Search

An antipeptide antibody (anti-VGR) was prepared by site-directed mutagenesis and was used to assess the structure of the amino terminus of the alpha1-subunit from sodium-potassium-adenosine triphosphatase (Na+-K+-ATPase). Immunoblot analysis of anti-VGR revealed a 100 kDa alpha-subunit. Binding of the subunit was observed in Escherichia coli containing an alpha1/troponin fusion proteins and in insects infected with baculovirus bearing alpha1 or alpha1T. Green monkey cells transfected with alpha1, however, did not show binding, suggesting that expression of alpha1 requires appropriate co- or posttranslational processing.

The reproducibility of mass spectrometry (MS) data collected using surface enhanced laser desorption/ionization-time of flight ( SELDI-TOF) has been questioned. This investigation was designed to test the reproducibility of SELDI data collected over time by multiple users and instruments. Five laboratories prepared arrays once every week for six weeks. Spectra were collected on separate instruments in the individual laboratories. Additionally, all of the arrays produced each week were rescanned on a single instrument in one laboratory. Lab-to-lab and array-to-array variability in alignment parameters were larger than the variability attributable to running samples during different weeks. The coefficient of variance (CV) in spectrum intensity ranged from 25% at baseline, to 80% in the matrix noise region, to about 50% during the exponential drop from the maximum matrix noise. Before normalization, the median CV of the peak heights was 72% and reduced to about 20% after normalization. Additionally, for the spectra from a common instrument, the CV ranged from 5% at baseline, to 50% in the matrix noise region, to 20% during the drop from the maximum matrix noise. Normalization reduced the variability in peak heights to about 18%. With proper processing methods, SELDI instruments produce spectra containing large numbers of reproducibly located peaks, with consistent heights. [ABSTRACT FROM AUTHOR]

The effects in the brain of selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have not yet been fully elucidated. Based upon the hypothesis that serotonin (5-HT)-steroid hormone interactions are important in mood regulation, we have compared six SERMs (tamoxifen, raloxifene, levormeloxifene, NNC 45-0781, NNC 45-0320, NNC 45-1506) with 17β-estradiol (E[sub 2] ) in terms of their ability to regulate mRNA levels of estrogen receptor (ER)α, ERβ, 5-HT[sub 1A] receptor, and 5-HT reuptake transporter (SERT) in the midbrain, amygdala, and hypothalamus of ovariectomized (OVX) rats. Female rats (n = 6/group, 8 groups total) were OVX and allowed to recover for 2 weeks. During the third post-OVX week, rats were injected subcutaneously with E[sub 2] (0.1 mg/kg) or one of the SERMs (5 mg/kg) once per day for 7 days. Twenty-four hours after the last injection, tissue was collected for the determination of mRNA levels by ribonuclease protection assay (RPA). E[sub 2] treatment significantly decreased mRNA levels for ERα, ERβ, and SERT in midbrain and ERα in hypothalamus. Tamoxifen increased ERβ mRNA levels in hypothalamus, while raloxifene increased ERβ mRNA levels in amygdala. NNC 45-0320 decreased ERα mRNA in hypothalamus and decreased ERβ mRNA in amygdala. These results suggest that while SERMs are not full estrogen receptor agonists in the brain, the agonist/antagonist profiles for individual SERMs may differ among brain areas. This raises the possibility of developing new SERMs for selective functions in specific brain areas.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The development of peptide/protein analyte assays for the purpose of diagnostic tests is driven by multiple factors, including sample availability, required throughput, and quantitative reproducibility. Laser Desorption/ionization mass spectrometry methods (LDI-MS) are particularly well suited for both peptide and protein characterization, and combining chromatographic surfaces directly onto the MS probe in the form of surface enhanced laser desorption/ionization (SELDI)-biochips has improved the reproducibility of analyte detection and provided effective relative quantitation. Here, we provide methods for developing reproducible SELDI-based assays by providing a complex artificial protein matrix background within the sample to be analyzed that allows for a common and reproducible ionization background as well as internal normalization standards. Using this approach, quantitative assays can be developed with CVs typically less than 10% across assays and days. Although the method has been extensively and successfully implemented in association with a protein matrix from E. coli, any other source for the complex protein matrix can be considered as long as it adheres to a set of conditions including the following: (1) the protein matrix must not provide interferences with the analyte to be detected, (2) the protein matrix must be sufficiently complex such that a majority of ion current generated from the desorption of the sample comes from the complex protein matrix, and (3) specific and well-resolved protein matrix peaks must be present within the mass range of the analyte of interest for appropriate normalization.