Response of the surrounding intestine to patch enteroplasty.
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Animals↗
Biomedical subjects
S K Saxena
Publications and source records attributed to S K Saxena.
Efficacy of milk-based diets in persistent diarrhea: a randomized, controlled trial.
OBJECTIVE: Previous studies have shown increased stool output when children with persistent diarrhea (PD) received milk as the predominant source of nutrition. METHODS: We evaluated the efficacy of milk given in modest amounts as a part of a mixed diet in children with PD. One hundred sixteen children 3 to 24 months of age with diarrhea for between 14 days and 12 weeks were allocated to milk-based (n = 60) or milk-free (n = 56) cereal dietary regimens. The two diets were isocaloric (86.9 calories/100 g for < or = 9 months; 95.6 cal/100 g for > 9 months) consisting of puffed rice cereal, sugar, and oil differing in only their source of protein, which was either milk or egg white, respectively. An average of 30% of the calories were constituted by milk in the milk-cereal diet. Both diets were offered at the rate of 150 kcal/kg per day. Children receiving milk-cereal consumed an average of 1.9 g/kg lactose per day. RESULTS: The baseline characteristics in the two groups were similar. Comparable amounts of diet were consumed in both groups. The milk-cereal group did not have higher median (range) stool output (g/kg/h) compared with the milk-free group during a 0- to 48-hour (milk-cereal, 1.7 [0.2 to 8.7]; milk-free, 1.5 [0.1 to 6.6]) or 0- to 120-hour (milk-cereal, 1.6 [0.4 to 7.2]; milk-free, 1.3 [0.1 to 7.6]) period. The percentage of weight gain was similar in the two groups, and there were no significant differences in the duration of diarrhea. Overall, 23 children had treatment failures, 10 (17%) in the milk-cereal and 13 (23.6%) in the milk-free groups. CONCLUSIONS: Our findings suggest that modest intakes of milk are well tolerated as a part of mixed diet during PD.
Animals↗
A study of the intracellular routing of cytotoxic ribonucleases.
Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and indicating that degradation of RNA is the intracellular lesion causing toxicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility.
Amino Acid Sequence↗
Epithelial cell proliferation and biodistribution of radiolabeled urogastrone in the gastrointestinal mucosa of young and old mice.
We have evaluated epithelial cell proliferation and biodistribution of radiolabeled recombinant human urogastrone/epidermal growth factor (125I rhUG/EGF) in the gastrointestinal mucosa of young (2+ months) and old (30+ months) mice. Animals were injected intraperitoneally with the metaphase arrest agent vincristine sulfate and intravenously with 125I rhUG/EGF. Animals were killed after two hours. Crypt cell production rate and uptake of radiolabeled urogastrone were measured in the same intestinal tissues. The results demonstrated that older animals had significantly greater crypt cell production rate as compared to young. However, the uptake of 125I rhUG/EGF was not significantly different (except in duodenum, where the uptake of 125I rhUG/EGF was significantly greater in young compared to old animals) between young and old animals. This suggests that increased epithelial cell proliferation in the aging animals is not associated with increased uptake of 125I rhUG/EGF. Thus epidermal growth factor/urogastrone may not be a primary factor for the intestinal kinetic differences which exist between young and old animals.
Aging↗
Acute viral cholecystitis in viral hepatitis.
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Acute Disease↗
Effect of systemic interleukin-3 administration on epithelial cell proliferation in mouse intestine.
The effect of interleukin-3 (IL-3) on the crypt cell production rate (CCPR) in the intestine of mice was studied using a stathmokinetic technique combined with crypt microdissection. Interleukin-3 (0.71 micrograms/injection) was administered subcutaneously (s.c.) as two injections per day for 7 successive days and small mucosal pieces (duodenum, jejunum, ileum and colon) removed at necropsy were organ cultured in the presence of the metaphase arrest agent vincristine sulfate for two hours. The number of metaphases was enumerated in dissected crypts and CCPR calculated. The results demonstrated that the CCPR was significantly increased in all mucosal segments in the IL-3 treated animals compared to saline injected controls. These results suggest that the growth promoting properties of IL-3 are not restricted to hematopoietic cells when used in vivo and may directly or indirectly increase epithelial cell turnover in gut mucosa.
Animals↗
Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily.
Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.
Amino Acid Sequence↗
Cell surface and intracellular functions for ricin galactose binding.
The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.
Animals↗
Association of radiolabeled urogastrone binding with regenerating intestinal mucosa and epidermal growth factor/urogastrone producing organs in rat.
In the present study, we have tested the hypothesis that the regeneration of intestinal epithelium is regulated by changes in the uptake of radiolabeled recombinant human urogastrone (125I rhUG) in the regenerating mucosa and epidermal growth factor/urogastrone (EGF/URO) producing organs in the rats. Operations were performed on rats to approximate the ileal mucosa to the serosal surface of the cecum. This procedure allows the regeneration of ileal mucosa onto the serosal surface of the cecum. Groups of 5 rats were killed on the 2nd, 4th, 8th and 12th post-operative days. Two hours before autopsy, rats were given 0.5 ml (50 microCi with 30 micrograms protein) of 125I rhUG intravenously and the following tissues were removed: regenerating mucosa, salivary gland, duodenum, liver and kidney. Results indicated that the uptake of 125I rhUG was significantly greater in the salivary gland and duodenum on the 2nd post-operative day which gradually tapered with increasing time after surgery. A similar pattern in the uptake of 125I rhUG was also evident in the regenerating mucosa. Further analysis revealed a significant correlation between the uptake of 125I rhUG in the salivary gland and duodenum vs rate of epithelialization and uptake of 125I rhUG in the regenerative mucosa. These results suggested that the endogenous EGF/URO produced in the salivary gland and duodenum may be a factor in the regulation of intestinal regeneration; however, the mechanism responsible for reflex stimulation of EGF/URO production in these organs is not known.
Animals↗
Role of epidermal growth factor in intestinal regeneration.
We have quantitated uptake of radiolabeled recombinant human urogastrone/epidermal growth factor (125I rhUG/EGF), crypt cell production rate, and rate of epithelialization on days 2, 4, 8, and 12 after operation in the ileum, mucosa adjacent to the wound, and neomucosa (regenerating mucosa) during healing of a full-thickness ileal defect in rats. The uptake of 125I rhUG/EGF in adjacent mucosa and neomucosa was highest on the day 2 (0.64% +/- 0.1% and 0.98% +/- 0.14% injected dose/gm 125I rhUG) and significantly decreased by postoperative day 8. The crypt cell production rate in the adjacent mucosa and neomucosa (23.8 +/- 4.2 and 21.9 +/- 2.1 cells/crypt/hr) peaked on the postoperative days 4 and 8, respectively. The epithelial cell migration rate was increased significantly in the neomucosa on the day 2 (33 +/- 2 mm/day) and tapered gradually until postoperative day 12 (14 +/- 1 mm/day). A linear correlation was evident between the rate of epithelialization and uptake of 125I rhUG/EGF in the neomucosa (r = 0.93; p less than or equal to 0.05). These findings suggest that epithelial cell migration occurs earlier than cell proliferation during regeneration of intestinal epithelium and is closely associated with changing concentration of 125I rhUG/EGF in the neomucosa. This suggests that endogenous EGF may play an important role in intestinal regeneration.
Animals↗
Lead poisoning due to environmental pollution.
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Acute Disease↗
Cytotoxic potential of ribonuclease and ribonuclease hybrid proteins.
Pancreatic RNase injected into Xenopus oocytes abolishes protein synthesis at concentrations comparable to the toxin ricin yet has no effect on oocyte protein synthesis when added to the extracellular medium. Therefore RNase behaves like a potent toxin when directed into a cell. To explore the cytotoxic potential of RNase toward mammalian cells, bovine pancreatic ribonuclease A was coupled via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M whereas greater than 10(-5) M native RNase was required to inhibit protein synthesis. Cytotoxicity requires both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Compounds that interfere with transferrin receptor cycling and compartmentalization such as ammonium chloride decreased the cytotoxicity of transferrin-RNase. After a dose-dependent lag period inactivation of protein synthesis by transferrin-RNase followed a first-order decay constant. In a clonogenic assay that measures the extent of cell death 1 x 10(-6) M transferrin-RNase killed at least 4 logs or 99.99% of the cells whereas 70 x 10(-6) M RNase was nontoxic. These results show that RNase coupled to a ligand can be cytotoxic. Human ribonucleases coupled to antibodies also may exhibit receptor-mediated toxicities providing a new approach to selective cell killing possibly with less systemic toxicity and importantly less immunogenicity than the currently employed ligand-toxin conjugates.
Ammonium Chloride↗
Comparison of RNases and toxins upon injection into Xenopus oocytes.
Several toxins abolish cellular protein synthesis by attacking specific sites in 28 S RNA. One of these toxins, alpha-sarcin, is an RNase that also cleaves nonspecifically on the 3' side of purines in deproteinized RNA. Several other RNases were injected into Xenopus oocytes, examined for their ability to abolish protein synthesis, and compared with alpha-sarcin and ricin. Surprisingly, pancreatic RNase A or B abolished oocyte protein synthesis at concentrations (approximately 0.03 nM) comparable to, or lower than, the amount of alpha-sarcin (approximately 2 nM) or ricin (approximately 0.07 nM) required to abolish protein synthesis. RNases S and T1 only inhibited oocyte protein synthesis when used at concentrations approximately 10 x higher than RNase A whereas RNases C, T2, U2, and nuclease P1 required concentrations approximately 100 times higher than RNase A to abolish protein synthesis. There was a direct correlation between the degradation of oocyte RNA and the inhibition of protein synthesis. The RNase inhibitors RNasin and Inhibit-Ace injected into the oocyte both prevented RNase A from hydrolyzing oocyte rRNA and abolishing protein synthesis. Enzymatically inactive oxidized RNase A did not inhibit protein synthesis when injected into the oocyte. None of the RNases or alpha-sarcin abolished protein synthesis when added to oocyte extracellular medium. Angiogenin is a human plasma protein that induces blood vessel formation in chick embryos, has 35% amino acid identity with RNase A, and cleaves 18 S and 28 S RNA in rabbit reticulocyte lysates (St. Clair, D. K., Rybak, S. M., Riordan, J. F. & Vallee, B. L. (1988) Biochemistry 27, 7263-7268, and references therein). Recombinant angiogenin injected into oocytes abolished protein synthesis, and this toxic effect was inhibited by RNasin but was not inhibited by Inhibit-Ace. Unlike RNase A and the other nucleases that hydrolyzed cellular rRNA, no cleavage of 18 or 28 S RNA by recombinant angiogenin was seen at concentrations 100 x greater than necessary to abolish protein synthesis. Recombinant angiogenin must selectively attack specific RNA(s) or another target in the cell.
Animals↗
Alanine-based oral rehydration solution: assessment of efficacy in acute noncholera diarrhea among children.
This randomized, double-blind trial determined whether adding 90 mmol/L of alanine with a reduction in glucose to 90 mmol/L (alanine ORS) improves the efficacy of the standard oral rehydration solution (WHO-ORS). One hundred twenty-nine males aged 3-48 months with weight for length greater than or equal to 70% of NCHS, diarrheal duration less than or equal to 96 h, and clinical signs of mild to moderate dehydration were randomly allocated to either treatment group. During 0-6 h of treatment, ORS was offered at 120 ml/kg for rehydration without food or water. Beyond 6 h, ORS was offered as a volume-to-volume replacement for stool losses and a mixed diet of uniform composition was offered in amounts standardized for body weight. The most frequently isolated pathogens in alanine ORS and WHO-ORS groups were rotavirus (42 and 48%, respectively) and enterotoxigenic Escherichia coli (15 and 12%, respectively). In the 0-6 h period when food was withheld, median urine output in ml/kg (8;5, p less than 0.05) and percentage decrease in total serum solids (9:7%, p = 0.06) was significantly greater in alanine ORS than in WHO-ORS; median ORS intake and stool output were marginally lower in the alanine group but the differences were statistically not significant. Between 0 h and recovery, although the median values for duration of diarrhea (56.5 and 65.0 h), ORS consumption (260 and 323 ml/kg), and stool output (188.4 and 216.3 g/kg) were lower in the alanine ORS group, these differences with the WHO-ORS group were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
Epithelial cell proliferation and uptake of radiolabeled urogastrone in the intestinal tissues following abdominal irradiation in the mouse.
We have evaluated the rate of crypt cell production and uptake of radiolabeled recombinant human urogastrone (125I-rhUG) in the intestinal tissues of the mouse at 3, 5, 7, 9, and 12 days following irradiation of the abdomen with 9 Gy. At autopsy, the animals were injected intraperitoneally with 1 microgram/g body weight of the metaphase arrest agent, vincristine sulfate, and 25 muCi of 125I-rhUG (specific activity 1.7 muCi/micrograms) to quantify the rate of crypt cell production and uptake of radiolabeled urogastrone, respectively. The results indicated that the rate of crypt cell production was increased significantly in the irradiated animals compared to the unirradiated animals and showed a peak on the 3rd and 5th postirradiation days in small intestine and colon, respectively. The uptake of 125I-rhUG was increased significantly on the 3rd postirradiation day in the intestinal tissues but showed a bimodal pattern with peaks on the 3rd and 9th postirradiation days. These results suggest that there may be a close association between epithelial cell proliferation and uptake of 125I-rhUG, particularly in the early part of recovery of intestinal mucosa following irradiation. However, these data do not discriminate whether the increased uptake of 125I-rhUG is the cause or the effect of proliferation induced by an irradiation stimulus. Further analysis also revealed that there was no relationship between crypt depth and 125I-rhUG uptake. However, crypt depth was inversely correlated with villus height in the proximal small intestine but not in the ileum. Villus height was correlated inversely with 125I-rhUG uptake in the ileum and jejunum but not the duodenum. The rate of crypt cell production was strongly correlated with crypt depth throughout the intestine and inversely correlated with villus height. This suggests that villus-to-crypt inhibitory feedback may be a primary regulator of cellular proliferation in the crypts and the association of 125I-rhUG uptake with proliferation indirectly reflects this interaction.
Animals↗
Ribozymes correctly cleave a model substrate and endogenous RNA in vivo.
The alpha-sarcin domain of 28 S RNA in Xenopus oocytes is attacked by several catalytic toxins (e.g. alpha-sarcin and ricin) that abolish protein synthesis. We synthesized 6 ribozymes targeted to the alpha-sarcin domain and to an oligoribonucleotide (34-mer) that mimics this domain. Sarcin ribozyme 5 (SR5) efficiently cleaved after the CUC site in the synthetic 34-mer in vitro at 50 degrees C. SR5 also cut the same site when both substrate and ribozyme were coinjected or injected separately into oocytes at 18 degrees C. Correct cleavage in vivo was shown by isolating and sequencing the large cleavage fragment. The cleavage reaction appeared to function equally well in the oocyte nucleus and cytoplasm. SR5 also correctly cleaved endogenous 28 S RNA in oocytes, although cutting was much less efficient than with alpha-sarcin. We therefore demonstrated that a ribozyme specifically cuts both a model substrate and a cellular RNA in vivo. Earlier work showed that certain injected deoxyoligonucleotides complementary to the alpha-sarcin region abolish protein synthesis. Oocyte protein synthesis was also abolished by an SR5 containing a single G----U substitution that inactivates RNA catalysis, indicating that SR5's translational suppression was perhaps due to antisense function rather than ribozyme cleavage.
Animals↗
Effect of age on intestinal regeneration in the rabbit.
We compared the rate of regeneration of full thickness serosa patched intestinal defects to determine whether this regenerative process is influenced by age. Twenty-five rabbits aged 3, 9, 15, 36 and 60 months were killed 3 weeks after patching. Epithelialization was slightly but significantly greater in the 3-month-old rabbits but there were no differences in contraction of the patched defect or villus development of the regenerating mucosa. Disaccharidase activity was greatest in the older animals but glucose uptake was similar at all ages. Proliferative activity was similar as well. Thus, intestinal regeneration in full thickness intestinal defects does not appear to be influenced significantly by the age of the animals.
Aging↗
Microinjected oligonucleotides complementary to the alpha-sarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes.
The integrity of the alpha-sarcin loop in 28 S ribosomal RNA is critical during protein synthesis. The toxins alpha-sarcin, ricin, Shiga toxin, and Shiga-like toxin inhibit protein synthesis in oocytes by attacking specific nucleotides within this loop (Ackerman, E.J., Saxena, S. K., and Ulbrich, N. (1988) J. Biol. Chem. 263, 17076-17083; Saxena, S.K., O'Brien, A.D., and Ackerman, E.J. (1989) J. Biol. Chem. 264, 596-601). We injected Xenopus oocytes with deoxyoligonucleotides complementary to the 17-nucleotide alpha-sarcin loop of Xenopus 28 S rRNA. Only injected oligonucleotides fully covering the alpha-sarcin loop or slightly beyond inhibited oocyte protein synthesis. Shorter alpha-sarcin domain deoxyoligonucleotides complementary to the alpha-sarcin and ricin sites but not spanning the entire loop were less effective inhibitors of protein synthesis. The alpha-sarcin domain oligonucleotides covering the entire loop were more effective inhibitors of protein synthesis than injected cycloheximide at equivalent concentrations. Control oligonucleotides complementary to nine other regions of Xenopus 28 S rRNA as well as universal M13 DNA sequencing primers had no effect on oocyte protein synthesis. Oligonucleotides complementary to the highly conserved alpha-sarcin domain therefore represent an alternative to catalytic toxins for causing cell death and may prove effective in immunotherapy.
Animals↗