The role of chromatin structure in cell migration

Chromatin dynamics play a major role in regulating genetic processes. Now, accumulating data suggest that chromatin structure may also affect the mechanical properties of the nucleus and cell migration. Global chromatin organization appears to modulate the shape, the size and the stiffness of the nucleus. Directed-cell migration, which often requires nuclear reshaping to allow passage of cells through narrow openings, is dependent not only on changes in cytoskeletal elements but also on global chromatin condensation. Conceivably, during cell migration a physical link between the chromatin and the cytoskeleton facilitates coordinated structural changes in these two components. Thus, in addition to regulating genetic processes, we suggest that alterations in chromatin structure could facilitate cellular reorganizations necessary for efficient migration.     

Early experimental obesity is associated with coronary endothelial dysfunction and oxidative stress

Obesity is independently associated with increased cardiovascular risk. However, since established obesity clusters with various cardiovascular risk factors, configuring the metabolic syndrome, the early effects of obesity on vascular function are still poorly understood. The current study was designed to evaluate the effect of early obesity on coronary endothelial function in a new animal model of swine obesity. As to method, juvenile domestic crossbred pigs were randomized to either high-fat/high-calorie diet (HF) or normal chow diet for 12 wk. Coronary microvascular permeability and abdominal wall fat were determined by using electron beam computerized tomography. Epicardial endothelial function and oxidative stress were measured in vitro. Systemic oxidative stress, renin-angiotensin activity, leptin levels, and parameters of insulin sensitivity were evaluated. As a result, HF pigs were characterized by abdominal obesity, hypertension, and elevated plasma lysophosphatidylcholine and leptin in the presence of increased insulin sensitivity. Coronary endothelium-dependent vasorelaxation was reduced in HF pigs and myocardial microvascular permeability increased compared with those values in normal pigs. Systemic redox status in HF pigs was similar to that in normal pigs, whereas the coronary endothelium demonstrated higher content of superoxide anions, nitrotyrosine, and NADPH-oxidase subunits, indicating increased tissue oxidative stress. In conclusion, the current study shows that early obesity is characterized by increased vascular oxidative stress and endothelial dysfunction in association with increased levels of leptin and before the development of insulin resistance and systemic oxidative stress. Vascular dysfunction is therefore an early manifestation of obesity and might contribute to the increased cardiovascular risk, independently of insulin resistance.     

Differentiation-Induced Internal Translation of c-sis mRNA: Analysis of the cis Elements and Their Differentiation-Linked Binding to the hnRNP C Protein

In previous reports we showed that the long 5' untranslated region (5' UTR) of c-sis, the gene encoding the B chain of platelet-derived growth factor, has translational modulating activity due to its differentiation-activated internal ribosomal entry site (D-IRES). Here we show that the 5' UTR contains three regions with a computer-predicted Y-shaped structure upstream of an AUG codon, each of which can confer some degree of internal translation by itself. In nondifferentiated cells, the entire 5' UTR is required for maximal basal IRES activity. The elements required for the differentiation-sensing ability (i.e., D-IRES) were mapped to a 630-nucleotide fragment within the central portion of the 5' UTR. Even though the region responsible for IRES activation is smaller, the full-length 5' UTR is capable of mediating the maximal translation efficiency in differentiated cells, since only the entire 5' UTR is able to confer the maximal basal IRES activity. Interestingly, a 43-kDa protein, identified as hnRNP C, binds in a differentiation-induced manner to the differentiation-sensing region. Using UV cross-linking experiments, we show that while hnRNP C is mainly a nuclear protein, its binding activity to the D-IRES is mostly nuclear in nondifferentiated cells, whereas in differentiated cells such binding activity is associated with the ribosomal fraction. Since the c-sis 5' UTR is a translational modulator in response to cellular changes, it seems that the large number of cross-talking structural entities and the interactions with regulated trans-acting factors are important for the strength of modulation in response to cellular changes. These characteristics may constitute the major difference between strong IRESs, such as those seen in some viruses, and IRESs that serve as translational modulators in response to developmental signals, such as that of c-sis.     

H-protein and X-protein. Two new components of the thick filaments of vertebrate skeletal muscle

With a view to obtaining a more complete view of the composition and structure of the thick filaments of vertebrate skeletal muscle, we have isolated and characterized two new myofibrillar components, H-protein and X-protein. These were purified by hydroxyapatite column chromatography of an impure C-protein preparation itself made from impure myosin extracted from rabbit back and leg muscles. H-protein is the protein responsible for band H on sodium dodecyl sulphate/polyacrylamide gel electrophoresis of crude myosin. X-protein, although present in such preparations in significant quantities, was not detected previously since it is difficult to resolve from C-protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Physical-chemical parameters have been determined for the new proteins and compared with those of C-protein. The apparent chain weight of H-protein estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis is 69,000, whereas that of X-protein (152,000) is only slightly greater than that of C-protein (140,000). The molecular weights of H- and X-proteins determined by sedimentation equilibrium centrifugation show that the molecules contain only a single polypeptide chain. The circular dichroism spectra indicate that the proteins have low alpha-helical contents. Both proteins, particularly H-protein, have a high proline content. Although X-protein is of similar chain weight to C-protein, the two show distinct differences in other properties. The sedimentation coefficient of X-protein is markedly lower than that of C-protein, suggesting X-protein is a more asymmetrical molecule. The amino acid compositions, although broadly similar, also show clear differences. Antibodies to H-protein, X-protein and C-protein have been raised in goats and shown not to cross-react.     

Axial arrangement of crossbridges in thick filaments of vertebrate skeletal muscle.

X-ray intensity data to 1.8 Å resolution were collected from native trigonal crystals of bovine trypsinogen. The orientation and position of the trypsinogen molecules within their crystal cells were determined by Patterson search techniques using the refined model of bovine trypsin (Bode &; Schwager, 1975), and by subsequent R factor refinement. The translation functions allowed discrimination between the enantiomorphic space groups P3₂21 and P3₁21. After one constrained crystallographic refinement cycle, which reduced the crystallographic reliability factor (R) from 35% to 31%, a preliminary difference Fourier map showed several interesting details. Several refinement cycles reduced the value of R to 23%. The overall chain folding is very similar to trypsin. The chain segments, including residues 184 to 193² and 217 to 223, which form the specificity pocket in trypsin, are flexible in trypsinogen. The autolysis loop is partially mobile between residues 142 and 152. There is no continuing electron density for the N terminal residues preceding Tyr20. This indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution.     

The interaction of C-protein with heavy meromyosin and subfragment-2.

C-protein has previously been shown to bind to the light-meromyosin region of the myosin tail. Examination of mixtures of C-protein with heavy meromyosin or subfragment-2 or subfragment-1 in the analytical ultracentrifuge shows that there is also a binding site for C-protein in the subfragment-2 region of the tail.     

Photoinducible DRONPA‐s: a new tool for investigating cell–cell connectivity

The development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the plant cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables tight control of symplasmic cell‐to‐cell transport. Here we report on an elegant non‐invasive method to quantify the passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA‐s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA‐s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA‐s fluorescence in selected cells of the root meristem. This enabled us to compare movement of DRONPA‐s from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the lowest efflux of DRONPA‐s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA‐s system generates reproducible data and is a valuable tool for studying symplasmic connectivity.     

Shape and flexibility of the myosin molecule

Molecules of rabbit skeletal myosin have been examined in the electron microscope after drying at low temperature from solutions containing ethylene glycol or glycerol and rotary-shadowing with platinum. Analysis of the structure has been assisted by stereo-photography. While the general appearance, two heads attached to a long tail, is similar to that described by Slayter & Lowey (1967), more detail about the shape and size of the heads can be discerned and new information has been obtained about the flexibility of the tail and the head-tail junction.The heads are 190 Å long and wider at their ends than near the junction with the tail; the shape resembles that of a pear. The length is appreciably greater than the generally accepted value for subfragment 1, the proteolytic fragment of myosin. The heads are flexibly attached to the tail and can assume a wide range of tilt angles.Because the point where the two heads join the tail can be identified, the length of the tail, 1560 (±50) Å, can be measured more accurately than formerly. While all parts of the tail are somewhat flexible, sharp bends often occur at a well-defined site 430 Å from the head-tail junction. The demonstration of hinges at the head-tail junction and in the tail provides strong support for H. E. Huxley's (1969) hypothesis for the mechanism of muscle contraction.