Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.     

Über den Informationsgehalt manueller Zielbewegungen

Summary It is investigated, wether or not informationcontent might be regarded as a suitable index of difficulty for manual aiming movements. Formulations proposed by Fitts, Crossman and Welford are used as a basis. The experiments include aiming movements to contact plates as well as transfer of pins to holes. Error percentage and movement time are recorded. It is shown, that the formulation (A =length of movement; W =aiming tolerance) including the information transmitted in the kinaesthetic and in the visual feedback loop separately, leads to a plausible result in the case of the contact-plate-experiment. After certain corrections regarding an “effective tolerance” the formulation is applicable to pin-hole-movements too. The maximal amount of information transmitted is 16 bit corresponding to an aiming tolerance of 1.4 mm. The rate of transmission of information varies between 11 and 35 bit/s according to the aiming tolerance. The results are to a certain extent in line with the results of comparable experiments.      

Anisotropic propagation of Ca2+ waves in isolated cardiomyocytes.

Digital imaging microscopy of fluor-3 fluorescence was used to study the propagation of intracellular Ca2+ waves in isolated adult rat cardiomyocytes from 17 to 37 degrees C. Ca2+ waves spread in both transverse and longitudinal direction of a myocyte. Transverse propagation was pronounced in waves starting from a focus at the edge of a myocyte and in waves following an irregular, curved path (spiral waves). For the former type of waves, propagation velocities were determined. Both transverse and longitudinal wave components propagated at constant velocity ranging from 30 to 125 micron/s. Myocytes were anisotropic with respect to wave propagation: waves propagated faster in the longitudinal than in the transverse direction. The ratio between longitudinal and transverse velocity increased from 1.30 at 17 degrees C to 1.55 at 37 degrees C. Apparent activation energies for transverse and longitudinal wave propagation were estimated to be -20 kJ/mol, suggesting that these processes are limited by diffusion of Ca2+. Direction-dependent propagation velocities are interpreted to result from the highly ordered structure of the myocytes, especially from the anisotropic arrangement of diffusion obstacles such as myofilaments and mitochondria.     

Horizontal Transfer of Archaeal Genes into the Deinococcaceae: Detection by Molecular and Computer-Based Approaches

Members of the Deinococcaceae (e.g., Thermus, Meiothermus, Deinococcus) contain A/V-ATPases typically found in Archaea or Eukaryotes which were probably acquired by horizontal gene transfer. Two methods were used to quantify the extent to which archaeal or eukaryotic genes have been acquired by this lineage. Screening of a Meiothermus ruber library with probes made against Thermoplasma acidophilum DNA yielded a number of clones which hybridized more strongly than background. One of these contained the prolyl tRNA synthetase (RS) gene. Phylogenetic analysis shows the M. ruber and D. radiodurans prolyl RS to be more closely related to archaeal and eukaryal forms of this gene than to the typical bacterial type. Using a bioinformatics approach, putative open reading frames (ORFs) from the prerelease version of the D. radiodurans genome were screened for genes more closely related to archaeal or eukaryotic genes. Putative ORFs were searched against representative genomes from each of the three domains using automated BLAST. ORFs showing the highest matches against archaeal and eukaryotic genes were collected and ranked. Among the top-ranked hits were the A/V-ATPase catalytic and noncatalytic subunits and the prolyl RS genes. Using phylogenetic methods, ORFs were analyzed and trees assessed for evidence of horizontal gene transfer. Of the 45 genes examined, 20 showed topologies in which D. radiodurans homologues clearly group with eukaryotic or archaeal homologues, and 17 additional trees were found to show probable evidence of horizontal gene transfer. Compared to the total number of ORFs in the genome, those that can be identified as having been acquired from Archaea or Eukaryotes are relatively few (approximately 1%), suggesting that interdomain transfer is rare.     

Effects of corallivory and coral colony density on coral growth and survival

A suite of processes drive variation in coral populations in space and time, yet our understanding of how variation in coral density affects coral performance is limited. Theory predicts that reductions in density can send coral populations into a predator pit, where concentrated corallivory maintains corals at low densities. In reality, how variation in coral density alters corallivory rates is poorly resolved. Here, we experimentally quantified the effects of corallivory and coral density on growth and survival of small colonies of the staghorn coral Acropora pulchra. Our findings suggest that coral density and corallivory have strong but independent effects on coral performance. In the presence of corallivores, corals suffered high but density-independent mortality. When corallivores were excluded, however, vertical extension rates of colonies increased with increasing densities. While we found no evidence for a predator pit, our results suggest that spatio-temporal variation in corallivore and coral densities can fundamentally alter population dynamics via strong effects on juvenile corals.

     

In vivo observations of timecourse and distribution of morphological dynamics in Xenopus retinotectal axon arbors

Changes in neuronal structure can contribute to the plasticity of neuronal connections in the developing and mature nervous system. However, the expectation that they would occur slowly precluded many from considering structural changes as a mechanism underlying synaptic plasticity that occurs over a period of minutes to hours. We took time-lapse confocal images of retinotectal axon arbors to determine the timecourse, magnitude, and distribution of changes in axon arbor structure within living Xenopus tadpoles. Images of axons were collected at intervals of 3 min, 30 min, and 2 h over total observation periods up to 8 h. Branch additions and retractions in arbors imaged at 3- or 30-min intervals were confined to shorter branches. Sites of additions and retractions were distributed throughout the arbor. The average lifetime of branches was about 10 min. Branches of up to 10 μm could be added to the arbor within a single 3-min observation interval. Observations of arbors at 3-min intervals showed rapid changes in the structure of branchtips, including transitions from lamellar growth cones to more streamlined tips, growth cone collapse, and re-extension. Simple branchtips were motile and appeared capable of exploratory behavior when viewed in time-lapse movies. In arbors imaged at 2-h intervals over a total of 8 h, morphological changes included longer branches, tens of microns in length. An average of 50% of the total branch length in the arbor was remodeled within 8 h. The data indicate that the elaboration of the arbor occurs by the random addition of branches throughout the arbor, followed by the selective stabilization of a small fraction of the new branches and the retraction of the majority of branches. Stabilized branches can then elongate and support the addition of more branches. These data show that structural changes in presynaptic axons can occur very rapidly even in complex arbors and can therefore play a role in forms of neuronal plasticity that operate on a timescale of minutes. © 1996 John Wiley & Sons, Inc.     

BRCA1-directed,enhanced and aberrant homologous recombination: Mechanism and potential treatment strategies

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.Key words: BRCT, DNA repair, peptide, radiation, RING, ubiquitylation     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.