Volatile constituents of male and female boll weevils and their frass

The volatile constituents of male and female boll weevils, Anthonomus grandis, and their frass were analysed by GLC-MS. The 4 previously identified components of the male pheromone were present only in the male volatile oil (3·9%) and in the male frass volatile oil (38·9%). The compounds found in one or both sexes and their frass include 26 carbonyls, 23 hydrocarbons, 12 alcohols, 6 phenols, 4 esters, 3 furans, 1 ether, and 1 lactone. Also found were 2 compounds containing nitrogen, 1 halogen, and 1 sulphur. There were 33 terpenes and 24 aromatic compounds. 3,7-Dimethyl-1-octanol comprised 15·6 per cent of the male frass oil. Carvone was found only in females (5·4%) and in female frass (6·8%). A series of monoterpene aldehydes (M⁺ 152) were found only in the female frass oil. A pheromone rôle for these components was suggested.     

Composition of turpentine from Pinus edulis wood oleoresin

Monoterpenoids and sesquiterpenoid hydrocarbons of Pinus edulis wood oleoresin were analyzed by chromatographic and spectroscopic methods. Monoterpenoid hydrocarbons (20·3%) were composed mainly of α-pinene, with camphene, β-pinene, 3-carene, sabinene, myrcene, limonene, β-phellandrene, trans-ocimene and terpinolene in secondary to trace amounts. Oxygenated terpenoids (0.28%) contained bornyl acetate and verbenone as major constituents, and linalool, camphor, terpinene-4-ol, citronellyl acetate, borneol, neral, α-terpineol, citronellol, nerol, and geraniol in smaller amounts. Oleoresin contained 1·1% of acetogenins, composed mainly of ethyl caprylate. Sesquiterpenoid hydrocarbons were high (5·7%) in oleoresin) and were composed of germacrene D as a major constituent (36·6%), of γ-amorphene, α-copaene, and longifolene as secondary constituents (5–20%), and β-farnesene, α- and γ-murolenes, β₁-, γ-, δ-, and ε-cadinenes, α-amorphene, δ-guaiene, sibirene, α-cubebene, β-copaene, β-ylangene, sativene, cyclosativene, β-bourbonene, α- and γ-humulenes, caryophyllene, α-longipinene and longicyclene in smaller amounts. Composition of P. edulis and of P. monophylla turpentines was found to be similar, with percentage of ethyl caprylate being the best distinguishing criterion.     

Unusual occurrence of aldehydes and ketones in the defensive secretion of the tenebrionid beetle, Eleodes beameri

The defensive secretion of the tenebrionid beetle, Eleodes beameri, is quite unlike the benzoquinone and 1-alkene secretion of other species of Eleodes and Tenebrionidae. Twenty-three compounds were isolated from the secretion by gas-liquid chromatography (GLC) and 13 of these were identified by infrared, nuclear magnetic resonance, ultraviolet, and mass spectroscopy. Identified compounds were: 1-nonene (3·2%), 1-undecene (<0·5%), n-hexanal (15·6%), n-heptanal (0·9%), n-octanal (4·5%), trans-2-hexenal (2·0%), trans-2-heptenal (1·5%), trans-2-nonenal (28·6%), trans-2-decenal (3·4%), n-3-nonanone (0·5%), n-1-nonen-3-one (16·8%), methyl-1,4-benzoquinone (22·0%), and 1-hexanol (<0·5%). 1-Nonen-3-one is unique to E. beameri. A number of minor components remain unidentified. The morphology and ultrastructure of the glands were similar to other species of Eleodes. The gland reservoirs are a pair of strongly bilobed sacs with narrow exit ducts opening between abdominal sternites 7 and 8. There are two types of secretory cell units: Type 1 consisting of cells closely attached to the reservoir intima, with large, central vesicles drained by highly convoluted tubules. Type 2 units are composed of a pair of cells functioning together, the cuticular organelle from the microvilli-filled vesicle of the distal cell (2a) passing through the vesicle of the proximal one (2b) and then draining more or less directly into the reservoir via a cuticular tubule.     

Carbohydrates of the inner bark of Pseudotsuga menziesii

A holocellulose fraction was isolated from the inner bark of Psedotsuga menziesii and analyzed. It was composed of acid-insoluble lignin (3·1 acid-soluble lignin (4·1%), l-arabinose (2·6%), d-xylose (6.3%). d-mannose(9·5%), d-galactose (2·3%), and d-glcose (61·1%). The presence of these sugars and their configurations were positively established by the preparation of crystalline derivatives. The holocellulose was fractionated into its component polysaccharides, a xylan, a galactoglucomannan, a glucomannan, and a glucan-rich residue.     

Chemical composition of volatiles from cortical oleoresin of Pseudotsuga menziesii

Pseudotsuga menziesii cortical oleoresin was found to contain 1·7% of oxygenated terpenoids and compounds of similar volatility composed of linalool, methylsalicylate, bornyl acetate, citronellol, geranyl acetate, methylthymol, citronellyl acetate, terpinen-4-ol, borneol, isopulegol, anethole, terpinen-4-ol acetate, camphor, geraniol, neryl acetate, and nerol. Sesquiterpenoid hydrocarbons were low (only 0·07%) and contained sibirene and longifolene as main constituents, with β-caryophyllene, γ-muurolene, γ-cadinene (identified by IR), and 20 additional compounds in small amounts. p-Cymen-8-ene was identified in monoterpene hydrocarbon fraction.     

THF INFLUENCE OF IRRADIANCE ON THF BIOCHEMICAL COMPOSITION OF THE PRYMNESIOPHYTE ISOCHRYSIS SP. (CLONE T-ISO)1

The effects of irradiance on the biochemical composition of the prymnesiophyte microalga, Isochrysis sp. (Parke; clone T-ISO), a popular species for mariculture, were examined. Cultures were grown under a 12:12 h light: dark (L:D) regime at five irradiances ranging from 50 to 1000 μE·m ²·s^?1 and harvested at late-logarithmic phase for analysis of biochemical composition. Gross composition varied aver the range of irradiances. The highest levels of protein were present in cells from cultures grown at 100 and 250 μE·m ³·s¹, and minimum levels of carbohydrate and lipid occurred at 50 μE·m^?2·s^?1. Because the cell dry weight was reduced at lower irradiances, different trends were evident when results were expressed as percentage of dry weights. Protein percentages were highest at Wand 100 μE·m^?2·s^?1 and carbohydrate at 100 μE·m^?2·s^?1. The composition of amino acids did not differ over the range of irradiances. Glutamate and aspartate were always present in high proportions (9.0–13.5%); histidine. methionine, tryptophan, cystine, and hydroxy-proline were minor constituents (0.0–2.6%). Glucose was the predominant sugar in all cultures, ranging from 23.0% (50 μE·m^?2·s^?1) to 45.0% (100 μE·m^?2·s^?1) of total polysaccharide. No correlation was found between the proportion of any of the sugars and irradiance. The proportions of the lipid class components and fatty acids showed little change with irradiance. The main fatty acids were 14:0, 16:0, 16:1(n-7), 18:1(n-9), 18:3(n-3). 18:4(n-3), 18:5(n-3), and 22:6(n-3). Proportions of 22: 6(n-3) increased, whereas l8:3(n-3). 18:3(n-6). and 18:4(n-3) decreased, with increasing irradiance. Pigment concentrations were highest in cultures grown at 50 μE·m^?2·s^?1, except for fucoxanthin and diadinoxanthin (100 μE·m^?2·s^?1). The concentrations of accessory pigments correlated with chlorophyll a, which decreased in concentration with increasing irradiance. On the basts of biochemical composition, an irradiance of 100 μE·m^?1·s^?1 (12:12 h L:D cycle)for the culture of Isochrysis sp. (clone T-ISO) may provide optimal nutritional value for maricultured animals, although feeding trials are now necessary to substantiate this.     

Respiration,energy balance and development during growth and starvation of Carcinus maenas L. larvae (Decapoda:Portunidae)

In all larval stages of Carcinus maenas L. oxygen consumption was measured at three temperatures (12,18,25 °C). Values increased during development and were in the range of 0.037 ± 0.01 (zoea-1, 12°C, $\text{x}\text{?}\text{± 95\% CL}$) to 0.734 ± 0.047 μl O₂ · h^?1 · ind^?1 (megalopa, 25 °C). Growing larvae showed temperature dependent trends in weight specific respiration rates (referred to dry wt; DW), with values between ≈2.4 and 9.4 μl O₂· h^?1·mg DW^?1. Increase in oxygen consumption of megalops did not differ much at temperatures between 18 and 25 °C. This points to an exceptional physiological position of this stage. Fed zoea-1 of C. maenas (18 °C) revealed growth rates in terms of 40% DW, 20% carbon (C), 30% nitrogen (N) and 65% hydrogen (H). At the same time larvae gained individual energy by 13% (J · ind^?1), while weight specific energy dropped by ≈ 19% (J · mg DW^?1) during the first day and remained constant until the moult. Starved zoea-1 of C. maenas (18 ° C) gained ≈ 20 % in DW through the first day, probably caused by inorganic salts which enter the organism after the moult of the prezoea. DW dropped to ≈ 25 % of initial value, when starvation continued. Single components decreased by ≈50% (C), 54% (N), 57% (J · ind^?1). Weight specific energy (J · mg DW^?1) decreased by 40% during the first 4 days of starvation, remaining constant thereafter. Individual respiration rate (R) dropped by 61 %, weight specific respiration rate (QO₂) by 55 %. Individual energy loss in starved zoea-1 was 0.077 J over a period of 11 days. In this period ≈ 9.3 μl O₂·ind^?1 were consumed. Thus effective oxygen capacity was lower than in growing larvae. It dropped to 5.3 J·mlO₂^?1 after 4 days and remained constant if starvation continued, i.e. 65 % of possible energy loss occurred during the first 4 days. Decrease in requirement for oxygen and its effective capacity were both recognized as independent components of survival during starvation. Partitioning of energy through individual larval development of C. maenas was investigated for all five larval stages. The cumulative budget could be calculated: consumption (C) = 28.23 J, growth (G) = 0.92 J, exoskeleton (Ex) = 0.20 J, metabolism (M) = 5.30 J, egestion and excretion (E) = 21.82 J. Mean gross and net growth efficiency were, K₁ = 3.3% and K₂ = 14.8%, respectively.     

Biogenesis of indole compounds from D- and L-tryptophan in segments of etiolated seedlings of cabbage,maize and pea

The metabolism of D-and L-tryptophan-3-¹⁴C (Try-3-¹⁴C) was studied and compared for three different plant species, cabbage, maize and pea. Apical segments of the seedlings were incubated for 6 hours in solutions of L- or D-Try-3-¹⁴C (1·5 μc/ml) with the addition of chloramphenicol (10^?4g/ml) and then allowed to stand for another 20 hours in moist chambers. The methanolic extract of the tissues was analyzed radiochromatographically and by paper electrophoresis in combination with biological tests. Chloramphenicol in a concentration of 10^?4 g/ml had little influence on the growth of the segments, though the antibiotic slightly decreased the uptake of L-Try, it did not prevent the formation of IAA from L-Try. In the segments of cabbage the following metabolites were formed from L-Try-3-¹⁴C (accounting for 52% of the activity of the chromatographically separated extract): glucobrassicin (26·0%), neoglucobrassicin (3·6%), a spot corresponding according to its Rf to 3-indolylacetamide (IAAmide—10·9%), β-glucoside of 3-indolylacetic acid (IAGluc—3·3%) and traces of 3-indolylacetonitrile (IAN), IAA and indole-3-carboxylic acid (total 5%). In maize segments L-Try-3-¹⁴C (53·0%) was transformed to several unidentified hydrophilic substances, one of them possessing auxin activity (total amount 6·9%), IAGlue (9·3%) accompanied by a small amount of tryptamine, a spot corresponding according to its Rf to IAAmide (16·5%), IAA and another unidentified hydrophobic substance (4·1%). In pea segments L-Try-3-¹⁴C (66·7%) gave a zone corresponding according to its Rf to IAAmide (20·0%), a substance similar to IAGluc (10·5%) and also hydrophobic substances (3·1%) containing traces of IAA, which could be demonstrated only by bioassay. D-Try is metabolised in the three plants by the virtually exclusive formation of malonyltryptophan.     

Improvement of Biomethane Production from Sewage Sludge in Co-digestion with Glycerol and Waste Frying Oil,Using a Design of Experiments

A central composite design circumscribed method was used to define the experimental conditions that improve the methane production rate (k_CH4, liters of methane per kilogram of VS of waste added and per day) and the cumulative methane production (cMP, liters of methane per kilogram of VS of waste added) of the co-digestion of sewage sludge (SS) with crude glycerol (cGly) and waste frying oil (WFO). Three factors were selected, i.e., SS concentration, global co-substrate concentration, and mass fraction of cGly (x_cGly) in a mixture of cGly and WFO (in chemical oxygen demand, COD). SS digestion without co-substrate reached a cMP of (294?±?6) L·kg^?1 and a k_CH4 of (64?±?1) L·kg^?1·d^?1, at standard temperature and pressure conditions and expressed relatively to the initial volatile solids. After statistical analysis, SS and co-substrate concentrations of 4.6 g·L^?1 and 8.8 g·L^?1 (in COD), respectively, with x_cGly of 0.8, were defined to simultaneously boost cMP (91 % more) and k_CH4 (3-fold increase). Application of these conditions would yield 214 MWh more in electricity per 1000 m³ of SS digested.     

Patterns of nitrogen utilization between the ambrosia beetle Xyleborus dispar and its symbiotic fungus

Some parameters of nitrogen utilization between the ambrosia beetle Xyleborus dispar in mutualistic association with its symbiotic fungus Ambrosiella hartigii, were examined. Qualitative and quantitative analyses of the major nitrogenous excretory products were made on the various life stages of X. dispar. The main nitrogenous product found in excreta and hindguts of beetles, larvae, and pupae, was uric acid (range 7·6–14·8 μg uric acid/beetle). No ninhydrin-positive compounds were located in excreta of the beetles. The concentration of ammonia-nitrogen in the various life stages averaged between 0·70 and 1·13 μg NH₃-N/beetle.Total nitrogen determinations were made on sapwood samples of Malus sylvestris (0·34 ± 0·005% N by dry weight), attacked wood, ‘pre-brood’ (0·31 ± 0·005% N by dry weight), and attacked wood ‘post-brood’ (0·17 ± 0·02% N). Similar determinations of the artificial medium (l-asparagine medium) indicated that a nitrogen requirement of about 0·08–0·1% N by dry weight was necessary before oviposition could occur.Fixation of atmospheric nitrogen by individual X. dispar beetles in vitro was not indicated using the acetylene ethylene reductase method. Proteolytic enzyme activity was not found on examination of diapause beetles, their excreta, larval and pupal excreta, and the ambrosial and mycelial forms of A. hartigii.Comparative concentrations of soluble proteins and free amino acids suggested that fungus in the mycangia was built up from free amino acids of the insects. At the period of emergence, flight, and attack of new hosts, the females were found to have a concentration of soluble proteins more than double that found in the beetles during the remainder of the year. The free amino acids were the lowest values recorded during this period (March–October).     

Xylanase recovery: effect of extraction conditions on the AOT-reversed micellar systems using experimental design

Xylanase recovery using AOT-reversed micelles was evaluated under different experimental conditions. A full factorial design with centre point was employed to verify the influence of different factors on the recovery. A mathematical model represented xylanase yield (Y) as a function of pH and temperature: Y=8·75−4·31A²−3·13C², where A=pH and C=temperature. The highest xylanase recovery, indicated by the model (10·2%), was attained at pH 6·3, AOT=0·57 M and temperature 46·8°C. A new xylanase extraction was performed under these conditions to test the model. The recovery yield obtained (9·1%) showed a close similarity between the experimental result and the value predicted by the model.     

Biosynthesis of geraniol and nerol in cell-free extracts of Tanacetum vulgare

Cell-free extracts from leaves of Tanacetum vulgare synthesised geraniol and nerol (3,7-dimethylocta-trans-2-ene-1-ol and its cis isomer) in up to 11·9 and 2·4% total yields from IPP-[4-¹⁴C] and MVA-[2-¹⁴C] respectively. Optimum preparations were obtained from plant material just before the onset of flowering. The ratio of the monoterpenols varied 28-fold for different preparations under conditions where these products or their phosphate esters were not interconverted. Similar extracts incorporated α-terpineol-[¹⁴C] and terpinen-4-ol-[¹⁴C] (p-menth-1-en-8- and -4-ol respectively) in 0·05 to 2·2% yields into a compound tentatively identified as isothujone (trans-thujan-3-one), and preparations from flowerheads converted IPP-[4-¹⁴C] in 2·7% yield into geranyl and neryl β-d-glucosides. Inhibitors of IPP-isomerase had little effect on the incorporation of IPP into the monoterpenols in cell-free systems from which endogenous compounds of low molecular-weight had been removed. The inference that a pool of protein-bonded DMAPP or its biogenetic equivalent was present was supported by the demonstration that geraniol and nerol biosynthesised in the absence of the inhibitors were predominantly (65 to 100%) labelled in the moiety derived from IPP.     

Chronic stress of rainbow trout Oncorhynchus mykiss at high altitude: a field study

The stress response of Oncorhynchus mykiss in high‐altitude farms in central Mexico was investigated over two seasons: the cool (9·1–13·7° C) dry winter season, and the warmer (14·7–15·9° C), wetter summer season. Fish were subjected to an acute stress test followed by sampling of six physiological variables: blood cortisol, glucose, lactate, total antioxidant capacity, haemoglobin concentration and per cent packed cell volume (V_PC%). Multivariate analyses revealed that lactate and total antioxidant capacity were significantly higher in the summer, when water temperatures were warmer and moderate hypoxia (4·9–5·3 mg l^?1) prevailed. In contrast, plasma cortisol was significantly higher in the winter (mean ± s.e .: 76·7 ± 4·0 ng ml^?1) when temperatures were cooler and dissolved oxygen levels higher (6·05–7·9 mg l^?1), than in the summer (22·7 ± 3·8 ng ml^?1). Haemoglobin concentrations (mg dl^?1) were not significantly different between seasons, but V_PC% was significantly higher in the summer (50%) than in the winter (35%). These results suggest that in summer, effects of high altitude on farmed fish are exacerbated by stresses of high temperatures and hypoxia, resulting in higher blood lactate, increased total antioxidant capacity and elevated V_PC% levels.     

Changes in monoterpene composition of Mentha aquatica produced by gene substitution from a high limonene strain of M. Citrata

The dominant gene Lm that causes 60–90% limonene/cineole was substituted into M. aquatica by four convergent backcrosses. The natural strain of M. aquatica has 7·7% cineole, 4·9% limonene, traces of terpinolene and pulegone, 0·1% menthone, 0.2% menthol, and 66·4% menthofuran. The two modified hybrid strains with dominant gene Lm have 53·8 and 78·7% limonene/cineole and a total of only 1·0-3·8% 3-oxygenated compounds in contrast to a total of 66·7% found in the natural strain. The postulate is made that the Lm gene largely prevents either the conversion of a-terpineol → terpinolene or of limonene → isopiperitenone and that in these strains the recessive cc genotype largely but not completely prevents the conversion of limonene → carvone resulting in limonene accumulation. Mentha species almost invariably have either 2-oxygenated or 3-oxygenated compounds, not both. Close coupling phase linkage of the genes Lm and C explains why the self-pollinated progeny of M. spicata or M. crispa C-Lm/c-lm have a ratio of 3 carvone/dihydrocarvone: 1 pulegone/menthone rather than a ratio of 9 carvone : 3 limonene : 3 carvone and menthone: 1 menthone which would be expected if the genes Lm and C were independently inherited     

Assessing the faecal source sensitivity and specificity of ruminant and human genetic microbial source tracking markers in the central Ethiopian highlands

This study tested genetic microbial source tracking (MST) methods for identifying ruminant- (BacR) and human-associated (HF183/BacR287, BacHum) bacterial faecal contaminants in Ethiopia in a newly created regional faecal sample bank (n = 173). BacR performed well, and its marker abundance was high (100% sensitivity (Sens), 95% specificity (Spec), median log₁₀ 8·1 marker equivalents (ME) g⁻¹ ruminant faeces). Human-associated markers tested were less abundant in individual human samples (median: log₁₀ 5·4 and 4·2 (ME + 1) g⁻¹) and were not continuously detected (81% Sens, 91% Spec for BacHum; 77% Sens, 91% Spec for HF183/BacR287). Furthermore, the pig-associated Pig2Bac assay was included and performed excellent (100% Sens, 100% Spec). To evaluate the presence of MST targets in the soil microbiome, representative soil samples were tested during a whole seasonal cycle (n = 60). Only BacR could be detected, but was limited to the dry season and to sites of higher anthropogenic influence (log₁₀ 3·0 to 4·9 (ME + 1) g⁻¹ soil). In conclusion, the large differences in marker abundances between target and non-target faecal samples (median distances between distributions ≥log₁₀ 3 to ≥log₁₀ 7) and their absence in pristine soil indicate that all tested assays are suitable candidates for diverse MST applications in the Ethiopian area.     

Chemical composition and synergistic effect of three Moroccan lavender EOs with ciprofloxacin against foodborne bacteria: a promising approach to modulate antimicrobial resistance

The aim of this study was to determine the chemical profile of the essential oils (EOs) of three Moroccan lavender species (Lavandula pedunculata, LP; Lavandula angustifolia, LA; and Lavandula maroccana, LM) and to investigate, for the first time, the synergistic effect of the optimal mixture of the EOs with conventional antibiotic ciprofloxacin against three pathogenic foodborne bacteria. Gas chromatography/mass spectrometry analysis showed that eucalyptol (39·05%), camphor (24·21%) and borneol (8·29%) were the dominant compounds of LA-EO. LP-EO was characterized by the abundance of camphor (74·51%) and fenchone (27·06%), whereas carvacrol (42·08%), camphor (17·95%) and fenchone (12·05%) were the main constituents of LM-EO. EOs alone or combined showed a remarkable antimicrobial activity against the tested bacteria with minimum inhibitory concentrations (MICs) ranging from 3·53 to 15·96 mg ml⁻¹. The optimal mixture, calculated using a mixture design, corresponded to 19% LA, 38% LP and 43% LM. All combination of the EOs and the best EO mixture with ciprofloxacin exhibited a total synergism with fractional inhibitory concentration index values ranging from 0·27 to 0·37. The best EO mixture showed the highest gain of 128-fold, especially against Salmonella spp., more than that found testing the EOs separately. These findings should be taken into consideration for a possible application in the pharmaceutical and food industries.     

Metabolism of l-β-lysine by a Pseudomonas: Purification and properties of 3-keto-6-acetamidohexanoate cleavage enzyme

Extracts of Pseudomonas B4 grown with l-β-lysine (3,6-diaminohexanoate) as the main energy source are shown to contain a 3-keto-6-acetamidohexanoate cleavage enzyme that converts 3-keto-6-acetamidohexanoate and acetyl · CoA reversibly to 4-acetamidobutyryl · CoA and acetoacetate. The enzyme catalyzes the third step in β-lysine degradation. In unfractionated extracts cleavage enzyme activity is generally assayed spectrophotometrically by coupling the forward reaction with excess 4-acetamidobutyryl · CoA thiolesterase, derived from the same organism, and measuring the rate of CoASH formation by reaction with 5,5-dithiobis(2-nitrobenzoic acid). Enzyme freed of thiolesterase is conveniently assayed by using 4-acetamidobutyryl · CoA and acetoacetate as substrates and measuring acetyl · CoA formation by means of citrate synthase reaction in the presence of 5,5-dithiobis(2-nitrobenzoic acid). The cleavage enzyme has been purified 38-fold to a specific activity of 237 mU/mg. The stoichiometry, equilibrium constant, molecular weight, and various kinetic properties of the enzymatic reaction have been determined. The substrate specificity of the Pseudomonas enzyme differs markedly from that of the analogous 3-keto-5-aminohexanoate cleavage enzyme of Clostridium subterminale strain SB4 and is broader. In the forward reaction 3-ketohexanoate can replace 3-keto-6-acetamidohexanoate, and propionyl · CoA can replace acetyl · CoA as a substrate. In the backward reaction, 4-acetamidobutyryl · CoA can be replaced by any of several CoA thiolesters including the butyryl, valeryl, 4-propionamidobutyryl, 3-acetamidopropionyl, and β-alanyl derivatives, and acetoacetate can be replaced by 2-methylacetoacetate. The products of these reactions have been characterized. Unlike the cleavage enzyme of Clostridium subterminale strain SB4, the Pseudomonas enzyme is not stimulated by Co²⁺ or Mn²⁺ and is not inhibited by EDTA, 5,5-dithiobis(2-nitrobenzoic acid), or p-chloromercuribenzoate. Tracer experiments indicate that carbon atoms 1 and 2 of acetoacetate are derived from carbon atoms 1 and 2 of 3-keto-6-acetamidohexanoate, and carbon atoms 3 and 4 of acetoacetate are derived from the acetyl group of acetyl · CoA. The cleavage enzyme is not formed in detectable amounts when Pseudomonas B4 is grown in a peptone-yeast extract medium.     

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N₂L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick‐end labelling (TUNEL)], plasma membrane integrity (SYBR‐14/PI) and mitochondrial membrane potential (ΔΨMMit, JC‐1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10⁷ spermatozoa oocyte^?1, by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d . DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (V_CL) 61·2 ± 17·4 µm s^?1; average‐path velocity (V_AP) 50·1 ± 17·3 µm s^?1; straight‐line velocity (V_SL) 59·1 ± 18·4 µm s^?1; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, V_CL, V_AP and V_SL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, V_CL and V_SL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with V_CL and V_SL (r = 0·59 and r = 0·55, respectively).     

N-(o-carboxybenzyl) chitosans: Novel chelating polyampholytes

The imine formed by chitosan with phthalaldehydic acid was reduced with sodium cyanoborohydride and the resulting N-(o-carboxybenzyl) chitosan (NCBC) was insolubilised with ethanol and acetone and obtained as a white, free-flowing powder, soluble in both acidic and alkaline solutions. A sample of NCBC with the following degrees of substitution: acetamido 42%±4%, N-(o-carboxybenzyl) amine 43%±3%, free amine 15%±1% and containing 16%±1% moisture, was characterised by IR and UV-Vis. spectrometry, titration and viscometry. The isoelectric point was 6·8; the pK_a values were 5·7 and 8·0. NCBC could be determined by UV-Vis. spectrophotometry in aqueous solutions at 274 nm; maximum viscosity of the solutions was observed at pH 4. Upon addition of NCBC to transition metal ion solutions (0·1–0·5 mm) chelation and insolubilisation took place immediately. The dependence of the collection percentage on pH, NCBC and metal ion concentrations was studied for nine metal ions.