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Biomedical subjects

M Kuehn

Publications and source records attributed to M Kuehn.

26 records · Page 2Linked to original sources
PubMed1992

Fimbriation of Pseudomonas cepacia.

Fimbriae (pili) on the surface of bacteria have been suggested to facilitate adherence to mucosal epithelial surfaces. Three Pseudomonas cepacia cystic fibrosis isolates were screened for their ability to agglutinate erythrocytes (HA), a characteristic of some fimbrial types. One strain, designated PC103, was HA+, while another, PC109, was HA-. A fimbriated (f+) HA+ derivative of PC109 (PC2(13)) was selected by repeated erythrocyte adsorption. The two HA+ strains were shown by transmission electron microscopy to possess fimbriae which averaged 4.8 +/- 1.36 nm in width and 200 to greater than 2,100 nm in length (PCE2(13)) and 3.4 to 11.4 nm in diameter and 280 to 720 nm in length (PC103). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins prepared from PC103, PC109, and PCE2(13) indicated that the putative fimbrial subunit had a mass of 16 kDa. Western blot (immunoblot) analysis of sheared cell supernatants indicated that the 16-kDa subunit from PC103 and PCE2(13) reacted with antibody to the P. aeruginosa PAK pilin subunit. Southern blot analysis of a SalI digest of PC103 DNA showed DNA fragments which hybridized to P. aeruginosa PAK probes containing either the pilin structural gene (pilA) or the pilin accessory genes (pilB, -C, and -D) but not the conserved N-terminal region of pilA. A 15-kb band was common to both hybridizations, indicating that this fragment contains the PC103 fimbrial gene cluster. These results indicated the presence of homology between P. aeruginosa PAK and PC103 fimbriae but also suggested that the P. cepacia fimbriae are not type IV-like. The importance of fimbriae in adherence to A549 cells (type II pneumocytes) was assessed with PC109 (f-) and PCE2(13) (f+). PCE2(13) had an approximately 20-fold-higher level of adherence to A549 cells than PC109. This suggested that fimbriation of P. cepacia is associated with increased adherence in vitro.

Animals↗
PubMed1991

Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s).

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the enzyme from MAP (0.008 microM) and MAP/47 (0.007 microM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).

Chromatography, Gel↗
PubMed1991

Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae.

A Haemophilus influenzae b (Hib) membrane protein with a molecular mass of 28 kDa bound polyclonal antisera raised against a highly purified Hib fimbrial subunit. We cloned the gene encoding this protein and found that the gene was expressed in Escherichia coli. DNA sequence analysis identified an 843-bp open reading frame which predicted a 26.78-kDa protein with an amino-terminal signal sequence and a mature protein with 70% similarity to the 28-kDa lipoprotein of E. coli (F. Yu, S. Inouye, and M. Inouye, J. Biol. Chem. 261:2284, 1986). Colony blot hybridization analysis with an intergenic probe of the cloned gene demonstrated that 29 of 32 H. influenzae strains hybridize with this gene. Insertion of a chloramphenicol acetyltransferase gene into the open reading frame inactivated expression of the 28-kDa protein in E. coli. Isogenic Hib strains were derived by marker exchange mutagenesis to generate mutants which no longer expressed the 28-kDa protein as recognized with Western immunoblot analysis. There was no difference in the rate of nasopharyngeal colonization of infant rats or monkeys by the isogenic mutants which lacked the 28-kDa protein compared with colonization by the wild-type strain. In contrast, the frequency of invasion and density of bacteremia in infant rats caused by the isogenic mutants were reduced relative to those caused by the wild-type Hib strain. We conclude that this 28-kDa outer membrane protein aids transepithelial invasion of type b strains but is not essential.

Amino Acid Sequence↗
PubMed1990

Amperometric determination of ammonium ions with a microbial sensor.

A sensor for NH+4 ions has been developed, which consists of immobilized micro-organisms (Bacillus subtilis, Pseudomonas aeruginosa, Trichosporon cutaneum) in combination with an electrochemical transducer. This sensor is based on the measurement of acceleration of respiration after addition of NH+4 in the presence of glucose. The physiological background of this signal and its connection with NH+4 ion uptake and/or metabolism is discussed. The response time of the sensor is about 5-10 s for NH+4 ions. A linearity was observed between 0.005 and 0.15 mmol dm-3 NH+4 ions. The sensitivity of the sensor remained almost constant for 12 days. The sensor was used to determine NH+4 ions in a microbial fermentation broth.

Bacillus subtilis↗
PubMed1984

Insertion of retrovirus into the first intron of alpha 1(I) collagen gene to embryonic lethal mutation in mice.

Integration of the Moloney murine leukemia virus (M-MuLV) into the germ line of Mov-13 mice blocked formation of stable alpha 1(I) collagen mRNA and led to an embryonic lethal mutation. A 14-kilobase fragment representing the integration site of the virus was molecularly cloned and identified as the alpha 1(I) collagen gene. Sequence and nuclease S1 mapping analyses were performed to characterize the position of the proviral genome in relation to the transcriptional map of the mutated gene. The results indicated that the virus has inserted into the first intron 19 base pairs downstream of the intron/exon boundary. Sequence comparison showed a striking homology of exon sequences and sequences up to 215 base pairs upstream of the mRNA start between the mouse and the human alpha 1(I) collagen gene. This indicates that the sequences upstream of the mRNA start are highly conserved during evolution, suggesting that this region has an important role in the control of tissue-specific collagen expression.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of the genetically labile repeated elements 5' to the origin of mouse rRNA transcription.

We have determined the complete nucleotide sequence of a cloned Balb/c mouse rDNA NTS fragment containing 13 tandem copies of a 135 bp subrepeating segment. This repetitious region (VrDNA) lies close to the origin of ribosomal RNA transcription. Analyses of these VrDNA subrepeats from Balb/c and a related species, Mus pahari, reveal regions of inverted repeat DNA as well as large poly T tracts, either of which may be significant to the generation of the high levels of VrDNA copy number variation found in wild and inbred mice and/or the modulation of rRNA synthesis. Unlike the highly homogeneous subrepeats in the Xenopus laevis NTS repetitious regions, the VrDNA subrepeats differ from one another on the average by about 13%. Sequence analysis and Southern hybridization studies have also shown that, unlike the Xenopus and Drosophila NTS, extensive duplications of sequences found surrounding the mouse rRNA initiation site are found neither in the VrDNA region nor 6 kb further upstream in the NTS.

Animals↗
PubMedDate not supplied

Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector.

Embryonic stem cells isolated directly from mouse embryos can be cultured for long periods in vitro and subsequently repopulate the germ line in chimaeric mice. During the culture period these embryonic cells are accessible for experimental genetic manipulation. Here we report the use of retroviral vectors to introduce exogenous DNA sequences into a stem-cell line and show that these modified cells contribute extensively to the somatic and germ-cell lineages in chimaeric mice. Compared with current methods for manipulation of the mouse genome, this approach has the advantage that powerful somatic-cell genetic techniques can be used to modify and to select cells with germ-line potential, allowing the derivation of transgenic strains with pre-determined genetic changes. We have by this means inserted many proviral vector sequences that provide new chromosomal molecular markers for linkage studies in the mouse and that also may cause insertional mutations.

Animals↗