Dynamic spin label study of the barstar-barnase complex

The dynamic spin label method was used to study protein-protein interactions in the model complex of the enzyme barnase (Bn) with its inhibitor barstar. The C40A mutant of barstar (Bs) containing a single cysteine residue was modified with two different spin labels varying in length and structure of a flexible linker. Each spin label was selectively bound to the Cys82 residue, located near the Bn-Bs contact site. The formation of the stable protein complex between Bn and spin labeled Bs was accompanied by a substantial restriction of spin label mobility, indicated by remarkable changes in the registered EPR spectra. Order parameter, S, as an estimate of rapid reorientation of spin label relative to protein molecule, was sharply increasing approaching 1. However, the rotational correlation time tau for spin-labeled Bs and its complex with Bn in solution corresponded precisely to their molecular weights. These data indicate that both Bs and its complex with Bn are rigid protein entities. Spin labels attached to Bs in close proximity to an interface of interaction with Bn, regardless of its structure, undergo significant restriction of mobility by the environment of the contact site of the two proteins. The results show that this approach can be used to investigate fusion proteins containing Bn or Bs.     

Feeding Competition in <Emphasis Type="Italic">Macaca fascicularis</Emphasis>: An Assessment of the Early Arrival Tactic

In primate species with unidirectional dominance relationships, rank order restricts the access of nondominant females to clumped resources. However, females might attempt to bypass the rank order by reaching feeding sites before the highest ranking individuals (early arrival tactic) when there are net benefits. We therefore analyzed the order of arrival to the feeding site of the adult members of a captive group of long-tailed macaques. We used 2 experimental conditions that differed in the spatial distribution of a fixed amount of food (large vs. small patch). Though each condition induced contest competition, it was stronger in the small-patch condition. Arrival order does not correlate with dominance rank in either experimental condition. The α-male and α-female reached the feeding site 10–30 s after the beginning of the test. Some females seized on opportunities to reach the feeding site before them, especially in the large-patch condition. They used the early arrival tactic when the risks of aggression were relatively low, which subjects accomplished either by being dominant or by being nondominant but tolerated by the α-male. Social tolerance may provide individuals with an alternative means to obtain resources. In sum, variation in food abundance and distribution may affect the extent to which rank order determines order of arrival to feeding sites. A higher rank may confer priority in the choice of tactics, but not necessarily priority of access to the resources themselves.     

The paleoenvironments of azhdarchid pterosaurs localities in the Late Cretaceous of Kazakhstan

Five pterosaur localities are currently known from the Late Cretaceous in the northeastern Aral Sea region of Kazakhstan. Of these, one is Turonian-Coniacian in age, the Zhirkindek Formation (Tyulkili), and four are Santonian in age, all from the early Campanian Bostobe Formation (Baibishe, Akkurgan, Buroinak, and Shakh Shakh). All so far collected and identifiable Late Cretaceous pterosaur bones from Kazakhstan likely belong to Azhdarchidae: Azhdarcho sp. (Tyulkili); Aralazhdarcho bostobensis (Shakh Shakh); and Samrukia nessovi (Akkurgan). These latter two taxa, both from the Bostobe Formation might be synonyms. Azhdarcho sp. from the Zhirkindek Formation lived in a tropical-to-subtropical relatively humid climate on the shore of an estuarine basin connected to the Turgai Sea. Known fossils were collected in association with brackish-water bivalves and so the overall paleoenvironment of this pterosaur was likely an estuarine marsh as indicated by the dominance of conifers and low relative counts of ferns and angiosperms. Aralazhdarcho bostobensis, from the Bostobe Formation, lived on a coastal fluvial plain along the Turgai Sea. This paleoenvironment was either floodplain (Akkurgan, Buroinak, and Shakh Shakh) or estuarine (Baibishe). In the Santonian – early Campanian, shallow waters near this coastal plain were sites for the intensive accumulation of phosphates under upwelling conditions caused by strong winds from the ancient Asian landmass. These winds also caused significant aridization of the climate during this time. We speculate that pterosaurs may have been attracted to this area by the abundant resources in the bio-productive estuaries and nearshore upwelling waters.     

Nonclassical and Endogenous Cannabinoids: Effects on the Ordering of Brain Membranes

The effects of several nonclassical cannabinoids and the endogenous cannabinoid ligand, anandam-ide on the lipid ordering of rat brain synaptic plasma membranes (SPM) were examined and compared to ⁹-tetrahydrocannabinol (⁹-THC). SPM order was determined using fluorescence polarization. All compounds tested affected membrane ordering. ⁹-THC, CP-55,940, CP-55,244 and WIN-55212 decreased lipid ordering in SPM. Some stereospecificity was observed with ⁹-THC and WIN-55212, but not other compounds. Anandamide also decreased lipid order as did its putative precursor, arachidonic acid. In contrast to these compounds, levonantradol increased SPM lipid order. Although all pharmacologically active cannabinoids affect SPM lipid order, potency on this measure does not correlate well with their pharmacological potency. The results of this study suggest that membrane perturbation (either increases or decreases in lipid order) may be a necessary characteristic for cannabinoid pharmacological activity, but it is not a primary or sufficient determinate of action for this class of drugs.     

Age and size at maturity in mountain and lowland populations of the expanding moss <Emphasis Type="Italic">Pogonatum dentatum</Emphasis>

The moss Pogonatum dentatum has expanded its distribution in Fennoscandia from mountainous areas into the lowlands. This recent expansion appears to be associated with changes in important life-history parameters in female shoots. We examined shoot age and size at first production of sex organs and mature spores in P. dentatum to investigate this phenomenon. Female shoots produced mature spores for the first time in the lowlands in their second year but in their third year in the mountains. However, sex organs were produced by second year plants in both areas. There was no size difference between the mountain and lowland female shoots at the time of spore production. Among mountain females reproducing for the first time, 41% of the shoots branched, making them potentially ‘iteroparous’. Branching was not observed among lowland females. Male shoots showed no difference in production of sex organs, and were produced by second year shoots in both areas. Female shoots in the lowlands have earlier spore production and exhibit ‘semelparous’ behaviour by not producing branches. This suggests that the lowland phenotypes of P. dentatum are more ‘invasive’ than the mountain phenotypes. Earlier studies showing high rates of diaspore establishment in lowland areas also support this observation.     

Movement patterns of yellow baboons (Papio cynocephalus): Central positioning of walking infants

According to DeVore and Washburn's protection theory of the spatial organization of moving baboon troops, walking infants, which are among the most vulnerable and least self-sufficient of all troop members, should tend to occupy the troop's center. The protection theory is an ultimate hypothesis from which persistently recurring behavior is expected. Two troops living in Mikumi National Park, Tanzania, were compared with troops studied at other locations. Walking infants tended to occupy the center of their troop and to be underrepresented primarily in the frontal portion of progressions and secondarily in the rear. The lead position of progressions was analyzed using 82 walking infants, 11 troops, three locations, two species, and three studies involving eight or more observers employing somewhat different procedures at the different study sites. Despite so many opportunities for variation, 1,317 observations from these 11 troops did not produce a single instance in which a walking infant led the troop and very few in which one was in the frontal twelfth.     

An alternative stochastic model of generation of oligodendrocytes in cell culture

According to our previous model, oligodendrocyte – type 2 (O-2A) astrocyte progenitor cells become competent for differentiation in vitro after they complete a certain number of critical mitotic cycles. After attaining the competency to differentiate, progenitor cells divide with fixed probability p in subsequent cycles. The number of critical cycles is random; analysis of data suggests that it varies from zero to two. The present paper presents an alternative model in which there are no critical cycles, and the probability that a progenitor cell will divide again decreases gradually to a plateau value as the number of completed mitotic cycles increases. In particular all progenitor cells have the ability to differentiate from the time of plating. The Kiefer-Wolfowitz procedure is used to fit the new model to experimental data on the clonal growth of purified O-2A progenitor cells obtained from the optic nerves of 7 day old rats. The new model is shown to fit the experimental data well, indicating that it is not possible to determine whether critical cycles exist on the basis of these experimental data. In contrast to the fit of the previous model, which suggested that the addition of thyroid hormone increased the limiting probability of differentiation as the number of mitotic cycles increases, the fit of the new model suggests that the addition of thyroid hormone has almost no effect on the limiting probability of differentiation. Received: 6 March 2000 / Revised version: 18 September 2000 / Published online: 30 April 2001     

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.     

Estimation of the reaction efficiency in polymerase chain reaction

Polymerase chain reaction (PCR) is largely used in molecular biology for increasing the copy number of a specific DNA fragment. The succession of 20 replication cycles makes it possible to multiply the quantity of the fragment of interest by a factor of 1 million. The PCR technique has revolutionized genomics research. Several quantification methodologies are available to determine the DNA replication efficiency of the reaction which is the probability of replication of a DNA molecule at a replication cycle. We elaborate a quantification procedure based on the exponential phase and the early saturation phase of PCR. The reaction efficiency is supposed to be constant in the exponential phase, and decreasing in the saturation phase. We propose to model the PCR amplification process by a branching process which starts as a Galton-Watson branching process followed by a size-dependent process. Using this stochastic modelling and the conditional least-squares estimation method, we infer the reaction efficiency from a single PCR trajectory.