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Biomedical subjects

R Crameri

Publications and source records attributed to R Crameri.

At least 91 records · Page 5Linked to original sources
PubMed1992

High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A2.

Bee venom phospholipase A2 (BV-PLA2) is a hydrolytic enzyme that specifically cleaves the sn-2 acyl bond of phospholipids at the lipid/water interface. The same enzyme is also believed to be responsible for some systemic anaphylactic reactions in bee venom sensitized individuals. To study the structure/function relationships of this enzyme and to define the molecular determinants responsible for its allergenic potential, a synthetic gene encoding the mature form of BV-PLA2 was expressed in Escherichia coli. This enzyme was produced as a fusion protein with a 6xHis-tag on its amino-terminus yielding 40-50 mg of fusion protein per 1 of culture after metal ion affinity chromatography. A kallikrein protease recognition site was engineered between the 6xHis-tag and the amino-terminus of the enzyme allowing isolation of the protein with its correct N-terminus. Recombinant affinity purified BV-PLA2 was refolded, purified to homogeneity, and cleaved with kallikrein, resulting in a final yield of 8-9 mg of active enzyme per 1 of culture. The enzymatic and immunological properties of the recombinant BV-PLA2 are identical to enzyme isolated from bee venom indicating a native-like folding of the protein.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test activity.

Aspergillus fumigatus secretes an 18-kDa nonglycosylated IgE-binding protein. This protein was previously shown to be a ribotoxin, like alpha-sarcin and mitogillin. A 686-bp long A. fumigatus cDNA encoding an 18-kDa ribotoxin was cloned and expressed in Escherichia coli as a fusion protein with six adjacent histidines (rAsp f I/a). rAsp f I/a was purified to homogeneity by Ni(2+)-chelate affinity chromatography and refolded. The recombinant protein was enzymatically active resulting in the cleavage of 28S rRNA within a universally conserved region. rAsp f I/a was cytotoxic for EBV immortalized or PHA stimulated human PBMC. Furthermore, rAsp f I/a was recognized by murine mAb made against an 18-kDa ribotoxin. IgE of individuals allergic to A. fumigatus bound to rAsp f I/a as shown by ELISA, dot blots, and Western blots. rAsp f I/a elicited positive immediate type I skin reactions in individuals allergic to A. fumigatus but not in healthy control individuals. The results show that rAsp f I/a has similar functional characteristics when compared to the native 18-kDa ribotoxin. rAsp f I/a expressed in E. coli can therefore be used as a standardized Ag/allergen for serologic and clinical diagnosis of A. fumigatus-associated diseases.

Allergens↗
PubMed1992

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus.

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.

Amino Acid Sequence↗
PubMed1992

Rabbit single domain antibodies specific to protein C expressed in prokaryotes.

VDJ genes were amplified by the polymerase chain reaction from mRNA isolated from peripheral blood B cells of rabbits immunized with protein C. The amplified genes were cloned into a lambda phage expression vector and packaged. A library of 6 x 10(5) recombinant phages was screened with labelled protein C and 30 positive clones were found. Three of them were plaque purified and the affinity of the single domain antibodies to the antigen determined to be 10(6)-10(7) l M-1. The data indicate the feasibility of generating single domain antibody, specific to protein antigen, from rabbit.

Animals↗
PubMed1991

Dynamics of Rn transport from the cellar to the living area in an unheated house.

Environmental parameters such as temperature and wind, occupant activities, and house-specific parameters such as subsoil geology, leakiness of the substructure to soil gas, and air exchange rate are the main factors influencing Rn entry into a building and its subsequent indoor behavior. Experiments performed in an unheated, uninhabited house showed a reproducible diurnal fluctuation of the indoor concentration of Rn decay products. Strong, long-term correlations between temperature differences indoor-outdoor (indoor temperature minus outdoor temperature) and pressure differences outdoor-indoor (outdoor pressure minus indoor pressure) were found. At positive temperature differences inside-outside, an average airflow velocity of about 0.05 m s-1 between ground floor and first floor was detected. This air movement was able to vertically transport Rn at a rate of approximately 11 kBq h-1 in a volume of air of about 5.5 m3 through a cross-sectional area of only 0.03 m2. For this specific house, stack effects were identified as the main driving force for Rn migration from the cellular to higher floors. The diurnal fluctuation of Rn progeny concentrations in the living area can be explained by temporal variations in the amount of Rn-rich air transported vertically from the cellar into the building as a consequence of stack effects.

Air Pollutants, Radioactive↗
PubMed1989

Indoor Rn levels in different geological areas in Switzerland.

The distribution of indoor Rn concentrations in different geological areas in Switzerland was studied using passive alpha-track detectors. Measurements involving a sample of 400 single-family homes were made in the cellar, on the ground floor and the first floor, respectively. On the basis of a pilot survey, the country was divided into four zones in which the Rn distribution in houses was analyzed separately. The indoor exposure to Rn and Rn decay products is quite variable from region to region. The geology of the different areas was found to be an important factor in determining the mean value Rn levels. In the basin north of the Alps, where the population centers are located, a median Rn gas level of 47 Bq m-3 for the living area was found. The arithmetic mean value of 60 Bq m-3 in this region leads to an annual effective dose equivalent of about 1.8 mSv. For the population living in alpine areas, an arithmetic mean value exceeding 200 Bq m-3 will lead to an annual effective dose equivalent in the range of 6 mSv. The estimated exposure to Rn and Rn decay products for the upper one-percentile of the homes in the most affected alpine region even exceeds the annual limit of 50 mSv effective dose equivalent for occupational exposure.

Air Pollutants↗
PubMed1989

Increased indoor Rn levels due to actinide containing mineral collections displayed in living areas.

Collection of crystals ranging from quartz to U or Th containing minerals is a widespread spare time activity in Switzerland. Radon emanation from stones displayed in showcases in the living area may contribute considerably to elevated Rn levels in the indoor air. Time-averaged Rn gas measurements in 35 homes of subscribers to a journal for mineral collectors showed an unexpected statistically significant increase of 98 Bq m-3 in the Rn level of the room containing the collections compared to the levels measured in a control room on the same floor. Using ICRP Publication 50 conversion factors for indoor exposure to Rn decay products, the additional effective dose equivalent contracted was estimated to amount to an average of 2.7 mSv y-1. Although the 220Rn emanation rate, as measured in the display cases, was considerable in several cases, the dose from 220Rn decay products in the living area remained always a small fraction of the Rn dose. Remedial actions for crystal collections containing considerable amounts of U- or Th-based minerals are suggested.

Air Pollutants↗
PubMed1987

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus.

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant plants.

Journal Article↗
PubMed1986

Increased production of aminoglycosides associated with amplified antibiotic resistance genes.

The 6'-N-acetyltransferase derived from Streptomyces kanamyceticus strain M1164 was cloned on to the high copy plasmid vector pIJ702 and introduced into S. kanamyceticus (ATCC 12853, a kanamycin producer) and S. fradiae (ATCC 10745, a neomycin producer). In both cases transformants containing the recombinant plasmid showed increased resistance to a number of aminoglycoside antibiotics and substantially increased production of kanamycin and neomycin. This demonstrates that specific amplification of gene products associated with antibiotic biosynthesis provides a means for improving antibiotic production.

Acetyltransferases↗
PubMed1985

Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions.

Streptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens. The repeated DNA units were 2.9 to 35 kb in length. No two sequences were identical. The amplified sequences occupied up to 45% of the total genomic DNA. Structural analysis of the cloned repeated DNA stretches by means of restriction enzymes and by cross hybridization revealed the presence of two chromosomal areas rich in DNA reiterations. In some cases reiterated regions were accompanied by nearby rearrangements.

Chimera↗
PubMed1985

Cloning and expression of an APH(3')-III phosphotransferase from Staphylococcus aureus in Streptomyces lividans.

An aminoglycoside 3' type III phosphotransferase derived from Staphylococcus aureus plasmid pRN1956 was cloned on the high copy number Streptomycetes vectors pIJ702 and pIJ704. Streptomyces lividans transformants carrying the hybrid plasmids show a resistance pattern towards aminoglycoside antibiotics comparable to the resistance pattern of S. aureus. The APH(3')-III with expanded spectrum of resistance, is a useful additional marker for gene cloning in Streptomycetes.

Cloning, Molecular↗
PubMed1984

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase.

The wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.

Chromosome Deletion↗
PubMed1984

Tyrosinase activity in Streptomyces glaucescens is controlled by three chromosomal loci.

In Streptomyces glaucescens the production of the enzyme tyrosinase (EC 1.14.18.1) is controlled by at least three loci: MELA, MELB, and MELC. Mutations in MELA and MELB are rare. They both map on the S. glaucescens chromosome in the interval between the markers leu-2 and ura-3. MELC, however, shows an exceptionally high mutation rate. MelC strains arise spontaneously with a frequency of about 0.1%, and they can be specifically induced with ethidium bromide, acridine orange, or by prolonged storage of spores or mycelium at 4 degrees C. Ultraviolet (UV) irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine under conditions which induce auxotrophic mutations, however, do not specifically induce the appearance of tyrosinase-negative strains. Most, but not all, of the melC strains acquire additional mutations which make them sensitive to streptomycin (strS) and unable to produce hydroxystreptomycin. Mapping of the mutations classified as melC proves to be difficult. Some of the matings indicate a map position between his-2 and pro-1, while others, all highly unbalanced crosses, can not be analyzed in a meaningful way because of abnormal segregation of the reference markers. This is probably due to some of the additional mutations which occur frequently with the melC mutations and not to extrachromosomal inheritance of MELC.

Alleles↗
PubMed1983

Chromosomal instability in Streptomyces glaucescens: mapping of streptomycin-sensitive mutants.

Streptomyces glaucescens strain GLAO (=ETH 22794) produces hydroxystreptomycin and has a high natural resistance to hydroxystreptomycin, dihydrostreptomycin and streptomycin. The wild-type strain gives rise spontaneously to streptomycin-sensitive (StrS-) variants at a frequency of 0 . 2 to 1 . 4%. These mutants lack streptomycin phosphotransferase activity responsible for the wild-type resistance to streptomycin group antibiotics and are unable to produce detectable amounts of hydroxystreptomycin. Mapping experiments showed that the strS marker lies between the chromosomal markers lys-2 and ura-3 on the linkage map of S. glaucescens. The molecular basis for instability of this marker is as yet unknown.

Alleles↗
PubMed1983

Hydroxystreptomycin production and resistance in Streptomyces glaucescens.

The wild-type strain of Streptomyces glaucescens produces hydroxystreptomycin and shows an inherent natural resistance to streptomycin group aminoglycoside antibiotics. Cell free extracts of the wild-type strain were able to inactivate streptomycin, hydroxystreptomycin and dihydrostreptomycin in the presence of ATP. The phosphotransferase did not inactivate other aminoglycoside antibiotics, including spectinomycin. Mutant strains were isolated, which were highly sensitive to streptomycin group aminoglycosides, had no measurable phosphotransferase activity and were unable to form detectable amounts of hydroxystreptomycin. This suggests either a correlation between phosphotransferase activity, streptomycin resistance and hydroxystreptomycin formation or defects in more than one gene in the strS mutant strains tested.

Anti-Bacterial Agents↗