A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H)-labeled proteins

Several HNCO-based three-dimensional experiments are described for the measurement of ¹³C(i–1)-¹³C(i–1), ¹⁵N(i)-¹³C(i–1), ¹⁵N(i)-¹³C(i), ¹⁵N(i)-¹³C(i–1), ¹H^N(i)-¹³C(i), ¹H^N(i)-¹³C(i–1), and ¹³C(i–1)-¹³C(i–1) scalar and dipolar couplings in ¹⁵N, ¹³C, (²H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superficially resembling an HNCO correlation spectrum, allowing accurate and simple measurement of couplings without introducing additional spectral crowding. Scalar and dipolar couplings are measured with good sensitivity from relatively large proteins, as demonstrated with three proteins: cardiac Troponin C, calerythrin and ubiquitin. Measurement of several dipolar couplings between spin-1/2 nuclei using spin-state selective 3D HNCO spectra provides a wealth of structural information.     

Intensity modulated HSQC and HMQC: Two simple methods to measure 3JHNH α in proteins

Two methods for the measurement of homonuclear ³J_HNH coupling constants are described. Both HSQC- and HMQC-type experiments employ `quantitative J-correlation', in which the coupling constant of interest is obtained from the intensity ratio of cross peaks of two spectra. The first spectrum is acquired with ³J_HNH evolution and the second with -proton decoupling. The resolution of these methods in the F₁-domain is not restricted.     

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between ¹H^N-¹³C, ¹⁵N-¹³C, ¹H^N-¹³C _i–1, ¹⁵N-¹³C _i–1, ¹H^N-¹³C_i–1, ¹⁵N-¹³C_i–1, and ¹³C_i–1-¹³C _i–1 without introducing nonessential spectral crowding compared with an ordinary two-dimensional ¹⁵N-¹H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of /-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure–activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.     

Spontaneous somatic embryogenesis and plant regeneration from root cultures of Peucedanum palustre

The regeneration of Peucedanum palustre (L.) Moench (milk parsley) was established for the first time via somatic embryogenesis from primary root cultures. Callus formation occurred on the root cultures and showed spontaneous embryogenic capability on B5 basal medium supplemented with a low concentration of indoleacetic acid (5.5 × 10^–7 M). 2,4-Dichlorophenoxyacetic acid was not needed for the initiation of embryogenesis. The somatic embryos germinated and formed plantlets on hormone-free B5 medium. These plantlets were easily transferable to pots, and are presently passing their second growing season in the greenhouse.Development of the somatic embryos progressed through the globular, heart-shaped, torpedo-shaped, and cotyledonary stages, typical of zygotic embryos. Synchronization performed by sieving the embryos did not affect the development time. The culture has retained its embryogenic capacity for 25 months.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA 3-indolebutyric acid - BAP 6-benzylaminopurine     

Culturing of human vascular endothelial cells strongly affects their endothelin-1 and prostacyclin production

Cultured human umbilical vein endothelial cells (HUVECs) are a widely used model to study the regulation of endothelial production of vasoactive substances such as endothelin-1 (ET-1) and prostacyclin (PGI₂) in human. As even short term culturing is known to affect the function of many cell types, we studied whether there are differences in the production of ET-1 and PGI₂ between freshly isolated HUVECs and HUVECs cultured for two passages, and whether variation in cell density affects the production of ET-1 and PGI₂ by these cells. At confluency, freshly isolated HUVECs produced only from one-tenth to one-fifth of ET-1, but 46-86 times more PGI2 (p < 0.001), when compared to respective productions by similar amounts of cultured HUVECs. When the cell density of freshly isolated HUVECs was lowered either by diluting the cell suspension or by plating the same amount of cells on different size wells, the production of ET-1 increased: lowering cell density to one-tenth led to 18 fold increase in ET-1 production (p < 0.001). PGI₂ production was not affected by cell density. Thus our data imply that the production of both ET-1 and PGI₂ are differently regulated in freshly isolated and cultured HUVECs, and that cell density is an important determinant in the regulation of ET-1 production.     

Cytolinker cross-talk: periplakin N-terminus interacts with plectin to regulate keratin organisation and epithelial migration

Periplakin is a cytoskeletal linker protein that participates in the assembly of epidermal cell cornified envelope and regulates keratin organisation in simple epithelial cells. We have generated a stably transfected MCF-7 subclone expressing HA-tagged periplakin N-terminus to identify molecular interactions of periplakin. Co-immunoprecipitation with anti-HA antibodies and mass spectrometry identified a 500-kDa periplakin-interacting protein as plectin, another plakin family member. Plectin-periplakin interaction was confirmed by immunoblotting of complexes immunoprecipitated by either anti-HA or anti-plectin antibodies. Transient transfections of periplakin deletion constructs indicated that first 133 amino acid residues of the N-terminus are sufficient for co-localisation with plectin at MCF-7 cell borders. Immunofluorescence analysis demonstrated that periplakin and plectin isoforms 1, 1f and 1k co-localise at cell borders of MCF-7 epithelia and that plectin-1f and 1k co-localise with periplakin in suprabasal epidermis. Ablation of plectin by siRNA in HaCaT keratinocytes resulted in aggregation of periplakin to small clusters. Scratch-wounded MCF-7 epithelia expressing periplakin N-terminus showed accelerated keratin re-organisation that was inhibited by siRNA knock-down of plectin. Finally, ablation of either periplakin or plectin, or both proteins simultaneously, impaired migration of MCF-7 epithelial sheets. Thus, we have identified a novel functional co-localisation between two plakin cytolinker proteins.     

Ecological succession as an energy dispersal process

Ecological succession is described by the 2nd law of thermodynamics. According to the universal law of the maximal energy dispersal, an ecosystem evolves toward a stationary state in its surroundings by consuming free energy via diverse mechanisms. Species are the mechanisms that conduct energy down along gradients between repositories of energy which consist of populations at various thermodynamic levels. The salient characteristics of succession, growing biomass production, increasing species richness and shifting distributions of species are found as consequences of the universal quest to diminish energy density differences in least time. The analysis reveals that during succession the ecosystem's energy transduction network, i.e., the food web organizes increasingly more effective in the free energy reduction by acquiring new, more effective and abandoning old, less effective species of energy transduction. The number of species does not necessarily peak at the climax state that corresponds to the maximum-entropy partition of species maximizing consumption of free energy. According to the theory of evolution by natural selection founded on statistical physics of open systems, ecological succession is one among many other evolutionary processes.     

Redistribution of rat renal allograft-responding leukocytes during rejection: 1. Model

We describe a model that enables us to trace the traffic of allograft-responding inflammatory leukocytes to and from the graft without handling of these cells in vitro. At different times after transplantation, the kidney transplant pedicle—including the artery, vein, and draining lymphatics—is clamped. The allograft-responding leukocytes are labeled by a [³H]thymidine pulse either in situ or in the systemic lymphoid organs of the recipient. Fifteen minutes later the pulse is chased with a 1000-fold excess of cold thymidine, and the clamp is opened. The animals are sacrificed 18 hr later, when a balance between the synthesis of new labeled leukocytes from the originally labeled ones and dilution of intracellular label has been achieved. This model was used to analyze the allograft-responding inflammatory cell traffic to and from a renal transplant performed across the major histocompatibility complex in the rat. A sizable traffic was observed to both directions: After systemic injection of label only 0.008 × 10⁶ labeled cells × hr^?1 were found to emigrate into a kidney allograft (control). Already on the third day after transplantation—when the in situ inflammatory response is still at its beginning—more than 0.3 × 10⁶ labeled cells × hr^?1 migrated from the host to the allograft and 1.6 × 10⁶ labeled cells × hr^?1 left the allograft to the recipient spleen. The first figure is several-fold higher than any previous estimate. The findings emphasize the systemic nature of the antiallograft inflammatory response.     

Reduced viability of human vascular endothelial cells cultured on Matrigel™

Optimal vascular homeostasis requires efficient control of both proliferation and elimination of vascular endothelial cells. Programmed cell death, or apoptosis, is the main mechanism controlling cell elimination, and it is an essential component of vascular formation. Human vascular endothelial cells die in vitro, if prevented from obligatory survival factors like growth factors or attachment and cell spreading, but very little is known about the mechanisms controlling endothelial cell elimination. Signaling from the extracellular matrix affects the behavior and functions of human umbilical vein endothelial cells (HUVECs), and we have recently demonstrated the beneficial effects of plating on the reconstituted extracellular matrix Matrigel™, on the inducible nitric oxide production of freshly isolated HUVECs. In this work we observed that cultured HUVECs formed typical capillary-like structures on Matrigel, but unexpectedly, after 24–48 hours their viability was gradually lost. Viability was measured with an assay based on mitochondrial reduction of reagent XTT. No decrease in viability was seen in freshly isolated HUVECs or in cultured fibroblasts during this time. It is known that cells often turn into apoptosis if they receive conflicting information from their surroundings, and apparently signaling from Matrigel to HUVECs, while at their in vitro proliferating phenotype, resulted in launching of the apoptotic machinery. Thus, proliferating and differentiated phenotypes of endothelial cells seemed to have different sensitivity to signals that induce apoptosis. J. Cell. Physiol. 176:92–98, 1998. © 1998 Wiley-Liss, Inc.