Hydrolase-catalyzed Michael addition of 1,3-dicarbonyl compounds to α,β-unsaturated compounds in organic solvent

A novel strategy to perform Michael additions between 1,3-dicarbonyl compounds and α,β-unsaturated compounds was developed by the catalysis of hydrolase. We found that 11 hydrolase could catalyze the enzymatic Michael addition reaction to form the carbon–carbon bond. In 2-methyl-2-butanol d-aminoacylase showed high Michael addition activity. The influence of substrate and Michael acceptor structure on Michael addition was evaluated systematically. Some control experiments demonstrated that the active site of d-aminoacylase was responsible for the enzymatic Michael addition reaction. This novel Michael addition activity of hydrolase is of practical significance in expanding the application of enzymes and in the evolution of new biocatalysts.     

Excretion of photosynthetically fixed organic carbon by metalimnetic phytoplankton

The effects of light intensity, oxygen concentration, and pH on the rates of photosynthesis and net excretion by metalimnetic phytoplankton populations of Little Crooked Lake, Indiana, were studied. Photosynthetic rates increased from 1.42 to 3.14 mg C·mg^–1 chlorophylla·hour^–1 within a range of light intensities from 65 to 150E·m^–2·sec^–1, whereas net excretion remained constant at 0.05 mg C·mg^–1 chlorophylla·hour^–1. Bacteria assimilated approximately 50% of the carbon released by the phytoplankton under these conditions. Excreted carbon (organic compounds either assimilated by bacteria or dissolved in the lake water) was produced by phytoplankton at rates of 0.02–0.15 mg C·mg^–1 chlorophylla·hour^–1. These rates were 6%–13% of the photosynthetic rates of the phytoplankton. Both total excretion of carbon and bacterial assimilation of excreted carbon increased at high light intensities whereas net excretion remained fairly constant. Elevated oxygen concentrations in samples incubated at 150E· m^–2·sec^–1 decreased rates of both photosynthesis and net excretion. The photosynthetic rate increased from 3.0 to 5.0 mg C·mg^–1 chlorophylla· hour^–1 as the pH was raised from 7.5 to 8.8. Net excretion within this range decreased slightly. Calculation of total primary production using a numerical model showed that whereas 225.8 g C·m^–2 was photosynthetically fixed between 12 May and 24 August 1982, a maximum of about 9.3 g C·m^–2 was released extracellularly.     

Highly Efficient Site-Directed RNA Cleavage by Imidazole-Containing Conjugates of Antisense Oligonucleotides

A method has been suggested for the synthesis of conjugates of oligodeoxyribonucleotides with chemical constructs mimicking the ribonuclease A active center for directed fragmentation of RNA. The method is based on sequential addition of a linker group, 9-(methylamino)anthracene, to the 5"- or 3"-terminal phosphate of oligonucleotide, and then an imidazole-containing construct by cycloaddition. The conjugates of oligonucleotides complementary to regions 44–61 (2B–R) and 60–76 (1C–R) of yeast phenylalanine tRNA proved able to cleave tRNA^Phe under physiological conditions preferentially at the sole phosphodiester bond (C63–A64 for 2B–R and C56–G57 for 1C–R, respectively). The half-time of tRNA^Phe hydrolysis in the presence of 2B–R conjugate was 30 min at a 2B–R concentration of 10 M and several minutes at conjugate concentration of 50 M.     

Benthic oxygen flux in the highly productive subarctic Lake Myvatn, Iceland: In situ benthic flux chamber study

In situ paired light and dark-stirred benthic flux chambers were used to estimate dissolved oxygen flux across the sediment–water interface in Lake Mývatn, Iceland. Three sampling stations were selected, each station reflecting a specific sedimentary environment, benthic communities, and water depth. During this study the phytoplankton density was low. Spatial and seasonal variations of bottom DO concentration and DO flux have been observed during this study. The oxygen consumption rate at all study sites had a mean of –89 (±44) mmol m^–2 d^–1 while the oxygen production rate due to benthic algae had a mean of 131 (±103) mmol m^–2 d^–1. There was a strong correlation (r=0.91) between oxygen consumption rate and temperature. This was presumably because of the temperature influence on rates of microbial and macrobenthic processes. The mean benthic primary production rate at all study sites was 1216 (±957) mg C m^–2 d^–1 between June 2000 and February 2001. Annual gross benthic primary production was estimated from the gross mean daily benthic DO production (P) and Redfield's C:O₂ ratio of 106:138 to be 420 g C m^–2 y^–1 at station HO, 250 g C m^–2 y^–1 at B2 and 340 g C m^–2 y^–1 at station 95. Thus, the mean gross benthic primary production was estimated as 1151 mg C m^–2 d^–1 at station HO, 685 mg C m^–2 d^–1 at station B2, and 932 mg C m^–2 d^–1 at station 95.     

MenD as a versatile catalyst for asymmetric synthesis

The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) from Escherichia coli K12, formerly known as SHCHC-synthase, catalyses the decarboxylation of α-ketoglutarate and the subsequent addition of the resulting succinyl-THDP to isochorismate. Here, the enzyme is tested for unphysiologial C–C bond-forming reactions.Condensation of α-ketoglutarate after decarboxylation to a broad range of aldehydes gave α-hydroxyketones with isolated yields from 26 to 87% and 94 to 98% ee for addition to aromatic aldehydes. MenD accepts a wide range of aldehydes as acceptor substrates to produce chiral α-hydroxyketones with conserved regioselectivity where the activated succinylsemialdehyde serves selectively as the donor. Regioselectivity is inverted only for condensation of α-ketoglutarate with pyruvate (activated acetaldehyde) as donor. Besides α-ketoglutarate, pyruvate and oxalacetate are accepted as donors in combination with benzaldehyde and 2-fluorobenzaldehyde as acceptors, however with decreased activity of C–C bond formation.The physiological 1,4-addition of α-ketoglutarate to isochorismate was investigated for acceptor substrate variability. (2S,3S)-2,3-Dihydroxy-2,3-dihydrobenzoate (2,3-CHD), which lacks the pyruvyl found in isochorismate, is converted to (5S,6S)-2-succinyl-5,6-dihydroxycyclohex-2-enecarboxylate. In contrast to the addition to carbonyls, the active site of MenD does appear to impose specific constraints on the acceptor substrate for 1,4-addition with α,β-unsaturated carboxylic acids.     

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (α, β-1, β-2, β-3, β-4 and β-5), PP2Cα was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2Cα was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2Cα is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.     

Thermoregulatory function of the parietal eye in the lizard Anolis carolinensis

Summary Animals were acclimitized to a daily cycle of 15–25°C and a photoperiod of LD 12:12. Parietalectomized animals in an experimental thermal gradient (15°C–40°C, LD 12:12) selected significantly higher temperatures than controls at all hours of the day except from 0800–1200. These results suggest that the lizard parapineal plays a direct role in thermoregulation in addition to the indirect function as an illuminometer.     

Effects of humid heat exposure in later sleep segments on sleep stages and body temperature in humans

This study sought to investigate the effects of humid heat exposure in later sleep segments on sleep stages and body temperature in humans. The subjects were eight healthy males, from whom informed consent had been obtained. The experiments were carried out under three different sets of conditions: a control climate [air temperature (Ta)=26°C, relative humidity (RH)=50%] (C); a humid heat climate (Ta=32°C, RH=80%) (H); and a humid heat exposure in later sleep segments (C for the first 3 h 45 min, followed by a 30-min transition to H, which was then maintained for the last 3 h 45 min) (C–H). Electroencephalogram, EOG, and mental electromyogram, rectal temperature (Tre), and skin temperature (Tsk) were continuously measured. The total amount of wakefulness was significantly increased in H compared to C–H or C. Compared to C, wakefulness in C–H and H was significantly increased during later sleep segments. Tre and mean Tsk were significantly higher in H than in C–H or C. In C–H, Tsk and Tre increased to levels equal to those observed in H after Ta and RH increase. Whole body sweat loss was significantly lower in C–H and C than in H. These results suggest that humid heat exposure in the later sleep segment reduces thermal load as compared to full-night humid heat exposure. In daily life, the use of air conditioning in the initial sleep hours can protect sleep and thermoregulation.     

Effect of seabuckthorn leaf extracts on circulating energy fuels, lipid peroxidation and antioxidant parameters in rats during exposure to cold, hypoxia and restraint (C–H–R) stress and post stress recovery

The present study was carried out to study mechanism of adaptogenic activity of seabuckthorn leaf extract, administered orally in rats both in single and five doses at a dose of 100 mg/kg body weight 30 min prior to C–H–R exposure. The efficacy of the extract was studied on circulating energy fuels, lipid peroxidation and anti-oxidant parameters in rats on attaining the T_rec 23 °C during C–H–R exposure and after recovery (T_rec 37 °C) from C–H–R induced hypothermia. Single dose treatment in rats restricted rise in blood malondialdehyde (MDA) levels and decrease in glutathione (GSH) and catalase (CAT) levels. Both single and five doses also restricted the rise in serum free fatty acids (FFA) and lactate dehydrogenase (LDH) levels on attaining T_rec 23 °C during C–H–R exposure, suggesting more efficient utilization of FFA for energy production and better maintained cell membrane permeability. This suggested that the adaptogenic activity of the extract might be due to its anti-oxidative activity, maintained blood glucose levels, better utilization of FFA and improved cell membrane permeability.     

Benthic bacterial biomass and production in the Hudson River estuary

Bacterial biomass, production, and turnover were determined for two freshwater marsh sites and a site in the main river channel along the tidally influenced Hudson River. The incorporation of [methyl-³H]thymidine into DNA was used to estimate the growth rate of surface and anaerobic bacteria. Bacterial production at marsh sites was similar to, and in some cases considerably higher than, production estimates reported for other aquatic wetland and marine sediment habitats. Production averaged 1.8–2.8 mg C·m^–2·hour^–1 in marsh sediments. Anaerobic bacteria in marsh sediment incorporated significant amounts of [methyl-³H]thymidine into DNA. Despite differences in dominant vegetation and tidal regime, bacterial biomass was similar (1×10³±0.08 mg C·m^–2) inTrapa, Typha, andNuphar aquatic macrophyte communities. Bacterial abundance and productivity were lower in sandy sediments associated withScirpus communities along the Hudson River (0.2×10³±0.05 mg C·m^–2 and 0.3±0.23 mg C·m^–2·hour^–1, respectively).     

Microbiological process for the removal of Cr(VI) from chromate-bearing cooling tower effluent

Summary A microbiological process using Pseudomonas mendocina was developed for the removal of Cr(VI) from cooling tower effluent. The process, when carried out in a 20 liter continuous stirred tank reactor removed 25–100 mg chromate/l in 4.5–8 h with >99.9% efficiency in the presence of sugarcane molasses as nutrient. The process could sustain wide variations in pH (6.5–9.5), temperature (25°C–40°C) and was unaffected by commonly used biocides.     

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Threonine synthase (TS), which is a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the γ-phosphate group from O-phospho-l-homoserine (OPHS) and the subsequent addition of water at Cβ to form l-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When l-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, l-threonine was formed with k_cat and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, α-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with l-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the Cβ of the PLP-α-aminocrotonate aldimine (AC) and the transaldimination to form l-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the Cβ of the analog. These results indicated that the phosphate ion released from OPHS by γ-elimination acts as the base catalyst for the addition of water at Cβ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/deprotonation at Cγ.     

A comparison of soil carbon pools and profiles in wetlands in Costa Rica and Ohio

Wetlands are large carbon pools and play important roles in global carbon cycles as natural carbon sinks. This study analyzes the variation of total soil carbon with depth in two temperate (Ohio) and three tropical (humid and dry) wetlands in Costa Rica and compares their total soil C pool to determine C accumulation in wetland soils. The temperate wetlands had significantly greater (P < 0.01) C pools (17.6 kg C m⁻²) than did the wetlands located in tropical climates (9.7 kg C m⁻²) in the top 24 cm of soil. Carbon profiles showed a rapid decrease of concentrations with soil depth in the tropical sites, whereas in the temperate wetlands they tended to increase with depth, up to a maximum at 18–24 cm, after which they started decreasing. The two wetlands in Ohio had about ten times the mean total C concentration of adjacent upland soils (e.g., 161 g C kg⁻¹ were measured in a central Ohio isolated forested wetland, and 17 g C kg⁻¹ in an adjacent upland site), and their soil C pools were significantly higher (P < 0.01). Among the five wetland study sites, three main wetland types were identified – isolated forested, riverine flow-through, and slow-flow slough. In the top 24 cm of soil, isolated forested wetlands had the greatest pool (10.8 kg C m⁻²), significantly higher (P < 0.05) than the other two types (7.9 kg C m⁻² in the riverine flow-though wetlands and 8.0 kg C m⁻² in a slowly flowing slough), indicating that the type of organic matter entering into the system and the type of wetland may be key factors in defining its soil C pool. A riverine flow-through wetland in Ohio showed a significantly higher C pool (P < 0.05) in the permanently flooded location (18.5 kg C m⁻²) than in the edge location with fluctuating hydrology, where the soil is intermittently flooded (14.6 kg C m⁻²).     

Diurnal changes in nitric oxide emissions from conventional tillage and pasture sites in the Amazon Basin: influence of soil temperature

We have investigated a subset of restoration practices applied to a degraded pasture at Fazenda Nova Vida, a 22000 ha cattle ranch in Rond^onia, Brazil. Nitric oxide (NO) and carbon dioxide (CO₂) emissions from soils were measured in conventional tillage and current pasture sites to assess N and C losses. Mean daily NO emissions from tilled plots were at least twice those from the pasture. Nitric oxide emissions from the tilled sites showed a strong diurnal pattern, while those from the pasture sites did not. Mean daytime NO emissions from the tilled sites were 9.7 g NO-N m^–2 h^–1, while mean nighttime emissions were 29.7 g NO-N m^–2 h^–1. In the pasture sites, NO emissions were 7.6 g NO-N m^–2 h^–1 during the day, and 7.7 g NO-N m^–2 h^–1 at night. Surface soil temperature was a good inverse predictor (r ²=0.75) of NO emissions from the tilled sites. Carbon dioxide emissions from the tilled sites were generally larger than CO₂ emissions from the pasture sites. The mean CO₂ emission rate from the tilled sites was 179 mg C m^–2 h^–1, while it was 123 mg C m^–2 h^–1 from the pasture sites. There was no distinct diurnal pattern for CO₂ emissions. We found that the very high temperatures measured at the soil surface in the tillage plots, in the range of 40–45°C, reduced the rate of NO emission. The reduction in NO emissions may be because of the sensitivity of autotrophic nitrifiers to high temperatures. This study provides insights on how land-use change may alter regional NO fluxes by exposing certain microbial communities to extreme environmental conditions. Future studies of NO emissions in tropical agricultural systems where soils are bare for extend periods need to make diurnal measurements or the daily fluxes will be substantially underestimated.     

Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from Nematoloma frowardii

The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus Nematoloma frowardii. The cell-free MnP system partially converted several aromatic compounds, including [U-¹⁴C]pentachlorophenol ([U-¹⁴C]PCP), [U-¹⁴C]catechol, [U-¹⁴C]tyrosine, [U-¹⁴C]tryptophan, [4,5,9,10-¹⁴C]pyrene, and [ring U-¹⁴C]2-amino-4,6-dinitrotoluene ([¹⁴C]2-AmDNT), to ¹⁴CO₂. Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [¹⁴C]2-AmDNT as an example. At [¹⁴C]2-AmDNT concentrations ranging from 2 to 120 μM, the amount of released ¹⁴CO₂ was directly proportional to the concentration of [¹⁴C]2-AmDNT. The formation of highly polar products was also observed with [¹⁴C]2-AmDNT and [U-¹⁴C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the ¹⁴COOH-glyoxalate and 9% of the ¹⁴CHO-glyoxalate were converted to ¹⁴CO₂, indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules, such as lignins and humic substances.     

Silicon-containing granules of rat liver,kidney and spleen mitochondria

Summary Isolated rat liver mitochondria containing granule aggregates (25–75 nm in diameter) and small (5–10 nm) electron opaque granules were examined by electron probe X-ray microanalysis. The granule aggregates gave an intense Si signal, while the small granules gave both Si and P signals. Isolated mitochondria of rat liver, spleen and kidney, subjected to detergent solubilization and differential centrifugation, produced two granule fractions: (1) a 10,000g fraction consisting predominantly of granule aggregates (25–75 nm) composed of smaller granules (5–10 nm in diameter), and (2) a 10,000–30,000 g fraction of non-aggregated small granules (5–10 nm). Thin sections of isolated granule aggregates gave Si X-ray signals similar to those obtained from in situ granules. In addition S, Cl, Mg, Cr and Fe X-ray signals were observed. Cr occurred only in the large kidney granules, while Fe occurred in both fractions of the spleen and kidney granules. The presence of Si in the granules was confirmed by chemical analysis of the isolated granules and in vivo radiolabeling of the granules with ³¹Si and ⁶⁸Ge. Contamination within the electron microscope was eliminated by a liquid nitrogen anticontamination device.Supported by Grant GM-08229-13-15 from the National Institutes of Health, USPHSWe are grateful to the Perkin-Elmer Corporation and to their Western Branch Manager, Mr. Michael E. Mullen and Microscopist, Mr. Minoru Saito, for use of and assistance with the Hitachi H 500 transmission electron microscope with the scanning attachment, and to the Kevex Corporation for the use of the Kevex X-ray Spectrometer. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and X-ray spectra photographs and Darlene Lum for technical assistance in the laboratory     

Functional Mapping of Brucella abortus Cyclic β-1,2-Glucan Synthase: Identification of the Protein Domain Required for Cyclization

Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that have been shown to play an important role in several symbiotic and pathogenic relationships. Cyclic β-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of CβG, is an integral membrane polyfunctional protein that catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CβG. Recently, we have identified the glycosyltransferase and the β-1,2-glucooligosaccharide phosphorylase domains of Brucella abortus Cgs. In this study, we performed large-scale linker-scanning mutagenesis to gain further insight into the functional domains of Cgs. This analysis allowed us to construct a functional map of the enzyme and led to the identification of the minimal region required for the catalysis of initiation and elongation reactions. In addition, we identified the Cgs region (residues 991 to 1544) as being the protein domain required for cyclization and demonstrated that upon cyclization and releasing of the CβG, one or more glucose residues remain attached to the protein intermediate that serves as a primer for the next round of CβG synthesis. Finally, our results indicate that the overall control of the degree of polymerization of CβG is the result of a balance between elongation, phosphorolysis, and cyclization reactions.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

The soil organic carbon in particle-size separates under different regrowth forest stands of north eastern Costa Rica

Despite the importance of the secondary forest (SF) in tropical areas, few studies have quantified the soil organic carbon (SOC) pool in Costa Rica. Most of the studies conducted to date in this country have focused mainly on changes in the soil C pool following conversion of forests to pastures, which is the predominant land use in the tropics. The aim of this study was to measure SOC concentration and pool in particle-size fractions down to 50 cm depth in four SF stands regenerating from different intensities of prior land use in loamy sand and sandy loam soils of northeast Costa Rica: (i) a gallery forest (GF), (ii) a 15-year-old SF enriched with commercially planted native trees (15SF), (iii) a 25-year-old SF (25SF), and (iv) an abandoned Theobromma cacao plantation >60 years old (60SF). Additional objectives were (1) to determine the relationship of SOC concentration with selected physical and chemical soil properties, and (2) to establish the key determinants of the depth distribution of SOC in order to identify meaningful trends in the SOC pool. The SOC pool was highest under the 60SF (221.4 Mg C ha⁻¹) followed by the 15SF (212.1 Mg C ha⁻¹), the 25SF (195.9 Mg C ha⁻¹) and the lowest in the GF (183.5 Mg C ha⁻¹). The SOC concentration decreased significantly from 59.7 to 94.1 g kg⁻¹ in the 0–10 cm layer down to 31.0 to 45.5 g kg⁻¹ in the 40–50 cm layer in all forest stands. The fine silt + clay fraction contained the highest values of SOC concentration in all forest stands. Soil texture and the age of the SF were identified as the main factors that explained the variability in SOC. The age of SF stand influenced the distribution of size class aggregates and SOC.