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Biomedical subjects

R J Grand

Publications and source records attributed to R J Grand.

At least 109 records · Page 6Linked to original sources
PubMed1990

Glycosylation of lactase-phlorizin hydrolase in rat small intestine during development.

Age-specific changes in glycosylation of rat intestinal lactase-phlorizin hydrolase were analyzed using enzyme immunoprecipitated from microvillus membranes of suckling, weaning, and adult rats, and carbohydrate moieties were examined by lectin affinity binding, metabolic labeling, and neuraminidase treatment. Lectin binding indicated the presence of N-linked and O-linked oligosaccharide chains containing mannose and galactose throughout development. An age-dependent shift in sialic acid and fucose was seen during the period of weaning; no fucose was detectable in lactase-phlorizin hydrolase until after the rats were 20 days of age, whereas sialic acid was reduced in adult lactase-phlorizin hydrolase. The presence of sialic acid in suckling intestines and fucose in adult was confirmed by metabolic labeling with appropriate radioactive precursors. Sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis of immunoprecipitated lactase-phlorizin hydrolase from the proximal and mid small intestine showed two bands of approximately 220 and 130 kilodaltons in all age groups. In the distal part of the adult small intestine, lactase-phlorizin hydrolase appeared as two bands of similar size to those found in the proximal and mid portions. In contrast, during the suckling and weaning periods, these distal bands were approximately 225 and 135 kilodaltons. [35S]-methionine labeling and fluorography of neonatal intestines confirmed these observations. The size difference between proximal and distal small intestines was virtually eliminated by neuraminidase treatment. These data indicate that the core structure of microvillus membrane lactase-phlorizin hydrolase, consisting of both N-linked and O-linked oligosaccharides, remains constant during development, although terminal sugars shift from predominantly sialic acid during the suckling period to fucose in adulthood. This alteration in glycosylation of the protein occurs in a different pattern from the postweaning decline in lactase specific activity. Consequently, age-dependent changes in glycosylation cannot account for the decrease in lactase-phlorizin hydrolase-specific activity observed during development.

Aging↗
PubMed1990

Integrated HPV 1 genomes in a human keratinocyte cell line can be transactivated by a SV40/BPV1 recombinant virus which expresses BPV1 E2 proteins.

This paper describes studies carried out on an HPV 1 carrying human keratinocyte cell line (SVD2) and two subclones of it. Although these lines contain multiple copies of HPV 1 genomes, in situ hybridization revealed that integration was restricted to band q33 on the long arms of chromosome 2. An E4 1.25-kb mRNA was specifically identified by Northern blotting and a PCR generated cDNA confirmed the presence of the E1/E4 spliced mRNA which is abundant in HPV 1 containing papillomas. Infection of an SVD2 subclone (SVD2 (cyst) sp) with a SV40/BPV1 recombinant virus which expresses the BPV1 E2 48-kb transactivator protein enhanced HPV 1 transcription at least 20-fold.

Cell Line, Transformed↗
PubMed1990

Azathioprine in the treatment of children with inflammatory bowel disease.

During a 6-year period, we treated 21 patients with azathioprine, 2 mg/kg/day, as an adjunct to their customary regimen. Nine patients had ulcerative colitis and 12 patients had Crohn disease; the patients' ages ranged from 3 to 17 years. The median duration of disease before the start of azathioprine therapy was 2 years, and median follow-up was 2 years. Sixteen patients seemed to respond to azathioprine therapy: six patients in each disease group had complete responses and four patients (one with ulcerative colitis and three with Crohn disease) had partial responses. Two patients with ulcerative colitis and three patients with Crohn disease did not respond. The median time until patients responded was less than 3 months for patients with ulcerative colitis and 4 months for those with Crohn disease. Reduction of corticosteroid dose was possible for all patients who responded to azathioprine therapy. Only minimal side effects were attributable to the drug. We conclude that azathioprine is an effective adjunctive agent for the treatment of inflammatory bowel disease in childhood, but because questions remain regarding its long-term safety, its use should be reserved for children with refractory disease or severe and unacceptable side effects of corticosteroids.

Administration, Oral↗
PubMed1990

The expression of the adenovirus 12 early region 1B 19K protein using a recombinant simian virus 40 system.

Using a simian virus 40/adenovirus 12 (Ad12)-recombinant virus, the Ad12 early region 1B (E1B) 19K protein has been produced at high levels after infection of Cos 1 cells. Expression of the 19K polypeptide reaches a maximum at about 48 h post-infection, declining at later times as host cell death occurs. Using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis, we have shown that the Ad12 E1B protein is a major species following infection of Cos 1 cells with recombinant virus. Two forms of 19K polypeptide can be distinguished following isoelectric focusing. Using subcellular fractionation of infected cells, it was found that a similar distribution of 19K protein occurred after recombinant virus and Ad12 infection, with the polypeptide being most abundant in nuclear and membranous fractions. Similarly, as in Ad12-infected cells, a certain proportion of the protein is located on the outside surface of the cell after recombinant viral infection. Immunohistochemical studies suggest that, at early times post-infection, the E1B 19K protein is located in the nuclear membrane, the Golgi apparatus and the endoplasmic reticulum. At later times, it can be seen to have spread to the cytoplasm as well as to the other organelles. These results are discussed in relation to the known functions of the 19K E1B protein.

Adenoviridae↗
PubMed1990

Lactose intolerance.

Lactose intolerance is a prevalent clinical problem. Low lactase levels result either from intestinal injury, or as in the majority of the world's adult population, from alterations in the genetic expression of lactase-phlorizin hydrolase. Progress is being made in the basic understanding of the molecular and cellular biology of this enzyme and of the scientific basis of clinical syndromes involving low lactase activity.

Animals↗
PubMed1990

Sensitivity to ionising radiation of transformed human cells containing mutant ras genes.

Measurement of colony forming ability following exposure to gamma-rays has been performed on human retinoblasts transformed with either adenovirus 5 or 12 early region 1 DNA, adenovirus early region 1A plus activated N- or H-ras DNA or SV40 DNA. In contrast to recently reported results (M.D. Sklar, 1988, Science, 239, 645-647), we found no general correlation between transformation with activated ras and increased radiation resistance. Similarly, there was no correlation between D0 values and the level of expression of ras p21 in transformed human retinoblasts as determined by liquid competition assay. Indeed, cell lines with very similar D0 values had ras contents varying by up to one hundred fold. Cell lines transformed with SV40 DNA were generally less sensitive to ionising radiation than adenovirus and/or ras transformants, but even so the variation in sensitivity within these encompassed the whole spectrum of values obtained for the ras transformants. It may be interesting to note, however, that two out of the three ras transformants which were least sensitive to gamma-rays were cell lines expressing the highest levels of p21.

Adenovirus Early Proteins↗
PubMed1989

Further studies of glycosylation and intracellular transport of lactase-phlorizin hydrolase in rat small intestine.

Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.

Alkaloids↗
PubMed1989

Conformational changes occurring in N-ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions.

Variations in susceptibility to proteolysis by trypsin and chymotrypsin have been used as indicators of conformational changes taking place in N-ras p21 in response to ligand binding. It has been observed that changes occur in undenatured protein, rendering it more resistant to degradation, in the presence of divalent cations such as Mg2+ and Ca2+ (suggesting direct binding of metals to the polypeptide) and even more markedly in the presence of GDP and/or Mg2+ GDP. Monovalent cations (Na+ or K+) cannot substitute for Mg2+ or Ca2+. Some capacity to bind guanine nucleotide is also retained by p21 treated with 7 M urea, as evidenced by increased resistance to proteolytic degradation, but the ability to bind divalent cations is irreversibly lost following denaturation. Protein prepared under denaturing conditions from a eukaryotic source, however, never regains the resistance to proteolysis shown by the bacterial p21 indicating irreversible changes in secondary and tertiary structure produced under these conditions.

Cell Line↗
PubMed1989

Structure-function relationships in cardiac troponin T.

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.

Animals↗
PubMed1989

Modulation of morphological differentiation of human neuroepithelial cells by serine proteases: independence from blood coagulation.

We have previously shown that a serum protein, termed differentiation reversal factor (DRF), is responsible for neurite retraction in differentiated cultures of an adenovirus 12 (Ad12) transformed human retinoblast cell line. Data is presented here to show that DRF is identical to the serine protease prothrombin. Both proteins have been immunoprecipitated using an antibody raised against purified prothrombin and have been shown to hydrolyse a specific thrombin substrate only after activation by the snake venom ecarin. Following addition to Ad12 HER 10 cells, which had previously been differentiated by culture in the presence of 2 mM dibutyryl cAMP in serum-free medium, thrombin and prothrombin caused half-maximal retraction of neurites at concentrations of 0.5 ng/ml and 20 ng/ml respectively. Interestingly, activation of prothrombin was shown to be unnecessary for biological activity. Using the inhibitor di-isopropylfluorophosphate (DIP), we have shown that abrogation of the proteolytic activity of thrombin also results in a loss (greater than 2000 fold) of differentiation reversal activity. Thrombin and its zymogen both stimulated the mitosis of differentiated Ad12 HER 10 cells to a similar extent. In addition, differentiation reversal was highly specific since, at physiologically significant concentrations, closely related serine proteases did not cause neurite retraction. Prothrombin and thrombin also reversed morphological differentiation in the SK-N-SH neuroblastoma cell line and in heterogeneous cultures of cells from various regions in the human foetal brain.

Axons↗
PubMed1989

Phosphorylation of the human papillomavirus type 1 E4 proteins in vivo and in vitro.

Following incubation of HPV 1-induced warts in the presence of [32P] phosphate several of the E4-encoded proteins were found to be radiolabeled. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 17K E4 polypeptides had incorporated [32P]phosphate whereas those of 16K were unlabeled. Purified E4 gene products were separated by ion exchange chromatography into a large number of different species, which were of similar size but of different charge due to varying extents of phosphorylated peptides have been isolated and identified. Phosphoserine and phosphothreonine were identified in all 16/17K E4 fractions but not phosphotyrosine. Both HPV 1 E4 16K and 17K fractions were phosphorylated in vitro by cAMP-dependent protein kinase but not by myosin light chain kinase or by phosphorylase kinase. Incubation with cAMP PK gave incorporation of approx. 0.5 mole phosphate/mol of protein indicating that the cAMP-dependent protein kinase site(s) was partially phosphorylated in vivo. This view was supported by the fact that species which were more heavily phosphorylated in vivo incorporated less phosphate after cAMP-dependent protein kinase phosphorylation. HPV 1 E4 was also phosphorylated at serine and threonine residues by a crude cytoplasmic extract prepared from cultured human keratinocytes and cultured human retinoblasts. These results are discussed in the light of the known effects of phosphorylation on the interactions of other keratinocyte-specific proteins.

Cell-Free System↗
PubMed1989

Purification of a serum factor which reverses dibutyryl cAMP induced differentiation.

We have previously shown [Grabham et al. (1988) Expt. Eye Res. 47, 123-133] that the adenovirus 12 transformed human retinoblast cell line (Ad 12 HER 10), like a number of other cell types of neuroepithelial origin, can be induced to differentiate in response to exposure to dibutyryl cAMP, and that this differentiation can be rapidly reversed by foetal calf serum. We present data here to show that a single protein, which we have termed differentiation reversal factor (DRF) and have isolated from serum, is responsible for this activity. Following reversal by DRF the growth rate of these cells was shown to be stimulated in serum-free medium. Using ammonium sulphate fractionation, gel filtration chromatography (Ultrogel AcA44), anion exchange chromatography (DEAE cellulose) and preparative gel electrophoresis, DRF has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis in the presence and absence of SDS. The protein has a mol. wt of 72,000 and appears to exist in vivo as a monomer. The concentration of DRF in serum is in the range 100-500 micrograms/ml and is capable of reversing cAMP-induced differentiation of various primary human neuroepithelial cells at physiological concentrations.

Blood Proteins↗
PubMed1989

New insights into lactase and glycosylceramidase activities of rat lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase, a small intestinal disaccharidase, has been considered mainly an enzyme important only for the hydrolysis of lactose. After weaning in most mammals lactase-specific activity falls markedly, and, functionally, adult mammals are considered to be lactase deficient. However, the persistence of low levels of lactase activity in adulthood has never been explained. In addition, it has been suggested that lactase-phlorizin hydrolase is associated with glycosylceramidase activity when the enzyme is prepared by column chromatography, but it is unclear whether this represents copurified activities or two catalytic sites on one peptide. The developmental patterns of lactase-phlorizin hydrolase and other disaccharidases were investigated in homogenates of total rat small intestine; lactase and several glycosylceramidases were measured in immunoprecipitates from these homogenates using a monoclonal antibody. The developmental pattern of total lactase activity showed a steady 2.3-fold increase to adult levels (specific activity decreased eightfold), whereas total phlorizin-hydrolase activity increased 10.7-fold (specific activity decreased threefold). As expected, levels of both total and specific sucrase and maltase activities increased during development. In lactating rats total lactase activity showed a significant increase compared with adult males. The developmental pattern of the enzyme activities for the glycolipid substrates was similar to that found for lactase, and the immunoprecipitated enzyme showed a 40- to 55-fold higher affinity for the glycolipids than for lactose. Galactosyl- and lactosylceramide inhibited lactose hydrolysis by 38%, without a competitive pattern, suggesting two different active sites for lactose and glycolipid hydrolysis, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1989

Suckling rat colon synthesizes and processes active lactase-phlorizin hydrolase immunologically identical to that from jejunum.

To identify potential tissue-specific characteristics of intestinal glycoprotein synthesis and processing, rat intestinal lactase-phlorizin hydrolase (L-Ph) was studied after pulse-labeling of colonic explants from 5-d-old suckling rats in organ culture and the data compared to similar studies in rat jejunum. Histologic sections of 5-d-old proximal colon showed villus-like structures lined with columnar epithelial cells. Lactase and phlorizin hydrolase activities showed tissue-specific developmental patterns. Using a MAb to small intestinal L-Ph, we were able to immunoprecipitate from colon at different ages a protein that hydrolyzed lactose and phlorizin, and whose activity was not inhibited by p-chloromercuribenzoate. After pulse-labeling for 60 min and chase for 30 min, immunoprecipitated L-Ph from total homogenates of rat colonic explants appeared on fluorography of SDS-PAGE as one band of approximately 205 kD. With increasing time of chase, it took 240 min before the precursor form was converted to the intermediate form (equivalent to the 180-kD form in jejunum) and the mature form (equivalent to the 130-kD form in jejunum), although these conversions in the jejunum were observed within 60 min of chase, and only 30 min of pulse labeling. When compared on SDS-PAGE to immunoprecipitated jejunal L-Ph, the precursor form in the colon had a slightly higher apparent mol wt than the corresponding precursor form found in the endoplasmic reticulum-Golgi fraction of the jejunum. The intermediate as well as the mature L-Ph forms in the colon were also both somewhat higher in apparent molecular weight than the same bands in the microvillus membrane fraction from jejunal explants. Removal of N-linked oligosaccharides from jejunum and colonic forms of L-Ph produced bands on SDS-PAGE with identical mobility, suggesting that the proteins were the same. The data demonstrate that, in neonatal colon, enzymatically active L-Ph undergoes biosynthetic and processing events similar to those in the jejunum. During early life, colonic L-Ph may function in the salvage of lactose not absorbed in the small intestine.

Animals↗
PubMed1989

The expression of Ad12 E1B 19K protein on the surface of adenovirus transformed and infected human cells.

Using radioimmunoassays we have confirmed earlier reports that the adenovirus 12 E1B 19K protein is expressed on the surface of transformed and infected human cells. Immunoprecipitation and radioimmunoassay of Ad 12 infected primary human embryo kidney cells demonstrated a considerable time lag in the translocation of the 19K protein to the external membrane after its expression within the cell. We have used antisera raised against synthetic octapeptides to detect and map the orientation of the polypeptide in the plasma membrane. The data obtained suggest that the C-terminus of the protein is exposed on the outside of the cell whereas the N-terminus is free in the cytoplasm. The predicted secondary structure of the 19K protein demonstrates a hydrophobic region which is highly conserved amongst Ad2, 5, 7 and 12 (amino acids 83-100 in Ad12) and we conclude that this forms the transmembrane domain. These observations are discussed in relation to the known functions of the Ad12 E1B 19K protein.

Acylation↗
PubMed1989

The binding of guanine nucleotide to N-ras p21--a phosphorous and proton magnetic resonance study.

One dimensional [1H] and [31P] nuclear magnetic resonance (NMR) studies have been carried out on purified wild type and mutant (Gly-12----Asp) N-ras protein expressed at high level in E. coli. Both proteins were isolated as stable 1:1 molar complexes with GDP with the upper limit for the first order rate constant for nucleotide dissociation 3 x 10(-4)s-1. From observation of the [31P] NMR spectrum after the addition of GTP it was concluded that the rate of nucleotide hydrolysis is appreciably greater than that of nucleotide exchange. Differences in the [31P] spectra of mutant and wild type proteins suggest that the mutation has a direct influence on the catalytic step. [1H] NMR spectra obtained for both mutant and wild type p21 were consistent with proteins of considerable stability and the addition of urea to concentrations of 4M appeared to cause little disruption in secondary structure. Additionally, the protein environment of the bound nucleotide remained well defined in the presence of a number of added reagents and over the pH range 5.8-9.5. The data are discussed in the light of the known crystal structure for H-ras p21 and indicate that the transforming mutation of aspartate for glycine-12 results in structural perturbations near the nucleotide binding site.

Binding Sites↗