Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.

Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants.

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid-located ferrochelatase (FeChII). A 1700-bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity-dependent formation of necrotic leaf lesions. In comparison with wild-type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.

Chronic myelogenous leukemia blast cell proliferation is inhibited by peptides that disrupt Grb2-SoS complexes.

Chronic myelogenous leukemia (CML) is commonly characterized by the presence of the p210(Bcr-Abl) oncoprotein. Many downstream effectors of Bcr-Abl have been described, including activation of the Grb2-SoS-Ras-MAP kinase (Erk) pathway. The precise contributions of these signal-transduction proteins in CML blast cells in human patients are not yet well defined. To gain further insight into the importance of Grb2 for CML, peptides that disrupt Grb2-SoS complexes were tested. These high-affinity Grb2-binding peptides (HAGBPs) can autonomously shuttle into cells and function by binding to the N-terminal SH3 domain of Grb2. The HAGBPs were analyzed for their effects on Bcr-Abl-expressing cell lines and freshly isolated CML blast cells from patients. They induced a dramatic decrease in the proliferation of CML cell lines. This was not observed with point-mutated control peptides with abolished Grb2SH3(N) binding. As expected, Grb2-SoS complexes were greatly diminished in the HAGBP-treated cells, and MAP kinase activity was significantly reduced as determined by an activation-specific phospho-MAPK antibody. Furthermore, cell fractions that are enriched for blast cells from CML patients with active disease were also incubated with the Grb2 blocker peptides. The HAGBPs led to a significant proliferation reduction of these cells in the majority of the isolates, but not in all patients' cells. These results show that, in addition to the direct targeting of Bcr-Abl, selective inhibition of Grb2 protein complexes may be a therapeutic option for a significant number of CML patients.

The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif.

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

Functional tethered lipid bilayers.

Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support. Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support. An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite. And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates. All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques. For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer. Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step. Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e. the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively. The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes. The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies. The results obtained for reconstituted H(+)-ATPase from chloroplasts and E. coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.

Initial experience in a community hospital with sentinel lymph node mapping and biopsy for evaluation of axillary lymph node status in palpable invasive breast cancer.

BACKGROUND AND OBJECTIVES: To determine the sentinel node detection rate and the accuracy with which the sentinel node histology reflects that of the axilla in a series of patients with palpable invasive breast cancer. METHODS: Forty-four patients with clinically node-negative palpable invasive T1 or T2 breast tumors underwent sentinel node biopsy using isosulfan blue dye, followed immediately by either local excision of the primary lesion with standard axillary lymph node dissection or modified radical mastectomy. All surgeries were performed at Northwest Hospital, Seattle, Washington, between January 1996 and October 1997. RESULTS: The sentinel node was successfully identified in 73% of the patients (32/44). The frequency of sentinel node detection was greater for tumors in the outer quadrants than the inner quadrants (z-test, P < 0.001). Of the 32 patients in whom a sentinel node was identified, 10 (31%) had histologically positive sentinel nodes: 5 (16%) by frozen section, 2 additional patients (6%) after permanent hematoxalin-eosin (H&E) stained sections, and the remaining 3 (9%) after immunohistochemical stains for cytokeratins when the FS and permanent H&E-stained sections were benign. Twenty patients had benign axilla. The sentinel node was falsely negative in 2 patients, yielding an accuracy of 93.8%, sensitivity of 83.3%, and negative predictive value of 91%. CONCLUSIONS: Lymphatic mapping is technically feasible for patients with small (T1 or T2) palpable invasive breast tumors. The sentinel node can be reliably identified in the majority of these patients, and its histology reflects that of the axilla with a high degree of accuracy. Immunohistochemical stains and permanent H&E-stained sections of the sentinel node increased the test's ability to correctly identify axillary metastases. Improving this sensitivity remains a primary goal, however, if benign sentinel node histology is to be used as a criterion to preclude axillary dissection.

Incorporation of the acetylcholine receptor dimer from Torpedo californica in a peptide supported lipid membrane investigated by surface plasmon and fluorescence spectroscopy.

The dimer species (M(r) 580,000) of the nicotinic acetylcholine receptor, isolated from the electric organ of Torpedo californica, was incorporated into a thiopeptide supported lipid bilayer. The incorporation was achieved by fusion of liposomes with reconstituted receptor onto a gold-supported thiopeptide lipid monolayer. Surface plasmon resonance spectroscopy (SPS) was used to monitor in real time the fusion process as well as the specific binding of the antagonist alpha-bungarotoxin. A recently developed extension of SPS offering enhanced sensitivity and specificity, surface plasmon fluorescence spectroscopy (SPFS), was then used to monitor subsequent binding of the monoclonal WF6 and polyclonal antibody, respectively. The latter was fluorescence labeled with Cy5. The different binding assays indicate the successful incorporation of the receptor in the lipid bilayer.

An interaction study with cimetidine and the new angiotensin II antagonist valsartan.

OBJECTIVE: This was a randomised, open, three-way crossover study in 12 healthy male volunteers to determine the effect of a single oral dose of cimetidine on the pharmacokinetics of a single oral dose of the angiotensin II receptor antagonist valsartan and vice versa. The volunteers received either valsartan alone (160 mg), or cimetidine alone (800 mg), or valsartan 1 h after cimetidine. The study was designed primarily to detect a possible influence of cimetidine on the rate and extent of absorption of valsartan. METHODS: Plasma concentrations of valsartan and cimetidine, measured by means of high-performance liquid chromatography, were used to calculate pharmacokinetic parameters. The rate of absorption of valsartan and the fraction of the dose absorbed and systemically available after oral administration were calculated using data from an i.v. study with valsartan in healthy young volunteers. RESULTS: The pharmacokinetics of cimetidine area under curve (AUC0-48 h), maximum concentration (Cmax), time to reach Cmax(tmax) and apparent terminal plasma half-life (t1/2) was not changed by co-administration of valsartan. For valsartan, the AUC0-48 h increased by 7% and the Cmax by 51% (ratio of geometric means) with co-administration of cimetidine. The higher value for Cmax was attributed to the initial increase in the rate of absorption of valsartan: ka was increased 2.7-fold and another indicator for the rate of absorption, Cmax/tmax, 2.2-fold. This effect was ascribed to inhibition of acid secretion by cimetidine, which leads to a higher gastric pH, thereby increasing the solubility of valsartan; the t1/2 of valsartan was not changed. After valsartan alone, 19% of the dose was absorbed, 23% with co-administration of cimetidine. It was estimated that only 2.2% of the possible change in AUC might be missed by giving a single high dose of cimetidine instead of multiple doses, with the aim to optimally inhibit formation of the inactive metabolite of valsartan. Cimetidine-related changes in the rate of elimination of valsartan were not anticipated, since the clearance from plasma occurs mainly by biliary excretion of unchanged valsartan; metabolism and renal excretion are only minor contributors. Therefore, even in the clinically relevant situation with multiple doses of valsartan and cimetidine, notable changes in the pharmacokinetics of valsartan, except for an increase in Cmax, are not to be expected. This increase in Cmax appears to be of no clinical significance. Valsartan alone and in combination with cimetidine was well tolerated by healthy subjects.

Continuous and direct infusion of drug solutions in the brain of awake animals: implementation, strengths and pitfalls.

One of the best strategies for understanding an animal's behavior is to study the function of the brain by experimentally modifying brain chemistry temporarily or on a long-term basis. This can be achieved by direct manipulation of neurochemistry of a targeted brain area with various drugs whose in vitro specificity and sensitivity are known. We assume that an animal's behavior is primarily controlled by the integrated performance of neural networks, rather than the action of a "superstar" single neuron which has narrowly tuned selectivity, in a specified brain region. Therefore, the former must be regulated by a large number of combinations of various transmitter/modulator receptors, hormones, growth factors, and other biochemically identifiable and yet unidentified substances. Under certain conditions, the activation of receptor-bound second messenger systems is thought to cause the enhanced expression of particular genes. Given the wide possibilities in manipulating brain chemistry, which may otherwise result in a variety of consequences, it is crucial to have a dependable means of sustaining the steady-state action of a drug for a sufficiently long time period at a targeted area in the brain of behaving animals. In most cases the continuous application of a drug is necessary to counteract its secondary mitigating effect, which is set in action through negative feedback loops and which in effect reduces the primary action of the drug in use. We have developed a technique to answer this need, using the Alzet osmotic minipump as the source of the continuous infusion force. A drug solution is continuously and directly infused, guided through a chronically implanted cannula, into a targeted area in the brain of behaving animals. The consequences of such an infusion are assessed, during as well as after the infusion, using various types of measurements in behavior, biochemistry, neurophysiology, pharmacology and morphology. The method has been successfully applied, for example, to the study of developmentally regulated neural plasticity in cat visual cortex. A few preconditions should be satisfied for the method to be properly applied to the brains of live animals. Those are: (1) manufacturing a suitable guide system, i.e., cannula-minipump assembly, for the infusion solution; (2) stereotaxic implantation of a cannula-minipump assembly into a selected brain region; and (3) estimating the concentration gradient of the continuously infused solution. This is crucial to assess the specificity and sensitivity of a drug for its assumed effects in vivo.

Clinical pharmacology of the new COMT inhibitor CGP 28,014.

CGP 28,014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28,014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28,014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28,014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28,014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.

A gas chromatographic/mass spectrometric assay for prenylamine suitable for pharmacokinetic studies of the racemate and the enantiomers.

A sensitive assay for prenylamine and dideuteroprenylamine (racemic or pseudo-racemate) has been developed and used in human pharmacokinetic studies. Plasma levels of prenylamine could be measured up to 50 h after a single oral therapeutic dose. The extracted drug was derivatized with pentafluoropropionic anhydride in acetonitrile. The dried samples were reconstituted in decane; an aliquot was injected into a fused-silica capillary in a cooled on-column injector. The base peaks in the electron impact mass spectra of the compounds--derived by loss of a benzyl radical--at m/z 384, 386 and 390 were measured for prenylamine, (D2)-prenylamine and the internal standard hexahydroprenylamine, respectively. The sensitivity of this assay--limit of detection 0.2 ng ml-1 plasma with a signal-to-noise ratio of 5:1--allowed measurement of the kinetics of the racemate and of both stereoisomers for the first time. In man, the (+)-isomer was eliminated considerably faster than the (-)-prenylamine; the area under the plasma concentration time curve (AUC) of the (+)-isomer was only about 1/4 of the AUC of (-)-prenylamine.

Urinary excretion of CGS 5,649 and its conjugated glucuronide and sulfate in man.

Experiments in animals have indicated that CGS 5,649 B [6-(2-isopropylaminopropyl)-3-pyridinol fumarate] might enhance memory and learning processes and might be a valuable drug for treatment of impairment of vigilance and mental performance in the elderly. CGS 5,649 (I) is extensively metabolized. Therefore, we investigated the excretion of the drug and its conjugates (I-SULF,I-GLUC) into urine after oral administration of 1200 mg of the fumarate salt of I to one healthy male volunteer. Three HPLC methods were developed to analyze the three compounds in urine: a. Solid-phase extraction and UV-measurement at 280 nm; b. Dansylation followed by fluorometric detection (lambda ex 340, lambda em 525 nm); c. Direct injection of diluted urine, gradient system with ion-pair reagent and UV-detection at 280 nm. The conjugates were measured after enzymatic hydrolysis with glucuronidase/sulfatase and sulfatase. I-GLUC was also measured directly since a synthetic sample had become available. Results from the different analytical methods showed agreement: about 100% of the administered dose was excreted within 24 h; the relative amounts of I, I-SULF and I-GLUC were about 1:2:2; after oral administration, CGS 5,649 B is rapidly and completely absorbed; it is eliminated from plasma by conjugation and direct excretion into urine.

Pharmacokinetics of prenylamine racemate and enantiomers in man.

Pharmacokinetics of racemic prenylamine were investigated in 6 healthy volunteers. Plasma levels were determined by gas chromatography/mass spectrometry. Concentration-time profiles were analyzed both by compartment-dependent and compartment-independent pharmacokinetic models. Terminal elimination half-life was 14.1 h (SD: 6.9 h). The apparent total clearance was 5.8 l/min. Mean residence time of racemic prenylamine was found to be 14.7 h (SD: 3.8 h). The relative bioavailability of prenylamine (Segontin 100) was 82.2% (SD: 9.9%) determined in six healthy volunteers. The volunteers received simultaneously the film tablet and 100 mg racemic dideuteroprenylamine as an aqueous solution of the lactate. This procedure is known to exclude intraindividual changes in absorption, first-pass metabolism or volume of distribution that might occur on sequential administration. The absolute bioavailability was estimated to be in the order of 15%. In a pilot study the pharmacokinetics of the enantiomers were investigated in 2 healthy volunteers. S-(+)-prenylamine was eliminated considerably faster from plasma than R-(-)-prenylamine suggesting a stereoselective metabolism. The AUC of the (+)-enantiomer was 20% of that of the R-(-)-prenylamine.

Does conditional discrimination learning by pigeons necessarily involve hierarchical relationships?

Four experiments demonstrate that when putative conditional and discriminative cues are presented simultaneously in the single reversal procedure, it is not possible to ascribe a uniquely conditional or uniquely discriminative function to either of the cues. In Experiment 1, pigeons were trained to respond to a blue key and not to a red key while the houselight was on; then in a different session they learned the reversal of this discrimination with the houselight off (single reversal). Separate groups were tested for color generalization with houselight conditions alternating in blocks of trials or for houselight intensity generalization with blue and red key colors alternating in blocks of trials. Both test procedures revealed a conditional relationship between houselight and key color conditions. Experiment 2 produced the same result following training in which the key colors were held constant across training sessions while the houselight and no houselight conditions varied within sessions. In Experiment 3, separate groups were trained with the two procedures but were tested with randomly ordered combinations of key colors and houselight intensities. The two groups yielded indistinguishable bidimensional generalization gradients with peaks at both previously reinforced stimulus combinations. In Experiment 4 the subjects were switched from one of these training procedures to the other with no decrement in their discriminative performance. We conclude that for successive discriminations between conditional- and discriminative-stimulus combinations, the notion of a hierarchical relation between conditional and discriminative stimuli must be extended to include a symmetrical relationship or the notion should be abandoned altogether.

High-performance liquid chromatographic determination of a new pyrazoloquinoline type benzodiazepine receptor antagonist and its hydroxy metabolite.

A rapid high-performance liquid chromatographic (HPLC) method for the determination of the novel benzodiazepine receptor antagonist 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one (CGS 8216) and its hydroxy metabolite is described. The method involves a solid-phase extraction with C18 disposable columns and quantification by HPLC with UV-detection. In plasma 20 ng/ml of the antagonist and 10 ng/ml of metabolite can be measured with a coefficient of variation of about 10%. The high sensitivity and selectivity of the assay makes it suitable for use in pharmacokinetic studies.

Diagnosis of the Beckwith-Wiedemann syndrome in the second trimester of pregnancy. A case report.

The diagnosis of Beckwith-Wiedemann syndrome was made in a 580-g, stillborn infant with an omphalocele. This case reemphasizes the importance of complete postmortem examinations. Appropriate counseling is dependent on an accurate diagnosis.

The genetic polymorphism of sparteine metabolism.

The formation of the two major metabolites of the antiarrhythmic and oxytocic drug sparteine (2- and 5-dehydrosparteine) exhibits a genetic polymorphism. Two phenotypes, extensive (EM) and poor metabolizers (PM) are observed in the population. The frequency of the PM phenotype in various populations (Caucasian and Japanese) ranges from 2.3 to 9%. The metabolism of sparteine is determined by two allelic genes at a single gene locus. PM subjects are homozygous for an autosomal recessive gene. The metabolism of sparteine is predominantly under genetic control as treatment with drugs such as antipyrine and rifampicin known to induce oxidative drug metabolism elicited only marginal changes in sparteine metabolism. The formation of 2-dehydrosparteine in human liver microsomes from EM and PM subjects showed a more than 40-fold difference in Km between EM and PM subjects. However, Vmax-values were almost identical in both groups. These data indicate that the basis of the differences in oxidative capacity between EM and PM subjects is more likely to be due to a variant isozyme with defective catalytic properties than to a decreased amount of the isozyme.

Fibrinopeptide A after strenuous physical exercise at high altitude.

To examine hemostasis after physical exercise at altitudes easily accessible to tourists by public transport, 20 young male volunteers were exposed to 3,457 m above sea level. Ten of them were subjected to an exhaustive exercise for about 8 min on a bicycle ergometer. The preexercise samples (n = 20) taken 1 h after arrival showed no significant alteration of coagulation compared with control values at 600 m. After the exercise the clotting times (P less than 0.001) and euglobulin lysis times (P less than 0.001) were shortened, whereas factor VIII activity (P less than 0.001) was elevated. There was, however, no significant difference in fibrinopeptide A levels between the exercise and the control group. Ethanol gelation test remained negative. We found no rise in fibrin(ogen) degradation products and fibrin(ogen) fragment E and thus conclude that there is no evidence for clinically relevant intravascular coagulation after short-term strenuous physical exercise at altitude.