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Biomedical subjects

R J Grand

Publications and source records attributed to R J Grand.

At least 127 records · Page 7Linked to original sources
PubMed1989

The expression of the retinoblastoma gene product Rb1 in primary and adenovirus-transformed human cells.

Polyclonal antibodies to the human retinoblastoma gene product (Rb1) have been produced in rats by immunisation with a fusion protein comprising part of Rb1 together with the E. coli beta-Gal sequence. We have used these antibodies in Western blotting studies to screen a number of human foetal tissues and organs and found approximately similar levels of expression of Rb1 in all of them. The protein seems to be somewhat more abundant in some cell lines produced by transfection of human embryo retinal (HER) cells with adenovirus 12 early region 1 (Ad 12 E1), Ad 5 E1, Ad 2 E1A + mutant N-ras or SV40 DNA. Using co-immunoprecipitation followed by Western blotting we have shown that the Rb1 protein binds to Ad 12 E1A 266 and 235 amino acid proteins. This interaction is ionic strength dependent but is unaffected by non-ionic detergent up to a concentration of at least 1%. In Ad 12 infected human cells it appears that less E1A is bound to Rb1 than in the transformants. These results are discussed in view of the known similarities and differences between the amino acid sequences of Ad 12 and Ad 5 E1A proteins.

Adenoviruses, Human↗
PubMed1988

Differentiation of normal and adenovirus-12 E1 transformed human embryo retinal cells.

Following the treatment with dibutyryl cyclic AMP (dbcAMP) in the absence of serum, a proportion of cultured human embryo retinoblasts will differentiate rapidly. This is characterized by cell rounding and the extension of neuritic-type processes. A transformed cell line (Ad 12 HER 10), developed by transfection of human embryo retinoblasts with adenovirus 12 (Ad 12) early region 1 (E1) DNA, forms retinoblastoma-like tumours in athymic nude mice and retains the ability to extend neuritic-like processes after treatment with dbcAMP in the absence of serum. This response, which occurs in almost all cells (over 98%), is accompanied by growth arrest and reverses rapidly after re-exposure to serum. Other agents known to increase intracellular cAMP also mediate differentiation. Ad 12 HER 10 cells were shown to contain the neuronal markers, neurone-specific enolase and protein gene product 9.5, but not the glial cell marker glial fibrillary acidic protein (GFAP). Activity of the neurotransmitter enzyme acetylcholinesterase was also detected and found to increase after differentiation. Interestingly, the expression of the Ad 12 E1 transforming proteins did not change throughout differentiation. These results show first, that the Ad 12 HER 10 cell line can differentiate in a similar manner to that observed in a proportion of primary retinoblasts and second, that the expression of transforming proteins does not preclude at least part of that differentiative capacity. This is the first demonstration of in vitro differentiation in an adenovirus-transformed human cell line, as well as offering a useful system for the study of factors controlling growth and differentiation of tumorigenic human retinoblasts.

Acetylcholinesterase↗
PubMed1988

Host range mutants of adenovirus type 12 E1 defective for lytic infection, transformation, and oncogenicity.

The human adenovirus type 12 (H12) E1A region encodes two early proteins of 266 amino acid residues (266R) and 235R whilst the H12 E1B promoter directs the synthesis of two major proteins of 163R and 482R. To determine the functions of E1A and E1B in lytic infection and oncogenic transformation we have isolated and characterized a series of H12 E1 mutants. Mutant H12 hr 700 contains a point mutation in exon 1 that alters a single amino acid common to both the 266 and 235R proteins. This mutant synthesized reduced levels of E1 and structural proteins at delayed times in HEK cells, transformed BRK cells, and induced tumors in newborn rats at reduced efficiency compared to wild-type virus. The mutation in H12 in 600 truncates the 266R protein in its unique sequences but this mutant synthesized the 235R, E1B, and structural proteins at delayed times in HEK cells. H12 in 600 was nontransforming but induced rare tumors in newborn rats. A third E1A mutant H12 in 601 synthesized no E1A proteins, reduced levels of E1B and structural proteins at delayed times in lytic infections, and was not a transforming or oncogenic virus. Three E1B mutants were studied in detail. Both H12 hr 703 and H12 in 602 encode N-terminal truncated 482R proteins whereas H12 del 620 encodes an in-frame internally deleted 482R protein. All three synthesized reduced amounts of E1A proteins and the E1B 163R protein, identifying a regulatory function for the 482R protein. None of the E1B mutants could transform and only H12 del 620 could induce rare tumors in newborn rats. These results show that H12 oncogenesis requires the coordinated expression of the E1 proteins.

Adenovirus Early Proteins↗
PubMed1988

Isolation and characterization of adenovirus type 12 E1 host-range mutants defective for growth in nontransformed human cells.

In order to define the functions of human adenovirus type 12 (Ad12) early region 1 (E1) products in lytic infection and oncogenic transformation we have isolated and phenotypically characterized a set of host-range (hr) mutants of this serotype. These mutants grow efficiently upon HER3 cells, which contain and express type 12 E1 genes, but are restricted for growth upon A549 carcinoma and HeLa cells. Inter- and intratypic complementation analysis, marker-rescue mapping, and DNA sequence analysis have assigned some of the mutations to E1A sequence, and some to the reading frame encoding the E1B 54-kDa (482R) protein. Phenotypic analysis of the E1B mutants in particular has revealed some interesting, and in some cases surprising, findings relating to the roles of that protein in virus-cell interactions. This Ad12 gene product is required, either directly or indirectly, for efficient viral DNA replication in A549 and HeLa cells, unlike its counterpart in type 5 virus. Surprisingly, however, despite the severe defect in viral DNA replication, the synthesis of a few species of viral late proteins continues in cells infected by some of the E1B mutants. In contrast, none of these mutants brings about the inhibition of host-cell protein synthesis characteristic of wild-type virus infection, and with some E1B mutants no viral late proteins are made. Further, in a separate study reported elsewhere, we have demonstrated that the E1B 54-kDa product may also be involved, either directly or indirectly, in positive regulation of both E1A and E1B 19-kDa (163R) gene expression. The molecular and/or physiological bases for these various effects remain to be determined, but our initial results suggest that the E1B 54-kDa protein may carry out multiple regulatory functions during the viral life cycle.

Adenoviruses, Human↗
PubMed1988

Pathogenesis of shigella diarrhea: evidence for a developmentally regulated glycolipid receptor for shigella toxin involved in the fluid secretory response of rabbit small intestine.

Shigella toxin reproduces the major manifestations of shigellosis in ligated intestinal loops from adult rabbits and binds to a microvillus membrane (MVM) glycolipid receptor, globotriaosylceramide (Gb3). Because neonatal human shigellosis is uncommon, we used the animal model for obtaining MVMs from rabbits of different ages to determine the presence of toxin receptors and Gb3 and to measure the fluid secretory response to toxin in ligated ileal loops. A single class of MVM receptors for 125I-labeled shigella toxin, first detected at 20 d of age, reached adult levels by 24 d (n = 1.7-23.8 X 10(10)/micrograms of protein; K = 1.1-3.8 X 10(9) M-1). Binding was specific for toxin subunit B. A toxin binding MVM glycolipid, identified as Gb3, was detected in animals greater than or equal to 16 d of age by high-performance thin-layer chromatography and autoradiography. Fluid secretion in response to shigella toxin in ligated small bowel loops occurred in temporal relation to the appearance of Gb3, a result thus indicating the involvement of Gb3 in mediating the toxin effects.

Aging↗
PubMed1988

Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A + ras.

The efficiency of transformation of primary human embryo retinal (HER) cells by the adenovirus E1 region (E1A + E1B) depended on the virus serotype whereas transformation by E1A alone was a rare event regardless of serotype. Activated human c-Ha-ras and N-ras genes co-operated differentially with different E1As for HER transformation but were ineffective without E1A. Ras + E1A co-transformants containing Ad 12 E1A established directly from foci, in contrast to those containing Ad 2 or Ad 5 E1A. A spectrum of activated ras gene expression was found in stable co-transformants with mRNA and protein levels being lower in Ad 12 E1A + N-ras than Ad 2 E1A + N-ras cell lines. Down regulation of E1A transcription in the absence of E1B was found in Ad 2 E1A + Ha-ras transformants only but E1A protein levels were similar to those in Ad 2 E1A + N-ras or Ad 5 E1A + E1B cell lines. HER cell transformants which contained Ad 12 E1A were more tumourigenic than those which contained the Ad 2 or Ad 5 E1A. This unique transformation system shows that stable malignant transformation of primary human cells in vitro is a complex process requiring the combined activities of two or more types of genes.

Adenovirus Early Proteins↗
PubMed1987

Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat.

The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.

Animals↗
PubMed1987

The level of expression of class-I MHC antigens in adenovirus-transformed human cell lines.

The level of expression of the class-I major histocompatibility (MHC) antigen was determined in a series of human embryo cell lines transformed with either adenovirus 12 (Ad 12) early region I (EI) or adenovirus 5 (Ad 5) EI DNA or with combinations of Ad 12 early region IA (EIA) or Ad 2 EIA with activated N-ras DNA. MHC class-I antigen expression was greatly reduced in all Ad 12 transformants, compared to primary cells. Expression was also reduced in the Ad 5 cell lines transformed with EIA and EIB DNA, but levels were near normal in those lines with only EIA DNA present. Amounts of MHC class-I antigen on the cell surface, as determined by RIA and FACS analysis, generally reflected total cellular levels as determined by Western blotting. Expression of beta 2 microglobulin was also much reduced in those cell lines with low levels of MHC class-I antigen. The treatment of primary cells and all the transformants with human gamma-interferon resulted in increased expression of HLA on the cell surface. Infection of primary human cells with Ad 12 or with a series of Ad 12 mutants did not have any effect on the MHC class-I antigens present.

Adenovirus Early Proteins↗
PubMed1987

Intramuscular vitamin E repletion in children with chronic cholestasis.

Progressive spinocerebellar degeneration was identified in six children with chronic cholestatic liver disease and attributed to severe vitamin E deficiency. In addition to areflexia, ataxia, dysmetria, and diminished vibratory and position sense, three patients had pigmentary retinopathy. Abnormalities were present on electromyography, nerve conduction studies, and electroretinography. Because the vitamin E deficiency was not corrected by oral administration of massive doses of vitamin E, vitamin E was administered by the intramuscular route. With doses of 50 to 100 mg of vitamin E every three to seven days, over a 32-month interval (range, 15 to 44 months), vitamin E deficiency and abnormal red blood cell peroxide hemolysis were corrected. Other than discomfort and occasional edema at the site of injection, there were no side effects of parenteral vitamin E therapy. In several other studies intramuscular vitamin E therapy has produced significant neurologic improvement in patients with similar characteristics. In this study clinical progression of spinocerebellar degeneration was arrested but improvement could not be demonstrated despite adequate vitamin E replacement.

Adolescent↗
PubMed1987

Pancreatitis in Kawasaki disease.

Two children with Kawasaki disease presented with vomiting, back and abdominal pain, elevated serum amylase values, and ultrasonographic findings consistent with pancreatitis. In both patients, pancreatitis appeared after approximately two weeks of illness, and both patients underwent extensive evaluations, which yielded negative results, for other disorders associated with pancreatitis. We suggest that in the absence of acute infection, and in the presence of appropriate diagnostic criteria, pancreatitis should be considered in all patients with Kawasaki disease who have severe abdominal pain.

Adolescent↗
PubMed1987

Pyoderma gangrenosum occurring at multiple surgical incision sites.

Ten weeks after subtotal colectomy and ileostomy for ulcerative colitis, a 16-yr-old girl developed wound drainage and back pain. Massive ulceration and skin separation occurred at the abdominal wound incision as well as at the incision sites of a previous central venous line. A diagnosis of pyoderma gangrenosum was made. High-dose steroid treatment induced prompt healing of the abdominal wound as well as the catheter sites. Pyoderma gangrenosum has rarely been seen in surgical wounds. To our knowledge, this is the first reported case of pyoderma gangrenosum occurring simultaneously in multiple surgical incision sites in a patient with ulcerative colitis.

Adolescent↗
PubMed1987

Abdominal abscesses in adolescents with Crohn's disease.

Little information is available about the development of abdominal abscesses in adolescents with Crohn's disease. We report the clinical presentation of five adolescents with Crohn's disease who developed this complication. The mean time from diagnosis until development of an abdominal abscess was 1.7 years. The admitting diagnosis was an acute abdomen in two patients and recurrent Crohn's disease in the other three. No features of the clinical presentation or laboratory data distinguished this group from other adolescents with Crohn's disease. The use of ultrasound and CT scanning was helpful in making this diagnosis preoperatively. Those patients with active Crohn's disease who do not respond promptly to medical therapy should be evaluated for the development of this complication.

Abdomen↗
PubMed1987

Evidence for the expression of protein IX in some rat cells transformed with adenovirus type 12 early region 1 DNA.

Sera were produced by injection of a particular rat cell line (HLBRK EcoC1), produced by transformation of baby rat kidney cells with adenovirus 12 early region 1 DNA, into the syngeneic host. Antibodies to a protein of Mr 15,200 as determined by SDS-PAGE, were detected in the sera from two rats bearing tumours. The 15K protein was identified as the viral structural component polypeptide IX by a number of criteria: (i) the protein was only observed in adenovirus 12-infected cells, (ii) it was produced at intermediate and late times after infection, (iii) it was immunoprecipitated from purified adenovirus 12 particles and (iv) it was immunoprecipitated from a purified protein preparation.

Adenoviridae↗