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Biomedical subjects

Bernhard H Geierstanger

Publications and source records attributed to Bernhard H Geierstanger.

13 recordsLinked to original sources
PubMed2006

Structural evaluation of a novel pro-apoptotic peptide coupled to CNGRC tumor homing sequence by NMR.

Hunter-killer peptides (HKPs) are synthetic peptides that target specific cell types for apoptosis. These studies report functional and structural characteristics of HKP9, an hunter-killer peptide that specifically targets tumor vasculature with a new apoptotic sequence. Vesicle leakage experiments were performed as a model for membrane perturbing activity. Placement of the homing sequence reduces both cell toxicity and vesicle leakage activity. NMR studies elucidate the conformation and orientation of HKP9 in micelles. The positively charged end of the HKP9 killing sequence is solvent exposed; however, the central portion of the peptide is helical and buried in dodecylphosphorylcholine micelles. The homing sequence is less solvent exposed than in a previously reported tumor-homing peptide. The results suggest that solvent accessibility of the homing sequence should be considered in design of future peptides.

Amino Acid Sequence↗
PubMed2006

Antibody microarrays using resonance light-scattering particles for detection.

Antibody microarray measurements show great potential for the simultaneous quantification of many proteins in small amounts of body fluids and extracts. Over the last few years, a micro-array platform centered around the concept of microarrays in microtiter wells was developed, and for the best assays we have achieved lower limits of detection in the femtomolar range using resonance light-scattering particles for the staining of biotinylated detection antibodies. Although conceptually simple, these multiplexed sandwich assays are technically challenging. Here we describe in detail our protocols and procedures for the manufacturing of antibody microarrays with up to 48 different antibodies and for performing plasma measurements.

Animals↗
PubMed2005

Automated microflow NMR: routine analysis of five-microliter samples.

A microflow CapNMR probe double-tuned for 1H and 13C was installed on a 400-MHz NMR spectrometer and interfaced to an automated liquid handler. Individual samples dissolved in DMSO-d6 are submitted for NMR analysis in vials containing as little as 10 microL of sample. Sets of samples are submitted in a low-volume 384-well plate. Of the 10 microL of sample per well, as with vials, 5 microL is injected into the microflow NMR probe for analysis. For quality control of chemical libraries, 1D NMR spectra are acquired under full automation from 384-well plates on as many as 130 compounds within 24 h using 128 scans per spectrum and a sample-to-sample cycle time of approximately 11 min. Because of the low volume requirements and high mass sensitivity of the microflow NMR system, 30 nmol of a typical small molecule is sufficient to obtain high-quality, well-resolved, 1D proton or 2D COSY NMR spectra in approximately 6 or 20 min of data acquisition time per experiment, respectively. Implementation of pulse programs with automated solvent peak identification and suppression allow for reliable data collection, even for samples submitted in fully protonated DMSO. The automated microflow NMR system is controlled and monitored using web-based software.

Image Processing, Computer-Assisted↗
PubMed2005

The pungency of garlic: activation of TRPA1 and TRPV1 in response to allicin.

Garlic's pungent flavor has made it a popular ingredient in cuisines around the world and throughout history. Garlic's health benefits have been elevated from folklore to clinical study. Although there is some controversy as to the efficacy of garlic, garlic products are one of the most popular herbal supplements in the U.S. Chemically complex, garlic contains different assortments of sulfur compounds depending on whether the cloves are intact, crushed, cooked, or raw. Raw garlic, when cut and placed on the tongue or lips, elicits painful burning and prickling sensations through unknown mechanisms. Here, we show that raw but not baked garlic activates TRPA1 and TRPV1, two temperature-activated ion channels that belong to the transient receptor potential (TRP) family. These thermoTRPs are present in the pain-sensing neurons that innervate the mouth. We further show that allicin, an unstable component of fresh garlic, is the chemical responsible for TRPA1 and TRPV1 activation and is therefore likely to cause garlic's pungency.

Animals↗
PubMed2005

Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: systematic variation between sample types and calibration of mass spectrometry data.

Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.

Blood Proteins↗
PubMed2004

Enzymatic synthesis and 19F NMR studies of 2-fluoroadenine-substituted RNA.

The production of isotopically labeled RNA remains critical to current NMR structural studies. One approach to obtain simple NMR spectra is to label with a nucleus that is not naturally occurring in RNA. Fluorine-19 can serve as a sensitive site-specific probe upon incorporation into RNA. Here we report the efficient in vitro enzymatic synthesis of 2-fluoroadenosine-5'-triphosphate and its incorporation into the HIV-2 transactivation region (TAR) of RNA by DNA template-directed transcription using phage T7 RNA polymerase. We provide unequivocal evidence for this 19F-substituted base analogue capability to selectively interact with uracil, forming 2F-A-U base pairs in RNA. The introduction of a 2-fluoroadenyl substitution is relatively nonperturbing and provides us with uniquely positioned, sensitive NMR reporter groups to monitor structural changes in the local RNA environment.

Adenine↗
PubMed2004

Evaluating sandwich immunoassays in microarray format in terms of the ambient analyte regime.

BACKGROUND: Conceptionally, antibody microarrays are simply multiplexed sandwich immunoassays in a miniaturized format. However, from the amounts of capture antibodies used, it is not apparent whether such assays are ambient analyte (Ekins. Clin Chem 1998;44:2015-30) or mass-sensing devices (Silzel et al. Clin Chem 1998;44:2036-43). We evaluated multiplexed microarray sandwich assays for 24 mouse serum proteins in these terms within the boundaries of our experimental setup and based on theoretical considerations of the law of mass action. METHODS: Capture antibodies for 24 mouse serum proteins were printed on planar microarray substrates. After incubation with mixtures of purified antigens for 1 or 18 h, mixtures of biotinylated detection antibodies were used. High assay sensitivity was achieved by use of resonance-light-scattering particles for signal generation. Titration curves were generated for assay volumes of 20, 40, and 80 microL, and detection limits were calculated and compared. The assays were modeled theoretically based on the amounts of capture antibodies and the assay volumes used. RESULTS: As predicted, experimental variations of the assay volume by up to fourfold did not appreciably affect detection. Even for the most sensitive assay, < 2% of the analyte molecules present in the sample were captured and generated signal at the detection limit. However, increasing the sample incubation time from 1 to 18 h on average lowered the detection limit threefold. CONCLUSIONS: In our experimental setup, all 24 sandwich microarray assays fulfill the criteria of the "ambient analyte" regime because depletion of analyte molecules from the assay volume is insignificant.

Animals↗
PubMed2004

Efforts to expand the genetic alphabet: identification of a replicable unnatural DNA self-pair.

Six unnatural nucleotides featuring fluorine-substituted phenyl nucleobase analogues have been synthesized, incorporated into DNA, and characterized in terms of the structure and replication properties of the self-pairs they form. Each unnatural self-pair is accommodated in B-form DNA without detectable structural perturbation, and all are thermally stable and selective to roughly the same degree. Furthermore, the efficiency of polymerase-mediated mispair synthesis is similar for each unnatural nucleotide in the template. In contrast, the efficiency of polymerase-mediated self-pair extension is highly dependent on the specific fluorine substitution pattern. The most promising unnatural base pair candidate of this series is the 3-fluorobenzene self-pair, which is replicated with reasonable efficiency and selectivity.

Base Pairing↗
PubMed2004

PNA-encoded protease substrate microarrays.

Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.

Humans↗
PubMed2004

Multiplexed sandwich assays in microarray format.

Antibody arrays have evolved into powerful tools for quantifying proteins and qualifying their state of activation in complex biological samples. This level of analysis holds tremendous promise as part of a diagnostic or prognostic platform. In particular, multiplex sandwich ELISA assays performed with microarrays in the wells of multi-well plates enable high-throughput analysis of multiple samples with multivariate readouts. Here, we compare and review recent advances in antibody microarray technology and describe its promises towards the systematic analysis of complex biological samples, ranging from profiling patient sera to studying intracellular signaling.

Animals↗
PubMed2004

Orientation and helical conformation of a tissue-specific hunter-killer peptide in micelles.

Hunter-killer peptides are chimeric synthetic peptides that selectively target specific cell types for an apoptotic death. These peptides, which are models for potential therapeutics, contain a homing sequence for receptor-mediated interactions and a pro-apoptotic sequence. Homing domains have been designed to target angiogenic tumor cells, prostate cells, arthritic tissue and, most recently, adipose tissue. After a receptor-mediated internalization, the apoptotic sequence, which contains D-enantiomer amino acids, initiates apoptosis through mitochondrial membrane disruption. We have begun structure and functional studies on a peptide (HKP1) that specifically targets angiogenic tumor cells for apoptosis. As a model for mitochondrial membrane disruption, we have examined peptide-induced leakage of a calcein fluorophore from large unilamellar vesicles. These experiments demonstrate more potent leakage activity by HKP1 than the peptide lacking the homing domain. Circular dichroism and 2D homonuclear NMR experiments demonstrate that this tumor-specific HKP adopts a left-handed amphipathic helix in association with dodecylphosphorylcholine micelles in a parallel orientation to the lipid-water interface with the homing domain remaining exposed to solvent. The amphipathic helix of the apoptotic domain orients with nonpolar leucine and alanine residues inserting most deeply into the lipid environment.

Animals↗
PubMed2003

Measurement and ab initio calculation of CSA/dipole-dipole cross-correlated relaxation provide insight into the mechanism of a H2-forming dehydrogenase.

Using transferred cross-correlated relaxation and DFT calculations, the conformation of the relevant conformation of N5,N10-methylenetetrahydromethanopterin, a reaction intermediate bound to the 80 kD H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase is determined. The conformation of the intermediate differs from the free form in solution and makes the reaction mechanism plausible.

Methanobacteriaceae↗