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Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 199 records · Page 11Linked to original sources
PubMed1992

In-vitro effects of liposome-encapsulated amphotericin B (AmBisome) and amphotericin B-deoxycholate (Fungizone) on the phagocytic and candidacidal function of human polymorphonuclear leucocytes.

Liposomal amphotericin B (AmBisome), at concentrations > or = 20 mg/L, and amphotericin B-deoxycholate (DC) (Fungizone), at concentrations > or = 1 mg/L, both caused a significant reduction in neutrophil uptake of Candida albicans blastospores following simultaneous addition of the drug with the blastospores. The reduction in uptake was seen also in tests in which blastospores were pre-treated with the drugs for 60 min, but was not detected in tests in which neutrophils were pre-treated for 60 min. Neither formulation affected neutrophil killing of C. albicans blastospores following simultaneous addition of the drug with the blastospores. However, previous treatment of the neutrophils with either formulation at a concentration of 20 mg/L led to enhanced killing of ingested blastospores. The results suggest that much higher concentrations of liposomal amphotericin B (AmBisome) are required to produce deleterious effects on neutrophil phagocytic function than with the conventional formulation of the drug.

Amphotericin B↗
PubMed1992

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.

Amino Acid Sequence↗
PubMed1992

The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication.

A major site of DNA bending is located 1.6 kb upstream of the P1 transcription start site of the human c-myc gene, near the center of a reported zone of initiation of DNA replication. A repeated, purine-rich element, termed PUR, at the bend site is specifically bound by a protein in HeLa cell nuclear extracts. This protein has specific affinity for the purine-rich single strand of the element. Methylation interference maps a pattern of specific contact points with guanosine bases in a 24-mer oligonucleotide containing the element. UV cross-linking reveals that contact is made by a polypeptide of approximately 28 kDa. The PUR element is present at origins of replication and in gene flanking regions in a variety of eukaryotes from yeasts through humans. The consensus sequence GGNNGAGGGAGARRRR has been derived. This element is present near centers of regions of two mammalian loci (human c-myc and hamster dhfr) recently reported as initiation zones for DNA replication. A 24-mer oligonucleotide representing the hamster dhfr version of the PUR element effectively competes with the human c-myc version for binding to Pur.

Animals↗
PubMed1991

Receptor-mediated retrograde transport in CNS neurons after intraventricular administration of NGF and growth factors.

Radiolabel tracer techniques were used to follow the distribution of nerve growth factor (NGF) and other neuromodulatory factors after intraventricular injection. Autoradiography showed that shortly after intraventricular injection of radio-iodinated NGF (125I-NGF), substantial amounts of radioactivity had penetrated the ventricular wall surfaces; this binding was transient and nonspecific. The 125I-NGF was progressively cleared from the central nervous system (CNS), presumably via the flow of cerebrospinal fluid (CSF) into the blood. A relatively small proportion of the injected 125I-NGF was taken up by NGF receptor-positive neurons in the CNS. Retrograde accumulation of radiolabel was observed within the basal forebrain cholinergic neurons at 5 hours after intraventricular injection. Labeling intensity was maximal at 18 hours and much reduced by 30 hours. This labeling was blocked by co-injection of an excess of unlabeled NGF. Specific and saturable retrograde labeling was also observed within other NGF receptor-bearing neurons, including the prepositus hypoglossal nucleus and the raphe obscurus nucleus. When epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-I), or IGF-II was radiolabeled and injected intraventricularly, specific labeling of neurons was observed for 125I-IGF-II and 125I-LIF within separate subpopulations of the dorsal and medial raphe. No retrograde accumulation within neurons was observed for EGF, TGF-beta 1, PDGF-AA, PDGF-BB, or IGF-I. This study describes an in vivo method for identifying putative neuromodulatory factors and their responsive neurons.

Animals↗
PubMed1991

Fibroblast growth factor receptor-bearing neurons in the CNS: identification by receptor-mediated retrograde transport.

Neurons that internalize and retrogradely accumulate acidic (aFGF) or basic (bFGF) fibroblast growth factor were identified by autoradiography after injections of 125 I-aFGF or 125I-bFGF into the adult rat central nervous system (CNS). Neuronal cell bodies within the lateral hypothalamus, pedunculpontine tegmental nucleus, laterodorsal tegmental nucleus, and the paracentral dorsal tegmental nucleus accumulated 125I-aFGF. Neurons in the hippocampus, subiculum, the centrolateral, paracentral, central medial, and parafascicular thalamic nuclei, the supramammillary nucleus, and substantia nigra compacta accumulated 125I-bFGF. The pattern of neuronal labeling with 125I-bFGF in adult rats was similar to that observed in newborn guinea pigs. No 125I-FGF labeling was observed in nerve growth factor (NGF) receptor-bearing neurons, including the basal forebrain cholinergic neurons. Time-course studies indicate that 125I-FGF was internalized at the terminals and retrogradely transported to the neuronal cell bodies. Neurons were retrogradely labeled either by injection of 125I-bFGF into the lateral ventricle or by injection into innervated target tissues. Co-injection of a 250-fold excess of unlabeled FGF with the 125I-FGF abolished the neuronal labeling. Co-injection of wheat germ agglutinin (WGA), which nonspecifically blocks binding of 125I-bFGF to its receptor, also prevented neuronal labeling. These studies demonstrate that specific neuronal populations within the CNS express functional receptors for aFGF and/or bFGF; in these neurons, aFGF and/or bFGF bind specifically to these receptors, are internalized and retrogradely transported to the neuronal soma in a manner analogous to NGF. The data indicate that FGF can provide trophic support to CNS neurons by both direct and indirect mechanisms.

Animals↗
PubMed1991

Evidence for endocytosis-dependent proteolysis in the generation of soluble truncated nerve growth factor receptors by A875 human melanoma cells.

We have identified nerve growth factor receptor (NGFR) on the cell surface and a truncated nerve growth factor receptor (NGFRt) in the conditioned medium of NGFR-negative cells that have been transfected with either the gene or the cDNA for the full-length receptor. By using cell surface iodination or metabolic labeling of A875 human melanoma cells, coupled with immunoprecipitation, we have determined the half-life of the cell-associated receptor to be approximately 7 h. Concomitant with receptor degradation is the accumulation of NGFRt in the extracellular medium. Approximately one-fifth of the labeled receptor can be recovered as the truncated species. These data support the hypothesis that NGFRt is generated by proteolysis of previously intact receptor. Furthermore, although no specific protease inhibitor assayed could affect this processing, NGFR degradation and truncation were retarded by treatment with: 1) the weak base amines, ammonium chloride or methylamine; 2) the carboxylic ionophore, monensin; or 3) the vacuolar ATPase inhibitor, bafilomycin A1, all agents that dissipate endosomal/lysosomal proton gradients via alternate mechanisms. Incubation of cells at 4 degrees C precluded NGFR degradation and truncation. The presence of ligand did not alter the time course of receptor truncation.

Ammonium Chloride↗
PubMed1991

Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic Na+ concentration changes evoked by angiotensin II and vasopressin in individual rat aortic smooth muscle cells. Use of microfluorometry and digital imaging.

Dual wavelength microfluorometry was used to characterize the changes in cytosolic free Ca2+ concentration [( Ca2+]i) in individual cultured rat aortic vascular smooth muscle cells (VSMC). Angiotensin II (ANG II) at 10(-8) M induced a transient rise in [Ca2+]i from 43 +/- 2 to 245 +/- 23 nM, lasting for approximately 60 s (n = 42). In half of the population, discrete oscillations in [Ca2+]i of smaller amplitude occurred after the initial [Ca2+]i peak, with a period of 58 +/- 8 s and a maximum height of 132 +/- 24 nM. A similar oscillatory pattern was observed with arginine vasopressin (AVP). The oscillations depended upon the presence of extracellular Ca2+. Cytosolic free Na+ concentration ([Na+]i) in VSMC was also measured using the fluorescent Na+ probe sodium-binding benzofuran isophthalate. ANG II induced a gradual and sustained elevation of [Na+]i, from 24.0 +/- 6.2 to 36 +/- 9.7 mM. In response to AVP, [Na+]i rose to 41.0 +/- 11.6 mM. Video imaging of individual VSMC, with on-line ratio calibration of [Ca2+]i, revealed an inhomogeneous distribution of Ca2+ within the cell. [Ca2+] in the nucleus was invariably lower than in the cytoplasm in resting cells. In the cytoplasm, there were small regions in which [Ca2+] was elevated, or "hot spots." In Ca(2+)-containing medium, the initial rise in [Ca2+]i triggered by ANG II and AVP appeared to emanate from the hot spots and to spread evenly throughout the cytoplasm. Between [Ca2+]i oscillations, Ca2+ retreated back to the original hot spots. This study demonstrates the cellular and subcellular heterogeneity of [Ca2+]i both in resting VSMC and during stimulation by ANG II and AVP and reports the direct measurement of [Na+]i in VSMC. The results suggest an action of Ca2+ in both the initial and sustained phases of the response in VSMC and a link between changes in [Ca2+]i and [Na+]i.

Angiotensin II↗
PubMed1991

A plastome mutation affects processing of both chloroplast and nuclear DNA-encoded plastid proteins.

Immunoblotting of a chloroplast mutant (pm7) of Oenothera showed that three proteins, cytochrome f and the 23 kDa and 16 kDa subunits of the oxygen-evolving subcomplex of photosystem II, were larger than the corresponding mature proteins of the wild type and, thus, appear to be improperly processed in pm7. The mutant is also chlorotic and has little or no internal membrane development in the plastids. The improperly processed proteins, and other proteins that are completely missing, represent products of both the plastid and nuclear genomes. To test for linkage of these defects, a green revertant of pm7 was isolated from cultures in which the mutant plastids were maintained in a nuclear background homozygous for the plastome mutator (pm) gene. In this revertant, all proteins analyzed co-reverted to the wild-type condition, indicating that a single mutation in a plastome gene is responsible for the complex phenotype of pm7. These results suggest that the defect in pm7 lies in a gene that affects a processing protease encoded in the chloroplast genome.

Amino Acid Sequence↗
PubMed1991

A gene delivery/recall system for neurons which utilizes ribonucleotide reductase-negative herpes simplex viruses.

We present data which show that ribonucleotide (RR)-negative herpes simplex virus type-1 (HSV-1) is a useful vector for gene delivery into neuronal cells. For these studies we used hrR3, a genetically engineered HSV-1 mutant which has an in-frame insertion of the bacterial lacZ gene into the HSV gene that encodes the large subunit (ICP6) of RR. After infection of rat primary sympathetic neuronal cultures with hrR3, the ICP6::lacZ chimeric gene was expressed, as shown by blue staining of the cells upon exposure to X-Gal, a chromogenic beta-galactosidase substrate. When the infection was performed in the presence of acyclovir, hrR3 appeared to become "latent"; neither infectious virus nor beta-galactosidase activity was detectable in these neuronal cultures at 3 weeks after the acyclovir was removed. However, beta-galactosidase activity was inducible in the "latent" cultures by superinfection with ICP6 delta (a RR-negative deletion mutant) without resulting in the "reactivation" of hrR3 and without apparent cytopathic effects. In contrast, superinfection with ICP6 delta + 3.1, a virus derived by marker rescue of ICP6 delta, resulted in the expression of lacZ, the release of hrR3 into the culture medium, and cytopathic effects. The introduction of a foreign gene into neuronal cells by a RR-negative herpes simplex virus, and the subsequent induction of gene expression by another noncomplementing virus, may constitute a prototype gene delivery/recall system for neurons.

Acyclovir↗
PubMed1991

An outbreak of aspergillosis in a general ITU.

Over a 5-month period, six patients in a general intensive therapy unit became colonized by Aspergillus species including Aspergillus fumigatus, and invasive infection occurred in at least two of them. Broncho-alveolar lavage was unhelpful in discriminating between infection and colonization. The source of infection was presumed to be disturbance of an accumulation of spores in fibrous insulation material above the perforated metal ceiling. Patients in such units without clearly identifiable defects of defence against infection may be at risk from aspergillosis. The risk can be reduced by improved hospital design, satisfactory ventilation and thorough regular cleaning of environmental surfaces.

Adult↗
PubMed1991

Regulation of phytochrome message abundance in root caps of maize.

In many cultivars of maize (Zea mays L.) red light affects root development via the photomorphogenetic pigment phytochrome. The site of perception for the light is the root cap. In the maize cultivar Merit, we investigated phytochrome-mediated events in the cap. We established that the message encoded by the phyA1 gene was most abundant in dark-grown tissue and was asymmetrically distributed in the root cap, with greatest expression in the cells which make up the central columella core of the cap. Phytochrome message was negatively autoregulated in a specific region within the root cap. This autoregulation was sensitive to very-low-fluence red light, and thus was characterized as a phytochrome-mediated, very-low-fluence event. The kinetics of message reaccumulation in the dark were also examined and compared to the kinetics of the light requirement for root gravitropism in this cultivar. Similarly, the degree of autoregulation present in two other maize cultivars with different light requirements for gravitropic sensitivity was investigated. It appears that the Merit cultivar expresses a condition of hypersensitivity to phytochrome-mediated light regulation in root tissues. We conclude that phytochrome regulates many activities within the cap, but the degree to which these activities share common phytochrome-mediated steps is not known.

Darkness↗
PubMed1991

Expression of FGF receptor gene in rat development.

We examined the expression of FGF-receptor (FGF-R) mRNA during rat development with in situ hybridization histochemistry. Embryonic tissues (E9, E12, E14, E17) and postnatal neural tissues (P1, P7, P14, adult) were examined. We detected significant levels of FGF-R mRNA in various tissues at different developmental stages. As postulated by previous studies using other methods, FGF-R gene expression was observed primarily in mesoderm- and neuroectoderm-derived tissues. In the nervous system, the pattern of gene expression was developmentally regulated; in embryos, FGF-R mRNA was mainly detected in the ependymal layer of the central nervous system (CNS). Postnatally, FGF-R transcripts were observed in specific neuronal populations, such as hippocampal neurons. FGF-R mRNA was also found in sensory systems such as trigeminal and dorsal root ganglia in late stage embryos; however, FGF-R mRNA decreased in the postnatal period. FGF-R mRNA expression was modulated in the developing retina: FGF-R messages were observed in the pigment epithelium and neuroblast layer at embryonic stages; in the postnatal period, they were found in the ganglion cell and inner granular layer. In non-neuronal embryonic tissues, a wide variety of organs expressed FGF-R message. Particularly, the prevertebral column, bone, kidney and skin showed high levels of expression. These observations reinforce the idea that FGF exerts effects on the development of various tissues.

Animals↗
PubMed1990

Expression of PiM-and PiZ-mutated forms of the human alpha 1-antitrypsin gene in transfected monkey COS1 cells.

The PiZ mutation of the gene coding for alpha 1-antitrypsin results in a serum deficiency of this protein leading to early onset emphysema and liver disease. The PiZ gene has a Z-specific point mutation in exon V together with a point mutation in exon III which is also present in some normal (PiM) individuals. There has thus far been no system to study the effects of PiZ point mutations in tissue culture. We constructed plasmids containing alpha 1-antitrypsin cDNA synthetically altered at either exon III or exon V mutation sites and linked to simian virus 40 promoter sequences. Such constructs with the exon V mutation were transfected into monkey COS1 cells followed by analysis of expression of alpha 1-antitrypsin gene products. COS1 cells normally synthesize virtually no alpha 1-antitrypsin mRNA or protein. alpha 1-Antitrypsin mRNA is transcribed at high levels in cells transfected with either M or Z plasmids. Immunologic staining of COS1 cells within 48 h of transfection localizes alpha 1-antitrypsin protein to specific regions of the cytoplasm. This extranuclear localization is also observed with human HepG2 hepatoma cells, which synthesize alpha 1-antitrypsin at high levels, and with human SK-Hep1 hepatoma cells transfected with an M plasmid. The cloned synthetically altered alpha 1-antitrypsin genes provide a system for dissecting contributions of distinct point mutations to the pathological effects of the PiZ protein.

Animals↗
PubMed1990

Developmental study of nerve growth factor receptor mRNA expression in the postnatal rat cerebellum.

Nerve growth factor receptor (NGFR) mRNA expression was examined in the developing rat cerebellum with in situ hybridization histochemistry. The probe used in this study was the 57-mer oligonucleotide complementary to the rat NGFR cDNA (bases 798-855). The specificity of the hybridization was determined with a 'sense' probe. From the age of postnatal day 0 (PND 0), hybridization signals were detected in the external granule cell layer, Purkinje cell layer, and meningeal cells of the cerebellum. Until PND 10, the external granular cell layer consistently expressed high levels of NGFR message, with the densest hybridization signal observed in the proliferative zone. NGFR mRNA level in this layer rapidly decreased at later ages and was barely detectable after PND 16. In the Purkinje cell layer beyond PND 4, the hybridization signal became more intense, reached a peak level around PND 10, and then gradually decreased. NGFR mRNA was detected until PND 30 in this layer. Meningeal cells expressed NGFR message from PND 0 to PND 8. These results suggest that NGF may be involved in cerebellar development as a mediator of morphogenesis and/or synaptogenesis.

Animals↗