Quantitative analysis of the dystrophin gene by real-time PCR

Duchenne and Becker muscular dystrophy (DMD/BMD) are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR® Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (ΔΔCt). The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07±0.20, and for carriers 0.56±0.11. This assay proved to be simple, rapid, reliable and cost-effective

Anthropogenetic variability in groups of children from regular and special schools from different localities in Serbia

This population-genetic study compares morpholophysiological and genetic variability in five control groups of individuals (children from five regular schools, N= 996) with children from that many special schools (N= 736) from Serbia, by using a test of determination of homozygously recessive characteristics in humans (HRC-test). Genetic homozygosity degree showed not only statistically significant difference between the mean values obtained for two groups of studied samples (control group 6.95± 0.07; children from special schools 8.63± 0.08 HRCs, out of 30 analyzed characteristics), but also differences in the type of distribution, as well as the presence of specific combinations of such traits. Results of comparisons done in different places (Kraljevo, Nis, Vranje, Leskovac, Pirot) showed the same tendency- the increase of genetic homozygosity and relative decrease of variability in samples of children from special schools. The number of HRCs among individuals from control groups varied from 2 to 15/30, and from 3 to 16/30 among children from special schools. It is possible that increased recessive homozygosity present in the group of children from special schools leads to increase of genetic loads, what may cause easier expression of some physiological and mental abilities that children from special schools have.A great individual variation in amount of genetic homozygosity that exists among human individuals may influence their potentials for different kinds of adaptation, including their mental abilities, physical capacities or resistance to different diseases. [Projekat Ministarstva nauke Republike Srbije, br. 175093

Comparative study on biochemical activity of the intestinal isolates Lactobacillus sp. V3 and Bifidobacterium sp. A71 in different substrates

The two intestinal isolates Lactobacillus sp. V3 and Bifidobacterium sp. A71 were selected for soymilk fermentation according to their acidification activity in soymilk. Beetroot juice and carrot juice were chosen for soymilk supplementation as additional sources of carbohydrates and brewer's yeast as an extra source of nitrogen. The fermentation was carried out for eight hours at 42°C. The fermentation was monitored by standard analytical and microbiological tests for changes of acidity (decreasing pH and increasing acid content), the contents of soluble dry substances, sugars and a-amino acids as well as changes in the number of viable cells. The samples were collected at the beginning and subsequently every two hours until the end of the fermentation. The results showed that there were differences between the tested isolates in terms of their ability to ferment soymilk. The mix with brewer's yeast had a better stimulating effect on the growth of both strains compared to those with juices alone. In addition, the carrot juice stimulated the growth of Bifidobacterium sp. A71 better than beetroot juice, while the opposite effect was found for the growth of Lactobacillus sp. V3

The influence of a cryoprotective medium containing glycerol on the lyophilization of lactic acid bacteria (NOTE)

The aims of liophilization (freeze-drying) of lactic acid bacteria are to preserve pure cultures or to prepare starters for the dairy industry. In both cases, the choice of the cryoprotectant is very important. In this work, samples of Bifidobacterium breve A71 and Bifidobacterium bifidum BbTD were freeze-dried in a new cryoprotective medium containing lactose, gelatine and glycerol (medium B). The reference medium contained saccharose, gelatine and skim milk (medium A). Before liophilization, the eutectic points of both media were determined, because the products must be cooled to a temperature below its freezing point. The success of the cryoprotectants was estimated in terms of the number of surviving organisms after lyophilization. Bifidobacterium breve A71 and Bifidobacterium bifidum BbTD freeze-dried in media A and B showed high survival rates. Bifidobacterium breve A71 showed a greater percentage survival in combination with medium B than with medium A. These results could be utilized in the manufacture of Bifidobacterium breve A71 as a starter in the diary industry because it is a human isolate which, except for acidification, has probiotic activity

Lipase catalyzed synthesis of flavor esters in non-aqueous media: Optimization of the yield of pentyl 2-methylpropanoate by statistical analysis

In this study, the synthesis of pentyl 2-methylpropanoate employing a commercial lipase from Candida rugosa was investigated, the emphasis being placed on analyzing the effects of various process conditions on the yield of ester. The response surface methodology (RSM) and five-level-five-factor central composite rotatable design (CCRD) were used to evaluate the effects of variables, namely the initial water content, 0.0–2.0 % (w/v), the reaction temperature, 35–75 °C, the enzyme concentration, 1.0–5.0 g dm-3, the acid/alcohol mole ratio, 1:2–5:2, and the reaction time, 4–48 h, on the yield (%) of ester. The production of pentyl 2-methylpropanoate was optimized and an ester yield response equation was obtained, enabling the prediction of ester yields from known values of the five main factors. It seems that the enzyme concentration, reaction time and acid/alcohol mole ratio predominantly determine the conversion process, while the amount of added water amount had no significant influence on the ester yield. Conversion of around 92 % of the substrate to ester could be realized using a concentration of lipase as low as 4.0 g dm-3 and in a relatively short time (26 h) at 35 °C, when a high substrate mole ratio of 2.5 was used

DIFFERENT EFFECTS OF THE ATROPINE UPON THE PENDULOUS MOVEMENTS OF THE PROXIMAL AND OF THE DISTAL COLON OF THE RABBIT

The influence of the non-selective blocker of the muscarinic receptors, namely of the atropine (1,7 x10-10 - 5x10-7). upon thependulousmovements of the proximal and of the distal colon of the rabbit was studied. In the correct proportion to the concentration the atropine reduces the amplitude of both the proximal (r=0,98; P<0,001) and of the distal colon (r= 0,97; P < 0,001). On the basis of the ED50 values - that amount to 7,7 ± 0,03 x 10-7 M for the proximal colon and 3,5 ± 0,06 x 10-5 M for the distal colon, the atropine recluces for twenty times the amplitude of the pendulousmovements of the rabbit's distal colon. Likewise, it reduces for dozen times the frequency of the pendulous movements of the distal colon (ED50 = 5 ± 0,02 x 10-6M) with respect to the proximal one (ED50 = 4,7 ± 0,06 x I0-5M). The atropine does not change the tone of the proximal colon (average relaxation is 5,47 ± 2,32 %) while it induces the concentrically-dependent relaxation of the distal colon (maximal relaxation amounts to 38.03 + 8,34). The ED50 values for the atropine while affecting the distal colon tone is 1,7 ± 0,03 x 10-6M

Relaxant Effect of the Ethanol Extract of Helichrysum plicatum (Asteraceae) on Isolated Rat Ileum Contractions

Helichrysum plicatum (Turkish Helichrysum) has been used in folk medicine for the treatment of gastric and hepatic disorders. The aim of the present study was to examine the relaxant activity of an extract of H. plicatum flowers on isolated rat ileum. Segments of ileum of rats were suspended in an organ bath. Cumulative concentrations of H. plicatum ethanol extract induced a relaxant effect on spontaneous rat ileum contractions. H. plicatum extract caused a mean contractile response of 81.68 ± 6.17% (at a dose of 0.01 mg/mL) and 30.08 ± 9.07% (at a dose of 1 mg/mL). A similar effect was observed with papaverine (0.01–3 μg/mL). H. plicatum extract (0.01–1 mg/mL) relaxed high K+ (80 mM) precontractions, an effect similar to that caused by papaverine (0.01–3 μg/mL). The plant extract (0.03–0.3 mg/mL) also induced a significant depression of the cumulative concentration response curve for acetylcholine (5–1500 nM) (p &lt; 0.01). Atropine (140 nM) abolished the acetylcholine effect. The extract (0.03–0.3 mg/mL) reduced the histamine (1–300 nM) and BaCl2 (3–900 μM) induced contractions (p &lt; 0.01). Our results showed the relaxant effect of the ethanol extract of Helichrysum plicatum flowers on the isolated rat intestine Extract of H. plicatum can inhibit the spontaneous ileum contractions and contractions induced by acetylcholine, histamine, barium and potassium ions

Efikasnost različitih metoda za određivanje ostataka organofosfata u fermentisanim uzorcima žita

In the present work, the efficiencies of three different sample preparation methods for GC/ MS determination of pirimiphos-methyl and chlorpyrifos-methyl residues in wheat (Triticum spelta) samples fermented by Lactobacillus plantarum were compared. All three methods were based on methanol:acetone=1:1 extraction, while further purification of the obtained samples was altered. First purification was through a column containing a mixture of aluminium oxide and activated charcoal slurry-packed and eluted with dichlormethane, the second was purification on a florisil column slurry-packed and eluted with ethyl acetate:acetone=4:1, while the third was based on a combination of the former two methods, i.e. clean-up through columns filled with a mixture of aluminum oxide and activated charcoal slurry-packed and eluted with ethyl acetate:acetone=4:1. The second method was found the most effective for obtaining satisfactory recoveries at four fortification levels. For pirimiphos-methyl, recoveries were in the range of 91.3-96.0% and had good reproducibility, i.e. RSD ranging from 2.2-4.1%, while the corresponding range for chlorpyrifos-methyl was 81.6-88.2%, and the RSD range 2.5-5.4%. The chosen method was further optimized in order to establish the optimum volume of elution solvent used during the clean-up procedures. The highest recoveries of 93.7±3.5% for pirimiphos- methyl and 85.3±2.5% for chlorpyrifos-methyl were obtained after elution with 25 ml volume of solvent. Considering all, simple, efficient and reliable GC/MS detection of pirimiphosmethyl and chlorpyrifos-methyl residues in wheat grain substrate altered by fermentation with L. plantarum was achieved by the two-steps extraction with 25 ml of methanol:acetone=1:1 solvent mix for 30 min, followed by a clean-up procedure through a glass column with florisil coupled with elution by 25 ml of ethyl acetate:acetone=4:1.Poređene su efikasnosti tri različite metode pripreme uzoraka za određivanje ostataka pirimifos-metila i hlorpirifos-metila u uzorcima pšenice (Triticum spelta) fermentisane sa Lactobacillus plantarum. Sve tri metode zasnivale su se na metanolsko-acetonskoj (1:1) ekstrakciji dok su se procedure prečišćavanja dobijenih ekstrakata razlikovale. Prva metoda bila je prečišćavanje kroz kolonu punjenu smešom aluminijum-oksida i aktivnog uglja u kombinaciji sa dihlormetanom kao eluentom, druga prečišćavanje kroz kolonu punjenu florisilom u kombinaciji sa smešom etil-acetata i acetona (4:1) kao eluentom, dok se treća zasnivala na kombinaciji prve dve, tj. prečišćavanju kroz kolonu punjenu smešom aluminijum-oksida i aktivnog uglja u kombinaciji sa smešom etil-acetata i acetona (4:1) kao eluentom. Druga testirana metoda pokazala se kao najefikasnija, pri čemu su dobijeni prinosi pirimifos-metila u opsegu 91,3-96,0% za četiri koncentraciona nivoa obogaćivanja, sa RSD% u opsegu 2,2-4,1%, dok su za hlorpirifos-metil ovi prinosi bili u opsegu 81,6-88,2% sa RSD% u opsegu 2,5-5,4%. Odabrana metoda je dodatno optimizovana variranjem različitih zapremina korišćenog eluenta. Najveći prinos metode za oba ispitivana pesticida postignut je pri zapremini eluenta od 25 ml (93,7±3.5% za pirimifos-metil i 85,3±2,5% za hlorpirifos-metil). Predložena metoda, bazirana na 30-minutnoj ekstrakciji sa 25 ml smeše metanol:aceton=1:1, prečišćavanju na florisilskoj koloni i eluiranju sa 25 ml smeše etilacetat: aceton=4:1, pokazala se kao efikasna, jednostavna i pouzdana metoda za određivanje ostataka ispitivanih organofosfata u fermentisanom žitnom supstratu

Efficiencies of different methods for determination of organophosphate pesticide residues in fermented wheat substrate

In the present work, the efficiencies of three different sample preparation methods for GC/MS determination of pirimiphos-methyl and chlorpyrifos-methyl residues in wheat (Triticumspelta) samples fermented by Lactobacillus plantarum were compared. All three methods werebased on methanol:acetone=1:1 extraction, while further purification of the obtained sampleswas altered. First purification was through a column containing a mixture of aluminiumoxide and activated charcoal slurry-packed and eluted with dichlormethane, the second waspurification on a florisil column slurry-packed and eluted with ethyl acetate:acetone=4:1, whilethe third was based on a combination of the former two methods, i.e. clean-up through columnsfilled with a mixture of aluminum oxide and activated charcoal slurry-packed and elutedwith ethyl acetate:acetone=4:1. The second method was found the most effective for obtainingsatisfactory recoveries at four fortification levels. For pirimiphos-methyl, recoveries werein the range of 91.3-96.0% and had good reproducibility, i.e. RSD ranging from 2.2-4.1%, whilethe corresponding range for chlorpyrifos-methyl was 81.6-88.2%, and the RSD range 2.5-5.4%.The chosen method was further optimized in order to establish the optimum volume of elutionsolvent used during the clean-up procedures. The highest recoveries of 93.7±3.5% for pirimiphos-methyl and 85.3±2.5% for chlorpyrifos-methyl were obtained after elution with 25 mlvolume of solvent. Considering all, simple, efficient and reliable GC/MS detection of pirimiphosmethyland chlorpyrifos-methyl residues in wheat grain substrate altered by fermentation withL. plantarum was achieved by the two-steps extraction with 25 ml of methanol:acetone=1:1solvent mix for 30 min, followed by a clean-up procedure through a glass column with florisilcoupled with elution by 25 ml of ethyl acetate:acetone=4:1