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Biomedical subjects

E Meyer

Publications and source records attributed to E Meyer.

At least 55 records · Page 3Linked to original sources
PubMed2002

Functional maturation during bovine granulopoiesis.

Granulocytic precursor cells undergo morphologic changes in the nucleus and the cytoplasm during the process of granulopoiesis, which takes place in the bone marrow. These changes are associated with the development of stage-specific proteins necessary for the highly specialized roles of polymorphonuclear leukocytes in phagocytosis, bacterial killing, and in mediating the inflammatory process. The objective of the current study was to sequence the various events that occur upon functional development of granulocytic bone marrow cells in the bovine species. Cells were obtained from the bone marrow of clinically healthy cows and separated into different stages of maturation using density gradient centrifugation. Three cellular fractions were obtained that were enriched for either early immature, late immature or mature granulocytic cells. Functions and receptor expressions assessed in the three maturation stages were:Fc-IgG2 receptor and CD11b expression, phagocytosis of Escherichia coli, respiratory burst activity, and cellular myeloperoxidase activity. Immature cells expressed already Fc-IgG2 receptor and CD11b on their cytoplasma membrane. Phagocytic ability was acquired in the myelocytic stage, but only the more mature forms were readily capable of phagocytosis. Promyelocytes, myelocytes and metamyelocytes showed no respiratory burst activity. Only band and segmented cells produced reactive oxygen species. Myeloperoxidase was present at all stages of maturity. Thus, each of the maturation stages was characterized by a selective expression of one or more functions and receptors. Therefore, sequential biochemical maturation is postulated during bovine granulopoiesis.

Animals↗
PubMed2001

Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, alpha-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and alpha-hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia beta-glucuronidase for 1 h at 56 degrees C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.

Benzodiazepines↗
PubMed2001

Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer.

Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.

Apoptosis↗
PubMed2001

Hepatocellular expression of a dominant-negative mutant TGF-beta type II receptor accelerates chemically induced hepatocarcinogenesis.

The potent growth-inhibitory activity of cytokines of the transforming growth factor-beta (TGF-beta) superfamily and their widespread expression in epithelia suggest that they may play an important role in the maintenance of epithelial homeostasis. To analyse TGF-beta mediated tumor suppressor activity in the liver, we generated transgenic mice overexpressing a dominant negative type II TGF-beta receptor in hepatocytes under control of the regulatory elements of the human C-reactive protein gene promoter. Transgenic animals exhibited constitutive and liver-specific transgene expression. The functional inactivation of the TGF-beta signaling pathway in transgenic hepatocytes was shown by reduced TGF-beta induced inhibition of DNA synthesis in primary hepatocyte cultures. Liver morphology and spontaneous tumorigenesis were unchanged in transgenic mice suggesting that interruption of the signaling of all three isoforms of TGF-beta in hepatocytes does not disturb tissue homeostasis in the liver under physiological conditions. However, following initiation with the carcinogen diethylnitrosamine and tumor-promotion with phenobarbital transgenic mice exhibited a moderate albeit significant increase in the incidence, size and multiplicity of both preneoplastic tissue lesions in the liver and of hepatocellular carcinomas. These results give in vivo evidence for a tumor suppressor activity of the endogenous TGF-beta system in the liver during chemical hepatocarcinogenesis.

Animals↗
PubMed2001

Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis.

Apoptosis in response to cellular stress such as treatment with cytotoxic drugs is mediated by effector caspases (caspase-3) which can be activated by different initiator pathways. Here, we report on a cell type specific triggering of death receptor and/or mitochondrial pathways upon drug treatment. In type I cells (BJAB), both the receptor and the mitochondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-X(L) only partially inhibited apoptosis. Drug treatment induced formation of a FADD- and caspase-8-containing CD95 death-inducing signaling complex (DISC) in type I cells resulting in activation of caspase-8 as the most apical caspase. In contrast, in type II cells (Jurkat), apoptosis was predominantly controlled by mitochondria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect. In these cells, caspases including caspase-8 were activated by mitochondria-driven signaling events and no DISC was detected despite expression levels of CD95, FADD and caspase-8 proteins comparable to type I cells. Likewise, drug-induced CD95 aggregation was predominantly found in type I cells. Bid was cleaved prior to mitochondrial alterations in type I cells providing a molecular link between caspase-8 activation and mitochondrial perturbations, whereas in type II cells, Bid was cleaved downstream of mitochondria. Our findings of a cell type specific response to cytotoxic drugs have implications for the identification of molecular parameters for chemosensitivity or resistance in different tumor cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Sensitive gas chromatographic--mass spectrometric screening of acetylated benzodiazepines.

GC-MS screening conditions were developed for 15 low-dosed benzodiazepines, covering alprazolam, flunitrazepam, flurazepam, ketazolam, lorazepam and triazolam, and the corresponding metabolites alpha-hydroxyalprazolam, 4-hydroxyalprazolam; 7-aminoflunitrazepam, desmethylflunitrazepam, 7-aminodesmethylflunitrazepam; hydroxyethylflurazepam, N-desalkylflurazepam; oxazepam and alpha-hydroxytriazolam, respectively. Benzodiazepines are analyzed on a polydimethylsiloxane column in both the scan and the multiple ion monitoring modes using on-column injection to attain maximal sensitivity. The reactive compounds are acetylated with pyridine and acetic anhydride for 20 min. The derivatives are stable for at least 4 days. The relative standard deviation observed with standard compounds at the low nanogram-level ranged from 1.13 to 4.87% within-day and from 1.12 to 4.94% between-day. Unequivocal identification potential, high chromatographic resolution and sensitivity are combined with minimal thermal degradation. The presented screening conditions provide the basis for a unique routine screening method for low-dosed benzodiazepines with a broad polarity range.

Acetylation↗
PubMed2001

Separation of bovine bone marrow into maturation-related myeloid cell fractions.

A prerequisite for studies on bovine myeloid cells in relation to maturity is a reliable separation method, in order to obtain enriched and partially purified cell fractions of different maturation stages. Since current techniques for bovine bone marrow cell isolation fall short of this requirement, a technique for fractionating bovine bone marrow using a three-layer discontinuous Percoll gradient was developed. Three maturation-dependent myeloid cell fractions were obtained at specific densities, as maturation of cells is accompanied with a progressive density increase. Early immature myeloid cells, i.e. myeloblasts and promyelocytes, were found at a density of 1.060g/ml. Late immature myeloid cells, i.e. myelocytes and metamyelocytes, were retrieved at 1.080g/ml. Bands and segmented cells, representing the mature fraction, accumulated in the high-density pellet (>1.080g/ml). Myeloid cell populations were identified in each fraction by flow cytometry based on their forward and side scatter pattern. Confirmation was provided by light microscopy of flow cytometrically sorted myeloid populations, using morphological characteristics. The developed method provides a unique tool for studying maturation-dependent functions in bovine bone marrow.

Animals↗
PubMed2001

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Animals↗
PubMed2001

Respiratory burst activity of blood and milk neutrophils in dairy cows during different stages of lactation.

The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0.001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0.001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0.001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1.7, 2.6 and 2.4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN. the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.

Animals↗
PubMed2001

Recombinant maxi-K channels on transistor, a prototype of iono-electronic interfacing.

We report on the direct electrical interfacing of a recombinant ion channel to a field-effect transistor on a silicon chip. The ion current through activated maxi-K(Ca) channels in human embryonic kidney (HEK293) cells gives rise to an extracellular voltage between cell and chip that controls the electronic source-drain current. A comparison with patch-clamp recording shows that the channels at the cell/chip interface are fully functional and that they are significantly accumulated there. The direct coupling of potassium channels to a semiconductor on the level of an individual cell is the prototype for an iono-electronic interface of ligand-gated or G protein-coupled ion channels and the development of screening biosensors with many transfected cells on a chip with a large array of transistors.

Cell Line↗
PubMed2000

Sensorimotor organization in patients who have undergone hemispherectomy: a study with (15)O-water PET and somatosensory evoked potentials.

To identify cortical structures that subserve residual motor and sensory function in patients with congenital hemiparesis due to a porencephalic cyst, we examined, using [(15)O]H2O, PET and somatosensory evoked potentials (SEPs) in three patients with left-sided hemiparesis who had undergone hemispherectomy. Motor stimulation of the affected hand produced ipsilateral activation in the premotor area in all patients, the SMA in two patients, and SII in two patients. Vibrotactile stimulation resulted in activation of the ipsilateral SII in all subjects. Median nerve stimulation of the affected hand produced ipsilateral long-latency SEPs in fronto-centro-parietal areas, whereas stimulation of the non-affected hand produced normal early cortical potentials in the contralateral hemisphere. Our results suggest that residual function in the paretic hand is warranted through non-primary motor and sensory areas, and higher order associative areas in the intact hemisphere.

Adult↗
PubMed2000

Extraction and amplification of authentic DNA from ancient human remains.

A total of 36 ancient human remains from 12 individuals (three tooth/bone samples each) and one sample each of three individuals from the newest time was typed in a blind study using the amelogenin sex test. Prior to this molecular sex determination the sex of the individual was determined morphologically. The success rate of the amelogenin amplifications was >90%. For every individual an unambiguous molecular sex typing result was obtained. Furthermore, the morphological and molecular sex determinations were in accordance with each other, giving evidence for the authenticity and ancient origin of the polymerase chain reaction (PCR) amplifications.

Amelogenin↗
PubMed2000

Effect of isoproterenol and dexamethasone on the lipopolysaccharide induced expression of CD11b on bovine neutrophils.

The present experiments investigate the changes in expression of CD11b on bovine neutrophils and its modulation by isopropylnoradrenaline (IPN, isoproterenol), dexamethasone (DX), phenylephrine (alpha-agonist) and clenbuterol (beta-agonist). Both IPN and DX caused a dose-dependent inhibition of LPS-induced CD11b expression. A combination of IPN and DX elicited a synergistical decrease of the CD11b expression. Clenbuterol mimicked the effect of IPN, whereas phenylephrine did not. The effect of IPN and DX could at least partly be mediated through a decreased TNF-alpha production by monocytes since tumor necrosis factor-alpha (TNF-alpha) is shown to mediate a dose-dependent CD11b up-regulation. Stimulation of stress hormone receptors partly immuno-suppresses neutrophil functions by inhibition of CD11b expression on the neutrophil surface upon LPS stimulation. This inhibition is probably related to a decrease in TNF-alpha production. A similar mechanism of immuno-suppression could contribute to the higher susceptibility of cattle to Gram-negative bacterial infections of the udder and lung during periods of stress.

Animals↗
PubMed2000

Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression.

Protein or RNA synthesis inhibitors are known to sensitize some resistant cells for death receptor-induced apoptosis. However, the molecular mechanism(s) involved in sensitization have not yet been defined exactly. Here, we report that metabolic inhibitors such as cycloheximide (CHX) or actinomycin D (ActD) sensitize for CD95-induced apoptosis by strongly down-regulating FLIP and RIP expression. Metabolic labeling studies revealed that CHX or ActD inhibited protein or RNA synthesis at concentrations required for sensitization. In contrast to Fas-associated death domain (FADD) or caspase-8, FADD-like interleukin 1-converting enzyme-inhibitory protein (FLIP) and RIP protein levels rapidly decreased upon treatment with CHX or ActD, indicating that both molecules have a high turnover rate. Selective down-regulation of FLIP expression by FLIP antisense oligonucleotides sensitized for CD95-induced apoptosis. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing the recruitment of caspase-8 to the DISC and caspase-8 activation. CHX- or ActD-mediated sensitization to CD95-induced apoptosis was predominantly found in type I cells in which FADD and caspase-8 are recruited to CD95 upon stimulation but not in type II cells in which no DISC formation is detected. Pretreatment with CHX or ActD sensitized for subsequent CD95 stimulation compared with cells without pretreatment. CHX or ActD also reduced XIAP expression and similarly sensitized for tumor necrosis factor-related apoptosis-inducing ligand- or tumor necrosis factor-alpha-induced apoptosis. Because blockade of death receptor triggering by FLIP overexpression has recently been implicated in tumorigenesis and treatment resistance in vivo, strategies to inhibit FLIP expression, e.g., by metabolic inhibitors, may prove to be a useful complementary tool for the treatment of cancer.

Apoptosis↗
PubMed2000

The structure of an insect chymotrypsin.

The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme.

Amino Acid Sequence↗
PubMed2000

Translating biomolecular recognition into nanomechanics.

We report the specific transduction, via surface stress changes, of DNA hybridization and receptor-ligand binding into a direct nanomechanical response of microfabricated cantilevers. Cantilevers in an array were functionalized with a selection of biomolecules. The differential deflection of the cantilevers was found to provide a true molecular recognition signal despite large nonspecific responses of individual cantilevers. Hybridization of complementary oligonucleotides shows that a single base mismatch between two 12-mer oligonucleotides is clearly detectable. Similar experiments on protein A-immunoglobulin interactions demonstrate the wide-ranging applicability of nanomechanical transduction to detect biomolecular recognition.

Animals↗
PubMed2000

alpha7 receptor-selective agonists and modes of alpha7 receptor activation.

The alpha7-selective agonists 3-(2, 4-dimethoxybenzylidene)-anabaseine (GTS-21), also known as DMXB, and 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) produce a variety of behavioral and cytoprotective effects that may be related to the activation of either large transient currents at high concentrations or small sustained currents at lower agonist concentrations. We are using acutely dissociated hypothalamic neurons, which express a central nervous system (CNS) alpha7-type receptor, to test a model for the concentration-dependent desensitization of alpha7-mediated responses. Our results confirm that 4OH-GTS-21 is a potent activator of neuronal alpha7 nicotinic-acetylcholine receptor. The rapid application of agonist leads to a brief period of maximal receptor-activation followed by desensitization. Rise rates, decay rates, and the degree to which current was desensitized were all concentration-dependent. Following the initial peak response to a 300-microM 4OH-GTS-21 application, current is reduced to baseline values within about 100 ms. Application of 30 microM 4OH-GTS-21 produced both a transient peak current and a sustained current that decayed only slowly after the removal of agonist. In the case of a 300-microM 4OH-GTS-21 application, after agonist was removed, we saw a rebound response up to the level of the 30-microM sustained current. The data, therefore, suggest that a sufficient level of agonist occupation can be retained on the receptor to promote activation for up to several hundred milliseconds.

Anabasine↗