Synthesis of type 2 Adenovirus DNA in the Presence of Cycloheximide

Adenovirus type 2 DNA synthesis, either in permissive human cells or nonpermissive monkey cells, becomes independent of protein synthesis after the appearance of progeny viral DNA. In the presence of cycloheximide, semiconservative replication and initiation of progeny molecules can occur.     

Macrophages and Apoptotic cells in Human Colorectal Tumours

The numerical relationship between tumour associated macrophages (TAM) and apoptotic cells in 12 human colorectal tumours was evaluated. TAM were labelled immunohistochemically and apoptotic cells were visualized by counterstaining with haematoxylin and eosin (H&E). The stereological techniques. Cavalieri's estimator of volume and the Disector were used to estimate both tumour volume and numerical density of both cell types. The occurrence of TAM per unit volume of tissue increased with increasing tumour volume to a maximum in a tumour of 110·5 cm³, after which numbers declined. Levels of apoptosis also increased with tumour volume though more erratically than levels of TAM and declined for tumour volumes greater than 80 cm³. This is the first report of an attempt to assess the relationship between apoptotic cells and TAM in human tumours.     

Changes in Eicosanoid and Tumour Necrosis Factor-alpha Production by Rat Peritoneal Macrophages During Carrageenin-Induced Peritonitis

Changes and correlations in cytokine and eicosanoid production by blood monocytes, non-purified and purified peritoneal cells during a carrageenin-induced peritonitis were investigated for a period of ten days. The cells were isolated and stimulated in vitro. Cytokine and eicosanoid production of the non-purified fraction increased steadily during peritonitis. During the whole episode of peritonitis the production capacity of granulocytes was very low and hardly any effect on the production capacity of macrophages (Mvarphi) was observed. Cytokine and eicosanoid production of the non-purified fraction was mainly due to the presence of Mvarphi. The production capacity of the peripheral blood monocytes was not similar to that of the peritoneal Mvarphi.     

PDT诱导的凋亡细胞对巨噬细胞NO合成的影响

NO作为细胞间信息传递的重要调节因子,在肿瘤的发生、发展以及转移过程中被广泛研究。一氧化氮合酶是合成NO的关键酶,诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)通常在应激、荷瘤等病理状态下被激活,产生大量NO。NO具有细胞毒性,与机体免疫反应及细胞凋亡有关,在许多致癌和抑癌机制中扮演着重要角色。实验探讨了光动力学疗法(photodynamic therapy,PDT)处理产生的小鼠乳腺癌凋亡细胞对巨噬细胞产生NO的影响,从而确定活化的巨噬细胞在肿瘤生长中的作用。     

Shiga Toxin 1 Induces on Lipopolysaccharide-Treated Astrocytes the Release of Tumor Necrosis Factor-alpha that Alter Brain-Like Endothelium Integrity

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS.     

Apoptotic Neutrophils Augment the Inflammatory Response to Mycobacterium tuberculosis Infection in Human Macrophages

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.     

Modulation of Apoptotic Pathways of Macrophages by Surface-Functionalized Multi-Walled Carbon Nanotubes

Biomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47^phox and p67^phox in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.     

Urographic Changes in Acute Papillary Necrosis in the Rat

Intravenous high-dosage urography was performed in rats which had renal papillary necrosis induced with ethyleneimine or renal tubular necrosis produced with mercuric chloride. In both groups, nephrograms were abnormally persistent. In animals treated with ethyleneimine dense selective opacification of the necrotic renal pyramid occurred. It is suggested that this selective opacification may be a valuable radiological sign of recent renal papillary necrosis.     

Changes in the H Reflex Preceding Voluntary Movements of the Contralateral Lower Limb in Humans

In electromyographic studies on healthy subjects, we recorded the H reflex from the right m. soleus and measured changes in the magnitude of this reflex response related to voluntary movements of the contralateral lower limb performed according to a visual signal. The effects of back and plantar flexions of the contralateral foot of the tested subject in the lying and standing positions were examined. Changes in the H reflex magnitude began to be recorded 60 to 90 msec prior to voluntary movements of the contralateral limb. When the subject was in the lying position, these changes looked like facilitation of the H reflex at both types of movement of the contralateral foot. When the subject stood, facilitation preceded back flexion of the foot of this extremity, while plantar flexion was preceded by inhibition of the tested H reflex. Our results show that the pattern of preliminary changes in the muscle tone of one of the lower limbs is determined by the type of future movement of another limb and peculiarities of the support function realized by this limb.     

Electrophysiological Changes Preceding the Onset of Atrial Fibrillation after Coronary Bypass Grafting Surgery

Background

The incidence of Post-CABG atrial fibrillation (AF) lies between 25% and 40%. It worsens morbidity and raises post-operative costs. Detection of incoming AF soon enough for prophylactic intervention would be helpful. The study is to investigate the electrophysiological changes preceding the onset of AF and their relationship to the preoperative risk.

Methods and Results

Patients were recorded continuously for the first four days after coronary artery bypass grafting surgery (CABG) with three unipolar electrodes sutured to the atria (AEG). The patients experiencing an AF lasting more than 10 minutes were selected and the two hours before the onset were analyzed. Four variables were found to show significant changes in the two hours prior to the first prolonged AF: increasing rate of premature atrial activation, increasing incidence of short transient arrhythmias, acceleration of heart rate, and rise of low frequency content of heart rate. The main contrast was between the first and last hour before AF onset. Preoperative risk was not predictive of the onset time of AF and did not correlate with the amplitude of changes prior to AF.

Conclusions

Post-CABG AF were preceded by electrophysiological changes occurring in the last hour before the onset of the arrhythmia, whereas none of these changes was found to occur in all AF patients. The risk was a weighted sum of factors related to the density of premature activations and the state of atrial substrate reflected by the sinus rhythm and its frequency content prior to AF. Preoperative risk score seems unhelpful in setting a detection threshold for the AF onset.     

Replication of Measles Virus: Continued Synthesis of Nucleocapsid RNA and Increased Synthesis of mRNA in the Presence of Cycloheximide

The effect of cycloheximide on virus specific RNA synthesis in Vero cells infected with either wild-strain (Edmonston) or subacute sclerosing panencephalitis strain measles virus was investigated. At 3 days postinfection, cells treated with cycloheximide (2.6 x 10(-4) M) and then exposed to [(3)H]uridine showed a marked increase in labeled virus-specific RNA. A major portion of this incremental labeled RNA was putative viral mRNA which sedimented at 16, 22, and 30S. Five distinct classes of polyribosomes, which were not evident in untreated cells, were found in cycloheximide-treated cells and each contained similar species of virus-specific RNA. Viral nucleocapsid RNA, 50 and 18S, was synthesized and encapsidated in the presence of cycloheximide. The latter observation is in apparent contrast to reports that cycloheximide inhibits replication of RNA of classical paramyxoviruses, and may indicate that mechanisms for replicating RNA of measles virus are different from those for replicating RNA of paramyxoviruses.     

Tim4- and MerTK-Mediated Engulfment of Apoptotic Cells by Mouse Resident Peritoneal Macrophages

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

Cycloheximide resistance in the Lipomycetaceae revisited

Cycloheximide (CYH) is a heterocyclic, glutarimide antibiotic that is a potent inhibitor of protein biosynthesis in most eukaryotes. This study demonstrated that yeasts from all species of the Lipomycetaceae, with the exception of Dipodascopsis spp., can grow in the presence of up to 5 g.L(-1) CYH -- a concentration that is five times higher than the accepted "highest" concentration of 1 g.L(-1) used in physiological tests for yeast identification. Lipomycetaceous yeasts are known to utilize heterocyclic nitrogen-containing compounds such as thymine as sole nitrogen source. CYH contains a glutarimide ring, which is chemically similar to thymine. We investigated the possibility that CYH resistance in the Lipomycetaceae might be due to an ability to degrade CYH and use it as the sole nitrogen source. However, we were unable to demonstrate significant growth on CYH as sole nitrogen source. When thymine was used as positive control, we could demonstrate its utilization as sole nitrogen source.     

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

Background

Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.

Methodology/Principal Findings

We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.

Conclusions/Significance

Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.     

Changes in Stem and Leaf Hydraulics Preceding Leaf Shedding in Castanea Sativa L.

This paper describes changes in leaf water status and in stem, petiole and leaf blade hydraulics preceding leaf senescence and shedding in Castanea sativa L. (chestnut). Measurements of maximum diurnal leaf conductance to water vapour (g_L), minimum water potential (_L), hydraulic conductance per unit leaf surface area of stems (K_SL), petioles (K_PL) and leaf blades (K_LL) and number of functional conduits and inside diameter distribution were performed in June, September and October 1999. In September, still green leaves had undergone some dehydration as indicated by decreased g_L (by 75 %) and _L with respect to June. In the same time, K_SL decreased by 88 %, while K_PL and K_LL decreased by 50 % and 20 % of the conduits of stems and 10 % of the petioles (all belonging to the widest diameter range) were no longer functioning, causing a decrease in the theoretical flow by 82 % in stems and 27 % in petioles. Stem xylem blockage was apparently due to tyloses growing into conduits. We advance the hypothesis that the entire process of leaf shedding and winter rest may be initiated by extensive stem embolism occurring during the summer.