Enzymatic synthesis of new aromatic and aliphatic esters of flavonoids using Candida antarctica lipase as biocatalyst

Enzymatic acylation of rutin and esculin with aromatic, aliphatic and aryl-aliphatic acids using Candida antarctica lipase in tert amyl alcohol as solvent was investigated under low water content. Whatever the acyl donor used, the conversion yields and initial rates for esculin were higher than for rutin. For a given flavonoid, the performance of these reactions depended on the acyl donor structures. For aliphatic acids, conversion yields and initial rates of both flavonoids were respectively in the ranges of 68-90% and of 9.5×10^-2-72×10^-2 mmol l^-1 h^-1. For aromatic acids, the reaction occurred only with the aryl subgroup (cinnamic, hydrocinnamic, 3,4-dihydroxyhydrocinnamic and 4-hydroxyphenyl acetic acids) and was drastically influenced by the presence of side chain and substitution patterns of the aromatic ring. Except for hydrocinnamic acid (75%, 23.4×10^-2 mmol l^-1 h^-1), with these acids the conversion yields and initial rates were lower and in the range of 10-45% and of 0.7×10^-2 to 12.1×10^-2 mmol l^-1 h^-1. Unsaturation of the side chain of the hydrocinnamic acid decreased the esculin conversion rate from 75 to 13% and initial rate from 23.4 to 1.76×10^-2 mmol l^-1 h^-1. The presence of hydroxyl or nitro-groups on the aromatic ring of the aryl aliphatic acid also reduced conversion yields and initial rates. Even without a spacer, the non-phenolic ring acid (quinic acid) was reactive and lead to conversion yields of about 20 and 23% respectively for rutin and esculin.     

Kinetics of Pyruvate Decarboxylase Deactivation by Benzaldehyde

Based on experimental data, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0-200 mM) in MOPS buffer (2.5 M) has been developed. An initial lag period prior to deactivation was found to occur. With first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration, a reaction time deactivation constant of 2.64×10^-3 h^-1 and a benzaldehyde deactivation coefficient of 1.98×10^-4 mM^-1 h^-1 were determined for benzaldehyde concentrations up to 200 mM. The PDC deactivation kinetic equations established in this study are an essential component in an overall model being developed to describe the enzymatic biotransformation of benzaldehyde and pyruvate to produce the pharmaceutical intermediate (R)-phenylacetylcarbinol (R-PAC).     

Efficacy of the Entomopathogenic Fungus, Culicinomyces clavisporus against Larvae of the Biting Midge, Culicoides nubeculosus (Diptera: Ceratopogonidae)

Culicinomyces clavisporus, a fungal pathogen of a wide range of mosquito species, was investigated in relation to potential pathogenicity against Culicoides nubeculosus biting midge larvae. Seven different C. clavisporus strains were assayed. Each showed some degree of activity against C. nubeculosus larvae with LC₅₀ values of between 3.2×10^-5 and 1.1×10^-6 spores/mL; these effects occurred in dose-dependent manners and tended to be delayed until 72-96 h post treatment. The results are discussed in relation to incorporation of C. clavisporus into biocontrol programmes for Culicoides spp.     

Differences in HPRT mutant frequency among middle-aged Flemish women in association with area of residence and blood lead levels

Biomarkers were measured in residents of Wilrijk and Hoboken, industrial suburbs of the city of Antwerp, and of Peer, a rural municipality in Flanders, Belgium. Persons with known occupational exposures to toxic compounds or commuting over long distances were excluded. Here, we report the hypoxanthine phosphoribosyltransferase gene (HPRT) variant frequencies for 99 non-smoking women aged 50-65 years. HPRT values above the detection limit (V_fpos values) were observed for 43 subjects (21 from Peer, 22 from Antwerp). The median (10th to 90th percentiles) HPRT variant frequency (V_fpos) in peripheral lymphocytes was 9.59 (3.44-56.99) for Peer and 3.57 (1.57-13.96) for Antwerp. The V_fpos value was significantly higher in Peer than in Antwerp, both in terms of crude data (p=0.011) and after correction for age, level of education, smoking status, serum level of selenium and body mass index through analysis of covariance (p=0.011). For the total study population, serum lead concentration showed a non-significant positive correlation with lnV_fpos. In addition, subjects with a blood lead concentration above the median tended to have higher V_fpos values (9.45×10^-6 for 'high' group versus 5.21×10^-6 for 'low' group; p=0.077 after correction for confounding). Subjects with a serum selenium level above the median tended to have lower V_fpos values (4.99×10^-6 for 'high' group versus 9.83×10^-6 for 'low' group; p=0.051 after correction for confounding). These data are consistent with an indirect genotoxic effect of lead and with an antimutagenic effect of selenium.     

Characterization of Aspergillus niger catalase

Aspergillus niger catalase has been characterized by a variety of physical techniques including gel filtration, sedimentation rate and equilibrium methods and photon correlation spectroscopy. The catalase has a sedimentation coefficient (S₂₀⁰) of 14.2 ± 0.08 S and diffusion coefficient (D₂₀⁰) of 4.14 ± 0.35 × 10⁻⁷ cm² s⁻¹. The average molecular weight of the catalase from all available data including current sedimentation equilibrium measurements and two previous literature values is 345 000. The frictional ratio of the molecule assuming a hydration parameter similar to that of bovine liver catalase (.3 g H₂O g⁻¹) is 1.103, suggesting that Aspergillus niger catalase has an asymmetric structure with an axial ratio of approximately 3 (the Stokes radius is 5.83 ± 0.49 nm). The titration curve and amino acid analysis indicate that in the native conformation only 23% of the ionizable amino acid residues are titratable between pH 3 and 10.5. Denaturation with sodium n-dodecylsulphate increases the number of titratable groups to 46%. The ratio of anionic to cationic amino acid residues in Aspergillus niger catalase is 2.46 and the isoelectric point is 6.5. The optimum pH for catalytic activity is approximately 7.     

琼东海域橙黄滨珊瑚骨骼生长特性及其主要影响因素

基于珊瑚骨骼X射线照片,使用CoralXDS软件,对海南岛(琼)东部海域的橙黄滨珊瑚生长特性进行研究,获取了百年来的年生长率(ER)、骨骼密度(D)和钙化速率(CR) 3种生长参数.结果表明: 该橙黄滨珊瑚ER的变化范围为0.49～1.10 cm·a^-1,年均值0.76 cm·a^-1;D的变化范围1.11～1.35 g·cm^-3,年均值1.22 g·cm^-3;CR的变化范围为0.55～1.41 g·cm^-2·a^-1,年均值0.94 g·cm^-2·a^-1.统计分析表明,研究海区表层水温(SST)是橙黄滨珊瑚骨骼生长参数变化的主要环境影响因素,对ER和CR的控制较强,对D的影响较弱,但总体上3项生长参数都随SST升高而增大.光照、盐度和水动力条件等是次要影响因素.台风和强热带风暴对琼东海域橙黄滨珊瑚骨骼密度条带分布的影响也较为显著.复杂的气候环境,赋予了橙黄滨珊瑚骨骼多变的生长模式.过去一个世纪以来,琼东海域年平均表层水温显著上升,气候倾向率为0.15 ℃·(10 a)^-1,SST上升分为两个阶段,20世纪40年代初和80年代初,人类活动加剧和全球气候变暖是导致琼东海域SST显著上升的原因.     

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P<0.10) and increased minor sperm defects (P<0.001) in Year 1. B. indicus bulls had greater (P<0.005) sperm concentration than B. taurus bulls in both years (1.7×10⁹/ml versus 1.2×10⁹/ml in Year 1 and 1.6×10⁹/ml versus 1.2×10⁹/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P<0.05) total (11.4×10⁹ versus 8.2×10⁹) and viable (6.7×10⁹ versus 4.9×10⁹) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P<0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3×10⁹ versus 9.1×10⁹ and 6.1×10⁹ versus 5.4×10⁹ in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P<0.10) to have more major sperm defects and had more (P<0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P<0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa.     

Cortisol and testosterone: Inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 × 10⁻⁶ and 10⁻⁴ M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 × 10⁻⁷ to 5 × 10⁻⁵ M and testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10⁻⁸ and 10⁻⁶ M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11β, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studies in our laboratory.     

One-electron reduction of selenomethionine oxide

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e^-_aq (k = 1.2 × 10¹⁰M^-1s^-1), CO^·-₂ (k = 5.9 × 10⁸ M^-1s^-1) and (CH₃)₂) C^·OH (k = 3.5 × 10⁷M^-1s^-1), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se_∴ N 2σ/1σ* three-electron bond, which is characterized by an optical absorption with λ_max at 375 nm, and a half-life of about 70 μs. The same transient is generated upon HO^· radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se_∴ N transient and GSH. The Se_∴ N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.     

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (<0.049×10⁻⁷). The frequency of HPRT exons 2+3 deletions was 0.10×10⁻⁷ per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%–70%) and previous results using the T-cell cloning assay (2–3×10⁻⁷ per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7×10⁸ leukocytes analyzed), and which occurred at frequencies from <1×10⁻⁷ (undetected) to 1.6×10⁻⁴ among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2+3 deletions in the leukocytes of the newborns, from 0.07×10⁻⁷ to 0.23×10⁻⁷. We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2+3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.     

Biological Control and Use of Adjuvants against Multiple Seeded Cocklebur (Xanthium strumarium) in Comparison with Several Other Cocklebur Types

Common cocklebur has several biotypes including multiple seeded cocklebur (MSC), NCC-TX, and NCC-MS. Alternaria helianthi applied at 2.5×10⁴ conidia mL^-1 in a 50% micro-emulsion of unrefined corn oil (MESUCO) or 0.2% Silwet L 77 caused 60-75% mortality on NCC-TX and MSC. Increasing the conidial concentration to 5×10⁴ mL^-1 increased mortality to 100% on MSC and NCC-TX, and 75% on NCC-MS. At 10×10⁴ conidia mL^-1, A. helianthi caused 100% mortality in all three biotypes. No mortality occurred in any biotype at inoculation rates of 2.5 and 5×10⁴ conidia mL^-1 when applied in water. Increasing the dew period from 0 to 12 h increased mortality from 0 to 100% on all three biotypes at a rate of 2.5×10⁴ conidia mL^-1 in Silwet and MESUCO. MSC appears to be the most sensitive biotype.     

Structure of 7S seed globulin from Phaseolus vulgaris L. in solution

The structure of the 7S globulin from Phaseoulus vulgaris L in dilatue solutions has been studied by small angle X-ray scattering (SAXS), by quasi-elastic light scattering (Q ELS), by circular dichroism spectroscopy (c.d.), and by precise density measurements. The molar mass, the radius of gyration, the volume, the maximum dimension and the diffusion coefficient were determined as M = 1.45 × 10⁵ g mol⁻¹, R_G = 4.05 nm, V = 300- nm³, L = 13.0 nm and D_20,w⁰ = 4.5 × 10⁻⁷ cm² s⁻¹, respectively. The molecule has an asymmetrical shape with the dimensions 12.5 × 12.5 × 3.75 nm. The secondary structure of the 7S globulin is characterized by a small portion of -helical structure (14%) and a marked content of β-structure (18%).     

Biphasic kinetics of Zn removal from Zn metallothionein by nitrilotriacetate are associated with differential reactivity of the two metal clusters

We investigated the kinetics of nitrilotriacetate (NTA) extraction of Zn²⁺ from Zn₇-metallothionein (MT) and a metal-hybrid derivative, Zn₄Ag₆MT, in which the Zn²⁺ and Ag⁺ ions occupy sites in the C-terminal and N-terminal β domains of the protein, respectively. Biphasic kinetics were observed for Zn₇MT under pseudo-first-order conditions. Rate constants were (5.2±0.6)×10⁻³ and (1.0±0.3)×10⁻⁴s⁻¹ in 20 mM phosphate, 100 mM KF, pH 7.5 at 23C. In contrast, Zn₄Ag₆MT showed a single kinetic step with a rate constant of (2.9±0.4)×10⁻³s⁻¹. These results indicate that the biphasic reactivity of Zn₇MT stems from differential susceptibility of the metal in the two metal–thiolate clusters to removal by competing ligands, with Zn²⁺ in the more stable -domain cluster reacting faster than that in the less stable β-domain cluster. Such behavior suggests that the structures of the two domains of mammalian MT may have evolved to assure that Cu binding does not compromise the structural characteristics that allow Zn to be rapidly transferred from MT to essential cellular ligands.     

Binding Specificity and Affinity of Acriflavine for Nucleic Acids

A set of conditions was developed for the specific binding of acriflavine to the DNA of intact squamous cells. This was achieved through a series of studies into the relative affinities for dye between DNA and various biopolymers by an agar gel diffusion technique. Specificity was ascertained by DNase and RNase treatment of the cells. The final conditions, based on an estimated DNA-to-dye ratio of 4:1, required a constant cell count of 100,000 and dye at a concentration of 0.0025 μg per ml in 10 ml of phosphate buffer, pH 6.0-7.4. These quantities were dictated by the sensitivity limitations of the analytical apparatus. To make use of standard fluorometric instrumentation, the whole cell population method for determining average values was followed. Free dye was analyzed after cell samples were stained, and the difference between this value and that of an aliquot of working dye was taken as the amount of bound dye. To ensure cell-free residues of dye, cells were removed by centrifugation through Teflon membrane filters. The average amount of dye which was bound to the DNA of normal squamous cells was 1.6 × 10^-7 μg (0.7 × 10^-15 mole) per cell, and the amount bound to HeLa cells was 2.3 × 10^-7 μg (1 × 10^-15 mole). These values were highly replicable, making it possible to use them as an expression of the DNA content of the cells. The mildness of the staining conditions, and the preservation of cellular integrity in this technique may permit quantitative measurements of the DNA content of living cells.     

The reactions of oxicam and sulfoanilide non steroidal anti-inflammatory drugs with hypochlorous acid: determination of the rate constants with an assay based on the competition with para-aminobenzoic acid chlorination and identification of some oxidation products

Hypochlorous acid (HOCl) is an oxygen-derived species involved in physiological processes related to the defence of the organism that may cause adverse effects when its production is insufficiently controlled. In order to examine its reactivity with potential scavenging molecules from the non steroidal anti-inflammatory drugs (NSAIDs) family, a competition assay based on para-aminobenzoic acid (PABA) chlorination was developed. The original optimised in vitro fluorimetric procedure offered the possibility to determine rate constants (k_s) for the reaction with HOCl in physiologically relevant conditions. The specificity of the system was improved by a liquid chromatography (LC) which allows the separation of the drugs and their oxidation products. After determination of the rate constant for PABA chlorination by HOCl (mean±SD in M_-1 s_-1: 4.3±0.3×10³), the applied mathematical model for a chemical competition permits to obtain linear curves from competition studies between several NSAIDs and PABA. Their slopes provided the following rate constants for the different studied drugs: tenoxicam: 4.0±0.7×10³, piroxicam: 3.6±0.7×10³, lornoxicam: 4.3±0.7×10³, meloxicam: 1.7±0.3×10⁴, nimesulide: 2.3±0.6×10². Meloxicam therefore reacted significantly faster than the other oxicams and nimesulide, which is the weakest scavenger of the studied series. The identification of some of the oxidation products by NMR or MS permitted to explore the reaction mechanism and to examine some aspects of the structure/activity relationships for the molecules of the same chemical family.     

Synthesis of graft copolymer (k-carrageenan-g-N,N-dimethylacrylamide) and studies of metal ion uptake, swelling capacity and flocculation properties

Graft copolymer of k-carrageenan and N,N-dimethylacrylamide has been synthesized by free radical polymerization using peroxymonosulphate/glycolic acid redox pair in an inert atmosphere. The grafting parameters i.e. grafting ratio, add on and efficiency decrease with increase in concentration of k-carrageenan from 0.6 to 1.4 g dm⁻³ and hydrogen ion from 3 × 10⁻³ to 7 × 10⁻³ mol dm⁻³, but these grafting parameters increase with increase in concentration of N,N-dimethylacrylamide from 16 × 10⁻² to 32 × 10⁻² mol dm⁻³, and peroxymonosulphate from 0.8 × 10⁻² to 2.4 × 10⁻² mol dm⁻³. The metal ion sorption, swelling behaviour and flocculation properties have been studied. The intrinsic viscosity of pure and grafted samples has been measured by using Ubbelohde capillary viscometer. Flocculation capability of k-carrageenan and k-carrageenan-g-N,N-dimethylacrylamide for both coking and non-coking coals has been studied for the treatment of coal mine waste water. The graft copolymer has been characterized by Infrared (IR) spectroscopy and thermogravimetric analysis.     

Microbial characterization during composting of municipal solid waste

This study investigates the prevailing physico-chemical conditions and microbial community; mesophilic bacteria, yeasts and filamentous fungi, bacterial spores, Salmonella and Shigella as well as faecal indicator bacteria: total coliforms, faecal coliforms and faecal Streptococci, present in a compost of municipal solid waste. Investigations were conducted in a semi-industrial pilot plant using a moderate aeration during the composting process. Our results showed that: (i) auto-sterilization induced by relatively high temperatures (60–55°C) caused a significant change in bacterial communities. For instance, Escherichia coli and faecal Streptococci populations decreased, respectively, from 2×10⁷ to 3.1×10³ and 10⁷ to 1.5×10³ cells/g waste dry weight (WDW); yeasts and filamentous fungi decreased from 4.5×10⁶ to 2.6×10³ cells/g WDW and mesophilic bacteria were reduced from 5.8×10⁹ to 1.8×10⁷ bacteria/g WDW. On the other hand, the number of bacterial spores increased at the beginning of the composting process, but after the third week their number decreased notably; (ii) Salmonella disappeared completely from compost by the 25th day as soon as the temperature reached 60°C; and (iii) the bacterial population increased gradually during the cooling phase. While Staphylococci seemed to be the dominant bacteria during the mesophilic phase and at the beginning of the thermophilic phase, bacilli predominated during the remainder of the composting cycle. The appearance of gram-negative rods (opportunistic pathogens) during the cooling phase may represent a serious risk for the sanitary quality of the finished product intended for agronomic reuse. Compost sonication for about 3 min induced the inactivation of delicate bacteria, in particular gram-negatives. By contrast, gram-positive bacteria, especially micrococcus, spores of bacilli, and fungal propagules survived, and reached high concentrations in the compost.     

Joint effect of nitrogen and phosphorous on glucose oxidase production by Aspergillus niger: Discussion of an experimental design with a risk of co-linearity

The combined effects of nitrogen and phosphorous on the production of glucose oxidase and gluconic acid by Aspergillus niger cannot be adequately described with Monod-type model, neither do they fit well to linear equations with interactions N × P, nor quadratic with N² and P² terms. On the other hand, the interactions of type N²P and NP², although common in real cases such as enzymatic kinetics in the presence of inhibitors, should be verified – if included in empiric models – by means of designs that can lead to artefactual results derived from the co-linearity. To avoid this risk we propose a procedure, based on the ‘bootstrap’ algorithm, which provided consistent results in the mentioned bioproductions. Applied together with methods of response surface and gradient, said procedure allowed to optimize the enzyme production as a function of the concentrations of N and P, to quintuple the initially obtained levels, and to explain other culture behaviours related with the sources of these nutrients.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

The short term effects of 5,7-dihydroxytryptamine on peripheral serotonin stores in Rhodnius prolixus and their long-term recovery

The serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) appears to affect invertebrate systems differently from vertebrate ones. The basis for toxicity in vertebrates appears to involve the intraneuronal actions of monoamine oxidase (MAO) upon the toxin. In insects, MAO is not present in appreciable amounts. In this study, we demonstrate that in vitro 5.7-DHT competitively inhibits the uptake of [³H]serotonin by serotonergic neurohaemal areas. The apparent K_M increases from 4.9 × 10⁻⁷ to 1.7 × 10⁻⁶ M. This neurotoxin also causes a significant release of previously accumulated [³H]serotonin in nominally Ca²⁺-free saline. While 5,7-DHT does not affect the uptake of [³H]tryptophan, it reduces the subsequent synthesis of [³H]serotonin. In vivo, the tissues appear to have recovered 2 weeks after toxin treatment, as determined by immunohistochemistry. At 24 h, 1 week and 2 weeks after injection, the tissues are able to take up and release [³H]serotonin normally. 1 and 2 weeks after injection, insects ingest a normal-sized blood meal, a behaviour acutely disrupted by 5,7-DHT treatment. The results of this and other invertebrate studies suggest that 5,7-DHT does not destroy serotonergic neurons, as it does in vertebrates. 5,7-DHT may be a more useful tool to study the functions of serotonin in invertebrates as one may transiently affect serotonin stores.