Cadmium‐induced cell death in BY‐2 cell culture starts with vacuolization of cytoplasm and terminates with necrosis

Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY‐2 cells treated with 50 µM CdSO₄. The cadmium‐induced alterations in cell morphology occurred gradually over a period of 3–4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium‐induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis.     

Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX⁺ foci), enhanced senescence-associated β-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the 'distended' morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.     

Two neuroactive steroids in midpregnancy as measured in maternal and fetal sera and in amniotic fluid

Allopregnanolone and pregnenolone sulfate, which are neuroactive steroids that differentially modulate the sensitivity of GABA(A) and NMDA receptors, were measured simultaneously in maternal and fetal sera and in amniotic fluid for the first time during the second trimester of gravidity. The study included 33 pregnant women, who underwent cordocentesis due to suspicion of fetal abnormality or alloimmunization. Allopregnanolone concentrations in maternal and fetal sera were similar and close to the previously found levels in healthy controls at 20 weeks of gestation The levels of pregnenolone sulfate in maternal serum were 2-3 times higher and in fetal serum, as much as 10-25 times higher than those found by others in the serum of healthy, non-pregnant women. A positive correlation between maternal and fetal allopregnanolone indicate similar 5alpha-reductase activities or the efficient transport of allopregnanolone between the two subjects. No correlation of pregnenolone sulfate levels between mother and fetus was found. This finding suggests the autonomous production of pregnenolone sulfate in mother and fetus.     

IrCL1 - the haemoglobinolytic cathepsin L of the hard tick, Ixodes ricinus

Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In our model tick, Ixodes ricinus, cathepsin L (IrCL1) is part of a gut-associated multi-peptidase complex; its endopeptidase activity is important in the initial phase of haemoglobinolysis. We present the functional and biochemical characterisation of this enzyme. We show, by RNA interference (RNAi), that cathepsin L-like activity that peaks during the slow feeding period of females is associated with IrCL1. Recombinant IrCL1 was expressed in bacteria and yeast. Activity profiling with both peptidyl and physiological protein substrates (haemoglobin and albumin) revealed that IrCL1 is an acidic peptidase with a very low optimum pH (3-4) being unstable above pH 5. This suggests an endo/lysosomal localisation that was confirmed by indirect fluorescence microscopy that immunolocalised IrCL1 inside the vesicles of digestive gut cells. Cleavage specificity determined by a positional scanning synthetic combinatorial library and inhibition profile indicated that IrCL1 has the ligand-binding characteristics of the cathepsin L subfamily of cysteine peptidases. A non-redundant proteolytic function was demonstrated when IrCL1-silenced ticks had a decreased ability to feed compared with controls. The data suggest that IrCL1 may be a promising target against ticks and tick-borne pathogens.     

Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ～5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.     

Luminescent properties of europium complexes with bis(diphenylphosphino)alkane dioxides

Eu(NO₃)₃?5H₂O and EuCl₃?6H₂O were allowed to react with bis(diphenylphosphino)alkane dioxides Ph₂P(O)(CH₂)_nP(O)Ph₂ (n = 2, 4, 6) to obtain polymeric and binuclear complexes. The prepared compounds were structurally characterized by X‐ray diffraction. Luminescence measurements (emission and excitation spectra, quantum yields, lifetimes) were compared with crystallographic data in order to find a relationship between luminescent properties of the Eu(III) complexes and their structures. The Eu(III) polymers, especially [Eu(dpphO₂)₂Cl₂]⁺Cl^–}_n, have shown extremely long luminescence lifetimes, up to 3.73 ms, as a result of a highly protecting hydrophobic shield. Copyright © 2011 John Wiley & Sons, Ltd.     

Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei

The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.     

A novel, cellulose synthesis inhibitory action of ancymidol impairs plant cell expansion

The co-ordination of cell wall synthesis with plant cell expansion is an important topic of contemporary plant biology research. In studies of cell wall synthesis pathways, cellulose synthesis inhibitors are broadly used. It is demonstrated here that ancymidol, known as a plant growth retardant primarily affecting gibberellin biosynthesis, is also capable of inhibiting cellulose synthesis. Its ability to inhibit cellulose synthesis is not related to its anti-gibberellin action and possesses some unique features never previously observed when conventional cellulose synthesis inhibitors were used. It is suggested that ancymidol targets the cell wall synthesis pathway at a regulatory step where cell wall synthesis and cell expansion are coupled. The elucidation of the ancymidol target in plant cells could potentially contribute to our understanding of cell wall synthesis and cell expansion control.     

Beobachtungen zu Biologie und Verhalten des Gro?en Kragenlaubenvogels (Chlamydera nuchalis)

Zusammenfassung Von Mai bis Juli 1969 wurde im Gebiet von Darwin und Arnhemland (Northern Territory, Australien) Biologie und Verhalten vonChlamydera nuchalis studiert.28 Lauben wurden gefunden. Einzelheiten des Laubenbaues und der hierzu verwendeten Materialien werden beschrieben. Das muß etwa 4–5000 Zweige zum Laubenplatz schaffen; die Wände der Laube bestehen aus etwa 1400 bis 1840 Zweigen.Die zur Ausschmückung der Laube verwendeten Gegenstände umfaßten Molluskenschalen, Knochen, Glasstückchen, Steine und kleine glänzende Metallobjekte. Das Gesamtgewicht des Laubenschmuckes betrug 6,6 bis 12,1 kg. Größe und Gewicht der Einzelobjekte schwankten zwischen 4–10 mm und 0,2–1,2 g bzw. 73 × 36 mm und 40 g. Ihre Gesamtzahl liegt zwischen 5000 und 12 000. Bevorzugt werden weiße und graue Objekte; auch grüne Glasstückchen wurden gefunden.Die Technik des Laubenbaus, die ausschließlich das ausführt, wird beschrieben. Der Rohbau nimmt etwa 3 Wochen in Anspruch, doch werden im Anschluß daran dauernd neue Objekte eingetragen. Anzeichen für das Ausmalen der Laube wurden nicht gefunden. Während der Balz singt das intensiv auf Bäumen oder am Boden. Die einzelnen Balzphasen werden beschrieben. Während des Höhepunktes versucht das durch Sprünge im Kreis das an der Laube zu halten. Begattung wurde außerhalb der Laube nahe dem Ausgang beobachtet. Offensichtlich hat ein mehrere während einer Balzperiode. Während der Balz wird die Laube dauernd ausgebessert und ihr Schmuck neu arrangiert.

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On the biology and behaviour of the Great Grey Bower Bird(Chlamydera nuchalis)

Summary From May to July, 1969 the author observed in the region of Darwin and Arnhem Land (Northern Territory, Australia) the biology and behaviour of the Great Grey Bower Bird.28 bowers were found, in three cases the building process was observed. Bower-building starts with cleaning up an area of 210 to 95 cm and with forming a 7.5 cm thick mat of small twigs intertwined in all directions. The walls are made of uniform twigs the measurements of which are on an average 29.2 mm in length and 2.4 mm in thickness at the lower end. A wall consists of 700 to 920 twigs placed parallel to each other (1400 to 1840 twigs on the two walls). The carries 4000 to 5000 twigs to the site of the bower. The paper gives the measurements of the bower and its orientation towards cardinal points.The bower is completed by a collection of ornaments which consists of land and sea shells of the generaXanthomelon, Venus andTelescopium, bone and glass fragments, stones (almost exclusively quartzite and limestone), and small pieces of glossy metal objects, tin seals etc. The lowest total weight of all objects collected was 6.2 kg, the highest was 12.1 kg. The size and weight of the objects varies between 4 to 10 mm and 0.2 to 1.2 g and 73 by 36 mm and 40 g respectively.Their total number was established at 5000 to 12000.Chlamydera nuchalis prefers white and grey objects with the exception of glass of which also green fragments were found. Some objects were experimentally painted blue, red, black, yellow and green. The discarded them successively and threw them out of the bower.The paper describes the technique of bower-building which is carried out solely by the . It takes the bird three weeks to finish the construction in the rough, but even afterwards the systematically continues to complete the bower, adding a growing number of ornamental objects. During the display period, however, a rebuilt a bower, which had been destroyed, within 2.5 days. The activities observed are shown in the table. Bower Birds may carry, on an average, 90 twigs and 28 glass fragments per hour to the bower. The bower wall painting behaviour ofChlamydera maculata andPtilonorhynchus violacaeus was not observed inChlamydera nuchalis. No traces of painting were found in any of the bowers observed. The author found an interesting behaviour pattern in two who stuck pieces of food into the cracks in the walls. This behaviour strikingly resembles that of passerines feeding their young.During the display period the sings ardently either on trees or on the ground. Display consists of alluring manifestations of the , including nape-crest presentation and bounds. At the bower, the shows the some objects on the opposite side of the passage, and after bowing and raising his head, he turns it so that the nape is directed toward the , and presents to her its lilac crest. During the culminating phase of display the attempts, by circling in leaps, to keep the at the bower. Mating was observed outside the bower, next to its exit. A apparently has several females during the display period. Also during display the rearranges and completes the bower.