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E Robbins

Publications and source records attributed to E Robbins.

At least 19 recordsLinked to original sources
PubMed1991

Quantitative non-radioactive in situ hybridization of preproenkephalin mRNA with digoxigenin-labeled cRNA probes.

Non-radioactive detection of mRNA with in situ hybridization histochemistry has emerged as an important new technology for the study of gene expression. Quantitative in situ hybridization studies have generally relied upon counting of autoradiographic grains in the emulsion overlying cells containing hybridized, radioactively labeled probe. However, such high resolution studies require tedious grain counting over individual cells, frequently in addition to weeks of exposure to nuclear emulsion. The present report describes a quantitative, non-radioactive approach to the detection of a specific mRNA in the brain with the advantages of comparatively rapid tissue processing and computerized image analysis. The validity of this approach was tested by measuring the haloperidol-induced increase in the level of preproenkephalin mRNA in striatal sections of the rat brain using an RNA probe labeled with digoxigenin-11-UTP. Detection of probe hybridized to tissue sections was carried out enzymatically following complex formation with an antidigoxigenin-alkaline phosphatase conjugate. Using computerized image analysis, it was found that chronic treatment of rats with haloperidol resulted in a 50 +/- 6% increase in striatal neuronal optical density, a value in good agreement with previous studies using low-resolution radioactive methods, showing a 30-80% increase in striatal preproenkephalin mRNA hybridization signal.

Animals↗
PubMed1991

Non-radioactive detection of nerve growth factor receptor (NGFR) mRNA in rat brain using in situ hybridization histochemistry.

Radioactively labeled RNA probes in conjunction with in situ hybridization histochemistry have become a useful method for studying gene expression in the central nervous system. We used digoxigenin-labeled uridine triphosphate to synthesize cRNA probes for localization of nerve growth factor receptor (NGFR) mRNA in the rat basal forebrain. Detection of cells containing digoxigenin-labeled NGFR mRNA was accomplished using a digoxigenin antibody conjugated with alkaline phosphatase. NGFR mRNA-positive cells were distributed in three major cell groups in the basal forebrain: the medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. This technique provides a rapid and sensitive method for high-resolution detection of mRNA species in the central nervous system, as well as the potential for co-localization of two different mRNA species within individual cells.

Animals↗
PubMed1990

Localization of nerve growth factor receptor mRNA in the rat basal forebrain with in situ hybridization histochemistry.

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.

Animals↗
PubMed1989

Characterization of striatal neurons expressing high levels of glutamic acid decarboxylase messenger RNA.

Two types of labelled cells are detected in sections of rat and mouse striata processed for in situ hybridization histochemistry with 35S-radiolabelled RNA probes complementary to the messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD), the synthesis enzyme for gamma-aminobutyric acid (GABA): numerous lightly, and fewer very densely labelled neurons. In order to determine whether the densely labelled cells correspond to the striatal somatostatinergic neurons with which they share morphological characteristics, the presence of GAD mRNA was examined in brain sections processed successively for dihydronicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, a marker of striatal somatostatinergic neurons, and in situ hybridization histochemistry. In addition, the distribution of GABAergic interneurons was analyzed with regard to striatal compartments (striosomes) indicated by patches of dense opiate binding sites. The results show that NADPH diaphorase activity and GAD mRNA do not co-exist in striatal neurons. Furthermore, in contrast to the somatostatinergic neurons which are almost exclusively located in the extrastriosomal matrix, densely labelled GAD cells were present both in the striosomes and the matrix, further suggesting that GABAergic and somatostatinergic neurons form two distinct interneuronal systems in the striatum of rats and mice.

Animals↗
PubMed1989

Regional differences in substance P-like immunoreactivity in the striatum correlate with levels of pre-protachykinin mRNA.

The distribution of neurons containing the messenger RNA encoding preprotachykinin, the precursor of the neuropeptides substance P and K, was examined in the striatum of the rat. The large majority of cells densely labelled by in situ hybridization histochemistry (using a 35S-radiolabelled RNA probe complementary of the beta-preprotachykinin mRNA) were located within the boundaries of areas of dense [3H]naloxone binding which delineate the striosomes, a striatal compartment distinct from the surrounding matrix with respect to its inputs, outputs and neurochemical content. In view of previous immunohistochemical data, the results suggest that tachykinin-containing neurons of the striosomes have a higher rate of tachykinin synthesis than those neurons located in the surrounding striatal matrix.

Animals↗
PubMed1989

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases.

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.

Amnion↗
PubMed1989

Effect of phosphatidylserine on the shape of McLeod red cell acanthocytes.

The rare McLeod blood group phenotype is characterized by weak Kell antigens, lack of the common Kx antigen, and acanthocytic morphology. Previous studies that did not detect membrane or cytoskeletal protein abnormalities suggested a lipid disturbance. In normal red cells, dimyristoyl phosphatidylserine (DMPS) is transported across the membrane by an enzymatic process and accumulates in the inner leaflet of the membrane bilayer causing discocyte to stomatocyte shape changes. Scanning electron microscopy of McLeod red cells shows a mixture comprised of 15% discocytes, 51% with irregular surfaces, and 34% acanthocytes. On incubation with various concentrations of DMPS at 37 degrees C for periods up to two hours, McLeod red cells transported DMPS across the membrane and caused irregularly shaped and acanthocytic McLeod red cells to attain normal discocyte shape and later to become stomatocytes. Chlorpromazine, which at 0 degrees C preferentially partitions into the inner monolayer of the membrane, had a similar effect on the shape of McLeod red cells. This suggests that in McLeod cells acanthocytosis is due to a lack of lipid in the inner leaflet of the membrane bilayer but that the imbalance is not caused by defective transport of phosphatidylserine across the membrane.

Acanthocytes↗
PubMed1987

Comparative analysis of two rapid automated methods and a semiautomated version of the urease method for determining aminoglycoside concentrations in serum.

The Syva EMIT Autolab 6000 and the Abbott TDX automated methods for measuring serum aminoglycoside concentrations were compared with each other and with a new semiautomated version of the urease technique. Each was evaluated for accuracy, reproducibility, cost, and ease of use. All three methods gave satisfactory results, although the Abbott TDX was the most accurate. Both automated methods gave results within minutes, whereas the new urease method, which involved more initial manipulation, took 1.25 h to complete (mainly incubation time). There was no difference in accuracy of performance between experienced and inexperienced operators. The new urease method was much cheaper in terms of initial capital expenditure and in cost per test and needed no provision of manufacturer's back-up maintenance services. This could prove invaluable in situations where financial resources are at a premium. Otherwise, the Abbott TDX is currently our method of choice, and reasons for its preference to the EMIT system are listed.

Anti-Bacterial Agents↗
PubMed1986

Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade.

To understand the role of proteinases in tumor invasion, the effects of inhibitors of metallo-, serine-, and cysteine-proteinases on this process were studied using 125I-iododeoxyuridine-labeled B16/BL6 cells grown on human amnion basement membrane. Cellular invasion was quantitated by measuring the radioactivity associated with the amniotic membrane after the B16/BL6 cells on the basement membrane were removed by lysis followed by scraping. The results obtained with proteinase inhibitors showed that inhibitors of collagenase and plasmin prevented invasion of the amnion. Tissue invasion was also blocked by antiurokinase antibodies. On the contrary, cysteine-proteinase inhibitors and anti-tissue plasminogen activator antiserum were ineffective. Mersalyl, a compound known to activate collagenase, stimulated invasion under conditions where plasmin formation or activity were inhibited. Evidence for the role of a plasminogen activator-plasmin-collagenase activation cascade in B16 invasion is provided.

Amnion↗
PubMed1986

A study of the HLA-D region in patients with classic Kaposi's sarcoma.

The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR beta and DQ beta. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ beta fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR beta fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group.

Alleles↗
PubMed1985

A systematic study of HLA class II-beta DNA restriction fragments in insulin-dependent diabetes mellitus.

DNA restriction fragments of the genes encoding HLA class II-beta antigens were compared in 34 patients with insulin-dependent diabetes mellitus and 34 HLA-DR-matched healthy individuals. Ninety-three fragments, determined by six restriction enzymes (EcoRI, EcoRV, HindIII, BamHI, Pvu II, and Taq I), were analyzed: (i) A DR Taq I 12.7-kilobase-pair fragment might be a marker for the extended haplotype HLA-B8, DR3. (ii) In controls, DR4 haplotypes are associated with two distinct clusters of DQ restriction fragments (DQR4 and DQR5). Almost all (94%) DR4 patients belong to the DQR4 and not to the DQR5 cluster. This suggests that, among HLA-DR4 haplotypes, only DQR4 haplotypes are involved in susceptibility to insulin-dependent diabetes mellitus. (iii) A DR Taq I 14.5-kilobase-pair fragment was found to be strongly associated with DQR4, mainly in DR3/DR4 heterozygous patients (P = 5 X 10(-4). However, these results must be interpreted with caution, taking into account the high number of statistical tests performed.

DNA Restriction Enzymes↗
PubMed1976

Pernicious anaemia.

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Anemia, Pernicious↗