Activation of extracellular signal-regulated kinase couples platelet-activating factor-induced adhesion and delayed apoptosis of human neutrophils

Platelet-activating factor (PAF) promotes adhesion of neutrophil granulocytes to the endothelium, which is also linked to neutrophil survival. Here we report that PAF can prolong neutrophil survival by suppressing spontaneous apoptosis. PAF induced concurrent activation of the Ras/Raf-1/mitogen-activated protein kinase kinase (MAPKK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt pathways. ERK activation tightly correlated with up-regulation of CD11b/CD18 expression and beta(2)-integrin-dependent homotypic adhesion. These actions of PAF were markedly attenuated by the MAPKK/ERK inhibitor PD98059, but not by the phosphatidylinositol 3-kinase inhibitor wortmannin. By contrast, concurrent activation of ERK and Akt was required to inhibit caspase-3 activation and consequently to delay apoptosis. Consistently, pharmacological inhibition of either ERK or Akt partially reversed the anti-apoptotic action of PAF; however, they did not produce additive inhibition. These results indicate that PAF-induced activation of ERK contributes to both the expression of the pro-adhesive phenotype and repression of neutrophil apoptosis, thereby amplifying the inflammatory response.     

Postischemic cardiac recovery in heme oxygenase‐1 transgenic ischemic/reperfused mouse myocardium

Heme oxygenase-1 (HO-1) transgenic mice (Tg) were created using a rat HO-1 genomic transgene. Transgene expression was detected by RT-PCR and Western blots in the left ventricle (LV), right ventricle (RV) and septum (S) in mouse hearts, and its function was demonstrated by the elevated HO enzyme activity. Tg and non-transgenic (NTg) mouse hearts were isolated and subjected to ischemia/reperfusion. Significant post-ischemic recovery in coronary flow (CF), aortic flow (AF), aortic pressure (AOP) and first derivative of AOP (AOPdp/dt) were detected in the HO-1 Tg group compared to the NTg values. In HO-1 Tg hearts treated with 50 μmol/kg of tin protoporphyrin IX (SnPPIX), an HO enzyme inhibitor, abolished the post-ischemic cardiac recovery. HO-1 related carbon monoxide (CO) production was detected in NTg, HO-1 Tg and HO-1 Tg + SnPPIX treated groups, and a substantial increase in CO production was observed in the HO-1 Tg hearts subjected to ischemia/reperfusion. Moreover, in ischemia/reperfusion-induced tissue Na⁺ and Ca²⁺ gains were reduced in HO-1 Tg group in comparison with the NTg and HO-1 Tg + SnPPIX treated groups; furthermore K⁺ loss was reduced in the HO-1 Tg group. The infarct size was markedly reduced from its NTg control value of 37 ± 4% to 20 ± 6% (P < 0.05) in the HO-1 Tg group, and was increased to 47 ± 5% (P < 0.05) in the HO-1 knockout (KO) hearts. Parallel to the infarct size reduction, the incidence of total and sustained ventricular fibrillation were also reduced from their NTg control values of 92% and 83% to 25% (P < 0.05) and 8% (P < 0.05) in the HO-1 Tg group, and were increased to 100% and 100% in HO-1 KO^−/− hearts. Immunohistochemical staining of HO-1 was intensified in HO-1 Tg compared to the NTg myocardium. Thus, the HO-1 Tg mouse model suggests a valuable therapeutic approach in the treatment of ischemic myocardium.     

Susceptibility of the common hamster (Cricetus cricetus) to Francisella tularensis and its effect on the epizootiology of tularemia in an area where both are endemic

Francisella tularensis is a highly infectious zoonotic agent causing the disease tularemia. The common hamster (Cricetus cricetus) is considered a pest in eastern Europe, and believed to be a source of human tularemia infections. We examined the role of the common hamster in the natural cycle of tularemia using serologic methods on 900 hamsters and real-time polymerase chain reaction (PCR) on 100 hamsters in an endemic agricultural area. We collected 374 Ixodes acuminatus ticks from the hamsters and tested them by real-time PCR. All tests were negative. To examine clinical signs, pathology, and histopathology of acute tularemia infection similar to the natural infection, two hamsters were infected with a large dose of a wild strain of F. tularensis ssp. holarctica. After a short period of apathy, the animals died on the eighth and ninth days postinfection. The pathologic, histopathologic, and immunohistochemical examination contributed to the diagnosis of septicemia in both cases. Our results confirmed previous findings that common hamsters are highly sensitive to F. tularensis. We conclude that although septicemic hamsters may pose substantial risk to humans during tularemia outbreaks, hamsters in interepizootic periods do not act as a main reservoir of F. tularensis.     

Ubiquitylation of Drosophila p54/Rpn10/S5a regulates its interaction with the UBA-UBL polyubiquitin receptors

Analysis of the in vivo ubiquitylation of the p54/Rpn10 polyubiquitin receptor subunit of the Drosophila 26S proteasome revealed that the site of ubiquitylation is the C-terminal cluster of lysines, which is conserved in higher eukaryotes. Extraproteasomal p54 was extensively multiubiquitylated, but only very modest modification was detected in the proteasome-assembled subunit. Ubiquitylation of p54 seriously jeopardizes one of its most important functions, i.e., the interaction of its ubiquitin-interacting motifs with the ubiquitin-like domain of Dsk2 and Rad23 extraproteasomal polyubiquitin receptors. This modification of p54 supports the previous notion that p54 is a shuttling subunit of the 26S proteasome with a specific extraproteasomal function. This assumption is supported by the observation that, while transgenic p54 can fully rescue the lethal phenotype of the Δp54 null mutation, its derivative from which the cluster of conserved lysines is deleted shifts the lethality from the early pupa to pharate adult stage but cannot rescue the Δp54 mutation, suggesting that ubiquitylated extraproteasomal p54 has an essential role in the pupa-adult transition.     

Saponin monomer 13 of dwarf lilyturf tuber (DT-13) protects serum withdrawal-induced apoptosis through PI3K/Akt in HUVEC

Dwarf lilyturf tuber is widely used in clinics to prevent cardiovascular diseases. DT-13, the saponin monomer 13 of dwarf lilyturf tuber, shows protective activities in anti-thrombosis, anti-inflammation, and cardioprotective. However, little is known about the cellular function of DT-13 in cardiovascular system. Vascular endothelial cells (EC) are important to maintain the integrity of the vasculature throughout entire body. Dysregulation of EC may lead to pathophysiological processes of numerous cardiovascular diseases. We thus tested the function of DT-13 in EC. In the present study, we are the first to report that DT-13 has anti-apoptosis activity on human umbilical vein endothelial cells (HUVEC), potentially through down regulation of cleaved caspase-3 and cleaved PARP expression. DT-13 also increased mitochondrial membrane potential. To explore the potential mechanism, we investigated the effect of DT-13 on Akt and MAPK pathways and found that DT-13 was involved in Akt signaling confirmed by using PI3 K/Akt inhibitor LY294002. Thus, DT-13 could improve survival of EC and therefore be a potential clinical use in the treatment of cardiovascular diseases.     

Design and synthesis of polyethylene glycol-modified biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties.     

Development of oxidative stress tolerance resulted in reduced ability to undergo morphologic transitions and decreased pathogenicity in a t-butylhydroperoxide-tolerant mutant of Candida albicans

We tested the hypothesis that adaptation of Candida albicans to chronic oxidative stress inhibits the formation of hyphae and reduces pathogenicity. Candida albicans cells were exposed to increasing concentrations of t-butylhydroperoxide (tBOOH), a lipid peroxidation-accelerating agent, and mutants with heritable tBOOH tolerance were isolated. Hypha formation by the mutants was negligible on Spider agar, indicating that the development of oxidative stress tolerance prevented Candida cells from undergoing dimorphic switches. One of the mutants, C. albicans AF06, was five times less pathogenic in mice than its parental strain, due to its reduced germ tube-, pseudohypha- and hypha-forming capability, and decreased phospholipase secretion. An increased oxidative stress tolerance may therefore be disadvantageous when this pathogen leaves blood vessels and invades deep organs. The AF06 mutant was characterized by high intracellular concentrations of endogenous oxidants, reduced monounsaturated and polyunsaturated fatty acid contents, the continuous induction of the antioxidative defense system, decreased cytochrome c-dependent respiration, and increased alternative respiration. The mutation did not influence growth rate, cell size, cell surface, cellular ultrastructures, including mitochondria, or recognition by human polymorphonuclear leukocytes. The selection of oxidative stress-tolerant respiratory Candida mutants may also occur in vivo, when reduced respiration helps the fungus to cope with antimycotic agents.     

Mass spectrometric identification of proteins in complex post-genomic projects. Soluble proteins of the metabolically versatile, denitrifying 'Aromatoleum' sp. strain EbN1

The rapidly developing proteomics technologies help to advance the global understanding of physiological and cellular processes. The lifestyle of a study organism determines the type and complexity of a given proteomic project. The complexity of this study is characterized by a broad collection of pathway-specific subproteomes, reflecting the metabolic versatility as well as the regulatory potential of the aromatic-degrading, denitrifying bacterium 'Aromatoleum' sp. strain EbN1. Differences in protein profiles were determined using a gel-based approach. Protein identification was based on a progressive application of MALDI-TOF-MS, MALDI-TOF-MS/MS and LC-ESI-MS/MS. This progression was result-driven and automated by software control. The identification rate was increased by the assembly of a project-specific list of background signals that was used for internal calibration of the MS spectra, and by the combination of two search engines using a dedicated MetaScoring algorithm. In total, intelligent bioinformatics could increase the identification yield from 53 to 70% of the analyzed 5,050 gel spots; a total of 556 different proteins were identified. MS identification was highly reproducible: most proteins were identified more than twice from parallel 2DE gels with an average sequence coverage of >50% and rather restrictive score thresholds (Mascot >or=95, ProFound >or=2.2, MetaScore >or=97). The MS technologies and bioinformatics tools that were implemented and integrated to handle this complex proteomic project are presented. In addition, we describe the basic principles and current developments of the applied technologies and provide an overview over the current state of microbial proteome research.     

Ultrastructural analysis of nematocyte removal in Hydra

A consecutive series of ultrathin sections through the distal one-third of a Hydra tentacle has revealed at least four categories of nematocytes: (1) normal, mounted nematocytes, in specific arrangements within the battery cells; (2) degenerating nematocytes, within the battery cells; (3) mature nematocytes, enclosed within endodermal cells; (4) a mature nematocyte, in the enteric cavity. The degenerating nematocytes within the battery cells and the nematocytes in the endoderm and enteric cavity appeared to be aging nematocytes undergoing death and removal. The results provide the first ultrastructural evidence for nematocyte degeneration within battery cells and also suggest phagocytosis of mature nematocytes by endodermal cells.