Effects of trichloroethylene,tetrachloroethylene and dichloromethane on enzymatic activities in soil

Summary Enzyme assays for -glucosidase, -acetylglucosaminidase, phosphatase, phosphodiesterase, and proteinase were made in soil samples incubated for two months after contamination with trichloroethylene, tetrachloroethylene, and dichloromethane. These volatile chlorinated hydrocarbons were added at doses of 10, 100, and 1000 g per 100 g dry soil, respectively. Almost no effect was observed in soil sample contaminated with 10 g of the chemicals when compared with control soil. When 100 g of the volatile chlorinated hydrocarbons was added, the activity of -glucosidase, -acetylglucosaminidase and, in part, also of proteinase, was reduced during the first 28 days of incubation but returned to the same or slightly higher level than in the control soil after 2 months. Trichloroethylene, tetrachloroethylene, and dichloromethane at a concentration of 1000 g per 100 g soil primarily inhibited activity of all enzymes under test. However, after two months, the enzymatic activities especially in soil samples contaminated with tetrachloroethylene and dichloromethane were found to be at the same or higher level than in the control soil.     

Dynamics of the base of ribosomal A-site finger revealed by molecular dynamics simulations and Cryo-EM

Helix 38 (H38) of the large ribosomal subunit, with a length of 110 Å, reaches the small subunit through intersubunit bridge B1a. Previous cryo-EM studies revealed that the tip of H38 moves by more than 10 Å from the non-ratcheted to the ratcheted state of the ribosome while mutational studies implicated a key role of flexible H38 in attenuation of translocation and in dynamical signaling between ribosomal functional centers. We investigate a region including the elbow-shaped kink-turn (Kt-38) in the Haloarcula marismortui archaeal ribosome, and equivalently positioned elbows in three eubacterial species, located at the H38 base. We performed explicit solvent molecular dynamics simulations on the H38 elbows in all four species. They are formed by at first sight unrelated sequences resulting in diverse base interactions but built with the same overall topology, as shown by X-ray crystallography. The elbows display similar fluctuations and intrinsic flexibilities in simulations indicating that the eubacterial H38 elbows are structural and dynamical analogs of archaeal Kt-38. We suggest that this structural element plays a pivotal role in the large motions of H38 and may act as fulcrum for the abovementioned tip motion. The directional flexibility inferred from simulations correlates well with the cryo-EM results.     

In vivo evaluation of bioprinted prevascularized bone tissue

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.     

High genetic diversity and low differentiation reflect the ecological versatility of the African leopard

1. Download : Download high-res image (204KB)
2. Download : Download full-size image

     

The metaphase chromosome ultrastructure : I. Acute angle metal deposition technique as an appropriate use of shadow casting in chromosome structure investigation

The ultrastructure of the metaphase chromosome, isolated by spreading and dried by the critical point method, has been studied by the technique of rotary shadow casting at very acute angles, equivalent to deposition of metal on the specimens at mere grazing incidence. Acute angle deposition visualizes a three-dimensional image of the chromosome periphery and the substructure of the basic chromosome fibre. This paper describes the technical details of acute angle deposition as a particular use of shadow casting. The technique seems to be adequate for the study of the three-dimensional aspects of relatively large subjects and represents an alternative to scanning electron microscopy when the details under study are in the 150 Å range.     

Autonomous folding of interdomain regions of a modular polyketide synthase

Domains within the multienzyme polyketide synthases are linked by noncatalytic sequences of variable length and unknown function. Recently, the crystal structure was reported of a portion of the linker between the acyltransferase (AT) and ketoreductase (KR) domains from module 1 of the erythromycin synthase (6-deoxyerythronolide B synthase), as a pseudodimer with the adjacent ketoreductase (KR). On the basis of this structure, the homologous linker region between the dehydratase (DH) and enoyl reductase (ER) domains in fully reducing modules has been proposed to occupy a position on the periphery of the polyketide synthases complex, as in porcine fatty acid synthase. We report here the expression and characterization of the same region of the 6-deoxyerythronolide B synthase module 1 AT-KR linker, without the adjacent KR domain (termed DeltaN AT1-KR1), as well as the corresponding section of the DH-ER linker. The linkers fold autonomously and are well structured. However, analytical gel filtration and ultracentrifugation analysis independently show that DeltaN AT1-KR1 is homodimeric in solution; site-directed mutagenesis further demonstrates that linker self-association is compatible with the formation of a linker-KR pseudodimer. Our data also strongly indicate that the DH-ER linker associates with the upstream DH domain. Both of these findings are incompatible with the proposed model for polyketide synthase architecture, suggesting that it is premature to allocate the linker regions to a position in the multienzymes based on the solved structure of animal fatty acid synthase.