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Biomedical subjects

K M Gottesdiener

Publications and source records attributed to K M Gottesdiener.

17 recordsLinked to original sources
PubMed2001

Dose proportionality of oral etoricoxib, a highly selective cyclooxygenase-2 inhibitor, in healthy volunteers.

To assess dose proportionality of etoricoxib across the anticipated clinical dose range, a single panel of 12 healthy subjects was administered single oral doses of etoricoxib of 5, 10, 20, 40, and 120 mg in an open, two-part, five-period crossover study. Plasma samples were collected aftereach dose and analyzed for etoricoxib concentrations. The pharmacokinetics of etoricoxib appear to be linear over the entire dose range examined, from 5 to 120 mg. Etoricoxib was found to be well tolerated across the 5 to 120 mg dose range.

Administration, Oral↗
PubMed1999

A Trypanosoma brucei bloodstream form mutant deficient in ornithine decarboxylase can protect against wild-type infection in mice.

A Trypanosoma brucei bloodstream mutant in which both copies of the ornithine decarboxylase (ODC) gene were knocked out (ODC mutant) was used to determine the biological functions of ODC in T. brucei. Growth of the mutant cells ceased within 12-24 h in regular culture medium deficient in polyamines, but could be rescued by supplementation with 1 mM putrescine. A mouse model of T. brucei infection was used to determine whether the mutant was still infective and was found to develop either extremely low or undetectable levels of parasitemia, suggesting that in T. brucei, ODC activity is essential for establishing an infection. Furthermore, when these mice were subsequently challenged with wild-type T. brucei cells expressing the same variant surface glycoprotein (VSG), they did not develop any parasitemia, indicating that inoculating the mice with the attenuated ODC mutant had conferred protection against challenge by wild-type cells. These results were reproduced in C57BL/6J mice deficient in alpha-beta and gamma-delta T-cell receptors. However, no protection was observed in rag-2 knockout mice deficient in both B and T lymphocytes or in C57BL/10J mice deficient only in B lymphocytes. The results thus suggest that the ODC mutant could induce a T-lymphocyte-independent but B-lymphocyte-dependent immunity against wild-type cells of the same VSG. Such a mechanism of immunity has been elicited only by live T. brucei cells, but not by isolated VSGs or radiation-killed trypanosomes. This ODC mutant may thus represent a genuinely attenuated T. brucei bloodstream form capable of immunizing mammals against infections by African trypanosomes of the same VSG subtype without causing detectable infection by itself. The observation also raises the interesting likelihood that the in vivo treatment of T. brucei bloodstream forms with alpha-DL-difluoromethylornithine is a de facto attenuation of the parasitic organisms, which may very well result in B-lymphocyte-dependent host immune responses to subsequent infections by parasites of the same VSG subtypes.

Africa South of the Sahara↗
PubMed1998

Trypanosoma brucei brucei: characterization of an ODC null bloodstream form mutant and the action of alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC) is a key enzyme in the polyamine synthesis pathway in African trypanosomes. We report here the characterization of an ODC null bloodstream form Trypanosoma brucei brucei mutant, created by replacing the ODC gene with antibiotic resistant marker genes through transfection and homologous recombination. The null mutant expresses no ODC mRNA or protein and does not have ODC enzymatic activity. We tested the attenuation of the bloodstream form ODC- mutants in mice, and showed that these mutants cannot multiply and are quickly cleared from the blood. We also tested the effect of DFMO on this ODC null mutant.

Animals↗
PubMed1997

Binding of trans-acting factors to the double-stranded variant surface glycoprotein (VSG) expression site promoter of Trypanosoma brucei.

Trypanosoma brucei evades its host's immune response by utilizing the system of antigenic variation, whereby the organism sequentially expresses antigenically distinct variant surface glycoproteins (VSGs). Actively expressed VSG genes are found in VSG expression sites (ESs), and transcription of these ESs is directed by a small promoter composed of two essential cis-acting elements, the VSG ES promoter upstream element (VUE) and VSG ES promoter downstream element (VDE). Using electrophoretic mobility shift assays, we have identified double-stranded DNA binding activity in bloodstream-form trypanosome nuclear extracts. This activity, the VEP complex, is specific for the VSG ES promoter, and requires the intact sequences of the VUE and VDE in the appropriate spacing. These requirements of VEP Complex formation parallel the requirements for promoter function, suggesting that the VEP complex may be composed of functionally significant trans-acting factors. Furthermore, the requirement of both elements suggests that the binding of factors to the promoter may be cooperative. However, subtly different binding characteristics were observed when we used nuclear extracts derived from procyclic trypanosomes.

Animals↗
PubMed1996

A detailed mutational analysis of the VSG gene expression site promoter.

The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. The parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than -67 bp 5' to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region -67/-60 and the region -35/-20, both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite.

Animals↗
PubMed1996

Analysis of a hybrid PARP/VSG ES promoter in procyclic trypanosomes.

The parasite Trypanosoma brucei changes its variant surface glycoprotein (VSG) coat to escape the host immune system. At a chromosomal locus, we analyzed the promoter that controls expression of VSG genes, using a system developed in collaboration with Urményi and Van der Ploeg (Urményi, T.P. and Van der Ploeg, L.H.T. (1995) Nucleic Acids Res. 23,1010-1016), and showed that the variant surface glycoprotein expression site (VSG ES) promoter directed < 6% the CAT activity produced by the procyclic acidic repetitive protein (PARP) promoter at the same locus. We identified a fragment from the PARP promoter (bp -743 to -111) that contained no intrinsic promoter activity. However, when this fragment was cloned 5' to 3' upstream of the VSG ES promoter, and this hybrid PARP/VSG ES promoter was stably integrated at the RNA polymerase (Pol) II largest subunit gene locus, expression from a CAT gene cassette increased 10-fold. Nascent RNA analysis independently showed that the relative efficiency of alpha-amanitin-resistant transcription directed by the hybrid PARP/VSG ES promoter was more than 6-fold higher than that directed by the wild-type VSG ES promoter. Furthermore, using nascent RNA protection assays, we mapped the transcription start site of the hybrid PARP/VSG ES promoter to the same initiation site as that of the wild-type VSG ES promoter. Finally, we evaluated the functional activity of the hybrid PARP/VSG ES mutant promoter at the dominant VSG gene expression site on the 1.5-Mb chromosome. At this locus, as well, the hybrid PARP/VSG ES promoter directed almost 3-times as much CAT activity as that of the wild-type VSG ES promoter.

Amanitins↗
PubMed1996

Procyclic Trypanosoma brucei cell lines deficient in ornithine decarboxylase activity.

Ornithine decarboxylase (ODC) is a rate limiting enzyme in the biosynthesis of polyamines. We report here the construction of ODC gene deficient Trypanosoma brucei brucei cell lines by homologous recombination and disruption of the two alleles of the ODC gene. With our first stable transfection vector, we replaced the 2.8 kb SacII ODC gene-containing fragment with a hygromycin-B-phosphotransferase gene (hph) cassette transcribed under the control of the endogenous promoter. For the second ODC allele knock-out, we stably transfected similar constructs that contained either the phleomycin or G418 resistance gene cassette, and included 1 mM putrescine in the media. These experiments resulted in two separate ODC- lines: one hygromycin and phleomycin resistant, the other hygromycin and G418 resistant. The two ODC gene knockout lines were verified by Southern and Northern hybridization, and confirmed by Western blot and enzymatic activity assay. There is no ODC expression in the two ODC- lines and the ODC messages in the single ODC gene knockouts were only half of that of the wild type. When grown in the presence of putrescine, the ODC- lines showed little difference, morphologically, from wild type trypanosomes. The growth rate of these lines varied greatly, depending on the concentration of the putrescine. Interestingly, when putrescine was completely withdrawn from the media, the ODC- trypanosomes soon reached a plateau phase and some cells remained viable for 7-8 weeks. The starved cells could be rescued by the addition of putrescine or introducing back the ODC gene. Cell cycle analysis suggested that putrescine is required for G1-S transition in the procyclic form T. brucei.

Alleles↗
PubMed1994

A new VSG expression site-associated gene (ESAG) in the promoter region of Trypanosoma brucei encodes a protein with 10 potential transmembrane domains.

In Trypanosoma brucei bloodstream variants 118 cl 1, 118a and 118b, the actively transcribed VSG gene expression site (ES) is located on a 1.5 Mb chromosome. The promoter region for this polycistronic transcription unit is unusual in that there are two, tandemly located, promoter repeats, each 2.1 kb in size, separated by 13 kb of intervening DNA. As previously shown, at inactivation of this ES, the promoter region was rearranged with the deletion of 15 kb of DNA. This result prompted us to search through the deleted DNA sequences to identify additional genes that might play a role in the inactivation of ESs. In this report, we identify a gene, encoding a putative transmembrane protein, that was deleted at this locus by the rearrangement event. This gene, which we tentatively call expression-site-associated-gene 10 (ESAG10), contains 10 potential transmembrane domains and had been located to T. brucei stock 427-60, ES-containing chromosomes.

Amino Acid Sequence↗
PubMed1992

A proposed mechanism for promoter-associated DNA rearrangement events at a variant surface glycoprotein gene expression site.

The expressed variant cell surface glycoprotein (VSG) gene of the protozoan parasite Trypanosoma brucei is invariably found at one of several telomeric VSG gene expression sites (ESs). The active ES in variant 118 clone 1 is found on a 1.5-Mb chromosome, and the promoter region is located more than 45 kb upstream of the VSG gene. We had previously shown that DNA rearrangement events occurred in the promoter region, specifically at inactivation of this ES (K. M. Gottesdiener, H.-M. Chung, S. L. Brown, M. G.-S. Lee, and L. H. T. Van der Ploeg, Mol. Cell. Biol. 11:2467-2477, 1991). In this report, we describe the cloning of the entire 17-kb promoter region, which revealed the presence of two identical 2.15-kb tandem promoter repeats separated by 13 kb of DNA. The two virtually identical promoter repeats both function efficiently in directing transcription in transient transfection assays in insect-form trypanosomes. We characterized the DNA rearrangement events that occur at ES inactivation, and by studying both of the reciprocal products of this recombination event, we infer that these result from direct (promoter) repeat recombination, formation of heteroduplex DNA, and a reciprocal exchange event that releases a circular DNA as a side product of the reaction. The finding of DNA recombinational events in a region of the VSG gene ES that encodes the promoter(s), and their relatively frequent occurrence at ES inactivation, suggests a possible role in ES control.

Base Sequence↗
PubMed1989

Transplanted infections: donor-to-host transmission with the allograft.

PURPOSE: To evaluate the transmission of infectious agents from organ donors to transplant recipients, and to assess risk factors for transmission, primarily in recipients of kidney, cornea, and heart allografts. DATA IDENTIFICATION: Computerized literature searches of MEDLINE and PAPERCHASE through January 1988, extensive review of references from identified articles, and review of major clinical and transplantation journals through June 1988. STUDY SELECTION: All case reports and studies that reported a possible donor-to-recipient transmission of infection were selected and reviewed. DATA EXTRACTION: Each case report or patient series of donor-to-recipient transmission was judged as possible, probable, or proven depending on the completeness of donor and recipient information available and the likelihood of alternate causes of infection. RESULTS OF DATA SYNTHESIS: True donor-transmitted infection can occur with viruses including human immunodeficiency virus, cytomegalovirus, herpes simplex, Epstein-Barr, rabies, the virus causing Creutzfeldt-Jakob disease, and with hepatitis B virus. It can also occur with many common aerobic bacteria, although allograft-transmitted bacterial infection is more often caused by contamination during harvesting and processing. Fungi and yeast, as well as toxoplasmosis, have been transmitted less frequently, and there have been rare instances when mycobacterial infection, malaria, trypanosomiasis, and strongyloidiasis have been transplanted with the donor organ. CONCLUSIONS: Infection can be transmitted with a donor organ to the recipient, but contamination of the organ during processing and harvesting is commoner and may lead to severe infection in the recipient, especially if contamination is by one of a subset of more virulent organisms. True donor-transmitted infection, although rare, can be reduced by careful donor screening, which should include clinical and epidemiologic assessment for evidence of infection, as well as judicious laboratory testing.

Bacterial Infections↗
PubMed1989

Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 427-60.

Separation of chromosome-sized DNA from the parasitic protozoan Trypanosoma brucei had previously resulted in the fractionation of DNA molecules that ranged in size from 50 kb up to roughly 1.5 Mb. The number of larger chromosomes and their size, accounting for 80% of the DNA of T. brucei remained unclear. We have now size separated these larger DNA molecules by pulsed field gel electrophoresis (PFG) and resolve a total of 20 bands, accounting for roughly 120 chromosomes, ranging in size from 50 kb up to the size of the largest, 5.7 Mb chromosome of Schizosaccharomyces pombe. Three different VSG gene expression sites were located to chromosomes of 430 kb, 1.5 Mb and 3 Mb, respectively. We have not been able to identify additional, previously cryptic DNA rearrangements, that could explain the activation or inactivation of the expression sites.

Animals↗
PubMed1989

Structure, expression and regulation of the murine 4F2 heavy chain.

The murine 4F2 molecule is a 125 kilodalton disulfide-linked heterodimeric cell-surface glycoprotein which has been shown to be involved in the processes of cellular activation and proliferation (1). To elucidate the structure, expression, and regulation of the 4F2 molecule, a murine 4F2 heavy chain (4F2HC) cDNA has been isolated and structurally characterized. The murine 4F2HC is a 526 amino acid (aa) type II membrane glycoprotein which is composed of a 75 aa N-terminal intracytoplasmic region, a single hydrophobic putative transmembrane domain, and a 428 aa C-terminal extracellular domain. Comparison with the human 4F2HC cDNA reveals the highest degree of sequence identity within the transmembrane and intracytoplasmic domains. Northern blot analyses have demonstrated that the 4F2HC gene is expressed at relatively high levels in adult testis, lung, brain, kidney, and spleen, and at significantly lower levels in adult liver and cardiac and skeletal muscle. Studies designed to elucidate the pattern of regulation of the murine 4F2HC gene have demonstrated that it is induced during the process of cell activation, but is subsequently expressed at constant levels throughout the cell cycle in exponentially growing cells.

Amino Acid Sequence↗
PubMed1988

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor alpha, beta, or gamma genes.

To test the hypothesis that the T-cell receptor (Tcr) gamma gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16+ lymphocytosis were analyzed for rearrangements and expression of the human Tcr alpha, beta, and gamma genes. Two of the clones displayed distinct rearrangements of their Tcr beta and gamma genes and expressed mature Tcr alpha, beta, and gamma RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gamma gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr alpha and beta gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.

Adult↗
PubMed1988

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth.

Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a lambda gt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggests that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution.

Amino Acid Sequence↗
PubMedDate not supplied

Cloning by functional complementation in Trypanosoma brucei.

A procyclic Trypanosoma brucei double-knockout mutant lacking the ornithine decarboxylase (ODC) gene was transfected with a T. brucei genomic library in the expression vector pTSO-HYG4, which utilizes the PARP promoter and replicates extrachromosomally by virtue of a minicircle origin of replication. Transfectants which grew in the absence of exogenous putrescine, the product of the ODC-catalyzed reaction, were obtained at a frequency of 1.6 x 10(-7) and shown to restore ODC protein synthesis and enzymatic activity. Restriction enzyme patterns and Southern blot analysis of plasmids recovered from these cells and propagated in E. coli showed that the inserts contained a single copy of the T. brucei ODC gene. These results demonstrate for the first time the feasibility of identifying novel T. brucei genes by direct complementation of mutant T. brucei cell lines.

Animals↗