Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin

Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin.F1.2 complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.     

Architecture of the budding yeast kinetochore reveals a conserved molecular core

How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.     

Quantitative isolation of microbial DNA from the different types of soils of natural and agricultural ecosystems

A novel procedure was developed for direct quantitative isolation of microbial DNA from soil. This technique was used to evaluate microbial DNA pools in soils of contrasting types (chernozems and brown forest soils) under different anthropogenic loads. A strong correlation was found between microbial biomass and DNA contents in soils of different types (R2 = 0.799). The ratio of soil CO2 emission rate to the amount of extractable DNA in the soil was shown to reflect physiological state of the soil microbial community; this ratio can be used as an ecophysiological parameter similarly to the metabolic quotient qCO2.     

Systematics of Halosarpheia based on morphological and molecular data

The genus Halosarpheia (Halosphaeriales) was established for marine ascomycetes with obpyriform to sub-globose, coriaceous, brown to black ostiolate ascomata with long necks; hamathecia of catenophyses; thin-walled, unitunicate, persistent asci with thick-walled apices; and ellipsoid, one septate, hyaline ascospores equipped with coiled, threadlike apical appendages that unfurl in water. Emphasis on ascospore appendage morphology has led to the inclusion in the genus of morphologically disparate fungi from a variety of marine and freshwater habitats. To better understand the evolutionary relationships of Halosarpheia species, phylogenetic analyses were conducted on 16 Halosarpheia species, 13 other species of Halosphaeriales and representatives of the Microascales, Hypocreales, Sordariales and Xylariales using 18S and 28S rDNA sequence data. All of the Halosarpheia species occurred on the Halosphaeriales clade. The type species of the genus, H. fibrosa, occurred on a well-supported clade with two morphologically similar species, H. trullifera and H. unicellularis. This clade, which phylogenetically was distant from the clades of other Halosarpheia species, represents the genus Halosarpheia sensu stricto. The other Halosarpheia species were distributed among eight other well-supported clades clearly separated from one another based on molecular data. New generic names are established for six of these clades, one new species is described, and one species is transferred to Aniptodera. A table (Table I) comparing the morphology, habitat, substrate and distribution of the genera of aquatic ascomycetes with coiled, threadlike apical appendages treated in this study is provided, along with a key for their identification.     

Confirmation of linkage of prostate cancer aggressiveness with chromosome 19q

Regions on chromosomes 7 and 19 were recently reported to contain susceptibility loci that regulate tumor aggressiveness of prostate cancer. To confirm these findings, we analyzed genome scan data from 161 pedigrees affected with prostate cancer. Using the Gleason score as a quantitative measure of tumor aggressiveness, we regressed the squared trait difference, as well as the mean-corrected cross product, on the estimated proportion of alleles shared identical-by-descent at each marker position. Our results confirm the previous linkage results for chromosome 19q (D19S902, P<.00001). In addition, we report suggestive evidence for linkage on chromosome 4 (D4S403, P=.00012). The results of previous findings, together with our results, provide strong evidence that chromosome 19 harbors a gene for tumor aggressiveness.     

The synergistic activation of Raf-1 kinase by phorbol myristate acetate and hydrogen peroxide in NIH3T3 cells

We have previously demonstrated that a 33kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H(2)O(2) induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmacological PKC activator PMA also induced a weak increase in Raf-1 kinase activity through PKC-epsilon activation as determined by the transient expression of dominant negative mutants of PKC-epsilon-K436R. Interestingly, H(2)O(2) produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H(2)O(2). This synergistic activation of Raf-1 kinase was further enhanced by cypermethrin (an inhibitor of protein phosphatase 2B) and dephostatin (tyrosine kinase inhibitor) implying an inhibitory role for these phosphatases in the Raf-1 signaling pathway. Taken together, our data suggest that the synergistic activation of Raf-1 kinase in response to PMA and H(2)O(2) occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-epsilon, along with a transient phosphorylation of both Raf-1 kinase and PKC.     

Isolation and characterisation of proline/arginine-rich cathelicidin peptides from ovine neutrophils

Cathelicidins are a family of gene-encoded antimicrobial peptides found in mammals. Seven cathelicidin genes have been identified in sheep, but up to now only two variants of one of these predicted peptides (OaBac5) have been purified from ovine neutrophils. In this work numerous proline/arginine-rich cathelicidin peptides were purified, including the originally predicted OaBac5 and another OaBac5 variant. As well as this, the C-terminus of the predicted OaBac7.5 and various truncated forms of OaBac11 were purified. Even though these peptides were much smaller than those predicted, they still displayed antimicrobial activity.     

Testing of the additivity-based protein sequence to reactivity algorithm

The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.     

Selective cell targeting with light-absorbing microparticles and nanoparticles

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.