Transcriptome Analysis Revealed Inflammation Is Involved in the Impairment of Human Umbilical Vein Endothelial Cells Induced by Post-hemorrhagic Shock Mesenteric Lymph.

Vascular endothelial injury caused by post-hemorrhagic shock mesenteric lymph (PHSML) return is an important manifestation during refractory hemorrhagic shock. Using human umbilical vein endothelial cells (HUVECs) and transcriptome analysis, this study sought to investigate the molecular mechanism underlying the adverse effect of PHSML on vascular endothelium. Post-hemorrhagic shock mesenteric lymph was collected from male rats after they underwent hemorrhagic shock and following resuscitation, while normal mesenteric lymph (NML) was harvested from sham rats.

XPD localizes in mitochondria and protects the mitochondrial genome from oxidative DNA damage.

Xeroderma pigmentosum group D (XPD/ERCC2) encodes an ATP-dependent helicase that plays essential roles in both transcription and nucleotide excision repair of nuclear DNA, however, whether or not XPD exerts similar functions in mitochondria remains elusive. In this study, we provide the first evidence that XPD is localized in the inner membrane of mitochondria, and cells under oxidative stress showed an enhanced recruitment of XPD into mitochondrial compartment. Furthermore, mitochondrial reactive oxygen species production and levels of oxidative stress-induced mitochondrial DNA (mtDNA) common deletion were significantly elevated, whereas capacity for oxidative damage repair of mtDNA was markedly reduced in both XPD-suppressed human osteosarcoma (U2OS) cells and XPD-deficient human fibroblasts.

RecQL4 helicase amplification is involved in human breast tumorigenesis.

Breast cancer occur both in hereditary and sporadic forms, and the later one comprises an overwhelming majority of breast cancer cases among women. Numerical and structural alterations involving chromosome 8, with loss of short arm (8p) and gain of long arm (8q), are frequently observed in breast cancer cells and tissues. In this study, we show that most of the human breast tumor cell lines examined display an over representation of 8q24, a chromosomal locus RecQL4 is regionally mapped to, and consequently, a markedly elevated level of RecQL4 expression.

An RNA-seq-based gene expression profiling of radiation-induced tumorigenic mammary epithelial cells.

Immortality and tumorigenicity are two distinct characteristics of cancers. Immortalization has been suggested to precede tumorigenesis. To understand the molecular mechanisms of tumorigenicity and cancer progression in mammary epithelium, we established a tumorigenic cell model by means of heavy-ion radiation of an immortal cell model, which was created by overexpressing the human telomerase reverse transcriptase (hTERT) in normal human mammary epithelial cells.

RecQL4 cytoplasmic localization: implications in mitochondrial DNA oxidative damage repair.

RecQL4, one of the five human RecQ helicases, is crucial for genomic stability and RecQL4 when mutated leads to premature aging phenotypes in humans. Unlike other human RecQ helicases, RecQL4 is found both in the nucleus and the cytoplasm. While the nuclear localization signal (NLS) and the retention domain at the N-terminus are responsible for the nuclear localization of RecQL4, the signal for its cytoplasmic localization is essentially unknown.

A multifunctional lentiviral-based gene knockdown with concurrent rescue that controls for off-target effects of RNAi.

The efficient, stable delivery of siRNA into cells, and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology. To overcome these drawbacks, we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell. To demonstrate the functional utility of this system, we performed RNAi-depleted α-actinin-1 (α-ACTNl) expression in human T cells.

Regulation of U6 promoter activity by transcriptional interference in viral vector-based RNAi.

The direct negative impact of the transcriptional activity of one component on the second one in cis is referred to as transcriptional interference (TI). U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector.

Regulation of p53 nuclear export through sequential changes in conformation and ubiquitination.

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation.

MDM2 binding induces a conformational change in p53 that is opposed by heat-shock protein 90 and precedes p53 proteasomal degradation.

p53 protein conformation is an important determinant of its localization and activity. Changes in p53 conformation can be monitored by reactivity with wild-type conformation-specific (pAb-1620) or mutant conformation-specific (pAb-240) p53 antibodies. Wild-type p53 accumulated in a mutant (pAb-240 reactive) form when its proteasome-dependent degradation was blocked during recovery from stress treatment and in cells co-expressing p53 and MDM2.

P53 and p73 differ in their ability to inhibit glucocorticoid receptor (GR) transcriptional activity.

Background: p53 is a tumor suppressor and potent inhibitor of cell growth. P73 is highly similar to p53 at both the amino acid sequence and structural levels. Given their similarities, it is important to determine whether p53 and p73 function in similar or distinct pathways.

Cdk2-dependent Inhibition of p21 stability via a C-terminal cyclin-binding motif.

p21 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors that includes p21, p27, and p57. Recent studies have suggested that Cdk2 activity may promote p21 degradation through a pathway similar to that for p27, although the mechanism by which this occurs has not been clarified. In the current report, co-expression with cyclin E and Cdk2 stabilized p21 in a manner that required the CDK-binding site of p21 and a cyclin-binding site (cy1) located in the p21 N terminus.

A link between p73 transcriptional activity and p73 degradation.

The p53 family of proteins includes three members, p53, p63, and p73. The levels and stability of p53 are controlled in large part by MDM2, which can bind the p53 N-terminus and promote its degradation. Because the MDM2 gene is transcriptionally activated by p53, it forms part of an autoregulatory feedback loop that directly links the transcriptional activity of p53 with its degradation.

Stalk region of beta-chain enhances the coreceptor function of CD8.

CD8 glycoproteins are expressed as either alphaalpha homodimers or alphabeta heterodimers on the surface of T cells. CD8alphabeta is a more efficient coreceptor than the CD8alphaalpha for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain.

Cloning and characterization of N4WBP5A, an inducible, cyclosporine-sensitive, Nedd4-binding protein in human T lymphocytes.

We have cloned and characterized a human cDNA, designated N4WBP5A, that belongs to the family of Nedd4-binding proteins. We originally identified N4WBP5A as an unknown expressed sequence tag (AA770150) represented in a cDNA microarray analysis that was up-regulated upon activation of T cells and inhibited by cell treatment with the calcineurin phosphatase inhibitors, cyclosporine (CsA) and tacrolimus (FK506). The predicted N4WBP5A amino acid sequence of 242 amino acid residues reveals an open reading frame of 729 nucleotides with a corresponding molecular mass of 27.

Inactivation of NF-kappaB-dependent cell survival, a novel mechanism for the proapoptotic function of c-Abl.

Using a system that expresses a constitutively kinase-active c-Abl protein [c-Abl(KA)], we identified the protein IkappaBalpha as a novel substrate of c-Abl. This kinase-substrate relationship is not only confirmed at the level of endogenous proteins but is also supported by a physical interaction between the two proteins. Interestingly, the association of c-Abl with IkappaBalpha, which is detectable in the form of nonphosphorylated proteins, is remarkably enhanced by an inducible binding of tyrosine-phosphorylated IkappaBalpha to the c-Abl SH2 domain.

Mutual dependence of MDM2 and MDMX in their functional inactivation of p53.

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments.