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Biomedical subjects

J E Johnson

Publications and source records attributed to J E Johnson.

At least 19 recordsLinked to original sources
PubMed1992

Comparison of the lipid regulation of yeast and rat CTP: phosphocholine cytidylyltransferase expressed in COS cells.

The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were over-expressed 20-30-fold in COS cells. Most of the CT activities were found in the cytosolic fraction. The regulation of the yeast CT activity (Y-CT) by lipids was characterized for the first time in comparison with the regulation of the well-studied rat CT (R-CT). Sonicated vesicles composed of egg phosphatidylcholine (PC) or 1-stearoyl-2-oleoyl PC had no effect on Y-CT and only slightly stimulated R-CT activity. Both CTs were activated 10-50-fold by the anionic lipids cardiolipin, phosphatidyl-glycerol, phosphatidylinositol and oleic acid. The effects of varying the vesicle concentration and the mol% of anionic lipid in PC vesicles were tested. The concentration optima for the activation of Y-CT by oleic acid or anionic phospholipids were 5-10-fold lower than those for R-CT. For example, the stimulation of Y-CT activity by phosphatidylglycerol vesicles was optimal between 5 and 15 microM and declined at higher concentrations, but R-CT activation by these vesicles saturated at approximately 25 microM. The positively charged aminolipid sphingosine antagonized the stimulation by oleic acid of both Y-CT and R-CT. Y-CT activity was insensitive to PC vesicles containing the neutral lipids diacylglycerol, monoacylglycerol or oleyl alcohol. However, R-CT was stimulated 10-20-fold by vesicles containing these neutral lipids. Translocation of the CTs to microsomal membranes enriched with anionic or neutral lipids was compared. Oleic acid enrichment promoted translocation of Y-CT and R-CT, whereas diacylglycerol promoted only R-CT translocation. These data show that the activity of Y-CT is lipid-sensitive. Y-CT is affected only by charged lipids, whereas R-CT responds to charged and neutral lipid activators. The data are consistent with different modes of interaction of the two CTs with lipids.

Animals↗
PubMed1992

Crystallization and preliminary data analysis of Flock House virus.

Flock House virus, purified from infected cultured Drosophila cells, crystallizes into three different forms under identical growth conditions. Two crystal forms grow in the trigonal space group R3, both with equivalent cell constants a = 323.6 A, alpha = 61.7 degrees. The difference between the two trigonal crystal forms is 1.1 degrees in the orientation of the virus particle as determined from the rotation function. Early crystal setups grew in one form, while recent crystals grew in the other form. The third space group, which accounts for 5% of the observed crystals and grows with both trigonal forms, is orthorhombic I222 with cell parameters a = 416.7, b = 332.1, c = 351.2 A. The trigonal crystal forms contain one virion per unit cell and the orthorhombic form contains two particles per cell. All three crystal forms diffract X-rays to 2.8 A resolution.

Animals↗
PubMed1992

Conformations, interactions, and thermostabilities of RNA and proteins in bean pod mottle virus: investigation of solution and crystal structures by laser Raman spectroscopy.

We report and interpret laser Raman spectra of the three virion components of bean pod mottle virus (BPMV). The top component of BPMV is an empty capsid; middle and bottom components package the RNA2 and RNA1 genome segments, respectively. All components were investigated as both single crystals and aqueous solutions, the latter over wide ranges of temperature and ionic strength. The isolated RNA2 molecule of BPMV middle component was also investigated in both H2O and D2O solutions. The results permit assessment of RNA and protein structures, their mutual interactions in the virions, and their conformational thermostabilities and comparison of these structural characteristics for solution and crystal states of the particles. The principal findings of this study are (i) The extent of ordered A-form backbone (74%) and of base pairing (38% AU + 22% GC) in unpackaged (aqueous) RNA2 are significantly altered by packaging. The A-form secondary structure of RNA2 is increased by 12 +/- 4%, and guanine base interactions are also substantially increased with packaging. (ii) The thermostability of Raman-monitored secondary structure of unpackaged RNA2 (Tm approximately 43 degrees C) is greatly increased in the packaged state (Tm approximately 53 degrees C). This increase corresponds to a stabilization of the A-form backbone geometry in 15 +/- 5% of genome nucleotides. (iii) Packaging of RNA2 in the middle component stabilizes subunit-subunit interactions of the capsid, as evidenced by a thermal denaturation temperature Td approximately 65 degrees C for the virion, compared with Td approximately 55 degrees C for the empty capsid. (iv) Raman marker-band shifts implicate the purine 7N sites of RNA2 and aromatic side chains of subunits as the principal targets for RNA-subunit interaction. (v) At the conditions of the present experiments (8 degrees C, pH approximately 7, moderate ionic strength), the subunit secondary structures observed for solutions of the top, middle, and bottom components are indistinguishable by Raman spectroscopy from secondary structures observed for corresponding crystalline samples. (vi) On the other hand, side chains of subunits in the top component (empty capsid) yield significantly different Raman intensities in crystalline and solution states. These differences are interpreted as the result of changes in a small number of side-chain environments between crystal and solution. (vii) Similarly, small differences exist between RNA Raman markers of crystalline and aqueous virions, which are attributed to altered environments of nucleotide residues and to a small increase in the amount of A-form backbone geometry upon going from the crystal to the solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1992

DNA binding and transcriptional regulatory activity of mammalian achaete-scute homologous (MASH) proteins revealed by interaction with a muscle-specific enhancer.

The MASH genes are vertebrate homologues of achaete-scute, genes required for neuronal determination in Drosophila. The sequence of MASH1 and MASH2 contains a basic helix-loop-helix (bHLH) motif that is present in other transcriptional regulators such as MyoD and E12. In the absence of an authentic target for the MASH proteins, we examined their DNA binding and transcriptional regulatory activity by using a binding site (the E box) from the muscle creatine kinase (MCK) gene, a target of MyoD. Like myogenic bHLH proteins, the MASH proteins form heterooligomers with E12 that bind the MCK E box with high affinity in vitro. Unexpectedly, however, MASH1 and MASH2 also activate transcription of both exogenous and endogenous MCK in transfected C3H/10T1/2 fibroblasts. However, they do not induce myogenesis. Myogenic activity is not exclusively a property of the MyoD basic region, as substitution of this domain fails to confer myogenic activity on MASH1. These data suggest that different bHLH proteins may activate overlapping but distinct sets of target genes in the same cell type.

Amino Acid Sequence↗
PubMed1992

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography.

Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 A resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few ångstroms by fitting atomic models of CPMV4 and Fab (Kol)5 to reconstructed density maps.

Antibodies, Monoclonal↗
PubMed1992

Comoviruses and enteroviruses share a T cell epitope.

An in vitro murine T cell proliferation assay was used to determine whether an antigenic epitope(s) recognized by enterovirus-immune T cells is held in common between plant comoviruses and human enteroviruses. Splenocytes isolated from C3H/HeJ mice infected with coxsackievirus B3 (CVB3) proliferated in vitro not only against a variety of enterovirus (CVB2, CVB3, CVB6, CVA16, PV1) antigens, but against comovirus (CPMV, BPMV) antigens as well. Splenocytes from mice inoculated with bean pod mottle virus (BPMV) also proliferated in response to comoviral and enteroviral antigens in vitro. However, if the viral inocula were highly purified prior to inoculation, then the splenocyte response was generated only against the group used to inoculate, suggesting that the epitope shared between the comoviruses and the enteroviruses resided in the nonstructural region. B (nonstructural) and M (structural) genomic segments of CPMV were translated in rabbit reticulocyte lysates and used as in vitro antigens. Splenocytes from mice inoculated with live CVB3 proliferated in response to the B-RNA-encoded but not the M-RNA-encoded polypeptides, confirming the nonstructural coding region location of the common epitope. Comparison of predicted amino acid sequences in the nonstructural coding regions of the comoviruses and picornaviruses suggested a potentially immunogenic linear epitope in protein 2C. The consensus peptide LEEKGI was synthezized and shown to be immunogenic for both BPMV- and CVB3-immune splenocytes.

Amino Acid Sequence↗
PubMed1992

Sequence and analysis of the capsid protein of Nudaurelia capensis omega virus, an insect virus with T = 4 icosahedral symmetry.

We have determined the nucleotide sequence of the RNA2 segment of the Nudaurelia capensis omega virus genome. It was found to consist of 2448 nucleotides and contained one long open reading frame (ORF) encoding the 644 residue capsid protein. The deduced amino acid sequence of this protein reveals a positively charged amino terminus, a characteristic exhibited by several other viral capsid proteins, that is thought to be important for interactions between the capsid and the genomic RNA. There are 366 and 150 bases of untranslated sequence on the 5' and 3' ends, respectively. The ORF encoding the capsid protein initiates at the second AUG from the 5' end. The 5' proximal AUG specifies a short ORF (30 codons) which terminates 1 base before the initiation codon for the coat protein. Our analysis also revealed the presence of a second, previously unidentified polypeptide associated with Nudaurelia capensis omega virus particles. The amino terminal sequence of this protein corresponds to a portion of the long ORF beginning at codon 571. The lack of an initiation codon near this sequence indicates that the small polypeptide is most likely produced as a carboxy terminal cleavage product from a 70-kDa capsid protein precursor, yielding the previously identified 62-kDa protein and the 8-kDa protein that we have observed. The putative cleavage site would be at an Asn/Phe pair, somewhat resembling known cleavage sites (Asn/Ala) in the T = 3 Nodaviridae. In addition, we have found that there is also a second polypeptide similar in size to that from Nudaurelia capensis omega virus associated with particles of Nudaurelia capensis beta virus, the type member of Tetraviridae.

Amino Acid Sequence↗
PubMed1992

Adipocytes of old rats produce a decreased amount of differentiation factor for preadipocytes derived from adipose tissue islets.

Adipose tissue of young rats (3.5 mo) contains cell formations ("islets") that, in culture, give rise to a variety of preadipocytes, or islet-derived cells (IDCs), that undergo rapid morphologic differentiation into adipocytes. Such adipose conversion depends on a differentiation-promoting factor produced by the mature adipocytes also present in the cultures. Here we report that cultured IDCs from epididymal fat of senescent rats (20 +/- 3 mo) show decreased adipose conversion compared to IDCs from 3.5 mo rats at 3 days of culture (14.2% vs 29.9%; p < .001). Both the number of co-cultured adipocytes and increased fragility with age were excluded as having a substantial role in explaining the decreased conversion. In order to determine whether the decrease differentiation of the IDCs of the old rats was intrinsic or due to reduced production of the differentiation factor by the old adipocytes, cross-over cultures of IDCs from young and old rats were grown in the presence of young or old adipocytes. When IDCs of old rats were cultured in the presence of young adipocytes, a significant twofold increase in differentiation was seen compared to old IDCs grown with old adipocytes (22.7% vs 10.3%; p < .001). This response was comparable to that of young IDCs grown with young adipocytes. Thus, old IDCs retain their ability to undergo extensive morphologic differentiation when appropriately stimulated. Reduced production of the differentiation-promoting factor by old adipocytes rather than the ability of IDCs to differentiate appears to be responsible for the decreased adipose conversion of IDCs seen in cultures of adipose tissue from senescent rats.

Adipose Tissue↗
PubMed1992

Detection of human immunodeficiency virus type 1 antibody by using commercially available whole-cell viral lysate, synthetic peptide, and recombinant protein enzyme immunoassay systems.

A total of 575 serum specimens received for human immunodeficiency virus type 1 (HIV-1) antibody determination were tested prospectively by enzyme immunoassay with a whole-cell viral lysate (VL) (Genetic Systems Corp.), a synthetic peptide (SP) (United Biomedical, Inc.), and a recombinant protein (RCP) (Syva Co.). Concordance of all three antigens was noted for 559 of 575 (97.2%) specimens tested. Of the specimens tested, 90 (15.7%) were positive and 469 (84.3%) were negative. Retrospective testing by SP and RCP of 86 specimens indeterminate for HIV-1 antibody by VL, Western blot (immunoblot), and immunofluorescence was also performed. The results of both phases of this study indicate that the specificity of the three antigens is RCP greater than SP greater than VL. As noted from the prospective phase, the sensitivities of the antigens appear to be equivalent, indicating that the RCP and SP systems could be used in place of, or to confirm, a VL enzyme immunoassay screening test.

Cell Fractionation↗
PubMed1992

Induction and repression of mammalian achaete-scute homologue (MASH) gene expression during neuronal differentiation of P19 embryonal carcinoma cells.

MASH1 and MASH2, mammalian homologues of the Drosophila neural determination genes achaete-scute, are members of the basic helix-loop-helix (bHLH) family of transcription factors. We show here that murine P19 embryonal carcinoma cells can be used as a model system to study the regulation and function of these genes. MASH1 and MASH2 display complementary patterns of expression during the retinoic-acid-induced neuronal differentiation of P19 cells. MASH1 mRNA is undetectable in undifferentiated P19 cells but is induced to high levels by retinoic acid coincident with neuronal differentiation. In contrast, MASH2 mRNA is expressed in undifferentiated P19 cells and is repressed by retinoic acid treatment. These complementary expression patterns suggest distinct functions for MASH1 and MASH2 in development, despite their sequence homology. In retinoic-acid-treated P19 cells, MASH1 protein expression precedes and then overlaps expression of neuronal markers. However, MASH1 is expressed by a smaller proportion of cells than expresses such markers. MASH1 immunoreactivity is not detected in differentiated cells displaying a neuronal morphology, suggesting that its expression is transient. These features of MASH1 expression are similar to those observed in vivo, and suggest that P19 cells represent a good model system in which to study the regulation of this gene. Forced expression of MASH1 was achieved in undifferentiated P19 cells by transfection of a cDNA expression construct. The transfected cells expressing exogenous MASH1 protein contained E-box-binding activity that could be super-shifted by an anti-MASH1 antibody, but exhibited no detectable phenotypic changes. Thus, unlike myogenic bHLH genes, such as MyoD, which are sufficient to induce muscle differentiation, expression of MASH1 appears insufficient to promote neurogenesis.

Animals↗
PubMed1992

Utilization of acute bronchodilator responses in stable COPD to predict the relative efficacy of individual agents.

A survey of four inhaled beta-agonist agents was evaluated as a means of selecting the optimum agent for chronic therapy in patients with stable COPD. Eighteen patients completed as protocol of prebronchodilator and postbronchodilator spirometry utilizing albuterol, metaproterenol, pirbuterol, and terbutaline daily in random order. Subsequently, each patient received treatment with either the greatest or least response-invoking agent for four weeks, followed by a second interval with the opposite agent. At the end of each interval, the results of repeat spirometry, arterial blood gas determinations, 12-min walks, dyspnea questionnaires, and self-monitored peak expiratory flow rates were recorded. Use of the greatest response-invoking agent resulted in significantly larger prebronchodilator and postbronchodilator FEV1 and FVC. No other study factor was significantly different. Acute bronchodilator surveys may have a role in medication selection in view of the improvement in spirometric volumes.

Bronchodilator Agents↗
PubMed1992

Quality improvement: the nurse's role.

Continuous quality improvement is a concept which includes: Quality assurance--the provision of services that meet an appropriate standard. Problem resolution--including all departments involved in the issue at hand. Quality improvement--a continuous process involving all levels of the organization working together across departmental lines to produce better services for health care clients. Deming (1982b) and others have espoused total system reform to achieve quality improvement--not merely altering the current system, but radically changing it. It must be assumed that those who provide services at the staff level are acting in good faith and are not willfully failing to do what is correct (Berwick, 1991). Those who perform direct services are in an excellent position to identify the need for change in service delivery processes. Based on this premise, the staff nurse--who is at the heart of the system--is the best person to assess the status of health care services and to work toward improving the processes by which these services are provided to clients in the health care setting. The nurse manager must structure the work setting to facilitate the staff nurse's ability to undertake constructive action for improving care. The use of quality circles, quality councils, or quality improvement forums to facilitate the coordination of quality improvement efforts is an effective way to achieve success. The QA coordinator assists departments in documenting that the quality improvement efforts are effective across all departments of the organization, and aggregates data to demonstrate that they meet the requirements of external regulatory agencies, insurers, and professional standards. The nurse executive provides the vision and secures the necessary resources to ensure that the organization's quality improvement efforts are successful. By inspiring and empowering the staff in their efforts to improve the process by which health care is provided, nurse managers participate in reshaping the health care environment. The professional nurse plays a vital role in the quality improvement of health care services. However, nurses cannot make these improvements in a vacuum; they must include other professionals and ancillary personnel in their efforts. Total quality commitment must include all levels of an organization's structure. Quality patient care services will be achieved as the result of positive interactions among departments working together to build a dynamic mechanism that continuously improves the processes and outcomes of health care services.

Humans↗