Developmental changes in gene expression and enzyme activities of anabolic and catabolic enzymes for storage carbohydrates in the honeybee, Apis mellifera.

Glycogen and trehalose are important sources of energy in insects. The expression of genes encoding the key metabolic enzymes-glycogen synthase (GS), glycogen phosphorylase (GP), trehalose-6-phosphate synthase (TPS-1), soluble trehalase (Tre-1) and membrane-bound trehalase (Tre-2)-was analyzed in 12 developmental stages of Apis mellifera worker brood. The content of GS and GP proteins, TPS activity, total trehalase activity, and the activity of Tre-1 and Tre-2 were determined. Transcript quantity was not always correlated with the content of the encoded GS or GP protein. The correlation was higher for GS (r = 0.797) than GP (r = 0.651). The expression of the glycogen synthase gene (gs) and the glycogen phosphorylase gene (gp) was high in 4- and 7-day-old larvae and in pupae, excluding the last pupal stage. The expression of the tps-1 gene was highest in the mid-pupal stage and contributed to higher enzyme activity in that stage. The expression of the tre-1 gene was higher than the expression of the tre-2 gene throughout development. In newly hatched workers, the expression of genes encoding catabolic enzymes of both carbohydrates, gp and tre-1, was higher than the expression of genes encoding anabolic enzymes. The results of this study suggest that sugar metabolism genes have somewhat different control mechanisms during larval development and metamorphosis.

Development of biodegradable polyesters with various microstructures for highly controlled release of epirubicin and cyclophosphamide.

In this study, "predominantly isotactic", disyndiotactic, and atactic polylactides (PLAs) and poly(ε-caprolactone)s (PCLs) were loaded with anticancer agents, epirubicin (EPI) and cyclophosphamide (CYCLOPHO), to investigate their properties as highly controlled delivery devices. It was found that the kinetic release of drugs from the obtained polyester matrices tested in vitro at 37°C and pH7.4 was strongly dependent on average molecular weight (Mn) of the polymers as well as the PLAs' microstructure. EPI and CYCLOPHO were released from various obtained matrices according to the diffusion, diffusion-degradation, and degradation mechanisms in a rather regular and continuous manner. Importantly, in some cases, the kinetics of the EPI and CYCLOPHO release was nearly zero-order, suggesting predominantly polymer degradation. It is shown that the drug release profiles can be tailored by a controlled design of the microstructure and Mn of polyesters, allowing use of the synthesized matrices for the development of highly controlled biodegradable anticancer drug delivery systems.

Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages of Anisakis simplex.

Extracts of Anisakis simplex third (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of alpha-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS-PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: alpha 1, alpha 2 and alpha 3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The alpha-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations of A. simplex larvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.

Trehalose catabolism enzymes in L3 and L4 larvae of Anisakis simplex.

The presence of trehalase and trehalose phosphorylase in L3 and L4 larvae of Anisakis simplex was demonstrated. The activity of trehalase and trehalose phosphorylase in L3 larvae was 6 and 10 times higher, respectively, than in L4 larvae. This suggests that trehalose metabolism is more important for L3 than LA larvae. Trehalases of L3 and L4 differ in their characteristics. The enzyme of L3 was present mainly in the lysosomes and cytosol, whereas in L4 the highest enzyme activity was measured in the lysosomal fraction. Trehalase activity was increased by 29% in L3 and 55% in L4 with the addition of Mg2+ (0.1 mmol). Tris inhibited trehalase in L3 larvae by 42% and in L4 by 25%. The enzymes differed in their reaction to EDTA, CaCl2, ZnCl2, and CH2ICOOH (all 0.1 mmol). High activity of trehalase from L3 larvae was measured within the pH range of 5.0 to 6.5, with an optimum pH of 6.1. The trehalase was a thermally tolerant enzyme from 25 C to 60 C. The enzyme lost half of its activity after preincubation without substrate above 75 C. The paper also discusses the similarities and differences in characteristics of trehalase from A. simplex larvae and presents the comparison to enzymes from other nematodes.

[The concentration of trehalose and acitvity of trehalase from Galleria mellonella larvae infected by Steinernema affinis, Bovien 1937 (Nematoda: Rhabditida: Steinernematidae)]. / Zawartosc trehalozy i aktywnosc trehalazy u larw Galleria mellonella zarazonych Steinernema affinis, Bovien 1937 (Nematoda: Rhabditida: Steinernematidae).

The experimental studies were conducted on caterpillars of wax moth Galleria mellonella infected with Steinernema affinis larvae. The concentration of trehalose and the activity of trehalase were measured during the invasion lasting 48h. The level of trehalose and activity of enzyme were slightly lower in infected insects in comparison to the control animals.

[The enzymes of carbohydrates metabolism from Cysttidicola farionis (Cystidicolidae)]. / Enzymy katabolizmu wwglowodanów z Cysttidicola farionis (Cystidicolidae).

The content of glycogen, glucose and trehalose was measured in larvae and adults of Cystidicola farionis, the parasite isolated from the swim bladder of Osmerus eperlanus from Vistula Lagoon. Activity of glycogen phosphorylase, alpha-amylase, glucoamylase, maltase, trehalase, and trehalose phosphorylase were measured. The highest activity was recorded for alpha-amylase 10.07 +/- 0.97 mu/mg and 7.47 +/- 0.24 mu/mg, next maltase 1.34 +/- 0.63 micromol/mg and 2.06 +/- 1.65 micronol/mg respectively for larvae and adults. The activity of glucoamylase was nearly the same for adults and larvae (about 0.20 micromol/mg). The trehalase activity was higher at adults (0.49 +/- 0.42 micromol/mg) than at larvae (0.18 +/- 0.12 micromol/mg). The activity of glycogen phosphorylase was much higher at larvae (3.58 +/- 1.49 micromol/mg) than at adults parasite (0.10 +/- 0.02 micromol/mg). The trehalose phosphorylase was present in both stages of parasite, but its activity was low. The content of glycogen and glucose was two-times higher in the adults' body than in larvae.

[The comparison of properties of alpha-amylase from the third and fourth stage of Anisakis simplex larvae]. / Porównanie wlasnosci alpha-amylazy z larw trzeciego i czwartego stadium Anisakis simplex.

alpha-Amylase is present in the third (L3) and in the fourth-stage (L4) of larvae from Anisakis simplex. The enzymes from both sources differ in some of their properties. alpha-Amylase from L3 showed a maximum at pH 7.8, enzyme from L4 stage at pH 6.5. The alpha-amylase from L3 was mainly lysosomal enzyme. The enzyme from L4 was located in the microsomal fraction. The L3 alpha-amylase showed the inhibition by EDTA and by -SH reagent iodoacetic acid. These agents did not change the activity of L4 enzyme. Both izoenzymes were unaffected by calcium and magnesium ions. Generally the alpha-amylase from L4 stage had higher activity (3.71 u/mg) than L3 one (2.29 u/mg).

Purification and characterization of alpha-amylases from the intestine and muscle of ascaris suum (Nematoda).

Alpha-Amylase (EC 3.2.1.1) was purified from the muscle and intestine of the parasitic helminth of pigs Ascaris suum. The enzymes from the two sources differed in their properties. Isoelectric focusing revealed one form of a-amylase from muscles with pl of 5.0, and two forms of amylase from intestine with pI of 4.7 and 4.5. SDS/PAGE suggested a molecular mass of 83 kDa and 73 kDa for isoenzymes of a-amylases from intestine and 59 kDa for the muscle enzyme. Alpha-Amylase from intestine showed maximum activity at pH 7.4, and the enzyme from muscle at pH 8.2. The muscle enzyme was more thermostabile than the intestinal alpha-amylase. Both the muscle and intestine amylase lost half of its activity after 15 min at 70 degrees C and 50 degrees C, respectively. The Km values were: for muscle amylase 0.22 microg/ml glycogen and 3.33 microg/ml starch, and for intestine amylase 1.77 microg/ml glycogen and 0.48 microg/ml starch. Both amylases were activated by Ca2+ and inhibited by EDTA, iodoacetic acid, p-chloromercuribenzoate and the inhibitor of a-amylase from wheat. No significant differences were found between the properties of a-amylases from parasites and from their hosts.

[Influence of zinc and lead ions on the development of eggs of Ascaris suum (Nematoda)]. / Wplyw jonów olowiu i cynku na rozwój jaj Ascaris suum (Nematoda).

The objective of this work was to examine the influence of heavy metals ions, zinc and lead, in external environment on the development of Ascaris suum to invasive stage. The culture mediums contained 25.5, and 0.5 mM Pb(NO3)2 or ZnSO4. After 6 weeks of development in 26 degrees C in the control 75.17% of eggs reached the invasive stage. Ions of lead and zinc, in a way depending on concentration inhibited development of embryos. The invasive stage in the presence of 25.5 and 0.5 mM Pb(NO3)2 were achieved by 37.89% , 44,75% and 64.63% of eggs and in ZnSO4 solutions with the same concentrations of ions 45.40%, 50.36% and 66.61% respectively. Many of eggs in 25 mM solutions of both metals stopped at the blastula stage.

[Enterobiosis in kindergarden and school children from selected institutions from Olsztyn and surroundings]. / Owsica u dzieci przedszkolnych i szkolnych z wybranych placówek Olsztyna i jego okolicy.

The studies were conducted during the school year of 1996/1997 among the children at the age of 5, 6 and 7 years old from Olsztyn and nearby municipality of Purda. In total, 151 children were examined by Graham's method. The presence of eggs of the parasite was confirmed in 13.3% of the total examined population. The infestation was lower in Olsztyn (12.0%) and the lowest in Purda (9.4%) but in the former state-owned Farm village Prajlowo it reached 27.8%. In general, the pinworms were found more frequently in 7 year old children (23.33%) than in case of the others (8.69% and 7.14% for 5 and 6 year old children respectively). In the study, younger boys were more frequently infested than the girls.

[The development of Ascaris suum eggs in the presence of moxidectin]. / Rozwój jaj Ascaris suum w obecnosci moksydektyny.

The development of Ascaris suum eggs maintained in the culture containing moxidectin (Cydectin, Cyanamid) in concentration 1, 5, 25, and 50 micrograms/ml was studied. The abnormalities and slowing down of the rate of eggs development, depending on the drug concentration, were observed. Exposure of eggs to moxidectin in concentration 1 and 5 microgram/ml resulted in one- or two-week delay of development. In the higher concentration of moxidectin (25 micrograms/ml) only 18% of eggs became invasive. In the presence of 50 micrograms/ml of the drug only few eggs developed to invasive stage after 7 weeks. The development of embryons was stopped more often in the blastula or gastrula stage. The drug's solvent in the highest concentration restricted the development of Ascaris eggs by about 10%. Unembryonated eggs of the worm appeared to be unaffected by 50 micrograms/ml of moxidectin treatment lasting 1-4 days, but for slowing down a little the development rate. The same concentration of the drug in the culture during the first four days of development resulted in 20% reduction of invasive stage in comparison to the control.

[The effect of cydectin on the survival of second stage larvae and mature Ascaris suum cultured in vitro]. / Wplyw Cydectinu na przezywalnosc larw drugiego stadium oraz osobników doroslych Ascaris suum w hodowli in vitro.

Larvae of Ascaris suum (L2) newly hatched from eggs with sodium hypochlorite, were placed (2000 larvae per 2 ml) of culture in EAGLE's medium, containing 10% calve serum or without it. Moxidectin (Cydectin) was introduced into medium in concentrations: 5, 10, 25 and 50 micrograms/ml. The cultures were incubated for 3 days, and there were controlled every 12 hours. The effect of drug on survival of larvae was slightly expressed. After 3 days survival rates were 87, 81, 80 and 78% in medium with serum, respectively to moxidectin concentrations. The lethality of larvae was higher in medium without serum, and amounted to 17, 23, 29 and 31% in comparison with control probe after 72 hours. Adult Ascaris were placed on ARS medium with glucose (0.1%) containing moxidectin in concentrations 1, 5, 50 and 100 micrograms/ml. The worms' condition was examined every day based on motility and turgor of their body. After 14 days of incubation in control probe 50% worms were alive, but their vitality was reduced. The behaviour of Ascaris in the presence of 50 micrograms/ml moxidectin was similar to those from the control. Only the highest concentration of drug (100 micrograms/ml) was lethal in 100% on the 8th day. The lower moxidectin concentrations (1 and 5 micrograms/ml) were not lethal to adult worms, they had even the positive effect on survival rate and condition of adult Ascaris. The eggs laid by females which were maintained in culture with drug were collected. There were no disturbances in their future development.

[The influence of various levels of carotene and protein in the diet on the index of invasion and digestive enzyme activity in chickens infected with Ascaridia galli]. / Wplyw zróznicowanego poziomu karotenu i bialka w diecie na wskazniki inwazji oraz aktywnosc enzymów trawiennych u kurczat zarazonych Ascaridia galli.

Chickens 10 days old were infected with 500 eggs of Ascaridia galli. They were fed with diet containing 11% or 19% protein. Some of them were given beta-carotene in a dose equivalent to daily requirement of vitamin A, others were given double dose of beta-carotene. The intensity and extensity of invasion, the body weight, and activities of alpha-amylase, lipase and trypsin in pancreas and duodenal content were measured. Application of beta-carotene (especially in a double dose) caused the decrease in the invasion index and moderated the changes in the activity of digestive enzymes, occurring in the infected chickens.

[Activity of alpha-amylase, trypsin and lipase in pancreas and duodenal contents of Ascaridia galli infected chickens fed with various proteins]. / Aktywnosc alfa-amylazy, trypsyny i lipazy w trzustce i tresci dwunastniczej kurczat zarazonych Ascaridia galli zywionych pasza o róznej zawartosci bialka.

The study was carried out on Astra S chickens which were grown on diets containing 11% and 19% proteins. In homogenized pancreas and duodenal contents from control animals and chickens infected with 500 invasive eggs of Ascaridia galli activities of alpha-amylase (Fennel method), lipase (Cherry-Crandall method) and trypsin (Anson method) were determined. After 7 weeks of the invasion the activities of these enzymes were higher in duodenal contents and lower in pancreas of infected birds in comparison with the control animals. The differences were significant for alpha-amylase and lipase activities in animals which were given 11% protein diet, and for trypsin activity of chickens groups fed with 19% protein diet.

[Effect of Hydrochloric acid on invasion of Ascaris suum; enzyme activity in the digestive system and serum of newborn piglets]. / Wplyw roztworu kwasu solnego na inwazje ascaris suum oraz aktywnosc enzymów trawiennych przewodu pokarmowego i surowicy prosiat osesków.

The studies were carried out on twenty newborn piglets. They were divided into four groups. The groups no 3 and no 4 were given intragastric 0.18% HCl from the 3th day of experiment. The groups no 2 and no 4 were infected on the 7th day with 10,000 invasive eggs of Ascaris suum. The presence of A. suum larvae in the lungs and liver was examined after one week lasting invasion by Baermann method. The total acidity in the gastric content was measured. The activity of alpha-amylase, lipase and proteases was determined in the extracts from pancreas and in the contents of stomach, duodenum and jejunum. The level of pepsinogens and alpha-amylase in the animals serum was studied. The intensity of Ascaris invasion was slightly higher in the group which was given HCl than in the infected group without HCl. The activity of digestive enzymes in the both groups was similar. Only in the stomach content from the 4th group the activity of pepsin was higher (p < 0.05), and alpha-amylase and lipase were lower (p < 0.01) than in the 2nd and in the 3th group. The level of pepsinogens was always lower and alpha-amylase higher in the serum of infected animals than in uninfected groups.

[Effect of vitamin A and protein levels in the diet of chickens infected with Ascaridia galli on activity of digestive enzymes in pancreas and small intestine]. / Wplyw poziomu witaminy a i bialka w diecie kurczat zarazonych nicieniem ascaridia galli na aktywnosc enzymów trawiennych w trzustce i jelicie cienkim.

Hybrid chickens Astra S fed vitamin A deficiency diet containing 11 or 19% protein were infected with 500 invasive eggs of Ascaridia galli. One part of them received vitamin A in a dose corresponding to daily requirement, other part in double dose. After 7 weeks the extensity and intensity of ascaridiosis were determined. In pancreas and intestinal contents the activities of alpha-amylase, lipase and trypsin were measured. The invasion index was the highest in the group of chickens fed the protein and vitamin A deficiency diet. Vitamin A in a requirement dose lowered the parasite index. The results with double dose of vitamin A were worse in comparison with single dose. The activities of digestive enzymes were lower in infected animals than in the control groups. Vitamin A acted as moderator of the enzymatic changes during ascaridiosis.

[In vitro study of anthelmintic influence on activities of digestive enzymes in extracts from the gut of Ascaris suum and pig's pancreas]. / Badania in vitro wplywu leków przeciwrobaczych na aktywnosc enzymów trawiennych w wyciagach z jelita Ascaris suum i trzustki wieprzowej.

The effect of eight anthelmintics (Rintal, Fenbesan, Telmin, Banminth, Pyrantel, Nilverm, Levamisol and Bioscardina) on the alpha- and gamma-amylases, trypsin, and lipase from pig's pancreas and gut of Ascaris suum was determined. In extracts from A. suum gut also the maltase, trehalase and saccharose activities were examined. All drugs tested did not influence on the host's alpha-amylase. Telmin and Bioscardina were inhibitors of gamma-amylase, Rintal and Telmin--of trypsin, Fenbesan and Bioscardina--of lipase from pig's pancreas. Among the parasite's enzymes the lipase was the most sensitive. Its activity was decreased (35-60%) by Telmin, Nilverm and both pyrantel derivatives. The activity of maltase and trehalase was reduced by Levamisole and Banminth, that of saccharose by Levamisole. It is concluded that the anthelmintics Levamisole and Banminth seemed the most efficient among the tested drugs because they did not alter the activity of host's enzymes and, showed the inhibitory effect on three of parasite's enzymes.

[Alpha-amylase release by pancreatic acini isolated from guinea pigs infected with invasive eggs of Ascaris suum]. / Uwalnianie alfa-amylazy przez komórki pecherzykowe izolowane z trzustek swinek morskich zarazonych inwazyjnymi jajami Ascaris suum.

The studies were carried out on dispersed acini prepared from pancreas of guinea pigs infected with 5000 invasive eggs of Ascaris suum. The basal amylase secretion and that stimulated with acetylcholine and carbachol or blocked with atropine was determined. The basal secretion of amylase was significantly higher in pancreatic acini isolated from infected animals. The stimulation by acetylcholine and carbachol of these acini was weaker than in the control group. These results suggest that cholinergic stimulation of pancreatic amylase secretion was changed during larval ascariasis.

[The influence of HCl administration on the acidity and the activity of enzymes in the stomach contents of suckling piglets]. / Wplyw podawania HCl na kwasowosc i aktywnosc enzymów w tresci zoladka nie odchowanych prosiat.

After HCl administration to 3-21 day old crossbred piglets (wbp x pbz x zlotnicka biala) the total acidity and output of acidity, the activity of pepsin, alpha-amylase and lipase were measured in the stomach content. The activity of pepsinogen was estimated in piglet's serum. The results do not indicate the significant influence of HCl on the acidity and the activity of enzymes in the stomach content.

[The influence of HCl administration on the activity of trypsin, alpha-amylase and lipase in different parts of the alimentary tract of piglets in the first three weeks of life]. / Wplyw podawania HCl na aktywnosc trypsyny, alfa-amylazy i lipazy w róznych odcinkach przewodu pokarmowego prosiat w pierwszych 3 tygodniach zycia.

The experiments were conducted using 3 to 21 days old piglets. The content of animals stomach was supplemented with 0.18% HCl starting from the 3rd, 7th and 10th day of their life. The animals were dissected on the 4th, 7th and 11th day after HCl administration. The activity of trypsin, alpha-amylase and lipase was measured in homogenate from pancreas and duodenum and ileum contents. The presented results do not confirm the existence of significant differences between control and experimental animals.