Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor, monitored by 2-dimensional electrophoresis and mass spectrometry.

The inclusion of protease inhibitors in serum or plasma samples has been found to significantly impact the isoform profile of selected plasma proteins as seen on 2-dimensional electrophoresis (2-DE) gels. With the addition of a protease inhibitor cocktail, several human plasma protein trains [depleted of albumin and immunoglobulin G (IgG)] exhibited higher isoelectric point (pI) isoforms. This shift was especially apparent for apolipoprotein A1 (apo A1), a relatively high abundance protein.

HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples.

There is a substantial list of pre-analytical variables that can alter the analysis of blood-derived samples. We have undertaken studies on some of these issues including choice of sample type, stability during storage, use of protease inhibitors, and clinical standardization. As there is a wide range of sample variables and a broad spectrum of analytical techniques in the HUPO PPP effort, it is not possible to define a single list of pre-analytical standards for samples or their processing.

Drug adverse events and system complications of intrathecal opioid delivery for pain: origins, detection, manifestations, and management.

Intrathecal drug delivery is effective for the treatment of cancer and nonmalignant pain in patients who do not respond well to oral opioids, in patients who cannot tolerate the side effects associated with opioids, or in patients who show a large, permanent increase in dosage. Although intrathecal drug delivery is associated with pharmacological side effects and complications, its benefits far outweigh its risks. There are three main categories of potential adverse events associated with intrathecal drug delivery: pharmacologic side effects, surgical complications, and device-related complications.

Neurologic sequelae of intraspinal drug delivery systems: results of a survey of american implanters of implantable drug delivery systems.

OBJECTIVES. The development of neurological sequelae subsequent to the placement of intraspinal drug delivery systems is particularly distressing. An attempt was made to determine the extent of this problem in both reported and in heretofore unreported cases.

Intraspinal analgesia for nonmalignant pain: a retrospective analysis for efficacy, safety and feasability in 50 patients.

Objectives. To test the efficacy and safety of intraspinal opioids for patients with nonmalignant pain. Design.

A DNA-binding element for a steroid receptor-binding factor is flanked by dual nuclear matrix DNA attachment sites in the c-myc gene promoter.

The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5'-GC-rich domain and a 3'-AT-rich domain, the latter of which has a homopurine/homopyrimidine structure.

Nuclear matrix acceptor binding sites for steroid hormone receptors: a candidate nuclear matrix acceptor protein.

Steroid/nuclear-hormone receptors are ligand-activated transcription factors that have been localized to the nuclear matrix. The classic model of hormone action suggests that, following activation, these receptors bind to specific "steroid response elements" on the DNA, then interact with other factors in the transcription initiation complex. However, evidence demonstrates the existence of specific chromatin proteins that act as accessory factors by facilitating the binding of the steroid receptors to the DNA.

Immunohistochemical localization of the avian progesterone receptor and its candidate receptor binding factor (RBF-1).

An avian oviduct nuclear matrix protein in the 6-10 kDa size range has been implicated to function in the cell-free nuclear binding of the avian oviduct progesterone receptor (PR). This protein, termed the receptor binding factor-1 (RBF-1), has been purified and partially characterized [Schuchard et al.: Biochemistry 30:4535-4542, 1991].

Steroid hormone regulation of nuclear proto-oncogenes.

The role of the nuclear proto-oncogenes as rapidly responding nuclear regulators in the cascade model for steroid hormone action is proposed. In this model, the nuclear proto-oncogenes respond within minutes to steroids and would code for regulatory proteins that in turn enter the nucleus to positively or negatively regulate "late" structural gene transcription and mRNA processing. The potential involvement of the nuclear matrix and one of its components, a receptor binding factor (RBF-1) for steroid receptors of these genes, is discussed.

Two-step "hot" PCR amplification of GC-rich avian c-myc sequences.

A new two-step cycle PCR method has been developed for amplification of GC-rich DNA sequences. Using this method, termed "hot PCR," 111 and 179 bp regions (GC contents of 74% and 76%, respectively) of the avian c-myc proto-oncogene were specifically amplified from cloned and genomic DNA. This method uses high-melting primers (Tm between 70 degrees and 74 degrees C), a two-step cycle that employs a 94 degrees C denaturation step and an annealing-elongation step between 70 degrees and 80 degrees C with or without formamide.

Purification of porcine heart latent protein phosphatase Fc.M.

Latent protein phosphatase, Fc.M, was purified from porcine heart extracts by a procedure involving precipitation at pH 5.0, DEAE-Sephacel chromatography, ammonium sulfate fractionation, chromatography on phenyl-Sepharose, Biogel-A 0.

High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity.

This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monitoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and appearance of products simultaneously.

Nuclear matrix localization and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor.

A chromatin acceptor protein for the avian oviduct progesterone receptor (PR), termed receptor binding factor 1 (RBF-1), has recently been shown to (1) be a component of the nuclear binding sites (acceptor sites) for PR and (2) generate high-affinity binding sites (termed the RBF-1 class of sites) on avian genomic DNA [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. A second class of sites and its associated protein (termed RBF-2) were also identified.

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor.

The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.

Functional role of the cysteine 451 thiol group in the M4 helix of the gamma subunit of Torpedo californica acetylcholine receptor.

Previous chemical modification studies of the acetylcholine receptor [Yee, A.S., Corey, D.

Salt stimulation of the activation of latent protein phosphatase, Fc.M, by Mn++ and by Mn/trypsin.

The activation of porcine heart latent protein phosphatase (Fc.M) by pretreatment with Mn++ followed by trypsin (Mn/trypsin) can be stimulated 2.5-fold by including NaCl or KCl in the activation mixtures.