Eimeria Spp.: Influence of coccidia on digestion (amylolytic activity) in broiler chickens

Loss in pancreatic weight and an overall decrease in amylolytic activity in the pancreas were seen in broiler chickens infected with Eimeria acervulina, E. maxima, and E. necatrix. Only E. acervulina and E. maxima reduced amylolytic activity in the surface mucosa of the regions they infected. Surface-bound amylolytic activity decreased as the severity of infection (as measured by lesion score) with E. acervulina increased. This decrease in activity was not related to the decreased feed consumption in infected birds. Little decrease in amylase activity was found in the lumenal contents with the exception of E. acervulina in the jejunum. When the effect of pH on enzyme activity was studied in vitro, a marked reduction in amylolytic activity was observed as the pH went below 5.0.     

New trifluoromethyl quinolone derivatives: Synthesis and investigation of antimicrobial properties

A series of quinolone derivatives, containing different heterocyclic amines were prepared. Synthesized compounds were evaluated for their in vitro antimicrobial activities against two Gram-positive bacteria, three Gram-negative bacteria as well as four fungi. All the derivatives showed good activity towards Gram-positive bacteria and less activity towards Gram-negative bacteria. They also showed moderate to comparable activity against Aspergillus niger and Candida albicans and low to moderate antifungal activity against Aspergillus fumigatus and Aspergillus flavus.     

Relationship of Phenylalanine Ammonia-lyase Activity to o-Hydroxycinnamic Acid Content in Melilotus alba

Phenylalanine ammonia-lyase activity was investigated in preparations representing various parts of sweetclover (Melilotus alba Desr.) plants of CuCu and cucu genotypes. In contrast to other plant parts, very young leaves and stems of CuCu plants displayed high phenylalanine ammonia-lyase activity. Initial leaf samples from CuCu plants were approximately 3 times as high in enzyme activity as leaves from cucu plants, but stems were only slightly higher in activity. Defoliation of the plants resulted in decreased enzyme activity, increased o-hydroxycinnamic acid content, and essentially no difference in enzyme activity between the genotypes. It appears that phenylalanine ammonia-lyase activity in leaves is not primarily controlled by the Cu/cu alleles and that the reaction catalyzed by this enzyme is not the limiting step in o-hydroxycinnamic acid synthesis.     

Nippostrongylus brasiliensis: Infection decreases plasma butyrylcholinesterase activity in rats

Plasma butyrylcholinesterase (BChE) hydrolyzes ester-containing compounds such as succinylcholine, as well as acting as a scavenger against neurotoxic organophosphates (OPs). We previously found that Nippostrongylus brasiliensis infection makes rats more susceptible to OP toxicity by decreasing serum paraoxonase-1 (PON1) activity. In the present study, we examined the effects of N.brasiliensis infection on acetylcholinesterase (AChE) activity in plasma, red blood cells (RBCs), brain and diaphragm, as well as serum PON1 activity, in rats at day 7 after infection. N.brasiliensis infection significantly decreased plasma BChE and PON1 activities without significantly altering AChE activity in RBCs, brain and diaphragm. These results provide further insight into the unusual deleterious effects of intestinal nematode infections on body homeostasis.     

Chloramphenicol acetyl transferase activity in Brassica spp.

Upon discovery that Brassica campestris leaf extracts harbour some chloramphenicol acetyl transferase (CAT) activity, a systematic screening of plant tissue for this activity, so far only reported for prokaryotic microorganism, has been conducted. Results were negative for three solanaceous plants as well as for the Cruciferae Arabidopsis thaliana and Orychophragmus violaceus. By contrast, the three tested species of the Cruciferae genus Brassica exhibit significant CAT activity. The Brassica CAT activity is much more heat labile than the enzyme encoded by the bacterial transposon, Tn9, that is commonly used as a reporter in gene fusion experiments.     

Expression of Maternally and Embryonically Derived Hypoxanthine Phosphoribosyl Transferase (Hprt) Activity in Mouse Eggs and Early Embryos

X-chromosome activity in early mouse development has been studied by a gene dosage method that involves measuring the activity level of the X-linked enzyme hypoxanthine phosphoribosyl transferase (HPRT) in single eggs and embryos from XO females and from females heterozygous for In(X)1H, a paracentric inversion of the X chromosome. The HPRT activity in oocytes increased threefold over a 24-hr period beginning after ovulation. Afterward, the activity plateaued in unfertilized eggs but continued to increase for at least 66 hr in presumed OY embryos. Both before and after ovulation, the level of activity in unfertilized eggs from In(X)/X females was twice that from XO females, and the distributions of activity in eggs for both sets of females remained unimodal. Beginning with the two-cell stage, distributions of activity for embryos from In(X)/X females were trimodal, which is evidence for embryonic activity. It is proposed that activation of a maternal mRNA or proenzyme is responsible for the HPRT activity increase in oocytes and early embryos and is supplemented by dosage-dependent activity of the embryonic Hprt gene as early as the two-cell stage.     

Hyaluronidase activity in the salivary glands of tabanid flies

Tabanids are haematophagous insects that act as biological and mechanical vectors of various diseases, including viruses, bacteria and parasites. The saliva of these insects contains strong anticoagulant and vasodilatory activities as well as immunoregulatory peptides. Here we demonstrate pronounced hyaluronidase (hyase) activity in ten tabanid species of the genera Chrysops, Haematopota, Hybomitra and Tabanus. Compared to other haematophagous insects, the ability of tabanid hyases to hydrolyze hyaluronic acid (HA) is extremely high, for example the enzyme activity of Hybomitra muehlfeldi was found to be 32-fold higher than the salivary hyase activity of the sand fly Phlebotomus papatasi. Hyases of all ten tabanid species tested also cleaved chondroitin sulfate A, another glycosaminoglycan present in the extracellular matrix of vertebrates. The pH optimum of the enzyme activity was measured in eight tabanid species; the hyase of Haemopota pluvialis was the only one with optimum at pH 4.0, while in the other seven species the activity optimum was at 5.0. SDS PAGE zymography showed the monomeric character of the enzymes in all tabanid species tested. Under non-reducing conditions the activities were visible as single bands with estimated MW between 35 and 52 kDa. The very high hyaluronidase activity in tabanid saliva might be related to their aggressive biting behavior as well as to their high efficiency as mechanical vectors. As they are supposedly involved in the enlargement of feeding hematomas, hyases might contribute to the mechanical transmission of pathogens. Pathogens present in vector mouthparts are co-inoculated into the vertebrate host together with saliva and may benefit from increased tissue permeability and the immunomodulatory activity of the salivary hyase.     

Enantiomeric trans β-aryl-δ-iodo-γ-lactones derived from 2,5-dimethylbenzaldehyde induce apoptosis in canine lymphoma cell lines by downregulation of anti-apoptotic Bcl-2 family members Bcl-xL and Bcl-2

For many years, studies focused on developing new natural or synthetic compounds with antineoplastic activity have attracted the attention of researchers. An interesting group of such compounds seem to be those with both lactone moiety and an aromatic ring which, in addition to antimicrobial or antiviral activity, also exhibit antitumor properties. The study shows antitumor activity of two enantiomeric trans isomers of 5-(1-iodoethyl)-4-(2′,5′-dimethylphenyl)dihydrofuran-2-one. Our aim was to determine their antitumor activity manifested as an ability to induce apoptosis in selected canine cancer cell lines as well as to evaluate differences in their strength depending on the configuration of their stereogenic centers. The enantiomers (+)-(4R,5S,6R)-1 and (?)-(4S,5R,6S)-2 were found to induce classical caspase-dependent apoptosis through downregulation of the expression of anti-apoptotic proteins Bcl-xL and Bcl-2. Although the mechanism of apoptosis induction was the same for both enantiomers, they differed in their strength, as stronger antineoplastic activity in vitro was exhibited by isomer (+)-(4R,5S,6R)-1.     

Comparison of the feeding deterrent activity of some sesquiterpene lactones and a lignan lactone towards selected insect storage pests

Four sesquiterpene lactones isolated from Eurpatorium cannabinum, Homogyne alpina and Petasites albus (all Compositae), together with two of their adducts, as well as one lignan lactone of dibenzylbutanolide type from Libocedrus yateensis (Cupressaceae), were tested for their feeding deterrent activity against the adults of Sitophilus granarius and Tribolium confusum, as well as against the larvae of Tribolium confusum and Trogoderma granarium. The strongest deterrent activity against all species was exhibited by the lignan lactone yatein and the sesquiterpene spirolactone bakkenolide A. Both may be included to the class of very good insect feeding deterrents. The comparison presented here showed that the conjugated α-exomethylene moiety of the lactone ring was not the decisive factor for the deterrent activity in the investigated test model.     

Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a K_m of 5 to 10 millimolar, stimulation being due to the anion, Cl⁻, and not the cation, K⁺, and was not inhibited by vanadate, but was inhibited by NO₃⁻. The activity is tentatively identified as the tonoplast ATPase.     

Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment

An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.     

Identification of Bacillus thuringiensis Delta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in Insect Specificity

Cry1C domain III amino acid residues involved in specificity for beet armyworm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against S. exigua but resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved in S. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.     

Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker

Previously, we isolated and characterized attacin from Spodoptera exigua and a coleoptericin-like protein from Protaetia brevitarsis seulensis. In this study, we fused these two genes encoding antimicrobial proteins to obtain a hybrid protein with enhanced antimicrobial activity. To fuse the two antimicrobial proteins, we employed helical and non-helical linker sequences that function as inter-domain linkers in proteins. We used the Gly–Gly–Gly–Gly–Ser peptide as a non-helical linker. The hybrid protein produced using this linker showed less antimicrobial activity against Escherichia coli, Bacillus subtilis, Burkholderia glumae, Pseudomonas corrugate, and Erwinia rhapontici than either of the two parental antimicrobial proteins. In addition, the MIC value of the hybrid protein was 23.1 μM, which indicates poor activity against E. coli. When we used three Glu–Ala–Ala–Ala–Lys (EAAAK) peptide sequences as a helical linker to fuse the two proteins, the resultant hybrid protein had much higher antimicrobial activity than the parental antimicrobial proteins. In particular, this hybrid protein had strong antimicrobial activity against P. corrugate. These results indicate that the EAAAK motif can be used to effectively separate two antimicrobial proteins and produce a hybrid protein with more antimicrobial activity than either of the parent proteins.     

Specificities and pH profiles of adenine and hypoxanthine–guanine–xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we suggest that the gene should be renamed gpT. Both enzymes exhibited unusual profiles of activity versus pH. The adenine PRTase showed the highest activity at pH 7.5–8.5, but had a distinct peak of activity also at pH 4.5. The 6-oxo PRTase showed maximal activity with hypoxanthine and guanine around pH 4.5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase.     

Phenolic accumulation and peroxidase activity inin vitro selected alfalfa callus cultures resistant to filtrate ofFusarium spp.

Changes in the phenylalanine ammonia-lyase (PAL) activity, accumulation of phenolic acids and ionically-bound peroxidase activity in thein vitro selected embryogenic and nonembryogenicMedicago sativa callus cultures resistant to the filtrate ofFusarium spp. were found. The PAL activity in bothin vitro selected cultures during a 4-week cultivation on a medium with phytotoxins was higher than in the control calli grown on a medium without toxin. The filtrate fromFusarium spp. evoked an increase in the contents of all determined phenolic acids in the selected calli. They occurred predominantly bound as esters. The most pronounced portions in the elevated acids level were of ester-bound p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids. The ionic cell wall-bound peroxidase activity in both selected calli cultivated on a medium with a filtrate was twice as high as the activity determined in the control cultures. The activity of soluble peroxidase was not influenced by challenge with a filtrate. No significant differences were found between thein vitro selected embryogenic and nonembryogenic alfalfa callus cultures in the response to the phytotoxic filtrate.     

Evaluation of Nostoc Strain ATCC 53789 as a Potential Source of Natural Pesticides

The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.     

Comparison of cytokinin activities of 9-substituted N6-benzyladenines in the Cucumis and Amaranthus bioassays

9-Substituted N⁶-benzyladenines were tested for their ability to eliminate the lag phase in and promote chlorophyll synthesis in Cucumis sativus cotyledons and for their effectiveness in eliciting the dark biosynthesis of betacyanin in Amaranthus tricolor cotyledon-hypocotyl explants. The following general relationships were established for dose-responses: (a) 9-ribosidation brought about little (in Amaranthus) or no (in Cucumis) decrease in activity relative to the free base, (b) the presence of a 9-ribose 5′-phosphate group moderately depressed activity in Amaranthus but slightly enhanced activity in Cucumis, (c) the presence of a 9-ribose 3′,5′- cyclic phosphate group depressed activity substantially in both systems, more so in Amaranthus, (d) 9-glucosylation greatly decreased activity, as did 7-glucosylation, while 3-glucosylation depressed activity to a much lesser extent, in both systems, (e) 9-substitution with cyclopentyl, methyl, methoxymethyl, and tetrahydropyranyl groups reduced activity, the first two substituents more so than the last two, and (f) alteration of the 9-riboside group to a 9-[2-O-β-hydroxyethylglycerol] moiety by oxidation- reduction led to complete (in Amaranthus) or nearly complete (in Cucumis) inactivation. Responses to hormone treatment were detectable after dark incubation times as short as 4 hr (in Cucumis) or 8 hr (in Amaranthus).     

Biochemical properties and level of expression of alcohol dehydrogenases in the allotetraploid plant Tragopogon miscellus and its diploid progenitors

This study demonstrates that homoeologous genes in two diploid plant species that specify different amounts of an enzyme maintain the same relative level of expression in an allotetraploid derivative. The three predominant alcohol dehydrogenase (ADH) isozymes (DD, DP, PP) in seeds of the recently evolved allotetraploid plant Tragopogon miscellus (Compositae) are dimers specified by Adh3-D and Adh3-P genes derived from its diploid progenitors T. dubius and T. pratensis. Seeds of T. pratensis contain twice as much ADH activity as those of T. dubius, while T. miscellus is intermediate. The three isozymes were similar in a number of catalytic properties; the densitometric ratio of the isozymes purified from T. miscellus was 1 DD:4DP:4PP for both ADH activity and protein; and dissociation-reassociation of the DP enzyme gave a 1:2:1 ratio of the three isozymes. Therefore, the enzymes were similar in specific activity, but twice as many P as D subunits were present in active enzymes in T. miscellus, precisely the difference in activity between the parents. In T. miscellus, the specific activity of ADH and its activity per mg tissue are intermediate to those of the diploids, because relative expression of the Adh gene in each genome is not influenced by the presence of the other genome.     

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.