Epidermal growth factor in saliva and serum of infants with necrotising enterocolitis.
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Enterocolitis, Pseudomembranous↗
Biomedical subjects
C R Erwin
Publications and source records attributed to C R Erwin.
Epidermal growth factor augments adaptation following small bowel resection: optimal dosage, route, and timing of administration.
BACKGROUND: In assorted animal models of small bowel resection (SBR), exogenous epidermal growth factor (EGF) has been shown to augment intestinal adaptation. This study was designed to elucidate the optimal dose, route, and timing of exogenous EGF to boost adaptation in our murine model of SBR. METHODS: Male ICR mice underwent either 50% proximal SBR or sham surgery (bowel transection with reanastomosis) and then randomized to receive either saline or human recombinant EGF (5, 50, 150, or 300 microg/kg/day) by twice daily intraperitoneal (i.p.) injection or orogastric gavage (o.g.). At 7 days, protein and DNA content, crypt depth, and villus height were determined in the ileum. The premium dose and route was then given for 1 week either during (1 week after SBR) or after the adaptive phase (1 month after SBR). Differences between group means were analyzed using ANOVA. A P < 0.05 was considered significant. RESULTS: EGF enhanced DNA and protein content, crypt depth, and villus height to the greatest extent at a dosage of 50 microg/kg/day by the o.g. route. EGF had no significant effect on enhancing adaptation when given after the adaptive response had already occurred. CONCLUSIONS: Intestinal adaptation is optimally enhanced by a specific dose and route of EGF. Exogenous EGF enhances adaptation only during the adaptive response to SBR and not after it has already taken place. Determination of the best circumstances for EGF administration will permit a systematic approach toward understanding a mechanism for the beneficial effect of EGF during intestinal adaptation.
Adaptation, Physiological↗
The EGF\EGF-receptor axis modulates enterocyte apoptosis during intestinal adaptation.
BACKGROUND: Adaptation after small bowel resection (SBR) is characterized by a new set point in the balance of enterocyte proliferation and apoptosis. Since epidermal growth factor (EGF) augments both proliferation and adaptation, we sought to determine the effect of EGF receptor manipulation on apoptosis following SBR. MATERIALS AND METHODS: Male ICR mice underwent 50% SBR or sham operation (bowel transection with reanastomosis) and then were given EGF (50 microg/kg/day) or saline by orogastric gavage. At 1 week, a proliferation index (PI) was measured in the ileum by BrdU uptake and an apoptosis index in crypts (cAI) and villi (vAI) scored by counting apoptotic bodies in enterocytes. In other experiments, AI was scored after SBR in mice with defective receptors (waved-2). Results are expressed as means +/- SE and evaluated statistically using ANOVA. # denotes P < 0.001. RESULTS: Following SBR, EGF increased PI (40 +/- 2% vs 50 +/- 1% BrdU + cells; #), villus height (252 +/- 4 micro(m) vs 401 +/- 15 micro(m); #), and crypt depth (77.3 +/- 1.5 micro(m) vs 120.8 +/- 5 micro(m); #). When compared with sham, SBR resulted in increased cAI (0.3 +/- 0.02 vs 2.0 +/- 0.1; #) and vAI (0.4 +/- 0.05 vs 1.1 +/- 0.1; #). EGF attenuated both cAI (0.5 +/- 0. 04) and vAI (0.5 +/- 0.03) following SBR. In the waved-2 mice, the highest levels of cAI (3.1 +/- 0.2) and vAI (3.6 +/- 0.3) were noted after SBR. CONCLUSIONS: Enterocyte apoptosis during adaptation is attenuated by EGF and exaggerated when the EGF receptor is defective. In addition to enhancing proliferation, suppression of apoptosis may provide a previously unrecognized mechanism for the beneficial effect of EGF during intestinal adaptation.
Adaptation, Physiological↗
Epidermal growth factor upregulates the expression of its own intestinal receptor after small bowel resection.
BACKGROUND/PURPOSE: Epidermal growth factor (EGF) binds to its enterocyte receptor and enhances intestinal adaptation after massive small bowel resection (SBR). To ascertain the mechanism for enhanced adaptation by EGF, we sought to determine the effect of EGF administration on in vivo expression of the intestinal EGF receptor after SBR. METHODS: Male ICR mice underwent a 50% proximal SBR and then were assigned randomly to EGF (150 microg/kg/d) or saline by twice daily intraperitoneal injection. After 3 days, the ileum was harvested and total protein and DNA content were measured. Northern hybridization and a ribonuclease protection assay were used to detect qualitative and quantitative expression of EGF receptor mRNA. The remaining ileum was pooled for each group and Western blotting used to determine expression of EGF receptor protein. RESULTS: EGF augmented adaptation after SBR as monitored by significant increases in ileal protein (2.7+/-0.08 saline versus 3.9+/-0.17 mg/cm EGF; P<.001) and DNA (55.8+/-1.6 saline versus 104+/-8.4 microg/cm EGF; P<.001) content. Northern blotting results showed a marked (>fivefold) increase in ileal EGF receptor mRNA, which was confirmed with the ribonuclease protection assay. Administration of EGF after SBR induced a similar expression of EGF receptor protein. CONCLUSIONS: EGF enhanced intestinal adaptation after SBR. This augmented response is associated with increased ileal expression of EGF receptor mRNA and protein. Increased EGF receptor expression and subsequent enhanced ligand/ receptor activity may be one important mechanism for the beneficial effect of EGF administration during intestinal adaptation.
Adaptation, Physiological↗
Intestinal adaptation is enhanced by epidermal growth factor independent of increased ileal epidermal growth factor receptor expression.
BACKGROUND/PURPOSE: Intestinal adaptation after massive small bowel resection (SBR) is augmented by epidermal growth factor (EGF) via an unknown mechanism. We recently have observed that EGF increases the expression of EGF receptor mRNA and protein content in the remnant ileum after SBR. The purpose of this study was to determine whether the magnitude of EGF-induced receptor expression correlates with intestinal adaptation. METHODS: A 50% proximal SBR or sham operation (bowel transection with reanastomosis) was performed on male ICR mice. Animals from each group were then selected randomly to receive either human recombinant EGF (150 microg/kg/d) or saline by twice daily intraperitoneal injections. The remnant ileum was harvested at 1 week, and parameters of adaptation measured as changes in protein content. Ileal EGF receptor mRNA was quantitated using a ribonuclease protection assay. Changes in the expression ileal EGF receptor protein were determined by Western blot after immunoprecipitation. Comparisons of mean values between groups was performed using analysis of variance (ANOVA) and a P value of less than .05 was considered significant. Values are presented as mean +/- SEM. RESULTS: EGF was mitogenic to the ileum after sham operation as monitored by increases in ileal protein content (2.21 +/- 0.002 mg/cm Sham v 2.97 +/- 0.25 mg/cm Sham +/- EGF; P < .05). After SBR, adaptation resulted in increased ileal protein content (4.45 +/- 0.27 mg/cm), which was substantially boosted by EGF (5.98 +/- 0.39 mg/cm; P < .05). No differences were detected in ileal EGF receptor mRNA or protein expression between Sham or SBR groups that did not receive EGF. However, EGF significantly enhanced the expression of ileal EGF receptor mRNA to an equal extent after both sham and SBR (approximately threefold). The magnitude of this increase in EGF receptor protein (four- to sixfold) was similar in both EGF groups as shown by Western blotting. CONCLUSIONS: Changes in ileal EGF receptor expression are not mandatory for adaptation to occur. EGF upregulates the expression of mRNA and protein for its own intestinal receptor in vivo. Because EGF-induced receptor expression was comparable after both SBR and Sham operation, the beneficial effect of EGF during adaptation is likely caused by other factors in addition to increased receptor expression.
Adaptation, Physiological↗
Adaptation after small bowel resection is attenuated by sialoadenectomy: the role for endogenous epidermal growth factor.
BACKGROUND: Epidermal growth factor (EGF) is likely involved during adaptation after small bowel resection (SBR) because some studies have shown enhanced adaptation by EGF administration. Because the major source of endogenous EGF in mice is the submandibular glands, we sought to determine the effect of submandibular gland excision (SAL) and luminal or systemic EGF replacement on adaptation after SBR. METHODS: A 50% proximal SBR or Sham-SBR (bowel transection and reanastomosis) was performed on male C57BL/6 mice after either SAL or gland mobilization only. Additional mice underwent both SBR and SAL and then received daily EGF or saline solution by intraperitoneal or orogastric administration. At 1 week, adaptation was characterized in the ileum as changes in villus height, DNA, and protein content. RESULTS: SAL significantly attenuated the increase in ileal villus height, total protein, and DNA content after SBR. Both systemic and oral EGF reversed these findings equally and significantly augmented all parameters of intestinal adaptation after SAL. CONCLUSIONS: Submandibular EGF is important for the adaptive response to massive SBR. As both luminal and systemic EGF equally reversed the findings following SAL and SBR, the specific site of action for endogenous EGF during adaptation is either the luminal or basolateral surface of the enterocyte.
Adaptation, Physiological↗
Epidermal growth factor receptor expression following small bowel resection.
BACKGROUND: Adaptation following massive small bowel resection (SBR) is an important compensatory response. While epidermal growth factor (EGF) has been shown to augment this response, the mechanism and role of EGF and its intestinal receptor (EGF-R) during adaptation are not known. The purpose of this study was to determine the effect of massive SBR and adaptation on intestinal expression of EGF-R. MATERIALS AND METHODS: Male Sprague-Dawley rats underwent either a 75% mid-SBR with primary reanastomosis or sham operation (bowel transection with reanastomosis). Ileal mucosa was harvested from animals of each group at 6 and 12 hr, 1, 3, and 5 days, and 1, 2, and 4 weeks after operation. Expression of EGF-R protein was studied by Western blotting. Expression of EGF-R mRNA was determined by quantitative reverse-transcriptase polymerase chain reactions normalized to beta-actin. RESULTS: When compared with sham, SBR resulted in a modest (32%) increase in the expression of EGF-R mRNA at 1 week (P < 0.003). A twofold greater expression of EGF-R protein corresponded to this time point. While not statistically significant, expression of EGF-R mRNA was slightly greater after SBR at every other time point measured. CONCLUSION: Following massive SBR, expression of both EGF-R mRNA and protein is slightly increased in the ileum with a more pronounced increase in protein. These findings, which have not previously been reported, suggest that changes in EGF-R signaling may not play a major role during the initiation and or progression of intestinal adaptation following massive SBR.
Animals↗
A defective EGF-receptor in waved-2 mice attenuates intestinal adaptation.
While the pathophysiology of intestinal adaptation following small bowel resection (SBR) is not well understood, there is evidence to suggest an important role for epidermal growth factor (EGF) in this process. In waved-2 mice, a naturally occurring mutation results in reduced EGF receptor protein tyrosine kinase activity. We tested the hypothesis that an intact EGF receptor is essential for adaptation by subjecting this strain of mice to SBR. A 50% proximal SBR or sham operation (bowel transection with reanastomosis only) was performed in waved-2, heterozygous, and wildtype mice. After 3 days, adaptation was characterized in the remnant ileum as changes in DNA and protein content per unit length. Villus height and crypt depth were measured, and crypt cell proliferation rates were determined by the percentage of crypt cells taking up 5-bromodeoxyuridine. Following sham surgery, all mice regained their preoperative weight by the third postoperative day. After SBR, all mice gained weight while the waved-2 mice did not. Ileal DNA and protein significantly increased after SBR in wild-type and heterozygous mice while these parameters were unchanged in the waved-2 mice. Villus height and crypt cell proliferation increased in response to SBR in all groups; however, the changes were less pronounced in the waved-2 mice. Adaptation after SBR is impaired in waved-2 mice. Signal transduction by the EGF receptor is a critical component of this response. These data endorse a crucial role for EGF and its receptor in the pathogenesis of intestinal adaptation.
Adaptation, Physiological↗
Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity.
Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less. For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates. On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster. Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower. In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster. Enzyme adsorption and the subsequent peptide hydrolysis are altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme. This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme. One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface. In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis.
Adsorption↗
Rapid analysis of single-cysteine variants of recombinant proteins.
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Animals↗
Intestinal adaptation following massive small bowel resection in the mouse.
BACKGROUND: Transgenic mice represent powerful tools for studying the role of genes and their expression under multiple conditions, and they may provide a unique model for studies of intestinal adaptation after massive small bowel resection (SBR). This study characterized a successful model for SBR and intestinal adaptation in the mouse. STUDY DESIGN: Sham operation (bowel transection with reanastomosis) or SBR was performed on male C57BL/6 mice. A solid or liquid diet, various sizes of monofilament suture for the anastomosis, and resection of 50 or 75 percent of the proximal small intestine were studied. In other studies, intestinal adaptation was characterized as changes in intestinal wet weight, DNA, protein, villus height, crypt depth, and crypt cell proliferation rates at 12 hours, 24 hours, three days, and one, two, and four weeks after 50 percent SBR. RESULTS: Survival was significantly improved with a liquid diet (8 percent compared with 88 percent; p < .001) and modestly improved by using the smallest suture for anastomosis (60 percent for 7-0 compared with 88 percent for 9-0; p = not significant). Mice did not tolerate more than 50 percent SBR (16 percent survival rate for 75 percent SBR compared with 85 percent survival rate for 50 percent SBR; p < .01). Small bowel resection augmented ileal wet weight, DNA and protein content, villus height, crypt depth, and crypt-cell proliferation rates. CONCLUSIONS: Provision of a liquid diet, using a small suture for anastomosis, and resection of no more than 50 percent of the proximal small intestine are important for survival. This model will permit researchers using transgenic mice to better understand critical genes during intestinal adaptation after SBR.
Adaptation, Biological↗
Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN' with enhanced surface stability.
Enzyme adsorption and inactivation at the solid/liquid interface for subtilisin BPN' show a strong dependence on the nature of the solid surface. Adsorption of BPN' at the solid/liquid interface is considerably greater for a hydrophobic surface than for a hydrophilic one. Likewise, the rate of inactivation of the wild-type BPN' is over five times greater when equilibrated with a hydrophobic surface than with a hydrophilic surface. The rate data from these enzyme inactivation experiments performed at 50 degrees C are best fit by a second-order kinetic equation, suggesting a bimolecular pathway to inactivation. The role of increased surface adsorption on this bimolecular inactivation is discussed in terms of two different mechanisms. Several site-specific variants of subtilisin BPN' have been made in an attempt to alter the surface-inactivation of the wild-type enzyme. The extent of adsorption on the model surfaces is significantly lowered by certain lysine to phenylalanine changes in BPN'. Consequently, the surface autolytic stability shows a 4-fold improvement. The change in surface autolytic stability is achieved even though the basic kinetic parameters (kcat and KM) of the variant enzymes are not significantly different on a soluble substrate. The results provide insights into the use of mutagenesis to probe the mechanism of protein interactions with surfaces.
Adsorption↗
Effects of engineered salt bridges on the stability of subtilisin BPN'.
Variants designed using PROTEUS have been produced in an attempt to engineer stabilizing salt bridges into subtilisin BPN'. All the mutants constructed by site-directed mutagenesis were secreted by Bacillus subtilis, except L75K. Q19E, expressed as a single variant and also in a double variant, Q19E/Q271E, appears to form a stabilizing salt bridge based on X-ray crystal structure determination and differential scanning calorimeter measurements. Although the double mutant was found to be less thermodynamically stable than the wild-type, it did exhibit an autolytic stability about two-fold greater under hydrophobic conditions. Four variants, A98K, S89E, V26R and L235R, were found to be nearly identical to wild-type in thermal stability, indicative of stable structures without evidence of salt bridge formation. Variants Q271E, V51K and T164R led to structures that resulted in varying degrees of thermodynamic and autolytic instability. A computer-modeling analysis of the PROTEUS predictions reveals that the low percentage of salt bridge formation is probably due to an overly simplistic electrostatic model, which does not account for the geometry of the pairwise interactions.
Bacillus subtilis↗
Cell-specific enhancers in the rat exocrine pancreas.
Pancreatic amylase, chymotrypsin B, and trypsin I genes are specifically expressed in the exocrine pancreas. The 5'-flanking regions of these genes direct preferential expression of a linked reporter function (chloramphenicol acetyltransferase) in the pancreatic exocrine cell line AR4-2J. The sequences upstream of the amylase and chymotrypsin genes that are required for this cell specific activity possess the characteristics of transcriptional enhancers. We have mapped the regions responsible for enhancer activity by deletion analysis. Modification of specific nucleotide sequences within these regions can alter or eliminate enhancer function. Comparison of the 5'-flanking regions of nine genes expressed in the exocrine pancreas identifies a family of short related sequences. These sequences are located within the enhancer regions that we have mapped and may play a role in the regulation of the expression of pancreatic exocrine-specific genes.
Acetyltransferases↗
Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones.
Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.
Amino Acid Sequence↗
Nucleotide sequence of the bovine growth hormone chromosomal gene.
A library of cloned bovine DNA fragments was constructed and screened for growth hormone sequences. The growth hormone gene was isolated from this library and its nucleotide sequence determined. The likely transcription initiation site was located using the S1 nuclease protection procedure. The bovine growth hormone gene contains approximately 1793 nucleotides and consists of five exons separated by four intervening sequences. The sequence TATAAA is found in the 5' flanking region and probably is involved in facilitating transcription initiation. Comparison of the bovine growth hormone gene to the known sequence of the rat and human genes reveals that the coding regions of the three genes are highly conserved. In general the intervening sequences are much less similar than the coding regions. Interestingly, all three growth hormone genes share a conserved (but nonidentical) 40 base pair region within the 5' flanking region. This conserved region may be an important sequence involved in the hormonal regulation of growth hormone gene transcription. Analysis of GH sequences present in total bovine DNA suggests that the bovine genome contains a gene similar to the cloned gene as well as a different, but related, gene. The functional significance of the two genes remains to be explored. Analysis of nuclear species of growth hormone mRNA has demonstrated the presence of RNAs of 2100, 1400 and 1000 nucleotides containing growth hormone sequences. These likely correspond to a polyadenylated primary transcript, a processing intermediate and mature growth hormone mRNA, respectively.
Amino Acid Sequence↗
Analysis of 5' flanking sequences and intron-exon boundaries of the rat prolactin gene.
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Animals↗
Structure of the rat prolactin gene.
The organization and sequence of the rat preprolactin gene has been investigated. Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon. Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments. The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments. The sequence of more than 1800 nucleotides of the cloned DNA has been determined. The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin. These coding regions are separated by an intervening sequence of 597 nucleotides. At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin. Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.
Amino Acid Sequence↗