests their probable crucial neuromodulatory and/or neurotransmitter function in rapid conduction of impulses to the brain.     

Apoptosis and regeneration of enterocytes in experimental atrophy of the small intestine mucosa

Apoptosis, one of the types of cell deaths, participates in regulating the size of regenerated tissue. Severe atrophy of small intestine mucosa in mice was caused by the administration of hydroxyurea solution. The degree of atrophy correlated with a lowering mitotic activity and DNA synthesis in the epithelium of crypts. Apoptotic bodies were situated above the basal membrane, in crypt lumen or were phagocytized by adjacent epithelial cells. The development of atrophy, as well as the regeneration of mucosa can be predicted by the relation between mitosis and apoptosis.     

Effect of protein synthesis inhibitors on sensitization of defence reaction and on cholinosensitivity of command neurons in Helix lucorum

We studied influence of protein synthesis inhibitors on short-term sensitization of Helix escape reaction and potentiation cholinosensitivity in command neurons. Inhibitor of protein synthesis anisomycin does not prevent behavioral sensitization. Anisomycin and irreversible inhibitor of protein synthesis saporin change the dynamics of cholinosensitivity potentiation in command neurons. The results Suggest that investigated sensitization of Helix escape reaction does not require synthesis of new proteins.     

Methiothepin-sensitive serotonin receptors are involved in postsynaptic mechanism of sensitization of Helix lucorum escape reaction

Tetanic electric stimulation of Helix foot evokes sensitization of escape reaction. This behavioral sensitization and posttetanic potentiation (PTP) of acetylcholine-induced inward current (ACh-current) in command Helix neurons of escape behavior were similar. Antagonist of serotonin receptors methiothepin prevents the PTP of the ACh-current and behavioral sensitization. Serotonin disrupts the PTP of the ACh-current. It is suggested that the increase in cholinosensitivity of the command neurons with the involvement of methiothepin-sensitive serotonin receptors may be the cellular postsynaptic mechanism of behavioral sensitization of Helix escape reaction.     

The functional morphology of the feeding appendages and gut of Hippolyte varians (Crustacea: Natantia)

The musculature of the feeding appendages are described and compared with those few early accounts of other Decapoda, particularly those of Pandalus danae and Astacus fluviatilis . While the musculature is very similar to that of the latter, the relative importance of the various muscles may differ markedly and a number of previously undescribed muscles have been found, while others present in Pandulus and Astacus are not present in Hippolyte . The alimentary canal is also described, together with its associated musculature. The function of the various appendages and the parts of the gut are described from observations of living specimens.     

Genetic and physiological variability among cultured cells and regenerated plants of Nicotiana tabacum

Summary Tobacco cell lines selected for resistance to picloram (4-amino-3,5,6-trichloropicolinic acid) and plants regenerated from these cell lines manifest several traits not shown by the parental strains. Genetics analyses of the regenerated plants have permitted the sources of this variability to be identified.Tricotyledenous seedlings appeared at a much higher frequency among the progeny of a heterozygous mutant plant (PmR1/+) regenerated from culture than they did among progeny of normal regenerated plants. In crosses with the regenerated heterozygous mutant plant and with homozygous progeny of this plant (PmR1/PmR1) the frequency of tricotyly was influenced more by the generation than by the genotype of the parent plant. Therefore, it is concluded that tricotyly is a physiological response to passage through cell culture.More than half of the picloram-resistant cell lines isolated were also resistant to hydroxyurea. Segregation of these two resistances was analyzed in progeny of crosses with regenerated plants. In all cases hydroxyurea-resistance was genetically stable and inherited as a single dominant nuclear mutation (designated HuR). In crosses with plants PmR1/+ and PmR7/+ the HuR and PmR mutations assorted independently. In contrast, the HuR mutation recovered from plant PmR6/+ was linked to the PmR6 mutation.     

Biosynthetic events of hydrid regeneration

Summary The results of a combined morphological and biochemical study of the role of DNA synthesis during distal regeneration inHydra oligactis revealed that a burst of³H-thymidine incorporation into DNA preceded the elaboration of each of the initial three tentacles. In addition, the relative level of each burst of precursor incorporation relfected the number of tentacles formed at that time. Cytological localization of concentrated amounts of labeled material in nuclei of the hypostome and tentacle regions provided corroborative evidence for the biochemical findings.Evidence that the increased DNA specific activity levels described above are associated with tentacle initiation derived from studies in which regenerating hydra were cultured in hydroxyurea and studies in which hydra regenerated proximally rather than distally. Hydra regenerating in 8 mg/ml (0.105 M) hydroxyurea developed morphologically recognizable hypostomes but no tentacles, and incorporated³H-thymidine into DNA at a level distinctly below that exhibited by uncut, untreated animals. Similarly, hydra regenerated a normal, functional basal disc in the absence of any increased DNA specific activity. Therefore, it is suggested that tentacle initiation inH. oligactis requires concomitant DNA synthesis and, as such, represents an epimorphic phenomenon.     

alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus.

1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2-macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2-macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin.     

Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.     

Double-strand break generation under deoxyribonucleotide starvation in Escherichia coli

Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo "replication fork reversal." This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.     

In silico and experimental improvement of bacteriorhodopsin production in Halobacterium salinarum R1 by increasing DNA-binding affinity of Bat through Q661R/Q665R substitutions in HTH motif

Extremophiles - DNA-binding motif of bacterioopsin activator (Bat) protein is a Helix–Turn–Helix motif, which binds to bop promoter and induces bacterioopsin (Bop) expression under...     

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Summary Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI 3. AI 3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2 Compared with AI 3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.