The thrombin high-affinity binding site on platelets is a negative regulator of thrombin-induced platelet activation. Structure-function studies using two mutant thrombins, Quick I and Quick II.

To elucidate the thrombin domains required for high-affinity binding and platelet activation, the platelet binding properties of thrombin and two mutant thrombins, thrombin Quick I and Quick II, were compared to their agonist effects in elevating intraplatelet [Ca2+]. In Quick I, a mutation within the fibrinogen binding groove results in decreased clotting and platelet aggregating activities, whereas in Quick II, a mutation in the primary substrate binding pocket abolishes both activities. Dysthrombin binding was decreased compared to thrombin. The fibrinogen binding groove appeared more important than the primary substrate pocket for high-affinity binding since Quick I showed drastically reduced, and Quick II only slightly reduced, binding affinity (Kd approximately 200 and approximately 10 nM, respectively). The deduced interaction of thrombin with its high-affinity binding site indicated that the thrombin catalytic site is directed toward the platelet surface and therefore, when bound, is proteolytically inactive. Quick I (0.5-5 nM) elicited intraplatelet [Ca2+] fluxes at concentrations where high-affinity binding was undetectable. Saturation of high-affinity binding sites with active-site-modified thrombin did not affect thrombin-induced (0.5 nM) or Quick I-induced (5 nM) responses. In contrast, addition of D-Phe-Pro-Arg chloromethyl ketone (FPRCK) subsequent to thrombin or Quick I stimulation of platelets abolished agonist-induced responses. Since Quick I was only 10-17% as effective as thrombin in increasing intraplatelet [Ca2+], our data support a model in which thrombin acts enzymatically on a platelet membrane "substrate", through an interaction mediated in part by the fibrinogen binding groove of thrombin. This conclusion is consistent with the inhibition observed with high concentrations (greater than 100 nM) of Quick II and FPRCK-modified thrombin (FPR-thrombin) in platelets stimulated with low concentrations of thrombin (less than 0.5 nM) or Quick I (less than 2 nM), consistent with inhibition by substrate depletion. In contrast, concentrations of FPR-thrombin or Quick II (less than 100 nM), which saturated predominantly the high-affinity binding sites, enhanced the platelet responses induced by thrombin (less than 0.5 nM). Thus, occupation of the high-affinity sites with inactive thrombin increased the concentration of active thrombin available for substrate interaction. Quick I-induced responses were not enhanced, consistent with its inability to interact with the high-affinity site. Since thrombin bound to the high-affinity site is proteolytically inactive, we hypothesize that the thrombin high-affinity binding site on platelets functions to alter thrombin activity and platelet activation.     

Concerted evolution of the cow epsilon 2 and epsilon 4 beta-globin genes

The nucleotide sequences of the cow epsilon 2 and epsilon 4 globin genes were determined. The sequences were 95% identical. These genes arose via a four-gene block duplication that also gave rise to the bovine fetal (gamma) and adult (beta) genes. Their deduced amino acid sequences are unlike any previously reported fetal or adult globins; rather, comparison to other mammalian globin genes indicates that they are embryonic in nature. The sequence data indicate that these two genes have converted each other during evolution. Pairwise comparison to the corresponding goat genes shows greater similarity between paralogues than between more directly related orthologues. This is in direct contrast to the situation between the cow and goat fetal and adult genes. These observations suggest that the frequency of DNA conversion or the fixation of conversion events may vary in different locations of the cow beta-globin cluster.      

Human responses to propionic acid. I. Quantification of within- and between-participant variation in perception by normosmics and anosmics

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra- threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri- threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.      

Population structure in the American oyster as inferred by nuclear gene genealogies

Multiple haplotypes from each of three nuclear loci were isolated and sequenced from geographic populations of the American oyster, Crassostrea virginica. In tests of alternative phylogeographic hypotheses for this species, nuclear gene genealogies constructed for these haplotypes were compared to one another, to a mitochondrial gene tree, and to patterns of allele frequency variation in nuclear restriction site polymorphisms (RFLPs) and allozymes. Oyster populations from the Atlantic versus the Gulf of Mexico are not reciprocally monophyletic in any of the nuclear gene trees, despite considerable genetic variation and despite large allele frequency differences previously reported in several other genetic assays. If these populations were separated vicariantly in the past, either insufficient time has elapsed for neutral lineage sorting to have achieved monophyly at most nuclear loci, or balancing selection may have inhibited lineage extinction, or secondary gene flow may have moved haplotypes between regions. These and other possibilities are examined in light of available genetic evidence, and it is concluded that no simple explanation can account for the great variety of population genetic patterns across loci displayed by American oysters. Regardless of the source of this heterogeneity, this study provides an empirical demonstration that different sequences of DNA within the same organismal pedigree can have quite different phylogeographic histories.      

Fungal degradation of lipophilic extractives in eucalyptus globulus wood

Solid-state fermentation of eucalypt wood with several fungal strains was investigated as a possible biological pretreatment for decreasing the content of compounds responsible for pitch deposition during Cl2-free manufacture of paper pulp. First, different pitch deposits were characterized by gas chromatography (GC) and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and different degradation patterns were observed. The results showed that some basidiomycetes that decreased the lipophilic fraction also released significant amounts of polar extractives, which were identified by thermochemolysis as originating from lignin depolymerization. Therefore, the abilities of fungi to control pitch should be evaluated after analysis of compounds involved in deposit formation and not simply by estimating the decrease in the total extractive content. In this way, Phlebia radiata, Funalia trogii, Bjerkandera adusta, and Poria subvermispora strains were identified as the most promising organisms for pitch biocontrol, since they degraded 75 to 100% of both free and esterified sterols, as well as other lipophilic components of the eucalypt wood extractives. Ophiostoma piliferum, a fungus used commercially for pitch control, hydrolyzed the sterol esters and triglycerides, but it did not appear to be suitable for eucalypt wood treatment because it increased the content of free sitosterol, a major compound in pitch deposits.     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).