Gut Homogenates Research Articles

Prodrugs of peptides. 17. Bioreversible derivatization of the C-terminal prolineamide residue in peptides to afford protection against prolyl endopeptidase

Various aliphatic N-acyl derivatives and an N-phthalidyl derivative of the model compound N- benzyloxycarbonyl-glycyl- l - prolineamide (Z-Gly-ProNH 2) were synthesized to assess their suitability as prodrug forms for the C-terminal prolineamide residue occurring in several peptides (e.g. TRH) with the aim of protecting the peptide against prolyl endopeptidase in the gut prior to absorption. Whereas Z-Gly-ProNH 2 was rapidly hydrolyzed in a rabbit gut homogenate, used as a source of prolyl endopeptidase, the N-acyl derivatives were found to afford protection by a factor of 1.5–6. The stability of the N-acyl derivatives in the gut homogenate decreased with increasing chain length within the acyl group. The N-phthalidyl derivative, on the other hand, degraded even faster than the parent compound. The derivatives were all converted quantitatively into the parent peptide in human plasma solutions via hydrolysis catalyzed by non-specific plasma esterases. The results suggest that by appropriate N-acylation it may be feasible to improve the stability of a C-terminal prolineamide moiety toward prolyl endopeptidase. The combination of increased stability in the intestine and higher lipophilicity of the N-acyl prodrugs might render it possible to improve the delivery characteristics of peptides containing a C-terminal prolineamide moiety.

Amylase activity in the gut homogenate of the kola weevil, Sophrorhinus insperatus Faust and its response to inhibitors from kola nuts

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 Cl, Ca2+ and Cd2+ ions and inhibited by Cu2+ and Hg2+ ions. 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.

A role for mucin in the absorption of inorganic iron and other metal cations: A study in rats

The steps involved in iron absorption are poorly understood. Although transferrin and ferritin are water soluble, most radioiron in gut homogenates after an intraluminal dose of radioiron is recovered in water-insoluble precipitates. Most radioiron in the precipitates was insoluble in detergents and organic solvents and was characterized as mucins. These isolates bound iron in vitro with a Kd of 9.09 × 10−5. Similar iron binding was observed with commercial mucins. Iron binding to mucin occurred at acid pH and maintained the iron available for absorption with alkalinization. Similar pH-dependent binding to mucin was observed with zinc, cobalt, and lead. Iron competitively inhibited binding of these metals to mucin. However, iron chelates of ascorbate, fructose, and histidine donated iron to mucin at neutral pH. These data provided a role for gastric HCl and intestinal mucin in absorption of iron and metal cations and partial explanation of the competition for absorption between certain metals from the gut lumen. It is postulated that intestinal mucin delivers inorganic iron to intestinal absorptive cells in an acceptable form for absorption.

A role for mucin in the absorption of inorganic iron and other metal cations

Abstract The steps involved in iron absorption are poorly understood. Although transferrin and ferritin are water soluble, most radioiron in gut homogenates after an intraluminal dose of radioiron is recovered in water-insoluble precipitates. Most radioiron in the precipitates was insoluble in detergents and organic solvents and was characterized as mucins. These isolates bound iron in vitro with a Kd of 9.09 × 10 −5 . Similar iron binding was observed with commercial mucins. Iron binding to mucin occurred at acid pH and maintained the iron available for absorption with alkalinization. Similar pH-dependent binding to mucin was observed with zinc, cobalt, and lead. Iron competitively inhibited binding of these metals to mucin. However, iron chelates of ascorbate, fructose, and histidine donated iron to mucin at neutral pH. These data provided a role for gastric HCl and intestinal mucin in absorption of iron and metal cations and partial explanation of the competition for absorption between certain metals from the gut lumen. It is postulated that intestinal mucin delivers inorganic iron to intestinal absorptive cells in an acceptable form for absorption.

Properties of glycosidases from the maize weevil, Sitophilus zeamais

A survey of glycosidase activity in adults of the maize weevil, Sitophilus zeamais Motschulsky, indicated a complex of enzymes qualitatively sufficient to hydrolyze the free di- and oligosaccharides in their cereal diets as well as the maltose and oligomaltodextrins produced by the action of α-amylase on ingested starch. Glycosidase activity was found primarily in the soluble fraction (105,000 g supernatant) of gut (foregut, midgut and contents) homogenates and was most active in buffers with slightly acidic pH. α-Glucosidase activity was detected by using p- nitrophenyl-α- d-glucopyranoside (NPαGlu), maltose, sucrose and melezitose as substrates. Based on differences in pH optima, there may be a specific α-trehalase. β-Glucosidase activity was detected with p- nitrophenyl-β- d-glucopyranoside and cellobiose. p- Nitrophenyl-α- d-galactopyranoside was not hydrolyzed but the slow hydrolysis of melibiose indicated the presence of an α-galactosidase. β-Galactosidase was detected with p- nitrophenyl-β- d-galactopyranoside . Raffinose was slowly hydrolyzed. The molecular mass of α-glucosidase, partially purified from adult weevils by ammonium sulfate precipitation and ion exchange chromatography, was estimated to be 130 kDa under non-dissociating conditions. α-Glucosidase activity was detected at R m 0.43 on 7.5% acrylamide gels with 4-methyl-umbelliferyl-α- d-glucoside as substrate. pI was estimated to be 4.9 by isoelectric focusing. Two fractions with activity against p- nitrophenyl-α- d-glucopyranoside were resolved by high performance liquid chromatography. One fraction (peak No. 2) was highly specific for maltose, had K m values of 12.9 mM for NPαGlu and 14 mM for maltose, and hydrolyzed oligomaltodextrins up to at least maltoheptaose

Thermal properties for digestive enzymes of a sub-antarctic beetle, hydromedion sparsutum (coleoptera, perimylopidae) compared to those in two thermophilic insects

1. 1. Proteolytic, lipolytic, amylolytic and cellulolytic activities were studied in adults of the phytophagous beetle, Hydromedion sparsutum, indigenous to the sub-Antarctic island of South Georgia. 2. 2. Gastric enzyme activities were measured at experimental temperatures of 5–40°C and results were compared with those obtained from two thermophilic insects, Gryllus bimaculatus and Tenebrio molitor. 3. 3. Protease and lipase activities in Hydromedion were 10–15 times lower than in Gryllus and Tenebrio. 4. 4. In the temperature range of 5–15°C, α-amylase activity from Hydromedion was only slightly lower than that from Gryllus. 5. 5. Hydromedion gut homogenates exhibited a distinct cellulolytic activity, even at a low temperature of 5°C. 6. 6. Cellulolytic activity in the digestive tract of Hydromedion was confirmed by the evolution of 14CO 2 after consumption of labelled cellulose. 7. 7. The thermal properties of digestive enzymes agree well with the role of Hydromedion as primary decomposer in its ecosystem.

Prodrugs of peptides. 8. In vitro study of intestinal metabolism and penetration of thyrotropin-releasing hormone (TRH) and its prodrugs

The feasibility of improving the poor oral bioavailability of the tripeptide TRH (pGlu-His-Pro-NH 2) was examined using the prodrug approach. The prodrugs studied were various N-alkoxycarbonyl derivatives formed by reacting TRH with the appropriate chloroformates at its imidazole moiety. In vitro metabolism studies with rabbit and rat intestinal homogenates showed that TRH and the prodrugs were rapidly degraded in the rabbit homogenate by virtue of prolyl endopeptidase which cleaves the C-terminal proline amide moiety. Whereas TRH showed a high stability in rat gut homogenates, the prodrug derivatives were readily degraded, partly due to prolyl endopeptidase and partly due to non-specific esterases. In vitro penetration studies using the modified Ussing chamber showed that the prodrugs did not improve the penetration of TRH across the jejunal, ileal and colonic segments of the rat. It is concluded that although the prodrugs are much more lipophilic than TRH in terms of octanol-buffer partition coefficients, the greater susceptibility of the derivatives to undergo enzymatic degradation more than the offsets the improved lipophilicity characteristics. Prodrugs suitable for improving the oral absorption of TRH should not only possess a certain lipophilicity, but should also be resistant towards the prolyl endopeptidase enzyme.

Characterization of two sulfate-reducing bacteria from the gut of the soil-feeding termite, Cubitermes speciosus

Two sulfate-reducing bacteria (SRB) were isolated from a mixed culture enriched with benzoate obtained from gut homogenate of the soil-feeding higher termite, Cubitermes speciosus. The organisms were vibrioid rods, staining Gram-negative, which performed incomplete substrate oxidation. They differed in several features. The smaller one, strain STp, was motile with a single polar flagellum. This strain differed from Desulfovibrio desulfuricans only by its inability to oxidize malate and pentanol. The bigger one, strain STg, differed from Desulfovibrio giganteus only by its nonmotility and a lower length. It is the first evidence of the presence of SRB in termite gut.

Development and infectivity of Anaplasma marginale in Dermacentor andersoni nymphs

SUMMARY The development of Anaplasma marginale was studied in Dermacentor andersoni nymphs after they had fed on a calf with ascending Anaplasma infection. Gut tissues were collected on day 4 of tick feeding, from newly replete (fed) nymphs and on postfeeding days (pfd) 5, 10, 15, 20, and were processed for light and electron microscopy to determine density of A marginale colonies. Homogenates of gut tissues were prepared from nymphs collected on the same days and inoculated into susceptible, splenectomized calves to test for infectivity. Anaplasma colonies were detected in gut cells on pfd 5, 10, 15, and 20. Although colony density appeared to be higher on pfd 10 and 15, differences were not significant. Nymphal type-1 colonies were detected in highest numbers on pfd 5 and 10, transitional colonies were seen in highest numbers at pfd 10 and 15, and nymphal type-2 colonies were observed only on pfd 20. Gut homogenates that were collected from ticks at 4 days of feeding, when newly replete, and on pfd 20 caused anaplasmosis when injected into susceptible calves, but homogenates made from ticks collected on pfd 5, 10, and 15 were not infective. The data indicate that of the colony types of A marginale that develop in replete nymphs, nymphal type-1 and transitional colonies may contain organisms that are not infective for cattle.

Partial characterization of proteinase activity in the larval midgut of the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae)

Protease activity in the alimentary tract of the European corn borer, Ostrinia nubilalis, can be attributed to at least three endoproteinases. A high alkaline trypsin with maximal hydrolysis of benzoyl arginine p-nitroanilide at pH values higher than 10.0, a low alkaline trypsin with maximal activity against benzoyl arginine ethyl ester at pH 9.0, and chymotrypsin, which hydrolyzed benzoyl tryosine ethyl ester at pH 7.5–8.0, were detected in gut homogenates. Total proteolysis, measured using azocasein, had maximal activity at pH 10.0 or higher. Corn borer chymotryptic activity had characteristics similar to the vertebrate enzyme. Both tryptic activities differed from vertebrate trypsin by being insensitive to ovomucoid trypsin inhibitor. High and low alkaline trypsin differed from each other by their pH optima. High alkaline trypsin was activated by magnesium and calcium, and low alkaline trypsin was not affected by inclusion of either chemical in the assay mixture.

Symbiont-independent digestion of cellulose and starch in Panesthia cribrata Saussure, an Australian wood-eating cockroach

Respiratory quotient values (i.e. carbon dioxide produced/oxygen consumed) of 0.98–1.03 were obtained when Panesthia cribrata was maintained on diets of wood, Avicel, filter paper or starch; this is consistent with only carbohydrate being metabolized for energy production by the cockroach. The rate of carbon dioxide production on various cellulose and starch diets at 22°C (2.9–6.9 μmol/h/g cockroach) indicates that glucose is being metabolised in vivo at 0.46–1.15 μmol/h/g cockroach. The in vitro rate of glucose production from crystalline cellulose in whole gut homogenates is 1.05 ± 0.09 μmol/h/g cockroach. Endogenous cellulase is secreted in the epithelium of the anterior ventriculus and is found predominantly (98%) in the foregut and the anterior ventriculus. The cellulase consists of two main components, an endo-β-1,4-glucanase (EC 3.2.1.4) M r, 2.4 × 10 4) which is active predominantly against carboxymethyl cellulose and a β-1,4-glucosidase (EC 3.2.1.21) (M r, 1.2 × 10 5) which is active predominantly against cellobiose. The β-1,4-glucosidase activity is inhibited by glucono-δ-lactone and is thus true cellobiase activity. The cockroaches are not dependent on protozoa for survival or for cellulase as cockroaches maintained on tetracycline-impregnated filter paper show no change in viability or in cellulase activity. α-Amylase (EC 3.2.1.1) (M r 5.9 × 10 4) activity is distributed in the gut in a similar way to cellulase activity and cockroaches can be maintained on starch for at least 12 weeks.

Mechanism of seed lectin tolerance by a major insect storage pest of Phaseolus vulgaris, acanthoscelides obtectus

AbstractThe bean weevil Acanthoscelides obtectus (Say) is a major storage peat of Phaseolus vulgaris L (kidney, haricot bean). The seeds of P vulgaris contain high levels (up to 30 mg g−1 DM) of lectin, which has been shown to be toxic towards larvae of the related bruchid storage pest of cowpea, Callosobruchus maculatus F. The lack of toxicity of this lectin towards larvae of A obtectus is demonstrated. Unlike the strong binding of lectin to the midgut epithelium observed in larvae of C maculatus, no binding of lectin molecules was found to occur in the gut epithelial cells of A obtectus. This observation provides the basis for a hypothesis explaining the lack of toxicity of P vulgaris lectin towards A obtectus. Assays of proteolytic activity in gut homogenates of C maculatus and A obtectus suggest that the difference in susceptibility of the two insects towards the toxic effects of the lectin is not due to differential inactivation by proteolysis. Besides its effects on larval development, the lectin has a further effect at pupation, causing disruption of adipose tissues in C maculatus but having no effect on A obtectus.

Proteolytic activity in the ectoperitrophic fluid of blood-fed Culex nigripalpus.

In blood-fed Culex nigripalpus Theobald, proteolytic activity appeared in the ectoperitrophic fluid after 3 h, but only after 6 h in a homogenate of the blood-filled midgut. The activity continued to be higher in ectoperitrophic fluid than in whole gut homogenate until about 40 h after the meal, when most of the intact clot had disappeared. Apparently, undigested blood inhibits proteolytic activity. The blood clot lacked activity and the inhibitor could not be removed by washing. The results are compatible with a hypothesis that the peritrophic membrane separates the digestion from the ingestion compartment.

Enzymatic activation of the Bacillus sphaericus mosquito larvicidal toxin

Bacillus sphaericus insecticidal toxin was activated by trypsin, α-chymotrypsin, and mosquito gut homogenates to a form which was cytotoxic to cultured mosquito cells. Gut extracts from highly resistant Aedes aegypti larvae were as effective in activating this toxin as extracts from the highly sensitive species Culex quinquefasciatus. Activation altered the apparent molecular weight of the toxin by ca. 2–4 kDa.

A model of acute infectious neonatal diarrhoea.

Oral inoculation of neonatal MFI mice with enterotoxigenic strains of Escherichia coli that possessed the K99 or F41 antigen or both resulted in severe diarrhoea with high mortality. The diarrhoea was associated with increased fluid in the gut, greatly increased numbers of E. coli in gut homogenates and reduced weight gain compared to control animals. Further studies with strain B44 demonstrated greatly increased numbers of E. coli on the surface of the intestinal mucosa and haemo-concentration. The infection was transmissible between litter-mates. There was no evidence of invasion of the intestinal tissue of infected animals. Gnotobiotic Balb C mice and endotoxin-resistant mice were susceptible to oral inoculation with bovine enterotoxigenic E. coli strains, but neonatal rats were not susceptible to infection with enterotoxigenic E. coli strains B44 or 431. Porcine strains of E. coli that possessed K88 or 987P antigen did not infect neonatal MFI mice but an "atypical" porcine strain (431) which possessed both K99 and F41 antigens caused diarrhoea and a high mortality. The disease in neonatal mice resembled acute diarrhoea caused by these bacteria in other species, particularly the calf, and the model should be of value in assessing the efficacy of therapeutic agents.

Leishmania major and L. donovani: Effects on proteolytic enzymes of Phlebotomus papatasi (Diptera, Psychodidae)

Phlebotomus papatasi is susceptible to Leishmania major which it transmits in nature, but is resistant to L. donovani. The present study compares the effect of L. major and L. donovani on the proteolytic activity of P. papatasi gut enzymes. The experiments measured digestion of C 14-labeled globin by gut homogenates of flies. Homogenates were prepared from flies fed on serum only (controls) or from flies fed serum containing promastigotes or their dried culture overlayer. In other experiments, the promastigotes or dried culture overlayers were added in vitro to the gut homogenate of control flies. Proteolytic activity of gut homogenate from flies infected with L. major was about one-third less than that of controls, while that from flies infected with L. donovani was one-third greater. Ingestion of L. major dried culture overlayer had an effect on flies similar to that of the promastigotes, while L. donovani dried culture overlayer produced no significant effect. When added to gut homogenate in vitro, promastigotes of both species promoted proteolysis as did dried culture overlayer of L. major. Dried culture overlayer of L. donovani, however, had an opposite effect. It is suggested that the observed reduction in proteolytic activity caused by L. major infection may result from inhibition of enzyme production.

Rapid immunoassay for detection of Crypto-sporidium oocysts

A direct immunological method by means of latex agglutination (LX) reaction was applied to the demonstration of Cryptosporidium oocysts from stools and gut homogenates. The LX method gave a positive diagnosis for all the specimens judged positive by the modified Ziehl-Neelsen technique, which served as a reference method, The results of the study indicate that the LX method can be adopted as a diagnostic tool for cryptosporidiosis, but further study concerning the specificity and sensitivity of the method is clearly warranted.

Effects of Bacillus sphaericus 1593 and 2362 spore/crystal toxin on cultured mosquito cells

An in vitro assay system for the toxin of Bacillus sphaericus strains 1593 and 2362 has been developed utilizing cultured Culex quinquefasciatus cells. The cytotoxic activity of extracts of B. sphaericus strain 1593 did not necessarily correlate with insecticidal activity. Cytotoxicity and larvicidal activity were neutralized by immune rabbit serum prepared against crude toxin extracts as well as by serum prepared against purified toxin from strain 2362. This purified toxin was also found to be cytotoxic. Activation with mosquito larval gut homogenates enhanced cytotoxicity of both 1593 extracts and purified toxin from 2362. The activity of cytotoxic preparations against three mosquito cell lines paralleled the activity of B. sphaericus spores against larvae of these mosquito species. The results suggest the presence of a protoxin and one or more cytotoxic proteins derived from it.

Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

AbstractMuch of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha‐casein, and N‐benzoyl‐DL‐arginine napthylamide‐(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p‐hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman‐Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L‐Trans‐epoxysuccinyl‐leucylamido‐(4‐guanidino)‐butane (E‐64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.

Carbohydrase and esterase activity in the gut of larvalCallosobruchus maculatus

Carbohydrase activity has been demonstrated in homogenates of the alimentary tracts of late instar larvae ofC. maculatus:β-D galactosidase, α-D glucosidase and N-acetylβ-D glucosaminidase activities were comparable and significantly greater than α-D galactosidase,β-D glucosidase and α-D mannosidase. The effects of pH and substrate concentration are reported. The presence and changes in pattern of non-specific esterase activity in larval and adult gut homogenates is also described.