Germination of fruit of common lambsquarters (Chenopodium album L.) from three out of four sources was enhanced by 9 to 41% by removal of the fruit coat, and by 21% by washing the fruit for 70 h. Brown-black seed germinated more rapidly than the black and brown seed after washing for 70 h. The washing treatment appeared to remove some unknown inhibitors. After washing, seed germination was found to be positively correlated with seed size.

The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus–oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein kinase (H-89; 10−9, 10−11 or 10−15 M concentration) and a specific adenylate cyclase inhibitor, 2′,3′-dideoxyadenosine (DDA; 10−8, 10−10 or 10−14 M concentration) were used to observe whether they could block the eGH effect. After 30 h of in vitro maturation at 38.5°C with 5% CO2 in air, oocytes were stained with 10 μg/ml of Hoechst to evaluate nuclear status. More mature oocytes (P < 0.05) were detected when COCs were incubated with eGH (29 of 84; 34.5%) than in the control group (18 of 82; 21.9%). The H-89 inhibitor used at a concentration of 10−9 M (4 of 29; 13.8%) decreased (P < 0.05) the number of oocytes reaching nuclear maturation when compared with eGH (11 of 29; 38%). The DDA inhibitor at a concentration of 10−8 M (2 of 27; 7.4%) also reduced (P < 0.05) the number of oocytes reaching maturity when compared with the eGH group (9 of 30; 30%). Results from the present study show that H-89 and DDA can be used in vitro to block the eGH effect on equine oocyte maturation.

The aim of the study was to evaluate the interference of acid and alkaline detergents employed in the cleaning of milking equipment of caprine dairy farms on the performance of microbial tests used in antibiotic control (BRT MRL, Delvotest MCS, and Eclipse 100). Eight concentrations of commercial detergents, five acid (0–0·25%) and five alkaline (0–1%) were add to antimicrobial-free goat's milk to evaluate the detergent effect on the response of microbial inhibitor tests. To evaluate the effect of detergents on the detection capability of microbial tests two detergents at 0·5 ml/l (one acid and one basic) and eight concentrations of four β-lactam antibiotics (ampicillin, amoxicillin, cloxacillin and benzylpenicillin) were used. Milk without detergents was used as control. The spiked samples were analysed twelve times by three microbial tests. The results showed that the presence of acid detergents did not affect the response of microbial tests for any of the concentrations tested. However, at concentrations equal to or greater than 2 ml/l alkaline detergents positive results were found in microbial tests (16·7–100%). The detection limits of the screening tests for penicillins were not modified substantially by the presence of detergents. In general, the presence of acid and alkaline detergents in goat's milk did not produce a great interference in the microbial tests, only high concentrations of detergents could cause non-compliant results, but these concentrations are difficult to find in practice if proper cleaning procedures are applied in goat dairy farms.

Glucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0·25 and 0·014 mm respectively. The rBmG6PD exhibited an optimum pH of 8·5 and temperature, 40 °C. Adenosine 5′ [γ-thio] triphosphate (ATP-γ-S), adenosine 5′ [β,γ-imido] triphosphate (ATP-β,γ-NH), adenosine 5′ [β-thio] diphosphate (ADP-β-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0·02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% β-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

Glycogen synthase kinase-3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. It has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. This enzyme has already been described in Rhipicephalus (Boophilus) microplus and the ovaries of females appeared to be the major site of GSK-3 transcription. The treatment with GSK-3 specific inhibitor (alsterpaullone, bromo-indirubin-oxime 6 and indirubin-3-oxime) caused a reduction in oviposition and egg hatching in completely engorged female ticks. The effect was more pronounced in partially engorged females when alsterpaullone was administrated by artificial capillary feeding. Moreover, GSK-3 gene silencing by RNAi in partially engorged females reduced significantly both oviposition and hatching. The study of tick embryogenesis and proteins that participate in this process has been suggested as an important means for the development of novel strategies for parasite control. GSK-3 is an essential protein involved in embryonic processes and for this reason it has already been suggested as a possible antigen candidate for tick control.

14C-glucose uptake by adult Trichuris globulosa is found to be a non-linear function of time and limiting substrate concentration. The uptake is a two component process, an initial rapid burst is followed by a lower steady state, implying a mediated process. The uptake is dependent on Na+ ions which cannot be replaced by K+, Li+ or choline. The uptake is also dependent on pH, being maximal at pH 7·4. 14C-glucose absorption is markedly inhibited by glucose, phlorizin, ouabain and to a smaller extent by a number of monosaccharides and other sugar-phosphates, nucleosides and metabolic inhibitors like p-nitrophenyl phosphate and iodoacetate. The inhibition constant for glucose, phlorizin and ouabain has been found to be 8 mM, 5 mM and 7 mM, respectively. A modified Dixon-plot shows that glucose is a completely competitive inhibitor for 14C-glucose uptake while the nature of competitive inhibition of phlorizin and ouabain are found to be partial.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0·27 μm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0·74 and 0·22 mm, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

Prolonged imbibition and incubation of lettuce seeds at a supraoptimal temperature induces secondary dormancy. Such seeds no longer germinate when returned to conditions optimal for germination of non-dormant seeds. The possibility that inhibitors are involved in the induction of thermodormancy was investigated.

Washing of thermodormant seeds restored germination to a small extent. However, continuous leaching of seeds during high-temperature treatment with distilled water, largely prevented the induction of thermodormancy. Such seeds were qualitatively similar to nondormant seeds, i.e. they required only light to germinate at a lower temperature and germinated in the dark if given GA3. The germination water from thermodormant seeds inhibited the germination of non-dormant lettuce seeds. The inhibition was not an osmotic effect. Absorbance in the ultraviolet region was higher in germination water from thermodormant seeds than that of non-dormant seeds. It is concluded that inhibitors are involved in the induction of thermodormancy of lettuce seeds.

Indigenous L-lactate dehydrogenase (LDH) in milk originates mainly from somatic cells, leucocytes and invading microorganisms. Its activity may be used for detection of mastitis. However, existing methods to measure LDH activity in milk both need pretreatment of the samples and still suffer from methodological problems. The present paper describes a fast, reliable method for determination of LDH activity, suitable for milk samples. The method is based on fluorometric determination of enzyme kinetics when L-lactate is converted to pyruvate. The assay uses raw milk without pretreatment and the method is easily adjustable to large-scale analyses on micro assay plates. Detection is based on (straight line) linear response within 4–7 min of initiation of the reaction. A substrate concentration of 35 mM in the reaction mixture was considered to be optimal for the assay. Intra plate assay precision was approx. 6% (CV) and the inter plate precision approx. 10%. Known inhibitors of LDH activity (oxidative direction), i.e., oxalic acid, oxamate, and pyruvate, were tested in different concentrations in order to verify the specificity of the response. The detailed kinetics of samples analysed indicated that the isoenzyme composition may have differed between milk samples, and that this composition may have been altered in high activity samples.

Considerable work still needs to be done to understand more fully the basic processes going on inside the non-photosynthetic plastid organelle of Plasmodium spp., the causative agent of malaria. Following an explosion of genomic and transcriptional information in recent years, research workers are still analysing these data looking for new material relevant to the plastid. Several metabolic and housekeeping functions based on bacterial biochemistry have been elucidated and this has given impetus to finding lead inhibitors based on established anti-microbials. Structural investigations of plastid-associated enzymes identified as potential targets have begun. This review gives a perspective on the research to date and hopes to emphasize that a practical outcome for the clinic should be an important focus of future efforts. Malaria parasites have become resistant to front-line anti-malarials that are widely used and were formerly dependable. This has become a worrying problem in many regions where malaria is endemic. The time lag between hunting for new inhibitors and their application as pharmaceuticals is so long and costly that a steady stream of new ventures has to be undertaken to give a reasonable chance of finding affordable and appropriate anti-malarials for the future. Attempts to find inhibitors of the plastid organelle of the malaria parasite should be intensified in such programmes.

Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibitor complexes was carried out using X-PLOR. The final round of refinement was done using SHELXL-97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H···X.

Haemolymph from the locusts Schistocerca gregaria and Locusta migratoria contains humoral factors such as lectin and phenoloxidase system. In vitro studies demonstrated that phenoloxidase system can be activated by LPS, laminarin, Malamoeba locustae cysts, trypsin and methanol. Haemolymph showed agglutination activity for mammalian erythrocytes and protozoa (M. locustae, Leishmania major and Trypanosoma brucei). A purified lectin was isolated from haemolymph by affinity chromatography. Analysis by SDS-PAGE gave a single band at 80 kD while on native PAGE, it gave a single band at 650 kD. Inhibitors of the two humoral factors were investigated. Lectin activity was inhibited by nitrophenyl a α D-galactopyranoside in both cases and by lectin antiserum. Inhibitors of the phenoloxidase system were found in plasma. Prospects for use of such inhibitors are discussed. They could be incorporated in biopesticide formulations in such a way that they could depress locust immune reaction and stimulate biocide activity.

The interaction between the race 1 of the cowpea rust fungus (Uromyces vignae Barclay) and the resistant cowpea (Vigna unguiculata (L.) Walp.) cv. Queen Anne is characterized by the deposition of callose around intracellular fungal structures. Ultrastructural examination of the early stages of infection by basidiospores of the fungus revealed two types of deposits induced by intracellular hyphae: a non-callose collar at the penetration site and a callose encasement around the invasion hypha. The callose encasement was developed from the site where the fungus encountered the inside of the plant cell wall and was separated from the fungus by the extrahyphal membrane and an extension of plant plasma membrane. The incidence of encasements was reduced in plants treated with inhibitors of transcription (actinomycin D), protein synthesis (cycloheximide), protein glycosylation (tunicamycin) and microfilament polymerization (cytochalasin E). Inhibitors of Golgi-associated vesicle transfer (monensin, brefeldin A) and anti-microtubule agents (colchicine, oryzalin) had no effect. When the fungus was killed by heat treatment in either the resistant or the susceptible cultivar, callose was deposited at various locations along the fungus, mostly in the extrahyphal matrix. The data suggest that unlike the extrahaustorial membrane surrounding D-haustoria of this fungus, the extrahyphal membrane is capable of generating callose. Since this process does not normally occur in the susceptible or the resistant cv. even when callose is deposited in the latter by regions of the plasma membrane not associated with the fungus, we conclude that the deposition of callose by the extrahyphal membrane is inhibited by the living fungus.

Some properties of the gut amylase of Sophrorhinus insperatus Faust have been studied. The enzyme has maximum activity at pH 5.0 and 45°C. It is activated by C−, Ca2+ and Cd2+ tons and inhibited by Cu2+ and Hg2+ lons. Low Michaelis constants of 0.26 mg/ml and 032 mg/ml were obtained for the larva and adult gut amylases. Enzymatic activity was found to be consistently higher in the larva than in adult stage, probably suggesting the larval stage as the most destructive stage. Extracts of Cola nitida (Vent) Schott and Endlicher and C. acuminata (Pal de Beauv) Schott and Endlicher, inhibited amylase activity in vitro at varying magnitudes. The higher level of inhibitors in C. acuminata is an index of its low susceptibility to weevil attack both on the field and at storage.