Identification, purification and characterization of a novel phosphatidylinositol-specific phospholipase C, a third member of the beta subfamily.

The partial sequence of a novel PtdIns-specific phospholipase C of the beta subfamily (PtdIns-PLC beta 3) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns-PLC beta 3 is membrane associated; purification (approximately 1000-fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 mumol.min-1.mg-1, with PtdIns 4,5-bisphosphate as substrate. Substrate specificity and Ca2+ dependence of this purified PtdIns-PLC are characteristic of the PtdIns-PLC beta subfamily.     

Secondary flow in the human common carotid artery imaged by MR angiography.

The blood flow in arteries affects both the biology of the vessels and the development of atherosclerosis. The flow is three-dimensional, unsteady, and difficult to measure or to model computationally. We have used phase-shift-based magnetic resonance angiography to image and measure the flow in the common carotid arteries of a healthy human subject. There was curvature of the vessels and thin-slice dynamic flow imaging showed evidence of the presence of secondary motions. Flexing the cervical spine straightened the vessels and reduced the asymmetry of the flow.     

Antigen presentation in acquired immunological tolerance

In acquired tolerance, previous exposure to antigen under certain conditions induces specific unresponsiveness instead of specific immunological memory. It has been studied as an approach to the mechanisms of self-tolerance that operate on immunocompetent T and B lymphocytes once they leave their sites of origin in the thymus and the bone marrow. Possible mechanisms involve induction of specific suppressor cells or inactivation of antigen-specific lymphocytes (clonal anergy) as a consequence of abortive antigen presentation, in which the antigen receptor is effectively engaged but certain poorly defined accessory signals the T lymphocytes require are lacking. We propose that small, resting B lymphocytes, which lack these accessory signals, are the inactivating antigen-presenting cells in acquired tolerance to proteins and to the class II transplantation antigens. B lymphocytes, which can use their antigen receptors to gather and process antigens that are present at very low concentrations, may play a role in self-tolerance. In addition, B lymphocytes and T lymphocytes rendered anergic by encounter with self antigens could persist as self-specific suppressor cells to block an autoimmune response of autoreactive clones that had escaped deletion or anergy.     

Purification and characterization of bovine brain type I phosphatidylinositol kinase

Investigation into the phosphatidylinositol kinase activities in bovine brain has revealed the presence of a type I PtdIns kinase activity. This classification is based upon potent inhibition by neutral detergent and the production of a phosphatidylinositol phosphate that can be distinguished from phosphatidyl-inositol-4-phosphate [PtdIns(4)P] by thin-layer chromatography. The enzyme has been substantially purified and the activity is associated with an 85-kDa polypeptide on SDS/polyacrylamide gel electrophoresis. Analysis of the product confirms the identification of the enzyme as a type I PtdIns kinase. The purified kinase has been characterized with respect to substrate dependence (Mg2+, ATP, PtdIns), substrate presentation (pure lipid versus mixed micelle) and specificity [PtdIns versus PtdIns(4)P and phosphatidylinositol 4,5-bisphosphate].     

pH-dependent denaturation of thrombin-activated porcine factor VIII

Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions.     

Properties of acetylcholine receptors translated by cat muscle mRNA in Xenopus oocytes.

Poly(A)+ mRNA, extracted from denervated skeletal muscles of the cat, directs the synthesis of acetylcholine receptors in Xenopus laevis oocytes. The receptors are inserted in the oocyte membrane where they form acetylcholine receptor-channel complexes which have properties like those of the native receptors in the muscle membrane.     

The asymmetric war of attrition

The paper, which has an informal discussion at the end, provides a game theoretical analysis of the asymmetric “war of attrition” with incomplete information. This is a contest where animals adopt different roles like “owner” and “intruder” in a territorial conflict, and where the winner is the individual prepared to persist longer. The term incomplete information refers to mistakes in the identification of roles. The idea by Parker & Rubenstein (1981) is mathematically worked out and confirmed that there exists only a single evolutionarily stable strategy (ESS) for the model with a continuum of possible levels of persistence and no discontinuities in the increase of cost during attrition. The ESS prescribes to settle the conflict according to “who has more to gain or less to pay for persistence”. The only evolutionarily stable convention is thus to give the player access to the resource who has the role which is favoured with respect to payoffs. By contrast, it was shown earlier (Hammerstein, 1981) for various asymmetric versions of the “Hawks-Doves” model that an ESS can exist which appears paradoxical with respect to payoffs. The nature of this contrast is further analyzed by introducing elements of discreteness in the asymmetric war of attrition. It turns out that some conditions must be satisfied in order to have the possibility of an alternative ESS which is not of the above simple commonsense type. First, a decision to persist (or escalate) further in a contest must typically commit a contestant to go on fighting for a full “round”, before he can give up without danger. Second, such a “discontinuity” must occur at a level of persistence where the contest is still cheap, and, finally, errors in the identification of roles must be rare.     

D-Lyxose as a substrate for Streptomyces D-xylose isomerase.

Results are presented which show that the D-xylose isomerases present in Streptomyces olivaceus and Streptomyces phaeochromogenes NRRL B-3559 are incapable of utilizing D-lyxose as a substrate. The implications of these findings as related to the use of D-lyxose in the selection of constitutive mutants are discussed.