Role of the intracellular domain of the human type I interferon receptor 2 chain (IFNAR2c) in interferon signaling. Expression of IFNAR2c truncation mutants in U5A cells

A human cell line (U5A) lacking the type I interferon (IFN) receptor chain 2 (IFNAR2c) was used to determine the role of the IFNAR2c cytoplasmic domain in regulating IFN-dependent STAT activation, interferon-stimulated gene factor 3 (ISGF3) and c-sis-inducible factor (SIF) complex formation, gene expression, and antiproliferative effects. A panel of U5A cells expressing truncation mutants of IFNAR2c on their cell surface were generated for study. Janus kinase (JAK) activation was detected in all mutant cell lines; however, STAT1 and STAT2 activation was observed only in U5A cells expressing full-length IFNAR2c and IFNAR2c truncated at residue 462 (R2.462). IFNAR2c mutants truncated at residues 417 (R2. 417) and 346 (R2.346) or IFNAR2c mutant lacking tyrosine residues in its cytoplasmic domain (R2.Y-F) render the receptor inactive. A similar pattern was observed for IFN-inducible STAT activation, STAT complex formation, and STAT-DNA binding. Consistent with these data, IFN-inducible gene expression was ablated in U5A, R2.Y-F, R2.417, and R2.346 cell lines. The implications are that tyrosine phosphorylation and the 462-417 region of IFNAR2c are independently obligatory for receptor activation. In addition, the distal 53 amino acids of the intracellular domain of IFNAR2c are not required for IFN-receptor mediated STAT activation, ISFG3 or SIF complex formation, induction of gene expression, and inhibition of thymidine incorporation. These data demonstrate for the first time that both tyrosine phosphorylation and a specific domain of IFNAR2c are required in human cells for IFN-dependent coupling of JAK activation to STAT phosphorylation, gene induction, and antiproliferative effects. In addition, human and murine cells appear to require different regions of the cytoplasmic domain of IFNAR2c for regulation of IFN responses.     

Black, Jewish, and Interracial: It's Not the Color of Your Skin but the Race of Your Kin, and Other Myths of Identity

Black, Jewish, and Interracial: It's Not the Color of Your Skin but the Race of Your Kin, and Other Myths of Identity. Katya Gibel Azoulay. Durham, NC: Duke University Press, 1997.220 pp.     

MAXIMUM SWIM SPEEDS OF CAPTIVE AND FREE-RANGING DELPHINIDS: CRITICAL ANALYSIS OF EXTRAORDINARY PERFORMANCE

Research into dolphin swimming historically was guided by false assumptions pertaining to maximum speed. Accurate measurements on swimming speed and duration of effort of free-ranging dolphins are rare. To examine the variance of maximum swimming speeds, nearly 2,000 speed measurements were obtained for both captive and free-ranging dolphins, including Tursiops truncatus, Pseudorca crassidens, Delphinus capensis , and Delphinus delpbis . Measurements were made from videotapes of dolphins trained to swim fast around a large pool or jumping to a maximum height, videotapes of captured wild dolphins immediately after release, and sequential aerial photographs of a school of free-ranging dolphins startled by a passing airplane. Maximum horizontal speeds for trained animals were 8.2 m/sec for T. truncatus , 8.0 m/sec for D. delphis , and 8.0 m/sec for P. crassidens . Maximum speeds for T. truncatus swimming upwards, prior to vertical leaps ranged from 8.2 to 11.2 m/sec. Wild T. truncatus demonstrated a maximum speed of 5.7 m/sec. Maximum swimming speed of free-ranging D. capensis responding to multiple passes by a low flying airplane was 6.7 m/sec. There was no evidence that the freeranging dolphins have superior swimming capabilities to captive animals. The results of this study imply that realistic maximum swimming speeds for dolphins are lower than previous reports which were based on sparse data and imprecise measurement techniques.     

Temperature-dependent kinetic model for nitrogen-limited wine fermentations

A physical and mathematical model for wine fermentation kinetics was adapted to include the influence of temperature, perhaps the most critical factor influencing fermentation kinetics. The model was based on flask-scale white wine fermentations at different temperatures (11 to 35 degrees C) and different initial concentrations of sugar (265 to 300 g/liter) and nitrogen (70 to 350 mg N/liter). The results show that fermentation temperature and inadequate levels of nitrogen will cause stuck or sluggish fermentations. Model parameters representing cell growth rate, sugar utilization rate, and the inactivation rate of cells in the presence of ethanol are highly temperature dependent. All other variables (yield coefficient of cell mass to utilized nitrogen, yield coefficient of ethanol to utilized sugar, Monod constant for nitrogen-limited growth, and Michaelis-Menten-type constant for sugar transport) were determined to vary insignificantly with temperature. The resulting mathematical model accurately predicts the observed wine fermentation kinetics with respect to different temperatures and different initial conditions, including data from fermentations not used for model development. This is the first wine fermentation model that accurately predicts a transition from sluggish to normal to stuck fermentations as temperature increases from 11 to 35 degrees C. Furthermore, this comprehensive model provides insight into combined effects of time, temperature, and ethanol concentration on yeast (Saccharomyces cerevisiae) activity and physiology.     

Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.     

Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia

This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O(2), respectively) or normoxia (21% O(2)) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2(-/-) mice compared with MMP-2(+/+) mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2(+/+) mice but not in MMP-2(-/-) mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.     

Homodimerization antagonizes nuclear export of survivin

Survivin plays separate roles during different phases of the cell cycle. In mitosis, Survivin is a key regulator of cell division, while in interphase, Survivin is able to protect cells from apoptosis. Survivin shuttles between nucleus and cytoplasm under the influence of one or more nuclear export signals (NESs). Paradoxically, our data show that Survivin poorly binds CRM1 in vitro because hydrophobic residues of the NES are occupied in homodimer contacts. We show that NES-preserving dimerization mutants behave as monomers in solution, show dramatically increased CRM1 binding and are more efficiently exported in vivo than wild-type Survivin. These data indicate that Survivin contains a monomer-specific NES and that dimerization modulates cytoplasmic access of the protein. Our findings have implications for both the mitotic and interphase roles of survivin.     

Kinematics of terrestrial locomotion in harbor seals and gray seals: Importance of spinal flexion by amphibious phocids

Pinnipeds are amphibious mammals with flippers, which function for both aquatic and terrestrial locomotion. Evolution of the flippers has placed constraints on the terrestrial locomotion of phocid seals. The detailed kinematics of terrestrial locomotion of gray (Halichoerus grypus) and harbor (Phoca vitulina) seals was studied in captivity and in the wild using video analysis. The seals exhibited dorsoventral undulations with the chest and pelvis serving as the main contact points. An anteriorly directed wave produced by spinal flexion aided in lifting the chest off the ground as the fore flippers were retracted to pull the body forward. The highest length‐specific speeds recorded were 1.02 BL/s for a gray seal in captivity and 1.38 BL/s for a harbor seal in the wild. The frequency and amplitude of spinal movement increased directly with speed, but the duty factor remained constant. Substrate did not influence the kinematics except for differences due to moving up or down slopes. The highly aquatic nature of phocids seals has restricted them to locomote on land primarily using spinal flexion, which can limit performance in speed and duration.     

The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity

Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQ_p, FtsB_p, and FtsL_p) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQ_p, FtsB_p, and FtsL_p individually and in combination. Upon co-expression, FtsQ_p was co-purified with FtsB_p and FtsL_p from E. coli extracts as a stable trimeric complex. FtsB_p was also shown to interact with FtsQ_p in the absence of FtsL_p albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQ_pB_pL_p complex and the FtsQ_pB_p subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.