In Vitro Induced Anaerobic Resistance to Metronidazole In Trichomonas Vaginalis

ABSTRACT. Resitance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10-02 and MRP-2) by cultivation at gradually increasing pressure of the drug (1-100 μ/ml) for 12 to 21 months. the resistant derivatives reproduced in anaerobic trypticase-yeast-extract-maltose medium at 100 μ/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556-1,600 μ/ml at 48-h exposure to the drug). Stepwise selection was necessary to develop the resistance in either strain. Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3-10 μ/ml) were unsuccessful. Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation. Development of stable resistance required further cultivation at 100 μ/ml metronidazole. Unstable substrains did not revert to original susceptibility. They retained a moderate level of resistance, being able to grow at 10 μ/ml metronidazole. the strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of [¹⁴C]-metronidazole. These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped.     

Some factors affecting the in vitro invasion of HeLa cells by Trypanosoma cruzi

The penetration of metacyclic forms of Trypanosoma cruzi into HeLa cells after different treatments was studied. When cell development was synchronized by two different processes, maximum rates of parasitization occurred during the S phase of cell cycle (29.48 and 24.3%). However, when cells were treated with trypsin (0.1%), parasitization rates appeared to be lower than controls, reaching values similar to controls 14 h after the beginning of the treatment. Infection values remained unaltered after treatment with colcemid (0.6 μg ml^?1). Cell treatment either with valinomycin (1 μg ml^?1) or with actinomycin D (250 μg ml^?1) caused a marked decrease in the percentage of parasitization. When cells were treated and infected in the presence of tunicamycin (100 ng ml^?1), parasitization rates were increased (14.7%) compared to control cells (6%). On the other hand, no differences in parasitization rates were observed when cells were treated with cycloheximide (100 μg ml^?1). Infection in a low redox medium (?100 mV) resulted in considerable increase in parasitization.     

The effect of mitomycin C treatment of infected erythrocytes on infection with Babesia microti and Babesia rodhaini in mice

In vitro treatment of Babesia microti infected erythrocytes with mitomycin C before their injection into mice prolonged the prepatent period of infection, reduced the levels of the infection in the ‘breakthrough’ parasitaemia and induced protection against reinfection. Treatment of B. microti with mitomycin C at a concentration of 25 μg ml^?1 resulted in a mean peak parasitaemia of 6.2% in the infected mice compared with 46.5% in control mice injected with untreated B. microti parasites. In addition, mice survived a normally fatal B. rodhaini infection if injected with 6.2 × 10⁷ infected erythrocytes treated with 25 μg ml^?1 mitomycin C and four of five mice survived infection with 6.2 × 10⁵ similarly treated infected erythrocytes. However, the degree of protection against B. rodhaini was dependent on the concentration of mitomycin C used to treat the parasites and treatment of 5 × 10⁷ infected erythrocytes with 50 μg ml^?1 resulted in survival of only four of the five infected mice. In addition, when 100 μg ml^?1 of mitomycin C was used to treat B. rodhaini parasites, the course of infection, although delayed, was indistinguishable from that seen in the control mice and all the mice died. The latter results and the lack of efficacy of comparable numbers of heat killed parasites suggested the necessity for sufficient, non-replicating, mitomycin C treated parasites to metabolize and produce and/or present protective antigens to the host.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Fasciculin, a powerful anticholinesterase polypeptide from Dendroaspis angusticeps venom

A powerful inhibition of mammalian acetylcholinesterase was detected in the venom of the snake Dendroaspis angusticeps (green mamba). The substances responsible for such inhibition were isolated and purified by gel filtration on Sephadex G-50 and ion exchange chromatography on Bio-Rex 70 and SP Sephadex C-25. These substances were polypeptides and were named, fasciculins.Upon intraperitoneal injection into mice fasciculins elicited severe, generalized, long-lasting muscle fasciculations with complete clinical recovery.In vitro preincubation with fasciculins at concentrations of 0.01 μg ml^?1 inhibited brain and muscle acetylcholinesterases up to 80%. Histochemical assay for acetylcholinesterase showed an almost complete disappearance of the black-brown precipitate at the neuromuscular end-plate after in vitro incubation with fasciculins.Fasciculins represent a new type of acetylcholinesterase inhibitors provoking muscle fasciculations through a powerful inhibition of enzyme activity at the neuromuscular end-plate, interfering with the normal degradative activity of the acetylcholine molecule. Fasciculins are also powerful inhibitors of brain acetylcholinesterases.     

The effect of butenolide on behavioral and morphological changes in two marine fouling species,the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC₅₀ = 0.82 μg ml^?1) and B. neritina (EC₅₀ = 3 μg ml^?1). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml^?1 and 10 μg ml^?1, butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

Content of cysteine, reduced and oxidized glutathione in spermatozoa of representatives of Acipenseriformes (Acipenser baerii and A. ruthenus) as well as teleosts (Perca fluviatilis and Sander lucioperca)

Glutathione belongs to a vital intra‐ and extra‐cellular protective antioxidant and is found almost exclusively in its reduced form. The ratio between its reduced and oxidized within cells is often used as a marker of cellular toxicity. The objectives of the study were to (i) determine both the reduced (GSH) and oxidized glutathione (GSSG) and cysteine (Cys) in the sperm of the Acipenser baerii and Acipenser ruthenus, as well as in Perca fluviatilis and Sander lucioperca, and (ii) to demonstrate the differences in concentration levels between representatives of acipenseriform and teleost species. High performance liquid chromatography with electrochemical detection was employed. The average content of the thiols determined in the sperm samples were as follows: Acipenser baerii (cysteine 55 ± 8 μg ml^?1; GSH 126 ± 19 μg ml^?1; GSSG 49 ± 7 μg ml^?1), Acipenser ruthenus (cysteine 62 ± 9 μg ml^?1; GSH 768 ± 115 μg ml^?1; GSSG 180 ± 16 μg ml^?1), Sander lucioperca (cysteine 251 ± 38 μg ml^?1; GSH 185 ± 28 μg ml^?1; GSSG 93 ± 14 μg ml^?1), Perca fluviatilis (cysteine 281 ± 42 μg ml^?1; GSH 496 ± 74 μg ml^?1; GSSG 138 ± 21 μg ml^?1). Based on the results obtained it can be concluded that this method is sensitive and selective for the determination of these compounds in real samples. Results revealed differences in cysteine content between species of the two systematic categories but also showed that ratios between GSH and GSSG can vary between species while potentially predict oxidative stress in fish sperm.     

Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico

Aims: Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. Methods and Results: Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed‐field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 μg ml^?1) and trimethoprim (MIC of >32 μg ml^?1); few were resistant to clindamycin (MIC₉₀ 4 μg ml^?1), erythromycin (MIC₉₀ 8 μg ml^?1) and tetracycline (MIC₉₀ 8 μg ml^?1); but none was resistant to azithromycin (MIC₉₀ 4 μg ml^?1), ciprofloxacin (MIC₉₀ 1 μg ml^?1) or gentamycin (MIC₉₀ 4 μg ml^?1). Most strains restricted with SmaI, but a combination of SmaI–KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST‐2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco‐2 cells, medium cytotoxicity with INT‐407 and Hep‐2 cells and high cytotoxicity with CHO cells. Conclusion: A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. Significance and Impact of the Study: This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.     

Feed intake and tissue distribution of florfenicol in cod (Gadus morhua) administered feed with different fat contents

This study was performed to examine the appetite and the corresponding plasma and tissue distribution of florfenicol when administered to healthy groups of cod using medicated and non‐medicated salmonid and marine feeds. Marine feed contains approximately 18% fat whereas salmonid feed contains approx. 30% fat. Two groups of fish were medicated with florfenicol at a dosage of 10 mg kg^?1 day^?1 for 10 consecutive days when the drug was administered either via marine or salmonid pellets. Two groups of fish also received either non‐medicated marine or salmonid pellets. Twenty‐four hours after giving the medicated marine feed, 14 out of 20 fish contained detectable concentrations of florfenicol with mean values (n = 14) of 4.67 ± 4.02 μg ml^?1 in plasma, 2.29 ± 2.11 μg g^?1 in muscle and 0.79 ± 0.69 μg g^?1 in the liver. In the fish given medicated salmonid feed, 18 of 20 fish contained detectable concentrations of florfenicol with mean values (n = 18) of 1.77 ± 1.84 μg ml^?1 in plasma, 0.75 ± 0.66 μg g^?1 in muscle and 0.30 ± 0.25 μg g^?1 in the liver. Decreased feed intake of the salmonid feed, both medicated and non‐medicated, was noted when compared to medicated and non‐medicated marine feed. No difference in feed consumption was registered between medicated and non‐medicated marine feed, however a difference was noted between the medicated and non‐medicated salmonid feed.     

Inhibition by amino acids of cytokinin-stimulated betacyanin synthesis inAmaranthus caudatus

A number of amino acids have been tested for their ability to inhibit the cytokinininduced synthesis of betacyanin inAmaranthus caudatus cotyledons. Under the conditions employed there was not any serious inhibition of pigment synthesis at amino acid concentrations belowca. 20 μg ml^-1. Amino acids such as methionine, γ-aminobutyric acid and leucine did not give rise to serious inhibition belowca. 200 μg ml^-1. At amino acid concentrations ofca. 2000 μg ml^-1, inhibitions of pigment synthesis was in all instances complete.     

Filtration and ingestion rates of Salpa fusiformis Cuvier (Tunicata : Thaliacea): Effects of size,individual weight and algal concentration

Filtration and ingestion rates of Salpa fusiformis Cuvier were determined while fed Phaeodactylumtricomutum Bohlin at concentrations of 2?64 × 10³ cells·ml^?1. Filtration and ingestion rates increase exponentially with increasing length and body protein. The relations between protein content and body length, and between filtration rate and weight are similar for blastozooids and oozooids. A capture efficiency of the order of 6–32% is calculated: Salpa fusiformis seems to have a low retention efficiency, but its very high filtration rate gives it pride of place amongst filter-feeders. Specific filtration rates are independent of weight; specific ingestion rates are independent of weight for blastozooids, but for oozooids they seem to diminish with increasing weight. The mean daily ration (μg C ingested · μg body C^?1) is 107% for a blastozooid and 117% for an oozooid. Specific filtration rates decrease exponentially as particle concentration rises, as for many other filter-feeders, and the specific ingestion rate follows an Ivlev relation.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina and Pseudoalteromonas haloplanktis

Previous studies have demonstrated that macroalgae from Brittany (France) contain products with antifouling activity against marine bacteria, fungi, diatoms, seaweeds and mussels. Little is known regarding the ecological function of these compounds and insufficient attention has been paid to evaluating the possible temporal variation in antifouling activity. Studies of chemical defenses in both terrestrial and marine organisms suggest that organisms vary widely in the production of chemical defenses associated with physical (temperature, light) and biological (e.g. grazing pressure) factors, season and geographical location. The present study aimed to investigate the antifouling activity of crude extracts of monthly collections of the brown alga, Bifurcaria bifurcata, against two marine bacteria, Cobetia marina and Pseudoalteromonas haloplanktis, and cypris larvae of the barnacle, Balanus amphitrite. The toxicity of the extracts was determined with a B. amphitrite nauplius assay.The antimicrobial activity of the extracts was found to be subject to seasonal variation, with the highest level of activity recorded from samples collected between April and September. Results of the anti-settlement experiments showed that the extracts of B. bifurcata (when tested from 0 to 100 μg/ml) can be divided into three groups on the basis of their minimum inhibitory concentrations (MICs): (1) extracts from plants collected from September to March reduced settlement at nontoxic concentrations (50-100 μg/ml); (2) extracts from plants collected from April to July (which were the most active extracts) reduced settlement significantly when tested at >5 μg/ml, but were toxic at 100 μg/ml; (3) the extract prepared from plants harvested in August was inhibitory at >25 μg/ml, but was toxic at 100 μg/ml. Toxicity tests on nauplii showed that LC₅₀ values of samples from the September to March collections were >100 μg/ml, demonstrating that they were nontoxic to nauplii. In contrast, samples obtained from the April to August collections were toxic to nauplii; the most toxic ones being from algae collected in May (LC₅₀=55.6 μg/ml) and in June (LC₅₀=38.3 μg/ml).The antifouling activity of extracts thus reached a peak in summer corresponding to maximal values for water temperature, light intensity and fouling pressure. It remains to be investigated whether this activity has an ecological role in the alga.     

Tritrichomonas foetus: Partly defined cultivation medium for study of the purine and pyrimidine metabolism

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose—the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.     

Durability of rubberwood (Hevea brasiliensis) treated with peppermint oil,eucalyptus oil,and their main components

Anti-fungal activities of two essential oils (peppermint oil and eucalyptus oil) and their main components (menthol and eucalyptol, respectively) against molds (Aspergillus niger, Penicillium chrysogenum, and Penicillium sp.) and a white-rot decay fungus (Trametes versicolor) identified from rubberwood surfaces were investigated. The broth dilution method and the agar diffusion technique were employed to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) using the concentration of substances between 100 and 800 μl ml^?1. Inhibitory effects of essential oils and their main components at the MICs against mold growth, fungal decay, and termite attack on rubberwood were further examined by means of the dip treatment method. It was found that MFC values against molds for all treatments examined were about 50–100 μl ml^?1 higher than MIC values. Peppermint oil and menthol exhibited high fungistatic and fungicidal activities, with MICs of 300 μl ml^?1 and 350 μl ml^?1, respectively, against the test molds and the decay fungus. Eucalyptus oil and eucalyptol were also effective against these microbes but at higher concentrations of 600 μl ml^?1 and 500 μl ml^?1, respectively. Only peppermint oil at the MIC was capable of providing a complete protection from mold growth on rubberwood for up to 12 weeks at storage conditions of 25 °C and 100% RH. Both peppermint oil and eucalyptus oil at the MICs showed moderate resistance to fungal decay and high resistance to termite attack.     

Different diatoms induce contrasting effects on the reproductive success of the copepod Temora stylifera

Four diatoms, Thalassiosira rotula (THA), Thalassiosira weissflogii (TWEI), Skeletonema costatum (SKE) and Phaeodactylum tricornutum (PHA), and one dinoflagellate Prorocentrum minimum (PRO, control diet), were tested during the course of 14-day laboratory experiments examining the effects of these algae on the fecundity and egg viability in the copepod Temora stylifera. All algae were provided at the same mean carbon concentrations of 0.98 μg C ml⁻¹ to normalize the effects due to differences in diatom size and nutrient concentrations, at least with regards to carbon, so that variations in egg production and hatching viability were presumably due to other chemical constituents of the cells. Our results show that only PRO supported high reproductive success, with stable egg production rates and egg viability close to 100%. By contrast, all four diatoms negatively impacted egg production and hatching success, with varying degrees of suppression. The two diets SKE and PHA induced dramatic effects on reproductive success; SKE was the worst diet with naupliar production ceasing altogether within only 3 days. With PHA, fecundity never reached zero, but at the end of the experiments, initial egg production rates had dropped to 6.6% of initial rates, and viability had declined to zero within 7 days. The two Thalassiosira species mainly affected egg viability, so that after 14 days of feeding, a decrease of 90% had occurred. However, egg production rates were high and were reduced only by 15-24% towards the end of the experiments. In terms of potential recruitment rates, even though PRO induced high mean naupliar production, a slight decrease was observed during the first 5 days, likely due to the time required for T. stylifera to acclimate to PRO. After day 5, potential recruitment rate was higher, comparable to in situ values. THA and TWEI inhibited potential recruitment up to 90-100%, but these effects were not evident over a short-term period, denoting the importance of introducing the time factor when evaluating diatom effects on copepod production. With the other two diatom diets, SKE and PHA, recruitment was zero after 2 and 6 days, respectively. Experiments testing the effects of unsaturated aldehydes in THA (2-trans-4-trans-decadienal) and SKE (2-trans-4-trans-octadienal and 2-trans-4-trans-heptadienal) on the hatching viability of T. stylifera indicate that diatom-derived aldehydes may differ in terms of biological activity. Of the three molecules tested, decadienal had somewhat stronger effects on hatching success, with total blockage occurring at concentrations of 2.0 μg ml⁻¹; the concentrations to induce total blockage by octadienal and heptadienal were somewhat higher and ≥2.5 μg ml⁻¹. We suggest that the greater biological activity of SKE in terms of cell blockage may thus be due to the presence of antimitotic compounds other than these unsaturated aldehydes.     

Seasonal nitrate physiology of Macrocystis integrifolia Bory

Ambient sea-water nitrate and tissue nitrogen (ethanol soluble nitrate and amino acids, as well as total nitrogen) of Macrocystis integrifolia Bory were monitored over a 2-yr period in Bamfield, Vancouver Island, British Columbia. Sea-water nitrate varied from a high of 12 μmol · 1^?1 (individual values as high as 23 μmol · 1^?1 were recorded) in late winter to below detection limits for most of the summer. Tissue nitrate and total nitrogen paralleled the ambient nitrate levels and showed summer minima and winter maxima (from 0 to 70 μmol · g fresh wt^?1 for nitrate and from 0.8 to 2.9% of dry wt for total N). The nitrate uptake capacity was inversely proportional to tissue nitrate concentration and, furthermore, was much higher for subapical surface blades (60–70 nmol · cm^?2 · h^?1) than for older, deeper blades (5–10 nmol · cm^?2 · h^?1). Nitrate uptake by subapical blade disks in summer is apparently higher in dark (1.0–1.7 μmol · g fresh wt^?1 · h^?1) than in light (0.6–1.3 μmol · g fresh wt^?1 · h^?1) and the data obtained in 36–108 h experiments indicate nitrate pool sizes of 30–90 μmol · g fresh wt^?1. These pools are $\text{2}\text{3}$ to nearly full in winter. Ammonium does not inhibit nitrate uptake. It is taken up and apparently utilized much faster than nitrate and it may well be an important source of nitrogen for marine macrophytes.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Use of Hantzsch reaction for quantitation of milnacipran as a magic treatment for fibromyalgia syndrome in human plasma and urine

A new fluorimetric procedure is described for analysis of milnacipran in its bulk, tablet dosage forms, as well as in biological human samples such as plasma and urine. The suggested method relies on the construction of a derivative with strong fluorescence called dihydropyridine derivative. This derivative resulted from the interaction of the primary amino group in the studied drug and acetylacetone/formaldehyde in McIlvaine buffer (pH 5). The fluorescent dihydropyridine derivative was measured at 470 nm. Influences of experimental variables namely pH, reagent concentration and temperature were examined and optimized. The calibration curve showed linearity over the range of 0.15–1.25 μg ml^?1 of milnacipran with an R² value of 0.9998. The detection limit was 0.02 μg ml^?1 and the determination limit was 0.07 μg ml^?1. The developed procedure was successfully used in the assay of the studied drug in Avermilan® tablets with excellent selectivity. In addition, the reaction was applied to estimate the drug in spiked human plasma and urine with mean percentage recoveries of 100.04 ± 1.61 and 99.78 ± 0.81% for urine and plasma, respectively.