Compartmentalisation of nitrogenase in a non-heterocystous cyanobacterium: Trichodesmium contortum

Abstract In Trichodesmium contortum , nitrogenase was detected in only a limited number (about 10%) of microscopically distinguishable, consecutively arranged cells in central regions of the trichomes. Cells with nitrogenase also contained the photosystem II associated pigment phycoerythrin. These cells were not distinguishable from other cells on a structural basis, but were clearly visible at low magnification microscopy as all in the zone were more compact and shorter than those on either side. The compartmentalisation of nitrogenase into a chain of cells and in a possibly photosynthetic environment represents a previously undescribed phenomenon. The nitrogenase containing cells apparently perform the O₂ protective function of heterocysts yet are different in several aspects.     

Metabolic activation and toxicity of a DDT-metabolite, 3-methylsulphonyl-DDE, in the adrenal zona fasciculata in mice

Whole-body autoradiography of 14C-labelled 3-methylsulphonyl-DDE (3-MeSO2-DDE) in female C57BL mice revealed a heavy accumulation in the adrenal cortex. Fairly high radioactivity appeared in the nasal mucosa and fat, while the labelling of the liver was intermediate. The adrenal radioactivity remained largely unextracted in tissue-sections treated with organic solvents. In the liver and intestinal contents the radioactivity was partly extracted, whereas in all other tissues almost completely extracted. According to light microscopic autoradiography, the tissue-bound adrenal radioactivity was confined to the zona fasciculata, leaving the other adrenal zones devoid of bound material. Incubation of 3-MeSO2-DDE with adrenal tissue (300 X g supernatant) revealed a dose- and time-dependent covalent binding to protein and formation of water-soluble metabolites. The cytochrome P-450 inhibitors metyrapone and carbon monoxide inhibited both covalent binding and polar metabolite formation. Addition of reduced glutathione decreased binding, while polar metabolite formation was increased. Histopathological examination of adrenals from 3-MeSO2-DDE-treated mice revealed extensive vacuolation and necrosis of the zona fasciculata 1-12 days after single doses down to 25 mg/kg. Degenerative changes were observed at 12.5 mg/kg. In contrast to 3-MeSO2-DDE, 14C-labelled 3,3'-bis(methylsulphonyl)-DDE was not accumulated in the adrenal cortex. 3-MeSO2-DDE is thus a persistent environmental pollutant with a unique ability to produce acute toxicity subsequent to metabolic activation in a mammalian tissue.     

The Nostoc-Nephroma symbiosis: localization, distribution pattern and levels of key proteins involved in nitrogen and carbon metabolism of the cyanobiont

The qualitative distribution and quantitative estimates of nitrogenase (EC 1.7.99.2), glutamine synthetase (EC 6.3.1.2), phycoerythrin and ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were studied in the cyanobacterium Nostoc residing in internal cephalodia of the tripartite lichen Nephroma arcticum L. Polyclonal antisera, raised in rabbit against the proteins, and goat anti-rabbit IgG conjugated to 10 nm gold were used as probes to detect the antigens by transmission electron microscopy. Western blot analyses demonstrated the monospecificity of the antisera. Nitrogenase was localized in heterocysts, with vegetative cells showing a label intensity comparable to the background. Distribution of the antigen within the heterocysts was uniform. Glutamine synthetase labelling was very low, but appeared to be distributed in both cell types. An intense phycoerythrin labelling was associated with the thylakoid region of the vegetative cells, whereas a much lower labelling was observed in the heterocyst. No significant differences were found between cyanobionts in younger and older cephalodia except for the nitrogenase labelling, which was higher in heterocysts of the cyanobiont in younger cephalodia. Most of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) label was present in vegetative cells. The Rubisco label was pronounced in the carboxysomes, whereas the label in the cytoplasm, on a unit area basis, was much lower. Heterocysts showed a label intensity similar to that of the vegetative cell cytoplasm. In Nostoc of the bipartite lichen Peltigera canina L., the Rubisco protein showed a comparable distribution pattern, but the average number of carboxysomes per vegetative cell was about 4 times higher.     

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Imipramine-fentanyl antinociception in a rabbit tooth pulp model

Antinociception of imipramine (I) and its effect in combination with fentanyl (F) was evaluated in rabbits using electrically-induced lick chew responses via tooth pulp stimulation as the model of nociception. Acute i.v. injections of I elicited a graded dose response comparable to i.v. morphine (M) with I ED 50 = 4.35 mg/kg (2.31-8.14, 95% CL) and M ED 50 = 1.81 mg/kg (1.11-3.90), with no differences in the slopes between the two curves. The lethal dose of I was 10 mg/kg. An i.v. dose of I twice the ED 50 elicited an antinociceptive effect of more than 50% maximum possible effect (MPE) for 90 minutes with peak effect of 82% MPE occurring at 15 minutes. These effects of I were not reversed by a morphine-reversal dose of naloxone (0.1 mg/kg i.v.) but were reversed with a ten fold dose of naloxone. F ED 50 values (mcg/kg) were lowered from 11.35 to 2.70, 0.74 and 0.33 with increasing pretreatment doses of I (1.0, 2.1 and 3.2 mg/kg). These magnitudes of potency increases of F were 4.2, 15.3 and 34.4 fold respectively. A single i.v. ED 50 dose of I extended the time to 50% MPE of an ED 90 dose of F from 26 minutes to 77 minutes; of a 2 X ED 50 dose of F from 17 minutes to 28 minutes. Data points for three different combinations of I and F fell significantly within the synergistic field of an ED 50 isobologram and a polynomial equation described the curve best fitting the data points. F alone (i.v. ED 50 dose) increased the PaCO2 values to 74% above controls and three different combinations with I showed no increases in PaCO2 values above controls. I alone did not significantly cause any change in PaCO2 values from controls.     

DNA replication and H5 histone exchange during reactivation of chick erythrocyte nuclei in heterokaryons

Fusion of terminally differentiated chick erythrocytes (CE) with replicating quail myoblasts or established L6J1 rat myoblasts results in reactivation of DNA synthesis in the dormant CE nuclei and in suppression of DNA synthesis in the myoblast nuclei. The nuclei of primary quail myoblasts are more effectively inhibited than the nuclei of established rat myoblasts. Inhibition of DNA replication occurs not only by preventing G1 nuclei from entering S-phase but also by blocking nuclei in S-phase and by delaying nuclei in G2 from undergoing mitosis and starting a new DNA replication cycle. No inhibition of DNA synthesis could be observed when mouse erythrocytes, i.e., erythrocytes lacking nuclei, were fused with rat myoblasts to generate mouse-globin-containing L6J1 cybrids. — Reactivation of CE nuclei is associated with a loss of the tissuespecific H5 histone variant. Complete elimination of H5 histone, however, does not seem to be a necessary prerequisite for the initiation or completion of DNA replication in CE nuclei since H5 antigens are found on reactivated G1, S, and G2 nuclei.     

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C₂H₂-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2–3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.Abbreviation GS glutamine synthetase     

Genetic factors influencing neurosensitivity to early phenobarbital administration in mice

This study was designed to assess the possible genetic determinants of neurosensitivity to early (neonatal) phenobarbital (PhB) administration and to conduct a strain comparison for the cerebellar histology of both inbred and outbred mice. HS/Ibg, C57BL/10 and DBA/1 pups were injected with 50 mg PhB/kg daily on neonatal days 2-21. On day 50, treated animals (B) of all strains had smaller brains than controls (C). Moreover, the cerebellar area was decreased in HS and C57 B mice but not in DBA mice, suggesting genotype-environment interaction. B mice from all strains had similar Purkinje cell losses. Strain comparison showed that control C57 mice had smaller brains than control HS, and DBA had smaller brains than both HS and C57. Similarly, C57 had smaller cerebellar layers than HS and DBA had smaller cerebellar layers than both HS and C57. DBA and C57 mice had fewer Purkinje cells than HS but did not differ from each other.