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Biomedical subjects

T Meyer

Publications and source records attributed to T Meyer.

At least 379 records · Page 21Linked to original sources
PubMed1989

A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-tumor activity.

Analogues of staurosporine were synthesized and their ability to inhibit protein kinases was examined. Staurosporine is a potent but non-selective inhibitor of in vitro protein kinase C(PKC) activity (IC50 6.0 nM). The derivative CGP 41 251 had reduced PKC activity with an IC50 of 50 nM but showed a high degree of selectivity when assayed for inhibition of cyclic AMP-dependent protein kinase (IC50 2.4 microM), S6 kinase (IC50 5.0 microM) and tyrosine-kinase-specific activity of epidermal growth factor receptor (IC50 3.0 microM). Staurosporine and CGP 41 251 exerted growth inhibition in the human bladder carcinoma line T-24, human promyelocytic leukemia line HL-60 and bovine corneal endothelial cells at concentrations which correlated well with in vitro PKC inhibition. In addition, both compounds inhibited the release of H2O2 from human monocytes pre-treated with 12-O-tetradecanoyl-phorbol-13-acetate at non-toxic concentrations. In vivo anti-tumor activity was examined in T-24 human bladder carcinoma xenografts in athymic nude mice. Tumor growth inhibition tests revealed significant anti-tumor activity (2p less than 0.001) at 1/10 of the maximum tolerated doses for both compounds. By contrast, a closely related derivative of staurosporine (CGP 42 700) was inactive at concentrations of over 100 microM in all in vitro enzyme and anti-proliferative assays as well as in animal tumor models. Our data suggest an association between PKC inhibition and anti-proliferative and anti-tumor activity.

Alkaloids↗
PubMed1989

A rapid phospholipase A2 bioassay using 14C-oleate-labelled E. coli bacterias.

Two methods of phospholipase A2 determination using 14C-labelled E. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved 14C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A2 regarding pH and Ca2+ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A2 levels, but that the filter method is superior in terms of sensitivity and workload.

Acute Disease↗
PubMed1989

Characterization of phospholipase A2 activity in aspirates of human pancreatic pseudocysts after isolation by reversed-phase high performance liquid chromatography.

Phospholipase A (PLA) is able to attack membrane phospholipids and thereby plays a putative role in the pathogenesis of pancreatic pseudocysts. We looked for PLA2-like activity in aspirates from human pancreatic pseudocysts. In material originating from one cyst which occurred shortly after an acute pancreatitis attack, hydrolyzing enzymatic activity measured by a sensitive bioassay system for PLA2 activity was found without prior trypsin activation (67 x 10(3) U/min/100 microliters). A biochemical characterization of this hydrolyzing enzymatic activity was provided after resolution of the respective proteins contained in the cyst fluid by HPLC. High hydrolyzing activities were found in correspondence to one specific, early eluting peak. The purified enzyme had pH optima at 3.5 and 6. Addition of EDTA (5 mM) to the test system abolished the enzymatic activity which mirrored the requirement for calcium ions. The activity was optimal at calcium concentrations ranging from 1-2 mM. Higher calcium concentrations reduced the enzymatic activity. The enzyme showed high heat stability. SDS-gel analysis of the peak showed one single band with a molecular weight of about 20,000 Daltons. Our findings demonstrate the possibility of activated, PLA-like activity in human pancreatic pseudocyst fluid. We speculate that an inappropriate activation of this enzyme in peri- or intrapancreatic "fluid collections" could account for pseudocyst formation after an acute pancreatitis attack.

Calcium↗
PubMed1989

Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs.

Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.

Animals↗
PubMed1989

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa.

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.

Amino Acid Sequence↗
PubMed1989

Genes coding for Shiga-like toxin and heat-stabile enterotoxin in porcine strains of Escherichia coli.

Besides diarrheagenic enterotoxigenic Escherichia coli (ETEC) that produce classical heat stable and/or heat labile enterotoxins (STs, LTs) and the class of Shiga-like toxin-producing entero-hemorrhagic E. coli (EHEC), a new category of E. coli is defined sharing similarities with ETEC and EHEC. DNA hybridization studies indicate that some E. coli serovars from porcine origin harbor genes encoding cytotonic ST and cytotoxic Shiga-like toxin. The presence of two potent toxins might contribute to the virulence of such strains and should be taken into consideration when bio-assays are performed.

Animals↗
PubMed1989

Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction.

By using a single synthetic oligonucleotide primer pair in the polymerase chain reaction, we amplified specific Shiga-like-toxin (SLT) gene segments from DNAs of 20 clinical Escherichia coli isolates, irrespective of whether they produce SLT-I, SLT-II, or heretofore uncategorized SLTs. These segments were not detectable in any of 20 nontoxigenic E. coli strains. The primers deduced from a conserved region among SLT genes are so-called degenerate-sequence primers; i.e., they contain intentionally introduced sequence ambiguities to overcome minor sequence variations within different SLT genes. In direct gel hybridization with genomic DNA, both primers recognized SLT-I and SLT-II DNA sequences. Amplified sequences of target DNA obtained by polymerase chain reaction were visualized after gel electrophoresis by ethidium bromide staining, and definitive identification of the amplification product as an SLT gene segment was achieved by hybridization to SLT-I- and SLT-II-specific 20-base oligonucleotide probes complementary to a portion of the amplified sequences but not to the primers. The detecting oligonucleotide probes shared only 30% base homology and were shown to recognize specifically SLT-I or SLT-II sequences within genomic DNA. Moreover, they were used to distinguish whether the amplified sequence originated from SLT-I or SLT-II genes. The PCR system with the primers described here is a powerful technique to amplify SLT sequences in E. coli strains that produce serologically distinct SLTs and will facilitate identification of these pathogens, particularly among a multitude of nonpathogenic E. coli strains.

Animals↗
PubMed1989

Evaluation of oligonucleotide probes for identification of shiga-like-toxin-producing Escherichia coli.

Four synthetic oligonucleotide probes representing different regions of the Shiga-like toxin I (SLT-I) structural genes and one oligonucleotide derived from the SLT-II gene of Escherichia coli serotype O157:H7 strain 933 were examined for the identification of E. coli strains that produce cytotoxins for Vero or HeLa cells. E. coli strains that synthesize SLT-I alone or O157:H7 isolates that coexpress SLT-I and SLT-II hybridized with all four probes that were complementary to the SLT-I genes, suggesting that they have toxin genes with great homology in all the regions examined. In colony hybridization tests, these oligonucleotide probes did not react with E. coli strains that were nontoxigenic for Vero cells or that produced cytotoxins belonging to the SLT-II family. The probe derived from the slt-IIA gene distinguished E. coli strains that produced SLT-II alone from SLT-I-producing strains and hybridized to all E. coli O157:H7 strains that produced both SLT-I and SLT-II. Using two of these oligonucleotide probes that were complementary to slt-IA or slt-IIA sequences, we identified 50 of 52 cytotoxin-producing strains, whereas none of 416 nontoxigenic E. coli strains was reactive. The colony blot hybridization with the oligonucleotide probes described here can serve as a specific and sensitive test with potential diagnostic value.

Animals↗
PubMed1989

[Late results with hybrid dentures in low-income patients. An investigation at the Cantonal Public Dental Clinic of Zurich].

72 patients who had received overdentures at the Cantonal Public Dental Clinic of Zurich during a period of ten years (1970-1980) were called for re-examination. Results obtained were evaluated according to an analogous survey which had been conducted 12 years previously at the same public dental clinic (Wiesmann 1975): The test subjects of both studies belonged exclusively to the lower-income population and treatment was performed by post-graduate dentists employed at the clinic. A comparison with Wiesmann's findings demonstrated a definite improvement in the general oral health conditions of these wearers of hybrid prosthesis. Since 1972 a recall system has been successively introduced which, for the greatest part, offers the most probable explanation for these positive findings. Each patient was summoned for at least one dental examination per year, thereupon presenting better results in regard to average plaque index, carious lesions, degree of gingival inflammation, probing depth and functionality of the prosthetic appliance, a marked difference to those patients who did not participate in such a dental maintenance program. Therefore, the regular motivation and supervision of patients is mandatory for long-term success of overdenture treatment.

Aged↗
PubMed1988

Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate.

The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.

Animals↗
PubMed1988

Comparison of the electrical properties of neocortical neurones in slices in vitro and in the anaesthetized rat.

Because neurones in isolated brain slices are abnormal in several respects, we have compared the electrical properties of neocortical neurones from the anaesthetized rat with neurones from slices of the same area of neocortex in vitro. Resting potentials were not significantly different but there was a small increase in mean spike amplitude and threshold in slices. The most striking observation was a doubling of the mean apparent input resistance (Rin) of neurones in slices (36 M omega cf. 18 M omega in vivo). To assess the extent to which the loss of surface membrane of neurones in slices might contribute to the raised Rin, we estimated the membrane capacity of neurones in the two samples from calculations of the membrane time constants. The mean membrane capacity was significantly lower in neurones from slices, being 2/3 that of the in vivo sample, showing that recordings were indeed mostly from smaller cells in the slices. Possible causes of the difference in mean input resistance not attributable to neuronal size are also discussed.

Action Potentials↗
PubMed1988

Particle counting by fluorescence correlation spectroscopy. Simultaneous measurement of aggregation and diffusion of molecules in solutions and in membranes.

A method for simultaneous determination of molar weights (M) and lateral diffusion constants (D) of particles in three- and two-dimensional systems is described. Spontaneous concentration fluctuations in space and time are analyzed, by monitoring fluctuations in the fluorescence from fluorescein-labeled molecules (1 dye/molecule is sufficient), excited by a rotating laser spot. For particles in solution, M values are determined over the range of 3 x 10(2) to 3 x 10(11) daltons, and D values can be determined from approximately 10(-7) to 10(-10) cm2/s. The time for a determination is approximately 1 min. Aggregation can be followed by changes of either M or D. This method is used to study the calcium dependence of vesicle aggregation or fusion, and the time course of aggregate formation of porin (an Escherichia Coli outer membrane protein) in lipid monolayers. Essential parameters for the development of the method are described. Equations to estimate the signal-to-noise ratios and to find the optimal free parameters for a specific application are derived. The theoretical predictions for the correlation function of the signal and for the signal-to-noise ratio are compared with observed values.

Chemical Phenomena↗
PubMed1988

Molecular model for receptor-stimulated calcium spiking.

Many cells exhibit periodic transient increases in cytosolic calcium levels rather than a sustained rise when stimulated by a hormone or growth factor. We propose here a molecular model that accounts for periodic calcium spiking induced by a constant stimulus. Four elements give rise to repetitive calcium transients: cooperativity and positive feedback between a pair of reciprocally coupled (crosscoupled) messengers, followed by deactivation and then by reactivation. The crosscoupled messengers in our model are inositol 1,4,5-trisphosphate (InsP3) and cytosolic calcium ions. The opening of calcium channels in the endoplasmic reticulum by the binding of multiple molecules of InsP3 provides the required cooperativity. The stimulation of receptor-activated phospholipase C by released calcium ions leads to positive feedback. InsP3 is destroyed by a phosphatase, and calcium ion is pumped back into the endoplasmic reticulum. These processes generate bistability: the cytosolic calcium concentration abruptly increases from a basal level to a stimulated level at a threshold degree of activation of phospholipase C. Spiking further requires slow deactivation and subsequent reactivation. In our model, mitochondrial sequestration of calcium ion prevents the cytosolic level from increasing above several micromolar and enables the system to return to the basal state. When the endoplasmic reticulum calcium store is refilled to a critical level by the Ca2+-ATPase pump, cooperative positive feedback between the InsP3-gated channel and phospholipase C begins again to give the next calcium spike. The time required for the calcium level in the endoplasmic reticulum to reach a threshold sets the interval between spikes. The amplitude, shape, and period of calcium spikes calculated for this model are like those observed experimentally.

Calcium↗
PubMed1988

Identification of rat pancreatic secretory proteins after separation by high-performance liquid chromatography.

In the present study an improved method of reversed-phase high-performance liquid chromatography (HPLC) for separation of rat pancreatic juice proteins is introduced. Aliquots of pancreatic juice were saved from conscious rats during basal secretion. The secretory proteins were separated on a wide-pore silica column by use of a multistep acetonitrile/water gradient. Up to 14 individual peaks could be separated by one run. Molecular weight analysis by sodium dodecyl sulfate (SDS)-gels allowed identification of peaks representing amylase, lipase, procarboxypeptidases, proelastase, chymotrypsinogen, and trypsinogen. Injection of pure rat amylase increased one specific peak which was assumed to represent amylase in the juice profile. Small amounts of residual enzymatic activities were measured for amylase, trypsin, and chymotrypsin in material of certain peaks. Activities of lipase, ribonuclease, and carboxypeptidases were not found, which reflected degradation of these enzymes by the separation procedure. High activities of phospholipase A2 were detected in one specific, early-eluting peak. Reversed-phase HPLC offers precise, reproducible, and rapid separation of the major proteins of rat pancreatic juice.

Animals↗
PubMed1988

ADP-ribosyl proteins formed by pertussis toxin are specifically cleaved by mercury ions.

Various types of ADP-ribosyl protein conjugates were synthesized and their chemical stability was compared with that of cysteine-linked ADP-ribosyl groups as formed by incubation of transducin or Gi/Go proteins with NAD and pertussis toxin. Treatment with 0.1 mM HgCl2 specifically cleaved the cysteine-linked conjugates. This may provide a tool for the quantitation of modified Gi/Go proteins as well as of other acceptors modified by ADP-ribose at cysteine residues in the presence of other ADP-ribosyl proteins.

Adenosine Diphosphate Ribose↗