Y-chromosomal Markers Suitable for Noninvasive Studies of Guenon Hybridization

We examine previously-published TSPY sequence data to identify synapomorphies useful for tracking Y-chromosomal gene flow between hybridizing guenon species. We then describe a set of PCR primers and protocols that amplify many of these variable sites from feces. Such Y-chromosomal markers are potentially very useful to conservation studies because they may offer an early sign of introgression as a threat to the genetic integrity of a rare species. Moreover, the ability to survey these markers from feces greatly expands the utility of noninvasive studies.     

Dissociation of thrombin from platelets by hirudin. Evidence for receptor processing.

Hirudin inhibited the binding of human 125I-alpha-thrombin to the saturable, but not the nonsaturable, sites on washed human platelets. When hirudin was added to a thrombin-platelet mixture, it caused a biphasic dissociation of bound thrombin. A partial dissociation was too rapid to measure and was followed by complete dissociation with a first order rate constant of about 10(-2) s-1. The fraction of bound thrombin in the more slowly dissociable form increased from essentially none after a 5-s preincubation of thrombin and platelets to as much as 75% of saturable binding after a 4-min preincubation. Transition to the slowly dissociable state was not accompanied by an increase in the amount bound and was not observed with active site serine-derivatized thrombin. This is the first evidence with intact platelets of a binding characteristic that depends, as does platelet stimulation, on catalytically active thrombin, suggesting that it may represent physiologically significant receptor processing.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

The reaction of thrombin with platelet-derived nexin requires a secondary recognition site in addition to the catalytic site

A protease nexin released by activated platelets forms stable complexes with alpha-thrombin. Active-site-blocked thrombin does not form the stable complex, but it inhibits formation of the stable complex by active alpha-thrombin. gamma-Thrombin, which has a damaged substrate recognition site (the anion-binding exosite), did not form the complex and did not inhibit formation of the stable complex by alpha-thrombin. Complex formation was inhibited by the C-terminal dodecapeptide of hirudin, which has been shown to bind to the anion-binding exosite. A monoclonal antibody that blocks reactions of thrombin that involve the anion-binding exosite also inhibited formation of a stable complex of alpha-thrombin and the platelet-derived protease nexin. It is concluded that the anion-binding exosite of thrombin, a site that confers a high degree of specificity for substrates with a complementary site, binds to the platelet nexin prior to reaction of the catalytic site with the serpin.     

Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation

To assess the possibility that hydrolysis of the platelet surface thrombin substrate, glycoprotein V, is a necessary step in thrombin-induced platelet activation, thrombin-catalyzed hydrolysis of glycoprotein V was correlated with thrombin-induced platelet activation. Hydrolysis of tritium-labeled glycoprotein V on washed human platelets was measured by the appearance of a labeled supernatant fragment, and platelet activation was measured as secretion of ATP. Hydrolysis of glycoprotein V was linear with respect to both thrombin concentration and time of incubation. The extent of platelet activation was correlated with the rate of hydrolysis but not with the amount hydrolyzed. Maximum platelet activation could be obtained with thrombin treatments resulting in hydrolysis of as little as 4% of glycoprotein V per min. Glycoprotein V was partially removed from platelets by pretreatment with either platelet calcium-dependent protease or chymotrypsin. The rate of thrombin-catalyzed hydrolysis of the remaining glycoprotein V from these pretreated platelets was as little as 1.5% the rate from control platelets, but there was no impairment of the extent of platelet activation. Thus, these protease-pretreated platelets compared with control platelets showed a different correlation of glycoprotein V hydrolysis with platelet activation. Glycoprotein V was also partially removed by pretreatment of prostacyclin-inhibited platelets with thrombin. After removal of thrombin and prostacyclin, these platelets were desensitized to subsequent activation by thrombin. Incubation of desensitized platelets with nonsaturating levels of thrombin led to less than 25% of the activation seen with control platelets but to a slightly greater hydrolysis of glycoprotein V. Thus, the desensitization to thrombin was not due to loss of ability of the activating thrombin to hydrolyze glycoprotein V. These results do not exclude a role for glycoprotein V as a component of the platelet thrombin receptor, but they indicate that there is no simple relationship between thrombin-induced hydrolysis of glycoprotein V and platelet activation.     

Hair Cell Interactions in the Statocyst of Hermissenda

Hair cells in the statocyst of Hermissenda crassicornis respond to mechanical stimulation with a short latency (<2 ms) depolarizing generator potential that is followed by hyperpolarization and inhibition of spike activity. Mechanically evoked hyperpolarization and spike inhibition were abolished by cutting the static nerve, repetitive mechanical stimulation, tetrodotoxin (TTX), and Co⁺⁺. Since none of these procedures markedly altered the generator potential it was concluded that the hyperpolarization is an inhibitory synaptic potential and not a component of the mechanotransduction process. Intracellular recordings from pairs of hair cells in the same statocyst and in statocysts on opposite sides of the brain revealed that hair cells are connected by chemical and/or electrical synapses. All chemical interactions were inhibitory. Hyperpolarization and spike inhibition result from inhibitory interactions between hair cells in the same and in opposite statocysts.