Impact of Antibiotics Associated with the Development of Toxic Epidermal Necrolysis on Early and Late-Onset Infectious Complications.

Toxic epidermal necrolysis (TEN) is a rare disease, which predominantly manifests as damage to the skin and mucosa. Antibiotics count among the most common triggers of this hypersensitive reaction. Patients with TEN are highly susceptible to infectious complications due to the loss of protective barriers and immunosuppressant therapy. The aim of this study was to investigate the potential relationship between antibiotics used before the development of TEN and early and late-onset infectious complications in TEN patients. In this European multicentric retrospective study (Central European Lyell syndrome: therapeutic evaluation (CELESTE)), records showed that 18 patients with TEN used antibiotics (mostly aminopenicillins) before the disease development (group 1), while in 21 patients, TEN was triggered by another factor (group 2). The incidence of late-onset infectious complications (5 or more days after the transfer to the hospital) caused by Gram-positive bacteria (especially by Enterococcus faecalis/faecium) was significantly higher in group 1 than in group 2 (82.4% vs. 35.0%, p = 0.007/p corr = 0.014) while no statistically significant difference was observed between groups of patients with infection caused by Gram-negative bacteria, yeasts, and filamentous fungi (p > 0.05). Patients with post-antibiotic development of TEN are critically predisposed to late-onset infectious complications caused by Gram-positive bacteria, which may result from the dissemination of these bacteria from the primary focus.

Anti-Atherogenic Properties of Allium ursinum Liophylisate: Impact on Lipoprotein Homeostasis and Cardiac Biomarkers in Hypercholesterolemic Rabbits.

The present investigation evaluates the capacity of Allium ursinum (wild garlic) leaf lyophilisate (WGLL; alliin content: 0.261%) to mitigate cardiovascular damage in hypercholesterolemic rabbits. New Zealand rabbits were divided into three groups: (i) cholesterol-free rabbit chow (control); (ii) rabbit chow containing 2% cholesterol (hypercholesterolemic, HC); (iii) rabbit chow containing 2% cholesterol + 2% WGLL (hypercholesterolemic treated, HCT); for eight weeks. At the zero- and eight-week time points, echocardiographic measurements were made, along with the determination of basic serum parameters. Following the treatment period, after ischemia-reperfusion injury, hemodynamic parameters were measured using an isolated working heart model. Western blot analyses of heart tissue followed for evaluating protein expression and histochemical study for the atheroma status determination. WGLL treatment mediated increases in fractional shortening; right ventricular function; peak systolic velocity; tricuspidal annular systolic velocity in live animals; along with improved aortic and coronary flow. Western blot analysis revealed WGLL-associated increases in HO-1 protein and decreases in SOD-1 protein production. WGLL-associated decreases were observed in aortic atherosclerotic plaque coverage, plasma ApoB and the activity of LDH and CK (creatine kinase) in plasma. Plasma LDL was also significantly reduced. The results clearly demonstrate that WGLL has complex cardioprotective effects, suggesting future strategies for its use in prevention and therapy for atherosclerotic disorders.

Wanderings in biochemistry.

My Ph.D. thesis in the laboratory of Severo Ochoa at New York University School of Medicine in 1962 included the determination of the nucleotide compositions of codons specifying amino acids. The experiments were based on the use of random copolyribonucleotides (synthesized by polynucleotide phosphorylase) as messenger RNA in a cell-free protein-synthesizing system. At Yale University, where I joined the faculty, my co-workers and I first studied the mechanisms of protein synthesis. Thereafter, we explored the interferons (IFNs), which were discovered as antiviral defense agents but were revealed to be components of a highly complex multifunctional system. We isolated pure IFNs and characterized IFN-activated genes, the proteins they encode, and their functions. We concentrated on a cluster of IFN-activated genes, the p200 cluster, which arose by repeated gene duplications and which encodes a large family of highly multifunctional proteins. For example, the murine protein p204 can be activated in numerous tissues by distinct transcription factors. It modulates cell proliferation and the differentiation of a variety of tissues by binding to many proteins. p204 also inhibits the activities of wild-type Ras proteins and Ras oncoproteins.

Conditionals by inversion provide a universal method for the generation of conditional alleles.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.

Memories of a senior scientist: on passing the fiftieth anniversary of the beginning of deciphering the genetic code.

2011 marked the fiftieth anniversary of breaking the genetic code in 1961. Marshall Nirenberg, the National Institutes of Health (NIH) scientist who was awarded the Nobel Prize in Physiology or Medicine in 1968 for his role in deciphering the code, wrote in 2004 a personal account of his research. The race for the code was a competition between the NIH group and Severo Ochoa's laboratory at New York University (NYU) School of Medicine, where I was a graduate student and conducted many of the experiments. I am now 83 years old. These facts prompt me to recall how I, together with Joe Speyer, an instructor in the Department of Biochemistry at NYU, unexpectedly became involved in deciphering the code, which also became the basis of my PhD thesis. Ochoa won the Nobel Prize in Physiology or Medicine in 1959 for discovering polynucleotide phosphorylase (PNP), the first enzyme found to synthesize RNA in the test tube. The story of how PNP made the deciphering of the code feasible is recalled here.

miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma.

Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3' untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.

The p200 family protein p204 as a modulator of cell proliferation and differentiation: a brief survey.

The expression of the murine p200 family protein p204 in numerous tissues can be activated by a variety of distinct, tissue-specific transcription factors. p204 modulates cell proliferation, cell cycling, and the differentiation of various tissues, including skeletal muscle myotubes, beating cardiac myocytes, osteoblasts, chondrocytes, and macrophages. This protein modulates these processes in various ways, such as by (1) blocking ribosomal RNA synthesis in the nucleolus, (2) inhibiting Ras signaling in the cytoplasm, (3) promoting the activity of particular transcription factors in the nucleus by forming complexes with them, and (4) overcoming the block of the activity of other transcription factors by inhibitor of differentiation (Id) proteins. Much remains to be learned about p204, particularly with respect to its expected involvement in the differentiation of several as yet unexplored tissues.

p204, a p200 family protein, as a multifunctional regulator of cell proliferation and differentiation.

The interferon-inducible p200 family comprises a group of homologous mouse and human proteins. Most of these have an N-terminal DAPIN domain and one or two partially conserved, 200 amino acid long C-terminal domains (designated as 200X domain). These proteins play important roles in the regulation of cell proliferation, tissue differentiation, apoptosis and senescence. p200 family proteins are involved also in autoimmunity and the control of tumor growth. These proteins function by binding to various target proteins (e.g. transcription factors, signaling proteins, oncoproteins and tumor suppressor proteins) and modulating target activity. This review concentrates on p204, a murine member of the family and its roles in regulating cell proliferation, cell and tissue differentiation (e.g. of skeletal muscle myotubes, beating cardiac myocytes, osteoblasts, chondrocytes and macrophages) and signaling by Ras proteins. The expression of p204 in various tissues as promoted by tissue-specific transcription factors, its distribution among subcellular compartments, and the controls of these features are also discussed.

p204 protein is a novel modulator of ras activity.

The murine p200 family protein, p204, modulates cell proliferation and tissue differentiation. Many of its activities are exerted in the nucleus. However, in cardiac myocytes, p204 accumulated in the cytoplasm. A yeast two-hybrid assay revealed a p204-cytoplasmic Ras protein interaction. This was confirmed (i) by coimmunoprecipitation of p204 with Ras in mouse heart extract and with endogenous or ectopic H-Ras and K-Ras in cell lysates as well as (ii) by binding of purified H-Ras-GTP to purified p204 in vitro. p204 inhibited (i) the cleavage of RasGTP to RasGDP by RasGAP; (ii) the binding to RasGTP of Raf-1, phosphatidylinositol 3-kinase, and Ral-GDS, effectors of Ras signaling; and (iii) activation by the Ras pathway of the phosphorylation and thus activation of downstream targets (e.g. MEK, Akt, and p38 MAPK). Oncogenic Ras expression triggered the phosphorylation and translocation of p204 from the nucleus to the cytoplasm. This is expected to increase the interaction between the two proteins. Translocation triggered by Ras oncoprotein was blocked by the LY294002 inhibitor of phosphatidylinositol 3-kinase. Ras did not promote phosphorylation or translocation to the cytoplasm of mutated p204 in which serine 179 was replaced by alanine. p204 overexpression inhibited the anchorage-independent proliferation of cells expressing Ras(Q61L) oncoprotein. Ras oncoprotein triggered in MEF3T3 cells the rearrangement of the actin cytoskeleton and the enhancement of cell migration through a membrane. Overexpression of p204 inhibited both. Ras oncoprotein or activated, wild-type Ras was described to increase Egr-1 transcription factor expression. We report that a sequence in the gene encoding p204 bound Egr-1, and Egr-1 activated p204 expression. Ras oncoprotein or activated wild-type Ras increased the expression in 3T3 cells of p204 together with that of Egr-1. Furthermore, the activation of expression of a single copy of K-ras oncogene in cultured murine embryonic cells induced the expression of a high level of p204 as well as its distribution between the nuclei and the cytoplasm. Thus, p204 may serve as a negative feedback inhibitor of Ras activity.

Multiple splicing results in at least two p203 proteins that are expressed in the liver and down-regulated during liver regeneration.

The interferon inducible p200 family proteins are expressed in a variety of cells and tissues. Intensive studies showed that they were involved in the regulation of cell proliferation and differentiation based on their ability to bind and modulate the activities of multiple transcription activators and inhibitors. Among p200 proteins, p203 has received the least attention and its function is unknown. In the present study, four multiple splicing isoforms of Ifi203, named temporally as Ifi203a/b-1, Ifi203a/b-2, Ifi203 a/b-3, and Ifi203a/b-4, were cloned from the interferon induced 10T1/2 cells. Anti-p203 antiserum was prepared and it could immunodetect a 46 kD and a 51 kD p203 proteins in a variety of cell lines. Unlike other p200 proteins, p203 was exclusively expressed in the liver in the adult C129/SvJ and C57BL/6 inbred strain mice. During the liver regeneration following a standard partial hepatectomy in C57BL/6 mice, the level of p203 decreased significantly in 6-24 h post-operation prior to the cell cycle progression through the G1/S transition.

The retinoblastoma protein is an essential mediator of osteogenesis that links the p204 protein to the Cbfa1 transcription factor thereby increasing its activity.

Bone formation requires the coordinated activity of numerous proteins including the transcription factor core-binding factor alpha1 (Cbfa1). Deregulation of Cbfa1 results in metabolic bone diseases including osteoporosis and osteopetrosis. The retinoblastoma protein (pRb) that is required for osteogenesis binds Cbfa1. We reported earlier that the p200 family protein p204, which is known to be involved in the differentiation of skeletal muscle myotubes, cardiac myocytes, and macrophages, also serves as a cofactor of Cbfa1 and promotes osteogenesis. In this study we established that suppression of p204 expression by an adenovirus construct encoding p204 antisense RNA inhibited osteoblast-specific gene activation by Cbfa1 in an osteogenesis assay involving the pluripotent C2C12 mesenchymal cell line. Using protein-protein interaction assays we established that Cbfa1, pRb, and p204 form a ternary complex in which pRb serves as a linker connecting p204 and Cbfa1. Chromatin immunoprecipitation assays revealed the binding of such a p204-pRb-Cbfa1 transcription factor complex to the promoter of the osteocalcin gene. The pRb requirement of the stimulation of Cbfa1 activity by p204 was established in experiments involving p204 mutants lacking one or two pRb binding (LXCXE) motifs. Such mutants failed to enhance the Cbfa1-dependent transactivation of gene expression as well as osteogenesis. Furthermore, as revealed in reporter gene and in vitro osteogenesis assays p204 synergized with pRb in the stimulation of Cbfa1-dependent gene activation and osteoblast differentiation.

Increased expression of Ifi202, an IFN-activatable gene, in B6.Nba2 lupus susceptible mice inhibits p53-mediated apoptosis.

Increased expression of p202 protein (encoded by the Ifi202 gene) in splenocytes derived from B6.Nba2 mice (congenic for the Nba2 interval derived from the New Zealand Black mice) was correlated with defects in apoptosis of splenic B cells and increased susceptibility to develop systemic lupus erythematosus. We have now investigated the molecular mechanisms by which increased expression of p202 in B6.Nba2 cells contributes to defects in apoptosis. In this study, we report that increased expression of p202 in the B6.Nba2 splenocytes, as compared with cells derived from the parental C57BL/6 (B6) mice, was correlated with increased levels of p53 protein and inhibition of p53-mediated transcription of target genes that encode proapoptotic proteins. Conversely, knockdown of p202 expression in B6.Nba2 cells resulted in stimulation of p53-mediated transcription. We found that p202 bound to p53 in the N-terminal region (aa 44-83) comprising the proline-rich region that is important for p53-mediated apoptosis. Consistent with the binding of p202 to p53, increased expression of p202 in B6.Nba2 mouse embryonic fibroblasts inhibited UV-induced apoptosis. Taken together, our observations support the idea that increased expression of p202 in B6.Nba2 mice increases the susceptibility to develop lupus, in part, by inhibiting p53-mediated apoptosis.

p204 is required for the differentiation of P19 murine embryonal carcinoma cells to beating cardiac myocytes: its expression is activated by the cardiac Gata4, Nkx2.5, and Tbx5 proteins.

Among 10 adult mouse tissues tested, the p204 protein levels were highest in heart and skeletal muscle. We described previously that the MyoD-inducible p204 protein is required for the differentiation of cultured murine C2C12 skeletal muscle myoblasts to myotubes. Here we report that p204 was also required for the differentiation of cultured P19 murine embryonal carcinoma stem cells to beating cardiac myocytes. As shown by others, this process can be triggered by dimethyl sulfoxide (DMSO). We established that DMSO induced the formation of 204RNA and p204. Ectopic p204 could partially substitute for DMSO in inducing differentiation, whereas ectopic 204 antisense RNA inhibited the differentiation. Experiments with reporter constructs, including regulatory regions from the Ifi204 gene (encoding p204) in P19 cells and in cultured newborn rat cardiac myocytes, as well as chromatin coimmunoprecipitations with transcription factors, revealed that p204 expression was synergistically transactivated by the cardiac Gata4, Nkx2.5, and Tbx5 transcription factors. Furthermore, ectopic p204 triggered the expression of Gata4 and Nkx2.5 in P19 cells. p204 contains a nuclear export signal and was partially translocated to the cytoplasm during the differentiation. p204 from which the nuclear export signal was deleted was not translocated, and it did not induce differentiation. The various mechanisms by which p204 promoted the differentiation are reported in the accompanying article (Ding, B., Liu, C., Huang, Y., Yu, J., Kong, W., and Lengyel, P. (2006) J. Biol. Chem. 281, 14893-14906).

p204 protein overcomes the inhibition of the differentiation of P19 murine embryonal carcinoma cells to beating cardiac myocytes by Id proteins.

We reported in the accompanying article (Ding, B., Liu, C., Huang, Y., Hickey, R. P., Yu, J., Kong, W., and Lengyel, P. (2006) J. Biol. Chem. 281, 14882-14892) that (i) the p204 protein is required for the differentiation of murine P19 embryonal carcinoma stem cells to beating cardiac myocytes, and (ii) the expression of p204 in the differentiating P19 cells is synergistically transactivated by the cardiac transcription factors Gata4, Nkx2.5, and Tbx5. Here we report that endogenous or ectopic inhibitor of differentiation (Id) proteins inhibited the differentiation of P19 cells to myocytes. This was in consequence of the binding of Id1, Id2, or Id3 protein to the Gata4 and Nkx2.5 proteins and the resulting inhibitions (i) of the binding of these transcription factors to each other and to DNA and (ii) of their synergistic transactivation of the expression of various genes, including atrial natriuretic factor and Ifi204 (encoding p204). p204 overcame this inhibition by Id proteins in consequence of (i) binding and sequestering Id proteins, (ii) accelerating their ubiquitination and degradation by proteasomes, and (iii) decreasing the level of Id proteins in the nucleus by increasing their translocation from the nucleus to the cytoplasm. Points (ii) and (iii) depended on the presence of the nuclear export signal in p204. In the course of the differentiation, Gata4, Nkx2.5, and p204 were components of a positive feedback loop. This loop arose in consequence of it that p204 overcame the inhibition of the synergistic activity of Gata4 and Nkx2.5 by the Id proteins.

The interferon-inducible p204 protein acts as a transcriptional coactivator of Cbfa1 and enhances osteoblast differentiation.

The differentiation of uncommitted mesenchymal cells into osteoblasts is a fundamental molecular event governing both embryonic development and bone repair. The bone morphogenetic proteins (BMPs) are important regulators of this process; they function by binding to cell surface receptors and signaling by means of Smad proteins. Core binding factor alpha-1 (Cbfa1), a member of the runt family of transcription factors, is an essential transcriptional regulator of osteoblast differentiation and bone formation, and this process is positively or negatively regulated by a variety of coactivators and corepressors. We report that p204, an interferon-inducible protein that was previously shown to inhibit cell proliferation and promote the differentiation of myoblasts to myotubes, is a novel regulator in the course of osteogenesis. p204 is expressed in embryonic osteoblasts and hypertrophic chondrocytes in the growth plate as well as in the calvaria osteoblasts of neonatal mice. Its level is increased in the course of the BMP-2-triggered osteoblast differentiation of pluripotent C2C12 cells. This increase is probably due to the activation of the gene encoding 204 (Ifi204) by Smad transcription factor, including Smad1, -4, and -5. Overexpression of p204 enhances the BMP-2-induced osteoblast differentiation in vitro, as revealed by elevated alkaline phosphatase activity and osteocalcin production. p204 acts as a cofactor of Cbfa1: 1) high levels of p204 augment, whereas the lowering of p204 level decreases, the Cbfa1-dependent transcription, and 2) p204 associates with Cbfa1 both in vitro and in vivo. Two nonoverlapping segments in p204 bind to Cbfa1, and the N-terminal 88-amino acid segment of Cbfa1 is required for binding to p204. p204, which is the first interferon-inducible protein found to associate with Cbfa1, functions as a novel regulator of osteoblast differentiation.

The mouse Ifi200 gene cluster: genomic sequence, analysis, and comparison with the human HIN-200 gene cluster.

The interferon-activatable Ifi200 gene cluster is located on mouse Chromosome 1q21-q23. We report here our analysis of two genomic regions encoding at least 10 closely related 200 family genes (Ifi201, Ifi202a, Ifi202b, Ifi202c, Ifi203a, Ifi203b, Ifi203c, Ifi203', Ifi204, and Ifi204') in 129/SvJ mice. Through a BAC-based sequencing approach, the exact structure and organization of these highly similar Ifi200 genes were obtained. A high degree of conservation (99% identity) was observed between Ifi202a and b and between Ifi203a and b. The presence of an additional transcribed region in intron 4 of Ifi203a and b suggests the possibility of alternative splicing, and a spliced variant of the Ifi204' mRNA exhibits 91% sequence identity with a related but unmapped D3 mRNA. Comparative analysis of the mouse and human clusters indicates an absence of significant sequence conservation in noncoding sequences, suggesting that the 200 family emerged prior to human-mouse speciation and subsequently diverged after gene duplication.

The interferon-inducible p202a protein modulates NF-kappaB activity by inhibiting the binding to DNA of p50/p65 heterodimers and p65 homodimers while enhancing the binding of p50 homodimers.

p202a is a member of the interferon-inducible murine p200 family of proteins. These proteins share 1 or 2 partially conserved 200 amino acid segments of the a or the b type. The known biological activities of p202a include among others the regulation of muscle differentiation, cell proliferation, and apoptosis. These biological activities of p202a can be correlated with the inhibition of the activity of several transcription factors. Thus, the binding of p202a results in the inhibition of the sequence-specific binding to DNA of the c-Fos, c-Jun, E2F1, E2F4, MyoD, myogenin, and c-Myc transcription factors. This study concerns the mechanisms by which p202a inhibits the activity of NF-kappaB, a transcription factor involved among others in host defense, inflammation, immunity, and the apoptotic response. NF-kappaB consists of p50 and p65 subunits. We demonstrate that p202a can inhibit in vitro and in vivo the binding to DNA of p65 homodimers and p50/65 heterodimers, whereas it increases the binding of p50 homodimers. Thus p202a can impair NF-kappaB activity both by inhibiting the binding to DNA of the transcriptionally active p65 homodimers and p50/p65 heterodimers and by boosting the binding of the repressive p50 homodimers. p202a can bind p50 and p65 in vitro and in vivo, and p202a can be part of the p50 homodimer complex bound to DNA. p50 binds in p202a to the a type segment, whereas p65 binds to the b type segment. Transfected ectopic p202a increases the apoptotic effect of tumor necrosis factor (at least in part) by inhibiting NF-kappaB and its antiapoptotic activity.

The MyoD-inducible p204 protein overcomes the inhibition of myoblast differentiation by Id proteins.

The murine p204 protein level is highest in heart and skeletal muscle. During the fusion of cultured myoblasts to myotubes, the p204 level increases due to transcription dependent on the muscle-specific MyoD protein, and p204 is phosphorylated and translocated from the nucleus to the cytoplasm. p204 overexpression accelerates myoblast fusion in differentiation medium and triggers this process even in growth medium. Here we report that p204 is required for the differentiation of C2C12 myoblasts. We propose that it enables the differentiation, at least in part, by overcoming the inhibition of the activities of the MyoD and E47 proteins by the Id proteins: Id1, Id2, and Id3. These are known to inhibit skeletal muscle differentiation by binding and blocking the activity of MyoD, E12/E47, and other myogenic basic helix-loop-helix (bHLH) proteins. Our hypothesis is based on the following findings. (i) A decrease in the p204 level in C2C12 myoblasts by antisense RNA (a) increased the level of the Id2; (b) inhibited the MyoD-, E12/E47-, and other bHLH protein-dependent accumulation of the muscle-specific myosin heavy-chain protein; and (c) inhibited the fusion of myoblasts to myotubes in differentiation medium. (ii) p204 bound to the Id proteins in vitro and in vivo. (iii) In the binding of p204 to Id2, the b segment of p204 and the HLH segment of Id2 were involved. (iv) Addition of p204 overcame the inhibition by the Id proteins of the binding of MyoD and E47 to DNA in vitro. (v) Overexpression of p204 in myoblasts (a) decreased the level of the Id proteins, even in a culture in growth medium, and (b) overcame the inhibition by the Id proteins of MyoD- and E47 dependent transcription and also overcame the inhibition by Id2 of the fusion of myoblasts to myotubes.