HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Infrared spectroscopy of proteins

This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.     

Versuch einer Kausalanalyse der geotropischen Reaktionskette im Chara-Rhizoid

Summary In the initial phase of the geotropical reaction of the Chara rhizoid the growth difference postulated by Sievers (1967c) between the physically upper, slightly subapical flank and the lower one is demonstrated. In horizontal exposure the growth of the extreme cell apex is continued, while the growth of the lower flank is inhibited and that of the upper one is promoted. In the end phase the cell apex shows a damped oscillation until it finally reaches the vertical growth direction. The statoliths follow the oscillating growth of the cell tip from one flank to the opposite one until they are statistically equally redistributed in their normal position.—In vertical exposure under reduced turgor pressure the statoliths fall down into the extreme cell apex, where they inhibit the growth of this part of the cell wall, while the subapical wall grows transversally.—It is concluded that the statoliths inhibit the growth of the cell wall area which they cover.—The physical phase of the reaction chain, the susception, is the gravity-induced downward displacement of the statoliths. The physiological phase starts with the diversion of the acropetal transport of the Golgi vesicles to the upper part of the cell, which is caused by the block of statoliths (perception). The greater rate of vesicle incorporation into the upper flank in comparison to the lower one causes the subapical growth difference which results in the curvature (reaction).—In the case of the Chara rhizoid Golgi- and statolith-apparatus function as a self-regulating cellular system.

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Herrn Prof. Dr. Dr. h. c. Kurt Mothes zum 70. Geburtstag.     

Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

The Legal Regulation of Floats and Gliders—In Quest of a New Regime?

This article analyzes the legal status of unmanned instruments (particularly, floats and gliders) for observation purposes in the ocean environment. These new kinds of instruments are being deployed by the thousands into the oceans, not the least as part of the Argo Project of the International Oceanographic Commission. Their uncontrolled drifting has raised legal questions, especially when such instruments enter waters subject to the jurisdiction of foreign states. The authors argue that the current international legal framework is insufficient to address the pertinent issues, and that a new legal regime is needed.     

An “orientation sphere” visualization for examining animal head movements

1. Animal behavior is elicited, in part, in response to external conditions, but understanding how animals perceive the environment and make the decisions that bring about these behavioral responses is challenging.
2. Animal heads often move during specific behaviors and, additionally, typically have sensory systems (notably vision, smell, and hearing) sampling in defined arcs (normally to the front of their heads). As such, head‐mounted electronic sensors consisting of accelerometers and magnetometers, which can be used to determine the movement and directionality of animal heads (where head “movement” is defined here as changes in heading [azimuth] and/or pitch [elevation angle]), can potentially provide information both on behaviors in general and also clarify which parts of the environment the animals might be prioritizing (“environmental framing”).
3. We propose a new approach to visualize the data of such head‐mounted tags that combines the instantaneous outputs of head heading and pitch in a single intuitive spherical plot. This sphere has magnetic heading denoted by “longitude” position and head pitch by “latitude” on this “orientation sphere” (O‐sphere).
4. We construct the O‐sphere for the head rotations of a number of vertebrates with contrasting body shape and ecology (oryx, sheep, tortoises, and turtles), illustrating various behaviors, including foraging, walking, and environmental scanning. We also propose correcting head orientations for body orientations to highlight specific heading‐independent head rotation, and propose the derivation of O‐sphere‐metrics, such as angular speed across the sphere. This should help identify the functions of various head behaviors.
5. Visualizations of the O‐sphere provide an intuitive representation of animal behavior manifest via head orientation and rotation. This has ramifications for quantifying and understanding behaviors ranging from navigation through vigilance to feeding and, when used in tandem with body movement, should provide an important link between perception of the environment and response to it in free‐ranging animals.

     

Protection of Mice against Lethal Coxsackievirus B3 Infection by Using DNA Immunization

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.     

Toll-like receptor 4 deficiency: Smaller infarcts,but nogain in function

Backgound  

It has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT).     

Coupled Diffusion of Peripherally Bound Peptides along the Outer and Inner Membrane Leaflets

Transmembrane signaling implies that peripheral protein binding to one leaflet be detected by the opposite leaflet. Therefore, protein recruitment into preexisting cholesterol and sphingolipid rich platforms may be required. However, no clear molecular picture has evolved about how these rafts in both leaflets are connected. By using planar lipid bilayers, we show that the peripheral binding of a charged molecule (poly-lysine, PLL) is detected at the other side of the bilayer without involvement of raft lipids. The diffusion coefficient, D_P, of PLL differed by a factor of √2 when PLL absorbed to one or to both leaflets of planar membranes. Fluorescence correlation spectroscopy showed that the changes of the lipid diffusion coefficient, D_M, were even more pronounced. Although D_M remained larger than D_P on PLL binding to the first membrane leaflet, D_M dropped to D_P on PLL binding to both leaflets, which indicated that the lipids sandwiched between two PLL molecules had formed a nanodomain. Due to its small area of ∼20 nm² membrane electrostriction or leaflet interaction at bilayer midplane can only make a small contribution to interleaflet coupling. The tendency of the system to maximize the area where the membrane is free to undulate seems to be more important. As a spot with increased bending stiffness, the PLL bound patch in one leaflet attracts a stiffening additive on the other leaflet. That is to say, instead of suppressing undulations in two spots, two opposing PLL molecules migrate along a membrane at matching positions and suppress these undulations in a single spot. The gain in undulation energy is larger than the energy required for the alignment of two small PLL domains in opposite leafs and their coordinated diffusion. We propose that this type of mechanical interaction between two membrane separated ligands generally contributes to transmembrane signaling.