Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency.

We examined the contribution of a cryptic plasmid, pRmeGR4b, to the nodulation of Medicago sativa by strain GR4 of Rhizobium meliloti. A 905-base-pair PstI DNA fragment in pRmeGR4b was found to hybridize DNA of the R. meliloti fixA promoter region as a probe. Sequence analysis of the PstI fragment showed a 206-base-pair region displaying high homology with the DNA upstream of the RNA start points of the P1 and P2 symbiotic promoters. Putative nif promoter consensus sequences were conserved in this DNA segment. Expression of DNA downstream of the nif promoterlike sequence, monitored by beta-galactosidase activity of different lacZ fusions, was demonstrated to depend on a functional nifA gene, both in microaerobically free-living cells and in nodules. Individual transposon Tn3-HoHo1 insertions in this DNA region caused a reduced nodulation competitiveness. This new symbiotic region, occupying approximately 5 kilobases of pRmeGR4b DNA, was called nfe (nodule formation efficiency).     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.     

Blocked and test-stimulus exposure effects in perceptual learning re-examined

Three experiments re-examined the effects of blocked or alternated exposure to the conditioning and test stimuli and the effect of simple exposure to the test stimulus, on stimulus generalization. In all experiments rats received conditioning where a compound flavor, AX, was paired with LiCl-induced illness. All rats were tested for generalization with another flavor, BX. In Experiment 1, rats that received alternating exposure to the two flavor compounds, AX and BX, prior to conditioning showed less generalization to BX than rats that received no exposure. Exposure to BX or AX alone was also somewhat effective in reducing generalization. In Experiment 2 blocked exposure to AX and BX prior to conditioning was effective in reducing generalization, as was alternated exposure, and extended exposure to BX was more effective than the other procedures. In Experiment 3, exposure to X alone prior to conditioning produced generalization equal to that produced by alternated or blocked exposure and replicated the effect of extended exposure to BX found in the previous experiment. The relevance of the results to the theories proposed by McLaren and Macintosh [Anim. Learn. Behav. 28 (2000) 211] and Hall [Q. J. Exp. Psychol. B 56 (2003) 43] is discussed.     

The disruption of a gene encoding a putative arylesterase impairs pyruvate dehydrogenase complex activity and nitrogen fixation in Sinorhizobium meliloti

Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the trichloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3' end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.     

The extinction context enables extinction performance after a change in context

One experiment with human participants determined the extent to which recovery of extinguished responding with a context switch was due to a failure to retrieve contextually controlled learning, or some other process such as participants learning that context changes signal reversals in the meaning of stimulus-outcome relationships. In a video game, participants learned to suppress mouse clicking in the presence of a stimulus that predicted an attack. Then, that stimulus underwent extinction in a different context (environment within the game). Following extinction, suppression was recovered and then extinguished again during testing in the conditioning context. In a final test, participants that were tested in the context where extinction first took place showed less of a recovery than those tested in a neutral context, but they showed a recovery of suppression nevertheless. A change in context tended to cause a change in the meaning of the stimulus, leading to recovery in both the neutral and extinction contexts. The extinction context attenuated that recovery, perhaps by enabling retrieval of the learning that took place in extinction. Recovery outside an extinction context is due to a failure of the context to enable the learning acquired during extinction, but only in part.