Sequence variation in the guillemot (Alcidae: Cepphus) mitochondrial control region and its nuclear homolog

We describe sequence variation in the mitochondrial control region and its nuclear homolog in three species and seven subspecies of guillemots (Cepphus spp.). Nuclear homologs of the 5' end of the control region were found in all individuals. Nuclear sequences were approximately 50% divergent from their mitochondrial counterparts and formed a distinct phylogenetic clade; the mitochondrial-nuclear introgression event must have predated the radiation of Cepphus. As in other vertebrates, the guillemot control region has a relatively conserved central block flanked by hypervariable 5' and 3' ends. Mean pairwise interspecific divergence values among control regions were lower than those in other birds. All individuals were heteroplasmic for the number of simple tandem nucleotide repeats (A(n)C) at the 3' end of the control region. Phylogenetic analyses suggest that black guillemots are basal to pigeon and spectacled guillemots, but evolutionary relationships among subspecies remain unresolved, possibly due to incomplete lineage sorting. Describing molecular variation in nuclear homologs of mitochondrial genes is of general interest in phylogenetics because, if undetected, the homologs may confound interpretations of mitochondrial phylogenies.      

Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72

Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out the SEK1 gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylation in unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72.     

The proteosomal degradation of fusion proteins cannot be predicted from the proteosome susceptibility of their individual components

It is assumed that the proteosome-processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome-degradable proteins does not necessarily result in a proteosome-degradable chimera. Conversely, fusion of proteosome-resistant proteins may result in a proteosome-degradable composite. We previously demonstrated that conserved influenza proteins can be unified into a single fusion antigen that is protective, and that vaccination with combinations of proteosome-resistant and proteosome-degradable antigens resulted in an augmented T-cell response. In the present study we constructed proteosome-degradable mutants of conserved influenza proteins NP, M1, NS1, and M2. These were then fused into multipartite proteins in different positions. The stability and degradation profiles of these fusion constructs were demonstrated to depend on the relative position of the individual proteins within the chimeric molecule. Combining unstable sequences of either NP and M1 or NS1 and M2 resulted in either rapidly proteosome degraded or proteosome-resistant bipartite fusion mutants. However, further unification of the proteosome-degradable forms into a single four-partite fusion molecule resulted in relatively stable chimeric proteins. Conversely, the addition of proteosome-resistant wild-type M2 to proteosome-resistant NP-M1-NS1 fusion protein lead to the decreased stability of the resulting four-partite multigene products, which in one case was clearly proteosome dependent. Additionally, a highly destabilized form of M1 failed to destabilize the wild-type NP. Collectively, we did not observe any additive effect leading to proteosomal degradation/nondegradation of a multigene construct.     

Implications from predictions of HLA-DRB1 binding peptides in the membrane proteins of Corynebacterium diphtheriae

The aerobic gram positive bacterium Corynebacterium diphtheriae causes diphtheria, a respiratory tract illness characterized by symptoms such as sore throat, low fever, and an adherent membrane on the tonsils, pharynx, and/or nasal cavity. Therefore, it is important to develop preventive vaccines for diphtheria. The availability of the 2,488,635 bp long complete sequence for the C. diphtheriae genome provides an opportunity to understand cell mediated immune response using Computational Biology tools from the bacterial proteome sequence data. We selected 355 membrane proteins from the C. diphtheriae proteome using annotation data to identify potential HLA-DRB1 binding short peptide using modeling, simulations and predictions. This exercise identified 30 short peptides in membrane proteins showing binding capability to HLA-DRB1 alleles. These peptides serve as outline for the understanding of cell mediated immune response to C. diphtheriae. It should be noted that the predicted data to be verified using binding assays for further consideration.     

Necrosis Is an Active and Controlled Form of Programmed Cell Death

In all studies on programmed cell death (PCD) and apoptosis as its most showy form, this process was considered to be a paradigmatic antithesis to necrotic cell death. On one hand, a concept on necrosis as a cellular cataclysm, an uncontrolled and passive phenomenon, had been provoked by an enormous bulk of experimental data on its inducibility by super-physiological exposures. On the other hand, much attention was attracted to a rapidly expanding (from nematodes) field of genetic studies on PCD. However, the findings accumulated which suggested a likeness rather than the opposition of the necrotic and apoptotic forms of elimination of unwanted cells. 1. Very diverse pathophysiological exposures (stimuli, stresses), such as heat, ionizing radiation, pathogens, cytokines cause both forms of cell death in the same cell population. 2. Antiapoptotic mechanisms (e.g., Bcl-2) can protect cells from both necrotic and apoptotic destruction. 3. Biochemical interventions (e.g., with inhibitors of poly-(ADP-riboso)-polymerase) into the signal and executive mechanisms of PCD can change the choice of the cell death form. 4. During both necrosis and epigenetic programs of apoptotic cell death that need no macromolecular synthesis (e.g., the CD95-dependent death), the nucleus plays a passive role. Therefore, necrosis, similarly to apoptosis, is suggested to be a form of the programmed cell death. However, for the whole body the physiological consequences of apoptosis and necrosis are quite different. In the case of apoptosis, all constituents of the nucleus and cytoplasm are isolated by an undamaged membrane and then by phagocytes together with the membrane-bound eat me markers (phosphatidylserine, etc.). In other words, the elimination of the cell which has realized its apoptotic program remains virtually unnoticed by the body. In the case of necrosis, the cytoplasmic content released into the intercellular space provokes an inflammatory response, i.e., an activation of resident phagocytes and attraction of leukocytes into the necrosis zone. It is suggested that under pathophysiological conditions, the necrotic cell destruction should amplify and catalyze pathological processes. The experimental data available now suggest that a disturbance in the body of optimal balance between the necrotic and apoptotic forms of PCD should be a crucial factor in the development of various pathophysiological processes associated with inflammation (diabetes, arthritis) or with aging (atherosclerosis, neurodegenerative diseases).     

Proteasome Failure Promotes Positioning of Lysosomes around the Aggresome via Local Block of Microtubule-Dependent Transport

Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species.     

Redox-sensing is the basis of photophobic responses in cyanobacteria

Abstract Cyanobacteria lack specialized photoreceptors for photophobic responses (PPR), the action spectrum for photosynthesis coinciding with that for PPR. In the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethyl urea) (DCMU), which blocks electron transfer prior to plastoquinone (PQ), turning on the light increased the membrane potential, but simultaneously evoked an unusual phobic response, presumably due to oxidation of PQ · H₂ via photosystem I (PS I). White light and the PQ reductant, (DQ · H₂), acted as attractants and produced methylation of a 40 kDa membrane peptide. Repellents, an uncoupler, carbonyl cyanide m -chlorophenylhydrazone (CCCP), and an oxidant, tetramethyl- p -hydroquinone(duroquinon) (DQ), caused demethylation. The results are interpreted in the framework of Häder's 'electron pool hypothesis', according to which PQ · H₂ level governs PPR. It is further concluded that the same system is the mechanism of pmf-sensing:      

Involvement of Ca2+ and cGMP in bacterial taxis

Abstract The level of cGMP in a suspension of Escherichia coli cells increased transiently upon the addition of chemoattractants. Ca²⁺ (1 mM), but not Mg²⁺, produced constant tumbling of cells in the presence of the ionophore A23187. The effect was observed either in stationary-state cells, or in a logarithmic culture treated with EDTA to increase permeability by A23187. Under the same conditions, Ca²⁺ decreased the cytoplasmic level of cGMP. In Phormidium uncinatum , rapid ⁴⁵Ca²⁺ accumulation followed a light-dark stimulus, or the addition of tetramethylquinone (TMQ), a chemorepellent. La³⁺, which increases the reversal rate, also enhanced the level of cytoplasmic Ca²⁺, presumably by blocking the outward Ca²⁺ flux. In both E. coli and P. uncinatum Ca²⁺ inhibited methylaccepting chemotaxis protein (MCP) methylation. It is concluded that cGMP and Ca²⁺ are secondary messengers in taxis information-processing.