Estrogen-induced spermatogenic cell apoptosis occurs via the mitochondrial pathway: role of superoxide and nitric oxide

The detrimental effects of estrogen on testicular function provide a conceptual basis to examine the speculative link between increased exposure to estrogens and spermatogenic cell death. Using an in vitro model, we provide an understanding of the events leading to estrogen-induced apoptosis in cells of spermatogenic lineage. Early events associated with estrogen exposure were up-regulation of FasL and increased generation of H(2)O(2), superoxide, and nitric oxide. The ability of anti-FasL antibodies to prevent several downstream biochemical changes and cell death induced by 17beta-estradiol substantiates the involvement of the cell death receptor pathway. Evidence for the amplification of the death-inducing signals through mitochondria was obtained from the transient mitochondrial hyperpolarization observed after estradiol exposure resulting in cytochrome c release. A combination of nitric oxide and superoxide but not H(2)O(2) was responsible for the mitochondrial hyperpolarization. Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an intracellular peroxynitrite scavenger, was able to reduce mitochondrial hyperpolarization and cell death. Although nitric oxide augmentation occurred through an increase in the expression of inducible nitric-oxide synthase, superoxide up-regulation was a product of estradiol metabolism. All of the above changes were mediated through an estrogen receptor-based mechanism because tamoxifen, the estrogen receptor modulator, was able to rescue the cells from estrogen-induced alterations. This study establishes the importance of the independent capability of cells of the spermatogenic lineage to respond to estrogens and most importantly suggests that low dose estrogens can potentially cause severe spermatogenic cellular dysfunction leading to impaired fertility even without interference of the hypothalamo-hypophyseal axis.     

Design,synthesis and biological evaluation of novel pyrazolo-oxothiazolidine derivatives as antiproliferative agents against human lung cancer cell line A549

An efficient, one-pot multicomponent reaction of novel pyrazolo-oxothiazolidine derivatives was achieved by condensation of 1-(benzofuran-2-yl)-3-(substituted-arylprop-2-en-1-ones, thiosemicarbazide and dialkyl acetylenedicarboxylates under the optimized reaction conditions. Synthesised compounds were evaluated for their antiproliferative activity against A549 human lung cancer cell line. Among all the tested compounds, 4a (IC₅₀ – 0.930?μg/mL), 4e (IC₅₀ – 1.207?μg/mL), 4f (IC₅₀ – 0.808?μg/mL), 4g (IC₅₀ – 1.078?μg/mL), 4h (IC₅₀ – 0.967?μg/mL) and 4j (IC₅₀ – 2.445?μg/mL) showed promising activity compared with standard drug Sorafenib (IC₅₀ – 3.779?μg/mL). Molecular docking studies indicated that compound 4f had the greatest affinity for catalytic site of receptors EGFR (PDB ID code: 1?M17) and VEGFR2 (PDB ID code: 4AGD, 4ASD). These novel pyrazolo-oxothiazolidine derivatives can be promising therapeutic agents for A549 human lung cancer cell line.     

α-Actinin Anchors PSD-95 at Postsynaptic Sites

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Gene expression profiling of coronary artery disease and its relation with different severities

Global gene expression profiling is a powerful tool enabling the understanding of pathophysiology and subsequent management of diseases. This study aims to explore functionally annotated differentially expressed genes (DEGs); their biological processes for coronary artery disease (CAD) and its different severities of atherosclerotic lesions. This study also aims to identify the change in expression patterns of DEGs in atherosclerotic lesions of single-vessel disease (SVD) and triple-vessel disease (TVD). The weight of different severities of lesion was estimated using a modified Gensini score. The gene expression profiling was performed using the Affymetrix microarray platform. The functional annotation for CAD was performed using DAVID v6.8. The biological network gene ontology tool (BiNGO) and ClueGO were used to explore the biological processes of functionally annotated genes of CAD. The changes in gene expression from SVD to TVD were determined by evaluating the fold change. Functionally annotated genes were found in an unique set and could be distinguishing two distinct severities of CAD. The biological processes such as cellular migration, locomotion, cell adhesion, cytokine production, positive regulation of cell death etc. enriched the functionally annotated genes in SVD, whereas, wound healing, negative regulation of cell death, blood coagulation, angiogenesis and fibrinolysis were enriched significantly in TVD patients. The genes THBS1 and CAPN10 were functionally annotated for CAD in both SVD and TVD. The 61 DEGs were identified, those have changes their expression with different severities of atherosclerotic lesions, in which 13 genes had more than two-fold change in expression between SVD and TVD. The consistent findings were obtained on validation of microarray gene expression of selected 10 genes in a separate cohort using real-time PCR. This study identified putative candidate genes and their biological processes predisposing toward and affecting the severity of CAD.     

A new role for ATM in selective autophagy of peroxisomes (pexophagy)

Peroxisomes are autonomously replicating and highly metabolic organelles necessary for β-oxidation of fatty acids, a process that generates large amounts of reactive oxygen species (ROS). Maintaining a balance between biogenesis and degradation of peroxisomes is essential to maintain cellular redox balance, but how cells do this has remained somewhat of a mystery. While it is known that peroxisomes can be degraded via selective autophagy (pexophagy), little is known about how mammalian cells regulate pexophagy to maintain peroxisome homeostasis. We have uncovered a mechanism for regulating pexophagy in mammalian cells that defines a new role for ATM (ATM serine/threonine kinase) kinase as a “first responder” to peroxisomal ROS. ATM is delivered to the peroxisome by the PEX5 import receptor, which recognizes an SRL sequence located at the C terminus of ATM to localize this kinase to peroxisomes. In response to ROS, the ATM kinase is activated and performs 2 functions: i) it signals to AMPK, which activates TSC2 to suppresses MTORC1 and phosphorylates ULK1 to induce autophagy, and ii) targets specific peroxisomes for pexophagy by phosphorylating PEX5 at Ser141, which triggers ubiquitnation of PEX5 at Lys209 and binding of the autophagy receptor protein SQSTM1/p62 to induce pexophagy.     

Powerful Sequence Similarity Search Methods and In-Depth Manual Analyses Can Identify Remote Homologs in Many Apparently “Orphan” Viral Proteins

The genome sequences of new viruses often contain many “orphan” or “taxon-specific” proteins apparently lacking homologs. However, because viral proteins evolve very fast, commonly used sequence similarity detection methods such as BLAST may overlook homologs. We analyzed a data set of proteins from RNA viruses characterized as “genus specific” by BLAST. More powerful methods developed recently, such as HHblits or HHpred (available through web-based, user-friendly interfaces), could detect distant homologs of a quarter of these proteins, suggesting that these methods should be used to annotate viral genomes. In-depth manual analyses of a subset of the remaining sequences, guided by contextual information such as taxonomy, gene order, or domain cooccurrence, identified distant homologs of another third. Thus, a combination of powerful automated methods and manual analyses can uncover distant homologs of many proteins thought to be orphans. We expect these methodological results to be also applicable to cellular organisms, since they generally evolve much more slowly than RNA viruses. As an application, we reanalyzed the genome of a bee pathogen, Chronic bee paralysis virus (CBPV). We could identify homologs of most of its proteins thought to be orphans; in each case, identifying homologs provided functional clues. We discovered that CBPV encodes a domain homologous to the Alphavirus methyltransferase-guanylyltransferase; a putative membrane protein, SP24, with homologs in unrelated insect viruses and insect-transmitted plant viruses having different morphologies (cileviruses, higreviruses, blunerviruses, negeviruses); and a putative virion glycoprotein, ORF2, also found in negeviruses. SP24 and ORF2 are probably major structural components of the virions.     

Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern

Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.     

Combination of covalent and hydrogen bonding in the formation of 3D uranyl-carboxylate networks

Four compounds containing uranyl cation [UO₂]²⁺ have been synthesized hydrothermally by reacting uranyl acetate and uranyl nitrate with various N/O donor ligands. The structure of all compounds was elucidated by single crystal X-ray diffraction study. Compound [(UO₂)(6-methylnicotinato)₃](H₃O)·4H₂O (1) is a discrete complex (0D), that gives rise in the crystal to hydrophilic channels, while [(UO₂)(OH)(μ₂-3-pyridylpropionato)]_n (2) and [(UO₂)(H₂O)(μ₃-4,4′-oxybis(benzoato)]_n (3) show the formation of 1D coordination polymers. Moreover, oxalate anions, formed in situ by using 5-methylisophthalic or 2,3-pyrazinedicarboxylic acid as reactant ligands, gave rise to a 2D coordinating network [(UO₂)₂(μ₂-oxalate)(μ₂-OH)₂(H₂O)₂]_n·nH₂O (4). All the complexes expanded their dimensionality to 3D through hydrogen bonds.     

Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.