Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.     

Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi,India

Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.     

Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission

Pluripotent stem cells are known to display distinct metabolic phenotypes than their somatic counterparts. While accumulating studies are focused on the roles of glucose and amino acid metabolism in facilitating pluripotency, little is known regarding the role of lipid metabolism in regulation of stem cell activities. Here, we show that fatty acid (FA) synthesis activation is critical for stem cell pluripotency. Our initial observations demonstrated enhanced lipogenesis in pluripotent cells and during cellular reprogramming. Further analysis indicated that de novo FA synthesis controls cellular reprogramming and embryonic stem cell pluripotency through mitochondrial fission. Mechanistically, we found that de novo FA synthesis regulated by the lipogenic enzyme ACC1 leads to the enhanced mitochondrial fission via (i) consumption of AcCoA which affects acetylation‐mediated FIS1 ubiquitin–proteasome degradation and (ii) generation of lipid products that drive the mitochondrial dynamic equilibrium toward fission. Moreover, we demonstrated that the effect of Acc1 on cellular reprogramming via mitochondrial fission also exists in human iPSC induction. In summary, our study reveals a critical involvement of the FA synthesis pathway in promoting ESC pluripotency and iPSC formation via regulating mitochondrial fission.     

Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1)

MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.     

AHR Over-Expression in Papillary Thyroid Carcinoma: Clinical and Molecular Assessments in a Series of Italian Acromegalic Patients with a Long-Term Follow-Up

Aim

Acromegaly reportedly carries an increased risk of malignant and benign thyroid tumors, with a prevalence of thyroid cancer of around 3–7%. Germline mutations in the aryl-hydrocarbon receptor (AHR) interacting protein (AIP) have been identified in familial forms of acromegaly. The molecular and endocrine relationships between follicular thyroid growth and GH-secreting pituitary adenoma have yet to be fully established. Our aim was to study the prevalence of differentiated thyroid cancer (DTC) in acromegaly, focusing on the role of genetic events responsible for the onset of thyroid cancer.

Methods

Germline mutations in the AIP gene were assessed in all patients; BRAF and H-N-K RAS status was analyzed by direct sequencing in thyroid specimens, while immunohistochemistry was used to analyze the protein expression of AIP and AHR. A set of PTCs unrelated to acromegaly was also studied.

Results

12 DTCs (10 papillary and 2 follicular carcinomas) were identified in a cohort of 113 acromegalic patients. No differences in GH/IGF-1 levels or disease activity emerged between patients with and without DTC, but the former were older and more often female. BRAF V600E was found in 70% of the papillary thyroid cancers; there were no RAS mutations. AIP protein expression was similar in neoplastic and normal cells, while AHR protein was expressed more in PTCs carrying BRAF mutations than in normal tissue, irrespective of acromegaly status.

Conclusions

The prevalence of DTC in acromegaly is around 11% and endocrinologists should bear this in mind, especially when examining elderly female patients with uninodular goiter. The DTC risk does not seem to correlate with GH/IGF-1 levels, while it may be associated with BRAF mutations and AHR over-expression. Genetic or epigenetic events probably play a part in promoting thyroid carcinoma.     

Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa.

In batch cultures of Pseudomonas aeruginosa, the maximum rate of exopolysaccharide synthesis occurred during exponential growth. In nitrogen-limited continuous culture, the specific rate of exopolysaccharide synthesis increased from 0.27 g g of cell-1 h-1 at a dilution rate (D) of 0.05 h-1 to 0.44 g g of cells h-1 at D=0.1 H-1. The yield of exopolysaccharide on the basis of glucose used was in the range of 56 to 64%. Exopolysaccharide was also synthesized in carbon-limited cultures at 0.19 g g of cell-1 h-1 at D=0.05 h-1 in a 33% yield. Nonmucoid variants appeared after seven generations in continuous culture and rapidly increased in proportion to the total number of organisms present.     

绵羊胞内单精子注射技术

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.