Integral kinetics of the cleavage of maltose catalysed by α-glucosidase fromSaccharomyces carlsbergensis

Summary A new, sensitive and continuous assay for -glucosidase is described exploiting the different angles of rotation for the substrate maltose and the product glucose. Kinetic experiments revealed a very pronounced product inhibition of -glucosidase fromSaccharomyces carlsbergensis with a K_i of 4.85·10^–3 M for glucose.The K_M of maltose was found to be 37.8·10^–3 M. Taking these values, an integral kinetic curve for the enzymatic hydrolysis of maltose was calculated, which is shown to fit the experimental data.Symbols used k₁ (min^–1) pseudo first-order rate constant (for enzymatic cleavage) - k₂ (min^–1) rate constant (for mutarotation reaction) - I, P (mol/1) inhibitor (product) concentration - k_i (mmol/1) inhibitor constant - K_M (mmol/l) Michaelis constant - [M] ₅₈₉ ³⁰ (degree/m · l/mol) molecular rotation at 30°C and 589 nm - s (mmol/l) substrate concentration - R (mmol/mg · min) reaction rate - V_max (mmol/mg · min) maximal rate - U (mol/min) activity unit (here at 30°C and pH=6.8) Indices O initial value - max maximal value     

Preparation of monoclonal antibody-ferritin conjugates of high specific activity

Summary ¹²⁵I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 11.4, contained over 90% ferritin-IgG monomers; 70–90% of the ¹²⁵I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.Dr. Rudick is the recipient of Teacher-Investigator Development Award PHS 1 KO7 NS 00791-01     

Effect of antagonists of ethylene action on binding of ethylene in cut carnations

Five hours after cut carnations had been treated with a pulse of 1 or 4 mM silver thiosulfate (STS), in vivo ethylene binding in petals was inhibited by 22 and 29%, respectively. When binding was measured 4 days after the 4-mM STS treatment, binding was inhibited by 81%. 2,5-Norbornadiene, which substantially delays carnation senescence, inhibited ethylene binding by 41% at a concentration of 1000 l/l. The Kd for ethylene binding in carnations was estimated to be 0.1 l/l in petals and 0.09 l/l in leaves. The concentration of binding sites was estimated to be 6.0×10^–9 mol/kg of petals and 2.0×10^–9 mol/kg of leaves     

Leaf photosynthetic characteristics of seedlings of actinorhizal Alnus spp. and Elaeagnus spp.

Single leaf photosynthetic characteristics of Alnus glutinosa, A. incana, A. rubra, Elaeagnus angustifolia, and E. umbellata seedlings conditioned to ambient sunlight in a glasshouse were assessed. Light saturation occurred between 930 and 1400 mol m^-2s^-1 PAR for all species. Maximum rates of net photosynthesis (Pn) measured at 25°C ranged from 12.8 to 17.3 mol CO₂m^-2s^-1 and rates of dark respiration ranged from 0.74 to 0.95 mol CO₂m^-2s^-1. These values of leaf photosynthetic variables are typical of early to midsuccessional species. The rate of Pn measured at optimal temperature (20°C) and 530mol m^-2s^-1 PAR was significantly (p<0.01) correlated with leaf nitrogen concentration (r=0.69) and negatively correlated with the mean area of a leaf (r=–0.64). We suggest that the high leaf nitrogen concentration and rate of Pn observed for Elaeagnus umbellata and to a lesser degree for E. angustifolia are genetic adaptations related to their crown architecture.Abbreviations Pn net photosynthesis     

Upregulation of (+)-7-Hydroxy-N,N-di-n-[3H]Propyl-2-Aminotetralin Binding Following Intracerebroventricular Administration of a Nitric Oxide Generator

Nitric oxide modulation of dopamine D₂ and D₃ receptor binding was examined using [¹²⁵I]epidepride (D₂) and (+)7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin([³H](+)-7-OH-DPAT, D₃). Nitric oxide, generated by i.c.v. injection of S-nitroso-N-acetyl-penicillamine (SNAP; 5 g or 10 g), significantly increased the density of [³H](+)-7-OH-DPAT binding sites (39% and 134%, respectively) in the striatum 24 hours post-injection in the absence of Gpp(NH)p, representing an upregulation of either D₃, receptors or high affinity D₂ receptors. In the presence of 10 M Gpp(NH)p, D₃ receptor upregulation was maintained in both the 5 g (increased 35%) and 10 g SNAP (increased 44%) groups. [³H](+)-7-OH-DPAT binding was reduced in both striatum and nucleus accumbens in the presence of 10 M Gpp(NH)p compared to binding in the absence of Gpp(NH)p, suggesting an upregulation of D₃ receptors. Administration of SNAP did not alter total specific [¹²⁵I]epidepride binding in either brain region. These data suggest that; 1) D₃ receptor density is modified following nitric oxide generation, and 2) the density of high affinity D₂ receptors identified by [³H](+)-7-OH-DPAT increases in the striatum, but decreases in the nucleus accumbens.     

Upregulation of NMDA Receptors in Hippocampus and Cortex in the Pentylenetetrazol-Induced “Kindling” Model of Epilepsy

Kindling is a phenomenon of epileptogenesis, which has been widely used as an experimental model of temporal lobe epilepsy. At the present work we investigated the contribution of NMDA receptors in the Pentylenetetrazol-induced kindling model in the mouse brain, by using quantitative autoradiography and the radioactive ligands [³H]MK801 and [³H]L-glutamate (NMDA-sensitive component). One week after establishment of kindling, a small but significant increase in [³H]MK801 as well as NMDA-sensitive [³H]glutamate binding was seen, being restricted to the molecular layer (ML) of the dentate gyrus (DG) and the CA3 region of the hippocampus. These binding augmentations persisted one month after establishment of kindling. A significant increase of NMDA receptor binding was also observed in the cortex-somatosensory and temporal one week after acquisition of the kindled state. The upregulation of NMDA receptors seen in DG and CA3 region of the hippocampus could be associated with the kindling process of this model especially with its maintenance phase, since it persists at long term, is area-specific and consistent with electrophysiological data. The increase of NMDA receptors seen in the cortex of the kindled animals could underlie the hyperexcitability detected by electrophysiological studies in this area.     

Extracellular Glucose Oxidase of Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1 using ultrafiltration membranes was developed. Two samples of the enzyme with a specific activity of 914–956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16°C was 2.74 × 10^–6 s^–1. This constant decreased significantly as the pH of the medium increased (4.0–10.0). The temperature optimum for glucose oxidase–catalyzed -D-glucose oxidation was in the range 30–65°C. At temperatures below 30°C, the activation energy for -D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase–catalyzed -D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Deficiency of activating Fcγ-receptors reduces hepatic clearance and deposition of IC and increases CIC levels in mercury-induced autoimmunity

Background

Inorganic mercury (Hg) induces a T-cell dependent, systemic autoimmune condition (HgIA) where activating Fcγ-receptors (FcγRs) are important for the induction. In this study we examined the influence of activating FcγRs on circulating levels and organ localization of immune complexes (IC) in HgIA.

Methods and Principal Findings

Mercury treated BALB/c wt mice showed a significant but modest increase of circulating IC (CIC) from day 12 until day 18 and day 35 for IgG2a- and IgG1- CIC, respectively. Mercury-treated mice lacking the trans-membrane γ-chain of activating FcγRs (FcRγ^−/−) had significantly higher CIC levels of both IgG1-CIC and IgG2a-CIC than wt mice during the treatment course. The hepatic uptake of preformed CIC was significantly more efficient in wt mice compared to FcγR^−/− mice, but also development of extrahepatic tissue IC deposits was delayed in FcRγ^−/− mice. After 35 days of Hg treatment the proportion of immune deposits, as well as the amounts was significantly reduced in vessel FcRγ^−/− mice compared to wt mice.

Conclusions

We conclude that mice lacking functional activating FcγRs respond to Hg with increased levels and altered quality of CIC compared with wt mice. Lack of functional activating FcγRs delayed the elimination of CIC, but also significantly reduced extrahepatic tissue localization of CIC.     

A phase I study of neuroblastoma with the anti-ganglioside GD2 antibody 14.G2a

Summary Nine patients with neuroblastoma stage IV were treated with the murine monoclonal antibody 14.G2a, directed against disialoganglioside GD2. The antibody was injected daily for 5–10 days and the total applied dosage ranged between 100 mg/m² and 400 mg/m². The peak serum levels of mAb 14.G2a ranged from 28 µg/ml to 61 µg/ml. Pharmacokinetic data obtained in three patients indicated that the serum elimination of mAb 14.G2a fits a two-compartment model, with an -half-time (t _1/2 ) between 0.66 h and 1.98 h and a -half-time (t _1/2 ) between 30.13 h and 53.33 h. All patients presented with a human anti-(mouse IgG) antibody response either during or shortly after therapy. Eight patients showed a continuous decrease in complement component C4 during therapy, as well as an initial decrease in C3c and an initial increase in C3a, all suggesting an activation of the complement cascade. Side-effects consisted of allergic reactions like pruritus, exanthema, urticaria and of severe pain, predominantly located in the abdomen and lower extremities, which required the use of continuous intravenous morphine. Four patients additionally developed a transient hypertension and one patient experienced a transient nephrotic syndrome. Three patients were treated in an adjuvant setting and are not evaluable for tumor response. Of the remaining six patients, two had a complete remission, two showed a partial remission, and two patients did not respond to treatment.Supported by the Deutsche Krebshilfe     

The dual influences of dissolved organic carbon on hypolimnetic metabolism: organic substrate and photosynthetic reduction

We investigated the effect of dissolved organic carbon (DOC) on hypolimnetic metabolism (accumulation of dissolved inorganic carbon (DIC) and methane (CH₄)) in 21 lakes across a gradient of DOC concentrations (308 to 1540 mol C L^–1). The highly colored nature of the DOC in these lakes suggests it is mostly of terrestrial origin. Hypolimnetic methane accumulation was positively correlated with epilimnetic DOC concentration (Spearman rank correlation = 0.67; p < 0.01), an indicator of allochthonous DOC inputs, but not with photic zone chlorophyll a concentration (Spearman rank correlation = 0.30; p = 0.22). Hypolimnetic DOC concentrations declined in 19 of 21 lakes during the stratified period at rates that ranged from 0.06 to 53.9 mmol m^–2 d^–1. The hypolimnetic accumulation of DIC + CH₄ was positively correlated with, and, in most cases of comparable magnitude to, this DOC decline suggesting that DOC was an important substrate for hypolimnetic metabolism. The percentage of surface irradiance reaching the thermocline was lower in high DOC lakes (0.3%) than in low DOC lakes (6%), reducing hypolimnetic photosynthesis (as measured by the depth and magnitude of the deep dissolved oxygen maxima) in the high DOC lakes. In June, the hypolimnia of lakes with < 400 mol L^–1 DOC had high concentrations of dissolved oxygen and no CH₄, while the hypolimnia of lakes with DOC > 800 mol L^–1 were completely anoxic and often had high CH₄ concentrations. Thus, DOC affects hypolimnetic metabolism via multiple pathways: DOC was significant in supporting hypolimnetic metabolism; and at high concentrations depressed photosynthesis (and therefore oxygen production and DIC consumption) in the hypolimnion.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Isolation of membrane bound light-harvesting-complexes from the dinoflagellates Heterocapsa pygmaea and Prorocentrum minimum

We have isolated Chl a-Chl c-carotenoid binding proteins from the dinoflagellates Prorocentrum minimum and Heterocapsa pygmaea grown under high (500 mol m^–2 s^–1, HL) and low (35 mol m^–2 s^–1, LL) light conditions. We compared various isolation procedures of membrane bound light harvesting complexes (LHCs) and assayed the functionality of the solubilized proteins by determining the energy transfer efficiency from the accessory pigments to Chl a by means of fluorescence excitation spectra. The identity of the newly isolated protein-complexes were confirmed by immunological cross-reactions with antibodies raised against the previously described membrane bound Chl a-c proteins (Boczar et al. (1980) FEBS Lett 120: 243–247). Spectroscopic analysis demonstrated the relatedness of these proteins with the recently described Chl-a-c ₂-peridinin (ACP) binding protein (Hiller et al. (1993) Photochem Photobiol 57: 125–131; Iglesias Prieto et al. (1993) Phil Trans R Soc London B 338: 381–392). The water-soluble peridinin-Chl a binding-protein (PCP) was not detectable in P. minimum. Two functional forms of ACP with different pigmentation were isolated. A variant of ACP which was isolated from high-light grown cells, that specifically binds increased amounts of diadinoxanthin was compared to the previously described ACPs that bind proportionately more peridinin.Abbreviations ACP Chl a-Chl c-peridinin binding protein - AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride - DDM dodecyl -d maltoside - Deriphat 160 N-lauryl-beta-iminopropionic acid - HEPES (N-2-hydroxyethylpiparizine-N-2-ethanesulphonic acid) - HL high light (500 mol m^–2 s^–1) - LL low light (35 mol m^–2 s^–1) - ₇₃₀ fluorescence yield (emission at 730 nm) - PCP peridinin-Chl a-binding protein - PMSF phenyl-methyl-sulfonyl-fluoride - PS I Photosystem I - PS II Photosystem II     

A Monomeric Chicken IgY Receptor Binds IgY with 2:1 Stoichiometry

IgY is the principal serum antibody in birds and reptiles, and an IgY-like molecule was the evolutionary precursor of both mammalian IgG and IgE. A receptor for IgY on chicken monocytes, chicken leukocyte receptor AB1 (CHIR-AB1), lies in the avian leukocyte receptor cluster rather than the classical Fc receptor cluster where the genes for mammalian IgE and IgG receptors are found. IgG and IgE receptors bind to the lower hinge region of their respective antibodies with 1:1 stoichiometry, whereas the myeloid receptor for IgA, FcαRI, and the IgG homeostasis receptor, FcRn, which are found in the mammalian leukocyte receptor cluster, bind with 2:1 stoichiometry between the heavy chain constant domains 2 and 3 of each heavy chain. In this paper, the extracellular domain of CHIR-AB1 was expressed in a soluble form and shown to be a monomer that binds to IgY-Fc with 2:1 stoichiometry. The two binding sites have similar affinities: K_a1 = 7.22 ± 0.22 × 10⁵ m⁻¹ and K_a2 = 3.63 ± 1.03 × 10⁶ m⁻¹ (comparable with the values reported for IgA binding to its receptor). The affinity constants for IgY and IgY-Fc binding to immobilized CHIR-AB1 are 9.07 ± 0.07 × 10⁷ and 6.11 ± 0.02 × 10⁸ m⁻¹, respectively, in agreement with values obtained for IgY binding to chicken monocyte cells and comparable with reported values for human IgA binding to neutrophils. Although the binding site for CHIR-AB1 on IgY is not known, the data reported here with a monomeric receptor binding to IgY at two sites with low affinity suggest an IgA-like interaction.Fc receptors link the specificity of the adaptive immune system with the effector mechanisms of innate immune cells. In birds and reptiles, IgY is the principal serum antibody, and both mammalian IgG and IgE have evolved from an IgY-like ancestor, so studies of IgY offer insights into their origins (1). The historical contribution of chicken immunology to a wider understanding of the subject has been considerable (2), and recently several chicken IgY-Fc receptors have been identified. In this paper, the chicken antibody, IgY, is shown to bind to a chicken leukocyte receptor, CHIR-AB1,⁴ in a different manner from that of its mammalian orthologues, IgG and IgE, to their respective Fc receptors.Phagocytosis, mediated in mammals by IgG, and passive cutaneous anaphylaxis, mediated by both IgG and IgE in mammals, have been observed in chickens (3, 4), presumably both effected by IgY. In vitro, IgY binds to monocyte cell lines (5, 6), and an IgY receptor (CHIR-AB1) has been identified that is able to mediate the influx of calcium into cells (5).The genes for the mammalian high affinity IgE receptor, and several IgG receptors, are located in the classical Fc receptor cluster, whereas in chickens, this cluster is represented by a single gene, the product of which has been expressed and found not to bind IgY (7). Intriguingly, the first IgY leukocyte receptor, CHIR-AB1, was found to be a member of the chicken leukocyte receptor cluster (LRC) (5), adjacent to over 100 genes with high intersequence homology (8). This finding, together with phylogenetic analysis of the orthologous Fc receptor gene clusters (7, 9), implies that during the evolution of the IgY-like ancestor of both IgG and IgE, antibody-Fc binding function migrated from proteins expressed in the LRC to those in the classical Fc receptor cluster. The human LRC is the site of FcαRI, the leukocyte receptor for IgA (an antibody involved in mucosal immunity), the fetal IgG receptor (FcRn, involved in adult IgG homeostasis), and also a number of natural killer cell receptors including the HLA-G ligand, KIR2DL4 (10). A further leukocyte receptor for chicken IgY, also related to LRC receptors, was identified recently, on chromosome 20 (11), and remains to be characterized.Typically, the stoichiometry of the receptor-antibody complex differs for receptors located in the classical Fc receptor cluster and the LRC. Crystal structures of IgG complexes with FcγRIII and of IgE with FcϵRI show 1:1 receptor:antibody stoichiometry, with the receptor binding across both heavy chains in the lower hinge (12). In contrast, the crystal structure of FcαRI complexed with IgA shows 2:1 stoichiometry (13) as does that of FcRn with IgG (14), with the two receptors binding between the heavy chain constant domains 2 and 3 on each heavy chain. The IgY/receptor interaction could have either stoichiometry; on the one hand, IgY is an orthologue of IgG and IgE, which can both show 1:1 stoichiometry, but on the other hand, the location of the IgY receptor, CHIR-AB1, in the same gene cluster as the IgA and FcRn receptors suggests the possibility of a 2:1 stoichiometry. Consistent with either of these binding modes, the crystal structure of IgY-Fc reveals that many of the residues located in the receptor-binding sites in human IgE, IgG, and IgA are present and accessible in IgY (15).The single extracellular domain of the chicken leukocyte IgY receptor, CHIR-AB1, has been expressed in insect cells by Arnon et al. (16), who showed that this preparation consists of a mixture of soluble monomer and dimer. Because of the heterogeneity of the protein, it was not possible to ascertain whether the observed 2:1 stoichiometry of receptor binding to antibody involved two monomers or a single dimer binding to IgY. Thus, it was not possible to answer the question of whether the antibody-receptor complex most resembles that of human IgA or of IgG and IgE. We have expressed the extracellular domain of CHIR-AB1 in human HEK cells. It is a monomer, and we report here that it binds to IgY and IgY-Fc with 2:1 stoichiometry.     

Photosynthesis and growth rates of Laurencia brongniartii J. Agardh (Rhodophyta, Ceramiales) in preparation for cultivation

Laurencia brongniartii is usually found at depths below 4 m, but can be found in shallow subtidal areas in crevices and on the walls of a coral reef in Amami Oshima Island, Kagoshima Prefecture, Japan, where irradiances were significantly lower than those at similar depths in open water. In preparation for the possible cultivation of this species for its antibiotic compounds, the effects of temperature and irradiance on photosynthesis and growth were measured. Photosynthesis and growth rates of L. brongniartii explants were highest at 26 and 28 °C, which closely corresponded to temperatures found during August to late December when it was most abundant. The estimated maximum photosynthesis rate (P _max) was 4.41 mol photon m^–2 s^–1 at 26 °C and 4.07 mol photon m^–2 s^–1 at 28 °C. Saturating irradiance occurred at 95 mol photon m^–2 s^–1 at 26 °C and 65 mol photon m^–2 s^–1 at 28 °C. In contrast, growth experiments at 41.7 mol photon m^–2 s^–1 caused bleaching of explants and the maximum growth rate observed during the study was 3.02 ± 0.75% day^–1 at 28 °C and 25 mol photon m^–2 s^–1. The difference in the saturating irradiance for photosynthesis and the irradiance that caused bleaching in growth experiments suggests that long-term exposure to high irradiance was detrimental and should be addressed before the initiation of large scale cultivation.     

Effect of soil characteristics on sequential reduction and methane production in sixteen rice paddy soils from China, the Philippines, and Italy

The potentials for sequential reduction of inorganic electron acceptors and production of methane have been examined in sixteen rice soils obtained from China, the Philippines, and Italy. Methane, CO₂, Fe(II), NO ₃ ^- , SO ₄ ² , pH, E_h, H₂ and acetate were monitored during anaerobic incubation at 30 °C for 120 days. Based on the accumulation patterns of CO₂ and CH₄, the reduction process was divided into three distinct phases: (1) an initial reduction phase during which most of the inorganic electron acceptors were depleted and CO₂ production was at its maximum, (2) a methanogenic phase during which CH₄ production was initiated and reached its highest rate, and (3) a steady state phase with constant production rates of CH₄ and CO₂. The reduction phases lasted for 19 to 75 days with maximum CO₂ production of 2.3 to 10.9 mol d^-1 g^-1 dry soil. Methane production started after 2 to 87 days and became constant after about 38--68 days (one soil >120 days). The maximum CH₄ production rates ranged between 0.01 and 3.08 mol d^-1 g^-1. During steady state the constant CH₄ and CO₂ production rates varied from 0.07 to 0.30 mol d^-1 g^-1 and 0.02 and 0.28 mol d^-1 g^-1, respectively. Within the 120 d of anaerobic incubation only 6--17% of the total soil organic carbon was released into the gas phase. The gaseous carbon released consisted of 61--100% CO₂, <0.1--35% CH₄, and <5% nonmethane hydrocarbons. Associated with the reduction of available Fe(III) most of the CO₂ was produced during the reduction phase. The electron transfer was balanced between total CO₂ produced and both CH₄ formed and Fe(III), sulfate and nitrate reduced. Maximum CH₄ production rate (r = 0.891) and total CH₄ produced (r = 0.775) correlated best with the ratio of soil nitrogen to electron acceptors. Total nitrogen content was a better indicator for available organic substrates than the total organic carbon content. The redox potential was not a good predictor of potential CH₄ production. These observations indicate that the availability of degradable organic substrates mainly controls the CH₄ production in the absence of inorganic electron acceptors.     

The Biogeochemical Cycle of Methane on the Northwestern Shelf of the Black Sea

Seasonal investigations of methane distribution and rates of its oxidation and generation in the water column and sediments of the Black Sea northwestern shelf were carried out within the framework of the interdisciplinary projects European River–Ocean Systems (EROS-2000, EROS-21) and Biogenic Gases Exchange in the Black Sea (BigBlack) in August 1995, May 1997, and December 1999. Experiments that involved the addition of ¹⁴CH₃COONa and ¹⁴CO₂ to sediment samples showed the main part of methane to be formed from CO₂. Maximum values of methane production (up to 559 mol/(m² day)) were found in coastal sediments in summer time. In winter and spring, methane production in the same sediments did not exceed 3.6–4.2 mol/(m² day). The ¹³C values of methane ranged from –70.7 to –81.8, demonstrating its microbial origin and contradicting the concept of the migration of methane from cold seeps or from the oil fields located on the Black Sea shelf. Experiments that involved the addition of ¹⁴CH₄ to water and sediment samples showed that a considerable part of methane is oxidized in the upper horizons of bottom sediments and in the water column. Nevertheless, it was found that, in summer, part of the methane (from 6.8 to 320 mol/(m² day)) arrives in the atmosphere.     

Light and temperature effects on the growth rate of three freshwater [2pt] algae isolated from a eutrophic lake

Effects of light and temperature, on the growth of three freshwater green algae isolated from an eutrophic lake and identified as Selenastrum minutum, Coelastrum microporum f. astroidea and Cosmarium subprotumidumwere studied in batch cultures under non-nutrient limited conditions. Experiments were performed to determine the growth rate over a wide range of light intensities (30–456 mol m^–2 s^–1) and temperature (15–35°C), using a 15/9 (light/dark) photoperiod cycle. The maximum growth rates and the optimum light intensities at a temperature of 35°C were 1.73 d^–1 and 420 mol m^–2 s^–1for Selenastrum minutum, 1.64 d^–1 and 400 mol m^–2 s^–1 for Coelastrum microporum and 1.00 d^–1 and 400 mol m^–2 s¹ for Cosmarium subprotumidum. The results were fitted with the mathematical models of Steele (1965), Platt & Jassby (1976) and Peeters & Eilers (1978). Steele's function and equation of Platt & Jassby don't describe correctly the relationship between the growth and light intensity. In the opposite, the equation of Peeters & Eilers provides the best fit for the three species.     

Interrelationships Between Nitrogen Supply and Photosynthetic Parameters in Vicia faba L.

We determined for Vicia faba L the influence of nitrogen uptake and accumulation on the values of photon saturated net photosynthetic rate (P _Nmax), quantum yield efficiency (), intercellular CO₂ concentration (C _i), and carboxylation efficiency (C _e). As leaf nitrogen content (N_L) increased, the converged onto a maximum asymptotic value of 0.0664±0.0049 mol(CO₂) mol(quantum)^–1. Also, as N_L increased the C _i value fell to an asymptotic minimum of 115.80±1.59 mol mol^–1, and C _e converged onto a maximum asymptotic value of 1.645±0.054 mol(CO₂) m^–2 s^–1 Pa^–1 and declined to zero at a N_L-intercept equal to 0.596±0.096 g(N) m^–2. fell to zero for an N_L-intercept of 0.660±0.052 g(N) m^–2. As N_L increased, the value of P _Nmax converged onto a maximum asymptotic value of 33.400±2.563 mol(CO₂) m^–2 s^–1. P _N fell to zero for an N_L-intercept of 0.710±0.035 g(N) m^–2. Under variable daily meteorological conditions the values for N_L, specific leaf area (_L), root mass fraction (R_f), P _Nmax, and remained constant for a given N supply. A monotonic decline in the steady-state value of R_f occurred with increasing N supply. _L increased with increasing N supply or with increasing N_L.     

Conformational Polymorphism and Extensibility of DNA Quadruplexes Formed by d(GT)nRepeats

We showed earlier that oligonucleotides 3"-d(GT)₅-pO(CH₂CH₂O)₃p-d(GT)₅-3" form bimolecular quadruplexes with parallel orientation of their strands, which are held by guanine quartets alternating with unpaired thymines (GT quadruplex). This work deals with the conformational polymorphism and extensibility of G quadruplexes in complex with molecules of an intercalating agent ethidium bromide (EtBr). A cooperative mechanism of EtBr binding to the GT quadruplex was revealed. The binding constant K= (3.3 ± 0.1)·10⁴M^–1, cooperativity coefficient = 2.5 ± 0.2, and maximal amount of EtBr molecules intercalated in GT quadruplex (N= 8) were determined. It was proved experimentally by analysis of adsorption isotherms and theoretically by mathematical modeling that the GT quadruplex is capable of double extension, which is indicative of the high elasticity of this four-stranded helix. Two most stable conformations of GT quadruplexes with thymine residues intercalated and/or turned outside were found by mechanico-mathematical modeling. The equilibrium is shifted toward the conformation with the looped out thymine residues upon intercalation of EtBr molecules into the GT quadruplex.