Cyclotosaurus cf. PosthumusFraas (Capitosauridae,Stereospondyli) from the Huai Hin Lat Formation (Upper Triassic), Northeastern Thailand: With a note on capitosaurid biogeography

Part of a large capitosaurid skull, similar to that of Cyclotosaurus posthumus from the Upper Triassic of Germany, has been discovered in the upper part of the Huai Hin Lat Formation near Chulabhorn (Nam Phrom) Dam. This discovery is consistent with the presumed Norian age of this formation. Although the phylogeny of the Capitosauridae is still unclear, the group of Upper Triassic Cyclotosaurus species to which C. posthumus belongs is monophyletic and seems to be known only from Laurasia or Northwestern Gondwana (Morocco). The occurrence of C. cf. posthumus in Thailand is consistent with the hypothesis previously put forward, that this part of Southeast Asia was bound to Laurasia in Mesozoic times.     

Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A₁ rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A₂₈₀ units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.     

Further characterization of a DNA polymerase activity in mouse sperm nuclei.

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.     

The carbamate reaction of glycylglycine, plasma, and tissue extracts evaluated by a pH stopped flow apparatus.

We have used a stopped flow rapid reaction pH apparatus to investigate the carbamate equilibrium in glycylglycine solutions and in three biological tissues, human plasma, sheep muscle, and sheep brain, as well as to investigate the kinetics of carbamate formation in glyclyglycine solution and in human plasma. The rapid reaction apparatus was equipped with a pH sensitive glass electrode in order to follow the time course of pH from 0.005 to 100 s after rapid mixing of a solution of amine or protein and CO2. Two phases of the pH curve were observed: a fast phase representing carbamate formation, and a slow phase due to the hydration of CO2 which was uncatalyzed since a carbonic anhydrase inhibitor was added to the biological solutions. From the time course of pH change during the fast phase K2, the R-NH2 ionization constant, and Kc, the carbamate equilibrium constant as well as the velocity constant for the formation of carbamate, ka could be calculated from data at different pH and pCO2. The carbamate formed in glycylglycine solutions over a wide range of pH and pCO2 was found consistent with the theory of carbamate formation and with published data. At ionic strength 0.16 and 37 degrees pK is 7.67. pKc 4.58. The heat of the carbamate reaction (deltaH) was calculated to be -3.2 kcal/mol between 20 degrees and 37 degrees. Kt of glycylglycine depends quantitatively on ionic strength as predicted by the Debye-Huckel theory. With ionic strength 0.16 ku was found to be 2,500 M1 S1 at 37 degrees. The activation energy of carbamate formation is 6.7 kcal/mol. Carbamate measurements in human plasma at pCO2 from 38 to 359 Torr. pH from 6.9 to 8.3, temperature 37 degrees, and ionic strength 0.15 provided evidence that two kinds of amino groups participate in carbamate formation. From the equilibrium constants computed for the two species they could be identified as alpha- and epsilon-amino groups. On the basis of a protein molecular weight of 69.000. 0.6 alpha-amino groups/molecule with pKz=7.0 and pKc=4.2, and 5.9 epsilon-amino groups/molecule with pKz=9.0 and pKc=4.3 contribute to carbamate formation. The velocity constant ka was estimated to be 4,950 M1 S1 for the alpha-amino groups and 13,800 M1 S1 for the epsilon-amino groups. Under physiological conditions (pCO2=40 Torr. pH=7.4). The concentration of carbamate in plasma is 0.6 mM and the half-time of carbamate formation is 0.05 s. In extracts prepared from sheep brain at 37 degrees pH=7 and pCO2=35 Torr. the carbamate formation was estimated to be 0.8 mM. With pCO2=70 Torr and the same pH and temperature the carbamate concentration in muscle approximates 0.3 mM and increases to 7 mM as pH rises to 8. It is concluded that, as in plasma, a considerable number of epsilon-amino groups appear to be available for carbamate formation in these tissues.     

Molecular aspects of the inactivation of tryptophanyl transfer ribonucleic acid synthetase by N-ethylmaleimide.

The tryptic maps of tryptophanyl-tRNA synthetase from beef pancreas show that the 8 cysteinyl residues of the enzyme subunit are located, 2 by 2, on four different peptides. The kinetics of the incorporation of radioactivity from N-[ethyl-14C]ethylmaleimide into these peptides are compared in this paper with the kinetics of the changes of the catalytic properties of the enzyme occurring during alkylation. This comparison allows the identification of (a) the peptide carrying the cysteinyl residues located on the surface of the molecule, (b) the peptide carrying the deeply buried residues unmasked by the dissociation of the subunits, and (c) the peptide carrying the --SH group located in the vicinity of the binding site of tryptophan. The fourth peptide is shown to have a great sensitivity to pH with respect to the reactivity of its cysteinyl residues toward N-ethylmaleimide. The same unusual pH dependence is found for the rate of quenching of the intrinsic fluorescence of the protein during the alkylation, suggesting a strong sensitivity of the conformation of tryptophanyl-tRNA synthetase to pH in the range of 7 to 9.     

Microbial invasion of a toxic medium is facilitated by a resident community but inhibited as the community co-evolves

Predicting whether microbial invaders will colonize an environment is critical for managing natural and engineered ecosystems, and controlling infectious disease. Invaders often face competition by resident microbes. But how invasions play out in communities dominated by facilitative interactions is less clear. We previously showed that growth medium toxicity can promote facilitation between four bacterial species, as species that cannot grow alone rely on others to survive. Following the same logic, here we allowed other bacterial species to invade the four-species community and found that invaders could more easily colonize a toxic medium when the community was present. In a more benign environment instead, invasive species that could survive alone colonized more successfully when the residents were absent. Next, we asked whether early colonists could exclude future ones through a priority effect, by inoculating the invaders into the resident community only after its members had co-evolved for 44 weeks. Compared to the ancestral community, the co-evolved resident community was more competitive toward invaders and less affected by them. Our experiments show how communities may assemble by facilitating one another in harsh, sterile environments, but that arriving after community members have co-evolved can limit invasion success.Subject terms: Microbial ecology, Microbial ecology     

Analysis of multiple basal cell carcinomas (BCCs) arising in one individual highlights genetic tumor heterogeneity and identifies novel driver mutations

Basal cell carcinoma (BCC) is the most common human cancer, especially in individuals with light skin phototypes (i.e., Fitzpatrick I-II skin type). Many affected develop multiple BCCs during their lifetime. It is not uncommon to observe elderly patients with >5 BCCs. In this study, we explored whether for patients diagnosed with multiple BCCs, analyzing the genomic mutations in one tumor could be sufficient to derive meaningful molecular/genetic conclusions regarding the other BCC tumors. Following the Genome Analysis Toolkit (GATK) best practices we have completed the study of 6 BCCs that occurred in an 83-year-old Caucasian male due to sun exposure. We have analyzed exome sequencing data of each BCC tumor and matched normal skin samples. We identified that BCCs from the same patient shared some of the key driver mutations, but they also displayed significant intertumoral heterogeneity. This finding may in part explain the different clinical progression/evolution of BCCs observed in the same patient. This work also highlights the value of characterizing multiple BCCs in one individual to identify patient-specific genetic events with a potential link to other malignancies and implications for personalized medicine.