Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. II. Effect of the nitrogen source on the nitrite reductase levels

The effect of different inorganic nitrogen sources on the cellular levels of nitrite reductase (NiR. EC 1.7.7.1) activity has been studied in the filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl,). Nitrate-grown cells gave the highest NiR in cell-free extracts [ca 165 nmol of nitrite reduced (mg protein)-1 min-], whereas no activity could be detected in extracts from ammonium-grown cells. The in vivo effect of ammonium on NiR was similar to that exerted by chloramphenicol, suggesting that de novo synthesis of protein was probably repressed by this ion. When ammonium was removed from the culture medium, a rapid increase of de novo synthetized NiR occurred, and the appearance of the enzyme was slightly stimulated by the presence in the medium of either nitrate or nitrite. However, remarkably high levels of NiR [around 1.2 μmol of nitrite reduced (mg protein) ⁻¹min⁻¹] could be routinely measured in nitrogen-deficient cells, indicating that the enzyme was ammonium-repressible rather than nitrate- or nitrite-inducible.     

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.MOMP³ is a key event in the intrinsic pathway of mammalian apoptosis, resulting in the release of several apoptogenic proteins from the mitochondrial intermembrane space into the cytosol (1). Released intraorganellar components, including cytochrome c, Smac/DIABLO, and AIF, then act as mediators for activating executioner caspase proteases or for other downstream events in the intracellular apoptosis cascade. MOMP is tightly regulated by BCL-2 family members, whose core components are proapoptotic BAX-type proteins that directly effect MOMP and antiapoptotic BCL-2-type proteins which inhibit MOMP (2, 3). In a currently popular model, a third subgroup of BCL-2 family proteins, the BH3-only proteins, trigger a set of conformational changes in BAX and/or its close homologue BAK that activates their permeabilizing function, thereby causing MOMP.Multiple proteins implicated in mitochondrial morphogenesis during normal growth conditions can cross-talk with BCL-2 family members to affect the mitochondrial pathway of apoptosis (4). For example, the large dynamin-like GTPase DLP1/Drp1 and hFis1, two essential components of the mitochondrial fission machinery, have been shown to modulate pro-apoptotic BAX function and mitochondrial cytochrome c release by acting at the level of the MOM (5–7). However, although excessive mitochondrial fragmentation is characteristic in mammalian apoptosis, controversy persists as to whether this phenomenon is merely coincident with or causatively linked to MOMP induction (4 – 8). In addition, a considerable body of evidence has amassed indicating that DLP1/Drp1 and hFis1 are multifunctional proteins that do not use the same mechanisms to reshape mitochondria in healthy conditions and to promote release of mitochondrial intermembrane space proteins during apoptosis (7–10).Endophilin B1/BAX-interacting factor 1 (Bif-1) is another protein linking mitochondrial morphological changes and BCL-2-regulated programmed cell death (4). On the one hand, Bif-1 is known to participate downstream of DLP1/Drp1, modulating normal MOM morphological dynamics in healthy cells (11). On the other hand, in response to specific apoptotic signals, a significant portion of Bif-1 binds BAX at the MOM in close temporal correlation with BAX conformational change and cytochrome c release (12). In addition, increasing the levels of Bif-1 has been shown to accelerate BAX conformational change, caspase activation, and apoptotic cell death, whereas loss of Bif-1 delays all these processes (12, 13). Together, these previous findings point to an important contributing role of Bif-1 in BAX-driven MOMP during apoptosis, but the underlying molecular mechanism remains unknown.As other members of the endophilin family, Bif-1 contains an N-BAR (Bin-amphiphysin-Rvs) domain that has been shown to confer ability to these proteins for transforming flat lipid bilayers into high curvature buds, tubules, and vesicles in vitro (14–17). Crystallographic studies of the N-BAR domain of endophilin A1, a close homologue of Bif-1, revealed a crescent-shape homodimer with a positively charged concave surface which is believed to act like a molecular scaffold that impresses its own curvature on binding to negatively charged membranes (16, 18). Another distinguishing feature of endophilin N-BAR domains is the presence of two distinct amphipathic segments referred to as “Helix 0” (H0) and “Helix 1 insert” (H1I) that penetrate only partway into the external leaflet and are thought to create a wedge effect that also increases membrane curvature. This dual curvature-generating mechanism has been linked to the shared capacity of endophilins to operate in membrane tubulovesicular dynamics during normal cell growth together with dynamin/dynamin-like proteins (16–18). However, exactly how the molecular-scale perturbation of membrane curvature induced by N-BAR domains translates into large scale membrane remodeling processes (e.g. tubulation and vesiculation) is not well understood (19–25). In addition, it is unclear whether the ability of Bif-1 to produce global changes in membrane morphology is functionally connected to its apoptotic mode of action.The complexity of the network of intermolecular interactions that controls the BCL-2-regulated MOMP pathway constitutes a major hurdle for gaining a molecular-level understanding of Bif-1 pro-death function in intact cells. Another complicating factor is that Bif-1 can interact with binding partners other than BAX at intracellular membranes distinct from the MOM depending on environmental conditions (14, 26–29). In previous studies this and other laboratories have shown that the BCL-2-regulated MOMP pathway can be reconstituted in a simplified system consisting of purified recombinant proteins and chemically defined MOM-like large unilamellar vesicles (LUV) in a manner that faithfully reflects the basic physiological functions of BCL-2 family proteins at the MOM (30–33). Here, we have used this minimal cell-free system to advance our understanding of the pro-death role of Bif-1. We provide strong evidence for a direct implication of Bif-1 in functional BAX activation at the membrane level and novel insights concerning the mechanism through which Bif-1 achieves this effect.     

DNA microarray technology: a new tool for the epidemiological typing of bacterial pathogens?

Genomic hybridization on whole genome arrays detects the presence or absence of similar DNA regions in sufficiently related microorganisms, allowing genome-wide comparison of their genetic contents. A whole genome array is based on a sequenced bacterial isolate, and is a collection of DNA probes fixed on a solid support. In a single hybridization experiment, the absence/presence status of all genes of the sequenced microbe in the queried isolate can be examined. The objective of this minireview is to summarize the past usage of DNA microarray technology for microbial strain characterizations, and to estimate its future utilization in epidemiological studies and molecular typing of bacterial pathogens. The studies reviewed here confirm the usefulness of microarray technology for the detection of genetic polymorphisms. However, the construction or purchase of DNA microarrays and the performance of strain to strain hybridization experiments are still prohibitively expensive for routine application. Future use of arrays in epidemiology is likely to depend on the development of more cost-effective protocols, more robust and simplified formats, and the adequate evaluation of their performance (efficacy) and convenience (efficiency) compared with other genotyping methods. It seems more likely that a more focused assay, concentrating on genomic regions of variability previously detected by genome-wide microarrays, will find broad application in routine bacterial epidemiology.