Structural and functional insights into IκB-α/HIV-1 Tat interaction

Protein-protein interactions play fundamental roles in physiological and pathological biological processes. The characterization of the structural determinants of protein-protein recognition represents an important step for the development of molecular entities able to modulate these interactions. We have recently found that IκB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) blocks the HIV-1 expression and replication in a NF-κB-independent manner by directly binding to the virus-encoded Tat transactivator. Here, we report the evaluation of the entity of binding of IκB-α to Tat through in vitro Surface Plasmon Resonance assay. Moreover, by designing and characterizing a set of peptides of the C-terminus region of IκB-α, we show that the peptide corresponding to the IκB-α sequence 262-287 was able to bind to Tat with high affinity (300 nM). The characterization of a number of IκB-α-based peptides also provided insights into their intrinsic folding properties. These findings have been corroborated by mutagenesis studies on the full-length IκB-α, which unveil that different IκB-α residues are involved in NF-κB or Tat recognition.     

Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.     

Interaction of Vault Particles with Estrogen Receptor in the MCF-7 Breast Cancer Cell

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.     

The inflammatory protein Pentraxin 3 in cardiovascular disease

The acute phase protein Pentraxin 3 (PTX3) plays a non-redundant role as a soluble pattern recognition receptor for selected pathogens and it represents a rapid biomarker for primary local activation of innate immunity and inflammation. Recent evidence indicates that PTX3 exerts an important role in modulating the cardiovascular system in humans and experimental models. In particular, there are conflicting points concerning the effects of PTX3 in cardiovascular diseases (CVD) since several observations indicate a cardiovascular protective effect of PTX3 while others speculate that the increased plasma levels of PTX3 in subjects with CVD correlate with disease severity and with poor prognosis in elderly patients.In the present review, we discuss the multifaceted effects of PTX3 on the cardiovascular system focusing on its involvement in atherosclerosis, endothelial function, hypertension, myocardial infarction and angiogenesis. This may help to explain how the specific modulation of PTX3 such as the use of different dosing, time, and target organs could help to contain different vascular diseases. These opposite actions of PTX3 will be emphasized concerning the modulation of cardiovascular system where potential therapeutic implications of PTX3 in humans are discussed.     

Effects of thyroid state on H2O2 production by rat heart mitochondria: sites of production with complex I- and complex II-linked substrates.

This work was designed to determine possible effects of altered thyroid states on rates and sites of H 2 O 2 production by rat heart mitochondria. Rates of O 2 consumption and H 2 O 2 release, capacities to remove the peroxide, lipid peroxidation, cytochrome oxidase activities and ubiquinone levels were determined in heart mitochondria from euthyroid, hypothyroid, and hyperthyroid rats. Hypothyroidism decreased, whereas hyperthyroidism increased the rates of O 2 consumption and H 2 O 2 release during both state 4 and state 3 respiration with Complex I- or Complex II-linked substrates. The percentage of O 2 released as H 2 O 2 was not significantly affected by thyroid state. However, the mitochondrial capacity to remove H 2 O 2 increased in the transition from hypothyroid to hyperthyroid state, which indicates that H 2 O 2 production did not modify in proportion to the rate of O 2 consumption. The thyroid-state-linked changes in H 2 O 2 production were well correlated with the levels of hydroperoxides. Rates of H 2 O 2 release in the presence of respiratory inhibitors indicated that changes in the H 2 O 2 production occurred at both sites at which H 2 O 2 was generated in euthyroid state. This result and the observation that ubiquinol levels and cytochrome oxidase activities increase in the transition from hypothyroid to hyperthyroid state suggest that the modifications of H 2 O 2 production are due to a modulation by thyroid hormone of mitochondrial content of autoxidisable electron carriers.     

Particulate nature of the unoccupied uterine estrogen receptor

Homogenization of rat uteri at 25 degrees C resulted in a particulate partitioning of the estrogen receptor. Homogenization at 0 degrees C, the use of frozen tissue, or the pre-exposure of the tissue to 0 degrees C prior to 25 degrees C homogenization induced soluble partitioning of the estrogen receptor. Binding of a radiolabeled monoclonal antibody indicated that, in absence of estradiol, the estrogen receptor is particulate and is associated with the nuclei-enriched fraction of the target cell. The presence of receptor in the soluble fraction thus appears to be an artifact of homogenization. The unoccupied receptor, loosely associated with the particulate fraction (cold-sensitive) represents the "native" form of receptor which, upon arrival of the hormone, becomes tightly associated (cold-insensitive). The transition from the cold-sensitive to the cold-insensitive status is accompanied by a modification of the electrical charge of the receptor.     

Hemorrhagic fever with renal syndrome complicated by orchitis

Hemorrhagic fever with renal syndrome (HFRS) is a disease caused by viruses of the family Bunyaviridae,genus Hantavirus.HFRS from Dobrava virus (DOBV) is a seldom reported disease in Albania.Clinically HFRS is manifested as mild,moderate,or severe.Therefore,the number of cases of Hantavirus'infection may be underestimated,and should be included in the differential diagnosis of many acute infections,hematologic diseases,acute abdominal diseases and renal diseases complicated by acute renal failure.We report here an atypical presentation of HFRS from Dobrava virus complicated by orchitis with a positive outcome.