Effect of different doses of GLP-2 (Teduglutide) on acute esophageal lesion due to acid-pepsin perfusion in male rats

Gastro-esophageal reflux currently is widespread disorders with dangerous complications. GLP-2 is a peptide that has trophic and anti-inflammatory effects on gastrointestinal mucosa. The aim of this study was to evaluate the protective role of GLP-2 in esophageal mucosa lesion due to perfusion acid-pepsin. Thirty-six male rats were used in this study and divided into six groups. They were control, acid-pepsin, GLP-2 20 μg, GLP-2 30 μg, GLP-2 40 μg and GLP-2 50 μg/kg groups. Esophageal blood flow, plasma NO metabolite, esophageal tissue NO metabolites and histological study of esophagus were performed as indicators of esophageal damage following acid-pepsin perfusion. Results showed that GLP-2 significantly increased plasma and tissue NO metabolites in comparison to acid-pepsin group. Also histological study showed significantly fewer lesions in the most effective dose GLP-2 30 μg in comparison to acid-pepsin group, our results show that GLP-2 could be useful for the treatment of esophageal in animal model.     

A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb alpha and beta chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.     

The influence of time and temperature on molecular gut content analysis： Adalia bipunctata fed with Rhopalosiphum padi

Gut content analysis is a useful tool when studying arthropod predator-prey interactions. We used polymerase chain reaction （PCR） technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR-based gut content analysis to field collected predators. Larvae of the two-spotted ladybeetle （Adalia bipunctata L.） were fed with the bird cherry-oat aphid （Rhopalosiphum padi L.） at either 21℃ or 14℃. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number ofA. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.     

An improved method for recovery of muscle-stage larvae from mice infected with Toxocara canis

We report a modified digestion method that improves the recovery of Toxocara canis larvae from skeletal muscle. Minced muscle tissue from infected mice was incubated in artificial gastric juice for 48 hr at 37 C, and ethanol was added for the second 24 hr. This procedure allowed the larvae to be identified and counted more quickly than with the standard digestion method. This method allows measurement of the total number of larvae present in muscle tissue following oral intubation of embryonated eggs, although it does not permit counting of live larvae.     

The Baermann technique for estimating Protostrongylus infection in bighorn sheep: effect of laboratory procedures

The modified Baermann funnel technique was evaluated to determine the effects of time of baermannization, fecal preparation, type and size of funnel, and type of filter on the number of first stage larvae of Protostrongylus spp. recovered from feces of Rocky Mountain bighorn sheep (Ovis canadensis). More larvae were recovered when fecal pellets were baermannized for 24 hr compared to 8 hr, and when feces were crushed than when left intact. Use of small funnels resulted in the recovery of more larvae per gram of feces than larger funnels, and glass funnels more than plastic ones. There was no difference in recovery of larvae between cheesecloth filters and cellulose filters.     

Total nitrogen determining for plant material containing nitrate

A nonfoaming method for semimicro Kieldahl determination of total nitrogen in plant samples containing appreciable amounts of nitrate was developed for use with a digestion block utilizing test tubes for digestion flasks. The sample (30 to 200 mg) is treated with 5 ml of a sulfuric acid:salicylie acid (30:1 $\text{v}\text{w}$) mixture at room temperature for 1 hr. Catalyst [K₂SO₄:CuSeO₃·2H₂O:pumice 970:19:11 w/w/w)] is added and the mixture is digested at 360 to 380°C for 1 hr after the mixture clears. Ammonium in the digest is determined by a suitable method. This semimicro Kjeldahl procedure results in a 95% or better recovery of nitrate, either from KNO₃ or from KNO₃ added to plant material.     

Effects of a juvenoid, DPE-28, on biology and behaviour of Culex quinquefasciatus, the human filariasis vector

Studies on the effect of a juvenoid, DPE-28 (2,4-dinitrophenyl-2',6'-di-tertiarybutyl phenyl ether) on biology and behaviour of Cx. quinquefasciatus showed that the developmental duration, sex ratio, mating success and blood feeding were considerably affected by the exposure of larvae and pupae to the compound. Exposure of fourth instar larvae to 0.007 (EI90) and 0.0019 (EI50) ppm of DPE-28 prolonged the duration of pupation by 58.6 and 52.4 hr and delayed the adult emergence by 35.4 and 17.7 hr in males and 36.8 and 21.1 hr in females respectively. Exposure of freshly ecdysed pupae to 10 and 5 ppm delayed the adult emergence with respect to the control by 54.3 and 32.4 hr in males and 55.2 and 33.2 hr in females respectively. The sex ratio of the adults emerged from treated larvae and pupae was also affected. The female mosquitoes that survived from the exposed fourth instar larvae and pupae exhibited a low blood engorgement ratio. This depression in blood feeding was more pronounced in adults emerged from treated pupae than that of treated fourth instar larvae. A significant proportion of adults emerged from treated larvae and pupae were able to feed only partially. Mating success of the treated populations declined considerably when crosses were made between the males and females emerged from treated fourth instar larvae and pupae. The adults emerged from treated larvae and pupae showed a significant reduction in the oviposition.     

Identification of intestinal bacteria from Japanese flounder (Paralichthys olivaceus) and their ability to digest chitin

AIMS: The aim of the present study was to clarify the taxonomic status of intestinal bacteria isolated from Japanese flounder (Paralichthys olivaceus) and describe their ability to digest chitin. METHODS AND RESULTS: Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences showed that 82 representative isolates were closely related to three major species of marine vibrios, Vibrio scophthalmi-Vibrio ichthyoenteri group (41 isolates), Vibrio fischeri (39 isolates) and Vibrio harveyi (two isolates), with similarities of 97.2-99.8%, 96.4-100% and 98.6-99.5% respectively. These findings indicate that V. scophthalmi-V. ichthyoenteri group is indigenous to the intestinal tract of Japanese flounder. Moreover, the ability of 82 isolates to digest chitin was examined using the agar plate method and PCR amplification of the chiA gene. The two V. harveyi isolates and 36 of 41 V. scophthalmi-V. ichthyoenteri isolates digested chitin and were chiA PCR positive, whereas all 39 V. fischeri isolates digested chitin but were chiA PCR negative. CONCLUSIONS: Intestinal bacteria from Japanese flounder were mainly composed of Vibrio scophthalmi-V. ichthyoenteri group and V. fischeri. Taken together, the results showed that 81 of 82 isolates could digest chitin. However, only 38 of these isolates possessed a chiA homologue which could be identified by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that Japanese flounder harbours bacteria of the V. scophthalmi-V. ichthyoenteri group, and these results are similar to what has been found for turbot (Scophthalmus maximus).     

Development of Haemonchus contortus in vitro and the stimulus from the host.

Infective larvae of Haemonchus contortus which have been exposed for about 9 hr to relatively high partial pressures of carbon dioxide at near-neutral pH, as they might be in the rumen, develop slowly to the fourth stage or not at all. But following this treatment, exposure to lower partial pressures of carbon dioxide and higher concentrations of hydrogen ions, such as might be encountered in the abomasal mucosa, brings about development at a rate comparable with that in the sheep.     

Further observations on the development of Gongylonema pulchrum in rabbits

Third-stage larvae of Gongylonema pulchrum from naturally infected dung beetles were inoculated orally into 24 rabbits. Worm recovery ranged from 54 to 91% (mean = 67.5%) during the period from 24 hr to 52 wk postinoculation (PI). Two hours PI, the larvae entered the mucosa at the junction of the stomach and esophagus and migrated upward. Early development occurred primarily in pharyngeal mucosa, tongue, and buccal mucosa. The third molt took place 11 days PI and the final molt at 36 days PI. Male worms reached sexual maturity at 7 wk PI and females at 9 wk PI. Adult worms were found mainly in the esophagus but also occurred in the tongue and the wall of the oral cavity after 30 wk PI. Embryonated eggs appeared in the feces of 3 rabbits inoculated with 50 or 100 larvae on days 72-81 PI. Morphologically, the cuticle in young fourth-stage larvae exhibited bosses on the anterior portion on day 11 PI, and the left spicule length : total body length exhibited no remarkable change between 9 and 52 wk PI. The latter finding confirms the utility of the ratio for identification of the nematode.     

Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes

The effects of post-ingestion and physical conditions under which killed mosquitoes are stored on the success of detecting blood meal DNA of Anopheles stephensi and Culex quinquefasiatus were investigated by polymerase chain reaction (PCR) amplification at the human mitochondrial DNA cytochrome b (cytB) gene. Host DNA extracted from the blood meal up to 33 h post-ingestion in both species acts as an efficient template for PCR amplification. However, more DNA concentration is needed for meals digested for a longer time, and successful PCR amplification from meals digested for 36 h,dropped to a faint band. There were no differences between PCR success rate for samples stored at +4 or -20 degrees C, but less successful products were observed in samples kept at 4 degrees C for the periods longer than 30 h digestion. The results of this study are important for conducting malaria epidemiological studies that provide information about the degree of contact between human hosts and mosquito vectors, impact of vector controls such as bed nets and repellents, and the transmission dynamics of human malaria and other vector-borne diseases.     

多重实时荧光PCR检测牛、山羊和绵羊源性成分

根据牛、山羊和绵羊线粒体细胞色素b基因序列, 设计特异性引物和以不同荧光素标记的Taqman探针。通过对PCR反应体系和反应条件的优化筛选, 建立能同时鉴别牛、山羊和绵羊源性成分的多重实时荧光PCR方法。采用本文方法与国标GB/T 20190-2006方法分别对17种不同源性动物DNA和200份不同来源样品DNA进行牛羊源性成分检测, 数据显示两者检测结果符合率达100%, 特异性相当。与国标方法相比, 本试验方法不需电泳、酶切和测序, 即可在一个PCR反应中同时鉴别检测牛、山羊和绵羊3种源性成分, 检测效率提高近3倍; 灵敏度更高, 比国标方法灵敏10倍; 适用性更广, 除了饲料, 还适用于肉品、奶品、生皮和动物油脂等动物产品的牛羊源性成分检测。     

Observations on the self-cure reaction and other forms of immunological responsiveness against Haemonchus contortus in sheep

Self-cure reactions and immunological responses preventing establishment of Haemonchus contortus in sheep may operate as separate entities. In one experiment, self-cure occurred when challenge infection with 5000 larvae was superimposed on an infection with 5000 larvae given to worm-free sheep 6 weeks previously. Resident worms were rejected and establishment of infection by incoming larvae was impeded. The latter effect was not observed in sheep treated similarly but with resident parasites removed by treatment with oxfendazole before challenge. In another experiment, younger worm-free sheep primed by three infections with 2000 larvae at intervals of 2 weeks or a single infection with 6000 larvae were challenged with 10,000 larvae 6 weeks after the first priming infection. Self-cure was not incited but establishment of infection was impeded in sheep primed with three divided doses of larvae whether or not priming infections had been removed by oxfendazole. Infection regimes used for priming did not influence numbers of arrested fourth-stage larvae derived from challenge infection. However, more arrested larvae were present when challenge was superimposed on extant infections, indicating that resident worms or a factor activated by their presence induced developmental arrest. In a third experiment, large burdens with H. contortus were established in sheep immunosuppressed with the corticosteroid, dexamethasone, at the time of infection. Self-cure was not triggered by a challenge infection given 32 days later either in these sheep, or in sheep with a smaller worm burden derived from infection given without immunosuppression. Faecal egg counts, however, indicated that development of the challenge infection was prevented in both groups of sheep.

Investigation of self-cure is restricted by lack of a predictable system for reproducing the phenomenon. Self-cure was induced by a single infection with 5000 larvae in mature sheep but not with 6000 larvae in immature sheep. Three infections with 3000 larvae given at intervals of 2 weeks to mature sheep did not prime for self-cure. Procedures aimed at heightening immediate hypersensitivity, i.e. treatment with pertussis vaccine or concurrent infections with Ostertagia circumcincta, did not promote self-cure reactivity in the latter situation.     

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.     

Modification of native sheep liver fructose-1,6-bisphosphatase by subtilisin.

Digestion of native sheep liver fructose-1,6-bisphosphatase by subtilisin resulted in a parallel decrease in activity and sensitivity to AMP inhibition at neutral pH and an increase in specific activity at alkaline pH. During the course of digestion the 35,500 subunit was progressively replaced by two peptides of approximately 29,000 and 6,000 molecular weight, respectively.A comparison of native and digested fructosebisphosphatase showed no significant changes in molecular weight or tryptophan content; however, their catalytic and regulatory properties were markedly different.     

Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples: comparison with isolation and biochemical characterization

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.     

A complex of genes involved in adaptation of Leptinotarsa decemlineata larvae to induced potato defense

The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.     

Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

Increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to Tribolium castaneum larvae, and the properties of digestive proteinases were compared in vitro. Cysteine proteinases were the major digestive proteinase class in control larvae, and serine proteinase activity was minor. Dietary serine proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine proteinase inhibitors, there was a dramatic shift from primarily cysteine proteinases to serine proteinases in the proteinase profile of the midgut. Moreover, a combination of cysteine and serine proteinase inhibitors in the diet prevented this shift from cysteine proteinase-based digestion to serine proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.     

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni

Abstract A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni . Amplimers of the FlaA gene obtained by PCR were digested with Alu I and Hin fI to distinguish C. coli from C. jejuni . With Alu I digestion C. jejuni -specific bands were observed at 110, 140 and 160 bp and C. coli -specific bands at 293 and 147 bp. C. jejuni -specific bands of 349 and 109 bp were found by Hin fI digestion but Hin fI did not digest the Fla A amplimer of C. coli . This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.