Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.     

GABA(B) receptors function as heterodimers

Our current understanding is that functional GABA(B) receptors exist as heterodimers of two related seven-transmembrane proteins, GABA(B)-R1 and GABA(B)-R2. GABA(B)-R1 requires GABA(B)-R2 to be expressed at the cell surface as a mature glycoprotein. Cloning of the GABA(B) receptor has failed to provide molecular evidence to support the existence of true receptor subtypes. The discovery of the heterodimeric nature of the GABA(B) receptor has already changed the way we think about GPCR function and it is likely that future studies will change our understanding about how receptor subtypes can be formed.     

Temporal integration in nasal lateralization and nasal detection of carbon dioxide

Two experiments examined time/concentration trading for the detection of carbon dioxide, an irritant with little or no odor. Experiment 1 employed the nasal lateralization method: subjects attempted to determine which nostril received carbon dioxide and which received pure air when presented simultaneously. Experiment 2 employed a temporal, two-alternative, forced-choice, detection paradigm with monorhinal stimulation. In both experiments, stimulus duration was varied at a number of fixed concentrations to determine the shortest, detectable pulse. Under both conditions, threshold pulse duration decreased as stimulus concentration increased. Power functions with exponents of less than negative one described the data quite well: More than a twofold increase in duration was needed to compensate for a twofold decrease in concentration. Thus, for carbon dioxide, the nasal trigeminal system functions as an imperfect integrator at threshold-level.     

Confirmation of data mining based predictions of protein function

MOTIVATION: A central problem in bioinformatics is the assignment of function to sequenced open reading frames (ORFs). The most common approach is based on inferred homology using a statistically based sequence similarity (SIM) method, e.g. PSI-BLAST. Alternative non-SIM based bioinformatic methods are becoming popular. One such method is Data Mining Prediction (DMP). This is based on combining evidence from amino-acid attributes, predicted structure and phylogenic patterns; and uses a combination of Inductive Logic Programming data mining, and decision trees to produce prediction rules for functional class. DMP predictions are more general than is possible using homology. In 2000/1, DMP was used to make public predictions of the function of 1309 Escherichia coli ORFs. Since then biological knowledge has advanced allowing us to test our predictions. RESULTS: We examined the updated (20.02.02) Riley group genome annotation, and examined the scientific literature for direct experimental derivations of ORF function. Both tests confirmed the DMP predictions. Accuracy varied between rules, and with the detail of prediction, but they were generally significantly better than random. For voting rules, accuracies of 75-100% were obtained. Twenty-one of these DMP predictions have been confirmed by direct experimentation. The DMP rules also have interesting biological explanations. DMP is, to the best of our knowledge, the first non-SIM based prediction method to have been tested directly on new data. AVAILABILITY: We have designed the "Genepredictions" database for protein functional predictions. This is intended to act as an open repository for predictions for any organism and can be accessed at http://www.genepredictions.org     

Nonhuman primate models of menopause workshop

The Nonhuman Primate Models of Menopause Workshop was held on the National Institutes of Health campus in January 2001. The purpose of this workshop, sponsored by the National Institute on Aging, was to review what is known about the female reproductive aging process in various species of monkeys (particularly rhesus, baboons, cynomolgus, and chimpanzees), including hormone profiles during the menopausal transition, occurrence of hot flashes, extent of age-related and menopause-associated changes in hormone levels on metabolism, bone loss, and impaired cardiovascular and cognitive function. Many aspects of the female reproductive aging process appear to be concordant between humans and these monkey species, but several important features may be species-specific. Those features that appear to parallel human menopause and aging include general similarity of hormone profiles across the menopausal transition, progression to cycle termination through irregular cycles, declining fertility with age, age-related gains in weight and percentage body fat content (with tendencies toward insulin resistance and glucose intolerance), increased low-density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol, declines in serum dehydroepiandrosterone, similarities in temperature-regulation systems, protective responses to estrogen replacement following ovariectomy in terms of bone metabolism, lipid profiles, and cognitive changes. Important differences include relatively short postmenopausal life span, timing in menopause-related changes in hormone secretion, and seasonal menstrual cycles. In addition, the question of whether ovariectomy in young adults is an appropriate model for the consequences of natural or surgical menopause in middle-aged and older adults is unresolved, and the numbers of older female animals available for research on menopause are very limited. The use of animal models is seen by workshop participants to be crucial for a mechanistic understanding of the human menopausal process and its connections to postmenopausal health problems; however, extensive in-depth and broad-based research is required to determine if nonhuman primates are appropriate models of human menopause.