Dark metabolism of carbon monoxide in lettuce leaf discs

In the dark, leaf tissue of crisphead lettuce (Lactuca sativa L.) metabolized ¹⁴CO to ¹⁴CO₂ and acid-stable products. Tissue incubated at 2.5°C for 3.5 hours and 48 hours converted about 1% and 17%, respectively, of the applied ¹⁴CO to ¹⁴CO₂, and incorporated about 0.04% and 0.6% of the ¹⁴C in acid-stable products. Examination of soluble acid-stable products from ¹⁴CO and ¹⁴CO₂-treated leaf tissue revealed that the labeling patterns of both treatments were identical during the 3.5-hour and the 48-hour incubation periods. Malate, citrate, and aspartate together comprised 70% or more of the soluble radioactivity from both treatments. Incorporation of radioactivity from CO into soluble acid-stable products during a 3-hour incubation period at 2.5°C was inhibited 90% by adding 3% nonradioactive CO₂. These results indicate that in head lettuce in the dark ¹⁴CO is metabolized primarily to ¹⁴CO₂ which is the precursor of acid-stable products. In leaf discs at 2.5°C, the apparent K_m for CO oxidation to CO₂ was 5.3 microliters per liter and the V_max was 9.7 nanoliters per gram per hour. The mitochondrial fraction of the leaf homogenate was the most active fraction to oxidize CO to CO₂, and this activity was heat-labile and cyanide-sensitive.     

The nature of formate metabolism in greening barley leaves

The metabolism of [³H]formate has been examined in etiolated and greening leaves of barley (Hordeum vulgare), dwarf bean (Phaseolus vulgarls), broad bean (Vicia faba) and corn (Zea mays). Tritium was extensively incorporated by primary leaves incubated for 20-min periods in light or dark. The organic acids and free amino acids were the principal products of formate metabolism but these and other products were more heavily labelled in green tissues. Time course experiments with barley leaves revealed a rapid labelling of serine, accompanied by increasing amounts of ³H in glycine and aspartate as the feeding period was extended. These amino acid products were formed throughout a 4-day greening period with an approximate doubling in total incorporation being due to large accumulations of tritiated glycine and aspartate. The involvement of tetrahydrofolate-dependent reactions in formate metabolism was indicated by inhibition of [¹⁴C] and [³H]formate incorporation by the folate antagonist, aminopterin. Labelling of glycine and serine was also strongly inhibited (up to 90%) when the leaves were incubated with increasing concentrations of isonicotinylhydrazide.     

Role of extracellular ligninases in biodegradation of benzo(a)pyrene by Phanerochaete chrysosporium

Phanerochaete chrysosporium oxidized benzo(a)pyrene rapidly to CO₂and several organic soluble and water soluble compounds in agitated pellet cultures during secondary metabolism. In 54 h the added benzo(a)pyrene was almost completely (99.5%) converted to metabolic products. After 10 days incubation in the presence of excess glucose 19% of the radiolabel was recovered as¹⁴CO₂. Maximal degradation rates calculated on the basis of evolved¹⁴CO₂were 15 nmol (3.7 μg) in a day by a 50 ml culture with 2 mg ml⁻¹dry weight fungal pellets. Extracellular ligninases were shown to be involved in the initial oxidation reactions. When ligninase preparation was added to the cultures simultaneously with benzo(a)pyrene, immediate accumulation of organic soluble and water soluble products occurred followed by evolution of CO₂. Without ligninase addition a lag period of 10–12 h was observed before meaningful CO₂. evolution started. When benzo(a)pyrene was incubated with ligninase and an H₂O₂generating system, three main organic soluble oxidation products were formed.     

Comparison of adenosine metabolism in leaves of several mangrove plants and a poplar species

A possible increased demand for ATP in salt- tolerant mangrove plants was studied by the comparison of metabolic fates of [8-¹⁴C] adenosine in leaf disks of several mangrove plants and of poplar. In mangrove trees, Rhizophora stylosa, Bruguiera gymnorrhiza, Kandelia candel and Sonneratia alba, 56–92% of [8-¹⁴C]adenosine taken up by leaf disks was converted during 3 h incubation to salvage products, i.e., nucleotides and RNA. Synthesis of nucleotides including ATP was stimulated by salt stress induced by 250 mM NaCl. In leaf disks of Avicennia marina, a mangrove shrub that produces glycinebetaine as compatible solutes, 46% of radioactivity entered salvage products when [8-¹⁴C] adenosine was continuously supplied to the leaf disks. Hydrolysis of adenosine to adenine was extremely active in this mangrove shrub. This is probably due to the high activity of adenosine nucleosidase (EC 3.2.2.7). In leaf disks of another mangrove shrub, Lumnitzera racemosa, only limited amounts of [8-¹⁴C]adenosine were metabolised (< ca. 30% taken up by leaf disks), but synthesis of ATP and ADP was stimulated by salt stress. In Pemphis acidula leaf disks, adenosine salvage activity was low and more than 30% of adenosine was hydrolysed to adenine. In leaf disks of poplar, a non-salt-resistant plant, ca. 40% of [8-¹⁴C] adenosine was converted to salvage products during 3 h of incubation, but the rate was slightly reduced by treatment with 250 mM NaCl. The present results suggest that large mangrove trees generally have efficient adenosine salvage ability, which is stimulated by salt. Lesser salvage activity is found in small size mangrove shrubs, although salt generally still enhances salvage activity.     

Cellulose Metabolism by the Termite Flagellate Trichomitopsis termopsidis

The end products of cellulose metabolism by the trichomonad flagellate Trichomitopsis termopsidis from the termite Zootermopsis sp. were investigated by growing axenic flagellates on [¹⁴C]cellulose. The growth of T. termopsidis resulted in the release of label into the supernatant fraction of the culture fluid, and > 75% was volatile under acid conditions. The label was analyzed for ¹⁴CO₂ and for [¹⁴C]volatile compounds by vacuum distillation under acid and alkaline conditions in disposable micro-distillation vessels. The distillate and undistilled culture supernatant fluid were chromatographed on cellulose thin layers to identify the labeled end product. T. termopsidis produced ¹⁴CO₂ and [¹⁴C]acetate which accounted for 25 to 30% and 55 to 60% of the labeled end products, respectively. The ratio of label in CO₂ to acetate suggests that they are produced in equimolar amounts. No neutral volatile compounds were produced. The remaining unidentified end product (10 to 20%) was not volatile nor extractable into ether. Hydrogen was produced by T. termopsidis, and the cells were killed by the drug metronidazole. Enzymatic activities were found which account for these end products: pyruvate:ferredoxin oxidoreductase and hydrogenase. The results indicate that acetate is the end product of T. termopsidis cellulose metabolism and is available to the termite for energy metabolism and biosynthesis.     

Anaerobic Biodegradation of the Lignin and Polysaccharide Components of Lignocellulose and Synthetic Lignin by Sediment Microflora

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses and [¹⁴C-polysaccharide]lignocelluloses were prepared from a variety of marine and freshwater wetland plants including a grass, a sedge, a rush, and a hardwood. These [¹⁴C]lignocellulose preparations and synthetic [¹⁴C]lignin were incubated anaerobically with anoxic sediments collected from a salt marsh, a freshwater marsh, and a mangrove swamp. During long-term incubations lasting up to 300 days, the lignin and polysaccharide components of the lignocelluloses were slowly degraded anaerobically to ¹⁴CO₂ and ¹⁴CH₄. Lignocelluloses derived from herbaceous plants were degraded more rapidly than lignocellulose derived from the hardwood. After 294 days, 16.9% of the lignin component and 30.0% of the polysaccharide component of lignocellulose derived from the grass used (Spartina alterniflora) were degraded to gaseous end products. In contrast, after 246 days, only 1.5% of the lignin component and 4.1% of the polysaccharide component of lignocellulose derived from the hardwood used (Rhizophora mangle) were degraded to gaseous end products. Synthetic [¹⁴C]lignin was degraded anaerobically faster than the lignin component of the hardwood lignocellulose; after 276 days, 3.7% of the synthetic lignin was degraded to gaseous end products. Contrary to previous reports, these results demonstrate that lignin and lignified plant tissues are biodegradable in the absence of oxygen. Although lignocelluloses are recalcitrant to anaerobic biodegradation, rates of degradation measured in aquatic sediments are significant and have important implications for the biospheric cycling of carbon from these abundant biopolymers.     

Evaluation of persistence and relative toxicity of some pest control products to adults of two native trichogrammatid species in Kenya

The utilization of native trichogrammatids for biocontrol of Helicoverpa armigera (Hbn.) and their potential integration with pesticide use are currently receiving attention. In this study the interaction of adults of Trichogramma sp. nr. mwanzai and Trichogrammatoidea sp. nr. lutea with commonly used pesticide products was investigated. The toxicity of eight pest control products commonly used in vegetable crops in Kenya, were evaluated by exposing the adults of the two species to detached potted tomato leaves at different intervals after spraying. Two biologically derived products, Achook^? (neem-based) and Dipel^? (Bt ssp. kurstaki)—were found to be harmless and had no persistent toxicity on both the trichogrammatid species. Two organophosphate products tested, dimethoate (Rogor^?) and malathion (Malathion^?) were found to be ‘slightly harmful’ and ‘moderately persistent’ respectively. Three other synthetic insecticides, lambdacyhalothrin (Karate^?), bifenthrin (Brigade^?) and alpha-cypermentrin (Fastac^?) were found to be ‘moderately harmful’ and ‘persistent’ respectively. Polytrin^? (mixture of cypermethrin and profenofos) was also found to be persistent but only ‘slightly harmful’. On the basis of five concentrations tested (0.032, 0.016, 0.008, 0.004 and 0.002 of the recommended field rates) the LC₅₀ values for adult T. sp. nr. mwanzai were estimated as 285, 411, 435 and 1,916 (μg active ingredient (a.i) ml^?1) for dimethoate; malathion; lambdacyhalothrin as well as cypermethrin?+?profenofos respectively. The corresponding values for T. sp. nr. lutea were 247, 251, 278 and 697 respectively. Further, Karate^? appeared to be the least toxic among the four products, across all the respective concentrations. The study was an attempt to adopt the methodologies of the IOBC (International Organization for Biological Control) on non-target risk assessment for pest control products to cater for the local needs of integrating the use of the trichogrammatids along with other pest control products.     

Horseradish peroxidase-catalyzed oxidation of chlorophyll a with hydrogen peroxide: Characterization of the products and mechanism of the reaction

Horseradish peroxidase was verified to catalyze, without any phenol, the hydrogen peroxide oxidation of chlorophyll a (Chl a), solubilized with Triton X-100. The 13²(S) and 13²(R) diastereomers of 13²-hydroxyChl a were characterized as major oxidation products (ca. 60%) by TLC on sucrose, UV-vis, ¹H, and ¹³C NMR spectra, as well as fast-atom bombardment MS. A minor amount of the 15²-methyl, 17³-phytyl ester of Mg-unstable chlorin was identified on the basis of its UV-vis spectrum and reactivity with diazomethane, which converted it to the 13¹,15²-dimethyl, 17³-phytyl ester of Mg-purpurin 7. The side products (ca. 10%) were suggested to include the 17³-phytyl ester of Mg-purpurin 18, which is known to form easily from the Mg-unstable chlorin. The side products also included two red components with UV-vis spectral features resembling those of pure Chl a enolate anion. Hence, the two red components were assigned to the enolate anions of Chl a and pheophytin a or, alternatively, two different complexes of the Chl a enolate ion with Triton X-100. All the above products characterized by us are included in our published free-radical allomerization mechanism of Chl a, i.e. oxidation by ground-state dioxygen. The HRP clearly accelerated the allomerization process, but it did not produce bilins, that is, open-chain tetrapyrroles, the formation of which would require oxygenolysis of the chlorin macrocycle. In this regard, our results are in discrepancy with the claim by several researchers that ‘bilirubin-like compounds’ are formed in the HRP-catalyzed oxidation of Chl a. Inspection of the likely reactions that occurred on the distal side of the heme in the active centre of HRP provided a reasonable explanation for the observed catalytic effect of the HRP on the allomerization of Chl. In the active centre of HRP, the imidazole nitrogen of His-42 was considered to play a crucial role in the C-13² deprotonation of Chl a, which resulted in the Chl a enolate ion resonance hybrid. The Chl enolate was then oxidized to the Chl 13²-radical while the HRP Compound I was reduced to Compound II. The same reactive Chl derivatives, i.e. the Chl enolate anion and the Chl 13²-radical, which are produced twice in the HRP reaction cycle, happen to be the crucial intermediates in the initial stages of the Chl allomerization mechanism.     

Anaerobic biohydrogen production from wheat stalk by mixed microflora: Kinetic model and particle size influence

This study investigated the influence of particle size on anaerobic biohydrogen production from wheat stalk by mixed microflora. In addition, the kinetic model for the formation of main products was also mentioned. The results demonstrated that all the cumulative productions of hydrogen, acetate and butyrate decreased as the particle size increasing from 1 to 10 mm at a constant TS value of 2%, 5% and 8%, respectively. However, this difference for aqueous products was not very obvious compared with hydrogen. A modified Gompertz equation was able to adequately describe the cumulative production of hydrogen, acetate and butyrate (R² higher than 0.989). The results also indicated that the formation of the main products were all associated with the degradation of cellulose and hemicellulose (R² higher than 0.855).     

Safety and quality of some chicken meat products in Al-Ahsa markets-Saudi Arabia

One hundred samples of 10 poultry meat products were collected from AL-Ahsa markets (Kingdom of Saudi Arabia). The samples were ranked from carcass cuts (chilled, frozen, fillet and thigh) to minced meat or further processed products as burger, nuggets, frankfurter and meat paste loaf. Samples were collected in triplicate for sensory, chemical and microbiological analysis to assure their quality and safety.The obtained results revealed variation in chemical composition; some products with high fat percentage had a high thiobarbituric acid value, which resulted in the appearance of an unacceptable flavor.Bacteriological analysis revealed that the mean total bacterial count was ranged from 2.7 × 10⁴ cfu/g for nuggets^A to 3.3 × 10⁷ cfu/g for burger^B and the other products in the range of 10⁵–10⁶ cfu/g. While Staphylococcus aureus mean count ranged from less than 10² cfu/g for all samples, accept 10⁴ and 10⁶ cfu/g for mince^B and frankfurter samples, respectively. Escherichia coli isolated from 70% of the samples and Salmonella arizona was isolated at once from thigh samples. Thirty percentages of samples not comply with Saudi Standards due to sensory unacceptability and 21% of samples nonconforming with bacteriological specifications.     

N-(m-[125I]iodophenyl)maleimide: an agent for high yield radiolabeling of antibodies

In an effort to radiolabel antibodies, N-(m-[¹²⁵I]iodophenyl)maleimide (m-[¹²⁵I]IPM) was prepared by the demetallation of an N-[m-tri-(n-butyl)stannylphenyl]maleimide intermediate. The unlabeled intermediate was synthesized in ⩾ 75% yield using a palladium catalyzed reaction of hexabutylditin with m-bromoaniline, followed by reaction with maleic anhydride and ring annulation. All products were confirmed by NMR and elemental analysis. Labeling with ¹²⁵I was carried out in a biphasic mixture containing chloramine-T (radiochemical yield ⩾ 70%). Rabbit IgG modified with the heterobifunctional crosslinking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and bovine serum albumin were conjugated with m-[¹²⁵I]IPM (yield: 40 and 80%, respectively). In addition, m-[¹²⁵I]IPM was conjugated to rabbit IgG subunits (HL) in 70% yield. The in vitro stability of the radiolabeled proteins in serum showed < 1% deiodination over 24 h.     

Aerobic Mineralization of Trichloroethylene, Vinyl Chloride, and Aromatic Compounds by Rhodococcus Species

Two Rhodococcus strains which were isolated from a trichloroethylene (TCE)-degrading bacterial mixture and Rhodococcus rhodochrous ATCC 21197 mineralized vinyl chloride (VC) and TCE. Greater than 99.9% of a 1-mg/liter concentration of VC was degraded by cell suspensions. [1,2-¹⁴C]VC was degraded by cell suspensions, with the production of greater than 66% ¹⁴CO₂ and 20% ¹⁴C-aqueous phase products and incorporation of 10% of the ¹⁴C into the biomass. Cultures that utilized propane as a substrate were able to mineralize greater than 28% of [1,2-¹⁴C]TCE to ¹⁴CO₂, with approximately 40% appearing in ¹⁴C-aqueous phase products and another 10% of ¹⁴C incorporated into the biomass. VC degradation was oxygen dependent and occurred at a pH range of 5 to 10 and temperatures of 4 to 35°C. Cell suspensions degraded up to 5 mg of TCE per liter and up to 40 mg of VC per liter. Propane competitively inhibited TCE degradation. Resting cell suspensions also degraded other chlorinated aliphatic hydrocarbons, such as chloroform, 1,1-dichloroethylene, and 1,1,1-trichloroethane. The isolates degraded a mixture of aromatic and chlorinated aliphatic solvents and utilized benzene, toluene, sodium benzoate, naphthalene, biphenyl, and n-alkanes ranging in size from propane to hexadecane as carbon and energy sources. The environmental isolates appeared more catabolically versatile than R. rhodochrous ATCC 21197. The data report that environmental isolates of Rhodococcus species and R. rhodochrous ATCC 21197 have the potential to degrade TCE and VC in addition to a variety of aromatic and chlorinated aliphatic compounds either individually or in mixtures.     

Thermophilic Anaerobic Biodegradation of [14C]Lignin, [14C]Cellulose, and [14C]Lignocellulose Preparations

Thermophilic (55°C) anaerobic enrichment cultures were incubated with [¹⁴C-lignin]lignocellulose, [¹⁴C-polysaccharide]lignocellulose, and kraft [¹⁴C]lignin prepared from slash pine, Pinus elliottii, and ¹⁴C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2–2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD^D485G variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad^D485G) ABE products resulted in a blend with nearly 50 wt%≤C₉ products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9 g L⁻¹ while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80 wt% of catalysis products having a carbon chain length≥C₁₁ which amounts to 9.8 g L⁻¹ of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.     

Factors affecting the microbial digestion of an industrial seaweed-based residue

Commercial preparation of a seaweed extract from the brown alga Ascophyllum nodosum for use as fertiliser and soil improver produces a sludge residue which requires remediation. This residue is rich in nutrients and offers the potential for other value-added products. The residue composition was analysed, a microbial digestion process for the residue was developed, and several factors affecting the digestion process were studied. The residue showed an alkaline pH (8.61?±?0.39) and 16% (w/w) total solids, which comprised 40.6% mineral, 29.5% fibre, 24.3% lipid, 4.9% protein and 0.5% polyphenols. The optimised digestion system included a 3-day anaerobic phase to decrease pH (from 8.96?±?0.40 to 7.72?±?0.38), the addition of an inoculum, followed by a 10-day aerobic phase where the insoluble material was digested. Every 3 days, the solubilised material was decanted and replaced with water to delay metabolite inhibition. The rate of digestion (decrease in insoluble material of 28.6?±?14.2% over 13 days) was influenced by the initial insoluble (R ²?=?0.773) and soluble (R ²?=?0.672) matter, the pH at the beginning of the aerobic phase (R ²?=?0.528) and by the accumulation of solubilised digestion products. A compositional analysis of the insoluble material after digestion showed that the lipid content of the residue was 96% digested and that the proportion of protein increased by 82.4%. Inocula and metabolite inhibition were critical features of A. nodosum residue digestion. Similar organic residues require a carefully chosen inoculum and a minimum initial insoluble content (65–70%) and/or a maximum soluble content (25.30%).     

Cellular reactions of O6-methylguanine, a product of some alkylating carcinogens

Cultures of a purine-requiring mutant of Chinese hamster ovary cells (CHO-104b), randomly bred hamster embryo cells, or Escherichia coli B_s−1 were treated with non-toxic doses of ³H-labelled O⁶-methylguanine. DNA and RNA were isolated and subjected to enzymic digestion to nucleosides at pH8. The products of digestion were analysed by ion-exchange chromatography on columns of Dowex 50 (NH₄⁺ form) at pH8.9. No ³H-labelled O⁶-methylguanosine was detected in nucleic acid digests. ³H-labelled O⁶-methylguanine was O-demethylated yielding [³H]guanine in CHO-104b cells. Radioactivity in nucleic acid digests was associated with thymidine, guanosine, deoxyguanosine and an unidentified early-eluting product. Reports of similar unidentified products from nucleic acids labelled with various agents are discussed.     

Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

Specific concerns have been raised that third-generation cephalosporin-resistant (3GC^r) Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COT^r) E. coli, 3GC^r Salmonella enterica, and nalidixic acid-resistant (NAL^r) S. enterica may be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n = 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GC^r Salmonella was detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NAL^r S. enterica was detected on only one hide. 3GC^r E. coli and COT^r E. coli were detected on 100.0% of hides during processing. Concentrations of 3GC^r E. coli and COT^r E. coli on hides were correlated with pre-evisceration carcass contamination. 3GC^r E. coli and COT^r E. coli were each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenic E. coli (ExPEC) virulence-associated markers. Only two COT^r E. coli isolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria.     

Regio- and enantioselective bioreduction of methyleneketoesters using both polymeric resin and cellulose matrix

Methyleneketoesters were prepared in >90% yield by performing an IBX oxidation of Morita–Baylis–Hillman adducts. A methodology was developed to achieve methyl 3-aryl-3-keto-2-methylenepropanoate reduction using a screening of yeast strains in three different reaction procedures to obtain products with both high yield and diastereoselectivity. The reactions conducted in water provided inferior yields (50%) for substrates 2b–c. Employing Amberlite^® XAD7HP which was a substrate reservoir that also immediately extracted the products from the reaction medium after their formation, syn-4a–c and anti-4a–c were isolated in 60–70% yield, with high stereoselectivity (98–99% ee). The best results were obtained using substrates adsorbed on filter paper which provided products yields above 70%, a 99% ee and a diastereomoeric ratio (syn-4: anti-4) 9:1. Cellulose matrix has excellent potential to be successfully employed in general biocatalytic reactions.     

A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity.

α-Amylases have been found to convert starch and glycogen, in part, to products other than hemiacetal-bearing entities (maltose, maltodextrins, etc.)—hitherto, the only products obtained from natural α-glucans by α-amylolysis. Glycosides of maltosaccharides were synthesized by purified α-amylases acting on starch or bacterial glycogen in the presence of p-nitrophenyl α- or β-d-glucoside. From a digest with crystallized B. subtilis var. amyloliquefaciens α-amylase, containing 4 mg/ml of [¹⁴C]glycogen and 40 mmp-NP β-d-glucoside, three pairs of correspondingly labeled glycosides and sugars were recovered: p-NP α-d-[¹⁴C]glucopyranosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]glucose; p-NP α-[¹⁴C]maltosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltose; p-NP α-[¹⁴C]maltotriosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltotriose. The three glycosides accounted for 11.4% of the [¹⁴C]glycogen donor substrate; the three comparable sugars, for 30.4%; higher maltodextrins, for 58.2%. Calculations based on the molar yields of all reaction products show that [¹⁴C]glycosyl moieties were transferred from donor to p-NP β-d-glucoside with a frequency of 0.234 relative to all transfers to water. This is a very high value considering the minute molar ratio (0.0007) of β-d-glucoside-to-water concentration. Less striking but similar findings were obtained with cryst. hog pancreatic and Aspergillus oryzae α-amylases. The results extend earlier findings (Hehre et al., Advan. Chem. Ser. (1973) 117, 309) in showing that α-amylases have a substantial capacity to utilize the C₄-carbinols of certain d-glucosyl compounds as acceptor sites.