CRISPR-based strategies for studying regulatory elements and chromatin structure in mammalian gene control

Mammalian Genome - The development of high-throughput methods has enabled the genome-wide identification of putative regulatory elements in a wide variety of mammalian cells at an unprecedented...     

alpha-Latrotoxin as an instrument for studying neurosecretions

alpha-Latrotoxin, a presynaptic toxin from black widow spider venom Latrodectus mactans tredecimguttatus, triggers exocytosis in a variety of neurosecretory cells both in the presence and absence of calcium in the medium. The toxin interacts with two types of membrane the receptors which belong to different families of neuronal proteins and have different structures. Calcium-dependent receptor of alpha-latrotoxin is identified as neurexin I alpha and belongs to the family of neurexins. This family is selectively expressed in nerve tissue. The calcium-independent receptor of alpha-latrotoxin belongs to the family of G-protein-coupled receptors and proteins which homologous to it are found in heart, lung, kidney and spleen tissues. As a result of alpha-latrotoxin interaction with membrane receptor in the calcium medium the toxin forms the ionic channels in plasmalemma and enhances its calcium permeability. The effects of alpha-latrotoxin on exocytosis in the calcium and calcium-free media and question concerning coupling of channel-forming and secretogenic properties of alpha-latrotoxin are discussed.     

The SPASIBA force field as an essential tool for studying the structure and dynamics of saccharides

The SPASIBA force field has been applied to the determination of the structure and dynamical properties of various disaccharides. It has been shown that the experimental properties (structure, dipole moment, conformational relative energies) are satisfactorily predicted. The anomeric and exo-anomeric effects are confidently reproduced without specific terms for the alpha and beta anomers and the type of glycosidic linkages.     

Use of histone antibodies for studying chromatin topography and the phosphorylation of chromatin subunits

Polyclonal and monoclonal antibodies specific for histones as well as sera directed against synthetic peptides of histones were used to probe the topography of chromatin subunits. In native chromatin, the regions corresponding to residues 130-135 of H3 and 6-18 of H2B were found to be exposed and able to interact with antibodies whereas the regions 26-35 and 36-43 of H2B and 80-89 and 85-102 of H4 were not. In vitro phosphorylation of H3 and H5 in native chromatin or of H3 in H1/H5-depleted chromatin led to a marked drop in the binding of antibodies specific for residues 130-135 of H3 and 6-18 of H2B. Phosphorylation of H1/H5-depleted chromatin also altered the degree of exposure of certain H2A epitopes but it did not affect the surface accessibility of residues 1-11 of H2B.     

Prestress as an optimal biomechanical parameter for needle penetration

Drug delivery requires precise intradermal and subcutaneous injections of formulations to clinically relevant penetration depths. However, penetration depth is confounded by skin deflection, which occurs prior to and during penetration as the skin surface deforms axially with the needle, and which varies profoundly due to differing intrinsic mechanical (e.g. viscoelastic) tissue properties, disease state, aging, and ethnicity. Herein, an ex vivo model was utilized to study factors that affect skin deflection and the efficacy of injection, including prestress applied at the tissue surface, needle gauge, velocity, and actuation depth. The application of prestress minimized skin deflection during needle penetration and allowed for needle actuation to the targeted penetration depths with minimum variability. The force required to achieve target penetration depths was found to increase with prestress and decrease with needle gauge. Our findings emphasize the need for prestress applied to the skin surface to minimize variation in skin properties and administer formulations for intradermal and subcutaneous treatments with maximum precision.     

Viscosity as an inseparable partner of muscle contraction

A simple model is presented where, by an iterative procedure, the forces delivered by the power strokes are summed up to overcome the load. The system is moderated by the viscous hindrance. The model reproduces the features of muscle contraction as defined by the data of He et al. [1997. ATPase kinetics on activation of permeabilized isometric fibres from rabbit and frog muscle: a real time phosphate assay. J. Physiol. 501, 125-148] on rabbit psoas muscle fibres. According to the model power strokes are random. Energy summation take place if the subsequent power stroke occurs before the energy delivered by the previous power stroke is completely used. In order the sarcomere to be competent to contract initial driving force must reach a threshold whose value increases with the load. The step size of the power stroke decreases with the increase of the load. The viscous regime is simulated by the equation, where 1/k measures the viscous hindrance of the system. The relationship between water activity, viscosity and stiffness is discussed. It is concluded that the three parameters vary cyclically and that when water activity decreases (sarcomere shortening, cross-bridge attachment) viscosity and stiffness increase.     

Aurintricarboxilic acid as a probe for the analysis of chromatin structure.

Aurintricarboxylic acid is shown to cause nuclear swelling, disaggregation of chromatin structure and release of histones from chromatin. The nuclear swelling is inhibited by Ca⁺⁺ and Mg⁺⁺. The potential usefulness of aurintricarboxylic acid as a probe in chromatin studies is suggested.     

A model for chromatin structure.

A model for chromatin structure is presented. (a) Each of four histone species, H2A (IIbl or f2a2), H2B (IIb2 or f2b), H3 (III or f3) and H4 (IV or f2al) can form a parallel dimer. (b) These dimers can form two tetramers, (H2A)2(H2b)2 and (H3)2(H4)2. (C) These two tetramers bind a segment of DNA and condense it into a "C" segments. (d) The adjacent segments, termed extended or "E" segments, are bound by histone H1 (I or fl) for the major fraction of chromatin; the other "E" regions can be either bound by non-histone proteins or free of protein binding. (e) The binding of histones causes a structural distortion of the DNA which, depending upon the external conditions, may generate the formation of either an open structure with a heterogeneous and non-uniform supercoil or a compact structure with a string of beads. The model is supported by experimental data on histone-histone interaction, histone-DNA interaction and histone subunit-DNA interaction.     

Assembly of an active chromatin structure during replication.

MSB cells were pulse labeled with 3H-thymidine and the isolated nuclei digested with either staphylococcal nuclease (to about 40% acid solubility) or DNase I (to 15% acid solubility). The purified, nuclease resistant single-copy DNA was then hybridized to nuclear RNA (nRNA). The results of these experiments show that actively transcribed genes are assembled into nucleosome-like structures within 5-10 nucleosomes of the replication fork and that they also acquire a conformation characteristic of actively transcribed nucleosomes (ie, a DNase I sensitive structure) within 20 nucleosomes of the fork. Assuming DNA sequence specific interactions are required for establishing a DNase I sensitive conformation on active genes during each round of replication, our results indicate that a specific recognition event can occur very rapidly and very specifically in eukaryotic cells. The results are discussed in terms of the possible mechanisms responsible for propagating active, chromosomal conformations from mother cells to daughter cells.     

Hymenolepis diminuta: mitochondrial transhydrogenase as an additional site for anaerobic phosphorylation

Employing adult Hymenolepis diminuta SMP and exogenous pyridine nucleotide-generating systems, reduced pyridine nucleotide-dependent net 32P incorporation into ATP was examined. NADH supported rotenone-sensitive 32P incorporation and this rate increased markedly with fumarate addition, in keeping with an active fumarate reductase. Interestingly, corresponding evaluations with NADPH did not result in detectable phosphorylation in the absence or presence of fumarate. However, with NAD addition, but without NAD generation, active NADPH-dependent phosphorylation occurred, thereby demonstrating mitochondrial transhydrogenase involvement, and 32P incorporation increased significantly with fumarate addition. More importantly, in the presence of rotenone and both NADPH and NAD generation, significant net 32P incorporation was noted, but was undetectable in the presence of DCCD or protonophores (e.g., niclosamide). Without NAD generation, minimal phosphorylation occurred. These data demonstrate that with ongoing NADPH and NAD generation, the H. diminuta, proton-translocating, mitochondrial transhydrogenase can serve as an additional anaerobic phosphorylation site. A model is presented.     

Caveolae as an additional route for influenza virus endocytosis in MDCK cells

Clathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is internalized via clathrin-coated pits and/or caveolae in Madin Darby canine kidney (MDCK) cells. By applying pharmacological manipulations to selectively disrupt the cell internalization pathways, we found that, in MDCK cells, the influenza virus may be internalized via caveolae in addition to entry by clathrin-mediated endocytosis. However, a small contribution by another mode of entry, as recently proposed, cannot be excluded.     

Spectral fluorescence: an ataxonomic tool for studying the structure of phytoplankton populations

Although early studies in plankton biology feature detailedanalyses of numbers of species, modern studies tend to emphasizebulk changes in biomass associated with rare processes. We arguethat the penalty for this approach will eventually be an incompleteunderstanding of seasonal and spatial changes in the patternof primary production of ocean waters. The ability of naturalpopulations to respond to changes in the marine environmentis due to adaptability at the cellular level (phenotypic) andchanges in whole species groups (genotypic). The inability totest the relative importance of these two strategies is dueto the incompatiblity of traditional taxonomic approaches andfield experimentation where rate processes are being measured.To counteract this difficulty we propose an ataxonomic techniquewhich utilizes the differences in light absorption by phytoplanktonfor photosynthetic processes. In this report we review how changesin the fluorescence spectral signatures reflect other biochemicalfeatures in natural populations. This is examined in concertwith physical and chemical changes associated with the characteristicsof ocean water masses with specific reference as to how thesehydrographic features influence the relative growth of phytoplanktonpopulations. ^*This paper is the result of a study made at the Group for AquaticPrimaiy Productivity (GAP). Second International Workshop heldat the National Oceanographic Institute, Haifa, Israel in April–May1984.