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R J Grand

Publications and source records attributed to R J Grand.

At least 55 records · Page 3Linked to original sources
PubMed1997

Differentiation is inhibited and a senescence pathway is activated when simian virus 40 tsA 58-transformed human retinoblasts are grown at the restrictive temperature.

Neonatal human retina cells transformed by the SV40 tumor antigens were shown to leave the cell cycle and differentiate following treatment with agents that raise intracellular levels of cyclic AMP. This was true for both precrisis and immortal cell lines. However, with time, some of the differentiated retinoblasts withdrew neurites and returned to the cell cycle. Attempts to inhibit this process by developing cell lines transformed using SV tsA 58 with a temperature-sensitive phenotype for growth did not enhance but inhibited retinoblast-differentiating capacity. Growth restriction at the nonpermissive temperature was found to activate a senescence pathway. We propose that at the nonpermissive temperature, stable SV40 T-ag-p53 complexes fragment releasing p53, which transactivates p21waf1/cip1/sdi1 with the subsequent accumulation of p21 culminating in growth inhibition and senescence.

Antigens, Polyomavirus Transforming↗
PubMed1997

Pancreatic metaplasia of the gastric mucosa in pediatric patients.

UNLABELLED: Antropyloric and intestinal-type metaplasia of gastric columnar epithelial cells occurs commonly in the setting of atrophic gastritis in both adults and pediatric patients. Pancreatic metaplasia of gastric mucosa, although less common, has been reported in a variety of clinical conditions in adult patients, but not in the pediatric population. OBJECTIVES: The aim of this retrospective review was to characterize the clinical and pathological findings in pediatric patients found to have histological and immunohistochemical evidence of pancreatic metaplasia of the gastric mucosa in biopsies taken at the time of upper endoscopy. METHODS: Patients with histological evidence of pancreatic metaplasia of the gastric mucosa were prospectively identified. Their individual medical records were then reviewed for presenting symptoms, pertinent laboratory data, gross findings on endoscopy, radiological features, diagnosis, and subsequent therapy. RESULTS: Of the six children (ages, 8-18 yr; mean, 13.0 yr), with pancreatic metaplasia of the gastric mucosa, two children each had gastroesophageal reflux disease and chronic abdominal pain, whereas one child had a duodenal ulcer and one child had nodular gastritis. Iron deficiency anemia was present in four of six patients; three of four patients with this finding presented with hematemesis. All biopsies were negative for Helicobacter pylori. CONCLUSIONS: Whereas pancreatic metaplasia of the gastric mucosa is strongly associated with chronic atrophic gastritis in adults, its occurrence in children who do not have atrophic gastritis raises the possibility that it may be a developmental phenomenon of gastric mucosal differentiation. The clinical significance of this finding remains to be determined; and its association with iron deficiency in children requires further study.

Abdominal Pain↗
PubMed1996

Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: relationship to the cell-cycle and role of the retinoblastoma protein.

Using a Burkitt lymphoma cell line to model human B-cell apoptosis in vitro, we observed that crosslinking, by antibody, of cell surface immunoglobulin induced G1 growth-arrest followed by apoptosis. By contrast, cells treated with the Ca(2+)-ionophore, ionomycin, generated apoptotic signals in G2/M as well as in G1. Both ionomycin and anti-immunoglobulin treatment induced rapid dephosphorylation of Rb prior to apoptosis. Apoptosis was repressed following exposure to CD40-ligand and was accompanied by hyperphosphorylation of Rb and cell-cycle progression but not Bcl-2 expression. Expression of Bcl-2 protein in stable bcl-2-transfectants, also resulted in repression of apoptosis and anti-immunoglobulin-treated cells no longer underwent growth-arrest. In Bcl-2-expressing cells in which apoptosis was repressed, Rb remained hyperphosphorylated, even during G1-arrest induced by ionomycin. TGF beta treatment of Bcl-2-expressing cells induced G1-arrest, de-phosphorylation of Rb and apoptosis. These results suggest that the functional activity of Bcl-2 in B-lymphoma cells is dependent upon, or leads to, sustained hyperphosphorylation of Rb and that Rb hyperphosphorylation can be uncoupled from cell-cycle progression.

Antibodies, Anti-Idiotypic↗
PubMed1996

Control of p53 expression by adenovirus 12 early region 1A and early region 1B 54K proteins.

The level of p53 is markedly increased in human cells in response to expression of the Ad12 E1A proteins and, quite separately, to the Ad12 E1B 54K protein. The behaviour of p53 in these two circumstances has been examined using A549 cells infected with Ad12 dl620 (a mutant virus which does not express the larger E1B protein and is replication-defective) and human skin fibroblasts expressing the Ad12 E1B 54K protein (HSF 54K). In normal and E1A-expressing A549 cells, p53 is located predominantly in the nucleus, whereas in the HSF 54K cells it is primarily cytoplasmic as is the Ad12 E1B 54K protein. The half-life of p53 is increased in Ad12 dl620-infected A549 cells from about 10 min (in uninfected cells) to 2 hr. The half-life of p53 in HSF 54K cells is even longer-probably in excess of 48 hr. The capacity of p53 to regulate transcription was assessed using a transfected CAT construct linked to p53-responsive elements. p53 transcriptional activity was very low in the HSF 54K cells and in human embryo kidney cells expressing the Ad12 E1B 54K protein (and p53) at high level. It was, however, dramatically increased in response to the p53 expressed as a result of E1A expression. Additionally, MDM2 was present at low level in the HSF 54K cell lines, whilst, as we have previously shown, it is overexpressed in response to infection with Ad12 dl620. We conclude that there are two distinct mechanisms for up-regulation of p53 attributable to the adenovirus E1 proteins. When E1A only is present the p53 is nuclear and transcriptionally active and can probably induce apoptosis in the absence of the E1B 19K protein. When the E1B 54K protein is present, however, p53 is transcriptionally inactive and does not induce apoptosis.

Adenovirus E1A Proteins↗
PubMed1996

Quantitative analysis of lactase-phlorizin hydrolase expression in the absorptive enterocytes of newborn rat small intestine.

At birth, the mammalian small intestine displays regional differences in morphology as well as complex proximal-to-distal (horizontal) patterns of protein distribution. Lactase-phlorizin hydrolase (LPH), an enterocyte-specific disaccharidase crucial for the digestion of lactose in milk, reveals a characteristic horizontal pattern of expression at birth. However, it is not certain whether this topographic pattern is due to variations in epithelial structure along the length of the small intestine or to regional differences in the transcription of the LPH gene. In order to understand the mechanisms that regulate the regionalization of LPH at birth, we characterized the epithelial structure along the horizontal axis using stereologic techniques and correlated these data with the patterns of lactase activity and LPH mRNA abundance in the small intestine of unsuckled, newborn rats. Epithelial volume and microvillar surface area per unit of intestinal length decreased three-to fourfold from duodenum to distal ileum. In contrast, lactase activity and LPH mRNA abundance were highest in proximal jejunum and lowest in the most proximal and distal ends of the small intestine. Mean lactase activity per cell in proximal duodenum, proximal jejunum, and distal ileum was estimated at 12.0, 26.7, and 5.6 nU/absorptive enterocyte, respectively, and paralleled the concentration of LPH mRNA in the same segments: 20, 45, and 15 molecules of LPH mRNA/absorptive enterocyte. Our data indicate that horizontal gradients of lactase activity in the newborn rat intestine do not depend on epithelial organization or on enteral factors, since the horizontal gradient is established before suckling. Each absorptive enterocyte along the small intestine expresses lactase activity in a position-dependent manner which is controlled at the level of mRNA abundance.

Absorption↗
PubMed1996

Combined use of cyclosporine and azathioprine or 6-mercaptopurine in pediatric inflammatory bowel disease.

The aim of this study was to assess whether in steroid-resistant patients with pediatric inflammatory bowel disease (IBD) a combination of cyclosporine and azathioprine (or 6-mercaptopurine) could induce remission and subsequently permit maintenance on azathioprine/6-mercaptopurine as the sole immunosuppressive agent. Two boys and six girls (six with ulcerative colitis and two with Crohn's disease; ages 3-17 years) received 100-200 micrograms/kg/day cyclosporine intravenously and then 4-10 mg/kg/day orally. Doses were adjusted to achieve trough serum cyclosporine levels of 100-200 mu/L (Abbott's TDX assay). Seven of the eight patients received azathioprine/6-mercaptopurine, and all were given a 5-aminosalicylate preparation and corticosteroids. The latter drugs were continued and then tapered off as clinical status allowed. Cyclosporine was continued for 3-10 months in those who responded. In seven of eight patients, there was a rapid clinical response; one patient showed a transient response, but recurrent bleeding led to total colectomy 9 days after starting cyclosporine. Of the seven responders, three were able to discontinue prednisone and cyclosporine and are doing well on azathioprine at long-term follow-up (2-5 years). One who did not receive azathioprine/6-mercaptopurine maintained remission for 2 years after cyclosporine was stopped, one experienced a disease flare-up 5 months after start of cyclosporine treatment and required colectomy, one who did not tolerate 6-mercaptopurine had a flare-up during cyclosporine tapering and underwent surgery at 6 months, and one started to flare up with cyclosporine tapering at 6 months and was scheduled for surgery. No significant complications of treatment were observed. Seven patients had an initial response and four of them have so far not required surgery. These preliminary findings suggest that azathioprine/6-mercaptopurine can be used safely to maintain cyclosporine-induced remission in children with IBD.

Adolescent↗
PubMed1996

Prenatal ethanol exposure alters the expression of intestinal hydrolase mRNAs in newborn rats.

To gain insight into the postnatal growth delay induced by ethanol in utero, we characterized functional impairments of the small intestine of neonatal rats prenatally exposed to ethanol using a well-described model of gestational alcoholism (25% ethanol w/v in the drinking water). Expression of the intestinal enzymes-lactase-phlorizin hydrolase (LPH) and intestinal alkaline phosphatase (IAP)-that are critical for enteral nutrition of neonates was studied. Characteristic patterns of LPH and IAP expression along the proximal-distal (horizontal) and crypt-villus (vertical) axes of the small intestine, as well as the intracellular localization of LPH and IAP mRNAs and immunoreactive proteins within absorptive enterocytes, were not altered by prenatal exposure to ethanol. However, a 10- to 15-fold increase in the number of LPH and IAP mRNA molecules per absorptive enterocyte was found throughout the intestine of ethanol-exposed neonates, compared with controls, whereas lactase and alkaline phosphatase activities per enterocyte remained unchanged. These findings suggest that ethanol in utero alters the mRNA abundance of epithelial enzymes in newborn rat small intestine. Changes in mRNA abundance could be an important aspect of enterocyte adaptation to high ethanol concentrations in gastrointestinal amniotic fluid of ethanol-exposed fetuses.

Alkaline Phosphatase↗
PubMed1996

A lamb-meat-based formula for infants allergic to casein hydrolysate formulas.

Ten infants with colitis due to milk protein allergy, presenting as hematochezia, whose symptoms did not resolve with the use of hydrolyzed cow-milk-based formulas, were treated with a modular lamb-meat-based formula (LOP). The patients were followed up for 3 months to 5 years. Prompt resolution of symptoms was achieved. In three patients, increased levels of creatinine and blood urea nitrogen and mild metabolic acidosis were noted, all returning to normal after the protein intake was lowered. All patients had normal growth. Seven patients were able to tolerate cow milk protein or soy at age 9-15 months. The LOP formula is well tolerated and is a safe alternative formula for infants allergic to cow milk hydrolysate formula.

Animals↗
PubMed1996

Therapeutic strategies for pediatric Crohn disease.

The main aims of therapy for inflammatory bowel disease (Crohn disease) in children and adolescents are (1) the induction and maintenance of remission, (2) the correction of nutrient deficits and (3) the restoration of growth and maturation. These goals are reached with the use of a combination of therapeutic methods, including pharmacologic agents, nutritional and psychological support, and surgical intervention. The commonly used drugs sulfasalazine, corticosteroids and metronidazole have all been shown to be safe and efficacious when given to children. Newer steroid preparations that are rapidly degraded either in the target tissue or elsewhere are being studied. Of these, budesonide currently shows promise as an efficacious drug with few side effects, but its use in children needs further study. Newer 5-amino-salicylate preparations such as Asacol have been shown to be effective in children, but the number of patients studied is small. Immunomodulatory drugs such as azathioprine and 6-mercaptopurine appear to be safe and efficacious for children; cyclosporine has been used infrequently to treat refractory Crohn disease in children. The use of other agents such as methotrexate, tacrolimus, monoclonal antibodies to cytokines, antibiotics and specific dietary products such as fish oils have not been intensively studied in children with Crohn disease. Nutritional therapy remains a mainstay of treatment because it corrects nutritional deficits, replaces losses and stimulates growth.

Adolescent↗
PubMed1995

Ras binding to a C-terminal region of GAP.

Using fluorescence spectroscopy we have identified a binding region for Ras on the GTPase activating protein (GAP) lying within residues 715-753. A synthetic peptide Y922, corresponding to residues 716-753 of GAP binds to wild type Ras showing 3.3-fold higher affinity for the GTP- over the GDP-bound forms of Ras. Binding is stabilised by Mg2+, although Y922 does not stimulate the GTPase activity of Ras. Peptide binding to the Y32A and Y40F Ras mutants showed equal affinity for both GDP- and GTP-bound forms, with binding to Y32A.GDP abolished in the absence of Mg2+. These results suggest that Y922 mimics the in vivo interactions shown by the intact p120GAP protein and provide the first direct demonstration of Ras interaction with GAP in the region 715-753.

Amino Acid Sequence↗
PubMed1995

The high levels of p53 present in adenovirus early region 1-transformed human cells do not cause up-regulation of MDM2 expression.

The cellular protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2 gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 98K protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.

Adenovirus E1B Proteins↗
PubMed1995

Regulation of neurite outgrowth from differentiated human neuroepithelial cells: a comparison of the activities of prothrombin and thrombin.

The mechanism by which thrombin and prothrombin control neurite retraction was studied in Ad12E1HER10 human neuroepithelial cells. Morphological changes in differentiated cells were apparent within minutes of the addition of very low concentrations of thrombin (3 pM). Higher concentrations (2 nM) of prothrombin were required to elicit a similar response. Doses of thrombin and prothrombin sufficient to cause neurite retraction stimulated protein tyrosine kinase activity. Protein tyrosine kinase activation also correlated positively with thrombin- and prothrombin-induced phosphoinositide 3-kinase activation and InsP6 dephosphorylation. However, thrombin-stimulated Ins(1,4,5)P3 generation and intracellular Ca2+ mobilization only occurred at concentrations in excess of those needed to induce retraction. No fluctuations in Ins(1,4,5)P3 were detected after stimulation with prothrombin, and no rapid synchronized release of Ca2+ was observed, even at very high concentrations. Prothrombin did, however, cause small oscillations in the intracellular Ca2+ concentration, similar to those produced by low concentrations of thrombin, after approximately 30 min. We conclude that prothrombin- and thrombin-induced neurite retractions are not dependent on PtdIns(4,5)P2 and Ca2+ mobilization, but are more probably mediated through an effector mechanism involving protein tyrosine kinase activation. No intracellular Ca2+ mobilization, protein tyrosine kinase activity or neurite retraction was observed after treatment of cells with proteolytically inactive mutant thrombin (S205-->A). Prothrombin-mediated intracellular Ca2+ mobilization and neurite retraction were inhibited by hirudin, which was shown to interact with thrombin but not prothrombin. It is concluded that cleavage of prothrombin to thrombin is a necessary prerequisite for biological activity on differentiated Ad12E1HER10 cells and that differences in agonist concentration are capable of coupling the thrombin receptor to different pathways within the cell.

Blotting, Western↗
PubMed1995

Effects of interferon-alpha on human B cells: repression of apoptosis and prevention of cell growth are independent responses of Burkitt lymphoma lines.

We have previously shown that interferon-alpha (IFN-alpha) can repress apoptosis in Burkitt lymphoma (BL) cells. In this study, we have compared this protective response with a further, well-established effect of IFN-alpha on BL cells, that of growth arrest. Of a panel of BL lines comprising (i) EBV-positive and -negative lines that retain the phenotype of the parental tumour cells and (ii) the prototype IFN-alpha-growth-inhibited line, Daudi, only Daudi cells were found to undergo substantial growth inhibition in response to the cytokine. By contrast, all lines, with the notable exception of Daudi, were protected by IFN-alpha from high-rate apoptosis initiated by the Ca2+ ionophore ionomycin. Ionomycin failed to elicit an IFN-alpha-repressible apoptotic response in either wild-type Daudi cells or IFN-resistant sublines that were refractory to the growth-arresting effects of the cytokine. Analysis of c-myc protein levels confirmed previous observations that repression of apoptosis in IFN-alpha-rescuable BL cells was associated with an early inhibition of myc that was followed by a return to high-level expression. Significantly, ionomycin alone induced a comparable transient inhibition of myc protein in Daudi cells. In Daudi cells, but not in IFN-alpha-rescuable BL cells, renewed expression of myc observed after the early, transient down-regulation was followed by sustained down-regulation of the protein, which paralleled growth arrest. Our results indicate that long-term growth arrest and repression of apoptosis in BL are distinct cellular responses to IFN-alpha.

Apoptosis↗
PubMed1995

Further characterization of the 5'-flanking region of the rat lactase-phlorizin hydrolase gene.

The lactase-phlorizin hydrolase gene is widely used as a marker of intestinal differentiation. Recent evidence demonstrating that transcription plays a major role in the regulation of this gene suggests that study of the 5'-flanking region will allow an understanding of how the expression of this gene is controlled. However, sequence, restriction, and primer extension analysis of a rat genomic clone has revealed that previously published data are incomplete. In the present study, we used a directed sequencing strategy to carefully analyze this region. Our expanded analysis of the 5'-flanking region of the lactase-phlorizin hydrolase gene should facilitate future studies of its structure and function.

Animals↗
PubMed1995

The quaternary structure of the adenovirus 12 early region 1B 54K protein.

The quaternary structure of the adenovirus early region 1B 54K protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 x 10(5). After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral protein could be distinguished in the nucleus-one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 x 10(6)). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component.

Adenovirus E1B Proteins↗
PubMed1995

A novel protein expressed in mammalian cells undergoing apoptosis.

Human and rodent cells undergoing apoptosis were observed to express high levels of a novel 45,000 M(r) protein. The protein, which we have termed apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca2+ ionophore, ionomycin, treatment with inhibitors of macromolecular synthesis (cycloheximide and actinomycin D), and cold shock. In BL cells treated with apoptotic stimuli, expression of the oncoprotein Bcl-2 was found to both protect from apoptosis and prevent expression of ASP. ASP was not detected either in viable cells or in cells dying passively by necrosis. Laser scanning confocal microscopy showed high levels of ASP in the cytoplasm of cells displaying the chromatin condensation and fragmentation patterns typical of apoptosis. Retention of ASP was observed even when DNA was no longer detectable, and two-color immunofluorescence staining indicated that the protein primarily colocalized with, but was clearly distinct from, non-muscle actin. These findings, together with the observation that biochemical extraction of ASP was only possible under conditions which caused solubilization of the cytoskeleton, leads us to conclude that ASP forms part of, or at least strongly associates with, a modified cytoskeleton unique to cells undergoing apoptosis. While elucidation of its function will require further work, ASP constitutes a powerful marker for the diagnosis and quantitation of apoptosis in vivo and in vitro.

Animals↗
PubMed1995

Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.

BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

Amino Acid Sequence↗