Direct measurement of histone peptide elongation rate in cleaving sea urchin embryos

Regulation of protein synthesis can be exercised at a number of levels. One of the more experimentally difficult levels to approach has been the measurement of peptide elongation rate. This paper presents a new application of the cyanogen bromide (CNBr) cleavage of proteins in a direct measurement of histone peptide elongation rate in cleaving sea urchin embryos (Strongylocentrotus purpuratus). The data indicate an elongation rate (at 15°C) for histones H2B and H1α of 0.69 and 0.80 codons per s, respectively. These values fall within the range of previously published values of average peptide elongation rate for total protein in these cells. This method should be generally applicable to many systems for which the measurement of peptide elongation rate may provide a key to the understanding of the regulation of protein synthesis.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5′-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.     

Molecular function of the dual-start motif in the λ S holin

The lambda S gene represents the prototype of holin genes with a dual-start motif, which leads to the synthesis of two polypeptides, S105 and S107. They differ at their N-terminus by only two amino acids, Met-1 and Lys-2, at the beginning of the longer product. Despite the minor difference, the two proteins have opposing functions in lysis, with protein S107 being an inhibitor and protein S105 being an effector of 'hole formation' in the inner membrane. Here, we have studied the molecular mechanism underlying the 'lysis clock' contributed by the dual-start motif. We have used protein fusions in which the secretory signal sequence of the M13 procoat protein VIII has been abutted to the N-terminal Met residues of S105 and S107 respectively. S-dependent 'hole formation' required removal of the signal sequence in both fusion proteins, as both the VIII-S105 and the VIII-S107 fusion proteins were non-functional when leader peptidase cleavage was inhibited. These results strongly supported the hypothesis that functional assembly of S proteins requires translocation of their N-terminus to the periplasm. Using signal sequence cleavage as a measure of translocation, we observed that the translocation kinetics of the N-terminus of the S107 moiety was reduced about threefold when compared with the N-terminus of the S105 moiety. Moreover, depolarization of the membrane resulted in an immediate cleavage of the signal sequence and 'hole formation' exerted by the S107 moiety of the VIII-S107 fusion protein. A model is presented in which S107 with a reversed topology of its N-terminus interacts with S105 and poisons 'hole formation'. Upon depolarization of the membrane, translocation of the N-terminus of S107 to the periplasm results in the functional assembly of S proteins, i.e. 'hole formation'.     

Dual targeting of higher-order DNA structures by azacryptands induces DNA junction-mediated DNA damage in cancer cells

DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.     

The hydrophilic C-terminal part of the lambda S holin is non-essential for intermolecular interactions

Available evidence indicates that oligomerization of the bacteriophage lambda S holin leads to a non-specific lesion in the cytoplasmic membrane which permits transit of the phage encoded transglycosylase to the periplasm. In an attempt to locate an intermolecular interaction domain in S a chimeric protein comprising the N-terminal 32 aa of phage PhiX174 lysis protein E and the last 75 aa of lambda S has been constructed. We report that the EΦS fusion protein is stable, membrane bound, and inhibits S-mediated lysis in trans. C-terminal truncations of the EΦS fusion protein indicated that the hydrophilic C-terminal end of S (i.e. the last 15 aa) is non-essential for oligomerization.     

Retinoic acid does not induce formation of cilia on the surface of wound epithelial cells in axolotls

It has been reported that vitamin A palmitate induces the production of cilia on the epidermal cells of the regenerating axolotl limb, and the formation of crevices in the epidermal surface. The aim of the present investigation was to reexamine under well defined conditions the potential of retinoids to evoke the above described metaplastic changes. In order to achieve our purpose we administered axolotls with retinoic acid for 2, 4, 6, 8, and 10 days after limb amputation. The young regenerates were inspected by scanning electron microscopy (SEM). The data obtained showed that the external layer of the wound epithelium and of the stump epidermis as well was quite normal without any sign of cilia formation. In some cases, crevices were observed even in control animals.     

Absence of egg discrimination in a suitable cuckoo Cuculus canorus host breeding away from trees

There is at present considerable variation in the level of antiparasite defences among different host species of avian brood parasites, but in many potential hosts some individuals reject poorly matching parasite eggs. Here we present unique absence of egg discrimination behaviour backed up by a lack of egg recognition abilities in a suitable common cuckoo Cuculus canorus host, the skylark Alauda arvensis. Skylarks did not show any clear rejection response to experimentally added highly non‐mimetic foreign eggs in any behavioural context, even before they had started laying or when the whole clutch was exchanged with foreign eggs. This absence of antiparasite defence can be explained by the breeding habitat of larks consisting of largely treeless open landscapes where cuckoos have little access to the nests, thereby eroding the possibility of coevolutionary interactions. Our results are strikingly consistent with the spatial habitat structure hypothesis proposed to explain the occurrence and extent of avian host‐parasite co‐adaptation.     

Marked differences in tissue-specific expression of chitinases in mouse and man.

Two distinct chitinases have been identified in mammals: a phagocyte-specific enzyme named chitotriosidase and an acidic mammalian chitinase (AMCase) expressed in the lungs and gastrointestinal tract. Increased expression of both chitinases has been observed in different pathological conditions: chitotriosidase in lysosomal lipid storage disorders like Gaucher disease and AMCase in asthmatic lung disease. Recently, it was reported that AMCase activity is involved in the pathogenesis of asthma in an induced mouse model. Inhibition of chitinase activity was found to alleviate the inflammation-driven pathology. We studied the tissue-specific expression of both chitinases in mice and compared it to the situation in man. In both species AMCase is expressed in alveolar macrophages and in the gastrointestinal tract. In mice, chitotriosidase is expressed only in the gastrointestinal tract, the tongue, fore-stomach, and Paneth cells in the small intestine, whereas in man the enzyme is expressed exclusively by professional phagocytes. This species difference seems to be mediated by distinct promoter usage. In conclusion, the pattern of expression of chitinases in the lung differs between mouse and man. The implications for the development of anti-asthma drugs with chitinases as targets are discussed.     

Diversity of neurodegenerative processes in the model of brain cortex tissue ischemia

Stroke is known to induce massive cell death in the ischemic brain. Either necrotic or apoptotic types of cell death program were observed in neurons in zone of ischemia. We suggest that spatial heterogeneity of glucose and oxygen distribution plays a crucial role in this phenomenon. In order to elucidate the role of glucose and oxygen in ischemic neurons choice of cell death pathway, conditions corresponding to different areas of insult were reproduced in vitro in the model of surviving brain cortex tissue slices. Three zones were modeled in vitro by varying glucose and oxygen concentration in surviving slices incubation media. Modeled ischemic area I (MIA I) was corresponded to the center of suggested ischemic zone where the levels of glucose and oxygen were considered to be extremely low. MIA II was assigned as intermediate area where oxygen concentration was still very low but glucose was present (this area was also divided into two sub-areas MIA IIa and MIA IIb with physiologically low (5 mM) and normal (10 mM) level of glucose respectively). MIA III was considered as a periphery area where glucose concentration was close to physiological level and high level of ROS production had been induced by reoxygenation after anoxia. Analysis of molecular mechanisms of cell death in MIA I, IIa, IIb and III was carried out. Cell death in MIA I was found to proceed by necrotic manner. Apoptosis characterized by cyt c release, caspase 3 activation and internucleosomal DNA fragmentation was observed in MIA III. Cell death in MIA II was accompanied by several (not all) hallmarks of apoptosis. Mechanisms of cell death in MIA IIa and MIA IIb were found to be different. Internucleosomal DNA fragmentation in MIA IIa but not in MIA IIb was sensitive to glycine (5 mM), inhibitor of NMDA receptor MK-801 (10 μM) and PTP inhibitor cyclosporine A (10 μM). Activation of caspase 3 was detected in MIA IIb but not in MIA IIa. However cytochrome c release was observed neither in MIA IIa nor in MIA IIb. In MIAs II–III apoptosis was accompanied by uncoupling of oxidative phosphorylation, which was induced by rise of intracellular Ca²⁺ and intensive ROS production. Results obtained in present study allow us to propose existence of at least four molecular pathways of cell death development in brain ischemic zone. The choice of cell death pathway is determined by oxygen and glucose concentration in the particular area of the ischemic zone.