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W Ross

Publications and source records attributed to W Ross.

At least 19 recordsLinked to original sources
PubMed1992

Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent.

Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.

Base Sequence↗
PubMed1992

Comparing in-patient classification systems: a problem of non-nested regression models.

Since 1983, hospitals in the United States have been receiving prospective payment for their in-hospital patient admissions covered under Medicare. Under such schemes each patient is placed in a group by a classification system, known as the Diagnosis Related Groups (DRG), and the hospital is reimbursed by the Health Care Financing Administration according to some predetermined group average, adjusted for hospital level characteristics, such as size, location and teaching activity. Recent interest has focused on refining the DRG system or considering totally different systems of classification. Studies designed to compare the ability of different systems to account for between-patient variability in resource consumption in the same dataset lead to the problem of model selection between large non-nested regressions, where resource consumption, measured by length of hospital stay or costs, is regressed on dummy-indicator variables representing different patient groups. We use a simple measure of fit to develop a symmetric test of the null hypothesis that the two systems account equally well for variability in resource consumption. With this method, unlike methods such as Akaike's AIC criterion, we can quantify the probability of a false positive, and thereby limit the probability of choosing one system over another when it is no better at accounting for variability in resource consumption.

Diagnosis-Related Groups↗
PubMed1992

Intracellular glutathione levels in T cell subsets decrease in HIV-infected individuals.

The authors have shown previously that intracellular glutathione (GSH) plays an important role in the regulation of human immunodeficiency virus (HIV) transcription and replication in vitro, through modulation of signal transduction by inflammatory cytokines. Moreover, intracellular GSH levels are known to regulate T-lymphocyte function. In multiparameter FACS studies presented here, we show that relative GSH levels in CD4+ and CD8+ T cells from HIV+ individuals are significantly lower than in corresponding subsets from uninfected controls. These studies define the relative intracellular glutathione (GSH) levels in CD4+ T cells, CD8+ T cells, B cells, and monocytes from 134 HIV-infected individuals and 31 uninfected controls. The greatest decreases in intracellular GSH occur in subsets of T cells in individuals in the later stages of the HIV infection. In AIDS patients, GSH levels are 63% of normal in CD4+ T cells (p less than 0.0001) and are 62% of normal in CD8+ T cells (p less than 0.0001). Similarly, in AIDS-related complex (ARC) patients, GSH levels are 66% of normal in CD4+ T cells (p less than 0.003) and are 69% of normal in CD8+ T cells (p less than 0.003). These findings suggest that low intracellular GSH levels may be an important factor in HIV infection and in the resulting immunodeficiency.

AIDS-Related Complex↗
PubMed1992

Involvement of Fis protein in replication of the Escherichia coli chromosome.

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.

Bacterial Proteins↗
PubMed1991

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.

Base Sequence↗
PubMed1990

Sequences upstream of the-35 hexamer of rrnB P1 affect promoter strength and upstream activation.

Transcription from Escherichia coli ribosomal RNA promoters is increased about 20-fold in vivo by a DNA sequence (the Upstream Activation Region, UAR) located upstream of the -35 conserved hexamer. The UAR stimulates transcription through two mechanisms: one which involves binding of the Fis protein to the UAR, and another mechanisms which functions in the absence of additional protein factors. We have previously constructed a collection of mutations in the region upstream of the -35 hexamer of rrnB P1. Most of these mutations have either no effect on promoter activity or decrease activity 2-5-fold in vivo (Gaal, T., Barkei, J., Dickson, R.R., De Boer, H.A., De Haseth, P.L., Alavi, H. and Gourse, R.L.(1989) J. Bacteriol. 171, 4852-4861). Two mutations leave both the -35 consensus hexamer and the Fis binding consensus sequence intact, yet have larger (14-50-fold) effects on transcription. One substitution just upstream of the -35 hexamer (a C to T change at position -37) primarily affects intrinsic promoter strength, leaving the UAR functional. On the other hand, a three base pair deletion (bases -38 through -40) severely reduces UAR-mediated activity. A substitution covering the three base pair deletion was constructed and found to be activated normally. UAR function appears dependent on its position relative to the RNA polymerase binding site, suggesting that a particular spatial geometry may be necessary for Fis-dependent and/or factor-independent activation to occur.

Base Sequence↗
PubMed1990

A photoactivatable probe for the Na+/H+ exchanger cross-links a 66-kDa renal brush border membrane protein.

Earlier studies on LLC-PK1 cells have demonstrated two pharmacologically distinct Na+/H+ exchangers in renal epithelia. In addition, the cDNA clone for the human Na+/H+ antiporter which is growth factor activatable has been isolated and expressed (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280). We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with 125I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na+/H+ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na+/H+ exchanger.

Affinity Labels↗
PubMed1990

E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.

An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20- to 30-fold over that of the same promoter lacking the UAR. We have used DNase footprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems. We find that purified Fis activates transcription from promoters containing these sites 10- to 20-fold in vitro at concentrations correlating with the filling of these sites. Three approaches indicate that Fis contributes to the function of the UAR in vivo. First, there is a progressive loss in the activity of rrnB P1-lacZ fusions as Fis binding sites are deleted. Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5-fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro. Third, upstream activation is reduced 5-fold in a Fis- strain. We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation. We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control. On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.

Base Sequence↗
PubMed1989

Transcriptional switching by the MerR protein: activation and repression mutants implicate distinct DNA and mercury(II) binding domains.

Bacterial resistance to mercuric compounds is controlled by the MerR metalloregulatory protein. The MerR protein functions as both a transcriptional repressor and a mercuric ion dependent transcriptional activator. Chemical mutagenesis of the cloned merR structural gene has led to the identification of mutant proteins that are specifically deficient in transcriptional repression, activation, or both. Five mutant proteins have been overproduced, purified to homogeneity, and assayed for ability to dimerize, bind mer operator DNA, and bind mercuric ion. A mutation in the recognition helix of a proposed helix-turn-helix DNA binding motif (E22K) yields protein deficient in both activation and repression in vivo (a-r-) and deficient in operator binding in vitro. In contrast, mutations in three of the four MerR cysteine residues are repression competent but activation deficient (a-r+) in vivo. In vitro, the purified cysteine mutant proteins bind to the mer operator site with near wild-type affinity but are variably deficient in binding the in vivo inducer mercury(II) ion. A subset of the isolated proteins also appears compromised in their ability to form dimers at low protein concentrations. These data, taken with the results in the preceding paper (Shewchuk et al., 1989), support a model in which DNA-bound MerR dimer binds one mercuric ion and transmits this occupancy information to a protein region involved in transcriptional activation.

Amino Acid Sequence↗
PubMed1989

AIDS dementia complex. Characteristics of a unique aspect of HIV infection.

A common complication of acquired immunodeficiency syndrome (AIDS) is being increasingly recognized. Known as AIDS dementia complex, the disorder is characterized by global impairment of intellectual and cognitive functions, personality and behavioral disturbances, and motor dysfunction manifested by impaired speech, gait, and coordination. AIDS dementia complex progresses rapidly and inexorably from psychomotor slowing to dementia, then to an akinetic mute state in which the patient becomes immobile and incapable of speaking, and finally to coma. Differentiation of AIDS dementia complex from a potentially treatable condition, such as reactive depression, is important. Zidovudine (Retrovir), the drug currently used to treat AIDS, has shown promise in the treatment of AIDS dementia complex. Clinical trials are now under way to determine its efficacy in this application.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Genetic analysis of transcriptional activation and repression in the Tn21 mer operon.

Transcription of the Tn21 mercury resistance operon (mer) is controlled by the toxic metal cation Hg(II). This control is mediated by the product of the merR gene, a 144-amino-acid protein which represses transcription of the structural genes (merTPCAD) in the absence of Hg(II) and activates transcription in the presence of Hg(II). We have used a mer-lac transcriptional fusion to obtain regulatory mutants in this metal-responsive system. Some mutants were defective in Hg(II)-induced activation while retaining repression function (a- r+), others were defective in repression but not activation (a+ r-), and some had lost both functions (a- r-). Mutations in three of the four cysteine residues of merR resulted in complete loss of Hg(II)-inducible activation but retention of the repressor function, suggesting that these residues serve as ligands for Hg(II) in the activation process. Other lesions adjacent to or very near these cysteines exhibited severely reduced activation and also retained repressor function. There were two putative helix-turn-helix (HTH) domains in merR, and mutants in each had very different phenotypes. A partially dominant mutation in the more amino-terminal region of the two putative HTH regions resulted in loss of both activation and repression (a- r-), consistent with a role for this region in DNA binding. Mutations in the more centrally located HTH region resulted only in loss of Hg(II)-induced activation (a- r+). Lesions in the central and in the carboxy-terminal regions of merR exhibited both Hg(II)-independent and Hg(II)-dependent transcriptional activation, suggesting that elements important in the activation mechanism may be widely distributed in this relatively small protein. The sole cis-acting mutant obtained with this operon fusion strategy, a down-promoter mutation, lies in a highly conserved base in the -35 region of the merTPCAD promoter.

Amino Acid Sequence↗
PubMed1988

Pleural pearl. An immature pleural ball?

An incidental, pearl-like body was attached to the visceral pleura of a young man who committed suicide. The lesion consisted of concentric laminations of collagen and mesothelial type cells, and was associated with a resolved pleurisy. The possible relationship of this lesion to previously reported "pleural balls" is discussed.

Adult↗
PubMed1987

Identification and expansion of human lymphokine-activated killer cells: implications for the immunotherapy of cancer.

Immunotherapy with lymphokine-activated killer (LAK) cells and interleukin 2 (IL-2) has been demonstrated to cause the regression of some human tumors. Expansion of peripheral blood lymphocytes (PBL) in recombinant or naturally produced IL-2, mitogens, and feeder cells results in a progressive expansion of cell number but a decline in lytic activity with time. Lytic activity for fresh tumor is best generated in IL-2 alone from specific purified subpopulations of lymphocytes. The precursor of the LAK cell has been isolated from a nonadherent, sheep erythrocyte receptor negative (E-) lymphocyte subpopulation by the selection of Leu 11+ and Leu 15+ cells by cell sorting. Separation resulted in a significant increase (P2 less than 0.05) in lytic activity against fresh tumor by the separated 11+15+ lymphocytes compared to the 11-/15- lymphocytes or PBL. Leu 11+/15+ lymphocytes could be expanded up to eightfold in IL-2 alone with maintenance of lytic activity. We identified two surface antigens on the LAK precursor cell, Leu 15 and Leu 11, and demonstrated a technique for obtaining purified populations of these cells. Purified and expanded LAK cells when administered to patients may have a role in increasing the potency and decreasing the toxicity associated with this therapy.

Cell Division↗
PubMed1987

Elimination of T cells from human peripheral blood and bone marrow using a cocktail of three anti-T cell immunotoxins.

Four anti-T cell monoclonal antibodies were coupled to ricin-A and tested for their ability to kill T cells in peripheral blood and bone marrow using a clonogenic assay to quantify T cell survival. The immunotoxins (IT) prepared from RFT11 (CD2) and WT1 (CD7) antibodies were the most toxic to peripheral blood T cells. The immunotoxin prepared from RFT1 (CD5) was the next most efficient toxin and the immunotoxin prepared from RFT8 (CD8) was the least toxic. When these reagents were applied to peripheral blood cells at 3 x 10(-8) M the number of T cell colonies was reduced by an average of 95%, 94%, 84% and 50%, respectively. Peripheral blood T cells from different donors showed marked variability in their sensitivity to ITs. However, a cocktail of three ITs prepared from RFT11, WT1 and RFT1 gave superior and consistent killing (mean 99.9%; range 99.8-100%) of peripheral blood T cells from six donors. When this cocktail was applied to bone marrow cells from six donors, an average of 99.6% (range 99.5-99.8%) of the T cells were killed. Under the same conditions there was little or no reduction in the number of normal haematopoietic progenitors (CFU-GM, CFU-GEMM, CFU-Meg and BFU-E).

Antibodies, Monoclonal↗
PubMed1987

Human lymphokine-activated killer cells: further isolation and characterization of the precursor and effector cell.

In a series of experiments we have demonstrated the progressive enrichment (5- to 40-fold) in lymphokine-activated killer (LAK) precursor activity by adherence depletion, sheep red cell rosetting, and depletion of CD3- and DR-positive lymphocytes. The LAK precursor cell thus appears to fall within the 'null' cell population. CD16 and CD11 are cell surface antigens expressed on the surface of the LAK precursor as demonstrated in sorting experiments. A 6- to 100-fold enrichment compared to unseparated peripheral blood was noted when sorted cells positive for CD16 and CD11 were tested. The LAK effector has been identified as being primarily CD3- and CD2+. Similar sorting equipment demonstrated a 7- to 500-fold difference in lytic activity for fresh tumor when comparing CD2+/CD3- and CD2+/CD3+ cells. The CD16+/CD11+ lymphocyte can proliferate in response to interleukin-2 (IL-2) alone in the absence of accessory cells and can be expanded in IL-2 alone with maintenance of lytic activity.

Antigens, Differentiation, T-Lymphocyte↗