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Biomedical subjects

R L Edelstein

Publications and source records attributed to R L Edelstein.

8 recordsLinked to original sources
PubMed2003

A simple pen-spotting method for arraying biomolecules on solid substrates.

We describe a simple, relatively inexpensive method for depositing biomolecules on a solid substrate using Rapidograph drafting pens. The pens can be used without modification to accurately deposit spots between approximately 100 and 600 microm in diameter. When mounted on a suitable microtranslation stage, the pens can be used to easily deposit tens of spots aligned with underlying substrate features such as microfabricated sensors. The pens are particularly convenient because pre-mixed solutions can be stored in the pens for multiple uses. We demonstrate the use of this approach to deposit DNA probes on a microsensor array.

Adsorption↗
PubMed2001

Stereochemical analysis of the reaction catalyzed by human protein geranylgeranyl transferase.

Protein geranylgeranyltransferase type I (PGGTase-I) catalyzes the nucleophilic substitution reaction between the C(20) geranylgeranyl diphosphate (GGPP) and a protein-derived thiol to form a thioether linkage. Here, we describe the stereochemical outcome, at the isoprenoid C1, of the reaction catalyzed by human PGGTase-I. To accomplish this, the pentapeptide N-dansyl-GCVLL was first enzymatically prenylated by human PGGTase-I with either (S)-[1-(2)H]farnesyl diphosphate or (S)-[1-(2)H]GGPP. The prenylated products were then degraded to dipeptides using carboxypeptidase Y. After HPLC purification, the prenylated dipeptide products were analyzed by (1)H NMR spectroscopy. The final spectra were compared with the spectra from the same product obtained via chemical synthesis to deduce the stereochemistry of the PGGTase-I-catalyzed reaction. This comparison showed that the reaction proceeds with inversion of configuration with no detectable (< 6%) racemization. These results are more consistent with an associative-type mechanism, but they cannot be used to rule out a dissociative mechanism involving a rigid, solvent-sequestered, tight ion pair.

Alkyl and Aryl Transferases↗
PubMed2000

The BARC biosensor applied to the detection of biological warfare agents.

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.

Biological Warfare↗
PubMed1997

Photoaffinity labeling of yeast farnesyl protein transferase and enzymatic synthesis of a Ras protein incorporating a photoactive isoprenoid.

Farnesyl protein transferase (FPTase) catalyzes the covalent attachment of a farnesyl (C15) group from farnesyl pyrophosphate (FPP) to a specific cysteine residue of Ras and several other proteins. In this report, photoactive farnesyl and geranylgeranyl pyrophosphate analogs 2-diazo-3,3,3-trifluoropropionyloxy-geranyl pyrophosphate (DATFP-GPP) and 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl pyrophosphate (DATFP-FPP) were used to study the active site of Saccharomyces cerevisiae FPTase. Both analogs are substrates for the enzyme, and upon irradiation, DATFP-GPP inhibits FPTase activity in a time-dependent manner. Photoinactivation by DATFP-GPP is prevented by the presence of the natural substrate FPP. Photolysis of radiolabeled DATFP-GPP results in preferential labeling of the beta subunit of FPTase, suggesting that this subunit is involved in recognition of FPP. Of particular importance, DATFP-GPP and DATFP-FPP were used to enzymatically transfer the photoactive isoprenoid moieties to peptides and to Ras; such molecules should be useful for identifying cellular components which specifically recognize farnesylated Ras and other prenylated proteins.

Affinity Labels↗
PubMed1982

Influence of various solvent-water mixtures on the extraction of dieldrin and methomyl residues from radishes.

The effect of organic solvent/water ratios on the extraction of field-incurred residues of dieldrin and methomyl from radishes was determined. 14C-Dieldrin and 14C-methomyl were applied separately to radishes in commercial formulations at rates of 0.2 and 0.9 kg/ha, respectively. Fourteen days post-application, the radishes were harvested and fresh root tissues were extracted using a Polytron homogenizer. Acetone, acetonitrile, and methanol containing 0, 10, 20, 30, 40, and 50% water were used as extraction solvents. For methomyl residues, the optimum water content of acetonitrile-water extraction mixtures was 40-50%; less than 40% water reduced the ability of acetonitrile to extract carbon-14. Methanol and acetone were nearly as efficient as 50% acetonitrile-water and were apparently not influenced by solvent/water ratios. For dieldrin, low water content slightly reduced the extraction efficiency of acetonitrile, with the optimum water content also being 40-50%. Percentage of water appeared to have little overall effect on methanol extraction efficiency. The extraction efficiency of acetone was lower than that of the other 2 solvents, and this effect was independent of the acetone/water ratio. Approximately 20% of the 14C-dieldrin residue was bound to radish roots 14 days post-application.

Acetone↗
PubMed1981

Degradation of methomyl residues in frozen strawberries.

14C-Methomyl (S-methyl N-[(methylcarbamoyl)-oxy]thioacetimidate) suspended in a commercial formulation was sprayed on strawberries. At 7 and 14 days post-application, mature berries were harvested in chopped. Fresh chopped berries were extracted with methanol using a Polytron homogenizer, and the crop marc was subsequently leached with methanol, freeze-dried, and re-extracted. Portions of the fresh chopped fruit were also frozen at-20 degrees C for 1, 30, and 102 days before extraction with methanol using a Polytron homogenizer, followed by subsequent leaching of the crop marc. For fresh berries, 93.7 and 88.9% of the 14C present at harvest was extractable at 7 and 14 days post-application, respectively. Based on thin layer chromatographic analysis, methomyl represented 83.5 and 68.2% of the 14C extracted from fresh berries at 7 and 14 days, respectively. Freezing of strawberries reduced extraction efficiency and appeared to degrade methomyl. At 7 and 14 days post-application, averages of 80.9 and 77.1%, respectively, of the 14C present were extractable after frozen storage regardless of storage time. Furthermore, only about 10% of the extracted 14C was methomyl; the remainder was of unknown composition.

Chromatography, Thin Layer↗