Accumulation,Allocation, and Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Soil-Brassica chinensis System

Farmland soil and leafy vegetables accumulate more polycyclic aromatic hydrocarbons (PAHs) in suburban sites. In this study, 13 sampling areas were selected from vegetable fields in the outskirts of Xi’an, the largest city in northwestern China. The similarity of PAH composition in soil and vegetation was investigated through principal components analysis and redundancy analysis (RDA), rather than discrimination of PAH congeners from various sources. The toxic equivalent quantity of PAHs in soil ranged from 7 to 202 μg/kg d.w., with an average of 41 μg/kg d.w., which exceeded the agricultural/horticultural soil acceptance criteria for New Zealand. However, the cancer risk level posed by combined direct ingestion, dermal contact, inhalation of soil particles, and inhalation of surface soil vapor met the rigorous international criteria (1×10⁻⁶). The concentration of total PAHs was (1052±73) μg/kg d.w. in vegetation (mean±standard error). The cancer risks posed by ingestion of vegetation ranged from 2×10⁻⁵ to 2×10⁻⁴ with an average of 1.66×10⁻⁴, which was higher than international excess lifetime risk limits for carcinogens (1×10⁻⁴). The geochemical indices indicated that the PAHs in soil and vegetables were mainly from vehicle and crude oil combustion. Both the total PAHs in vegetation and bioconcentration factor for total PAHs (the ratio of total PAHs in vegetation to total PAHs in soil) increased with increasing pH as well as decreasing sand in soil. The total variation in distribution of PAHs in vegetation explained by those in soil reached 98% in RDA, which was statistically significant based on Monte Carlo permutation. Common pollution source and notable effects of soil contamination on vegetation would result in highly similar distribution of PAHs in soil and vegetation.     

Morphology of echovirus 22

Purified preparations of echovirus 22 were examined in the electron microscope. The virus was found to possess 32 capsomers arranged at the vertices of either a pentakis dodecahedron or a rhombic triacontahedron. The size of the virions ranges from 22 × 10⁻³ to 32 × 10⁻³ μm with a mean of 27 × 10⁻³ μm and a mode of 28 × 10⁻³ μm.     

Concentrations,Source and Risk Assessment of Polycyclic Aromatic Hydrocarbons in Soils from Midway Atoll,North Pacific Ocean

This study was designed to determine concentrations of polycyclic aromatic hydrocarbons (PAHs) in soil samples collected from Midway Atoll and evaluate their potential risks to human health. The total concentrations of 16 PAHs ranged from 3.55 to 3200 µg kg⁻¹ with a mean concentration of 198 µg kg⁻¹. Higher molecular weight PAHs (4–6 ring PAHs) dominated the PAH profiles, accounting for 83.3% of total PAH mass. PAH diagnostic ratio analysis indicated that primary sources of PAHs in Midway Atoll could be combustion. The benzo[a]pyrene equivalent concentration (BaP_eq) in most of the study area (86.5%) was less than 40 µg kg⁻¹ BaP_eq and total incremental lifetime cancer risks of PAHs ranged from 1.00×10⁻¹⁰ to 9.20×10⁻⁶ with a median value of 1.24×10⁻⁷, indicating a minor carcinogenic risk of PAHs in Midway Atoll.     

Laser activation of rapid absorption changes in spinach chloroplasts and chlorella

The kinetics of the 520 mμ absorption change in spinach chloroplasts and Chlorella vulgaris following a flash from the ruby laser have been determined as follows: rise halftime ≤ 0.3 × 10⁻⁶ second; rapid recovery halftime = 5 to 6 × 10⁻⁶ second; intermediate recovery halftime = 4 × 10⁻⁴ second (spinach chloroplasts only); slow recovery halftime = 12 to 170 × 10⁻³ second, dependent on the measuring light intensity and aerobicity of the suspension.

The rapid phase of the 520 mμ reaction is approximately independent of temperature, from 295° to 77° Absolute.

With increasing oxygenation of the sample, the extent of the rapid phase decreases, the extent of the slow phase increases, while the extent of the intermediate phase in spinach chloroplasts remains constant.

In spinach chloroplasts, no recovery halftime of the 3 recovery phases for the 520 mμ absorption change was observed to correspond to the halftime for oxidation of cytochrome f (t_½ = 1.3 × 10⁻³ second).

     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Bacterioplankton in Antarctic Ocean Waters During Late Austral Winter: Abundance, Frequency of Dividing Cells, and Estimates of Production

Bacterioplankton productivity in Antarctic waters of the eastern South Pacific Ocean and Drake Passage was estimated by direct counts and frequency of dividing cells (FDC). Total bacterioplankton assemblages were enumerated by epifluorescent microscopy. The experimentally determined relationship between in situ FDC and the potential instantaneous growth rate constant (μ) is best described by the regression equation ln μ = 0.081 FDC − 3.73. In the eastern South Pacific Ocean, bacterioplankton abundance (2 × 10⁵ to 3.5 × 10⁵ cells per ml) and FDC (11%) were highest at the Polar Front (Antarctic Convergence). North of the Subantarctic Front, abundance and FDC were between 1 × 10⁵ to 2 × 10⁵ cells per ml and 3 to 5%, respectively, and were vertically homogeneous to a depth of 600 m. In Drake Passage, abundance (10 × 10⁵ cells per ml) and FDC (16%) were highest in waters south of the Polar Front and near the sea ice. Subantarctic waters in Drake Passage contained 4 × 10⁵ cells per ml with 4 to 5% FDC. Instantaneous growth rate constants ranged between 0.029 and 0.088 h⁻¹. Using estimates of potential μ and measured standing stocks, we estimated productivity to range from 0.62 μg of C per liter · day in the eastern South Pacific Ocean to 17.1 μg of C per liter · day in the Drake Passage near the sea ice.     

Analysis of arsenic induced physiological and biochemical responses in a medicinal plant,Withania somnifera

Withania somnifera has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India. However, these grow as weeds mostly in the wastelands, which receive contaminated water from municipal and industrial sources. In the present investigation, plants of Withania somnifera were exposed to various concentrations of arsenate (AsV) and arsenite (AsIII) (0, 10, 25, 50, 100 μM) for 10 days and analysed for accumulation of arsenic (As) and physiological and biochemical changes. Plants showed more As accumulation upon exposure to AsIII (320 μg g⁻¹ DW in roots and 161 μg g⁻¹ DW in leaves) than to AsV (173 μg g⁻¹ DW in roots and 100 μg g⁻¹ DW in leaves) after 10 days of treatment. Consequently, AsIII exposure caused more toxicity to plants as compared to that AsV, as evaluated in terms of the level of photosynthetic pigments and oxidative stress parameters (superoxide, hydrogen peroxide and lipid peroxidation), particularly at higher concentrations and on longer durations. Plants could tolerate low concentrations (variable for AsIII and AsV) until longer durations (10 days) and high concentrations for shorter durations (1–5 days) through increase in antioxidant enzymes and by augmented synthesis of thiols. In conclusion, As tolerance potential of Withania plants on one hand advocates its prospective use for remediation under proper supervision and on the other demonstrates possible threat of As entry into humans due to medicinal uses.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.     

Highly Sensitive Detection of Melamine Using a One-Step Sample Treatment Combined with a Portable Ag Nanostructure Array SERS Sensor

There is an urgent need for rapid and reliable methods able to detect melamine in animal feed. In this study, a quick, simple, and sensitive method for the determination of melamine content in animal feed was developed using surface-enhanced Raman spectroscopy on fabricated Ag nanorod (AgNR) array substrates with a one-step sample extraction procedure. The AgNR array substrates washed by HNO₃ solvent (10⁻⁷ M) and methanol and showed the good stability within 6 months. The Raman shift at △ν = 682 cm⁻¹ was used as the characteristic melamine peak in the calculations. Sufficient linearity was obtained in the 2–200 μg·g⁻¹ range (R² = 0.926). The limits of detection and quantification were 0.9 and 2 μg·g⁻¹, respectively. The recovery rates were 89.7–93.3%, with coefficients of variation below 2.02%. The method showed good accuracy compared with the tradition GC-MS analysis. This new protocol only need 2 min to fininsh the detection which could be developed for rapid onsite screening of melamine contamination in quality control and market surveillance applications.     

Influence of Cyanobacterial Hyperscum on Heterotrophic Activity of Planktonic Bacteria in a Hypertrophic Lake

The response of the planktonic heterotrophic bacterial community to the buildup and breakdown of a semipermanent, crusted, floating cyanobacterial mat, or hyperscum, that covered 1 to 2 ha was studied in a hypertrophic lake (Hartbeespoort Dam, South Africa). The initial response of bacteria in the main basin to the release of dissolved organic carbon (DOC) from the hyperscum 1 km away was an increase in activity per cell from 35 × 10⁻¹² to 153 × 10⁻¹² μg of C cell⁻¹ h⁻¹ for total cell counts, while activity per cell for metabolically active cells increased from 19 × 10⁻¹¹ to 85 × 10⁻¹¹ μg of C cell⁻¹ h⁻¹. No major population growth occurred at this stage. Later, with the continuous supply of DOC from the hyperscum, total bacterial numbers increased from 6.6 × 10⁶ to 20 × 10⁶ cells ml⁻¹, while the activity per cell declined. Metabolically active bacteria followed the same trend. Shorter-term DOC increases caused only increases in bacterial activity per cell. The data from Hartbeespoort Dam demonstrate an interesting and little-documented mechanism by which aquatic bacteria respond to increased DOC concentration and which may be universal for aquatic systems.     

NO? Binds Human Cystathionine β-Synthase Quickly and Tightly

The hexa-coordinate heme in the H₂S-generating human enzyme cystathionine β-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO^• or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO^• was reported to bind at the CBS heme with very low affinity (K_d = 30–281 μm). This discrepancy was herein reconciled by investigating the NO^• reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO^• binds tightly to the ferrous CBS heme, with an apparent K_d ≤0.23 μm. In line with this result, at 25 °C, NO^• binds quickly to CBS (k_on ∼ 8 × 10³ m⁻¹ s⁻¹) and dissociates slowly from the enzyme (k_off ∼ 0.003 s⁻¹). The observed rate constants for NO^• binding were found to be linearly dependent on [NO^•] up to ∼ 800 μm NO^•, and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO^• quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO^•. The novel findings reported here shed new light on CBS regulation by NO^• and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO^•, CO, and H₂S in cell signaling.     

Protozoan Grazing and Bacterial Production in Stratified Lake Vechten Estimated with Fluorescently Labeled Bacteria and by Thymidine Incorporation

In stratified Lake Vechten, The Netherlands, protozoan grazing was estimated on the basis of uptake of fluorescently labeled bacteria and compared with bacterial production estimated on the basis of thymidine incorporation. By using a grazer-free mixed bacterial population from the lake in continuous culture, an empirical relationship between cell production and thymidine incorporation was established. Thymidine incorporation into total cold-trichloroacetic-acid-insoluble macromolecules yielded a relatively constant empirical conversion factor of ca. 10¹⁸ (range, 0.38 × 10¹⁸ to 1.42 × 10¹⁸) bacteria mol of thymidine⁻¹ at specific growth rates (μ) ranging from 0.007 to 0.116 h⁻¹. Although thymidine incorporation has been assumed to measure DNA synthesis thymidine incorporation appeared to underestimate the independently measured bacterial DNA synthesis by at least 1.5- to 13-fold, even if all incorporated label was assumed to be in DNA. However, incorporation into DNA was found to be insignificant as measured by conventional acid-base hydrolysis. Methodological problems of the thymidine technique are discussed. Like the cultures, Lake Vechten bacteria showed considerable thymidine incorporation into total macromolecules, but no significant incorporation into DNA was found by acid-base hydrolysis. This applied not only to the low-oxygen hypo- and metalimnion but also to the aerobic epilimnion. Thus, the established empirical conversion factor for thymidine incorporation into total macromolecules was used to estimate bacterial production. Maximum production rates (141 × 10⁶ bacteria liter⁻¹ h⁻¹; μ, 0.012 h⁻¹) were found in the metalimnion and were 1 order of magnitude higher than in the epi- and hypolimnion. In all three strata, the estimated bacterial production was roughly balanced by the estimated protozoan grazing. Heterotrophic nanoflagellates were the major consumers of the bacterial production and showed maximum numbers (up to 40 × 10⁶ heterotrophic nanoflagellates liter⁻¹) in the microaerobic metalimnion.     

Assessing Phytoplankton and Bacterioplankton Production During Early Spring in Lake Erken, Sweden

The spring development of both phytoplankton and bacterioplankton was investigated between 18 April and 7 May 1983 in mesotrophic Lake Erken, Sweden. By using the lake as a batch culture, our aim was to estimate, via different methods, the production of phytoplankton and bacterioplankton in the lake and to compare these production estimates with the actual increase in phytoplankton and bacterioplankton biomass. The average water temperature was 3.5°C. Of the phytoplankton biomass, >90% was the diatom Stephanodiscus hantzchii var. pusillus, by the peak of the bloom. The ¹⁴C and O₂ methods of estimating primary production gave equivalent results (r = 0.999) with a photosynthetic quotient of 1.63. The theoretical photosynthetic quotient predicted from the C/NO₃ N assimilation ratio was 1.57. The total integrated incorporation of [¹⁴C]bicarbonate into particulate material (>1 μm) was similar to the increase in phytoplankton carbon determined from cell counts. Bacterioplankton increased from 0.5 × 10⁹ to 1.52 × 10⁹ cells liter⁻¹ (~0.5 μg of C liter⁻¹ day⁻¹). Estimates of bacterioplankton production from rates of [³H]thymidine incorporation were ca. 1.2 to 1.7 μg of C liter⁻¹ day⁻¹. Bacterial respiration, measured by a high-precision Winkler technique, was estimated as 4.8 μg of C liter⁻¹ day⁻¹, indicating a bacterial growth yield of 25%. The bulk of the bacterioplankton production was accounted for by algal extracellular products. Gross bacterioplankton production (production plus respiration) was 20% of gross primary production, per square meter of surface area. We found no indication that bacterioplankton production was underestimated by the [³H]thymidine incorporation method.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Design and green synthesis of novel quinolinone derivatives of potential anti-breast cancer activity against MCF-7 cell line targeting multi-receptor tyrosine kinases

A new set of 4,6,7,8-tetrahydroquinolin-5(1H)-ones were designed as cytotoxic agents against breast cancer cell line (MCF-7) and synthesised under ultrasonic irradiation using chitosan decorated copper nanoparticles (CS/CuNPs) catalyst. The new compounds 4b, 4j, 4k, and 4e exhibited the most potent cytotoxic activity of IC₅₀ values (0.002 − 0.004 µM) comparing to Staurosporine of IC₅₀; 0.005 μM. The latter derivatives exhibited a promising safety profile against the normal human WI38 cells of IC₅₀ range 0.0149 − 0.048 µM. Furthermore, the most promising cytotoxic compounds 4b, 4j were evaluated as multi-targeting agents against the RTK protein kinases; EGFR, HER-2, PDGFR-β, and VEGFR-2. Compound 4j showed promising inhibitory activity against HER-2 and PDGFR-β of IC₅₀ values 0.17 × 10⁻³, 0.07 × 10⁻³ µM in comparison with the reference drug sorafenib of IC₅₀; 0.28 × 10⁻³, 0.13 × 10⁻³ µM, respectively. In addition, 4j induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells.     

Muramic Acid Measurements for Bacterial Investigations in Marine Environments by High-Pressure Liquid Chromatography

Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 × 10⁻¹⁰ to 5.3 × 10⁻¹⁰ μg per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 10⁸ to 10¹¹ cells per liter. The muramic acid content in natural marine water decreased from 5.3 × 10⁻¹⁰ to 1.6 × 10⁻¹⁰ μg per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis.     

Potential for aflatoxin B1 and B2 production by Aspergillus flavus strains isolated from rice samples

In this study, we investigated the potential for aflatoxin B₁ (AFB₁) and B₂ (AFB₂) production in rice grain by 127 strains of Aspergillus flavus isolated from rice grains collected from China. These strains were inoculated onto rice grains and incubated at 28 °C for 21 days. AFB₁ and AFB₂ were extracted and quantified by high-performance liquid chromatography coupled with fluorescence detection. Among the tested strains, 37% produced AFB₁ and AFB₂ with levels ranging from 175 to 124 101 μg kg⁻¹ for AFB₁ and from not detected to 10 329 μg kg⁻¹ for AFB₂. The mean yields of these isolates were 5884 μg kg⁻¹ for AFB₁ and 1968 μg kg⁻¹ for AFB₂. Overall, most of the aflatoxigenic strains produced higher levels of AFB₁ than AFB₂ in rice. The obtained information is useful for assessing the risk of aflatoxin contamination in rice samples.     

A Large-Scale Genetic Analysis Reveals a Strong Contribution of the HLA Class II Region to Giant Cell Arteritis Susceptibility

We conducted a large-scale genetic analysis on giant cell arteritis (GCA), a polygenic immune-mediated vasculitis. A case-control cohort, comprising 1,651 case subjects with GCA and 15,306 unrelated control subjects from six different countries of European ancestry, was genotyped by the Immunochip array. We also imputed HLA data with a previously validated imputation method to perform a more comprehensive analysis of this genomic region. The strongest association signals were observed in the HLA region, with rs477515 representing the highest peak (p = 4.05 × 10⁻⁴⁰, OR = 1.73). A multivariate model including class II amino acids of HLA-DRβ1 and HLA-DQα1 and one class I amino acid of HLA-B explained most of the HLA association with GCA, consistent with previously reported associations of classical HLA alleles like HLA-DRB1^∗04. An omnibus test on polymorphic amino acid positions highlighted DRβ1 13 (p = 4.08 × 10⁻⁴³) and HLA-DQα1 47 (p = 4.02 × 10⁻⁴⁶), 56, and 76 (both p = 1.84 × 10⁻⁴⁵) as relevant positions for disease susceptibility. Outside the HLA region, the most significant loci included PTPN22 (rs2476601, p = 1.73 × 10⁻⁶, OR = 1.38), LRRC32 (rs10160518, p = 4.39 × 10⁻⁶, OR = 1.20), and REL (rs115674477, p = 1.10 × 10⁻⁵, OR = 1.63). Our study provides evidence of a strong contribution of HLA class I and II molecules to susceptibility to GCA. In the non-HLA region, we confirmed a key role for the functional PTPN22 rs2476601 variant and proposed other putative risk loci for GCA involved in Th1, Th17, and Treg cell function.     

Distribution of Sulfate-Reducing and Methanogenic Bacteria in Anaerobic Aggregates Determined by Microsensor and Molecular Analyses

Using molecular techniques and microsensors for H₂S and CH₄, we studied the population structure of and the activity distribution in anaerobic aggregates. The aggregates originated from three different types of reactors: a methanogenic reactor, a methanogenic-sulfidogenic reactor, and a sulfidogenic reactor. Microsensor measurements in methanogenic-sulfidogenic aggregates revealed that the activity of sulfate-reducing bacteria (2 to 3 mmol of S²⁻ m⁻³ s⁻¹ or 2 × 10⁻⁹ mmol s⁻¹ per aggregate) was located in a surface layer of 50 to 100 μm thick. The sulfidogenic aggregates contained a wider sulfate-reducing zone (the first 200 to 300 μm from the aggregate surface) with a higher activity (1 to 6 mmol of S²⁻ m⁻³ s⁻¹ or 7 × 10⁻⁹ mol s⁻¹ per aggregate). The methanogenic aggregates did not show significant sulfate-reducing activity. Methanogenic activity in the methanogenic-sulfidogenic aggregates (1 to 2 mmol of CH₄ m⁻³ s⁻¹ or 10⁻⁹ mmol s⁻¹ per aggregate) and the methanogenic aggregates (2 to 4 mmol of CH₄ m⁻³ s⁻¹ or 5 × 10⁻⁹ mmol s⁻¹ per aggregate) was located more inward, starting at ca. 100 μm from the aggregate surface. The methanogenic activity was not affected by 10 mM sulfate during a 1-day incubation. The sulfidogenic and methanogenic activities were independent of the type of electron donor (acetate, propionate, ethanol, or H₂), but the substrates were metabolized in different zones. The localization of the populations corresponded to the microsensor data. A distinct layered structure was found in the methanogenic-sulfidogenic aggregates, with sulfate-reducing bacteria in the outer 50 to 100 μm, methanogens in the inner part, and Eubacteria spp. (partly syntrophic bacteria) filling the gap between sulfate-reducing and methanogenic bacteria. In methanogenic aggregates, few sulfate-reducing bacteria were detected, while methanogens were found in the core. In the sulfidogenic aggregates, sulfate-reducing bacteria were present in the outer 300 μm, and methanogens were distributed over the inner part in clusters with syntrophic bacteria.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.