Search PubMed⌕ Search

Biomedical subjects

F Fischer

Publications and source records attributed to F Fischer.

At least 55 records · Page 3Linked to original sources
PubMed1999

Impairment of HIV polymorphonuclear leukocyte transmigration across T84 cell monolayers: an alternative mechanisms for increased intestinal bacterial infections in AIDS?

Our objective was to study the influence of HIV infection of polymorphonuclear leukocytes (PMN) on transepithelial migration. To date, reports of functional PMN chemotaxis in AIDS are contradictory. This is the first attempt to assess this function via an in vitro model allowing transmigration of neutrophils through an intestinal epithelial barrier. PMN were isolated from 45 HIV-infected patients and 45 healthy volunteers. PMN transmigration across T84 epithelial cells was initiated by applying either various concentrations of formyl-met-leu-phe peptide (f-MLP) or interleukin-8 and assayed by quantification of myeloperoxidase activity. CD11b, CD18, and CD47 expression on PMN was compared before and after transepithelial migration by flow cytometry analysis. CD11b expression was studied by electron microscopy. Apoptosis of transmigrated HIV PMN and control PMN was investigated by morphology and DNA fragmentation characterization. Compared to control PMN, HIV PMN exhibited a decrease in transepithelial migration that directly correlated with CD4+ counts. Basal and transepithelial migration-mediated expression of CD11b, CD18, and CD47 were unmodified in HIV PMN compared to control PMN. Electron microscopy labeling confirmed no difference in CD11b expression on HIV and control PMN. The index of apoptosis in transmigrated HIV PMN and control PMN was identical. These data provide evidence of a defect in the f-MLP-induced chemotaxis of PMN from HIV-infected patients across an intestinal epithelial barrier. This defective migration is not due to a quantitative modification of CD11b, CD18 and CD47 on HIV PMN suggesting a more subtle alteration. The impairment in the transmigration function may contribute in vivo to an increased susceptibility to intestinal bacterial infection in HIV-infected patients.

Adult↗
PubMed1999

Regulatory changes at high altitudes in comparison with post-space-flight reactions.

This report will compare results of applications of the psychophysiological method "Assessment of Autonomic Outlet Type" (AOT) in space as well as at high altitudes. The aim was to find out whether it is possible to differentiate a subject's reaction to different environments by this method (Johannes et.al. 1995). This assessment as described below was first applied to clinically well defined cohorts and to healthy subjects. By means of cluster analysis it could be demonstrated that different reaction patterns exist. These different patterns were correlated to two groups of normal controls ([I] = non-responder/voice-responder and [II] = heart-responder) and two groups of patients with different clinical diagnoses (rheumatic diseases ([III] = autonomic-responder) and [IV] = hypertensives). Based on these clinical reference data the method provides for each "new" experiment a common classification of the complex regulatory reaction pattern of the autonomic nervous system (ANS) and the Cardiovascular system (CVS) to psychological stressors by means of a discriminant function. Since August 1997 the experiment has been applied to all Russian cosmonauts continuously each month. (Johannes et.al. 1998). At the same time numerous terrestrial experiments have been running parallel for comparison with the results from space. This report will focus on a study at altitudes of 3600 m.

Acclimatization↗
PubMed1998

A chemical dosimeter for the determination of the photodynamic activity of photosensitizers.

Uric acid a known singlet oxygen scavenger, was investigated as a chemical dosimeter in physiological aqueous solution for use in photodynamic therapy. The uric acid test takes the decrease in uric acid (UA) absorbance at 293 nm after laser light irradiation of a solution containing UA and a photosensitizer as a rapid evaluation of relative photodynamic activities of the photosensitizer. A uric acid test standard procedure was defined. To compare photodynamic activity of different photosensitizers or irradiation conditions a proposal for a photodynamic activity scale based on the uric acid test is given. Examples of uric acid test operation are given by comparing the changes in UA absorbance decrease with respect to irradiation wavelength and to photosensitizer concentration of Photofrin II with that of two other photosensitizers (5,10,15,20-tetrakis-[4-hydroxyphenyl]-21H,23H-porphyrin (TOP) and 5,10,15,20-tetrakis-[4-carboxyphenyl)-21H, 23H-porphyrin (TCPP), both derivatized with methoxypolyethyleneglycol (TOP-MPV and TCPP-AMP) as a macromolecular carrier). The photodynamic activity of the three photosensitizers using the proposed photodynamic activity scale is given.

Dihematoporphyrin Ether↗
PubMed1998

The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties.

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase alpha subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.

Azospirillum brasilense↗
PubMed1998

Blood cell damage after in vitro irradiation of fresh whole blood with 630 nm laser light.

A study on blood cell damage after irradiation of fresh whole blood with 630 nm laser light was carried out in vitro. Various fluence rates of laser light were used with and without cooling of blood. Damage to the blood was assessed by blood cell counts, osmotic fragility measurements and examination of blood films. Exposure of a 1 mm blood layer to 630 nm laser light without cooling led to changes in blood counts first detected at fluence rates of 130 mW/cm2. Changes in osmotic fragility first became evident at 210 mW/cm2. Increasing cell damage with increasing fluence rates was evident in blood films. Using the cooling device changes in whole blood after irradiation first occurred at a fluence rate of 293 mW/cm2. Measurement of the fluence rates at which cell damage begins is important in laser induced fluorescence diagnostics and photodynamic therapy applications in blood or blood products using photosensitizers.

Adult↗
PubMed1998

Construction of a mouse hepatitis virus recombinant expressing a foreign gene.

The genome of the coronavirus mouse hepatitis virus (MHV) contains genes which have been shown to be nonessential for viral replication and which could, in principle, be used as sites for the introduction of foreign sequences. We have inserted heterologous genetic material into gene 4 of MHV in order (i) to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the N gene; (ii) to develop further genetic tools for mutagenesis of structural genes other than N; and (iii) to examine the feasibility of using MHV as an expression vector. A DI-like donor RNA vector containing the MHV S gene and all genes distal to S was constructed. Initially, a derivative of this was used to insert a 19-nucleotide tag into the start of ORF 4a of MHV-A59 using the N gene deletion mutant A1b4 as the recipient virus. Subsequently, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4. This heterologous gene was shown to be expressed by recombinant viruses but not at levels sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected displacement of the mobility, relative to those of wild-type, of all subgenomic mRNAs larger than mRNA5. An unexpected result of the Northern analysis was the observation that GFP recombinants also produced an RNA species the same size as that of wild-type mRNA4. RT-PCR analysis of the 5' end of this species revealed that it was actually a collection of mRNAs originating from a cluster of 10 different sites, none of which possessed a canonical intergenic sequence. The finding of these aberrant mRNAs, all of nearly the same size as wild-type mRNA4, suggests that long range structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.

Animals↗
PubMed1998

Photodynamic therapy as a tool for suppressing the haematogenous dissemination of tumour cells.

The chance of most cancer patients surviving their disease is to a high degree dependent on the status of the metastatic processes. One general route of cancer-cell dissemination is passive transport in the blood stream, i.e., haematogenous dissemination. In this study we try to find an answer to the following question: is it possible to use photodynamic therapy for suppressing the haematogenous dissemination of cancer cells? In first in vitro experiments we incubated CX1 cells (colon carcinoma cells) with two photosensitizers, Photofrin II and mesotetra(hydroxyphenyl)chlorin (mTHPC). We added the cells to fresh whole blood and irradiated the blood with suitable laser light in a flow-through irradiation system. The tumour-cell survival fraction (SF) was determined with plating efficiency. Using Photofrin II we observed a minimal tumour-cell survival in blood of SF = 3.5% and using mTHPC we measured SF = 0.02%. These results encourage further investigations concerning the use of photodynamic therapy for suppressing haematogenous dissemination.

Culture Media↗
PubMed1998

The impact of vessel and catheter position on the measurement accuracy in catheter-based quantitative coronary angiography.

BACKGROUND: The calculation of absolute artery dimensions in quantitative coronary angiography is usually carried out by catheter calibration. It is based on the proportional comparison of the dimension of the imaged artery segment to the dimension of the imaged angiographic catheter of known size. This calibration method presumes an identical radiographic magnification between angiographic catheter and artery segment of interest. However, due to the different intrathoracic location of both objects the radiographic magnification or calibration factor is often not identical for a given angiographic projection. The aim of this study was to quantify the magnification error (out-of-plane magnification error) for the major coronary artery segments imaged in frequently used angiographic projections. METHODS: The intrathoracic spatial location of 468 coronary segments (RCA 196, LAD 156, LCX 116) and their respective coronary catheters were established with biplane angiography and known imaging geometry data. The error in the radiographic magnification or calibration factor was then calculated for all 936 monoplane projections using the spatial coordinates and imaging geometry data. RESULTS: The mean magnitude of magnification error was 4% within all 936 measurements. The magnitude and direction of error varied with the lesion localization and the angiographic projection angle (range -12.6% to +10.6%). The error characteristics could be described with six typical error groups by stratifying the data according to the three main coronaries and two angiographic planes. In 24% of measurements, the magnification error exceeded the 5.2% error limit acceptable for reference vessel sizing. Measurements of left coronary arteries were mainly affected by it. CONCLUSION: The magnification error contributes to the calibration error in measuring arterial dimensions by quantitative angiography. This error may affect the reliability of clinical studies and the proper sizing of interventional devices. These findings could be used to improve current error correction algorithms in order to reduce the effect of the magnification error in measuring arterial dimensions.

Algorithms↗
PubMed1998

Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly.

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

Amino Acid Sequence↗
PubMed1998

Assessment of arrhythmia under micro-g by mental loading tests.

Based on data of former clinical studies (Johannes et al. 1994) the experiment regulation was designed to assess and classify the actual psychophysiological reactivity of the cosmonauts. Beside this scientific main goal of the experiment it was for us of interest to check the data whether the development of arrhythmia can be seen under micro-g conditions when flight related stressful conditions prevail. This report will focus on the ECG analysis of one subject in whom this was actually the case.

Arrhythmias, Cardiac↗
PubMed1997

Evaluation of the ABX Cobas Vega automated hematology analyzer and comparison with the Coulter STKS.

An evaluation of the new automated hematology analyzer was performed in comparison with the Coulter STKS on 1,694 blood samples coming from the different departments of Nice University Hospital. The Cobas Vega showed very satisfactory results in terms of repeatability, reproducibility and linearity. Correlation with the STKS was excellent with the exception of the following parameters: red blood cell distribution index and the absolute values for eosinophils and basophils. Two qualities were particularly appreciable: absence of leukocyte carryover, and stability of the complete blood count and leukocyte differential count over a long period. Analysis of qualitative flags showed that the overall blood smear review rate was 47% for the Cobas Vega, not forgetting that optical microscopy detects 37% of all abnormalities. The STKS's review rate was 49.5%. Flags commonly concerned the granulocytic lineage, 61% for the STKS and 48% for the Vega, with a false positive rate of 43.4% for the STKS compared with 22% for the Vega. The opposite phenomenon was observed with the flag for atypical lymphocytes which represented 11% of flags for the STKS and 25.6% for the Vega, with a false positive rate of 25.5% for the STKS and 34% for the Cobas Vega. This may be explained by the fact that lymphocyte abnormalities sometimes generated "granulocytic" flags on the STKS. Studies of the false negative rate carried out using light microscopy on 505 blood samples without flags on either system, detected the presence of a slight myelemia, and a few hyperbasophilic lymphocytes or plasmocytes in 18.6% of all cases. Finally, the Cobas Vega's practicality was greatly appreciated and there was no trouble with breakdowns throughout the whole period of its use.

Autoanalysis↗
PubMed1997

Comparative in-vitro validation of eight first- and second-generation quantitative coronary angiography systems.

BACKGROUND: It is known that first-generation quantitative coronary angiography (QCA) systems overestimate small vessel sizes owing to the point-spread function of the respective X-ray imaging chain. With second-generation systems new algorithms were introduced to correct for this source of error. OBJECTIVE: To evaluate the efficiency of the modified contour detection algorithms. METHODS: Six second-generation QCA systems (CMS, QANSAD, AWOS, CAAS II, Cardio 500, and Angioimage) were validated and compared with first-generation systems (CAAS and ARTREK). By using an arterial phantom consisting of stenotic and nonstenotic glass tubes (of diameters 0.5-5.0 mm) the accuracy and precision of each analysis system, as well as their additional accuracy and precision values for phantom diameters < or = 1.0 mm were determined. RESULTS: All systems had high accuracy and precision values, but first-generation systems overestimated small vessel diameters. With second-generation systems a significantly improved accuracy in the submillimeter range (an accuracy within +/-0.028 mm) was obtained. This improvement was accompanied by a moderate reduction in precision in the submillimeter range. CONCLUSION: The new algorithms of the second-generation QCA systems allow accurate and reliable measurements of small coronary dimensions and, therefore, precise analysis of coronary stenoses of moderate-to-high grade seems feasible with the improved accuracy of the new systems.

Algorithms↗
PubMed1997

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.

Animals↗
PubMed1997

Analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription.

We have inserted heterologous genetic material into the nonessential gene 4 of the coronavirus mouse hepatitis virus (MHV) in order to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the nucleocapsid (N) gene and to develop further genetic tools for site-directed mutagenesis of structural genes other than N. Initially, a 19-nucleotide tag was inserted into the start of gene 4a of MHV strain A59 with the N gene deletion mutant Alb4 as the recipient virus. In further work, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4, creating the currently largest known RNA virus. The expression of GFP was demonstrated by Western blot analysis of infected cell lysates; however, the level of GFP expression was not sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected alteration of the pattern of the nested MHV subgenomic mRNAs. Surprisingly, though, GFP recombinants also produced an RNA species that was the same size as wild-type mRNA4. Analysis of the 5' end of this species revealed that it was actually a collection of mRNAs originating from 10 different genomic fusion sites, none possessing a canonical intergenic sequence. The finding of these aberrant mRNAs suggests that long-range RNA or the ribonucleoprotein structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.

Animals↗