A connecter-like factor, CacA, links RssB/RpoS and the CpxR/CpxA two-component system in Salmonella

BACKGROUND: Bacteria integrate numerous environmental stimuli when generating cellular responses. Increasing numbers of examples describe how one two-component system (TCS) responds to signals detected by the sensor of another TCS. However, the molecular mechanisms underlying this phenomenon remain poorly defined. RESULTS: Here, we report a connector-like factor that affects the activity of the CpxR/CpxA two-component system in Salmonella enterica serovar Typhimurium. We isolated a clone that induced the expression of a cpxP-lac gene fusion from a high-copy-number plasmid pool of random Salmonella genomic fragments. A 63-amino acid protein, CacA, was responsible for the CpxA/CpxR-dependent activation of the cpxP gene. The CpxR-activated genes cpxP and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean cacA deletion mutant strain in comparison to wild-type. From 33 response regulator (RR) deletion mutants, we identified that the RssB regulator represses cacA transcription. Substitution mutations in a conserved -10 region harboring the RNA polymerase recognition sequence, which is well conserved with a known RpoS -10 region consensus sequence, rendered the cacA promoter RpoS-independent. The CacA-mediated induction of cpxP transcription was affected in a trxA deletion mutant, which encodes thioredoxin 1, suggesting a role for cysteine thiol-disulfide exchange(s) in CacA-dependent Cpx activation. CONCLUSIONS: We identified CacA as an activator of the CpxR/CpxA system in the plasmid clone. We propose that CacA may integrate the regulatory status of RssB/RpoS into the CpxR/CpxA system. Future investigations are necessary to thoroughly elucidate how CacA activates the CpxR/CpxA system

Ethanol-induced Stress Leads to Apoptosls Via Endoplasmic Reticulum Stress in SK-Hepl Cells

Alcoholic liver disease causes oxidative stress and induces apoptosis during alcohol metabolism. Ethanol causes endoplasmic reticulum (ER) stress in hepatocytes, stimulating the unfolded protein response (UPR) pathway and/or Ca2+-dependent calpain and caspase-4 activities. However, it is poorly understood whether ethanol-induced oxidative stress directly leads to apoptosis promoted by ER stress-associated pathways. This study investigated this question in human liver adenocarcinoma (SK-Hep1) cells, which were treated with 200 mM ethanol for 5 hours in the presence or absence of the antioxidant N-acetyl-cysteine (NAC). We found that treatment with ethanol significantly increased ROS production and cellular apoptosis in the SK-Hep1 cells, and that this response was significantly suppressed by pretreatment with NAC. Furthermore, pretreatment with NAC significantly reduced the observed increases in the mRNA expressions of Bip, Chop, and sXbp-1, and the activity of caspase-3 in ethanol-induced apoptotic cells. However, pretreatment with NAC did not attenuate the transient rise in cytosolic Ca2+ nor the activities of caspase-4 and calpain induced by ethanol. Together, these results revealed that ethanol-induced stress promotes apoptosis not only through mitochondria-mediated pathways, but also via ER stress. The findings further suggested that ethanol-induced oxidative stress and non-oxidative stress both stimulate the pathway regulating ER stress-mediated apoptosis

Increase in Matrix Metalloproteinase-2 and 9 in the Liver of Nonalcoholic Steatohepatitis (NASH) Model Rats

Nonalcoholic steatohepatitis (NASH) is regarded as a hepatic manifestation of the metabolic syndrome which can progress to hepatic cirrhosis and hepatocellular carcinoma. It is thought that matrix metalloproteinases (MMPs) play an important role in hepatic fibrosis and we previously reported a correlation between oxidative stress and MMP-9 expression. However, the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the progression of NASH is unclear. In this study we used spontaneously hypertensive and hyperlipidemic rats (SHHR) fed a high-fat diet and 30% sucrose solution (HFDS) as a model for NASH, in order to clarify the relationships between oxidative stress, liver weight (LW), MMPs and TIMPs at various time-points in the progression of NASH. Male SHHR and Sprague-Dawley (SD) rats were divided into four groups: SHHR-normal diet (ND), SHHR-HFDS, SD-ND and SD-HFDS. Hepatic fibrosis was clearly increased at 13 months in SHHR-HFDS, resembpling NASH. LW and oxidative stress markers in plasma were increased in SHHR-HFDS compared to the other groups. Oxidative stress was correlated with LW in all rats. Expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA, measured by real-time polymerase chain reaction, was increased in the liver of SHHR-HFDS at 13 months. This study suggests that oxidative stress, MMPs and TIMPs may play an important role in the progression of NASH

A connecter-like factor, CacA, links RssB/RpoS and the CpxR/CpxA two-component system in <it>Salmonella</it>

Abstract Background Bacteria integrate numerous environmental stimuli when generating cellular responses. Increasing numbers of examples describe how one two-component system (TCS) responds to signals detected by the sensor of another TCS. However, the molecular mechanisms underlying this phenomenon remain poorly defined. Results Here, we report a connector-like factor that affects the activity of the CpxR/CpxA two-component system in Salmonella enterica serovar Typhimurium. We isolated a clone that induced the expression of a cpxP-lac gene fusion from a high-copy-number plasmid pool of random Salmonella genomic fragments. A 63-amino acid protein, CacA, was responsible for the CpxA/CpxR-dependent activation of the cpxP gene. The CpxR-activated genes cpxP and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean cacA deletion mutant strain in comparison to wild-type. From 33 response regulator (RR) deletion mutants, we identified that the RssB regulator represses cacA transcription. Substitution mutations in a conserved -10 region harboring the RNA polymerase recognition sequence, which is well conserved with a known RpoS -10 region consensus sequence, rendered the cacA promoter RpoS-independent. The CacA-mediated induction of cpxP transcription was affected in a trxA deletion mutant, which encodes thioredoxin 1, suggesting a role for cysteine thiol-disulfide exchange(s) in CacA-dependent Cpx activation. Conclusions We identified CacA as an activator of the CpxR/CpxA system in the plasmid clone. We propose that CacA may integrate the regulatory status of RssB/RpoS into the CpxR/CpxA system. Future investigations are necessary to thoroughly elucidate how CacA activates the CpxR/CpxA system.</p

(-)-Epigallocatechin-3-gallate Inhibits Differentiation and Matrix Metalloproteinases Expression in Osteoclasts

The osteoclast is a multinucleated giant cell differentiated from monocyte macrophages that has an important role in bone resorption. Several studies have reported a relationship between tea consumption and decreased risk of bone fracture. Matrix metalloproteinases (MMPs) play an important role in the degeneration of bone and cartilage matrix. Regulation of osteoclast activity is essential in the treatment of bone disease. Moreover, MMPs are associated with osteoclast formation and differentiation. We have reported previously that (-) -epigallocatechin-3-gallate (EGCG) inhibits MMP-2 and MMP-9 expression and activity. However, the effects of EGCG on osteoclasts and other MMPs are not clear. Therefore, in the present study we examined whether EGCG affects MMP expression, as well as osteoclast formation, differentiation and activity, in vitro. We used bone marrow cells from the femur and tibial bones of male ddY mice. Bone marrow cells were cultured in the presence of 1-100µM EGCG for 6 or 8 days. EGCG decreased the number of mature osteoclasts, as determined by tartrate-resistant acid phosphatase staining. Concentrations as low as 1µM EGCG clearly inhibited the differentiation of osteoclasts from bone marrow cells. EGCG also inhibited the number of osteoclasts with an actin-ring, as determined by rhodamine phalloidin staining, as well as osteoclast activity, as determined by the pit formation assay. Furthermore, EGCG concentration-dependently decreased MMP-9 and membrane type 1-MMP mRNA expression in mouse osteoclasts. However, EGCG had no changing on mRNA levels of tissue inhibitor of metalloprotease (TIMP)-1 and TIMP-3. Together, the results suggest that EGCG may be a suitable agent or lead compound for the development of treatments for bone resorption diseases associated with MMPs

Risk factors predicting osteosarcopenia in postmenopausal women with osteoporosis: A retrospective study.

There is growing interest in "osteosarcopenia" as the coexistence of osteoporosis and sarcopenia exacerbates negative outcomes. However, limited information is available regarding the risk factors of osteosarcopenia development in patients with osteoporosis. Therefore, we retrospectively reviewed 276 consecutive patients with postmenopausal osteoporosis who regularly visited Showa University Hospital. Patients were eligible for the study if they were ≥65 years of age and underwent dual-energy X-ray absorptiometry, blood sampling, and physical performance assessment. Patients were divided into the osteosarcopenia and osteoporosis alone groups according to the diagnostic criteria of the Asian Working Group for Sarcopenia. Of the 276 patients with osteoporosis, 54 patients (19.6%) had osteosarcopenia. Patients in the osteosarcopenia group had a greater risk of frailty than did those in the osteoporosis alone group (odds ratio 2.33; 95% confidence interval, 1.13-4.80, P = 0.028). Low body mass index seemed to be the strongest factor related to the development of osteosarcopenia, and none of the patients in the osteosarcopenia group were obese (BMI ≥27.5 kg/m2). Multiple logistic analyses revealed that patients aged 65-74 years who had comorbidities such as kidney dysfunction and high levels of HbA1c were at risk of developing osteosarcopenia. Thus, we strongly recommend the assessment of the key components of the diagnosis of osteosarcopenia in an osteoporosis clinic for patients with low body mass index. Furthermore, appropriate assessments, including comorbidities, will help in identifying patients at greater risk of developing osteosarcopenia

Multicenter Retrospective Analysis of Intradiscal Condoliase Injection Therapy for Lumbar Disc Herniation

Background and Objectives: Intradiscal injection of Condoliase (chondroitin sulfate ABC endolyase), a glycosaminoglycan-degrading enzyme, is employed as a minimally invasive treatment for lumbar disc herniation (LDH) and represents a promising option between conservative treatment and surgical intervention. Since its 2018 approval in Japan, multiple single-site trails have highlighted its effectiveness, however, the effect of LDH types, and influences of patient age, sex, etc., on treatment success remains unclear. Moreover, data on teenagers and elderly patients has not been reported. In this retrospective multi-center study, we sought to classify prognostic factors for successful condoliase treatment for LDH and assess its effect on patients &lt; 20 and &ge;70 years old. Materials and Methods: We reviewed the records of 137 LDH patients treated through condoliase at four Japanese institutions and assessed its effectiveness among different age categories on alleviation of visual analog scale (VAS) of leg pain, low back pain and numbness, as well as ODI and JOA scores. Moreover, we divided them into either a &ldquo;group-A&rdquo; category if a &ge;50% improvement in baseline leg pain VAS was observed or &ldquo;group-N&rdquo; if VAS leg pain improved &lt;50%. Next, we assessed the differences in clinical and demographic distribution between group-A and group-N. Results: Fifty-five patients were classified as group-A (77.5%) and 16 patients were allocated to group-N (22.5%). A significant difference in Pfirrmann classification was found between both cohorts, with grade IV suggested to be most receptive. A posterior disc angle &gt; 5&deg; was also found to approach statical significance. In all age groups, average VAS scores showed improvement. However, 75% of adolescent patients showed deterioration in Pfirrmann classification following treatment. Conclusions: Intradiscal condoliase injection is an effective treatment for LDH, even in patients with large vertebral translation and posterior disc angles, regardless of age. However, since condoliase imposes a risk of progressing disc degeneration, its indication for younger patients remains controversial