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Biomedical subjects

S Becker

Publications and source records attributed to S Becker.

At least 91 records · Page 5Linked to original sources
PubMed2000

Therapeutic monitoring of sirolimus in human whole-blood samples by high-performance liquid chromatography.

BACKGROUND: Sirolimus is a macrolide antibiotic isolated from Streptomyces hygroscopicus that has demonstrated immunosuppressive activity. Human and animal studies have shown a good correlation of trough sirolimus concentrations with immunosuppressive efficacy. OBJECTIVE: This report describes a reverse-phase high-performance liquid chromatography (HPLC) method used for therapeutic drug monitoring of sirolimus. METHODS: A reverse-phase C18 column method was developed using an automated HPLC system and ultraviolet (UV) detection. Whole-blood samples collected in ethylenediamine-tetraacetic acid (EDTA) are first hemolyzed, and an internal standard (desmethoxysirolimus) is added to 1.0 mL of sample, which is then extracted with 1-chlorobutane and, after the organic layer is removed, evaporated to dryness. The residue is reconstituted in a 70% methanol/water mixture. Reconstituted extracts are analyzed by HPLC at a column temperature of 60 degrees C and a flow rate of 1.0 mL/min. Typically, chromatography requires 35 minutes between each sample injection. The UV detector is set at 278 nm with a response sensitivity of 0.010 AUFS (absorbance units full scale). Standards and controls prepared in hemolyzed EDTA-anticoagulated whole blood are extracted and run in parallel. Identification of peaks of interest is by retention time; quantification of sirolimus in controls and clinical samples uses a peak-height ratio (sirolimus/internal standard). RESULTS: The assay's precision (coefficients of variation, 5.7%-14.4%) and sensitivity (2.5 ng/mL) were found to be appropriate for therapeutic monitoring purposes. Analytical recovery of 88.0% to 106.3% was observed throughout the assay's linear range (2.5-150.0 ng/mL). Stability studies at 20 degrees C to 25 degrees C and 2 degrees C to 8 degrees C showed an estimated recovery of sirolimus ranging from 85% to 110% of target concentrations (10-90 ng/mL). In a study comparing the results of 194 samples from kidney transplant recipients assayed by the HPLC-UV assay and by a microparticle enzyme immunoassay, the HPLC-UV method provided approximately 10% lower values. CONCLUSION: The HPLC-UV assay is analytically capable of providing useful data for the clinical assessment of patients receiving sirolimus.

Biotransformation↗
PubMed2000

High intensity dependence of auditory evoked dipole source activity indicates decreased serotonergic activity in abstinent ecstasy (MDMA) users.

Neurotoxic damage of central serotonergic systems has been demonstrated in numerous animal studies after exposure to methylenedioxyamphetamines (ecstasy). A high intensity dependence of auditory evoked potentials and, particularly, of the tangential N1/P2 source activity has been associated with low levels of serotonergic neurotransmission in humans. We performed an auditory evoked potentials study in 28 abstinent recreational ecstasy users and two equally sized groups of cannabis users and nonusers. The ecstasy users exhibited an increase of the amplitude of the tangential N1/P2 source activity with higher stimulus intensities; whereas, both control groups failed to exhibit this feature. These data are in line with the hypothesis that abstinent ecstasy users present with diminished central serotonergic activity. This feature of information processing is probably related to the well-recognized neurotoxic potential of ecstasy. Our data indicate that recreational ecstasy use may cause long-term alterations in the function (and possibly structure) of the human brain.

Adolescent↗
PubMed2000

Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies.

We previously demonstrated that a member of the Hedgehog gene family, Indian hedgehog (Ihh), is expressed in the visceral endoderm of EC and ES cell embryoid bodies and mouse embryos. Overexpression studies suggested that Ihh was involved in visceral endoderm differentiation. We now provide evidence for a Hh response in the embryoid body core and in the mesothelial layer of the visceral yolk sac. We also demonstrate that treatment of ES embryoid bodies with the Hh antagonists cAMP and forskolin results in downregulation of the Hh response and altered embryoid body differentiation. The outer endoderm layer undergoes a transition to parietal endoderm while formation of an embryonic ectoderm layer surrounding a cavity is inhibited. These treatments also result in a decrease in the expression of markers for the mesoderm derivatives, blood and endothelial cells. We present a model to explain how Ihh and BMP signaling may regulate extraembryonic endoderm and embryonic ectoderm differentiation.

Animals↗
PubMed2000

Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes.

Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i.e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulation. Until recently, others and we have used immuno-topochemical and biochemical methods to demonstrate the integration of the channel into the cell membrane and endoplasmic reticulum of vertebrate cells. In the present study, we used molecular biological methods to induce the heterologous expression of tagged human type-1 porin in oocytes of Xenopus laevis and to illustrate its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminus of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indicating a strong influence of the porin N-terminus on protein trafficking to the plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. Here, plasmalemmal expression was observed using anti-FLAG M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver enhancement through scanning electron microscopy. In contrast to the EGFP-porin fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plasma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are essential for future electrophysiological studies on the channel.

Animals↗
PubMed2000

The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels.

Recent patch-clamp studies have shown that anti-porin antibodies, applied to the external side of excised plasma membrane patches of mammalian astrocytes, close chloride channels that are thought to be engaged in cell volume regulation. Frog oocytes are often used to study this basic cell function. Here we document the localisation of endogenous porin voltage-dependent anion-selective channels in Xenopus laevis oocyte plasma membranes. In confocal laser microscopy images a disjunctive pattern of fluorescing spots appear about 10 microm apart. Labelling was prevented by preabsorption of the antibodies with synthetic peptides comprising the epitope of the antigen. Immuno-gold marking of oocyte surfaces followed by silver enhancement of the gold particles lead to a plasma membrane labelling corresponding to that obtained by the confocal laser approach. The data suggests the presence of voltage-dependent, anion-selective channels in oocyte plasma membranes. This data should be borne in mind when frog oocytes are used to study the characteristics of endogenous or heterologously expressed ion channels or regulatory proteins.

Amino Acid Sequence↗
PubMed2000

Gene therapy of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.

Animals↗
PubMed2000

Role of glutathione in the formation of the active form of the oxygen sensor FNR ([4Fe-4S].FNR) and in the control of FNR function.

The oxygen sensor regulator FNR (fumarate nitrate reductase regulator) of Escherichia coli is known to be inactivated by O2 as the result of conversion of a [4Fe-4S] cluster of the protein into a [2Fe-2S] cluster. Further incubation with O2 causes loss of the [2Fe-2S] cluster and production of apoFNR. The reactions involved in cluster assembly and reductive activation of apoFNR isolated under anaerobic or aerobic conditions were studied in vivo and in vitro. In a gshA mutant of E. coli that was completely devoid of glutathione, the O2 tension for the regulatory switch for FNR-dependent gene regulation was decreased by a factor of 4-5 compared with the wild-type, suggesting a role for glutathione in FNR function. In isolated apoFNR, glutathione could be used as the reducing agent for HS- formation required for [4Fe-4S] assembly by cysteine desulfurase (NifS), and for the reduction of cysteine ligands of the FeS cluster in FNR. Air-inactivated FNR (apoFNR without FeS) could be reconstituted to [4Fe-4S].FNR by the same reaction as used for apoFNR isolated under anaerobic conditions. The in vivo effects of glutathione on FNR function and the role of glutathione in the formation of active [4Fe-4S].FNR in vitro suggest an important role for glutathione in the de novo assembly of FNR and in the reductive activation of air-oxidized FNR under anaerobic conditions.

Bacterial Proteins↗
PubMed2000

In vivo visualization of the neuromuscular junction before and after autologous nerve transfer in the rat.

In vivo visualization of the neuromuscular junction with epifluorescence imaging techniques has become a successful method of observing the ongoing process of re-occupation by regenerating motor axons of former post-synaptic sites after nerve injury. By using a light-integrating video camera for digital documentation, all parts of the neuromuscular junction can be visualized, as detailed as when documented with high-speed film, but with a minimum light intensity to prevent damage of neural or muscular structures. Results from comparisons of pre- and post-synaptic staining indicate a non-reoccupation rate up to 37 percent at a 55-day interval after nerve transfer, and up to 34 percent at a 66-day postoperative interval. Morphologic findings suggest that these high non-reoccupation rates are caused jointly by intramuscular missprouting, an insufficient intramuscular guidance apparatus, and intramuscular microneuroma formation at the insufficient neuromuscular junction.

Animals↗
PubMed2000

Soluble components of Utah Valley particulate pollution alter alveolar macrophage function in vivo and in vitro.

Water-soluble extracts of Utah Valley dust (UVD) have been found to cause inflammatory injury of the lung in both humans and rodents. The degree of lung damage found correlated with the metal content in the extracts. In the present study, extracts of a set of UVD PM(10) filters collected over a 3-yr span, varying in total metal content with yr 1 = yr 3 > yr 2, were used to assess effects on human alveolar macrophage (AM) function. The phagocytic activity and oxidative response of AM was investigated 24 h after segmental instillation of UVD, or after overnight in vitro culture of the extracts with AM. Using flow cytometry analysis, AM phagocytosis of fluorescently (FITC)-labeled Saccharomyces cerevisiae was inhibited following instillation of UVD1 (61%) but not by yr 2 and 3. Neither baseline oxidant activity nor phorbol ester-induced oxidant generation was affected by the dust extracts in vivo. Overnight culture of AM with UVD1 resulted in a significant decrease in the percentage of AM phagocytizing particles (30%), while no significant effect on this function was found with the other two extracts. Furthermore, only UVD1 caused an immediate oxidative response in AM, although both UVD1 and UVD3 inhibited oxidant activity in AM when the cells were incubated with the extracts overnight. The detrimental effects on AM host defenses could be due to apoptosis, which was evident in cells exposed to the UVD1 and to a much lesser extent with AM exposed to yr 2 and 3. The component(s) responsible for the toxic effects on AM in vitro were removed by pretreatment of the UVD extracts with a polycation chelating resin, chelex-100. However, since yr 1 and 3 are similar in their soluble metal content, but differ in their effects on AM phagocytosis, it is possible that the metals may not be the culprit in effects of particulate matter on AM host defense.

Adolescent↗
PubMed2000

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.

Animals↗
PubMed2000

Selected topics in the epidemiology of reproductive outcomes.

In summary, progress in research on reproductive epidemiology will depend on the increased use of biologic measurement, a focus on multiple outcomes, and an increased use of the couple or sexual network as the unit of study, with appropriate statistical methods for analysis of correlated data. The combination of these approaches holds great promise for research in the 21st century.

Biomarkers↗
PubMed2000

PCR bias in ecological analysis: a case study for quantitative Taq nuclease assays in analyses of microbial communities.

Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69-75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 10(4)-fold excess of phylogenetically similar DNA and of an up to 10(7)-fold excess of dissimilar DNA.

Bacteria↗
PubMed2000

Ultrastructural organization of recombinant Marburg virus nucleoprotein: comparison with Marburg virus inclusions.

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP) have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins.

Animals↗
PubMed2000

Impaired cognitive performance in drug free users of recreational ecstasy (MDMA)

OBJECTIVES: Ecstasy (3,4-methylenedioxymethamphetamine (MDMA) and related congerers: MDA, MDEA) is the name given to a group of popular recreational drugs. Animal data raise concern about neurotoxic effects of high doses of ecstasy on central serotonergic systems. The threshold dose for neurotoxicity in humans is not clear and serotonin is involved in several functions including cognition. The purpose of this study was to investigate cognitive performance in a group of typical recreational ecstasy users. METHODS: A comprehensive cognitive test battery was administered to 28 abstinent ecstasy users with concomitant use of cannabis only and to two equally sized matched groups of cannabis users and non-users. The sample consisted of ecstasy users with a typical recreational use pattern and did not include very heavy users. RESULTS: Ecstasy users were unimpaired in simple tests of attention (alertness). However, they performed worse than one or both control groups in the more complex tests of attention, in memory and learning tasks, and in tasks reflecting aspects of general intelligence. Heavier ecstasy and heavier cannabis use were associated with poorer performance in the group of ecstasy users. By contrast, the cannabis users did not differ significantly in their performance from the non-users. CONCLUSIONS: The present data raise concern that use of ecstasy possibly in conjunction with cannabis may lead to cognitive decline in otherwise healthy young people. Although the nature of the emerging cognitive disturbance is not yet clear, an impairment of working memory might be the common denominator underlying or contributing to declines of performance in various tasks. The cognitive disturbance is likely to be related to the well recognised neurotoxic potential of ecstasy. The data suggest that even typical recreational doses of ecstasy are sufficient to cause neurotoxicity in humans.

Adolescent↗
PubMed2000

Adhesion and pollution particle-induced oxidant generation is neither necessary nor sufficient for cytokine induction in human alveolar macrophages.

Adhesion of human monocytes (MOs) results in the rapid transcriptional activation of cytokine genes that are dependent on nuclear factor (NF)-kappaB. Several pathways leading to activation of NF-kappaB have been described, including those involving reactive oxygen intermediates (ROIs) and members of the mitogen-activated protein (MAP) kinase superfamily. To investigate the involvement of tyrosine phosphorylation (TP) and oxidant generation in interleukin (IL)-8 and GRO messenger RNA induction, MOs and human alveolar macrophages (AMs) were adhered to plastic or exposed to a particulate pollutant, residual oil fly ash (ROFA). Both stimuli caused rapid TP and ROI production in MOs and AMs. However, neither NF-kappaB translocation nor IL-8 gene induction occurred in adhered or ROFA-exposed AMs. Analysis of MAP kinase activation found phosphorylation of Jun amino-terminal kinase (JNK) and p38 in the AMs, but not of extracellular regulated kinase/MAP kinase (ERK/MAPK). AMs stimulated with lipopolysaccharide activated ERK/MAPK, in addition to JNK and p38, and showed translocation of NF-kappaB. In contrast to AMs, MO adhesion or exposure to ROFA particles in suspension rapidly activated p38, JNK, and ERK/MAPK, and activated NF-kappaB binding as well as IL-8 mRNA expression. Pretreatment with the tyrosine kinase inhibitors genistein or herbimycin A before adherence had no effect on transcriptional activation in MOs, whereas adherence and ROFA-induced oxidant generation was inhibited in both MOs and AMs. Taken together, these data indicate that NF-kappaB activation or generalized transcriptional activation of cytokine genes are independent of changes in oxidant stress imposed on phagocytes by adhesion. Furthermore, the data suggest that certain environmental responses in AMs may be uncoupled from activation of NF-kappaB.

Adolescent↗
PubMed2000

Maternal cigarette smoking during pregnancy and infant ponderal index at birth in the Swedish Medical Birth Register, 1991-1992.

OBJECTIVES: This study examined the effect of maternal smoking during pregnancy on infant body proportion. METHODS: The ponderal index, defined as birthweight divided by crown-heel length cubed, was examined in 207,607 infants from the Swedish Medical Birth Register for 1991 and 1992. RESULTS: Infant ponderal index was used as the outcome variable in an ordinary least squares continuous regression, which included early pregnancy smoking status, gestational age, and birthweight among the predictors. Ponderal index increased by 0.030 (+/- 0.0014) among infants of moderate smokers and by 0.040 (+/- 0.0017) among infants of heavy smokers, showing a dose response. CONCLUSIONS: Smoking differentially alters the trajectory of weight vs length growth in the fetus.

Adult↗
PubMed2000

Literacy promotion in primary care pediatrics: can we make a difference?

BACKGROUND: Reading skills are critical to children's success in school and the increasingly technologic workforce. Children from low-income families are at risk for home environments that fail to promote emergent literacy and for reading failure. A home environment that encourages learning and parents who are involved in their children's education are important factors in school achievement. OBJECTIVE: To evaluate the effects of a literacy promoting intervention delivered by pediatric providers as part of well-child care on parent attitudes and behaviors and on child language. DESIGN/METHODS: A multicultural group of 205 low-income families with 5- to 11-month-olds were prospectively enrolled, interviewed, and randomized to intervention (n = 106) or control (n = 99) groups. Families in the intervention group received developmentally appropriate children's books and educational materials and advice about sharing books with children, while those in the control group received no books or materials relevant to literacy. After an average of 3.4 well-child visits in both groups, 153 (75%) were reinterviewed and the children's receptive and expressive vocabulary was tested using a modified version of the MacArthur Communication and Development Inventory (Short Form). Parents were asked if their child understood (receptive vocabulary) or said (expressive vocabulary) each of 100 words, half of which were in the books given. Families were found to have a Child-Centered Literacy Orientation if they mentioned reading aloud as one of their child's favorite activities or as one of their own favorite joint activities or if they usually read together at bedtime. At follow-up toddlers were 18.4 months old on average. RESULTS: Intervention and control groups had similar literacy related characteristics at baseline. There was a 40% increase in Child-Centered Literacy Orientation among intervention families compared with 16% among controls. Intervention families read more with their toddlers (4.3 vs 3.8 days/week). Both receptive and expressive vocabulary scores were higher in older intervention toddlers (18-25 months old; n = 88), but not in younger intervention toddlers (13-17 months old; n = 62). This significant effect of the intervention on vocabulary scores in older toddlers was found for both the 50 words in the books and those not in the books. After parent education, foreign birth and language proficiency, and child age were statistically controlled, the intervention remained significantly associated with higher language outcomes in older toddlers. However, when reading aloud was added to the multivariate analysis, the influence of the intervention was no longer evident, suggesting the intervention's effect on child language was mediated through increased shared reading with these toddlers. CONCLUSION: This simple and inexpensive intervention, delivered as part of well-child care, changed parent attitudes toward the importance of reading with their infants and toddlers. These intervention parents and their children read more together and this was associated with enhanced language development in older toddlers in this diverse group of low-income families.

Books↗