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1.
Food Funct ; 4(8): 1148-84, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23760593

ABSTRACT

Several antioxidants and agents having similar antioxidant effects are known to exert beneficial effects in ameliorating the injurious effects of hyperglycemia on liver in different diabetic in vitro and in vivo models. The review deals with some of the agents which have been shown to exert protective effects on liver against hyperglycemic insult and the various mechanisms involved. The different classes of agents which protect the diabetic liver or decrease the severity of hyperglycemia mediated injury include flavonoids, catechins, and other polyphenolic compounds, curcumin and its derivatives, certain vitamins, hormones and drugs, trace elements, prototypical antioxidants and amino acids. Some of the pronounced changes mediated by the antioxidants in liver exposed to hyperglycemia include decreased oxidative stress, and alterations in carbohydrate and lipid metabolism. Other mechanisms through which the agents ameliorate hyperglycemia mediated liver injury include decrease in oxidative DNA and protein damage, restoration of mitochondrial structural and functional integrity, decrease in inflammation and improved insulin signaling. Thus, antioxidants may prove to be an important mode of defense in maintaining normal hepatic functions in diabetes.


Subject(s)
Antioxidants/administration & dosage , Diabetes Mellitus/drug therapy , Hyperglycemia/drug therapy , Liver/injuries , Oxidative Stress/drug effects , Animals , Diabetes Mellitus/metabolism , Humans , Hyperglycemia/metabolism , Liver/drug effects , Liver/metabolism
2.
Mol Cell Biochem ; 378(1-2): 83-9, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23504145

ABSTRACT

The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.


Subject(s)
Brain/enzymology , Intramolecular Lyases/metabolism , Protein Processing, Post-Translational , Amino Acid Motifs , Animals , Antibodies/chemistry , Intestines/enzymology , Intramolecular Lyases/chemistry , Intramolecular Lyases/immunology , Isoenzymes/metabolism , Male , Molecular Weight , Organ Specificity , Peptide Fragments/chemistry , Peptide Fragments/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/metabolism , Phosphorylation , Rats , Rats, Sprague-Dawley , Serine/chemistry , Serine/metabolism , Testis/enzymology
3.
J Dev Orig Health Dis ; 1(1): 67-74, 2010 Feb.
Article in English | MEDLINE | ID: mdl-25142933

ABSTRACT

Maternal under-nutrition (MUN) during gestation results in growth-restricted newborns with reduced glomerular number and subsequent hypertension. We investigated dysregulation of glial derived neurotrophic factor (GDNF) and MAPK-ERK (mitogen-activated protein kinase-extracellular signal-regulated protein kinase) signal pathway gene expression following MUN. MUN rats were 50% food restricted from embryonic day 10 till postnatal day 1. Kidneys were harvested at embryonic day (E)20, and postnatal days (P)1 and 21. Kidney protein expression was determined by Western blot. At E20, protein expression of growth factor receptor alpha 1 (GFRα1) and phosphorylated ERK1/2 and mitogen-activated protein kinase kinase (MEK)1/2 were reduced significantly, and immunohistochemistry confirmed reduction of phosphorylated ERK (pERK) with maintenance of pERK localization. Total MEK and ERK were unchanged. At P1, only GFRα1 and pERK1/2 were reduced significantly while at P21, expression of all growth factors except total MEK was unchanged. Total MEK was increased. Glomerular number was decreased by 19% in P21 kidneys and blood pressure was increased in 12-week-old rats. In conclusion, GDNF and MAPK-ERK signaling are dysregulated during active nephrogenesis in fetal and early newborn offspring kidneys in the MUN model. This may be a key mechanism in reduced offspring nephrogenesis and programmed hypertension.

4.
J Perinatol ; 28 Suppl 3: S8-13, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19057616

ABSTRACT

To use sheep and rat models and demonstrate that stressors activate fetal glucocorticoid (GC) system, corticotrophin-releasing factor (CRF) system and cholinergic neurotransmitter system (ChNS) leading to propulsive colonic motility and in utero meconium passage. Immunohistochemical studies (IHS) were performed to localize GC-Receptors, CRF-receptors and key molecules of ChNS in sheep fetal distal colon. CRF expression in placenta and enteric endocrine cells in fetal rat system were examined and the effects of acute hypoxia on in utero meconium passage was tested. IHS confirmed localization and gestation dependent changes in GC-Rs, CRF-Rs and cholinergic markers in sheep fetal colon. Rat placenta and enteric endocrine cells express CRF and gastrointestinal tract express CRF-Rs. Hypoxia is a potent inducer of meconium passage in term fetal rats. Stress is a risk factor for in utero meconium passage and laboratory animal models can be used to develop pharmacotherapy to prevent stress-induced in utero meconium passage.


Subject(s)
Gastrointestinal Motility , Hypoxia/complications , Meconium/physiology , Stress, Physiological , Adrenocorticotropic Hormone/physiology , Animals , Disease Models, Animal , Glucocorticoids/physiology , Humans , Rats , Risk Factors , Sheep
5.
Neurogastroenterol Motil ; 20(12): 1328-39, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19019035

ABSTRACT

Meconium passage is frequently observed in association with feto-maternal stress factors such as hypoxia and infection, but the triggering mechanism is unknown. We hypothesize that differential regulation of corticotrophin-releasing factor (CRF) receptors during gestation play an important role in determining the susceptibilities of the fetus to stress-induced in utero meconium passage at term. We examined the innervation patterns of CRF-receptor type 1 (CRF-R1), a stimulator of gastrointestinal motility and CRF-receptor type II (CRF-R2), an inhibitor of gastrointestinal motility in ovine fetal distal colonic segments from very preterm to term gestation. Both CRF-R1 and CRF-R2 receptors were present in muscularis mucosa as well as in longitudinal and circular smooth muscle layers in fetal distal colonic segments at all gestational ages. Quantitative image analysis indicated a 42% increase in CRF-R1 receptor immunoreactivity in muscularis mucosa and a 30% in longitudinal smooth muscle layers from very preterm to term. In contrast, CRF-R2 receptor immunoreactivity in muscularis mucosa as well as in longitudinal and circular smooth muscle layers decreased by 38%, 55% and 51%, respectively, at term. The percentage of enteric ganglia and the number of enteric neurons expressing CRF-R1 receptors were high at term. Western blot analysis identified 235 and 50 kDa molecular species of CRF-R1 receptors and 37 and 28 kDa molecular species of CRF-R2 receptors. In summary, we speculate that downregulation of CRF-R2 receptor abundance with concurrent increases in CRF-R1 receptor levels in myenteric-smooth muscle unit with advancing gestation sensitizes the colonic motility responses to stressors.


Subject(s)
Colon/embryology , Colon/innervation , Colon/metabolism , Receptors, Corticotropin-Releasing Hormone/biosynthesis , Animals , Blotting, Western , Fetus , Fluorescent Antibody Technique , Image Processing, Computer-Assisted , Immunohistochemistry , Microscopy, Confocal , Myenteric Plexus/metabolism , Sheep
6.
Neuroscience ; 151(4): 1184-97, 2008 Feb 19.
Article in English | MEDLINE | ID: mdl-18222042

ABSTRACT

The mechanisms underlying lithium's therapeutic efficacy in the chronic treatment of bipolar disorder are not clearly understood. Useful insights can be obtained by identifying genes that are differentially regulated during chronic lithium treatment. Toward this end, we have used microarray technology to identify mRNAs that are differentially expressed in a human neuronal cell line that has been continuously maintained in therapeutic levels of lithium for 33 days. Significantly, unlike other transcriptomes where predominantly rodent cells were used and a limited number of genes probed, we have used human cells probed with more extensive 44,000 gene microarrays. A total of 671 differentially regulated transcripts, after correcting for false discovery rates, were identified, of which 347 and 324, respectively, were found to be up- and downregulated. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, was the most upregulated while tribbles homolog 3 (TRB3), a pro-apoptotic protein, was the most downregulated, implying a beneficial effect of lithium on neuronal cells. Several of the most highly regulated genes are novel, uncharacterized and encode proteins of unknown function. Differentially expressed genes associated with phosphoinositide metabolism include those encoding phosphatidyl inositol 4-phosphate 5-kinase type II alpha (PIP5K2A), WD repeat domain, phosphoinositide interacting 1 protein (WIPI49), tribbles homolog 3 (TRB3) and sorting nexin 14 (SNX14). A protein interactome using some of the saliently regulated genes identified protein kinase C (PKC) as a major target for lithium action while a global analysis of all 671 differentially expressed genes identified the mitogen-activated protein kinase pathway as the most regulated. The list of highly regulated genes, besides encoding putative targets for antimanic agents, should prove useful in defining novel pathways, or to better understand the mechanisms, underlying the mood stabilization process.


Subject(s)
Antipsychotic Agents/pharmacology , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression/drug effects , Lithium/pharmacology , Cell Line, Tumor , Gene Expression/physiology , Humans , Neuroblastoma/pathology
7.
Exp Dermatol ; 9(4): 283-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10949551

ABSTRACT

The cellular localization of NGF mRNA and its translation products have been identified in ovine hair follicles. NGF mRNA was detected in the proliferating cells of the follicle bulb and differentiating cells of the suprabulbar region, but was absent from the outer root sheath. Western analysis revealed the presence of a 73 kDa NGF prohormone in extracts of ovine flank skin, but the mature 13 kDa NGF was absent. Immunohistochemical analysis with antibodies specific to mouse NGF and a pro-NGF specific domain localized the NGF prohormone to outer root sheath cells in the upper bulb region of the follicle, adjacent to the zone of keratinization. Antibody binding was also associated with the luminal epithelium of the apocrine sweat gland and the pilary canal of the follicle at its junction with the epidermis. These observations, together with the reported presence of high- and low-affinity NGF receptors in the follicle, implicate the NGF prohormone-responsive neuronal system in the regulation of hair growth.


Subject(s)
Hair Follicle/metabolism , Nerve Growth Factor/genetics , Nerve Growth Factor/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Animals , Blotting, Western , Gene Expression Regulation, Developmental , Hair Follicle/growth & development , Immunohistochemistry , In Situ Hybridization , Male , Protein Biosynthesis , Protein Precursors/genetics , Protein Precursors/metabolism , Sheep , Skin/innervation , Skin/metabolism
8.
J Neurochem ; 74(5): 2127-33, 2000 May.
Article in English | MEDLINE | ID: mdl-10800958

ABSTRACT

Examination of commercial recombinant human beta-nerve growth factor (rh-beta-NGF) preparations with polyclonal antibodies specific to 13-kDa NGF and pro-NGF-specific domains revealed the presence of high-molecular-mass immunoreactive proteins, including a 60-kDa NGF prohormone. On incubation with a mixture of N- and O-specific glycosidases, the 60-kDa NGF pro-hormone generated a 32-kDa protein corresponding to the molecular size of NGF precursor predicted by the cloned human NGF cDNA. Highly sensitive chemiluminescence immunoblot analysis of adult rat dorsal root ganglia, spinal cord, and colon tissues with NGF- and pro-NGF domain-specific antibodies also revealed the presence of high-molecular-mass proteins, including the 60-kDa NGF prohormone. Based on the presence of the 60-kDa NGF prohormone in dorsal root ganglia and its efferent tissues, we suggest that proteolytically unprocessed, glycosylated NGF prohormone may mediate interactions between neurons and the tissues they innervate.


Subject(s)
Ganglia, Spinal/chemistry , Nerve Growth Factor/analysis , Nerve Growth Factor/chemistry , Protein Precursors/analysis , Animals , Colon/chemistry , Humans , Immunoblotting , Luminescent Measurements , Molecular Weight , Protein Precursors/chemistry , Rats , Rats, Sprague-Dawley , Recombinant Proteins/chemistry , Spinal Cord/chemistry
9.
Scand J Gastroenterol ; 35(11): 1174-80, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11145289

ABSTRACT

BACKGROUND: Epidermal growth factor and related proteins share some structural homology and bind to one common receptor. We have shown previously that exogenously applied EGF protects colonic mucosa against injury in an experimental model of colitis in rats and that the endogenously expressed ligands for the EGF-receptor are predominantly transforming growth factor alpha precursors. The aim of our present study was to evaluate the EGF-receptor expression in response to mucosal injury in the same model of colitis. METHODS: The trinitrobenzene sulphonic acid (TNBS)/ethanol-induced model of colitis in rats was used and EGF-receptor expression was evaluated using ribonuclease protection assay and Western blot analysis. The extent of mucosal injury and inflammation was characterized by using a microscopic and macroscopic damage score and by estimation of the myeloperoxidase activity in colonic specimens. RESULTS: Irritation of the colonic mucosa leads to severe colonic inflammation with tissue oedema, erosions and mucosal ulcers and to a significant increase in myeloperoxidase activity expressed by neutrophil granulocytes and macrophages. A significant increase in EGF-receptor mRNA expression was obtained at 8-24 h followed by an increased expression of the EGF-receptor protein at 1-5 days after the induction of colitis. On Western blot analysis only one immunoreactive band with a molecular weight of approximately 170 kDa was detected. CONCLUSIONS: Mucosal inflammation leads to a significant increase in the EGF-receptor expression in the early phases of colitis. These findings support our hypothesis that EGF and related proteins and their common receptor play a pivotal role in mucosal defence and repair.


Subject(s)
Colitis/metabolism , Colon/metabolism , ErbB Receptors/metabolism , Actins/metabolism , Animals , Blotting, Western , Colitis/pathology , Colon/pathology , Male , Nuclease Protection Assays , Peroxidase/metabolism , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley
10.
Scand J Gastroenterol ; 35(11): 1181-7, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11145290

ABSTRACT

BACKGROUND: It has been reported that epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) play an important role in colonic mucosal defense and repair. Waved-2 (wa-2) mice harboring a defect EGF-R and phenotypically similar to TGF-alpha knockout mice provide a novel approach to study the role of EGF-R ligands in the maintenance and repair of colonic mucosa. METHODS: Acute colonic mucosal injury was induced by oral administration of dextran sodium sulfate (DSS: 5 g%) given for 6 days ad libitum to wa-2 homozygotes and their genetic controls (n = 10, each group), as well as to wa-2 mice with and without exogenous EGF administration. Severity of colonic injury was assessed histologically of the entire colon and graded. A crypt damage score (CDS) reflecting all three grades of mucosal pathology was calculated. Decrease in total body weight, colon length and colonic blood content was determined for all groups. RESULTS: Thirty-eight percent of the entire colonic mucosa was destroyed in wa-2 animals compared to 15% in control mice. The CDS was 16.0 +/- 1.4 and 9.6 +/- 0.8 in wa-2 and control mice, respectively. EGF application to wa-2 mice did not reduce the severity of mucosal injury (CDS: 18.9 +/- 1.7 and 19.4 +/- 2.1 in EGF and vehicle injected mice, respectively). CONCLUSIONS: The increased susceptibility of wa-2 mice to DSS demonstrates the pivotal role of EGF-R ligands such as EGF and TGF-alpha in preserving the integrity of the colonic mucosa against mucosal injury. The missing beneficial effect of exogenous EGF administration in these mice further underlines the importance of an intact ligand/EGF-R pathway.


Subject(s)
Colitis/genetics , Dextran Sulfate , ErbB Receptors/genetics , Animals , Colitis/chemically induced , Colitis/metabolism , Colitis/pathology , Colon/pathology , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Female , Genetic Predisposition to Disease , Homozygote , Intestinal Mucosa/pathology , Mice , Mice, Knockout , Mice, Mutant Strains , Transforming Growth Factor alpha/genetics
11.
Int J Radiat Oncol Biol Phys ; 45(3): 653-6, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10524419

ABSTRACT

PURPOSE: Hyperfractionation has been advocated to improve local tumor control by increasing radiation dose without increasing late normal tissue complications. The aim of this study was to determine if hyperfractionation decreased late bowel complications. METHODS AND MATERIALS: Thirty patients with Stage II and III cervical cancer were randomized to receive either hyperfractionation or conventional fractionation. Patients were followed for 5 years and monitored for tumor control, recurrence, and bowel complications. The relative risks of tumor control and bowel complications were computed at 1 year and 5 years of follow-up. Kaplan-Meier survival curves were plotted to determine probabilities of being tumor-free and bowel complication-free. RESULTS: There were 15 patients in each group. At 1 year of follow-up, 2 patients in the hyperfractionation group (13%) and 7 patients in the conventional treatment group (45%) had tumor (relative risk [RR] 0.3; 95% confidence interval [CI] 0.1, 1.1; p = 0.054). Delayed bowel complications were seen in 8 patients in the hyperfractionation group and 1 patient in the conventional treatment group (RR 7.5; 95% CI 1.1, 52; p = 0.014). At 5 years, 2 patients in the hyperfractionation group and 8 patients in the conventional treatment group had tumor (RR 0.3; 95% CI 0.1, 1.1; p = 0.04). Delayed bowel complications (Grades 2 and 3) occurred in 9 women in the hyperfractionation group and 2 patients in the conventional group (RR 5.4; 95% CI 1.5, 19.5; p = 0.0006). Kaplan-Meier analysis showed that the hyperfractionation group had significantly more bowel complications over the 5 years of follow-up (p = 0.024). CONCLUSION: Hyperfractionation may result in better tumor control both at 1 year and at 5 years following treatment of cervical cancer. However, hyperfractionation could lead to increased late bowel complications and must be used judiciously in the treatment of cervical cancer.


Subject(s)
Dose Fractionation, Radiation , Intestinal Diseases/etiology , Radiation Injuries/etiology , Uterine Cervical Neoplasms/radiotherapy , Adult , Aged , Confidence Intervals , Female , Follow-Up Studies , Humans , Middle Aged , Neoplasm Staging , Survival Analysis , Uterine Cervical Neoplasms/pathology
12.
Br J Psychiatry ; 174: 254-8, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10448452

ABSTRACT

BACKGROUND: Resources to address the needs of parents of intellectually disabled children in developing countries are limited. AIMS: The efficacy of interactive group psychoeducation on measures of parental attitude towards intellectual disability was assessed in southern India. METHOD: Fifty-seven parents randomised to 10 weeks of experimental and control therapy were assessed using the Parental Attitude Scale towards the Management of Intellectual Disability. The pre- and post-intervention measurements were done by a single-blinded rater and compared. RESULTS: The intervention group had a statistically significant increase in the outcome scores and clinical improvement in the total parental attitude score, orientation towards child-rearing knowledge towards intellectual disability and attitude towards management of intellectual disability, but no change in attitude towards the intellectual disability subscale. CONCLUSIONS: Interactive group psychoeducation is effective for changing the attitude of parents with intellectually disabled children, and is a viable option to be developed in situations where resources are limited.


Subject(s)
Parents/psychology , Persons with Mental Disabilities/rehabilitation , Adult , Attitude to Health , Family Health , Female , Health Education/methods , Humans , India , Male , Persons with Mental Disabilities/psychology
13.
Indian J Cancer ; 36(2-4): 109-19, 1999.
Article in English | MEDLINE | ID: mdl-10921214

ABSTRACT

This is a retrospective study of cutaneous lymphomas (CL) seen over a ten year period. (January '88-December '97) in Christian Medical College & Hospital, Vellore, S. India. The aims of the study were to describe the clinicopathological profile of CL and correlate it with results of immunophenotyping. Thirty three patients were included of whom 31 had non-Hodgkin's lymphoma (NHL) and two Hodgkin's disease. REAL scheme was used for classification. In the NHL group there were 20 patients with cutaneous T cell lymphoma (CTCL), 7 with cutaneous B cell lymphoma (CBCL), 1 with anaplastic large cell lymphoma (ALCL) and 3 patients were unclassifiable. Mycosis fungoides was the most common CTCL (55.5%). Three (15%) of patients had subcutaneous panniculitic T cell lymphoma (SPTL). The duration of the disease was significantly longer (P < .05) in patients with MF as compared to non MF CTCL and CBCL. One patient with Sezary's syndrome was HTLV 1 positive. There was no difference in survival (p = 0.86) after diagnosis in the three main groups.


Subject(s)
Lymphoma/mortality , Skin Neoplasms/mortality , Adolescent , Adult , Aged , Child , Female , Humans , Immunophenotyping , India/epidemiology , Lymphoma/classification , Lymphoma/pathology , Male , Middle Aged , Retrospective Studies , Skin Neoplasms/classification , Skin Neoplasms/pathology , Survival Analysis
14.
Gut ; 43(1): 64-70, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9771407

ABSTRACT

BACKGROUND: Transforming growth factor alpha (TGF-alpha) knockout mice have increased susceptibility to dextran sodium sulphate (DSS) induced colitis. AIM: To substantiate the findings that TGF-alpha is a key mediator of colonic mucosal protection and/or repair mechanisms by evaluating the susceptibility of mice overexpressing TGF-alpha to DSS induced colitis. METHODS: TGF-alpha overexpression was induced in transgenic mice by ZnSO4 administration in drinking water (TG+). Three groups were used as controls: one transgenic group without ZnSO4 administration (TG-), and two non-transgenic littermate groups receiving ZnSO4 (Non-TG+) or only water (Non-TG-). Acute colitis was induced in all groups by administration of DSS (5%, w/v) in drinking water for six days and libitum. RESULTS: About 35-39% of the entire colonic mucosa was destroyed in Non-TG-, Non-TG+, and TG- animals compared with 9% in TG+ mice. the crypt damage score was 18.7 (0.9), 18.2 (1.0), 18.9 (0.8), and 6.8 (1.5) (means (SEM)) in Non-TG-, Non-TG+, TG-, and TG+ mice respectively. Mucin and bromodeoxyuridine staining were markedly enhanced in colons of TG+ mice compared with controls, indicating increased mucosal protection and regeneration. CONCLUSIONS: The significantly reduced susceptibility of mice overexpressing TGF-alpha to DSS further substantiates that endogenous TGF-alpha is a pivotal mediator of protection and/or healing mechanisms in the colon.


Subject(s)
Colitis/metabolism , Transforming Growth Factor alpha/physiology , Acute Disease , Animals , Body Weight , Cell Division , Colitis/chemically induced , Colitis/pathology , Colon/metabolism , Colon/pathology , Dextran Sulfate , Disease Susceptibility , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Mice , Mice, Transgenic , Radioimmunoassay , Statistics, Nonparametric , Transforming Growth Factor alpha/analysis , Zinc/administration & dosage
15.
J Neurosci Methods ; 76(1): 75-81, 1997 Sep 05.
Article in English | MEDLINE | ID: mdl-9334942

ABSTRACT

Highly sensitive chemiluminescence immunoblot analysis was utilized to examine the purity of mouse 2.5S-, beta- and 7S nerve growth factors as well as that of recombinant human beta-nerve growth factor obtained from commercial vendors. Three polyclonal antisera and two monoclonal antibodies to 13 kDa nerve growth factor (2.5S NGF and beta-NGF) were employed for assessing the purity of each preparation. In addition, polyclonal antisera against two prepro-NGF specific domains were used for immunoblotting analysis to ascertain the identity of high molecular weight nerve growth factor immunoreactive proteins as prohormones. Both the mouse and human NGF preparations contained 53 and 60 kDa immunoreactive proteins. Of these, the mouse 60 kDa and the human 53 kDa proteins strongly immunoreacted with both prepro-nerve growth factor specific domain antibodies suggesting that they are two NGF prohormone isoforms. In addition, both the mouse and human nerve growth factor preparations contained proteins that were immunoreactive to polyclonal antisera and monoclonal antibodies to mouse 2.5S and/or beta-NGF. High molecular weight aggregates of prohormones were also observed in mouse and human nerve growth factor samples. In summary, none of the ten NGF samples examined were pure as stated. Our study cautions investigators in the field to be aware of the presence of nerve growth factor prohormones and other proteins in various mouse and human nerve growth factors sold commercially.


Subject(s)
Nerve Growth Factors/chemistry , Animals , Blotting, Western , Humans , Isomerism , Luminescent Measurements , Mice , Molecular Weight , Recombinant Proteins/chemistry
16.
Gastroenterology ; 113(3): 825-32, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9287974

ABSTRACT

BACKGROUND & AIMS: There is indirect evidence that transforming growth factor alpha (TGF-alpha) is an important mediator of mucosal defense and repair. TGF-alpha knockout mice and TGF-alpha-deficient mice (wa-1) provide novel approaches to evaluate the role of TGF-alpha in preserving the integrity of the colon. METHODS: Colitis was induced by oral administration of dextran sodium sulfate (DSS, 5 g/dL) to knockout mice, their genetic controls (GC), wa-1 mice, and BALB/c mice. TGF-alpha was also administered intraperitoneally to wa-1 mice to evaluate the effect of exogenous TGF-alpha in DSS colitis. RESULTS: In response to DSS, nearly 60% of the entire colonic mucosa was destroyed in knockout and wa-1 mice, compared with 22% in GC mice and 16% in BALB/ c mice. Body weight loss was doubled in knockout (28%) and wa-1 mice (23%) compared with GC (11%) and Balb/c mice (12%). TGF-alpha application to wa-1 mice reduced the severity of mucosal injury by almost 70% compared with controls. CONCLUSIONS: The marked susceptibility of TGF-alpha knockout and wa-1 mice to DSS and the obvious amelioration of the colonic injury by exogenous TGF-alpha application in wa-1 mice suggest that TGF-alpha is a mediator of protection and/or healing mechanisms in the colon.


Subject(s)
Colitis/chemically induced , Colitis/genetics , Dextran Sulfate , Mice, Knockout/genetics , Transforming Growth Factor alpha/genetics , Animals , Colitis/pathology , Colon/pathology , Drinking , Female , Genetic Predisposition to Disease , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Reference Values , Solutions , Transforming Growth Factor alpha/deficiency , Transforming Growth Factor alpha/pharmacology
17.
Gut ; 41(2): 195-202, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9301498

ABSTRACT

BACKGROUND AND AIM: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), members of the EGF family of growth factors, protect rat gastric and colonic mucosa against injury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was to evaluate the endogenously expressed ligand mediating the protective effect of EGF/TGF-alpha in vivo. METHODS: In an experimental model of trinitrobenzene sulphonic acid (TNBS)/ethanol induced colitis in rats EGF and TGF-alpha expression was evaluated using a ribonuclease protection assay, northern blot analysis, western blot analysis, and immunohistochemistry. RESULTS: TGF-alpha mRNA increased 3-4 times at 4-8 hours after induction of colitis and returned to control levels within 24 hours. TGF-alpha immunoreactive protein with a molecular size of about 28 kDa representing TGF-alpha precursors increased markedly after induction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-alpha protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the rat colon and no EGF protein was observed by immunohistochemistry or western blot analysis. CONCLUSIONS: TGF-alpha precursors are the main ligands for the EGF receptor in acute colitis. It is hypothesised that TGF-alpha precursors convey the biological activity of endogenous TGF-alpha peptides during mucosal defence and repair.


Subject(s)
Colitis/metabolism , Epidermal Growth Factor/metabolism , Transforming Growth Factor alpha/genetics , Acute Disease , Animals , Blotting, Northern , Blotting, Western , Colitis/chemically induced , DNA Primers , Epidermal Growth Factor/genetics , Ethanol , Gene Expression Regulation , Immunohistochemistry , Male , Protein Precursors/metabolism , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Transforming Growth Factor alpha/metabolism , Trinitrobenzenesulfonic Acid
18.
Regul Pept ; 69(3): 121-5, 1997 Apr 30.
Article in English | MEDLINE | ID: mdl-9226395

ABSTRACT

We report TGF alpha immunoreactivity in neurons of the myenteric plexus and in nerve fibers in the muscle and submucosal layers of the rat stomach. Association of TGF alpha staining nerve fibers to vessels and smooth muscle cells gives morphological evidence that EGF/TGF alpha's actions to increase mucosal blood flow and gastric motility may be mediated by TGF alpha derived from neural structures. These data suggest that TGF alpha plays a role in the neural control of the gastric function.


Subject(s)
Gastric Mucosa/metabolism , Stomach/innervation , Transforming Growth Factor alpha/metabolism , Animals , Blood Flow Velocity/physiology , Epidermal Growth Factor/metabolism , Gastric Mucosa/blood supply , Gastric Mucosa/innervation , Immunohistochemistry , Male , Myenteric Plexus/metabolism , Nerve Fibers/metabolism , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Transforming Growth Factor alpha/physiology
19.
Gastroenterology ; 110(4): 1077-83, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8612996

ABSTRACT

BACKGROUND & AIMS: Keratinocyte growth factor (KGF) is known to enhance tissue repair in the skin; however, its role in the gastrointestinal tract is largely unknown. The aim of this study was to evaluate the effects of exogenous KGF in an experimental model of colitis in rats. METHODS: KGF was administered before or after induction of colitis with 2,4,6-trinitrobenzenesulfonic acid/ethanol. In the first two study groups, KGF (5 mg/kg) was administered intraperitoneally 24 hours and 1 hour before induction of colitis; animals were killed 8 hours (n=10) and 1 week (n=10) after injury. In subsequent study groups, KGF or vehicle treatment was begun 24 hours after the induction of colitis at doses of 5 (n=20), 1 (n=10), and 0.1 (n=10) mg/kg intraperitoneally and continued once daily for 1 week. Colonic tissue samples were evaluated macroscopically and microscopically for mucosal injury and assayed for myeloperoxidase activity. RESULTS: Administration of KGF after but not before induction of colitis significantly ameliorated tissue damage. Macroscopic necrosis and microscopic ulcerations were reduced by 40%-50% at KGF doses of 1 and 5 mg/kg. CONCLUSIONS: Exogenous KGF has a key role in mucosal healing in an experimental model of colitis in rats.


Subject(s)
Colitis/pathology , Fibroblast Growth Factors , Growth Substances/pharmacology , Intestinal Mucosa/pathology , Animals , Cell Division , Colitis/metabolism , Disease Models, Animal , Fibroblast Growth Factor 10 , Fibroblast Growth Factor 7 , Intestinal Mucosa/metabolism , Male , Mucins/metabolism , Mucus/metabolism , Necrosis , Peroxidase/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Ulcer/pathology
20.
Gastroenterology ; 107(1): 12-7, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8020654

ABSTRACT

BACKGROUND/AIMS: The role of epidermal growth factor (EGF) in the maintenance of mucosal integrity in the lower gastrointestinal tract is unknown. The aim of this study was to determine the effect of EGF in experimental colitis. METHODS: Colitis was induced with 2,4,6-trinitrobenzenesulfonic acid/ethanol enemas. Rats were pretreated with intraperitoneal administration of recombinant human EGF (600 micrograms/kg) or vehicle 1 hour before induction of colitis and daily thereafter until killed at 8 hours, 48 hours, and 1 week. A separate group received an identical dosage and administration of EGF or vehicle for 1 week with treatment initiated 24 hours after the induction of colitis. Colonic tissue was evaluated macroscopically, histologically, and for myeloperoxidase activity. RESULTS: Pretreatment with EGF reduced microscopic erosions at 8 and 48 hours by 74% and 54%, respectively (P < 0.05). At 1 week, microscopic ulcerations and myeloperoxidase activity were reduced by 65% in the EGF-pretreated group (P < 0.05). No significant difference in macroscopic injury, histological damage, or myeloperoxidase activity was noted when EGF treatment was initiated after the induction of colitis. CONCLUSIONS: Systemic EGF administration reduces mucosal damage and inflammation in a trinitrobenzenesulfonic acid/ethanol model of colitis in rats through a mechanism involving mucosal protection.


Subject(s)
Colitis/prevention & control , Epidermal Growth Factor/pharmacology , Animals , Colitis/chemically induced , Colitis/drug therapy , Disease Models, Animal , Epidermal Growth Factor/physiology , Epidermal Growth Factor/therapeutic use , Ethanol/adverse effects , Gastric Mucosa/enzymology , Gastric Mucosa/pathology , Gastric Mucosa/physiology , Male , Peroxidase/analysis , Rats , Rats, Sprague-Dawley , Trinitrobenzenesulfonic Acid/adverse effects
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