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W Ross

Publications and source records attributed to W Ross.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1986

Hemoglobin H disease with hemoglobin Constant Spring in a child of Laotian extraction.

In alpha-thalassemia, deletion or inactivation of one, two, three, or all four alpha-genes causes, respectively, silent carrier state (-alpha/alpha alpha); alpha-thalassemia trait (--/alpha alpha in Orientals, and -alpha/-alpha in blacks); Hb H disease (--/-alpha); and Hb Bart's hydrops fetalis (--/--). We have described a case of Hb H disease with Hb Constant Spring (--/alpha alpha cs) in a Laotian child whose father had alpha-thalassemia trait and whose mother was a carrier of Hb CS, a mutant hemoglobin produced in minimal amounts. Since alpha-thalassemia is highly prevalent in Southeast Asia, physicians should become alert to the potential occurrence of Hb H disease and Hb Bart's hydrops fetalis in the new Oriental immigrants and their progeny.

Genetic Carrier Screening↗
PubMed1986

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.

Bacterial Proteins↗
PubMed1986

Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity.

Recent evidence indicates that type II DNA topoisomerases mediate epipodophyllotoxin-induced DNA damage and may be intrinsic to the drug's antitumor effects. Using an epipodophyllotoxin-resistant cell line, we have now further defined the relationship between DNA damage and cell death and delineated the significance of certain drug-enzyme interactions. When compared to wild-type cells, the mutant Chinese hamster ovary cell line, VpmR-5, exhibits marked resistance to both the cytotoxic and DNA cleavage activities of etoposide (VP-16). Steady-state concentrations of radiolabeled VP-16 are identical in both cell lines. Catalytic activity in crude nuclear extracts from wild-type and VpmR-5 cells is equal and is equally sensitive to inhibition by VP-16. However, using an assay that specifically measures generation of 5' protein-linked breaks in 32P-labeled 3' DNA, we have found that DNA cleavage activity in nuclear extract from the VpmR-5 line is profoundly resistant to stimulation by VP-16. Further, a somatic cell hybrid line of VpmR-5 cells and drug-sensitive EOT-3 cells exhibits recovery of VP-16 sensitivity in concert with reconstitution of DNA cleavage activity. These data indicate that stimulation of enzyme-mediated DNA cleavage, rather than loss of normal topoisomerase function, is responsible for epipodophyllotoxin-induced cytotoxicity.

Aminoacridines↗
PubMed1986

Cross-resistance to intercalating agents in an epipodophyllotoxin-resistant Chinese hamster ovary cell line: evidence for a common intracellular target.

Several intercalating agents, as well as the epipodophyllotoxins, appear to effect DNA damage through their interaction with type II DNA topoisomerases. However, the relationship of this phenomenon to anti-tumor activity remains unproven. Our studies with an epipodophyllotoxin-resistant cell line not only provide additional evidence that the enzyme is a multidrug target but also serve to implicate it as a mediator of cytotoxic effect. When compared to wild-type cells, the epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, exhibits cross-resistance to both the cytotoxic and DNA cleavage activities of 4',9-acridinylaminomethanesulfon-m-anisidide, mitoxantrone, and Adriamycin. Steady-state concentrations of radiolabeled-4',9-acridinylaminomethanesulfon-m-anisidide and daunomycin are identical in both cell lines. Sharp plateaus in the VpmR-5 dose-response curves for Adriamycin-induced DNA strand breaks and cytotoxicity appear to be related to interference with type II topoisomerase-mediated cleavage of DNA at high concentrations of the intercalator. These data support a direct role for DNA strand scission in cell death and also suggest that multidrug resistance may be acquired by a qualitative change in type II topoisomerase that alters interaction of drug with the enzyme or enzyme-DNA complex.

Aminoacridines↗
PubMed1985

Inhibition of epipodophyllotoxin cytotoxicity by interference with topoisomerase-mediated DNA cleavage.

This laboratory and others previously proposed that the antitumor effects of the epipodophyllotoxin compounds are based on their abilities to stimulate DNA cleavage by a DNA topoisomerase. To explore this relationship further, we studied the intercalating agent ethidium bromide and found that it blocked epipodophyllotoxin-induced DNA cleavage by DNA topoisomerase II in vitro as well as in vivo. Using an in vitro assay consisting of purified calf thymus DNA topoisomerase II, end-labeled DNA, and the epipodophyllotoxin teniposide, we found that ethidium bromide markedly interfered with the enzyme-mediated DNA cleavage. Furthermore, ethidium bromide also blocked the formation of DNA single- and double-strand breaks in mouse L1210 cells when exposed to the epipodophyllotoxin etoposide. This effect cannot be explained by alterations in drug accumulation since steady-state drug concentrations were unchanged, and the effect was also observed in isolated nuclei. In addition to its effects on epipodophyllotoxin-mediated DNA breakage, ethidium bromide also potently inhibited the cytotoxic effects of etoposide but only when present during drug treatment. Thus, we believe that ethidium bromide may be a useful tool to investigate drug-induced perturbations of topoisomerase activity and their relationship to antitumor effect. Our data strongly support the hypothesis that the antitumor activity of epipodophyllotoxins is based on the ability to stimulate the formation of a cleavable complex between DNA topoisomerase and DNA.

Animals↗
PubMed1985

Intensive short-term chemotherapy for patients with acute myelogenous leukemia: long-term follow-up.

Our pilot study addresses the problem of early relapse from complete remission in young adults with acute myelogenous leukemia (AML). Twelve patients with AML, 16-58 years of age, were entered in a study of four intense courses of cytotoxic chemotherapy using the following drugs: cytarabine, daunorubicin, 5-azacitidine, and 6-thioguanine. They received no maintenance therapy. Nine of 12 patients achieved complete response. With a minimum follow-up of 35 months, the observed disease-free survival at 2 years was 67% (14 +/- SE) and the actuarial disease-free survival at 4 years was 38% (17 +/- SE). It appears that brief intensive chemotherapy early in the management of AML can produce prolonged remission without the need for maintenance therapy.

Actuarial Analysis↗