The Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores and Changes in the Oxidation-Reduction Potential of Cultures

S ummary . The E_h fall observed during incubation of Clostridium butyricum spores occurred during germination and emergence, not during the log phase; it is attributed to the H₂ tension resulting from metabolism. When the O₂ tension in the medium was increased, the E_h fell only after a few spores outgrew and replicated; germination of remaining spores then followed. It is suggested that the few cells able initially to metabolize can (a) elaborate NADH etc. which reduce the O₂ tension to a level non-inhibitory for the remaining spores, and (b) produce the H₂ tension recorded by the Pt electrode.     

Possible involvement of phospholipase A2 in light signal transduction of guard cells of Commelina communis

Polyunsaturated fatty acids induce stomatal opening (Y. Lee, H. Lee, R. C. Crain, A. Lee and S. J. Korn. 1994. Cell Signal. 6: 181–186), but it is not known whether they function as second messengers in guard cells exposed to signals that open stomata. To test the hypothesis that phospholipase A₂ (PLA₂), which produces fatty acids and lysophospholipids, is involved in light signal transduction in guard cells, we treated epidermal peels of Commelina communis L. with PLA₂ inhibitors and followed the changes in stomatal apertures in response to light. Stomatal opening by white, blue, or red light was inhibited by 2–3 different PLA₂ inhibitors in concentration ranges that have been reported to inhibit PLA₂ activity. However, the PLA₂ inhibitors could not block stomatal opening induced by a polyunsaturated fatty acid. These results suggest that PLA₂ functions as a signal transducer for both blue and red light in guard cells.     

Effects of aluminium on ATP pools and utilization in the cyanobacterium Anabaena cylindrica: a model for the in vivo toxicity

ATP pools extracted from the cyanobacterium Anabaena cylindrica , grown in the absence or presence of AlCl₃, were measured using the luciferin-luciferase assay. Addition of low concentrations of AlCl₃ (3.6–36 μ M ) increased the ATP pool 20–40% within 24 h, the effect being more marked with time. When using the Tris-EDTA boiling technique for extraction of cellular ATP, the ATP from aluminium-exposed cells appeared more stable during the extraction than the ATP from untreated cells. The higher ATP pools in aluminium-exposed cells were also evident after dark treatment and addition of the phosphorylating inhibitors carbonylcyanide m -chloro-phenylhydrazone (CCCP) and N,N-dicyclohexylcarbodiimide (DCCD). The formation of elevated ATP pools in cells exposed to aluminium was curtailed by high concentrations of cellular phosphate and postincubation at high pH (>8). These results favour the hypothesis that intracellular aluminium binds to ATP by competing with Mg²⁺ and, as a consequence, the stable Al³⁺-ATP complex formed is no longer available for cellular metabolism. The cyanobacterium is assumed to compensate by increasing the total pool of ATP. At high AlCl₃-concentrations, and in particular at low phosphate: aluminium molar ratios (<1), aluminium apparently also interferes with the membranes in A. cylindrica as indicated by inhibited O₂ production, reduced ATP production and cell lysis.     

Cell Surface Sialoglycoproteins of Cultured Rat Cerebellar Interneurons

Abstract: The sialoglycoproteins of cultures of relatively pure rat cerebellar interneurons were labelled by NaIO₄ oxidation/NaB ³H₄ reduction. The labelled molecules were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate followed by fluorography. Faint labelling could be detected in three components if cells were labelled without any oxidation. In young cultures, oxidation by galactose oxidase alone failed to reveal any additional bands. After oxidation by NaIO₄ or galactose oxidase in the presence of neuraminidase, many more components were labelled. After NaIO₄ oxidation, about 80% of the cell-associated radioactivity could be removed by treating the cells with neuraminidase, which left the cells more than 95% viable. The majority of the bands seen after neuraminidase treatment were substantially reduced when compared with untreated controls, supporting a surface localisation of these molecules. Reproducible developmental changes were seen in the profiles of bands labelled by NaIO₄/NaB ³H₄ in time course studies of cultures up to 8 days in vitro . Some bands became more prominent, and others disappeared. The gel profiles of the neuron cultures were quite distinct from those of cerebellar astrocyte cultures, which contain all the cell types likely to be contaminants of the neuron cultures.     

Peroxide inducible phage reactivation in Bacteroides fragilis

Abstract Reactivation of UV-irradiated phage b-1 was induced by H₂O₂ and UV in Bacteroides fragilis . The characteristics of H₂O₂ and UV induced phage reactivation differ from a previously reported oxygen induced reactivation system. The survival of B. fragilis cells after UV irradiation was also increased by pretreatment with H₂O₂. DNA synthesis was not inhibited in the host cells exposed to H₂O₂ concentrations which induced phage reactivation. The pattern of DNA degradation and synthesis after UV irradiation with and without H₂O₂ differed from the effect of O₂ on DNA synthesis in irradiated B. fragilis cells.     

The regulation of photosynthetic rate and activation of extracellular carbonic anhydrase under CO2-limiting conditions in the marine diatom Skeletonema costatum

In the marine diatom Skeletonema costatum , carbonic anhydrase activity exterior to the plasma membrane (CA_ext) was detected only when the available CO₂ concentration was less than 5·0 mmol m^–3, this activity being unaffected by the total dissolved inorganic carbon concentration. The inhibition of CA_ext by dextran bound sulphonamide (DBS) demonstrated the key role of this enzyme in maintaining photosynthetic rate under CO₂-limited conditions. Treatment with trypsin followed by affinity chromatography on p-aminomethylbenzene-sulphamide agarose and subsequent SDS-PAGE analysis revealed a polypeptide from carbon-replete cells of identical molecular mass to the CA_ext released by trypsin from CO₂-limited cells. Redox activity in the plasma membrane of intact cells was measured by following the light-dependent reduction of ferricyanide or NADP, the greatest activity being shown by CO₂-limited cells. Overall the results suggest that high rates of redox activity under conditions of CO₂-limitation were required for the activation of CA_ext.     

Degradation activity of adhered and suspended Pseudomonas cells cultured on 2,4,6-trichlorophenol, measured by indirect conductimetry

The degradation activity (expressed as specific CO₂ production rates) of adhered and suspended Pseudomonas cells, strains SP1 and SP2, during the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), was compared using indirect conductimetry technique. This technique is defined as the measurement of CO₂ ionization in an alkaline solution and expressed as the negative conductance change values of such solution. The attachment surfaces were porous glass and silicone rubber. The 2,4,6-TCP concentrations ranged from 10 to 500 mg 1⁻¹. Specific respiration rates were determined from CO₂ evolution rates and biomass yields of both suspended and adhered cell cultures. CO₂ evolution rates were determined after conversion of conductance change values into CO₂ produced values. Results indicate that glass-adhered cells reached a higher maximum specific CO₂ evolution rate ( Q _CO2max) than both suspended and silicone rubber-adhered cells. However, suspended cells showed a lower saturation constant ( K_s ) than the adhered cells. These results suggest that depending on support nature the respiration activity of adhered cells could be higher than of suspended cells. Moreover, the indirect conductimetry technique could efficiently be used by measurements of respiration activities of both attached or suspended xenobiotic-degrading micro-organisms.     

Prostaglandin E2 Induces Interleukin-6 Synthesis in Human Astrocytoma Cells

Abstract: Prostaglandins (PGs) and cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), have been implicated in the etiopathology of various inflammatory and degenerative disorders, including Alzheimer's disease (AD) and prion diseases. Nonsteroidal antiinflammatory drugs (NSAIDs), potent inhibitors of PG synthesis, appear to be beneficial in the treatment of AD. To assess whether PGs are able to induce IL-6 synthesis in cells of the CNS, IL-6 mRNA and protein syntheses were measured in a human astrocytoma cell line after stimulation with different PGs. PGE₁ and PGE₂, but not PGD₂ and PGF_2α, led to a rapid and transient induction of IL-6 mRNA, followed by IL-6 protein synthesis. Furthermore, PGE₂ potentiated IL-1β-induced IL-6 mRNA synthesis. These results are discussed with respect to the participation of PGs in neurodegenerative diseases (and its inhibition by NSAIDs) by affecting cytokine expression.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

Effect of inhibitors on ammonium assimilation in Chlorella sorokiniana in light and darkness

In N-sufficient cells of Chlorella sorokiniana Shihira and Krauss strain 211/8K (CCAP of Cambridge University), assimilation of ammonium was strictly dependent on light and CO₂, and was severely inhibited by 100 μ M atrazine or 10 μ M 3-(3,4-dichlorophenyl)-1, l-dimethylurea (DCMU). In N-limited cells, assimilation of NH₄⁺ took place at similar rates in both light and darkness, which were 1.6-fold higher than the rate of light-dependent assimilation by N-sufficient cells. Assimilation by N-limited cells was inhibited by l -methionine- dl -sulfoximine (MSX), but not by atrazine or DCMU.
The rate of photosynthetic O₂ evolution was 2.9±0.9 mmol ml⁻¹ packed cell volume (PCV) h⁻¹ in N-sufficient cells, and 0.64±0.12 mmol ml⁻¹ PCV h⁻¹ in N-limited cells. In the latter resupply of ammonium resulted in a rapid activation by 22%;, followed by a time-dependent increase of the photosynthetic O₂ evolution, which after 12 h reached the same rate as in N-sufficient cells.
Respiratory consumption of oxygen in darkness in N-sufficient and N-limited cells was 0.10±0.03 and 0.11±0.02 mmol ml⁻¹ PCV h⁻¹, respectively. Addition of ammonium was without effect on respiration of N-sufficient cells, but resulted in a 4-fold stimulation of respiration of N-limited cells. Such stimulation took place also in cells treated with DCMU, atrazine, or MSX, and it was also promoted by methylammonium. The stimulation of respiration lasted for several hours.     

Effects of thermal stress on respiratory responses to hypoxia of a South American Prochilodontid fish, Prochilodus scrofa

Oxygen consumption (o₂) and respiratory variables were measured in the Prochilodontid fish, Prochilodus scrofa exposed to graded hypoxia after changes in temperature. The measurements were performed on fish acclimated to 25°C and in four further groups also acclimated to 25°C and then changed to 15, 20, 30 and 35°C. An increase in o₂ occurred with rising temperature, but at each temperature o₂ was kept constant over a wide range of O₂ tensions of inspired water ( Pi o₂). The critical oxygen tensions ( Pc o₂) were Pi o₂= 22 mmHg for 25°C acclimated specimens and after transfer from 25°C to 15, 20, 30 and 35°C the Pc o₂ changed to Pi o₂= 28, 22, 24 and 45 mmHg, respectively. Gill ventilation ( _G ) increased or decreased following the changes in o₂ as the temperature changed and was the result of an accentuated increase in breath frequency. During hypoxia the increases in _G were characterized by larger increases in breath volume. Oxygen extraction was kept almost constant at about 63% regardless of temperature and ambient oxygen tensions in normoxia and moderate hypoxia ( P o₂∼70 mmHg). P. scrofa showed high tolerance to hypoxia after abrupt changes in temperature although its survival upon transfer to 35°C could become limited by the capacity of ventilatory mechanisms to alleviate hypoxic stress.     

Biosynthesis of Prostacyclin in Rat Cerebral Microvessels and the Choroid Plexus

Abstract: Microvessels, predominantly capillaries, were isolated from rat cerebrum by a modification of published procedures. The morphology and purity of the preparations were monitored by light and electron microscopy and by enrichment in alkaline phosphatase, γ-glutamyl transpeptidase, and prostacyclin synthetase. A reversed-phase high-pressure liquid chromatographic method was used in the purification of prostaglandins after extraction from aqueous incubation solutions. Prostacyclin synthesis in brain is localized in cerebral blood vessels and capillaries. The endogenous biosynthetic capacity of the isolated cerebral capillary fractions for prostacyclin, measured as its chemically stable breakdown product, 6-keto-prostaglandin F_1α, was 11 ng/mg protein/10 min. Choroid plexus and intact surface vessels synthesized 6-keto-prostaglandin F_1α at 37 and 35 ng/mg protein/10 min, respectively. The prostacyclin-synthesizing enzyme of the cerebral capillaries also converted the exogenously added prostaglandin endoperoxides to 6-keto-prostaglandin F_1α. Comparison of the synthesis of prostaglandins 6-keto-F_1α, E₂, and F_2α showed that 6-keto-prostaglandin F_1α was the major prostaglandin formed in the microvessels, in the larger surface vessels, and in the choroid plexus. Prostaglandin D₂ was not detected. Prostacyclin synthesis by the cerebral vasculature is similar to that in other blood vessels and cultured human endothelial cells. Possible physiological roles of prostacyclin in the cerebral microvasculature are discussed with special regard to the autoregulation of cerebral blood flow.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

Regulation of nitrate uptake into Chara corallina cells via NH+4 stimulation of NO−3 efflux

Abstract. Net NO₃ uptake by NO⁻₃ deficient Chara cells was used to calculate [NO⁻₃]_c assuming that the cytoplasm occupies 10% total volume and that nitrate reduction and storage are negligible (i.e. maximum [NO⁻₃]_c was calculated). A linear relationship was found between NO⁻₃ efflux and [NO⁻₃]_c. There was an initial burst of NO⁻₃ efflux when NH⁺₄ was added, followed by a slower efflux rate which matched influx rate such that net NO⁻₃ uptake was zero. Over 50% of NO⁻₃ that had been taken up in 2 h was lost within the first 5 min of NH⁺₄ addition. The Nernst equation was used to predict the direction of the electrochemical driving force for NO⁻₃ entry. Under the experimental conditions used NO⁻₃ efflux is actively transported. The differential involvement of both NO⁻₃ influx and NO⁻₃ efflux in the regulation of NO⁻₃ uptake is discussed and a model is proposed to account for these results which envisages discrete NO⁻₃ influx and NO⁻₃ efflux carriers.     

Appearance of a reversible hydrogenase activity in Anabaena cylindrica grown in high light

In vivo H₂ evolution by Anabaena cylindrica Lemm. strain PCC 7122 grown in the presence of ammonia at low and high light intensities was studied. We found that after 2 h of anaerobic incubation, H₂ evolution [at a rate of 0.5 μmol (mg dry weight)¹ h⁻¹] via reversible hydrogenase occurred in high light grown cells, while this kind of activity was not found in low light grown cells. H₂ evolution was inhibited by 3-(3'. 4'-dichlorophenyl-1, 1-dimethylurea (DCMU). Illuminating the cells in the phycocyanin absorption region resulted in a higher rate of H₂ evolution than illuminating the cells in the chlorophyll absorption region. The results indicate that reversible hydrogenase receives reducing equivalents from photosynthetic water photolysis and that both photosystem II and photosystem I participate in the H₂ production. Hydrogenase activity was found in the soluble fraction after mild sonication in the case of low light grown cells. After this treatment high light grown cells retained 70% of their hydrogenase activity in the particulate fraction, but released it into the soluble fraction in the presence of 2% deoxycholic acid.
In vitro H₂ evolution did not differ significantly in the low and high light grown cells. Hence, the differences in the in vivo H₂ evolution reflect the different availability of endogenous reductants for hydrogenase in the two kinds of cells. On the basis of our results it is suggested that high light grown Anabaena cells eliminate part of the photosynthetically produced excess electrons via an induced reversible hydrogenase activity. This is the first report of H₂ evolution associated with water photolysis and catalyzed by hydrogenase in cyanobacteria.     

Variability of ecdysteroid-induced cell cycle alterations in Drosophila Kc sublines

Abstract. The cell cycle of two lines isolated from Drosophila Kc cells was followed by flow cytofluorometry and cell counting. The first line is the 8-9K clone which grew in a medium supplemented with 5% serum; the second, named subline Kc0, grew in a serum-free medium. The stationary phase is characterized by a G₂ cell accumulation: 73% in the 8-9K clone and 50% in the Kc0 subline.
When the medium was supplemented with the steroid moulting hormone 20-hydroxyecdysone, more than 90% of 8-9K cells and 65% of Kc0 cells were progressively arrested in G₂. In the continuous presence of 20-hydroxyecdysone, most of the 8-9K cells remain G₂-arrested; no massive G₂ release into M was observed and only a few cells were able to divide. When treated for only 3 or 7 days, a transient release into M and proliferation occurred after hormone-free medium renewal, largely masked by G₂ cell death. These results are discussed in comparison with other reports on cell cycle alteration induced by ecdysteroids.     

Constitutive Activity and Structural Instability of the Wild-Type Human H2 Receptor

Abstract: Stable expression of the human H₂ receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H₂ receptor. Burimamide, a neutral antagonist at the rat H₂ receptor, behaved as a weak partial agonist at the human H₂ receptor. Burimamide competitively antagonized both the histamine-induced increase in cAMP and the cimetidine-induced reduction of the basal cAMP level with apparent K _B values that were similar to its H₂ receptor affinity. Investigation of the modulation of receptor expression after long-term drug treatment revealed that at low concentrations histamine induced a significant reduction in H₂ receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an upregulation of the human H₂ receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H₂ receptor in transfected Chinese hamster ovary cells. Occupation of the H₂ receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.     

N-Methyl-d-Aspartate-Sensitive Glutamate Receptors Induce Calcium-Mediated Arachidonic Acid Release in Primary Cultures of Cerebellar Granule Cells

Abstract: In primary cultures of cerebellar granule cells, glutamate, aspartate, and N -methyl-d-aspartate (NMDA) induced a dose-dependent release of [³H]arachidonic acid ([³H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist-induced [³H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A₂ (PLA₂) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor-mediated activation of PLA₂ rather than to the action of PLC followed by diacylglycerol lipase. The receptor-mediated [³H]AA release was dependent on the presence of extracellular Ca²⁺ and was mimicked by the Ca²⁺ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor-mediated [³H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA-sensitive glutamate receptors leads to the activation of PLA₂ that is mediated by Ca²⁺ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin-sensitive GTP-binding protein can be excluded.     

SPERMATOGONIAL STEM CELL RENEWAL IN THE MOUSE: II AFTER CELL LOSS

The repair of the mouse seminiferous epithelium after cell loss has been studied in seminiferous tubules mounted in toto . Cell loss was inflicted by injection of Myleran in a dose of 10 mg/kg body weight. In stages 7–8, in which we mainly counted, the numbers of A_isolated (A_is), A_paired (A_pr), A_aligned (A_al) and A₁ spermatogonia and resting primary spermatocytes decreased after injection. After about 24 days normal numbers of A₁ spermatogonia were found again. Thereafter a substantial overshoot in the number of A₁ spermatogonia was found.
While normally most of the A_pr and A_al cells differentiate into A₁ spermatogonia in stages 3 and 4 and do not divide until stage 9, during repair they pass through one more division during stages 6 and 7. Normally, during these stages divisions of these spermatogonia are rare. Owing to this extra division the transformation of A_pr and A_al into A₁ spermatogonia is delayed from stage 3 or 4 to stage 8, i.e. still before stage 9, in which A₁ spermatogonia divide. From 16 days after the injection onwards the extra division takes place less generally and more and more cells transform into A₁ spermatogonia at the normal time.     

Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii

Phosphate addition to P-limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O₂ consumption. The respiration rate continued to increase for several minutes after the addition of P₁. Similar patterns of P₁ stimulation of respiratory O₂ consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O₂ consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP-dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P₁, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P₁, whereas exhaustion of extracellular P₁ resulted in a rapid decline in the O₂ consumption rate. These results are consistent with control of respiration in P-limited cells occurring largely at the level of glycolysis.