Marni E. Cueno

A-kinase interacting protein 1 (AKIP1) acts as a molecular determinant of the role of PKA in NF-κB signaling

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-κB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-κB activity. In this study, we have demonstrated the effect of PKA on NF-κB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-κB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-κB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-κB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-κB activation; PKA-activating agents enhanced NF-κB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-κB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-κB-induced breast cancer cell proliferation and multiple NF-κB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-κB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

Role of the histone H3 lysine 9 methyltransferase Suv39 h1 in maintaining Epsteinn–Barr virus latency in B95–8 cells

The ability of Epstein–Barr Virus (EBV) to establish latent infection is associated with infectious mononucleosis and a number of malignancies. In EBV, the product of the BZLF1 gene (ZEBRA) acts as a master regulator of the transition from latency to the lytic replication cycle in latently infected cells. EBV latency is primarily maintained by hypoacetylation of histone proteins in the BZLF1 promoter by histone deacetylases. Although histone methylation is involved in the organization of chromatin domains and has a central epigenetic role in gene expression, its role in maintaining EBV latency is not well understood. Here we present evidence that the histone H3 lysine 9 (H3K9) methyltransferase suppressor of variegation 3–9 homolog 1 (Suv39 h1) transcriptionally represses BZLF1 in B95–8 cells by promoting repressive trimethylation at H3K9 (H3K9me3). Suv39 h1 significantly inhibited basal expression and ZEBRA‐induced BZLF1 gene expression in B95–8 B cells. However, mutant Suv39 h1 lacks the SET domain responsible for catalytic activity of histone methyl transferase and thus had no such effect. BZLF1 transcription was augmented when Suv39 h1 expression was knocked down by siRNA in B95–8 cells, but not in Akata or Raji cells. In addition, treatment with a specific Suv39 h1 inhibitor, chaetocin, significantly enhanced BZLF1 transcription. Furthermore, chromatin immunoprecipitation assays revealed the presence of Suv39 h1 and H3K9me3 on nucleosome histones near the BZLF1 promoter. Taken together, these results suggest that Suv39 h1–H3K9me3 epigenetic repression is involved in BZLF1 transcriptional silencing, providing a molecular basis for understanding the mechanism by which EBV latency is maintained.

Middle-aged rats orally supplemented with gel-encapsulated catechin favorably increases blood cytosolic NADPH levels

Green tea catechins are primarily known to function as free radical scavengers and have several beneficial uses. Orally supplemented catechin (OSC) was previously shown to increase mitochondrial heme and catalase levels in rat heart blood, however, its effect in the cytosol has not been elucidated. Here, we determined the effects of OSC in the rat heart blood cytosol. We used middle-aged (40 week-old) and young (4 week-old) rats throughout the study. We isolated blood cytosol, verified its purity, and determined heme, hydrogen peroxide (H2O2) levels, catalase (CAT) activities, gp91(phox) amounts, NADP and NAD pools, sirtuin 1 (SIRT1) and glutathione reductase (GR) activities, and free fatty acids (FFA). We established that OSC is associated with decreased heme-dependent H2O2 amounts while increasing heme-independent CAT activity. Moreover, we found that OSC-related decrease in NAD(+) amounts among middle-aged rats is associated to increased NADPH levels and SIRT1 activity. In contrast, we associated OSC-related decrease in NAD(+) amounts among young rats to decreased NADPH levels and increased SIRT1 activity. This highlights a major difference between catechin-treated middle-aged and young rats. Furthermore, we observed that cytosolic FFA and GR levels were significantly increased only among OSC-treated middle-aged rats which we hypothesize are related to increased NADPH levels. This insinuates that OSC treatment allows higher catechin amounts to enter the bloodstream of middle-aged rats. We propose that this would favorably increase NADPH amounts and lead to the simultaneous decrease in NADPH-related pro-oxidant activity and increase in NADPH-related biomolecules and anti-oxidant activities.

Re-discovering periodontal butyric acid: New insights on an old metabolite

The oral microbiome is composed of detrimental and beneficial microbial communities producing several microbial factors that could contribute to the development of the oral microbiome and, likewise, may lead to the development of host diseases. Metabolites, like short-chain fatty acids, are commonly produced by the oral microbiome and serve various functions. Among the periodontal short-chain fatty acids, butyric acid is mainly produced by periodontopathic bacteria and, attributable to the butyrate paradox, is postulated to exhibit a dual function depending on butyric acid concentration. A better understanding of the interconnecting networks that would influence butyric acid function in the oral cavity may shed a new light on the current existing knowledge and view regarding butyric acid.

Neuraminidase-producing oral mitis group streptococci potentially contribute to influenza viral infection and reduction in antiviral efficacy of zanamivir

Influenza is a serious respiratory disease among immunocompromised individuals, such as the elderly, and its prevention is an urgent social issue. Influenza viruses rely on neuraminidase (NA) activity to release progeny viruses from infected cells and spreading the infection. NA is, therefore, an important target of anti-influenza drugs. A causal relationship between bacteria and influenza virus infection has not yet been established, however, a positive correlation between them has been reported. Thus, in this study, we examined the biological effects of oral mitis group streptococci, which are predominant constituents of human oral florae, on the release of influenza viruses. Among them, Streptococcusoralis ATCC 10557 and Streptococcus mitis ATCC 6249 were found to exhibit NA activity and their culture supernatants promoted the release of influenza virus and cell-to-cell spread of the infection. In addition, culture supernatants of these NA-producing oral bacteria increased viral M1 protein expression levels and cellular ERK activation. These effects were not observed with culture supernatants of Streptococcus sanguinis ATCC 10556 which lacks the ability to produce NA. Although the NA inhibitor zanamivir suppressed the release of progeny viruses from the infected cells, the viral release was restored upon the addition of culture supernatants of NA-producing S. oralis ATCC 10557 or S. mitis ATCC 6249. These findings suggest that an increase in the number of NA-producing oral bacteria could elevate the risk of and exacerbate the influenza infection, hampering the efficacy of viral NA inhibitor drugs.

Orally supplemented catechin increases heme amounts and catalase activities in rat heart blood mitochondria: A comparison between middle-aged and young rats

Orally-administered catechin has long been known to have several beneficial effects on the mammalian host, however, the effects of orally supplemented catechin on the host through gingival tissues have not yet been established. Here, we elucidated the effects of orally supplemented catechin in the rat heart blood mitochondria. We used middle-aged (40 week-old) and young (4 week-old) rats throughout the study. We indirectly verified blood serum catechin levels by measuring O-methyl catechin derivatives using HPLC. Interestingly, we observed higher blood serum O-methyl levels in middle-aged rats as compared to young rats. Subsequently, we isolated blood mitochondria, verified its purity, and measured heme, hydrogen peroxide, and catalase (CAT) levels. We found that catechin induces an increase in blood mitochondrial heme amounts and is associated with an increase in blood mitochondrial CAT activity which is surprisingly higher in middle-aged rats as compared to young rats. This would imply that orally supplemented catechin induces heme increase that preferentially favours CAT activity and is more beneficial to the middle-aged rats.

Cytokinin dehydrogenase differentially regulates cytokinin and indirectly affects hydrogen peroxide accumulation in tomato leaf

Cytokinin dehydrogenase (CKX) catalyzes the irreversible degradation of cytokinins (CKs). CKs play a role in the regulation of hydrogen peroxide (H(2)O(2)) accumulation while H(2)O(2) is involved in chlorophyll degradation. Here, we elucidated how CKX differentially regulates representative CK levels and indirectly influence H(2)O(2) accumulation in tomato leaves. We induced drought, salt and iron-deficiency stresses in tomato plants and found that chlorosis-inducing stresses (salt and iron-deficiency) induced lower chlorophyll levels as compared to drought stress and control. Protein gel blot analysis detected two CKXs in tomato leaves: a 35 kDa protein (CKX35) found in chlorotic leaves associated with a reduction in representative CK levels and high H(2)O(2) concentrations, and a 37 kDa protein (CKX37) found in green leaves associated with representative CKs and H(2)O(2) at normal levels. In summary, CKX isoforms in the tomato leaf appear to have distinct roles in differentially regulating CK levels and indirectly influencing H(2)O(2) accumulation.

Various cellular stress components change as the rat ages: An insight into the putative overall age-related cellular stress network

&NA; Cellular stress is mainly comprised of oxidative, nitrosative, and endoplasmic reticulum stresses and has long been correlated to the ageing process. Surprisingly, the age‐related difference among the various components in each independent stress pathway and the possible significance of these components in relation to the overall cellular stress network remain to be clearly elucidated. In this study, we obtained blood from ageing rats upon reaching 20‐, 40‐, and 72‐wk.‐old. Subsequently, we measured representative cellular stress‐linked biomolecules (H2O2, glutathione reductase, heme, NADPH, NADP, nitric oxide, GADD153) and cell signals [substance P (SP), free fatty acid, calcium, NF‐&kgr;B] in either or both blood serum and cytosol. Subsequently, network analysis of the overall cellular stress network was performed. Our results show that there are changes affecting stress‐linked biomolecules and cell signals as the rat ages. Additionally, based on our network analysis data, we postulate that NADPH, H2O2, GADD153, and SP are the key components and the interactions between these components are central to the overall age‐related cellular stress network in the rat blood. Thus, we propose that the main pathway affecting the overall age‐related cellular stress network in the rat blood would entail NADPH‐related oxidative stress (involving H2O2) triggering GADD153 activation leading to SP induction which in‐turn affects other cell signals. HighlightsCellular stress components vary throughout the ageing process.Independent stress pathways are interrelated.Ageing affects the overall cellular stress network.

Ab initio modeling approach towards establishing the structure and docking orientation of the Porphyromonas gingivalis FimA.

Porphyromonas gingivalis FimA is a major aetiological agent in periodontal disease development, however, its structure has never been determined. Here, we established the mature P. gingivalis FimA ab initio model of all six FimA variants. We determined the conserved amino acid sequences of each FimA variant and generated mature FimA models. Subsequently, we validated their quality, protein empirical distribution, and radius of gyration. Similarly, structural comparison and topological orientation were elucidated, and the probable protein-protein docking was investigated. We found that the putative mature FimA model is β-sheet-rich and, likewise, we observed that each mature FimA model has varying levels of structural differences which can be topologically subdivided into the upper, middle, and lower FimA sections. Moreover, we found that the FimA epithelial cell-binding domain (EBD) is structurally conserved within the middle FimA section of all variants and FimA-FimA docking suggests that the FimA EBDs are oriented in opposite and alternating directions of each other.

Structural Insights on the Potential Significance of the Twin Asn-Residue Found at the Base of the Hemagglutinin 2 Stalk in All Influenza A H1N1 Strains: A Computational Study with Clinical Implications

Influenza hemagglutinin (HA) is a homotrimeric glycoprotein responsible for binding to sialic acids found in the host cell surface. HA has a prominent 75 Å-long α-helix (HA2 stalk) that contributes to overall HA structural stability. Among the H1N1 strains, a high level of predicted disorder is found at the base of the HA2 stalk, predominantly containing Asn residues. Surprisingly, the significance of Asn residues at the base of the HA2 stalk has not been elucidated. In this study, we analyzed the HA2 stalk base of 2830 amino acid sequences of the influenza H1N1 subtype obtained from human, swine, and avian strains throughout 1918-2012. We detected a structurally conserved twin Asn-residue (N145(2)-N146(2)) present at the HA2 stalk base in all H1N1 strains. In addition, we found that the twin Asn-residue maintains both a 2.13 Å salt bridge and 11.74 Å 110-helix:B-loop distance measurement in all H1N1 strains studied. Both observations were consistent with known HA crystal structures. Interestingly, amino acid substitutions in either or both residues 145(2) and 146(2) were found to alter these measurements and, likewise, shift the empirical distribution of HA amino acid residues. Thus, we hypothesize that both the N145(2) and N146(2) residues found at the HA2 stalk base in all H1N1 strains provide the necessary structural requirements to stabilize the HA protein. More importantly, this would imply that the twin-Asn-residue is an ideal target for anti-influenza therapies.