Biomedical subjects

M Uhlén

Publications and source records attributed to M Uhlén.

At least 19 recordsLinked to original sources

PubMed1992

Analysis of a polyketide synthesis-encoding gene cluster of Streptomyces curacoi.

A gene cluster homologous to the beta-ketoacyl synthase-encoding gene, actl, was cloned by Southern blot hybridization from Streptomyces curacoi, and a 5469-bp fragment was sequenced. Analysis of the sequence revealed seven open reading frames (ORFs). A striking similarity to the whiE locus (encoding spore pigment synthesis) of Streptomyces coelicolor was found throughout the whole cluster, with conservation of the size and position of the seven ORFs. The structure of these two gene clusters suggests that they have a common origin, although the lower similarity in the noncoding regions suggests that the regulatory regions have diverged through evolution.

Amino Acid Sequence↗

PubMed1992

T cell receptor diversity and activation markers in the V delta 1 subset of rheumatoid synovial fluid and peripheral blood T lymphocytes.

In the present study we have characterized the gamma/delta T cell receptor (TcR) population in synovial fluid (SF) and peripheral blood (PB) of patients with chronic inflammatory arthritis. By double staining we have shown that (a) synovial V delta 1+ cells have a high expression of activation markers CD45R0 ("memory cells") and HLA-DR as compared to PB, indicating a preactivated population of V delta 1-carrying T cells in vivo and (b) interleukin 2-induced expansion of synovial cells yields a high proportion of gamma/delta in most samples expressing predominantly the V delta 1 TcR. Junctional sequence analysis of the TcR delta chain from interleukin 2-expanded PB cell lines demonstrated a polyclonal V delta 1 population in three out of three samples. In SF cell lines three out of four samples were polyclonally expanded. In SF from one patient, however, a limited repertoire of expressed V delta 1 genes was found. Altogether, our data demonstrate the presence of preactivated V delta 1-expressing cells in the synovial compartment. This V delta 1 population is predominantly polyclonal, except in one patient where oligoclonally expanded V delta 1 cells were detected.

Antigens, CD↗

PubMed1992

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.

Automation↗

PubMed1992

Colorimetric detection of Plasmodium falciparum and direct sequencing of amplified gene fragments using a solid phase method.

A rapid colorimetric assay for the detection of DNA from Plasmodium falciparum malaria is described, allowing direct sequencing of amplified fragments in the positive samples. The method is based on amplification by the polymerase chain reaction (PCR), with incorporation of biotin and a lac operator sequence in the amplified target DNA. The PCR product was immobilized on streptavidin-coupled magnetic beads, and detected by the specific binding of an Escherichia coli lac repressor beta-galactosidase fusion protein. Positive samples were subsequently treated with alkali to generate single stranded templates, which were used for solid phase genomic sequencing. As targets for amplification and sequencing we selected a region of the gene for the antigen Pf155/RESA and a region of the parasite dihydrofolate reductase gene (PfDHFR/TS). We show here that both of these gene targets can be used for specific detection of P. falciparum in patient blood samples. Genomic sequencing of five patient isolates revealed no variation in the Pf155/RESA gene fragment. In a comparison of this sequence with conserved protein domains, a marked similarity to the src homology region 3 was detected. A point mutation was found in the PfDHFR/TS gene fragment of one of the clinical samples, replacing Ser108 with Asn. This mutation has earlier been described in pyrimethamine and cycloguanile-resistant strains of P. falciparum.

Amino Acid Sequence↗

PubMed1992

Fusion proteins in biotechnology.

Gene fusion techniques allow the production of recombinant proteins featuring the combined characteristics of the parental products. Originally, these techniques were used to probe transcriptional and translational activity, to translocate proteins across cell membranes, and to facilitate the recovery of proteins. Recently, new applications have emerged in areas such as protein refolding, immunology, drug targeting and protein display. A slightly modified version of this review is also published in Current Opinion in Structural Biology 1992, 2:569-575.

Biotechnology↗

PubMed1992

Loss of synergistic response to combinations containing AZT in AZT-resistant HIV-1.

The use of combination chemotherapy for the treatment of HIV-1 infection offers promise for overcoming the problems of toxicity and development of resistance. Primary HIV-1 isolates from three patients before and after treatment with azidothymidine (AZT) were examined for sensitivity to AZT and synergistic response to three different combinations of drugs: AZT+fluorothymidine (FLT), AZT+dideoxyinosine (ddI), and FLT+ddI. All three patients initially harbored AZT-sensitive virus, but posttherapy isolates were resistant to AZT. The pretreatment, AZT-sensitive virus from each patient showed synergistic inhibition by the combinations of AZT+FLT, AZT+ddI, and FLT+ddI. In contrast, the posttreatment, AZT-resistant virus showed only addition or antagonism by the combinations containing AZT, whereas the synergistic response to the combination of FLT+ddI was preserved. Our study argues for early intervention with combination chemotherapy, since after development of resistance, AZT no longer showed synergy but addition or antagonism in combination with other drugs. After resistance to AZT has developed, combination chemotherapy not involving AZT may offer advantages over continued mono- or combination therapy involving AZT.

Antiviral Agents↗

PubMed1992

Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT)-treated patients.

The emergence of azidothymidine (AZT)-resistant human immunodeficiency virus (HIV) variants in clinical samples was studied by a direct genomic sequencing method. Sequential lymphocyte samples from four patients, who had been treated with AZT for up to 27 months, were shown to gradually accumulate multiple nucleotide changes, some of which are known to be associated with AZT resistance. Several samples were shown to contain mixtures of wild-type and mutated genomes, indicating gradual rather than sudden changes in the HIV-1 quasispecies. These results demonstrate for the first time that automated solid-phase DNA sequencing is a rapid and useful tool for investigation of antiviral drug resistance and suggest that DNA sequencing may be important in routine clinical diagnostics in the future.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Immunomagnetic recovery of Chlamydia trachomatis from urine with subsequent colorimetric DNA detection.

There is need for integrated systems that provide rapid and sensitive diagnosis of Chlamydia trachomatis. Here we describe for the first time that specific antibodies to a C. trachomatis surface protein can be used for magnetic recovery of bacteria directly from crude urine samples. The combination of immunomagnetic separation (IMS), where the bacterial cells are both purified and concentrated, with a colorimetric PCR assay makes the method sensitive and suitable for routine clinical screening in microtiter format. No filtrations, centrifugations, extractions, or electrophoretic techniques are required throughout the whole procedure. Using magnetic beads as the solid phase, the colorimetric detection of immobilized amplified nucleic acids (DIANA) also allow direct DNA sequencing of positive samples for more detailed analysis. Of the 27 urine samples analyzed, eight were found to be positive using this method. Only five out of these eight were determined to be positive by cell culture.

Adult↗

PubMed1992

Cloning, analysis, and heterologous expression of a polyketide synthase gene cluster of Streptomyces curacoi.

Streptomyces curacoi produces curamycin, an antibiotic based on a modified orsellinic acid skeleton that is synthesized by the polyketide pathway. We have cloned, characterized, and partly sequenced a polyketide synthase gene cluster of S. curacoi. The sequence data reveal an organization of open reading frames that is similar to those of other polyketide synthetic clusters, although the biosynthetic products differ considerably in size and structure. We propose that one of the predicted open reading frames (curA) encodes polykeptide synthase, on the basis of its homology with other enzymes with similar functions. Expression of the cloned chromosomal fragment in the heterologous host S. lividans leads to the production of a brown pigment in large quantities. The analysis and expression of the cur genes for detailed molecular studies of the mechanism of polyketide biosynthesis is discussed.

Amino Acid Sequence↗

PubMed1992

Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus.

An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.

Amino Acid Sequence↗

PubMed1992

Variations in the cytomegalovirus major immediate-early gene found by direct genomic sequencing.

An assay to detect and sequence DNA from human cytomegalovirus (HCMV) immediate-early gene region 1 has been developed; it involves in vitro amplification by the polymerase chain reaction and direct solid-phase sequencing of the amplified material. Urine samples from 16 patients tested positive for HCMV DNA in both a colorimetric assay for the detection of immobilized amplified nucleic acids and a standard polymerase chain reaction assay with agarose gel electrophoresis. Ten urine samples from healthy people tested negative in the same assays. Analysis of 106-bp fragments from seven patients and two laboratory HCMV strains (Ad 169 and Towne) demonstrated that the viral sequences were conserved in samples collected at different times from the same patient and in tissue-cultured samples. Two of the patient strains had variations in the amplified region, with a total of seven nucleotide substitutions yielding five amino acid alterations in the coding sequence.

Amino Acid Sequence↗

PubMed1992

Human immunodeficiency virus type 1 is present in the cerebrospinal fluid of a majority of infected individuals.

Cerebrospinal fluid (CSF) specimens from 63 patients with different severities of human immunodeficiency virus (HIV-1) infection, including asymptomatic virus carriers, were examined for the presence of HIV-1 by using polymerase chain reaction (PCR) and virus isolation. Polyadenylated RNA, presumably associated with virus particles, was extracted and reverse transcribed, and the pol region was amplified in a nested PCR. Virus could be detected in 90% of the CSF specimens examined by PCR, and data on isolation of virus from CSF were in agreement with these figures. In fact, when several CSF specimens from the same individual were studied, HIV-1 could be isolated from 80% of the patients. The presence of the viral RNA in CSF was independent of the clinical stage of infection and of neurological symptoms. These results show that the spread of HIV-1 to the brain represents an early event during infection and occurs in the majority of asymptomatic individuals.

Blood↗

PubMed1992

Persistence of azidothymidine-resistant human immunodeficiency virus type 1 RNA genotypes in posttreatment sera.

The rate of reversion from azidothymidine (zidovudine; AZT) resistance was studied by direct sequencing of human immunodeficiency virus type 1 (HIV-1) virion RNA in sera from four patients who discontinued long-term treatment. Before cessation of treatment, all four patients harbored HIV-1 with multiple mutations reported to confer AZT resistance. In three patients, slow reversions of these mutations starting after 9, 9, and 18 months were detected. The slow reversions indicate that AZT-resistant HIV-1 variants are likely to have an unaltered replicative capacity and pathogenic potential. Furthermore, there were discrepancies between the in vivo RNA sequences and the sequences of virus isolates, indicating that the isolation procedure may select for nonrepresentative virus variants.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Differential degradation of a recombinant albumin-binding receptor in Escherichia coli.

The degradation in Escherichia coli of the recombinant serum-albumin-binding receptor derived from streptococcal protein G was investigated using a dual-affinity fusion approach. The proteolytic degradation of the receptor was characterized when fused to human proinsulin and human secretin. Several cleavages occurred at sequences not normally regarded as proteolytically sensitive, such as the dipeptide sequences Ile-Gly, Val-Ser and Ser-Ala. Depending on the fusion partner, large differences in the degradation of the albumin-binding domain were observed. Thus, susceptibility to proteolysis of a recombinant protein can be affected by a neighbouring domain.

Amino Acid Sequence↗

PubMed1991

Characterization of an extended form of recombinant human insulin-like growth factor II.

To investigate the biological role of variants of human insulin-like growth factor II (IGF-II), an extended form designated IGF-IIE21, with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein. After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIE21 and IGF-II have similar or identical activities according to radioimmunoassay and radioreceptor assay. However, IGF-IIE21 showed only 1% growth promotion activity as compared with IGF-II in a clonal expansion assay using human K562 cells which lacks IGF-I receptors. These results suggest that this extended variant of IGF-II can bind to the receptor but has limited growth promoting activity.

Amino Acid Sequence↗

PubMed1991

Structural and functional analysis of the human IgG-Fab receptor activity of streptococcal protein G.

Streptococcal protein G (SPG) shows specific binding activity to IgGs and serum albumins from various species. In order to investigate the structural domains of SPG responsible for the specific interaction with human IgG-Fab, the binding characteristics of a collection of recombinant receptors were analysed. The study includes receptors comprising different parts of the SPG molecule as well as chimeric receptors containing IgG-binding domains of staphylococcal protein A (SPA) fused to the N-terminal AB-region of SPG, which has been claimed to interact with human IgG-Fab. Purified defined gene products were allowed to compete for the binding to human IgG, human IgG-F(ab')2 fragments and human serum albumin (HSA) in several sets of competitive binding experiments. The results demonstrate that the C-terminal C domains have both IgG-Fc- and IgG-Fab-binding capacities, whereas the N-terminal AB region is responsible for the HSA-binding only. These results, which are in conflict with previous work, demonstrate that the binding to both the IgG-Fc and the IgG-Fab region is mediated by the same structurally distinct receptor region of SPG.

Bacterial Proteins↗

PubMed1991

Genetic analysis of the polymorphism of the human apolipoprotein E using automated solid-phase sequencing.

A direct sequencing approach has been used to analyze the polymorphism in the human apolipoprotein E gene. A method is described, in which the DNA is amplified by the polymerase chain reaction, immobilized, and sequenced by a semi-automatic procedure adaptable to clinical diagnosis. The three alleles of the apolipoprotein E gene, which differ from each other by two nucleotide substitutions and which influence serum cholesterol levels, were analyzed. The solid-phase method was able to resolve the correct nucleotide sequence in samples from both homozygous and heterozygous individuals. No cloning steps are needed and the immobilization and separation of the DNA is accomplished using magnetic beads.

Apolipoproteins E↗

PubMed1991

Facilitated in vitro refolding of human recombinant insulin-like growth factor I using a solubilizing fusion partner.

We describe a new approach to refolding recombinant proteins in which an affinity fusion partner, consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A, is used to solubilize misfolded molecules before, during and after reduction and reoxidation. We show that human insulin-like growth factor I (IGF-I) can be refolded as a fusion protein at a concentration as high as 1-2 mg/ml without the use of denaturing agents. A process scheme suitable for large scale application is described in which the yield of correctly folded human IGF-I with full biological activity is substantially increased.

Immunoglobulin G↗