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D M Rothstein

Publications and source records attributed to D M Rothstein.

At least 55 records · Page 3Linked to original sources
PubMed1990

Sulfadiazine crystalluria revisited. The treatment of Toxoplasma encephalitis in patients with acquired immunodeficiency syndrome.

Toxoplasma gondii encephalitis is an important opportunistic infection in the acquired immunodeficiency syndrome, estimated to occur in 20,000 to 40,000 patients with acquired immunodeficiency syndrome in the United States by 1991. The combination of sulfadiazine and pyrimethamine is regarded as the treatment of choice. Acute renal failure due to crystal deposition in the urinary tract was well described 30 to 40 years ago and is likely to resurface as a clinical entity if appropriate prophylactic measures are not taken. We describe two cases of sulfadiazine-induced crystalluria and renal failure in patients with acquired immunodeficiency syndrome, review the pertinent literature, and discuss the pathogenesis. Recommendations are made for the prophylaxis and treatment of sulfadiazine-related renal toxic reaction. Physicians using this "new" drug must be aware of the potential danger of sulfonamide-induced injury to the urinary tract.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Effect of activation of protein kinase C on CD45 isoform expression and CD45 protein tyrosine phosphatase activity in T cells.

The T200/leukocyte common antigen (CD45) is a family of at least five large-molecular weight glycoproteins, which are differentially expressed on T cell subsets. The CD45 antigen consists of a variable heavily glycosylated exterior domain, a single membrane-spanning region, and a large cytoplasmic domain that has protein tyrosine phosphatase (PTPase) activity. In this study, we examined the effects of activation of protein kinase C (PKC) on the phosphorylation and expression of CD45 isoforms and PTPase activity in human T cells. After activation of PKC by phorbol 12-myristate 13-acetate (PMA), CD45RA expression rapidly increased within the first 24 h, whereas CD45R0 expression did not change within this time. However by 48 h, expression of CD45R0 also began to increase. Metabolic labeling showed that the rapid increment in CD45RA expression observed after PMA stimulation is primarily due to increased de novo synthesis of the 205-kDa and not the 220-kDa molecule. PMA treatment resulted in the phosphorylation of each CD45 isoform to a degree corresponding to its relative surface expression. Significantly, we found that the phosphorylation of CD45 by PKC activation down-regulated CD45 PTPase activity.

Antigens, Differentiation↗
PubMed1990

The functional heterogeneity of CD8+ cells defined by anti-CD45RA (2H4) and anti-CD29 (4B4) antibodies.

The monoclonal antibodies, anti-2H4(CD45RA), and anti-4B4(CD29), along with UCHL1-(CD45RO), identify reciprocal populations of CD4 cells with distinct suppressor inducer (CD45RA+CD29-CD45RO-) and helper inducer (CD45RA-CD29+CD45RO+) functions. Although the CD8+ population is known to contain precytotoxic, cytotoxic, suppressor, and some natural killer cells, the exact phenotypic identities of these functional CD8 subsets has not been established. In this study, we tried to determine whether these monoclonal antibodies could distinguish functionally distinct subsets of cells within the CD8+ population. For this purpose, whole T cells or fractionated T cells were sensitized with irradiated allogeneic non-T cells for 6 days, following which, CD8+ or CD8+CD11b- cells were isolated and cellular functions such as suppressor, killer precursor, and killer effector activity were assessed. The results showed that both class I-restricted alloantigen-specific killer effector and killer precursor cells belonged to the CD8+CD11b-CD45RA-CD29+ population. Moreover, these killer effector cells expressed the CTL-associated S6F1 molecule, an epitope of the LFA-1 antigen. In contrast, suppressor effector cells belonged to the CD8+CD11b-CD45RA+CD29- cell population. Although the UCHL1 antigen has been reported to define the CD4+CD29+ helper inducer cell, over 90% of allo-activated CD8+ cells expressed this antigen, whereas only 40-60% of these cells expressed either CD45RA or CD29 antigens. These results suggest that anti-CD45RA and anti-CD29 antibodies may provide useful tools for distinguishing between suppressor effector versus killer effector and killer precursor cells within the CD8+CD11b- population.

Antibodies, Monoclonal↗
PubMed1990

CD4+CD45RA+ and CD4+CD45RA- T cell subsets in man maintain distinct function and CD45RA expression persists on a subpopulation of CD45RA+ cells after activation with Con A.

We have previously shown that Con A-induced suppressor T cells belong to the CD45RA+ subset. After unseparated T cells are activated with Con A, CD45RA expression increases to a maximum (Day 2), and then decreases significantly, but does not disappear entirely (Day 9), while CD29 expression increases steadily. In the present study, we examined the fate of these cell surface molecules on isolated CD4+CD45RA+ and CD4+CD45RA- cells following activation with Con A, and their relationship to the regulatory functions of these subsets. After activation of CD4+CD45RA+ cells with Con A, CD45RO and CD29 antigen expression rapidly increases (greater than 90%). While CD45RA expression is downregulated, approximately 40% of the cells continue to express low-density CD45RA in a stable fashion through Day 21. Despite these phenotypic changes, cells originally CD45RA+ continue to suppress IgG synthesis and provide only minimal B cell help. Furthermore, when cells originally CD45RA+ were sorted on the basis of continued presence, or loss of CD45RA antigen 14 days after activation, both populations demonstrated potent suppression and minimal help. In contrast, after activation with Con A, CD4+CD45A- cells maintain stable phenotype and provide significant help and minimal suppression. Immunoprecipitation of the CD45RA antigen from Day 14 activated CD4+CD45RA+ cells confirms the continued presence of the 205-kDa isoform, but reveals a significant decrease in the 220-kDa isoform. These results suggest that after activation with Con A, cells originally CD45RA+ remain functionally distinct from cells originally CD45RA-, and that CD45RA antigen persists on a subpopulation of CD45RA+ cells after activation with Con A.

Antigens, CD↗
PubMed1990

Sensitive biological detection method for tetracyclines using a tetA-lacZ fusion system.

A sensitive microbiological detection system for tetracyclines, utilizing an Escherichia coli strain containing a cloned tetA-lacZ gene fusion, is described. Expression of beta-galactosidase by the fusion plasmid pUB3610 remained subject to regulatory control by the TetR repressor protein, with the presence of tetracyclines in the growth medium leading to a 12-fold induction of beta-galactosidase synthesis. Because synthesis of beta-galactosidase was influenced to a small extent by the carbon source and the addition of cyclic AMP to the medium, cells were grown in the presence of cyclic AMP to enhance the sensitivity of the assay. All commonly marketed tetracyclines and some derivatives at concentrations as low as 0.1 ng/ml could be detected in the growth medium. A plate assay utilizing the fusion plasmid that detects 1 ng of tetracycline has also been developed.

Chromosomes, Bacterial↗
PubMed1989

Functional characterization of the CD45R (2H4) molecule on CD8 (T8) cells in the autologous mixed lymphocyte reaction system.

In the present study, we have investigated the molecular basis for the immunoregulatory function of CD8 cells after autologous mixed lymphocyte reaction (AMLR) activation. We demonstrated that the CD8+CD45R+, but not the CD8+CD45R- subset of cells effected suppression following AMLR activation. In contrast, cytotoxic activity against alloantigens resided in both the CD8+CD45R+ and CD8+CD45R- subsets of cells. Biochemical analysis showed that on CD8 cells, the 220-kDa isoform of the LCA/T200 antigen family was better represented than the 200-kDa isoform, when compared to CD4 cells. The density of the CD45R antigen increased on CD8 cells following activation in AMLR and treatment of AMLR-activated CD8 cells with either anti-CD45R antibody or anti-CD3 antibody abolished the suppressor function of these cells. In contrast, treatment of AMLR-activated CD4 cells with anti-CD45R, but not anti-CD3 antibody, abolished the suppressor/inducer function of these cells. The results suggest that the CD45R antigen as well as CD3 T cell receptor complex have an important role in the suppressor function of AMLR-activated CD8 cells.

Antigen-Antibody Reactions↗
PubMed1989

Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences.

We demonstrated previously that the 0.4-kilobase DNA fragment from Micromonospora echinospora contains multiple tandem promoters, P1a, P1b, P1c, and P2, which are also functional when cloned into Streptomyces lividans. We now show by in vitro transcription with Streptomyces RNA polymerase that each of these promoters is an authentic initiation site, rather than a processing site for transcripts which initiate further upstream. The DNA sequence requirements for the closely spaced promoters P1a, P1b, and P1c, which are coordinately induced during stationary phase in M. echinospora, were examined by deletional analysis in S. lividans. The P1a and P1b promoters were functional despite deletion of native sequences 5 and 17 base pairs upstream of each initiation site, respectively. Thus, P1a and P1b had greatly reduced upstream DNA sequence requirements compared with typical procaryotic promoters. In contrast, transcription from promoter P1c was significantly decreased when native sequences 34 base pairs upstream were replaced.

Base Sequence↗
PubMed1989

Requirements for the induction of allospecific CD8+ suppressor T cells in the rat primary mixed lymphocyte response. CD4+, CD45R+ T cells, or supernatant factor.

We examined the requirements for the induction of the MLR-generated allospecific CD8+ suppressor T cells in the rat. Depleting the responder population of CD4+ T cells before initiating the primary MLR abrogates the generation of day-5 CD8+ T suppressor effectors. Readdition of at least 10% CD4+ T cells to the CD4+ depleted primary MLR reconstitutes suppressor cell generation. Using the anti-CD45R monoclonal antibody OX22, we also show that the T suppressor inducer cells are CD4+ CD45R+. Using a dual chamber Transwell culture system, which allows cells to be co-incubated without direct cell-to-cell contact, we show that a soluble factor/s, produced during the course of the primary MLR, is capable of inducing naive CD8+ T cells to become suppressor effectors but only when these CD8 T cells are in direct contact with allogeneic stimulators. Allospecificity is conferred by the stimulator cells and not by the suppressor-inducer factor. The supernatant of day-5 primary MLR is also capable of inducing antigen-specific suppressor effectors from naive CD8+ T cells, and also only in the presence of allogeneic stimulator cells. Recombinant human IL-2, in doses that are up to five times the amount present in the supernatant cultures, is unable to induce suppressor-effector cells from naive CD8+ T cells. We conclude that, to become allospecific suppressor effectors, naive CD8+ T cells require contact with allogeneic stimulator cells and either CD4+ CD45R+ suppressor inducer cells or suppressor inducer factor/s produced during the course of the primary MLR.

Animals↗
PubMed1988

Temporally regulated tandem promoters in Micromonospora echinospora.

A collection of promoters from the Micromonospora echinospora strain that produces the calichemicin antitumor antibiotics was identified by the use of the promoter-probe vector pIJ486 in Streptomyces lividans. A 0.4-kilobase-pair Micromonospora DNA fragment was found to contain multiple tandem promoters which were characterized by S1 nuclease protection, Northern blotting, and DNA sequence determination. Analysis of RNA isolated from timed Micromonospora cultures revealed two classes of promoters within the 0.4-kilobase-pair fragment. The P2 promoter was maximally active during the exponential phase. In contrast, the P1 promoter cluster, consisting of three closely spaced start sites located 80 base pairs upstream of P2, was maximally active during the stationary phase. Because P1 was strongly induced in synchrony with calichemicin drug production, P1 is of potential utility in expressing cloned genes specifically during the stationary phase.

DNA, Bacterial↗
PubMed1987

Transcription efficiency along the tissue-plasminogen-activator cDNA gene in Escherichia coli.

Transcription along the human tissue-plasminogen activator gene (tpa) cloned in Escherichia coli was studied by inserting the 4.5S RNA gene at various locations within tpa, and measuring the accumulation of 4.5S RNA. A six-fold decrease in transcription of 4.5S RNA was observed as the distance from the Ptrc promoter to the 4.5S RNA gene increased. Independent measurements indicated that the quantity of initiations from the Ptrc promoter could not account for the differences observed. Because the 4.5S RNA is stable, the decrease in transcription was assumed to be due to premature transcription-termination within the tpa gene. This polar effect could contribute to poor expression of tpa in E. coli.

Cloning, Molecular↗
PubMed1987

Mutations that create new promoters suppress the sigma 54 dependence of glnA transcription in Escherichia coli.

Escherichia coli rpoN mutants lack sigma 54 and are therefore unable to initiate the transcription of glnA at glnAp2, which is required for the production of a high intracellular concentration of glutamine synthetase. We have found that the dependence on sigma 54 can be overcome by mutations that have apparently created a new sigma 70-dependent promoter. The position -35 RNA polymerase contact site of this new promoter overlaps glnAp2. The initiation of transcription at the new promoter is inhibited by sigma 54-RNA polymerase even in the absence of nitrogen regulator I-phosphate, the activator required for the initiation of transcription at glnAp2. The results suggest that in cells growing with an excess of nitrogen and therefore lacking nitrogen regulator I-phosphate, sigma 54-RNA polymerase is bound at glnAp2.

Base Sequence↗
PubMed1986

Expression of alpha-amylase in Bacillus licheniformis.

In Bacillus licheniformis, alpha-amylase production varied more than 100-fold depending on the presence or absence of a catabolite-repressing carbon source in the growth medium. alpha-Amylase was produced during the growth phase and not at the onset of the stationary phase. Induction of alpha-amylase correlated with synthesis of mRNA initiating at the promoter of the alpha-amylase gene.

Bacillus↗
PubMed1985

Thermoinducible transcription system for Bacillus subtilis that utilizes control elements from temperate phage phi 105.

We describe a thermoinducible-expression system for Bacillus subtilis which utilized an early promoter-operator sequence from temperate phage phi 105 and the thermolabile prophage repressor from the phage variant phi 105 cts23. The system operated at the transcriptional level to control expression in B. subtilis of the cat-86 gene derived from Bacillus pumilis. Details of the strategies used to isolate the early phage promoter are described. This promoter lay in close proximity to the prophage repressor gene on the phi 105 genome. The sequence of the early promoter differed from that of the vegetative B. subtilis consensus promoter by 1 base pair in both the -10 and -35 regions. We also present evidence that our phage-derived expression system could function in Escherichia coli to effect thermoinducible expression of the galK gene.

Bacillus subtilis↗
PubMed1983

Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region.

We have determined the nucleotide sequences of the Klebsiella pneumoniae nifL (regulation of N2 fixation genes) and the Escherichia coli glnA (glutamine synthetase) promoters. We compared these sequences with the published sequences of three other promoters that, like the nifL and glnA promoters, are activated by the general nitrogen regulators glnF (ntrA) and glnG (ntrC). The three promoters are the argTr (arginine transport) and dhuA (histidine transport) promoters of Salmonella typhimurium and the nifH (nitrogenase) promoter of Rhizobium meliloti. All five sequences (with at most one mismatch) contain the heptameric consensus sequence T-T-T-T-G-C-A. In the R. meliloti nifH and K. pneumoniae nifL promoters, in which the transcription initiation sites have been determined, the consensus sequence is situated in the -15 region. We recently reported that the K. pneumoniae nifA product, which activates nif genes, can substitute for the glnG (ntrC) product in activating promoters of several genes involved in nitrogen assimilation, including the nifL, the glnA, and the R. meliloti nifH promoters. It is likely that nifA also activates the S. typhimurium argTr and dhuA promoters. In contrast, the glnG product cannot substitute for the nifA product in the activation of the K. pneumoniae nifH (nitrogenase) promoter. Consistent with this latter observation, and supporting the conclusion that the T-T-T-T-G-C-A sequence is a regulatory site for glnG product activation, the K. pneumoniae nifH promoter (C-C-C-T-G-C-A) has only partial similarity with the T-T-T-T-G-C-A consensus sequence in the -15 region.

Amino Acid Sequence↗
PubMed1982

Complex glnA-glnL-glnG operon of Escherichia coli.

The glnG gene product is both a positive regulator and a negative regulator of the expression of glnA, the structural gene for glutamine synthetase, as well as a positive regulator of the expression of a number of genes whose products are involved in the uptake and degradation of nitrogen-containing compounds. The regulation of beta-galactosidase in various strains containing a Mu d1 (lac bla) insertion within glnG leads to the following conclusions regarding the expression of this gene: (i) like the synthesis of glutamine synthetase, the synthesis of the glnG product is regulated in response to the nitrogen source; (ii) high-level expression of glnG under nitrogen-limiting growth conditions depends on transcription initiated at the glnA promoter; and (iii) there is a second, glnA-distal promoter for glnG, whose activity is negatively controlled by the glnG product. Thus, the glnG product regulates the synthesis of the glnG product at two distinct promoters (positively and negatively at the glnA promoter and negatively at the glnA-distal promoter). In addition, a high level of glnG product, corresponding to the level produced by initiation of transcription at the glnA promoter under nitrogen-limiting conditions, is necessary for activation of histidase synthesis. The lower level of glnG product originating from transcription initiated at the glnA-distal promoter is not sufficient to activate histidase synthesis, but is sufficient to activate fully and to repress glnA expression.

Bacteriophage mu↗
PubMed1980

Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase.

One of the suspected regulators of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] in enteric bacteria is glutamine synthetase itself. We isolated Escherichia coli strains carrying fusions of the beta-galactosidase structural gene to the promoter of the glutamine synthetase gene, with the aid of the Casadaban Mud1 (ApR, lac, cts62) phage. Some aspects of regulation were retained in haploid fusion strains despite the absence of glutamine synthetase, whereas other aspects required glutamine synthetase catalytic or regulatory activity or both. The direction of transcription of the glutamine synthetase gene was also determined.

Escherichia coli↗