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Biomedical subjects

D C Johnson

Publications and source records attributed to D C Johnson.

At least 19 recordsLinked to original sources
PubMed2005

NifU and NifS are required for the maturation of nitrogenase and cannot replace the function of isc-gene products in Azotobacter vinelandii.

In recent years, it has become evident that [Fe-S] proteins, such as hydrogenase, nitrogenase and aconitase, require a complex machinery to assemble and insert their associated [Fe-S] clusters. So far, three different types of [Fe-S] cluster biosynthetic systems have been identified and these have been designated nif, isc and suf. In the present work, we show that the nif-specific [Fe-S] cluster biosynthetic system from Azotobacter vinelandii, which is required for nitrogenase maturation, cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins, such as aconitase. The results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.

Azotobacter vinelandii↗
PubMed2003

Acid neutralisation capacity of accelerated carbonated stainless steel slag.

The acid neutralisation capacity test is widely used to assess the long-term performance of waste materials prior to disposal. Samples of fixed mass are exposed to increasing additions of nitric add in sealed containers and the resultant pH is plotted as a titration curve. In this work, the add neutralisation capacity test was used in the assessment of an accelerated carbonated stainless steel slag. Difficulties arose in applying the test procedure to this material. This was largely because of the raised pressure from significant volumes of released carbon dioxide trapped in the sealed sample containers, causing an alteration to leachate pH values. Consequently, the add neutralisation capacity test was modified to enable testing of samples in equilibrium with the atmosphere. No adverse effects on the results from testing of a carbonate free material were recorded.

Carbon Dioxide↗
PubMed2003

Solidification of stainless steel slag by accelerated carbonation.

On exposure to carbon dioxide (CO2) at a pressure of 3 bars, compacts formed from pressed ground slag, and 12.5 weight percent water, were found to react with approximately 18% of their own weight of CO2. The reaction product formed was calcium carbonate causing the slag to self-cement. Unconfined compressive strengths of 9MPa were recorded in carbonated compacts whereas strengths of < 1 MPa were recorded in non-carbonated slag compacts. As molten stainless steel slag containing dicalcium silicate (C2S) cools it can undergo several phase transitions. The final transformation from the beta-polymorph to gamma-C2S is accompanied by a volume change that causes the slag to self-pulverise or 'dust'. As a consequence of this the fine grained portion of the slag contains more of this phase whilst the coarser particles of the slag contain more of the calcium magnesium silicates that contribute the bulk of the waste. The fine fraction (< 125 microm) of the slag when ground is found to react to the same extent as the ground bulk slag and produces compacts with equivalent strength. A coarser fraction (4-8 mm) when ground to a similar grading does not react as extensively and produces a weaker product. Additions of ordinary Portland cement (OPC) at 5 and 10 percent by weight did not alter the degree of reaction during carbonation of the bulk slag or ground fine fraction, however the strength of the 4-8 mm fraction was increased by this change.

Calcium Carbonate↗
PubMed2002

Inhibition of the MHC class II antigen presentation pathway by human cytomegalovirus.

Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals. Normally, anti-HCMV immune response controls virus replication following reactivation from latency. However, HCMV, like other large herpesviruses, encodes immune evasion proteins that allow the virus to replicate, for a time or in specific tissues, and produce viral progeny in the face of robust host immunity. HCMV glycoproteins US2, US3, US6 and US11 all inhibit different stages of the MHC class I antigen presentation pathway and can reduce recognition by CD8+ T lymphocytes. Here, we discuss two novel inhibitors of the MHC class II antigen presentation pathway, HCMV glycoproteins US2 and US3. Both US2 and US3 can inhibit presentation of exogenous protein antigens to CD4+ T lymphocytes in in vitro assays. US2 causes degradation of MHC class II molecules: HLA-DR-alpha and HLA-DM-alpha, as well as class I heavy chain (HC), but does not affect DR-beta or DM-beta chains. Mutant forms of US2 have been constructed that can bind to DR-alpha and class I HC but do not cause their degradation, separating the binding step from other processes that precede degradation. We also found evidence that US2-induced degradation of class I and II proteins involves a cellular component, other than Sec61, that is limiting in quantity. Unlike US2, US3 binds newly synthesized class II alpha/beta complexes, reducing the association with the invariant chain (Ii) and causing mislocalization of class II complexes in cells. US3 expression reduces accumulation of class II complexes in peptide-loading compartments and loading of peptides. Since US2 and US3 are expressed solely within HCMV-infected cells, it appears that these viral proteins have evolved to inhibit presentation of endogenous, intracellular viral antigens to anti-HCMV CD4+ T cells. This is different from how the MHC class II pathway is normally viewed, as a pathway for presentation of exogenous, extracellular proteins. The existence of these proteins indicates the importance of class II-mediated presentation of endogenous antigens in signalling virus infection to CD4+ T cells.

Animals↗
PubMed2001

Composition dependence of the nucleation energy of iron antimonides from modulated elemental reactants.

Modulated elemental reactants containing iron and antimony were found to react at low temperature (T < 200 degrees C) forming either FeSb(2) or FeSb(3) depending on both the layer thicknesses and the overall composition of the initial reactant. For films containing 75% antimony and 25% iron, the metastable compound FeSb(3) was observed to nucleate and grow if the layer thickness was below approximately 35 A. Above this critical thickness for the modulation, annealing led to the formation of FeSb(2). This, combined with low-angle diffraction data, suggests that the initial interdiffusion between iron and antimony layers in an elementally modulated reactant results in the formation of an amorphous reaction intermediate if the layering thickness is less than 35 A. For modulated reactants with composition between 70 and 90 atomic % antimony and below this critical layer thickness, the metastable compound FeSb(3) formed. In more iron-rich modulated reactants FeSb(2) nucleates. The nucleation temperature and the nucleation energy of FeSb(3) were found to be a function of the composition of the amorphous intermediate, while those of FeSb(2) were found to be relatively independent of composition.

Journal Article↗
PubMed2001

Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread.

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

Adaptor Protein Complex 1↗
PubMed2001

Herpes simplex virus with highly reduced gD levels can efficiently enter and spread between human keratinocytes.

The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

Cell Line↗
PubMed2001

Cytoplasmic domain of herpes simplex virus gE causes accumulation in the trans-Golgi network, a site of virus envelopment and sorting of virions to cell junctions.

Alphaherpesviruses express a heterodimeric glycoprotein, gE/gI, that facilitates cell-to-cell spread between epithelial cells and neurons. Herpes simplex virus (HSV) gE/gI accumulates at junctions formed between polarized epithelial cells at late times of infection. However, at earlier times after HSV infection, or when gE/gI is expressed using virus vectors, the glycoprotein localizes to the trans-Golgi network (TGN). The cytoplasmic (CT) domains of gE and gI contain numerous TGN and endosomal sorting motifs and are essential for epithelial cell-to-cell spread. Here, we swapped the CT domains of HSV gE and gI onto another HSV glycoprotein, gD. When the gD-gI(CT) chimeric protein was expressed using a replication-defective adenovirus (Ad) vector, the protein was found on both the apical and basolateral surfaces of epithelial cells, as was gD. By contrast, the gD-gE(CT) chimeric protein, gE/gI, and gE, when expressed by using Ad vectors, localized exclusively to the TGN. However, gD-gE(CT), gE/gI, and TGN46, a cellular TGN protein, became redistributed largely to lateral surfaces and cell junctions during intermediate to late stages of HSV infection. Strikingly, gE and TGN46 remained sequestered in the TGN when cells were infected with a gI(-)HSV mutant. The redistribution of gE/gI to lateral cell surfaces did not involve widespread HSV inhibition of endocytosis because the transferrin receptor and gE were both internalized from the cell surface. Thus, gE/gI accumulates in the TGN in early phases of HSV infection then moves to lateral surfaces, to cell junctions, at late stages of infection, coincident with the redistribution of a TGN marker. These results are related to recent observations that gE/gI participates in the envelopment of nucleocapsids into cytoplasmic vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000) and that gE/gI can sort nascent virions from cytoplasmic vesicles specifically to the lateral surfaces of epithelial cells (D. C. Johnson, M. Webb, T. W. Wisner, and C. Brunetti, J. Virol. 75:821-833, 2000). Therefore, gE/gI localizes to the TGN, through interactions between the CT domain of gE and cellular sorting machinery, and then participates in envelopment of cytosolic nucleocapsids there. Nascent virions are then sorted from the TGN to cell junctions.

Adenoviridae↗
PubMed2000

Importance of adjusting carbon monoxide diffusing capacity (DLCO) and carbon monoxide transfer coefficient (KCO) for alveolar volume.

The volume dependence of single breath carbon monoxide diffusing capacity (DLCO) and carbon monoxide transfer coefficient (KCO) was determined in 24 healthy subjects. The change in DLCO [fraction of DLCO measured at total lung capacity (TLC)] to change in alveolar volume [fraction of alveolar volume (VA) at TLC] closely fitted a simple linear regression and matched a theoretical model. As VA decreased, DLCO fell linearly and KCO increased as expected from the relation of DLCO to VA. The equations for adjustment of predicted DLCO and KCO for alveolar volume are: DLCO/DL COtlc = 0.58 + 0.42 VA/VAtlc, KCO/KCOtlc = 0.42 + 0.58/(VA/VAtlc). DLCO and KCO were evaluated in 2313 patients. Subgroups of patients with asthma, emphysema, extrapulmonary lung disease, interstitial lung disease and lung resection were identified. Unadjusted DLCO and KCO percent predicted values showed large differences and much variability, so can be misleading. As expected, KCO and DLCO percent predicted values adjusted for alveolar volume were nearly identical. Subgroups have characteristic patterns of VA and unadjusted and adjusted DLCO and KCO. Changes in DLCO and KCO with alveolar volume are relevant for accurate interpretation of diffusion in patients with low lung volumes. Adjusting predicted DLCO and KCO for alveolar volume provides a better assessment of lung function.

Adolescent↗
PubMed2000

Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8(+) cytotoxic T lymphocytes.

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8(+) CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.

Animals↗
PubMed2000

The extracellular domain of herpes simplex virus gE is sufficient for accumulation at cell junctions but not for cell-to-cell spread.

Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or cell-to-cell spread.

Alanine↗
PubMed1999

The closed heart MAZE: a nonbypass surgical technique.

BACKGROUND: The MAZE-III is the surgical treatment of choice for medically refractory atrial fibrillation. Although a number of nonsurgical techniques are evolving to duplicate the transmural atrial lesions of the MAZE-III, the surgical atriotomy remains the gold standard for conduction block. It was the objective of this study to surgically create the atrial incisions of the MAZE-III without the use of cardiopulmonary bypass. METHODS: A technique was developed to create and intersect the linear incisions of the MAZE-III on 10 beating canine hearts without the use of cardiopulmonary bypass using a "tunnel" of atrial tissue. The effectiveness of the procedure was tested by atrial burst pacing. RESULTS: This technique was successfully performed on 10 mongrel dogs without operative mortality. Preoperatively, sustained atrial fibrillation (>30 seconds) was induced in all animals. Postoperatively, all the animals remained in sinus rhythm even after burst pacing. CONCLUSIONS: In an experimental canine model, the MAZE-III can be performed on beating hearts without the assistance of cardiopulmonary bypass using a "tunnel" technique. This technique allows for the immediate assessment of electrophysiologic and mechanical function after the MAZE-III, or any other type of procedure using the "maze principle" and may find future application in the clinical arena.

Animals↗
PubMed1999

Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-alpha and DM-alpha. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.

Adenoviridae↗