Transcriptional heterogeneity of catabolic genes on the plasmid pCAR1 causes host-specific carbazole degradation.

To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the Pant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The Pant promoter regulates the transcription of both the car and ant operons, which are responsible for converting carbazole into anthranilate and anthranilate into catechol, respectively. In the presence of anthranilate, transcription of the Pant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the Pant promoter followed by gfp was inserted into the chromosomes of KTPC and CA10. After adding anthranilate, GFP expression in the population of CA10 showed an unimodal distribution, whereas a small population with low GFP fluorescence intensity appeared for KTPC. CA10 has a gene, antRCA, that encodes an iso-functional homolog of AntR on its chromosome. When antRCA was disrupted, a small population with low GFP fluorescence intensity appeared. In contrast, overexpression of pCAR1-encoded AntR in KTPC resulted in unimodal expression under the Pant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the Pant promoter. Raman spectra of single cells collected using deuterium-labeled carbazole showed that the C-D Raman signal exhibited greater variability for KTPC than CA10. These results indicate that heterogeneity at the transcriptional level of the Pant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazole-degrading capacity of host bacterial cells.IMPORTANCEHorizontally acquired genes increase the competitiveness of host bacteria under selective conditions, although unregulated expression of foreign genes may impose fitness costs. The "appropriate" host for a plasmid is empirically known to maximize the expression of plasmid-borne traits. In the case of pCAR1-harboring Pseudomonas strains, P. resinovorans CA10 exhibits strong carbazole-degrading capacity, whereas P. putida KT2440 harboring pCAR1 exhibits low degradation capacity. Our results suggest that a chromosomally encoded transcription factor affects transcriptional and metabolic fluctuations in host cells, resulting in different carbazole-degrading capacities as a population. This study may provide a clue for determining appropriate hosts for a plasmid and for regulating the expression of plasmid-borne traits, such as the degradation of xenobiotics and antibiotic resistance.

The effects of palatinose on attention and cerebral blood flow in healthy adults: A randomized double-blind placebo-controlled crossover study.

OBJECTIVE: The primary energy source for the brain is glucose, and a continuous supply is required for the brain to work longer. This study aimed to verify the effects of palatinose on attention and cerebral blood flow in healthy adults. METHODS: This randomized, double-blind, placebo-controlled crossover study included 64 healthy Japanese adults. Participants performed the Digit Vigilance Task (DVT) 60 min pre-ingestion (14:00) and 0 (15:00), 60 (16:00), 120 (17:00), and 180 (18:00) min after ingestion of 10 g of either palatinose or glucose. Cerebral blood flow was measured using a wearable 2CH functional near-infrared spectrometer (fNIRS) during each DVT. The participants underwent the Uchida-Kraepelin (UK) test between each DVT to control for fatigue. RESULTS: DVT reaction times with palatinose intake were significantly shorter than those with glucose intake at 16:00, 17:00, and 18:00 (p = 0.0015, p < 0.001, and p < 0.001, respectively). The change in cerebral blood flow as a function of total hemoglobin level was significantly higher in the palatinose group than in the glucose group (p = 0.018). Regarding the post-UK mood questionnaire, "physically fatigued" and "annoyed" were significantly lower in the palatinose intake group compared to the glucose intake group at 17:00 (p = 0.0445 and p = 0.0318, respectively). Furthermore, "physically fatigued" was significantly lower, and "seriously" was higher in the palatinose intake compared to the glucose intake group at 18:00 (p = 0.00652 and p < 0.001, respectively). CONCLUSION: These findings suggest that 10 g of palatinose has favorable effects on attention and cerebral blood flow. TRIAL REGISTRATION: UMIN000046182.

Tyrosine phosphorylation of p145met mediated by EGFR and Src is required for serum-independent survival of human bladder carcinoma cells.

Here we address the molecular mechanism of serum-independent survival and growth of human bladder carcinoma cell line 5637. Serum starvation promoted tyrosine phosphorylation of a 145-kDa protein and activation of the tyrosine kinase Src and the receptor for epidermal growth factor (EGFR) over a slow time course (>8 hours). The phosphorylated 145-kDa protein was identified as the beta-subunit of c-Met/hepatocyte growth factor (HGF) receptor, p145(met), in which tyrosine residues 1003, 1234, and 1235 were phosphorylated. Inhibitors of Src (PP2, SU6656) or EGFR (AG99), but not p145(met) (K252a), effectively blocked tyrosine phosphorylation of p145(met) and promoted cell death accompanied by activation of caspase-like proteases. Conditioned medium from the serum-starved 5637 cells or purified EGF readily promoted the activation of Src and EGFR, and tyrosine phosphorylation of p145(met) in normally grown 5637 cells, suggesting that autocrine signaling of EGFR ligands is responsible for signal transduction events in serum-starved cells. Consistent with this idea, a monoclonal antibody against EGFR that would interfere with the ligand binding to EGFR blocked tyrosine phosphorylation events and promoted the caspase activation and cell death in serum-free conditions. Such apoptotic cell death was also induced by pretreatment of cells with a high concentration of HGF that downregulated endogenous p145(met). Nevertheless, Cu2+ ions, competitive inhibitors for HGF-binding to p145(met), did not show any effect on cellular functions in serum-free conditions. These results suggest that the serum-independent growth of 5637 cells involves the transmembrane signaling cascade via EGFR ligand(s) (but not HGF), EGFR, Src and p145(met).