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Biomedical subjects

S Grosse

Publications and source records attributed to S Grosse.

At least 19 recordsLinked to original sources
PubMed2004

Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells.

To transfer genes into airway epithelial cells, we have generated auxotrophic dap Escherichia coli BM2710 mutant that expresses the invasin of Yersinia pseudotuberculosis and the listeriolysin of Listeria monocytogenes. E. coli BM2710 harboring a plasmid carrying the gfp gene was incubated with immortalized normal or cystic fibrosis (CF) airway epithelial cells or with primary bronchial epithelial cells grown as an explant-outgrowth cell culture model. Approximately 2% of immortalized cells expressed GFP. Few primary cells were transfected that were always poorly differentiated and located at the edge of the outgrowth. This was consistent with the expression of beta1-integrins only on these cells and with the required interaction for cell entry of E. coli expressing the invasin with beta1-integrins. The subsequent intracellular trafficking of E. coli BM2710 studied by confocal and electronic microscopy showed that the E. coli-containing phagosomes rapidly matured into phagolysosomes. This is the first demonstration that recombinant bacteria are able to transfer genes into primary airway epithelial cells, provided that they are able to invade the cells.

Adhesins, Bacterial↗
PubMed2003

Glycofection: facilitated gene transfer by cationic glycopolymers.

Mammalian cells express several types of lectins involved in intracellular trafficking, including endocytosis, interorganelle routing and putatively nuclear import. In order to enhance the gene transfer efficiency, glycosylated cationic polymers have been used as nonviral vectors. We developed a simple method to convert reducing saccharides into glycosynthons. Glycosynthons are used to synthesize cationic glycopolymers, called Glycofectins. Glycofectins interact with a plasmid to give a glycoplex, a compacted form of a polymer/DNA complex. The high glycoplex efficiency depends on the sugar involved in the uptake and in the intracellular trafficking of glycoplexes. The present paper deals with glycoplexes, with gene transfer into cystic fibrosis airway epithelial and gland serous cells, and with some of the problems that have to be solved before clinical trials.

Animals↗
PubMed2002

Targeting of cell receptors and gene transfer efficiency: a balancing act.

Vectors conjugated with ligands recognized by cell surface receptors are of interest for cystic fibrosis gene therapy since these vectors would allow cell-specific targeting. However, an efficient and specific uptake may be abrogated by a subsequent intracellular trafficking leading to an inefficient gene transfer. This has been shown for polylysine substituted with mannose residues. While mannose-specific membrane lectins are predominantly expressed at the surface of airway cells and mannosylated complexes are the most efficiently incorporated glycosylated complexes in these cells, mannosylated complexes lead to a low gene transfer efficiency because of an inefficient exit from endosomal compartments, a high accumulation in lysosomes and an inefficient nuclear import. In contrast, the entry of low amounts of lactosylated complexes is balanced by more efficient intracellular trafficking, leading to an efficient gene transfer. This emphasizes that for a successful gene transfer, it is necessary to find the balance between efficient and specific uptake, and intracellular trafficking that overcomes the various cellular barriers and enables the plasmid to reach the nucleus.

Biological Transport↗
PubMed2002

Intracellular rate-limiting steps of gene transfer using glycosylated polylysines in cystic fibrosis airway epithelial cells.

To identify the intracellular barriers to efficient gene transfer, we studied the intracellular trafficking of biotinylated plasmid DNA complexed with either fluorescein-conjugated lactosylated or mannosylated polylysine by confocal microscopy. Both are known to be taken up by cystic fibrosis airway epithelial cells (SigmaCFTE29o- cells), but their gene transfer efficiencies differ markedly: lactosylated polylysine is the most efficient glycosylated polylysine while mannosylated polylysine is quite inefficient for gene transfer. Mannosylated complexes appeared to stay longer in endosomes labeled by anti-transferrin receptor antibody than lactosylated complexes (from 30 min to 3 h and from 10 min to 30 min, respectively). At 24 h, higher percentages of mannosylated than lactosylated complexes were localized inside lysosomes labeled by anti-LAMP-1 antibody (41.8 +/- 6.6% versus 19.8 +/- 5.2%, respectively, P < 0.05). Between 30 min and 8 h, complexes reached the nuclei labeled by anti-lamin A/C antibody and no difference was observed between the nuclear amounts of mannosylated and lactosylated complexes. However, as analyzed by a nuclease S1 transcription assay, initiation of transcription was prevented when plasmid DNA was complexed to mannosylated polylysine. Our results indicate that the major limiting steps for mannosylated versus lactosylated polylysine transfer of plasmid DNA are delayed exit from endosomes, high accumulation in lysosomes and limited transcription of the complexed plasmid DNA.

Cystic Fibrosis↗
PubMed2002

Solubilization of low-rank coal by Trichoderma atroviride: evidence for the involvement of hydrolytic and oxidative enzymes by using 14C-labelled lignite.

The deuteromycete Trichoderma atroviride is able to solubilize lignite in dependence on a given carbon source for growth. When cultivated on media containing glutamate, this mold excreted a set of different enzymes with hydrolytic activity. Addition of lignite to the growth media induced the synthesis of extracellular lignite-specific esterase activity but no evidence has been provided for its direct involvement in the process of lignite solubilization. Hence, the basic capability of T. atroviride enzymes to degrade a variety of ester and ether bonds at the surface or within the bulky lignite structure was tested using coal following its direct labelling with 14C-alkyl iodide. The participation of hydrolytic and oxidative enzymes in lignite degradation was assessed by measuring the release of 14C radioactivity from selectively alkylated carboxylic and phenolic OH groups. T. atroviride cleaved both carboxylic esters using esterases and the phenolic ether bonds by using oxidative enzymes, most likely laccases.

Carbon Radioisotopes↗
PubMed2001

Effect of selenite on growth and protein synthesis in the phototrophic bacterium Rhodobacter sphaeroides.

The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides.

Aerobiosis↗
PubMed2000

How useful is positron emission tomography for lymphnode staging in non-small-cell lung cancer?

UNLABELLED: The introduction of positron emission tomography (PET) raises the question of the new method's capabilities in the staging of mediastinal lymphnodes, since PET differentiates between metabolically active and inactive tissues. 80 patients with histologically confirmed non-small-cell lung cancer (NSCLC) underwent PET scanning with 18-F-marked fluorodeoxyglucose (FDG). Extensive dissection of mediastinal lymphnodes (18-28 lymphnodes recovered) was performed in 78 cases. Metastasis to mediastinal lymphnodes were observed in 25 patients (N2: 22; N3: 3). RESULTS: Primary Tumor: FDG-PET showed significant enhancement of the primary tumor in 78 of 80 patients (sensitivity: 97%). Lymphnode Involvement: FDG-PET was positive in 23 of 25 patients with surgically confirmed lymphnode involvement (sensitivity: 92%). After a median follow up interval of 18 months, 11 patients with false positive lymphnode uptake were still alive; 10 of them showed no tumor recurrency. On the basis of these findings, enlarged mediastinal lymphnodes visualized at CT, but negative at FDG-PET are free of metastatic involvement with a sensitivity of 92%. FDG uptake of mediastinal lymphnodes at PET, however, should not be interpreted as proof of malignancy.

Adult↗
PubMed2000

FDG positron emission tomography in the diagnosis of peripheral pulmonary focal lesions.

Positron emission tomography (PET) using fluoride-18-marked fluoride deoxyglucose (FDG) represents a metabolically based imaging technique capable of providing information on the potential malignancy of peripheral pulmonary focal lesions. In the present prospective study, we investigated the effectiveness of FDG-PET in determining the dignities of 67 such lesions in 35 patients. Findings of FDG-PET were compared with those of computed tomography (CT), as well as with surgical and histological reports, and the value of FDG-PET as a diagnostic method evaluated. FDG-PET correctly identified 38 lesions as positive for malignancy, 18 correctly as negative, 7 incorrectly as negative, and 4 incorrectly as positive. Based on lesions, this yields a sensitivity of 84.4% and a specificity of 81.8%. All malignant focal lesions with a diameter of over 1.2 cm were correctly identified (sensitivity: 100%). In cases of intense FDG uptake, differentiation between a primary lesion, a metastasis, and an acute inflammation is often not possible.

Adolescent↗
PubMed1999

Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp. strain WI 14.

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 microM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an alpha(2)beta(2)gamma(2) structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg(2+) ions (with a total loss of enzyme activity at 0.01 mM Hg(2+)) and Cu(2+), Zn(2+), and Ni(2+) ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).

Alphaproteobacteria↗
PubMed1998

Purification and properties of methanol dehydrogenase from Methylosinus sp. WI 14.

Similarly to the recently described methanol dehydrogenase (MDH) from Methylocystis sp. GB 25 (Grosse et al. 1997) MDH from Methylosinus sp. WI 14 is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified about 9-fold to electrophoretic homogeneity and is localised in the soluble fraction. The relative molecular mass of the native enzyme has been determined to be 140 kDa. It is composed of two identical subunits of relative molecular mass 70 kDa. The amino terminal sequence shows a strong similarity (a match of 80% over the first 20 amino acids) to the MDH from Methylocystis sp. GB 25. PQQ could be detected as the prosthetic group of MDH in the purified enzyme fraction by using the apoenzyme of a membrane-bound glucose dehydrogenase from Pseudomonas aeruginosa. A PQQ ratio of 1.3 per mole MDH was estimated. The purified enzyme has an optimum activity at pH 9.0 and at 57 degrees C. MDH from Methylosinus sp. WI 14 oxidises only primary alcohols up to octanol and several aldehydes. The estimated K(m)-values vary between 0.18 mM for the sorbic alcohol and 6.3 mM for butanol and show no dependence upon the chain length.

Alcohol Oxidoreductases↗
PubMed1997

Purification and properties of methanol dehydrogenase from Methylocystis sp. GB 25.

Methanol dehydrogenase (MDH) from Methylocystis sp. GB 25, which belongs to the group II of methanotrophic bacteria, is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified 20-fold by a 5 step procedure to electrophoretic homogeneity. After cell disruption by French press, about 95% of MDH-activity was found in the soluble fraction. The relative molecular mass of the native enzyme has been estimated to be 122 kDa by gel filtration and 115 kDa by the method of Hedrick and Smith (1968). It seems to be composed of two identical subunits with a relative molecular mass of 62 kDa (estimated by SDS gel electrophoresis). The isoelectric point was found to be about 8.3. The amino terminal sequence shows a strong similarity to the alpha-chain of MDH from the facultative methylotrophic bacterium Methylobacterium extorquens AM1. PQQ, the probable prosthetic group of MDH, could be detected in the supernatant of the culture by using the apoenzyme of a membrane-bound glucose dehydrogenase from Pseudomonas aeruginosa but not absolutely in the absorption spectra of the enzyme after DEAE-chromatography. The purified MDH has an optimum activity at pH 9.0 and at 45 degrees C. MDH of Methylocystis sp. GB 25 oxidises only primary alcohols from methanol to heptanol and aldehydes from formaldehyde to propionaldehyde and the glutaraldehyde, respectively. The estimated Km-values show no dependence upon the chain length of substrates.

Alcohol Oxidoreductases↗
PubMed1990

Altered condylar morphology associated with disc displacement in TMJ dysfunction: observations by MRI.

Magnetic resonance images of 276 TM joints in 138 symptomatic patients were analyzed in a retrospective study to determine the condylar shape and size and to correlate it with internal derangement. Internal derangement by MRI was noted in 66% of the TM joints. Our study demonstrates that the regressive condylar changes in TM joints with internal derangement were more common (61%) than proliferative bony changes (39%). On the converse, none of the TM joints with regressive condyles revealed normal disc. The altered bony morphology also correlated with the severity of internal derangement, i.e. bony changes in TM joints with anterior closed lock were noted in 64% compared to 45% with reducible disc. The cause and effect relationship of the regressive condylar remodeling and disc abnormality is not clear and needs further study.

Adolescent↗