Real-time imaging reveals endothelium-mediated leukocyte retention in LPS-treated lung microvessels.

Endotoxemia, a major feature of sepsis, is a common cause of acute lung injury and initiates rapid accumulation of leukocytes in the lung vasculature. Endothelial mechanisms that underlie this accumulation remain unclear, as current experimental models of endotoxemia are less suitable for targeted activation of the endothelium. Toward elucidating this, we used the isolated blood-perfused rat lung preparation. With a microcatheter inserted through a left atrial cannula, we cleared blood cells from a small lung region and then infused lipopolysaccharide (LPS) into microvessels. After a Ringer's wash to remove residual LPS, we infused fluorescently-labeled autologous leukocytes and imaged their transit through the treated microvessels. Image analysis revealed that leukocytes infused 90 min after LPS treatment were retained more in treated venules and capillaries than untreated vessels. Further, pretreatment with either the intercellular adhesion molecule-1 (ICAM-1) mAb or polymyxin-B blunted LPS-induced leukocyte retention in both microvessel segments. In addition, retention of leukocytes treated ex vivo with LPS in LPS-treated microvessels was higher compared to retention of untreated leukocytes. In situ immunofluorescence experiments revealed that LPS significantly increased microvessel ICAM-1 expression at 90 min post treatment. Polymyxin pretreatment inhibited this increase. Taken together, the data suggest that LPS increased leukocyte retention in both venules and capillaries and this response was mediated by the increased expression of endothelial ICAM-1. Thus, endothelial mechanisms may themselves play a major role in LPS-induced leukocyte retention in lung microvessels. Blunting the endothelial responses may mitigate endotoxin-induced morbidity.

A Novel Therapeutic Approach With Sodium Pyruvate on Vital Signs, Acid-Base, and Metabolic Disturbances in Rats With a Combined Blast and Hemorrhagic Shock.

Background: Blast injuries from improvised explosive devices (IEDs) are known to cause blast traumatic brain injuries (bTBIs), hemorrhagic shock (HS), organ damage, mitochondrial dysfunction, and subsequent free radical production. A pre-citric acid cycle reagent, pyruvate, is suggested to improve mitochondrial ATP production through the activation of the mitochondrial gatekeeper enzyme "pyruvate dehydrogenase complex (PDH)." Our study aimed to investigate the role of physiologic, metabolic, and mitochondrial effects of hypertonic sodium pyruvate resuscitation in rats with a combined blast and HS injury. Methods: A pre-clinical rat model of combined injury with repetitive 20 PSI blast exposure accompanied with HS and fluid resuscitation (sodium pyruvate as metabolic adjuvant or hypertonic saline as control), followed by transfusion of shed blood was used in this study. Control sham animals (instrumental and time-matched) received anesthesia and cannulation, but neither received any injury nor treatment. The mean arterial pressure and heart rate were recorded throughout the experiment by a computerized program. Blood collected at T0 (baseline), T60 (after HS), and T180 (end) was analyzed for blood chemistry and mitochondrial PDH enzyme activity. Results: Sodium pyruvate resuscitation significantly improved the mean arterial pressure (MAP), heart rate (HR), pulse pressure (PP), hemodynamic stability (Shock index), and autonomic response (Kerdo index) after the HS and/or blast injury. Compared with the baseline values, plasma lactate and lactate/pyruvate ratios were significantly increased. In contrast, base excess BE/( HCO 3 - ) was low and the pH was also acidotic <7.3, indicating the sign of metabolic acidosis after blast and HS in all animal groups. Sodium pyruvate infusion significantly corrected these parameters at the end of the experiment. The PDH activity also improved after the sodium pyruvate infusion. Conclusion: In our rat model of a combined blast and HS injury, hypertonic sodium pyruvate resuscitation was significantly effective in hemodynamic stabilization by correcting the acid-base status and mitochondrial mechanisms via its pyruvate dehydrogenase enzyme.

p53 binding to nucleosomal DNA depends on the rotational positioning of DNA response element.

The sequence-specific binding to DNA is crucial for the p53 tumor suppressor function. To investigate the constraints imposed on p53-DNA recognition by nucleosomal organization, we studied binding of the p53 DNA binding domain (p53DBD) and full-length wild-type p53 protein to a single p53 response element (p53RE) placed near the nucleosomal dyad in six rotational settings. We demonstrate that the strongest p53 binding occurs when the p53RE in the nucleosome is bent in the same direction as observed for the p53-DNA complexes in solution and in co-crystals. The p53RE becomes inaccessible, however, if its orientation in the core particle is changed by approximately 180 degrees. Our observations indicate that the orientation of the binding sites on a nucleosome may play a significant role in the initial p53-DNA recognition and subsequent cofactor recruitment.

Restoration of DNA-binding and growth-suppressive activity of mutant forms of p53 via a PCAF-mediated acetylation pathway.

Tumor-derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell-type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re-induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild-type p53 is modulated by various acetyl-transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl-transferase is down-regulated in tumors harboring p53 mutants, where its re-expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA-binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53-regulated promoters and transcriptional activity in vivo. These data suggest that PCAF-mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis-regulation of PCAF activity is a pre-requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53.

DNA dependent protein kinase (DNA-PK) enhances HIV transcription by promoting RNA polymerase II activity and recruitment of transcription machinery at HIV LTR.

Despite reductions in mortality from the use of highly active antiretroviral therapy (HAART), the presence of latent or transcriptionally silent proviruses prevents HIV cure/eradication. We have previously reported that DNA-dependent protein kinase (DNA-PK) facilitates HIV transcription by interacting with the RNA polymerase II (RNAP II) complex recruited at HIV LTR. In this study, using different cell lines and peripheral blood mononuclear cells (PBMCs) of HIV-infected patients, we found that DNA-PK stimulates HIV transcription at several stages, including initiation, pause-release and elongation. We are reporting for the first time that DNA-PK increases phosphorylation of RNAP II C-terminal domain (CTD) at serine 5 (Ser5) and serine 2 (Ser2) by directly catalyzing phosphorylation and by augmenting the recruitment of the positive transcription elongation factor (P-TEFb) at HIV LTR. Our findings suggest that DNA-PK expedites the establishment of euchromatin structure at HIV LTR. DNA-PK inhibition/knockdown leads to the severe impairment of HIV replication and reactivation of latent HIV provirus. DNA-PK promotes the recruitment of Tripartite motif-containing 28 (TRIM28) at LTR and assists the release of paused RNAP II through TRIM28 phosphorylation. These results provide the mechanisms through which DNA-PK controls the HIV gene expression and, likely, can be extended to cellular gene expression, including during cell malignancy, where the role of DNA-PK has been well-established.

Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR.

Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication.

SLC25A1, or CIC, is a novel transcriptional target of mutant p53 and a negative tumor prognostic marker.

Mutations of the p53 gene hallmark many human cancers. Several p53 mutant proteins acquire the capability to promote cancer progression and metastasis, a phenomenon defined as Gain of Oncogenic Function (GOF). The downstream targets by which GOF p53 mutants perturb cellular programs relevant to oncogenesis are only partially known. We have previously demonstrated that SLC25A1 (CIC) promotes tumorigenesis, while its inhibition blunts tumor growth. We now report that CIC is a direct transcriptional target of several p53 mutants. We identify a novel interaction between mutant p53 (mutp53) and the transcription factor FOXO-1 which is responsible for regulation of CIC expression levels. Tumor cells harboring mutp53 display higher CIC levels relative to p53 null or wild-type tumors, and inhibition of CIC activity blunts mutp53-driven tumor growth, partially overcoming GOF activity. CIC inhibition also enhances the chemotherapeutic potential of platinum-based agents. Finally, we found that elevated CIC levels predict poor survival outcome in tumors hallmarked by high frequency of p53 mutations. Our results identify CIC as a novel target of mutp53 and imply that the employment of CIC inhibitors may improve survival rates and reduce chemo-resistance in tumors harboring these types of mutations, which are among the most intractable forms of cancers.

An intrinsically disordered region of the acetyltransferase p300 with similarity to prion-like domains plays a role in aggregation.

Several human diseases including neurodegenerative disorders and cancer are associated with abnormal accumulation and aggregation of misfolded proteins. Proteins with high tendency to aggregate include the p53 gene product, TAU and alpha synuclein. The potential toxicity of aberrantly folded proteins is limited via their transport into intracellular sub-compartments, the aggresomes, where misfolded proteins are stored or cleared via autophagy. We have identified a region of the acetyltransferase p300 that is highly disordered and displays similarities with prion-like domains. We show that this region is encoded as an alternative spliced variant independently of the acetyltransferase domain, and provides an interaction interface for various misfolded proteins, promoting their aggregation. p300 enhances aggregation of TAU and of p53 and is a component of cellular aggregates in both tissue culture cells and in alpha-synuclein positive Lewy bodies of patients affected by Parkinson disease. Down-regulation of p300 impairs aggresome formation and enhances cytotoxicity induced by misfolded protein stress. These data unravel a novel activity of p300, offer new insights into the function of disordered domains and implicate p300 in pathological aggregation that occurs in neurodegeneration and cancer.

Clinical significance of P53 and Bcl-2 in acute myeloid leukemia patients of Eastern India.

The frequency of p53 and Bcl-2 protein expression in 100 newly diagnosed and 10 relapsed acute myeloid leukemia (AML) patients was analyzed by immunocytochemistry (ICC). The Kaplan-Meier method was used for univariate and multivariate statistical analysis to assess the relationship between p53, Bcl-2 and clinico-hematologic feature with respect to overall survival (OS) using SPSS statistical software. No statistical significance was found in univariate analysis (P=0.60). However, when the subgroups of patients (+1, +2, +3 and +4) were compared, expression of p53 and Bcl-2 protein (1-10%, 11-30%, 31-50% and >50%) was statistically significant (P<0.05). However, in multivariate analysis, p53, immunopositivity was independently associated with a shorter overall survival (OS) (P=0.038) while Bcl-2 immunopositivity was associated with longer overall survival (OS) (P=0.002). Our finding shows that p53 and Bcl-2 protein overexpression is a strong indicator of response to chemotherapy and overall survival. This study reports for the first time AML in patients from Eastern India.