Cardiac Fibroblasts Contribute to Myocardial Dysfunction in Mice with Sepsis: The Role of NLRP3 Inflammasome Activation

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.     

Methods of tracing hydrogen in polyketide biosynthesis: High field NMR spectroscopy of citrinin produced in a D2O based medium

Penicillium citrinum cultures have been germinated on an H₂O-based medium, resuspended on a D₂O-based medium and treated with [l,2-¹³C₂] acetate. The resulting citrinin (1) has been analysed by²H and¹³C nuclear magnetic resonance spectroscopy and information about the metabolism of hydrogen in citrinin biosynthesis has been deduced.     

Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

8-Hydroxydeoxyguanosine in DNA from TPA-Stimulated Human Granulocytes

8-Hydroxydeoxyguanosine (8-OHdG) is now widely used as a sensitive marker of oxidative damage to DNA. When human granulocytes are stimulated with TPA, they release a large quantity of reactive oxygen species (superoxide, hydrogen peroxide) which might be expected to generate hydroxyl radicals (OH-) which in turn could produce 8-OHdG in the DNA. There had been considerable debate as to whether OH -is detectable in stimulated granulocytes; most workers now agree that none can be detected, unless exogenous iron is added. An earlier report had described that 8-OHdG (a marker of OH -) was increased in the DNA of TPA-stimulated, compared to control, granulocytes. We have repeated this experiment and have been unable to reproduce this Finding. We conclude that the amount of 8-OHdG produced in the DNA of TPA-stimulated human ganulocytes is indistinguishable from that seen in control (unstimulated) cells (less than one 8- OHdG/10⁵ dG).     

Corticosteroid-induced cleft palate in mice and H-2 haplotype: Maternal and embryonic effects

This report confirms and expands on the original preliminary observations made by Bonner and Slavkin that corticosteroid-induced cleft palate in mice is associated with H-2 haplotype. Using three congenic strains, B10, B10.A, and B10.D2, our studies demonstrate that B10.A (H-2 ^b) is most susceptible and B10.D2 (H-2 ^d) is least susceptible, B10 (H-2 ^b) being intermediate. Variation in fetal loss among strains accounts for less than 1 percent of the variation in cleft-palate frequency among strains; variation in H-2 haplotype, however, accounts for more than 60 percent of the variation in cleft-palate frequency. With regard to all possible reciprocal F₁ hybrids, our results indicate that while there is a significant maternal effect, maternal haplotype can account for only 11 percent of the variation in cleft-palate frequency among crosses. Embryonic haplotype accounts for 17 percent of the variation, which is indicative of an important embryonic effect. Finally, our studies suggest that susceptibility to corticosteroid-induced cleft palate is associated with the K end of the H-2 complex.     

Translocation(X;Y)(p22.33;p11.2) in XX males: Etiology of male phenotype

Summary Analysis of G-banded prometaphase chromosomes from three XX males revealed extra bands on the distal end of one X short arm. These bands were similar both in size and staining properties to the distal Y short arm of their fathers (in the two cases examined) and also to other chromosomally normal males. The extra material on the abnormal X chromosomes was not C-or G-11 positive in the two cases examined, suggesting that the proximal Y long arm was not present.Previous karyotype-phenotype correlations with structurally altered Y chromosomes provided evidence for localization of male determinants on the Y short arm. The present findings in XX males provide support for more precise localization, to bands p11.2pter of Y short arm.     

Studying the uptake of cell-penetrating peptides

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.     

Species relationships in the genus Vasconcellea (Caricaceae) based on molecular and morphological evidence

Validity of the taxa currently recognized in the genus Vasconcellea was analyzed by investigating morphological and molecular data from 105 specimens of this genus and six specimens of the related genus Carica. Taxon identification of these specimens was compared with clustering in two phenetic dendrograms generated with 36 morphological characters and 254 amplified fragment length polymorphic (AFLP) markers. Moreover, cytoplasmic haplotypes were assessed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of one mitochondrial and two chloroplast DNA regions. Results show that the morphological data set, containing mainly vegetative characteristics, merely reveals external resemblance between specimens, which is not directly associated with genetic relationships and taxon validity. Phenotypic plasticity and intercompatibility between several species are likely to confuse morphological delimitation of the taxa. Based on the results of our study, several specimens that could not be identified with the currently used identification key (1) could be attributed to a known taxon, which should be extended to include a higher range of morphological variability or (2) could be hypothesized to be of hybrid origin. Because of the high intraspecific variation within V. microcarpa and V. × heilbornii, revision of these taxa is recommended.     

Synergistic anti-proliferative and pro-apoptotic activity of combined therapy with bortezomib, a proteasome inhibitor, with anti-epidermal growth factor receptor (EGFR) drugs in human cancer cells

The proteasome plays a pivotal role in the turnover of regulatory transduction proteins induced by activated cell membrane growth factor receptors. The epidermal growth factor receptor (EGFR) pathway is crucial in the development and progression of human epithelial cancers. Proteasome inhibition may sensitize human cancer cell lines to EGFR inhibitors. We investigated the growth inhibitory and pro-apoptotic effects of the proteasome inhibitor bortezomib in combination with anti-EGFR drugs, such as gefitinib, vandetanib, and cetuximab in EGFR-expressing human cancer cell lines. Bortezomib determined dose-dependent growth inhibition in a nine cancer cell line panel (IC(50) values, range 6-42 nM). A significant synergistic growth inhibitory effect was observed with the combination of bortezomib and each EGFR inhibitor in all cell lines (combination index, CI, range 0.10-0.55), which was accompanied by a significant induction in apoptosis by the combined treatment with bortezomib, cetuximab and vandetanib. In HCT-116 colon cancer and A549 lung adenocarcinoma cells, bortezomib plus EGFR inhibitor treatment induced a more effective inhibition of EGFR-activated down-stream signals, including a marked suppression in activated, phosphorylated Akt (P-Akt). In contrast, overexpression of a constitutively active P-Akt protected A549 cells by cell growth inhibition and apoptosis following treatment with bortezomib and EGFR inhibitors. The combined treatment with bortezomib and EGFR inhibitors has a synergistic growth inhibitory and pro-apoptotic activity in different human cancer cells which possess a functional EGFR-dependent autocrine growth pathway through to a more efficient and sustained inhibition of Akt.