Megaichnus igen. nov.: Giant Paleoburrows Attributed to Extinct Cenozoic Mammals from South America

In the last ten years, more than 1,500 large burrows have been discovered in southern and southeastern Brazil, dug in rocks that include weathered granitic and basaltic rocks, sandstones, and other consolidated sediments. Their presence in geological units of Plio-Pleistocene age suggests that large extinct mammals produced these structures. The internal walls exhibit scratches and grooves left by the animals that inhabited these structures. The burrows are straight or slightly sinuous tunnels that measure up to tens of meters in length. One smaller type measures up to 1.5 meter in diameter, and the larger type can reach 2 meters in height and 4 meters in width, suggesting that such structures have been produced by at least two kinds of organisms. This contribution proposes a classification for these ichnofossils under the generic designation Megaichnus igen. nov., consisting of two ichnospecies identified so far: M. major and M. minor ispp. nov. Although the exact identity of the producers of the burrows is yet unknown, the dimensions and morphology point to ground sloths and giant armadillos.     

A biomechanical mathematical model for the collagen bundle distribution-dependent contraction and subsequent retraction of healing dermal wounds

A continuum hypothesis-based, biomechanical model is presented for the simulation of the collagen bundle distribution-dependent contraction and subsequent retraction of healing dermal wounds that cover a large surface area. Since wound contraction mainly takes place in the dermal layer of the skin, solely a portion of this layer is included explicitly into the model. This portion of dermal layer is modeled as a heterogeneous, orthotropic continuous solid with bulk mechanical properties that are locally dependent on both the local concentration and the local geometrical arrangement of the collagen bundles. With respect to the dynamic regulation of the geometrical arrangement of the collagen bundles, it is assumed that a portion of the collagen molecules are deposited and reoriented in the direction of movement of (myo)fibroblasts. The remainder of the newly secreted collagen molecules are deposited by ratio in the direction of the present collagen bundles. Simulation results show that the distribution of the collagen bundles influences the evolution over time of both the shape of the wounded area and the degree of overall contraction of the wounded area. Interestingly, these effects are solely a consequence of alterations in the initial overall distribution of the collagen bundles, and not a consequence of alterations in the evolution over time of the different cell densities and concentrations of the modeled constituents. In accordance with experimental observations, simulation results show furthermore that ultimately the majority of the collagen molecules ends up permanently oriented toward the center of the wound and in the plane that runs parallel to the surface of the skin.

     

In vitro models to study compressive strain-induced muscle cell damage

Skeletal muscle tissue is highly susceptible to sustained compressive straining, eventually leading to tissue breakdown in the form of pressure sores. This breakdown begins at the cellular level and is believed to be triggered by sustained cell deformation. To study the relationship between compressive strain-induced muscle cell deformation and damage, and to investigate the role of cell-cell interactions, cell-matrix interactions and tissue geometry in this process, in vitro models of single cells, monolayers and 3D tissue analogs under compression are being developed. Compression is induced using specially designed loading devices, while cell deformation is visualised with confocal microscopy. Cell damage is assessed from viability tests, vital microscopy and histological or biochemical analyses. Preliminary results from a 3D cell seeded agarose model indicate that cell deformation is indeed an important trigger for cell damage; sustained compression of the model at 20% strain results in a significant increase in cell damage with time of compression, whereas damage in unstrained controls remains constant over time.     

Simultaneous measurement of protein one-bond and two-bond nitrogen-carbon coupling constants using an internally referenced quantitative J-correlated [(15)N,(1)H]-TROSY-HNC experiment

A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone ¹ J _NC, ¹ J _NC, and ² J _NC coupling constants are obtained for the two [¹⁵N;¹³C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [²H;¹⁵N;¹³C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J _NC values on protein backbone torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated ¹⁵N-¹³C residual dipolar couplings.     

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4°C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2′-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2′-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.     

Fission of a Spotted Hyena Clan: Consequences of Prolonged Female Absenteeism and Causes of Female Emigration

We describe the fission of one large East African clan of spotted hyenas (Crocuta crocuta). During an 8-mo period from late 1989 to early 1990, 8 of 22 adult females vanished from the clan, along with their juvenile offspring. 6 of these 8 adult females formed a new clan in an area adjacent to the parent clan's home range. The 6 adult female dispersers belonged to three mid- or low-ranking matrilines in the parent clan. Two juvenile males returned to the parent clan for temporary visits, and three female members of one matriline rejoined the parent clan after absences of over 8 mo. Returning females, but not males, were targets of severe aggression by adult female residents of the parent clan, and by juvenile residents of both sexes. Returning females fell from their previous mid-ranking positions to the bottom of the adult female hierarchy. Our data suggest that clan fission was promoted by the simultaneous occurrence of low food availability, an unusually high density of higher-ranking conspecific competitors, and the availability of neighboring vacant habitat.     

Inhibition of pain responses by activation of CB(2) cannabinoid receptors

Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence demonstrates that CB(1) cannabinoid receptor activation inhibits pain responses. Recently, the synthesis of CB(2) cannabinoid receptor-selective agonists has allowed testing whether CB(2) receptor activation inhibits pain. CB(2) receptor activation is sufficient to inhibit acute nociception, inflammatory hyperalgesia, and the allodynia and hyperalgesia produced in a neuropathic pain model. Studies using site-specific administration of agonist and antagonist have suggested that CB(2) receptor agonists inhibit pain responses by acting at peripheral sites. CB(2) receptor activation also inhibits edema and plasma extravasation produced by inflammation. CB(2) receptor-selective agonists do not produce central nervous system (CNS) effects typical of cannabinoids retaining agonist activity at the CB(1) receptor. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB(2) receptors. CB(2) receptor agonists may have promise for the treatment of pain and inflammation without CNS side effects.     

Detection of activity of telomerase in tumor cells using fiber optical biosensors

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.     

Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.     

Multifaceted roles of Lys166 of ribulose-bisphosphate carboxylase/oxygenase as discerned by product analysis and chemical rescue of site-directed mutants.

Ab initio calculations [King, W. A., et al. (1998) Biochemistry 37, 15414-15422] of an active-site mimic of D-ribulose-1,5-bisphosphate carboxylase/oxygenase suggest that active-site Lys166 plays a role in carboxylation in addition to its functions in the initial deprotonation and final protonation steps. To test this postulate, the turnover of 1-(3)H-labeled D-ribulose 1,5-bisphosphate (RuBP) by impaired position-166 mutants was characterized. Although these mutants catalyze slow enolization of RuBP, most of the RuBP-enediol undergoes beta-elimination of phosphate to form 2,3-pentodiulose 5-phosphate, signifying deficiencies in normal carboxylation and oxygenation. Much of the remaining RuBP-enediol is carboxylated but forms pyruvate, rather than 3-phospho-D-glycerate, due to incapacity in protonation of the terminal aci-acid intermediate. As a further test of the postulate, the effects of subtle perturbation of the Lys166 side chain on the carboxylation/oxygenation partitioning ratio (tau) were determined. To eliminate a chemically reactive site, Cys58 was replaced by a seryl residue without any loss of activity. The virtually inactive K166C-C58S double mutant was chemically rescued by aminoethylation or aminopropylation to reinsert a lysyl-like side chain at position 166. Relative to the wild-type value, tau for the aminoethylated enzyme was increased by approximately 30%, and tau for the aminopropylated enzyme was decreased by approximately 80%. Thus, two lines of experimentation support the theoretically based conclusion for the importance of Lys166 in the reaction of RuBP-enediol with gaseous substrates.