Integrative analysis of cell cycle control in budding yeast

The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo.     

Extended rat liver slice survival and stability monitored using clinical biomarkers

Precision-cut liver slices are reportedly limited as toxicity models by their short half-life in culture. We used traditional clinical chemistry biomarkers and histology to assess a newer procedure for improved liver slice maintenance. Slices from Sprague-Dawley rat livers were well maintained in a roller culture system for up to 10 days based on protein content (60-70% or higher of initial values) and biomarker retention and verified by histological examination of the tissues showing morphological integrity and viability of hepatocyte and biliary regions. Exposure of the slices to geldanamycin (GEL) resulted in loss of slice LDH and transaminase content, with associated depression in ALP and GGT levels and elevated bilirubin, indicating that GEL affects both cell types as occurs in vivo with this hepatobiliary toxicant. Thus, we conclude that liver slices merit further investigation as a general model for chronic as well as acute toxicity studies.     

Contiguous deletion of the X-linked adrenoleukodystrophy gene (ABCD1) and DXS1357E: a novel neonatal phenotype similar to peroxisomal biogenesis disorders

X-linked adrenoleukodystrophy (X-ALD) results from mutations in ABCD1. ABCD1 resides on Xq28 and encodes an integral peroxisomal membrane protein (ALD protein [ALDP]) that is of unknown function and that belongs to the ATP-binding cassette-transporter superfamily. Individuals with ABCD1 mutations accumulate very-long-chain fatty acids (VLCFA) (carbon length >22). Childhood cerebral X-ALD is the most devastating form of the disease. These children have the earliest onset (age 7.2 +/- 1.7 years) among the clinical phenotypes for ABCD1 mutations, but onset does not occur at <3 years of age. Individuals with either peroxisomal biogenesis disorders (PBD) or single-enzyme deficiencies (SED) in the peroxisomal beta-oxidation pathway--disorders such as acyl CoA oxidase deficiency and bifunctional protein deficiency--also accumulate VLCFA, but they present during the neonatal period. Until now, it has been possible to distinguish unequivocally between individuals with these autosomal recessively inherited syndromes and individuals with ABCD1 mutations, on the basis of the clinical presentation and measurement of other biochemical markers. We have identified three newborn boys who had clinical symptoms and initial biochemical results consistent with PBD or SED. In further study, however, we showed that they lacked ALDP, and we identified deletions that extended into the promoter region of ABCD1 and the neighboring gene, DXS1357E. Mutations in DXS1357E and the ABCD1 promoter region have not been described previously. We propose that the term "contiguous ABCD1 DXS1357E deletion syndrome" (CADDS) be used to identify this new contiguous-gene syndrome. The three patients with CADDS who are described here have important implications for genetic counseling, because individuals with CADDS may previously have been misdiagnosed as having an autosomal recessive PBD or SED     

Sniffers,buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell

The physiological responses of cells to external and internal stimuli are governed by genes and proteins interacting in complex networks whose dynamical properties are impossible to understand by intuitive reasoning alone. Recent advances by theoretical biologists have demonstrated that molecular regulatory networks can be accurately modeled in mathematical terms. These models shed light on the design principles of biological control systems and make predictions that have been verified experimentally.     

LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum

Lhs1p is an Hsp70-related chaperone localized in the endoplasmic reticulum (ER) lumen. Deltalhs1 mutant cells are viable but are constitutively induced for the unfolded protein response (UPR). Here, we demonstrate a severe growth defect in Deltaire1Deltalhs1 double mutant cells in which the UPR can no longer be induced. In addition, we have identified a UPR- regulated gene, SIL1, whose overexpression is sufficient to suppress the Deltaire1Deltalhs1 growth defect. SIL1 encodes an ER-localized protein that interacts directly with the ATPase domain of Kar2p (BiP), suggesting some role in modulating the activity of this vital chaperone. SIL1 is a non-essential gene but the Deltalhs1Deltasil1 double mutation is lethal and correlates with a complete block of protein translocation into the ER. We conclude that the IRE1-dependent induction of SIL1 is a vital adaptation in Deltalhs1 cells, and that the activities associated with the Lhs1 and Sil1 proteins constitute an essential function required for protein translocation into the ER. The Sil1 protein appears widespread amongst eukaryotes, with homologues in Yarrowia lipolytica (Sls1p), Drosophila and mammals.     

Mutational spectrum in the cardioauditory syndrome of Jervell and Lange-Nielsen

Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessive syndrome characterised by profound congenital sensorineural deafness and prolongation of the QT interval on the electrocardiogram, representing abnormal ventricular repolarisation. In a study of ten British and Norwegian families with JLNS, we have identified all of the mutations in the KCNQ1 gene, including two that are novel. Of the nine mutations identified in this group of 10 families, five are nonsense or frameshift mutations. Truncation of the protein proximal to the recently identified C-terminal assembly domain is expected to preclude assembly of KCNQ1 monomers into tetramers and explains the recessive inheritance of JLNS. However, study of a frameshift mutation, with a dominant effect phenotypically, suggests the presence of another assembly domain nearer to the N-terminus.     

Field Flight Dynamics of Hummingbirds during Territory Encroachment and Defense

Hummingbirds are known to defend food resources such as nectar sources from encroachment by competitors (including conspecifics). These competitive intraspecific interactions provide an opportunity to quantify the biomechanics of hummingbird flight performance during ecologically relevant natural behavior. We recorded the three-dimensional flight trajectories of Ruby-throated Hummingbirds defending, being chased from and freely departing from a feeder. These trajectories allowed us to compare natural flight performance to earlier laboratory measurements of maximum flight speed, aerodynamic force generation and power estimates. During field observation, hummingbirds rarely approached the maximal flight speeds previously reported from wind tunnel tests and never did so during level flight. However, the accelerations and rates of change in kinetic and potential energy we recorded indicate that these hummingbirds likely operated near the maximum of their flight force and metabolic power capabilities during these competitive interactions. Furthermore, although birds departing from the feeder while chased did so faster than freely-departing birds, these speed gains were accomplished by modulating kinetic and potential energy gains (or losses) rather than increasing overall power output, essentially trading altitude for speed during their evasive maneuver. Finally, the trajectories of defending birds were directed toward the position of the encroaching bird rather than the feeder.     

Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization

Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ～10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.