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Biomedical subjects

G Ott

Publications and source records attributed to G Ott.

At least 145 records · Page 8Linked to original sources
PubMed1992

Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons.

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.

AIDS Vaccines↗
PubMed1992

[Detection of Epstein-Barr virus genomes in various entities of low and high grade T-cell lymphomas].

72 T-cell lymphomas were analyzed for the presence of Epstein-Barr virus DNA using Southern or dot blot hybridization. EBV DNA was found in 25 peripheral T-cell lymphomas and in none of 6 T-lymphoblastic tumors. A high prevalence of EBV was detected in AILD type, Lennert's, pleomorphic medium and large cell nodal lymphomas and in upper aerodigestive tract lymphoma biopsies. Clonotypic analysis revealed monoclonal as well as oligoclonal viral populations. The possible influence of EBV in lymphomagenesis, the impact of virus-mediated interactions in the B- and T-cell system and factors involving site of tumor development are discussed.

Biopsy↗
PubMed1992

[Classification and virus expression of primary cerebral lymphomas].

Fourty-three primary cerebral lymphomas (PCL) were histologically classified and examined for genome expression of Epstein Barr Virus (EBV) and human herpes virus 6 (HHV6) using dot blotting, polymerase chain reaction, and Southern blotting. Only 20 tumors (16 high grade and 4 low grade lymphomas) could be suitably placed into a category of the Updated Kiel Classification, whereas the non-classified 23 tumors were highly malignant B-lymphomas and referred to as small-cell (SC) or large-cell (LC) blastic PCL. Most of the LC PCL showed a tumor-like infiltration pattern with high cellular density and little remaining parenchyma, whereas the SC PCL more often showed an inflammation-like pattern characterized by loose arrangement of tumor cells and marked astrocytic, microglial and T-lymphocytic reaction. EBV genome was found in 3/3 AIDS cases, but in none of 40 immunocompetent cases, while HHV6 was detected in 2 tumors of immunocompetent patients. We conclude that (1) the Updated Kiel Classification is not applicable to a majority of PCL, and (2) EBV and HHV6 do not appear to play a major role in the pathogenesis of PCL in immunocompetent subjects.

Autopsy↗
PubMed1992

[99mTc-anti-granulocyte antibodies (BW 250/183) in the detection of appendicitis].

Scintigraphy with 99mTc-labeled anti-granulocyte antibodies (AGAb) was performed in 50 patients with suspected appendicitis. Sequential and static imaging as well as SPECT of the pelvis and abdomen was performed 2 h p.i. In all patients the diagnosis was confirmed either histologically or by long-term follow-up. 13 patients had histologically proven acute appendicitis. In 11 patients the appendix scan had been positive and in 2 patients the scan had shown no significant tracer uptake in the right lower abdomen. The remaining 37 patients turned out not to have acute appendicitis. 29 out of these patients had negative and 3 had positive scan findings. In 5 patients the scan was equivocal. Out of these patients 2 had pathologic findings on the left side of the abdomen which turned out to be acute diverticulitis in one patient and acute peritonitis in the other. The remaining 3 patients with unclear scintigraphic findings had no acute appendicitis. Scintigraphy with AGAb is fast and easy to perform and thus superior to cell labeling methods for diagnosing acute appendicitis. Sensitivity for the diagnosis of acute appendicitis was 85% with a specificity of 91%. Chronic or scarred non-granulocytic appendicitis--in which there is often no definite indication for surgery--was negative in our study except for two cases.

Acute Disease↗
PubMed1991

Characterization of the new bladder cancer cell line HOK-1: expression of transitional, squamous and glandular differentiation patterns.

The new continuous cell line HOK-1 derived from a grade-III transitional-cell bladder carcinoma with foci of squamous and glandular differentiation was shown to retain this phenotypical heterogeneity for more than 45 passages in vitro. Electron microscopy revealed transitional as well as a considerable proportion of squamous carcinoma and adenocarcinoma cells. PAS-positive mucus was detected in numerous cells. These features were principally maintained when grown as multicellular spheroids and in nude mice. More pronounced signs of differentiation (i.e., expression of cytokeratins 10 and 11, formation of glandular structures) were found in xenograft tumours. Independently, cytokeratins 13, 18 and 19 were detected in vitro and in vivo, reflecting the urothelial origin. The line forms distinct colonies in soft agar, expresses Lewis-x and Lewis-y antigens and reacts with monoclonal antibodies (MAbs) against CEA, beta-HCG and URO-5. Cytogenetic analysis revealed several related clones with a rearrangement at chromosome 1 and loss of one X chromosome as common karyotypic changes in all clones. DNA content, as quantified by image analysis, showed a DNA stemline close to 2c. The new cell line HOK-1 can be used as an in vitro model to study the mechanisms of heterogeneous differentiation patterns in bladder cancer.

Aged↗
PubMed1991

Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA.

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.

Electrophoresis, Agar Gel↗
PubMed1990

Interaction of the isolated domain II/III of Thermus thermophilus elongation factor Tu with the nucleotide exchange factor EF-Ts.

The middle and C-terminal domain (domain II/III) of elongation factor Tu from Thermus thermophilus lacking the GTP/GDP binding domain have been prepared by treating nucleotide-free protein with Staphylococcus aureus V8 protease. The isolated domain II/III of EF-Tu has a compact structure and high resistance against tryptic treatment and thermal denaturation. As demonstrated by circular dichroism spectroscopy, the isolated domain II/III does not contain any alpha-helical structure. Nucleotide exchange factor, EF-Ts, was found to interact with domain II/III, whereas the binding of aminoacyl-tRNA, GDP and GTP to this EF-Tu fragment could not be detected.

Circular Dichroism↗
PubMed1990

Ternary complexes of Escherichia coli aminoacyl-tRNAs with the elongation factor Tu and GTP: thermodynamic and structural studies.

The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions. The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA. The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP. The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues. An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support. It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.

Anticodon↗
PubMed1990

The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0.

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.

Anti-Bacterial Agents↗
PubMed1990

The role of modified purine 64 in initiator/elongator discrimination of tRNA(iMet) from yeast and wheat germ.

The role of 2'-ribosylated adenosine 64 in tRNA(iMet) from yeast in initiation/elongation discrimination was investigated. As measured by in vitro translation in rabbit reticulocyte lysate, the specific removal of the 2'-ribosylphosphate at adenosine 64 via periodate oxidation allows tRNA(iMet) to read internal AUG codons of the globine messenger RNA. Yeast Met-tRNA(iMet) lacking the modification of nucleoside 64 forms ternary complexes with GTP and elongation factor Tu from Escherichia coli. The lack of modification at position 64 does not prevent tRNA(iMet) from participating in the initiation process of in vitro protein synthesis. Wheat germ tRNA(iMet) has a 2'-ribosylated guanosine at position 64. Removal of this modification from the wheat germ tRNA(iMet) enables it to read internal AUG codons of globine and tobacco mosaic virus messenger RNA in reticulocyte and wheat germ translation systems, respectively.

Adenosine↗
PubMed1990

The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy.

A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for [AEDANS-s2C]Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.

Escherichia coli↗
PubMed1990

Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli.

Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M. Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP. This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec). We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.

Chromatography, Affinity↗
PubMed1989

The selenocysteine-inserting opal suppressor serine tRNA from E. coli is highly unusual in structure and modification.

Selenocysteine is cotranslationally incorporated into selenoproteins in a unique pathway involving tRNA mediated suppression of a UGA nonsense codon (1-3). The DNA sequence of the gene for this suppressor tRNA from Escherichia coli predicts unusual features of the gene product (4). We determined the sequence of this serine tRNA (tRNA(UCASer]. It is the longest tRNA (95 nt) known to date with an acceptor stem of 8 base pairs and lacks some of the 'invariant' nucleotides found in other tRNAs. It is the first E. coli tRNA that contains the hypermodified nucleotide i6A, adjacent to the UGA-recognizing anticodon UCA. The implications of the unusual structure and modification of this tRNA on recognition by seryl-tRNA synthetase, by tRNA modifying enzymes, and on codon recognition are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1989

Interaction of elongation factor Tu from Escherichia coli with aminoacyl-tRNA carrying a fluorescent reporter group on the 3' terminus.

Transfer ribonucleic acids containing 2-thiocytidine in position 75 ([s2C]tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3'-shortened tRNA using ATP(CTP):tRNA nucleotidyltransferase. [s2C]tRNA was selectively alkylated with fluorescent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) on the 2-thiocytidine residue. The product [AEDANS-s2C]aminoacyl-tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore. From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast [AEDANS-s2C]Tyr-tRNATyr, yeast Tyr-tRNATyr, and the homologous E. coli Phe-tRNAPhe, respectively, interacting with E. coli elongation factor Tu.GTP. The measurement of the association and dissociation rates of the interaction of [AEDANS-s2C]Tyr-tRNATyr with EF-Tu.GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol-1 K-1 for delta S degrees' and -34.7 kJ mol-1 for delta H degrees' of this reaction.

Cytidine↗
PubMed1989

Diagnostic value of combined bronchoalveolar lavage and transbronchial lung biopsy.

The aim of our study was to assess the diagnostic value of combined transbronchial lung biopsy (TBB) and bronchoalveolar lavage (BAL) in non-immunosuppressed patients, who underwent routine bronchoscopy for evaluation of interstitial lung disease. We examined routinely sampled and processed material from 87 patients from two peripheral pulmological centers and compared the lavage and biopsy results to the final clinical outcome. 22 of these patients showed no evidence of interstitial lung disease during the course of follow-up. Sufficient lung parenchyma was present in 81 of 91 biopsy specimens (including four repeat biopsies), and 79 of 91 BAL samples were considered adequate. The combination of BAL and TBB was more efficient (efficiency 80.3% vs. 67.9% in TBB and 64.6% in BAL) than either method alone. Sensitivity showed a similar increase from 58.8% in TBB and 73.9% in BAL to 79.5% for both combined. Specificity for BAL was a low 60.7% due to contamination by inflammatory cells from upper airways, whereas specificity for TBB was 100%. We conclude that the combination of bronchoalveolar lavage and transbronchial lung biopsy is a valuable tool in the evaluation of interstitial lung disease and should be employed whenever possible.

Adult↗
PubMed1988

The effect of adjuvants on the efficacy of a recombinant herpes simplex virus glycoprotein vaccine.

A recombinant, truncated HSV type 1 glycoprotein D secreted by Chinese hamster ovary cells (rgD1) was used to compare the ability of several adjuvants to stimulate protective immunity in guinea pigs. Adjuvants tested included CFA, aluminum hydroxide (alum), a lipophilic derivative of muramyl tripeptide (MTP-PE), and a muramyl dipeptide (MDP) covalently conjugated to rgD1. Animals were immunized three times with rgD1 plus the various adjuvants and antibody titers were determined by ELISA. Four weeks after the last immunization, the animals were challenged intravaginally with HSV type 2 and were monitored daily for clinical signs of disease, including frequency and severity of herpetic lesions, incidence of urinary retention, and mortality during the 14-day post-challenge observation period. Animals immunized in the foot-pad with rgD1 formulated with CFA showed the highest antibody titers. Animals immunized in the footpad with rgD1 using MTP-PE in a 4% squalene formulation, alum, or rgD1 conjugated to MDP showed mean antibody titers that were 57, 16, and 13% of the CFA titers, respectively. Immunization with rgD1 plus MTP-PE, alum, or rgD1-MDP conjugate by the i.m. route elicited lower antibody titers than the footpad route of immunization. Results of the viral challenge indicated that clinical symptoms of the groups immunized with rgD1 with CFA or MTP-PE as adjuvant were similar in magnitude and were markedly reduced compared with unimmunized control groups. Animals immunized with rgD1 combined with alum or rgD1-MDP conjugate showed clinical symptoms significantly more severe than the CFA or MTP-PE groups. The protective immunity observed after i.m. immunization of animals with rgD1 and MTP-PE was only slightly lower than animals immunized with the same Ag-adjuvant combination in the footpad. The results indicate that MTP-PE is an effective adjuvant for the recombinant herpes gD vaccine.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1988

Study of mortality among chemical workers in the Kanawha Valley of West Virginia.

To assess the mortality experience of a cohort of chemical workers in the Kanawha Valley of West Virginia, 29,139 males who worked at any one of three facilities over a 39-year period were followed-up for vital status. The facilities include two chemical manufacturing plants and a research and development center. From this cohort, 5,785 men were found to have died as of the study end, December 31, 1978. This was less than the 6,148.5 men expected to have died, based upon the United States white male population (standardized mortality ratio (SMR) = 94, 95% confidence interval (CI) = 92-96). Eighty-six specific causes of death were examined. Statistically significant increased deaths were observed for two causes; cancers of the liver (not specified as primary or secondary) (SMR = 174; CI = 102-280) and lympho- and reticulosarcoma (SMR = 140; CI = 104-187). When all biliary and liver cancer was examined by duration and time since initial employment, the SMR for those who worked at least 25 years and whose deaths occurred 30 years or more after first employment was 301 (95% confidence limit = 168-497). The identification and follow-up of this complete cohort provides the basis for future study of subcohorts with specific chemical and process exposures and case control studies of specific causes of death.

Adult↗