Differential requirements for AP-2 in clathrin-mediated endocytosis

AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.     

Modeling domino effects in enzymes: molecular basis of the substrate specificity of the bacterial metallo-beta-lactamases IMP-1 and IMP-6

Metallo-beta-lactamases can hydrolyze a broad spectrum of beta-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme-substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect: upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site.     

Generation and characterization of a transgenic mouse with a functional human TSPY

To generate an animal model that is suitable for the analysis of regulation and expression of human testis-specific protein, Y-encoded TSPY, a transgenic mouse line, TgTSPY9, harboring a complete structural human TSPY gene was generated. Fluorescence in situ hybridization and Southern analyses show that approximately 50 copies of the human TSPY transgene are integrated at a single chromosomal site that maps to the distal long arm of the Y chromosome. The transgene is correctly transcribed and spliced according to the human pattern and is mainly expressed in testicular tissue, with spermatogonia and early primary spermatocytes (leptotene and zygotene) as expressing germ cells. TSPY transgenic mice are phenotypically normal, and spermatogenesis is neither impaired nor enhanced by the human transgene. The present study shows that a human TSPY gene integrated into the mouse genome follows the human expression pattern although murine tspy had lost its function in rodent evolution millions of years ago.     

Population size and identity influence the reaction norm of the rare,endemic plant Cochlearia bavarica across a gradient of environmental stress

Habitat degradation and loss can result in population decline and genetic erosion, limiting the ability of organisms to cope with environmental change, whether this is through evolutionary genetic response (requiring genetic variation) or through phenotypic plasticity (i.e., the ability of a given genotype to express a variable phenotype across environments). Here we address the question whether plants from small populations are less plastic or more susceptible to environmental stress than plants from large populations. We collected seed families from small (<100) versus large natural populations (>1,000 flowering plants) of the rare, endemic plant Cochlearia bavarica (Brassicaceae). We exposed the seedlings to a range of environments, created by manipulating water supply and light intensity in a 2 x 2 factorial design in the greenhouse. We monitored plant growth and survival for 300 days. Significant effects of offspring environment on offspring characters demonstrated that there is phenotypic plasticity in the responses to environmental stress in this species. Significant effects of population size group, but mainly of population identity within the population size groups, and of maternal plant identity within populations indicated variation due to genetic (plus potentially maternal) variation for offspring traits. The environment x maternal plant identity interaction was rarely significant, providing little evidence for genetically- (plus potentially maternally-) based variation in plasticity within populations. However, significant environment x population-size-group and environment x population-identity interactions suggested that populations differed in the amount of plasticity, the mean amount being smaller in small populations than in large populations. Whereas on day 210 the differences between small and large populations were largest in the environment in which plants grew biggest (i.e., under benign conditions), on day 270 the difference was largest in stressful environments. These results show that population size and population identity can affect growth and survival differently across environmental stress gradients. Moreover, these effects can themselves be modified by time-dependent variation in the interaction between plants and their environment.     

Human immunodeficiency virus-1 Tat protein interacts with distinct proteasomal alpha and beta subunits

The human immunodeficiency virus-1 (HIV-1) Tat protein was previously reported to compete the association of PA28 regulator with the alpha rings of the 20S proteasome and to inhibit its peptidase activity. However, the distinct interaction sites within the proteasome complex remained to be determined. Here we show that HIV-1 Tat binds to alpha4 and alpha7, six beta subunits of the constitutive 20S proteasome and the interferon-gamma-inducible subunits beta2i and beta5i. A Tat-proteasome interaction can also be demonstrated in vivo and leads to inhibition of proteasomal activity. This indicates that Tat can modulate or interfere with cellular proteasome function by specific interaction with distinct proteasomal subunits.     

Adopting a database as a solution to managing electron image data

A database was used for data management and interprogram communication in an image processing and three-dimensional reconstruction program suite for biological bundles. The programs were modified from the MRC crystallographic package. The database server works with local and remote programs and data sets, allows simultaneous requests from multiple clients, and maintains multiple databases and data tables within them. It has built-in security for the data access. Several graphical user interfaces are available to view and/or edit data tables. In addition, FORTRAN interface and function libraries are written to communicate with image processing software. The data management overhead is inexpensive, requiring only narrow bandwidth from the network. It easily handles several data tables with over 1000 entries.     

The impact of intraoperative manipulation of the prostate on total and free prostate-specific antigen

OBJECTIVES: It is well documented that mechanical manipulation of the prostate can elevate total PSA (t-PSA) levels in serum. However, less is known about its effects on free PSA (f-PSA) and the free-to-total PSA ratio (f/t-PSA). We therefore examined the impact of prostate manipulation on t-PSA and f-PSA during surgical procedures involving the prostate. METHODS: Intraoperative blood samples for t-PSA and f-PSA measurement (Hybritech) were collected every 15 min during 14 radical retropubic prostatectomies (RRP) and 10 radical cystoprostatectomies (RCP). RESULTS: Prostatic manipulation induced significant elevations in t-PSA and f-PSA during RRP and RCP. Postmanipulatory peaks were markedly higher for f-PSA than for t-PSA. The mean maximum f-PSA levels showed a 4.3- (RRP) and 7.9-fold (RCP) increase, followed by a rapid decline after prostate removal. t-PSA increased 1.2- (RRP) and 1.3-fold (RCP), and declined more slowly. Postmanipulatory f/t-PSA ratios also increased significantly, reaching mean elevations of +0.29 and +0.28 over preoperative ratios during RRP and RCP, respectively. CONCLUSIONS: Prostate manipulation can induce transient increases in t-PSA, f-PSA and f/t-PSA in benign and malignant prostates. The extent of these alterations and their course over time must be taken into account when postmanipulatory changes in PSA forms are investigated. Timing of postmanipulatory venipunctures and the molar response ratio of t-PSA assays used (equimolar versus nonequimolar) seem to have substantial impact on the results of such studies.     

Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.     

Enhanced values of the RBE and H ratio for cytogenetic effects induced by secondary electrons from an X-irradiated gold gurface

The low-energy secondary electrons emerging from the entrance surface of an X-irradiated gold foil increase the dose to cells in contact with or at micrometer distances from this surface (Radiat. Res. 150, 92-100, 1998). We examined the effect of the spectrum of these low-energy electrons on the RBE for cytogenetic effects and showed that this RBE was increased. A monolayer of surface-attached human T lymphocytes was exposed to 60 kV X rays in the absence or presence of a gold foil positioned immediately behind the cell layer or separated from it by a Mylar foil 0.9 or 2 microm thick. The enhancement of dose in the cell nuclei caused by the photoelectrons and Auger electrons emerging from the entrance surface of the gold foil was measured by TSEE dosimetry. Dose enhancement factors of 55.7, 46.6 and 37.5 were obtained with 0, 0.9 and 2 microm of Mylar inserted between the gold surface and the cell layer. This large enhancement results from the photoelectric effect in the gold foil, as shown by the accompanying Monte Carlo calculations of the secondary electron spectra at the gold surface. Auger electrons from the gold foil generally were not able to penetrate into the cell nuclei except for that fraction of the cells that had a very thin (< 0.7 microm) layer of cytoplasm and membranes between gold surface and cell nucleus. The dose-yield curves for dicentric chromosomes plus centric rings and for acentric fragments obtained after exposures without or with the gold foil were linear-quadratic. The coefficient alpha, the slope of the linear yield component, was increased in the presence of the gold foil and showed RBE values ranging from 1.7 to 2.2 compared to exposures in absence of the gold foil. The ratio of the yield of interstitial deletions and dicentrics (H ratio) was significantly increased from about 0.17 in the absence of the gold foil to about 0.22 in the presence of the gold foil. The increases in the RBE and the H ratio are interpreted in microdosimetric terms: The preferred occurrence of electron track ends in the vicinity of the gold surface causes an increase in the dose-mean restricted linear energy transfer in cell nuclei exposed to the photoelectrons and Auger electrons.