Bioactivity of two major constituents isolated from the essential oil of Artemisia judaica L

The essential oil of Artemisia judaica L., grown on Sinai Peninsula of Egypt, was extracted via hydrodistillation. Chromatographic separation on repeated silica gel columns led to isolate two compounds namely piperitone and trans-ethyl cinnamate. Insecticidal, antifeedant and antifungal properties of the isolated compounds were examined. Both compounds showed pronounced insecticidal and antifeedant activity against the third instar larvae of Spodoptera littoralis (Boisd). trans-Ethyl cinnamate (LD₅₀ = 0.37 μg/larva) was more toxic than piperitone (LD₅₀ = 0.68 μg/larva). The two isolated compounds revealed antifeedant activity in a concentration dependent manner, with complete feeding inhibition at a concentration of 1000 μg/ml. When tested for antifungal activity against four plant pathogenic fungi, the isolated compounds exhibited a moderate to high activity.     

BIOACTIVITIES AND MECHANISM OF SPIRO ENOL ETHER ANALOGUES AGAINST PIERIS RAPAE

Abstract  Nineteen kinds of spiro enol ether analogues were screened with larvae of Pieris rapae for antifeedant activity. The results showed that the antifeedant activity of compounds No.20 and No. 12 was higher than others. In non-choice test, AFC50 values within 24 h of compounds No.20 and No. 12 against 3rd instar larvae of P. rapae were 226.93 μg/mL and 370.00 μg/mL, and that in choice test against 4th larvae were 280.54 μg/mL and 398.88 μg/mL, respectively. Compd. No.20 could prolong the eggs hatch time and reduce the haemolymph content and the protein content in haemolymph of 4th instar larvae obviously. Compd. No.20 could protect tested leaves and control larvae of P. rapae effectively.     

Inhibitory effect of methyl jasmonate and its related compounds on kinetin-induced retardation of oat leaf senescence

The senescence-promoting activities of methyl jasmonate and its related compounds were compared with respect to structure-activity relationships. The activities were assayed by using oat ( A vena saliva L. cv. Victor) leaf segments in the presence of 2 μg/ml kinetin. Dextrorotatory methyl jasmonate prepared from an authentic sample of the racemate mixture was less active than the naturally occurring levorotatory form especially at its low concentrations (0.1 to 2.5 μg/ml). The activity of jasmonic acid, the free acid form of methyl jasmonate, was much less than the methyl ester, and this relationship was true for the other compounds tested. The reduction of the unsaturated bond in the substituent at the C-2 position and the keto group at the C-3 position greatly reduced the activity. The length of the n -alkyl substituents at the C-2 position had also a significant effect on the activity. From these results, it is concluded that the important functional groups for the high senescence-promoting activity of the methyl jasmonate related compounds are the methyl acetate substituent at the C-l position, the 2' cis -pentenyl or n -pentyl group at the C-2, position and the keto group at the C-3 position in methyl jasmonate.     

Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris , opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70-90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30-45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.     

Hemagglutinating activity of vibrio cholerae enterotoxin

Abstract The hemagglutinating activity and carbohydrate specificity of cholera toxin (cholera enterotoxin) was studied using hemagglutination and hemagglutination inhibition. Hemagglutination was obtained with cholera toxin at >108 μg/ml for human types A, B, and O erythrocytes, >216 μg/ml for chicken erythrocytes, and >865 μg/ml for sheep erythrocytes. When the erythrocytes were treated with either neuraminidase or pronase, the hemagglutinating activity of cholera toxin was enhanced about 8- to 32-fold. Hemagglutination of pronase-treated human type B erythrocytes induced by cholera toxin was inhibited by lactose, galactose, melibiose and l -arabinose. Lactose was the most effective of the mono-, di-, and polysaccharides used as inhibitors, being a slightly better inhibitor than galactose, and much more potent than melibiose. These results suggest that cholera toxin is a bacterial lectin specific for galactose and/or lactose.     

Khayanolides from African mahogany Khaya senegalensis (Meliaceae): A revision

Five khayanolides (1-O-acetylkhayanolide B 1, khayanolide B 2, khayanolide E 3, 1-O-deacetylkhayanolide E 4, 6-dehydroxylkhayanolide E 5) were isolated from the stem bark of African mahogany Khaya senegalensis (Meliaceae). Their structures and absolute configurations were determined through extensive spectroscopic analyses including MS, NMR, and single-crystal X-ray diffraction experiments. The results established that two previously reported khayanolides, 1α-acetoxy-2β,3α,6,8α,14β-pentahydroxy-[4.2.1^10,30.1^1,4]-tricyclomeliac-7-oate 6 and 1α,2β,3α,6,8α,14β-hexahydroxy-[4.2.1^10,30.1^1,4]-tricyclomeliac-7-oate 7, were, in fact, 1-O-acetylkhayanolide B 1 and khayanolide B 2, and that the two reported phragmalin derivatives, methyl 1α-acetoxy-6,8α,14β,30β-tetrahydroxy-3-oxo-[3.3.1^10,2.1^1,4]-tricyclomeliac-7-oate 8 and methyl 1α,6,8α,14β,30β-pentahydroxy-3-oxo-[3.3.1^10,2.1^1,4]-tricyclomeliac-7-oate 9, were, in fact, khayanolide E 3 and 1-O-deacetylkhayanolide E 4, respectively. Based on the results from this study and consideration of the biogenetic pathway, the methyl 6-hydroxyangolensate in African mahogany K. senegalensis should have a C-6 S configuration while methyl 6-hydroxyangolensate in genuine mahogany Swietenia species should have a C-6 R configuration.     

Phytotoxic Activity of Tenuazonic Acid Isolated from Alternaria alternata (Fr.) Keissler Causing Leaf Blight of Datura innoxia Mill. and its Effect on Host Metabolism

Tenuazonic acid isolated from Alternaria alternata (Fr.) Keissler causing leaf blight of Datura innoxia Mill. showed significant phytotoxic activity when tested on monocot and dicotyledonous plants. The toxin induced chlorosis and necrosis on leaves of D. in noxia, D. stramonium, D. metel , belladonna, cowpea, wheat, rye, cabbage, cauliflower and maize at 200μg/ml and wilting of seedlings of D. innoxia at 100μg/ml concentration. It also caused complete inhibition of root and shoot elongation of germinating seeds of D. innoxia , wheat, rye, green gram and lettuce at 100μg/ml concentration. It was a nonspecific phytoxin and appeared to have significant role during pathogenesis.
Tenuazonic acid did not cause any significant change in the rate of respiration and in sugar, carbohydrate, total phenol and nitrogen contents of D. innoxia leaves. But its treatment on host plant induced 64 % reduction of chlorophyll content in leaves after 72 hours and 40 % reduction in protein content after 24 hours.     

Rings D-seco and B,D-seco tetranortriterpenoids from root bark of Entandrophragma angolense

Investigation of the root bark extract of Entandrophragma angolense led to identification of two gedunin-type limonoids 5-hydroxy-7-deacetoxy-7-oxogedunin and 5,6-dehydro-7-deacetoxy-7-oxogedunin, and three methyl angolensate derivatives, 6-deacetoxydomesticulide D, 6-deacetoxydomesticulide D 21-methylether, and entangosin, together with known compounds, methyl angolensate, 6-acetoxymethyl angolensate and secomahoganin. Their structures were established by extensive NMR experiments in conjunction with mass spectrometry. Entangosin is a rare example of a limonoid derivative having a fully O-substituted furan moiety.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

Identification and Phytotoxicity of 3-methylthiopropanoic and Trans-3-methylthiopropenoic Acids Produced in Culture by Xanthomonas campestris pv. vitians

Two phytotoxic metabolites were isolated from culture filtrates of Xanthomonas campestris pv, vitians , the causal agent of lettuce leaf spots and headrot. The two compounds were identified as 3-methylthiopropanoic (1) and trans-3-methylthiopropenoic (2) acids by chemical and spectroscopic methods. Toxic effects of the two compounds on leaf tissues and protoplasts of lettuce and cabbage were investigated. Solutions of 1 and 2 induced chlorosis and necrosis on lettuce leaves at minimum concentrations of 300 and 50 μg/ml, respectively. Infiltration in cabbage leaves did not produce any symptoms. The LD₅₀ values for 1 and 2 against lettuce protoplasts were 15 and 16 μg/ml, respectively. Activity of the two metabolites against cabbage protoplasts was very low (LD₅₀ > 500 μg/ml).     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

The toxicity of a number of different bactericides to Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] and to the tomato plant, Lycopersicon esculentum

Twenty-seven proprietary products and pure chemicals were tested in vitro against cells of Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] (the cause of bacterial canker of tomato) and also for their phytotoxicity to tomato plants. The most bactericidal of these, with a minimum cidal concentration (MCC) range of > 10-< 100 μg/ml, were a phenolic product called Applied 3–78, two quaternary ammonium compounds (benzalkonium chloride and cetrimide), and a silver colloid compound. Of these, only Applied 3–78 was not phytotoxic at values of 10 μg/ml or less, although it was phytotoxic at 10000 μg/ml. Copper oxychloride and sodium hypochlorite were amongst the group with a middle range of bactericidal properties, their MCC range being from > 1000 to < 10000 μg/ml. They were phytotoxic at 1000 μg/ml or less. When organic matter, a dead yeast suspension, was added to Applied 3–78, Kohrsolin and Panacide, only the activity of Applied 3–78 was relatively unchanged. The MCC ranges were: Applied 3–78, >80–< 100 μg/ml; Kohrsolin, > 800-< 1000 μg/ml; and Panacide, > 1000 μg/ml. Phytotoxicity tests on 10 different tomato cultivars confirmed that Applied 3–78 was the least phytotoxic of these three products. Field trials on tomato crops showed that when Applied 3–78 was sprayed on the plants once, and Kohrsolin was either sprayed on or they were drenched with it once at 1000 μg/ml, no phytotoxicity symptoms developed.     

Regulated interactions of the norepineprhine transporter by the actin and microtubule cytoskeletons

One role of the actin cytoskeleton is to maintain the structural morphology and activity of the pre-synaptic terminal. We sought to determine if the actin cytoskeleton plays a role in regulating interactions between the norepinephrine transporter (NET) and alpha-Synuclein (α-Syn), two proteins expressed in the pre-synaptic terminal. In cells transfected with either 0.5 μg/mL or 3 μg/mL of α-Syn and 1 μg/mL of NET DNA, treatment with cytochalasin D, an actin depolymerizing agent, caused a dose-dependent decrease and increase, respectively, in [³H]-NE uptake. Protein interactions between NET, β-actin, and α-Syn were modified, along with levels of surface transporters. Treatment of primary brainstem neurons and frontal cortex synaptosomes with cytochalasin D caused a 115% and 28% increase, respectively, in NET activity. Depolymerization of both actin and microtubules did not alter NET activity in cells with 0.5 μg/mL α-Syn, but caused an increase in [³H]-NE uptake in cells transfected with 3 μg/mL of α-Syn and primary neurons. This is the first direct demonstration of NET activity being regulated via actin and modulated by interactions with α-Syn.     

Sulfation of Dopamine and Other Biogenic Amines by Human Brain Phenol Sulfotransferase

Abstract: Phenol sulfotransferase was isolated in 100,000g supernatant fractions prepared from postmortem samples of human brain. Since phenol sulfotransferase (PST) has been shown to conjugate the amine neurotransmit-ters in vivo , the abilities of eight different biogenic amines and structurally related compounds to act as substrates for PST were studied. These experiments demonstrate that at a concentration of 20 μM, dopamine (DA) was the best substrate examined and was followed in decreasing order of activity by 3-methoxytyramine (3-MT), tyramine, norepinephrine, 3-methoxy-4-hydroxyphenylethyleneglycol, octopamine, 5-hydroxytryptamine and dihydroxyphenylethyleneglycol. At a substrate concentration of 100 /UM the relative order of activity was altered, so that tyramine became the most rapidly conjugated substrate while the activity of DA and 3-MT relative to the other substrates tested was diminished. This change in substrate affinity with differing substrate concentrations can be explained, at least for DA, by the occurrence of apparent substrate inhibition at concentrations above 25 to 30 μM. Using PST isolated in 100,000g supernatant fractions from human brain, the K_m value for DA was found to be 5.0 μM, while the K_m value for the sulfate-donor 3'-phosphoadenosine-5'-phosphosulfate was 0.25 μM. The ratio of 3- O - to 4- O -DA-sulfate formed in vitro by human brain PST was found to be about 4: 1. In addition, both the 3- O - and 4- O -esters were found not to be deaminated by human brain mitochondrial MAO. The relative role of PST with respect to MAO and catechol- O -methyltransferase in the degradation of the biogenic amine neurotransmitters in human brain is discussed.     

Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

Procedures were examined for labelling enterotoxin isolated from Clostridium perfringens type A. with ¹²⁵I using chloramine T as the oxidizing agent. The iodination method was evaluated critically to establish the optimal conditions for the preparation of iodinated enterotoxin with a high specific radioactivity and without impairing the immunospecificity and biological activity. The use of 250 μg/ml of chloramine T in the reaction mixture. 500–1000 μCi of Na¹²⁵I/10 μg of enterotoxin and a reaction time of 40 s at pH 7–0 produced ¹²⁵I-enterotoxin of both high specific radioactivity and immunospecificity which retained its biological activity. No damage or aggregate formation due to the iodination process was observed. Enterotoxin labelled with high specific activity (135 μCi μg) showed extensive dissociation of ¹²⁵I when stored at 4°C and—20°C. In contrast, toxin labelled with low specific activity (7 μgCi/μg) was stable for as long as two months. The immunoreactivity of all labelled preparations was essentially unchanged after storage for one month.     

Antifeedant activity and toxicity of two alkaloids from Adhatoda vasica Nees leaves against diamondback moth Plutella xylostella (Linn.) (Lepidoptera: Plutellidae) larvae

Two natural alkaloids viz., Vasicine acetate and 2-Acetyl benzylamine, isolated from Adhatoda vasica leaves, showed antifeedant, larvicidal and moult inhibiting properties against diamondback moth Plutella xylostella in laboratory experiments. Maximum antifeedant activity of 98.5% was recorded at 1000 ppm concentration of Vasicine acetate treatment, whereas as 2-Acetyl benzyl amine recorded only 71.4% antifeedant activity at 1000 ppm concentration. Azadirachtin treatment presented 82% antifeedant activity at the highest concentration (1000 ppm). Both the active compounds of A. vasica showed lethal toxicity on larvae and pupae. The highest larvicidal and pupicidal activities were recorded in 2-Acetyl benzylamine treatment at 125 ppm concentration. The two A. vasica compounds also affected the normal growth and development and moulting process of P. xylostella. Final moulting of larvae into pupae was disrupted by the treatments, which resulted in larval–pupal intermediates and abnormal pupae. Treatments also produced small-size pupae and malformed adults with poorly developed wings.     

The reaction of young coho Oncorhynchus kisutch to declining oxygen levels during long-term exposure

The respiration of coho salmon, Oncorhynchus kisutch , weighing between 20 and 45 g was measured at gradually declining oxygen levels and at temperatures ranging between 14 and 17 °C. The maximum and minimum oxygen concentrations tested were 235 and 41 μmol/L, respectively. Respiration rates were measured for 24 h at 235 μmol/L before the oxygen concentration was lowered stepwise to 157 and 81 μmol/L. In one single trial, the oxygen level was lowered to 66, 53, and 41 μmol/L. Respiration was highly variable in time. Peak activities always occurred during the night. The standard metabolic rate at normoxic conditions was estimated to be around 4 μmol oxygen/g/h. The highest rates reached values close to 15 μmol oxygen/g/h. At reduced oxygen levels the standard oxygen demand slightly increased to 4.5 μmol oxygen/g/h, indicating a higher demand for vital metabolic functions. Due to the decrease of swimming activity, the maximum oxygen uptake rates dropped to < 8 μmol oxygen/g/h below 81 μmol/L oxygen concentration. Under long-term conditions, physiological and behavioural adaptations play an important role for survival and need to be considered for the design and operation of fish farm facilities.     

Insecticidal activity influence of destruxins on the pathogenicity of Paecilomyces javanicus against Spodoptera litura

Abstract:  The bioactivities of destruxins (dtx), depsipeptides isolated from Metarhizium anisopliae , against Spodoptera litura were tested in laboratory. For contacting toxicities, dtx-E was more effective than dtx-A and dtx-B. The LC₅₀s values of dtx-A, B and E were 197.98, 292.00 and 113.99 mg/l at 48 h after treatment, while the LT₅₀s were 42.65, 59.45 and 23.68 h at 300 mg/l. In the experiment of antifeedant activity, dtx-A, dtx-B and dtx-E at five concentrations (200, 100, 50, 25 and 12.5 mg/l) were bioassayed. Destruxins in a dose-dependent manner gave an apparent antifeedant activity. Generally, dtx-A, over dtx-B and dtx-E had the significant (P < 0.05) larger choice and no-choice antifeedant indexes (CAIs and NCAIs). At the concentration of 200 mg/l, the CAIs or NCAIs of dtx-A, dtx-B and dtx-E were 96.78, 84.93 and 85.90 or 89.75, 62.42 and 72.28 respectively. Furthermore, the synergistic activity of crude destruxin (CD) for pathogenicity of Paecilomyces javanicus strain Pj01 was detected. The LC₅₀s values of single Pj01 and the mixtures of Pj01 plus CD at 100 or 200 mg/l (Pj01-CD100 or Pj01-CD200) were respectively 474.63 × 10⁵, and 197.45 × 10⁵ or 113.11 × 10⁵ spores/ml at the fifth day after treated. Meanwhile, Pj01, Pj01-CD100 and Pj01-CD200 gave the LT₅₀s values of 6.99 day, 5.49 day and 4.21 day at 100 × 10⁵ spores/ml. Clearly, dtx decreased the values of LC₅₀ and LT₅₀ of the strain Pj01.     

Inhibitory effect of oregano and thyme essential oils on moulds and foodborne bacteria

The essential oil of oregano ('origanum oil'; thymol type oil from Origanum vulgare) inhibited completely the mycelial growth of Aspergillus niger and A. flaous at 400 μg/ml, while A. ochraceus was inhibited at 600 μg/ml. At 700 μg/ml, thyme oil inhibited the mycelial growth of A. flavus and A. niger but not that of A. ochraceus . Fungal spore germination was inhibited by 600 μg/ml of origanum oil and (with the exception of A. ochraceus) by 700 μg/ml of thyme oil. Under aerobic conditions, the essential oils of oregano (250 μg/ml) and thyme (350 μg/ml) inhibited to some extent the growth of Staphylococcus aureus and Salmonella typhimurium. Pseudomonas aeruginosa was not affected by either oregano or thyme oil at concentrations up to 500 μg/ml. The origanum oil was very effective against Campylohacter jejuni and Clostridiurn sporogenes and thyme oil was very effective against C. jejuni. The antagonistic effect of the two oils on Staph. aureus and Salm. typhimuriutn was greatly enhanced when those organisms were incubated in atmospheres of low oxygen tensions     

In vitro antiplasmodial activity of extract and constituents from Esenbeckia febrifuga, a plant traditionally used to treat malaria in the Brazilian Amazon

Esenbeckia febrifuga (Rutaceae) is a plant traditionally used to treat malaria in the Brazilian Amazon region. Ethanol extract of stems displayed a good antiplasmodial activity against Plasmodium falciparum strains W-2 (IC₅₀ 15.5±0.71 μg/ml) and 3 D7 (IC₅₀ 21.0±1.4 μg/ml). Two coumarins (bergaptene 1 and isopimpinellin 2), five alkaloids (flindersiamine 3, kokusaginine 4, skimmiamine 5, γ-fagarine 6 and 1-hydroxy-3-methoxy-N-methylacridone, 7), besides a limonoid (rutaevine 8), have been isolated for the first time from this species. Antiplasmodial activity of compounds 3, 5–8 has been evaluated in vitro against P. falciparum strains (W-2 and 3D7) and the furoquinolines 5 and 6 were the most potent displaying IC₅₀ values <50 μg/ml; flindersiamine (3) showed a weak activity while alkaloid 7 and rutaevine (8) were inactive (IC₅₀>100 μg/ml).