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Biomedical subjects

D Bucher

Publications and source records attributed to D Bucher.

At least 55 records · Page 3Linked to original sources
PubMed2000

Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling.

We show here, using locust wholemount ganglia as an example, that scaling artifacts in three-dimensional reconstructions from confocal microscopic images due to refractive index mismatch in the light path and tissue shrinking, can account for dramatic errors in measurements of morphometric values. Refractive index mismatch leads to considerable alteration of the axial dimension, and true dimensions must be restored by rescaling the Z-axis of the image stack. The appropriate scaling factor depends on the refractive indices of the media in the light path and the numerical aperture of the objective used and can be determined by numerical simulations, as we show here. In addition, different histochemical procedures were tested in regard to their effect on tissue dimensions. Reconstructions of scans at different stages of these protocols show that shrinking can be avoided prior to clearing when dehydrating ethanol series are carefully applied. Fixation and mismatching buffer osmolarity have no effect. We demonstrate procedures to reduce artifacts during mounting and clearing in methyl salicylate, such that only isometric shrinkage occurs, which can easily be corrected by rescaling the image dimensions. Glycerol-based clearing agents produced severe anisometric and nonlinear shrinkage and we could not find a way to overcome this.

Animals↗
PubMed1993

Small virus-like particles bud from the cell membranes of normal as well as HIV-infected human lymphoid cells.

Electron microscopy (EM) of cell sections showed cell associated virus-like particles (VLP), 50-60 nm in diameter, budding from the membrane of human lymphoid cells in culture. The particles had an envelope continuous with the cell membrane and a dense core that almost filled the particle. Particles 70-80 nm in diameter with prominent external spikes were found in the culture medium by negative staining (medium-associated VLP). Cell-associated VLP were also present in cord lymphocytes, both on initial separation and after culture with or without foetal calf serum, and therefore were considered to be endogenous to the cells and were not bovine diarrhoea virus. VLP were observed in most of the lymphoid cell lines examined. VLP were also found less frequently in established human tumour and nontumour cell lines. Both cell-associated and medium-associated VLP were also present in HIV infected cell cultures, and they could be distinguished from HIV by their characteristic morphology and smaller size. It was not determined whether the 2 types of particle represented the same entity, or whether they were defective virus particles or a cellular secretory product.

Cell Line↗
PubMed1993

Oral immunization of rats with proteinoid microspheres encapsulating influenza virus antigens.

Influenza virus antigen microspheres were prepared by a pH-dependent process using a protein-like polymer (proteinoid) made by thermal condensation of amino acids. The efficacy of these preparations to induce specific IgG responses when used as oral vaccines in rats was evaluated. A single enteric dose of M1 entrapped in proteinoid microspheres was able to induce a significant IgG response to M1 as early as 2 weeks postdosing, while rats dosed orally with the same M1 total dose (no microspheres) showed no detectable antibody response. An unencapsulated hemagglutinin and neuraminidase (HA-NA) preparation induced a moderate anti HA-NA IgG response. A single enteric dose of HA-NA spheres induced a response in 33% of the rats; this response was up to eight times higher than that observed in the rats dosed with unencapsulated antigen.

Administration, Oral↗
PubMed1992

Subtype H7 influenza viruses: comparative antigenic and molecular analysis of the HA-, M-, and NS-genes.

Antigenic analysis of the haemagglutinin and matrix protein with corresponding sets of monoclonal antibodies as well as sequence analysis of HA-, M-, and NS-genes were carried out to establish antigenic and genetic relationships between four fowl plague virus (FPV) strains of H7 subtype. The data obtained revealed close genetic relatedness between the oldest known influenza A virus, A/chicken/Brescia/1902 (H7N7), and two FPV strains, A/FPV/Dobson (H7N7) and A/FPV/Weybridge (H7N7). These three strains apparently differ in all genes investigated from the A/FPV/Rostock isolate.

Amino Acid Sequence↗
PubMed1992

Host range determination and functional mapping of the nucleoprotein and matrix genes of influenza viruses using monoclonal antibodies.

Construction and comparison of phylogenetic trees, the standard approach to determining the host-specific lineage of influenza A virus genes is tedious and expensive. In this study, panels of monoclonal antibodies (Mabs) produced against the matrix proteins (M1) of A/WSN and A/PR/8/34 and the nucleoprotein (NP) of A/WSN were assessed for their value in identifying the hosts of origin of the M1 and NP genes in influenza virus isolates and in mapping the proteins' functional domains. Using ELISA against a broad spectrum of reference viruses, we found two Mabs against the NP (150/4 and 469/6) to be useful in determining host-specific lineage. Comparative sequence analysis placed five amino acids within the antigenic domains recognized by Mab 150/4 and two amino acids within the domains recognized by 469/6. One Mab against the NP (5/1) recognized a conserved epitope that is present on each of the 36 influenza A viruses tested. This epitope may be a type-specific determinant for influenza A viruses and an RNA binding site. Monoclonal antibodies to M1 did not discriminate among species, but they did contribute information to the construction of a functional map of M1. These results demonstrate that Mabs to defined protein epitopes can provide useful information on the molecular epidemiology of influenza viruses.

Animals↗
PubMed1992

Absence of infectious human immunodeficiency virus type 1 in "natural" eccrine sweat.

Although human immunodeficiency virus type 1 (HIV-1) has been found in numerous body fluids, there are no reports of attempts to demonstrate this virus in eccrine sweat, a fluid frequently encountered during person-to-person interactions. "Natural" eccrine sweat samples and blood from 50 HIV-1-seropositive patients and 2 HIV-1-seronegative controls were cultured for HIV-1 by a cocultivation method. Polymerase chain reaction for HIV-1 RNA and proviral DNA was done on 40 sweat samples (39 patients, 1 control). HIV-1 was isolated from peripheral blood mononuclear cells of 39 (78%) of 50 patients but from none of 52 sweat samples. No HIV-1 viral DNA or RNA was detected in the 40 sweat samples tested. With present methodology, infectious HIV-1 cannot be demonstrated in "natural" eccrine sweat samples from HIV-infected patients.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Analysis of internal proteins of influenza A (H2N2) viruses isolated from birds in East Germany in 1983.

Proteins and RNAs of influenza A (H2N2) viruses isolated from birds in 1983 in East Germany were compared antigenically with those of H2N2 human strains. The electrophoretic mobility of the viral proteins and of the S1-treated double-stranded RNAs from two human and six avian strains, as well as the results of EIA-tests using monoclonal antibodies to their matrix protein and nucleoproteins indicate an antigenic relationship between the avian isolates and human strains of H2N2 subtype. One of the avian strains had a reduced amount of matrix protein.

Animals↗
PubMed1989

M protein (M1) of influenza virus: antigenic analysis and intracellular localization with monoclonal antibodies.

A panel of 16 monoclonal antibodies recognizing M protein (M1) of influenza virus was generated. Competition analyses resulted in localization of 14 monoclonal antibodies to three antigenic sites. Three monoclonal antibodies localized to site 1B recognized a peptide synthesized to M1 (residues 220 to 236) with enzyme-linked immunosorbent assay titers equivalent to or greater than that seen with purified M1; therefore, site 1B is located near the C terminus of M1. Sites 2 and 3 localize to the N-terminal half of M1. Antigenic variation of M proteins was seen when the monoclonal antibodies were tested against 14 strains of type A influenza viruses. Several monoclonal antibodies showed specific recognition of A/PR/8/34 and A/USSR/90/77 M proteins and little or no reactivity for all other strains tested. Immunofluorescence analysis with the monoclonal antibodies showed migration of M protein to the nucleus during the replicative cycle and demonstrated association of M protein with actin filaments in the cytoplasm. Use of a vaccinia virus recombinant containing the M-protein gene demonstrated migration of M protein to the nucleus in the absence of synthesis of gene products from other influenza virus RNA segments.

Amino Acid Sequence↗
PubMed1989

Microenvironmental toxicity of azidothymidine: partial sparing with hemin.

Azidothymidine (AZT) is a useful drug in management of AIDS. Nevertheless, its hematologic toxicity such as anemia and neutropenia present further complications to an already compromised hematopoietic state in patients. We studied the effects of AZT on human and murine bone marrow (BM) colony growth as determined by assays of CFU-E, BFU-E, CFU-GM, and fibroblastoid stromal (CFU-Fb) colonies. Cultures were grown in methylcellulose with growth factors and scored after three- to 14-day incubation. In general, murine marrow cultures were more sensitive to AZT as compared with human marrow. Furthermore, interindividual variation in toxicity to AZT was observed between marrow samples; 1 mumol/L AZT inhibited murine CFU-E, BFU-E, and CFU-GM by 98% to 100%, whereas human marrow was inhibited by 52%, 87%, and 65%, respectively. Lower concentrations of AZT (0.1 mumol/L) inhibited murine erythroid colony growth by 85% to 90%, whereas human growth was inhibited by only 39% to 52%. Myeloid colony inhibition was similar for human and murine systems. CFU-Fb growth was markedly suppressed (75%) by 1 mumol/L AZT. Hemin, at a concentration of 10 mumol/L, overcame some of the inhibitory effects of 1 to 0.1 mumol/L AZT without hindering antiviral activity. Inhibition of human CFU-E growth was completely overcome with hemin, whereas CFU-GM growth was recovered to 66% to 74% of control. A similar but less pronounced effect was observed for BFU-E. Furthermore, hemin does not decrease AZT's effects of HIV antigen content in vitro. We conclude that anemia and neutropenia, occurring as a result of AZT, may not be as pronounced in the presence of hemin. Furthermore, CFU-Fb was significantly reduced in the presence of low concentrations of AZT. This may indicate a major target site for BM toxicity since the stromal microenvironment may be responsible for maintaining short- and long-term hematopoiesis.

Animals↗
PubMed1989

Human intrinsic factor. Its primary structure compared to the primary structure of rat intrinsic factor.

Human intrinsic factor was digested by trypsin and the resulting peptides purified by gelfiltration. Two peptides were sequenced to a total of 61 amino acid residues. Including the sequence for the N-terminal peptide and four cyanogen bromide peptides previously reported, we have now determined a total of 163 amino acid residues, that is a fraction of about 0.40 of the primary structure of human intrinsic factor. The 110 of the 163 residues known of human intrinsic factor are identical to the sequence of rat intrinsic factor.

Amino Acid Sequence↗
PubMed1988

Renal tubular function in patients treated with high-dose cisplatin.

The effect of three cycles of high-dose cisplatin (40 mg/m2 day for 5 days) on renal tubular function was evaluated in 30 patients. A significant impairment of proximal tubular salt and water reabsorption rates was observed, but also distal tubular function seemed to be affected. These changes were also present 6 months after termination of treatment. Sodium and magnesium clearance increased significantly during treatment. Magnesium clearance normalized shortly after treatment but sodium clearance was significantly elevated 6 months after treatment. Proteinuria, albuminuria, and amino aciduria, together with an increase of beta 2-microglobulin and N-acetyl-beta-D-glucosaminidase (NAG) excretion rates, were observed during each treatment cycle. A good correlation was registered between the increase in urinary excretion rates of protein, NAG, and magnesium and the decrease in proximal tubular salt and water reabsorption during cisplatin administration.

Acetylglucosaminidase↗
PubMed1988

Increased hepatic amino nitrogen conversion after elective cholecystectomy in man.

1. The effect of elective, uncomplicated cholecystectomy on plasma clearances of amino acids and on amino acid-stimulated urea synthesis was investigated in 10 patients, pre-operatively and on the first post-operative day, and compared with six controls treated identically apart from the surgery. 2. A mixture of amino acids was given as a prime-continuous infusion. Steady-state concentrations 75% higher than basal were attained and were maintained for 90 min. The clearances of amino acids were calculated as the ratios between amino acid infusion rate and the concentration. The urea synthesis rate was calculated as urinary excretion corrected for accumulation and intestinal loss. 3. After surgery the fasting plasma concentrations of alanine, arginine, glutamine plus glutamate, glycine, proline, lysine and threonine decreased by 20-30%, but were unchanged in the control group. The plasma clearance of alpha-amino nitrogen increased from 5.1 +/- 1.2 ml/s before surgery (mean +/- SD) to 6.1 +/- 1.1 ml/s (P less than 0.05, paired t-test) after surgery due to increased clearances of the above-mentioned amino acids. In the control group, the clearance decreased from 6.4 +/- 1.6 to 5.9 +/- 1.1 (P less than 0.05, paired t-test). The amino acid-stimulated urea synthesis rate after surgery was 37 +/- 9 mumol of N/s vs 30 +/- 6 (P less than 0.01, paired t-test) in the controls despite a lower alpha-amino nitrogen concentration (4.5 +/- 0.5 mmol/l vs 5.1 +/- 0.5 mmol/l, P less than 0.05, paired t-test). The post-operative urea synthesis rate exceeded the amino nitrogen infusion by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1986

Cerebrospinal fluid glutamine in intracranial hemorrhage in the newborn.

Cerebrospinal fluid concentration of glutamine was measured in 43 premature and mature babies suffering from intracranial hemorrhage. Compared with a control group of 38 infants we found elevated levels in all infants with intraparenchymal and subarachnoid hemorrhages. Two out of ten children with subependymal hemorrhages and approximately half of the children with intraventricular hemorrhages also had elevated levels. Elevated levels of cerebrospinal fluid glutamine persisted for up to three weeks and maximum levels were reached two days to two weeks after birth. The analysis of cerebrospinal fluid glutamine concentration might be useful in the early diagnosis of intraparenchymal and subarachnoid hemorrhage in the newborn infant.

Cerebral Hemorrhage↗
PubMed1982

Elimination of infused amino acids from plasma of control subjects and of patients with cirrhosis of the liver.

In four control subjects and four patients with cirrhosis of the liver a multiple amino acid mixture was infused for 12 h at a constant rate of 68 and 56 mumol alpha-amino N/s, respectively. Before infusion the plasma amino N concentration was 2.4 +/- 0.2 (mean +/- SD) mmol/l in control subjects and 3.5 +/- 0.7 mmol/in patients (P less than 0.025). The concentration of alanine, proline, arginine, tyrosine, and citrulline was significantly increased in the cirrhosis group. 12 h after the infusion began approximately constant amino N concentrations of 11.4 +/- 1.8 mmol/l in controls and 13.7 +/- 3.9 mmol/l in patients were attained, and the urea N synthesis rate was 63 +/- 17 and 44 +/- 8 mumol/s, respectively (P less than 0.05). After correction for loss of amino acids in urine this means that on the average 94 per cent of the N load was recovered as urea. The plasma clearance of infused amino acids, calculated as the ratio between infusion rate and steady state concentration, was 6.0 +/- 1.2 and 4.1 +/- 0.9 ml/s for amino N in the control and cirrhosis group, respectively (P less than 0.025). The clearance of individual amino acids ranged between 2.5 and 28 ml/s. The clearance of most amino acids was decreased in the cirrhosis groups, and of glycine, proline, lysine, threonine, and arginine significantly so (P less than 0.05), reflecting accumulation of amino acids in patients. This indicates that a primary defect in the conversion of amino N in cirrhosis is the reduced urea synthesis.

Adult↗
PubMed1982

Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed for the titration of antibodies in human sera to influenza virus neuraminidase, employing partially purified N1 neuraminidase. Specificity of the test was demonstrated, and the test was more sensitive than either the conventional neuraminidase inhibition or plaque size reduction tests in detecting anti-neuraminidase antibody.

Antibodies, Viral↗
PubMed1982

Isolation and partial characterisation of three major allergens in an extract from cow hair and dander.

Three 'major allergens' of an extract from cow hair and dander were isolated by means of anion-exchange chromatography and hydrophobic chromatography. Two of the isolated 'major allergens' were immunochemically pure and the third contained less than 1% impurities. The molecular weights were 2.0, 2.2 and 2.4 x 10(4) daltons, and pI was about 4.0. The amino acid content revealed no specific traits. The protein part of the major allergens constituted about 60% (w/w). The allergenic activity of the isolated allergens was verified in RAST-inhibition experiments and in prick tests.

Allergens↗