Isolation and characterization of heterocysts from Anabaena sp. strain CA

A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (_max 615 nm) and allophycocyanin (_max 645 nm, shoulder 620 nm). Isolated heterocysts under H₂ showed acetylene reduction rates of 57 nmol C₂H₄/mg dry wt·min (342 mol C₂H₄/mg chl a·h), whereas intact filaments reduced at the rate of 18 nmol C₂H₄/mg dry wt·min (108 mol C₂H₄/mg chl a·h). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H₂ for more than 3 h. Addition of as little as 5% H₂ under argon stimulated the C₂H₂ reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O₂ either under H₂ or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C₂H₂ reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.     

Ecological rationale for H2 metabolism during aquatic blooms of the cyanobacterium Anabaena

Summary Nitrogenase-produced H₂ serves to remove excess intracellular O₂ during vigorous growth periods (blooms) of the nuisance cyanobacterium Anabaena. In two naturally-occurring species, A. oscillarioides and A. spiroides, nitrogen fixation (acetylene reduction) showed a high degree of resistance to O₂ inactivation. Under the influence of supersaturated O₂ concentrations, commonly encountered in lake blooms, elevated cellular ATP levels and enhanced uptake hydrogenase and nitrogenase activities were observed in actively growing filaments. Oxygen enhancement of nitrogenase activity appears mediated through localized uptake hydrogenase reactions. Hydrogen assimilated by hydrogenase is combined with O₂ in a Knallgas reaction, leading to the formation of H₂O and ATP via a respiratory chain. This combination of activities appears poised at O₂ removal and allows Anabaena to dominate O₂ supersaturated surface waters while maintaining optimal nitrogenase activity. Hence, instead of being a wasteful dissipation of reducing power, H₂ evolution via nitrogenase ultimately affords protection from O₂ while constituting a source of ATP through subsequent H₂ metabolism.     

Interchange-trisomics in barley

Observations on morphology, sterility and cytology of some interchangetrisomics obtained from the progeny of radiation induced interchangeheterozygotes in a 6-rowed barley (Hordeum vulgare L.) var. K₁₂ are described. The different interchange trisomics show distinct phenotypic expression and have been classified as bushy, slender, robust, semi-erect and pseudonormal on the basis of their gross morphology, leaf characteristics, awn and spike lengths. All interchange trisomics show pollen and ovule sterility which varies between plants. Meiotic behaviour is described. The five different interchange trisomics appear to have different extra chromosomes.     

Sensitivity of Nostoc commune UTEX 584 (Cyanobacteria) to water stress

Cells of Nostoc commune UTEX 584 from liquid cultures expressed an upshift in nitrogenase activity when immobilised on inert supports and exposed to matric water potentials between -1.10 and -99.5 MPa. Cells incubated at 0.10 MPa (a_w=c 1.0) maintained increased activity for at least 48 h following immobilization. At water potentials below -23.1 MPa (a_w=0.85), the upshift was transitory. Nitrogenase activity decreased rapidly when immobilised cells were incubated at lower values of _m.Desiccated cells stored at -99.5 MPa (a_w=0.50) underwent an upshift in nitrogenase activity, and in the size of the intracellular ATP pool, when rewetted with either distilled water or liquid MB_o medium (_o =-0.18 MPa). The upshift in nitrogenase activity was chloramphenicol-sensitive and was preceeded by a lag. The duration of the lag depended on the time taken to equilibrate cells to-99.5 MPa, the time desiccated, and the conditions of storage and rewetting. Cells that had no, or very low, nitrogenase activity when rewetted in air, showed a marked stimulation of nitrogenase activity in the presence of 5% v/v CO₂ under both aerobic and anerobic conditions.When rewetted in the presence of 1% w/v glucose (_o =-0.14 MPa), vegetative cells remained intact, but heterocysts underwent autolysis and nitrogenase activity was not detected, even in the presence of 5% v/v CO₂.Abbreviations TTC 2,3,5-triphenyl-2-tetrazolium chloride - _m matric water potential - _o osmotic water potential - a_w water activity     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

Growth,acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

A comparative study was conducted on the effect of NH₄Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (P-N), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (P-N) or with 0.2 g NH₄Cl/l (P+N). Strain Cp1.2 as may other Frankia strains, showed on P+N medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on P-N medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in P+N media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH ₄ ⁺ -grown cells. The regulation of induction of nitrogenase in Frankia by NH₄Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH ₄ ⁺ -grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.     

Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins

Photon yields of oxygen evolution at saturating CO₂ were determined for 44 species of vascular plants, representing widely diverse taxa, habitats, life forms and growth conditions. The photonyield values on the basis of absorbed light ( ^a) were remarkably constant among plants possessing the same pathway of photosynthetic CO₂ fixation, provided the plants had not been subjected to environmental stress. The mean ^a value ±SE for 37 C₃ species was 0.106±0.001 O₂·photon^-1. The five C₄ species exhibited lower photon yields and greater variation than the C₃ species ( ^a=0.0692±0.004). The ^a values for the two Crassulaceanacid-metabolism species were similar to those of C₃ species. Leaf chlorophyll content had little influence on ^a over the range found in normal, healthy leaves. Chlorophyll fluorescence characteristics at 77 K were determined for the same leaves as used for the photon-yield measurements. Considerable variation in fluorescence emission both at 692 nm and at 734 nm, was found 1) among the different species; 2) between the upper and lower surfaces of the same leaves; and 3) between sun and shade leaves of the same species. By contrast, the ratio of variable to maximum fluorescence emission at 692 nm (F_v/F_M, 692) remained remarkably constant (The mean value for the C₃ species was 0.832±0.004). High-light treatments of shade leaves resulted in a reduction in both ^a and the F_v/F_M, 692 ratio. The extent of the reductions increased with time of exposure to bright light. A linear relationship was obtained when ^a was plotted against F_v/F_M, 692. The results show that determinations of the photon yield of O₂ evolution and the F_v/F_M, 692 ratio can serve as excellent quantitative measures of photoinhibition of overall photosynthetic energy-conversion system and of photochemistry of photosystem II, respectively. This is especially valuable in field work where it is often impossible to obtain appropriate controls.Abbreviations and symbols CAM Crassulacean acid metabolism - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F_o, F_M, F_v instantaneous, maximum, variable fluorescence emission - absorptance - ^a photon yield (absorbed light) - ⁱ photon yield (incident light) C.I.W.-D.P.B. Publication No. 923     

Niche dimensions of New England cottontails in relation to habitat patch size

We examined physical condition, niche dimensions, and survival of New England cottontails (Sylvilagus transitionalis) that occupied 21 habitat patches of different sizes during winter. Rabbits on small patches (2.5 ha) were predominantly males, and both sexes had lower body mass than individuals on large patches (5.0 ha). Niche indices (, where ranges from 0 to 1. and values approaching 1 indicate generalized resource use) of habitat use revealed that rabbits on small patches used a greater variety of microhabitats (based on understory stem density: _s, and proximity to cover: _c) than rabbits occupying large patches (_s=0.65, _c=0.66). Rabbits on small patches also consumed low quality forage more often and fed at sites farther from escape cover than rabbits on large patches. There were no significant correlations between rabbit densities and niche dimensions. Niche expansion was not a result of compertitive release or relaxation of predator pressure. Rabbits on small patches apparently modified their niche dimensions in response to resource limitations. This response included occupying sites with limited understory cover that apparently resulted in rabbits on small patches having a lower survival rate (0.35) than rabbits on large patches (0.69) during a 10-week monitoring period. Skewed sex ratios and low survival rates among rabbits on small patches suggest that these habitats act as sinks to dispersing, juveniles from large (source) patches. As a result, local populations of New England cottontails may become vulnerable to extinction if larte patches of habitat are not maintained.     

Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1

Under diurnal 16/8-h light-dark cycles, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) at 1 mM completely blocked the appearance of rhythmic N₂-fixing activity in Synechococcus RF-1. Ca²⁺ at 2 mM, when supplied either together with or several hours after the EGTA application, restored the nitrogenase activity, whereas, when Ca²⁺ was supplied several hours later, the peak of nitrogenase activity was shifted from the dark to the light period in which the activity is normally suppressed. Sr²⁺ also reversed the inhibition by EGTA, but only partially. When O₂ in the gas phase above the culture was below 1%, the inhibition of nitrogenase activity by EGTA was reduced to less than 20% of the control value without EGTA. Thus Ca²⁺ appears to be required by the cell to protect its nitrogenase from inactivation by O₂. In media without EGTA, a close correlation between nitrogenase activity and concentrations of Ca²⁺ was also observed.Abbreviation EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Nitrogen fixation and nitrogen metabolism in heliobacteria

Three species of anoxygenic phototrophic heliobacteria, Heliobacterium chlorum, Heliobacterium gestii, and Heliobacillus mobilis, were studied for comparative nitrogen-fixing abilities and regulation of nitrogenase. Significant nitrogenase activity (acetylene reduction) was detected in all species grown photoheterotrophically on N₂, although cells of H. mobilis consistently had higher nitrogenase activity than did cells of either H. chlorum or H. gestii. Nitrogen-fixing cultures of all three species of heliobacteria were subject to switch-off of nitrogenase activity by ammonia; glutamine also served to switch-off nitrogenase activity but only in cells of H. mobilis and H. gestii. Placing photosynthetically grown heliobacterial cultures in darkness also served to switch-off nitrogenase activity. Dark-mediated switch-off was complete in lactate-grown heliobacteria but in pyruvate-grown cells substantial rates of nitrogenase activity continued in darkness. In all heliobacteria examined ammonia was assimilated primarily through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway although significant levels of alanine dehydrogenase were present in extracts of cells of H. gestii, but not in the other species. The results suggest that heliobacteria, like phototrophic purple bacteria, are active N₂-fixing bacteria and that despite their gram-positive phylogenetic roots, heliobacteria retain the capacity to control nitrogenase activity by a switch-off type of mechanism. Because of their ability to fix N₂ both photosynthetically and in darkness, it is possible that heliobacteria are significant contributors of fixed nitrogen in their paddy soil habitat.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H₂- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.Abbvreviations ATCC American Type Culture Collection - Diaminodurol (DAD) 2,3,5,6-tetramethyl-p-phenylenediamine dihydrochloride - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol - E Einstein (mol photons) - FNR ferredoxin - NADP oxidoreductase (EC 1.18.1.2) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Metronidazole 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole     

On the relationship between chlorophyll fluorescence quenching and the quantum yield of electron transport in isolated thylakoids

The relationship between the empirical fluorescence index F/Fm and the quantum yield of linear electron flow, ^s, was investigated in isolated spinach thylakoids. Conditions were optimised for reliable determination of F/Fm and ^s with methyl viologen or ferricyanide as electron acceptors under coupled and uncoupled conditions. Ascorbate in combination with methyl viologen was found to stimulate light-induced O₂-uptake which is not reflected in F/Fm and interpreted to reflect superoxide reduction by ascorbate. In the absence of ascorbate, the plot of F/Fm vs. ^s was mostly linear, except for the range of high quantum yields, i.e. at rather low photon flux densities. With ferricyanide as acceptor, use of relatively low concentrations (0.1–0.3 mM) was essential for correct Fm-determinations, particularly under uncoupled conditions. Under coupled and uncoupled conditions the same basic relationship between F/Fm and ^s was observed, irrespective of ^s being decreased by increasing light intensity or by DCMU-addition. The plots obtained with methyl viologen and ferricyanide as acceptors were almost identical and similar to corresponding plots reported previously by other researchers for intact leaves. It is concluded that the index F/Fm can be used with isolated chloroplasts for characterisation of such types of electron flow which are difficult to assess otherwise, as e.g. O₂ dependent flux. The origin of the non-linear part of the relationship is discussed. An involvement of inactive PS II centers with separate units and inefficient Q_A-Q_B electron transfer is considered likely.Abbreviations AsA - ascorbate - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MDA - monodehydroascorbate - MV - methyl viologen - PAR - photosynthetically active radiation - SOD - superoxide dismutase This paper is dedicated to David Walker who after 40 years in the field of photosynthesis is now retiring from his duties at Sheffield University.     

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.     

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Cell differentiation and pigment composition in Anabaena cylindrica

Summary Cell differentiation in Anabaena cylindrica is accompanied with characteristic changes in the pigment composition of heterocysts and spores. In both the absence of phycocyanin is consistent with the lack of CO₂-fixing ability previously reported. The presence of chlorophyll and -carotene suggests a functional photosystem I in heterocysts. In the spores chlorophyll is largely replaced by pheophytin. The quantitative distribution of carotenoids is also affected. An increase in the proportion of -carotene is characteristic of heterocysts while spores show a larger proportion of xanthophylls compared with the intact filament.     

Secretion of either of a pair of immunoglobulins,IgM or IgX,in somatic hybrid cells derived by fusion of a B-cell lymphoma cell line carrying both immunoglobulin isotypes

The I.29 cell line is a nonsecreting B-cell leukemia which bears two different immunoglobulin isotypes on its surface, IgM and IgX. The I.29 cells were hybridized with nonsecreting myeloma cells giving rise to dozens of immunoglobulin secreting hybridomas. These fall into three groups differing in the class of immunoglobulin they secrete. Cells of the first group secrete pentameric IgM (, ), those of the second group secrete an unknown immunoglobulin, IgX, which may constitute an allotype of IgA, and those of the third group produce light chains only. The two complete immunoglobulins, IgM and IgX, have the same idiotype, as revealed by serological cross-reactivity of an exhaustively absorbed rabbit anti-idiotype serum.The molecular sizes of the heavy chains of the secreted IgM and IgX are slightly smaller than the and chains, respectively, which are derived from the surface of normal B cells as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Abbreviations used in this paper Ig immunoglobulin - NMS normal mouse serum - NaDodSO₄ sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - 2ME 2-mercaptoethanol - BPB bromophenol blue - NP40 nonidet P40 This particular immunoglobulin heavy chain has not been fully characterized. It is neither , , nor but is related to, although not identical with, . Because this immunoglobulin has unique properties, it is referred to as IgX.     

Nitrite reduction in Bacteroids of Rhizobium “hedysari” strain HCNT 1

Ex planta, bacteroids of the sulla-symbiont Rhizobium hedysari strain HCNT 1 terminated reduction of nitrite at nitrous oxide irrespective of the presence or absence of acetylene. Nitrate was not reduced during the experimental period, but slight nitrate reductase activity occurred if incubation with nitrate was prolonged (up to 15 h). As was observed in free-living cells, exposure of the bacteroids to the metal chelator, diethyldithiocarbamate, prevented reduction of nitrite, indicating the presence of a copper-containing nitrite reductase. Pulses of 10–75 M nitrite transiently impeded O₂ uptake in bacteroids, which resumed consumption of O₂ when the nitrite had been reduced. Exposure to >1.0 mM nitrite for 24h greatly inhibited nitrogenase activity (assayed as acetylene reduction activity) of bacteroids in planta. Exposure to the same concentrations of nitrite after 1h of incubation in the presence of acetylene almost completely stopped ongoing ethylene production in bacteroids of strain HCNT 1 extracted from nodules. Free cells of the non-nitrite-reducing R. hedysari strain CC 1335 were lacking in nitrogenase (acetylene-reduction) activity, whereas identically cultured (low-oxygen) strain HCNT 1 cells reduced both nitrite and acetylene.Abbreviations PMS phenazine methosulfate - DDC diethyldithiocarbamate     

The rapid switch-off of nitrogenase activity in obligate methane-oxidizing bacteria

Nitrogenase activity in the obligate methaneoxidizing bacterium Methylococcus capsulatus (Bath) was added ammonia. This observation was extended to include other ammonia. This observation was extended to include other representative N₂-fixing species of methanotrophs. The ammonia switch-off of nitrogenase in M. capsulatus (Bath) was reversed on washing cells to remove excess ammonia, in the presence of chloramphenicol, suggesting that a form of covalent modification of nitrogenase may occur. Replacing the oxidizable substrate methanol with formaldehyde, formate, ethanol or hydrogen had no effect on nitrogenase switch-off. A number of potential nitrogen sources or intermediates of nitrogen metabolism such as glutamine, asparagine, glutamate and alanine when tested, did not effect switch-off. However, the rapid inhibition of nitrogenase activity of M. capsulatus (Bath) could be achieved by adding the uncoupler carbonylcyanide m-chlorophenylhydrazone or nitrite. The glutamine synthetase inhibitor methionine sulphoximine blocked the switch-off effect of ammonia, indicating that the metabolism of ammonia may be essential for switch-off to occur. Inhibitors of glutamate synthase did not alleviate the ammonia switch-off response. Methionine sulphoximine did not alleviate the rapid inhibition of nitrogenase by carbonylcyanide m-chlorophenylhydrazone indicating that the shortterm regulation of nitrogenase by uncouplers and ammonia proceed via different mechanisms.Abbreviations MSX methionine-DL-sulphoximine - DON 6-diazo-5-oxo-L-norleucine - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase (glutamate synthase) - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Utilization of Energy Nutrients by Cerebellar Slices

We performed an ontogenetic study about the utilization of glycine, glutamine, -hydroxybutyrate and glycerol as energy nutrients by rat cerebellum slices. Production of CO₂ from glycerol and glutamine increased with the animals' age and glutamine was the most used nutrient for CO₂ production. In adult age, glutamine oxidation to CO₂ was 15 to 35 times higher than all other nutrients studied. CO₂ production from glycine decreased markedly with age and 10 day-old rats showed an oxidation 7.5 times higher than that of adult rats. At fetal age and at 10 postnatal days, glycine oxidation to CO₂ was only 2 times lower than glutamine oxidation to CO₂. Lipid synthesis from -hydroxybutyrate was highest in adult rats. We did not observe any difference in the utilization of -hydroxybutyrate between slices of cerebral cortex and cerebellum at the ages of 10 days and adult. The main nutrients used for lipid synthesis were glycerol and -hydroxybutyrate.