Synthetic ceramides inhibit CD36 expression in U937 cells through a reduction in cytosolic peroxide

Ceramide (a sphingolipid) and reactive oxygen species (ROS) are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. It has been reported that synthesis of ceramide and ROS are intimately linked, and show reciprocal regulation. The levels of ceramide are reported to be elevated in atherosclerotic plaques providing circumstantial evidence for a pro-atherogenic role for ceramide. Indeed, LDL may be important sources of ceramide from sphingomyelin, where it promotes LDL aggregation. Using synthetic, short chain ceramides to mimic the cellular responses to fluctuations in natural endogenous ceramides, we have investigated ceramide effects on both intracellular redox state (as glutathione and ROS) and redox-sensitive gene expression, specifically the scavenger receptor CD36 (using RT-PCR and flow cytometry), in U937 monocytes and macrophages. We describe that the principal redox altering properties of ceramide are to lower cytosolic peroxide and to increase mitochondrial ROS formation, where growth arrest of U937 monocytes is also observed. In addition, cellular glutathione was depleted, which was independent of an increase in glutathione peroxidase activity. Examination of the effects of ceramide on stress induced CD36 expression in macrophages, revealed a dose dependent reduction in CD36 mRNA and protein levels, which was mimicked by N-acetyl cysteine. Taken together, these data suggest that ceramides differentially affect ROS within different cellular compartments, and that loss of cytosolic peroxide inhibits expression of the redox sensitive gene, CD36. This may attenuate both the uptake of oxidised LDL and the interaction of HDL with macrophages. The resulting sequelae in vivo remain to be determined.

Articular cartilage glycosaminoglycans inhibit the adhesion of endothelial cells

Articular cartilage undergoes severe loss of proteoglycan and its constituent glycosaminoglycans (GAGs) in osteoarthritis. We hypothesize that the low GAG content of osteoarthritic cartilage renders the tissue susceptible to pathological vascularization. This was investigated using an in vitro angiogenesis model assessing endothelial cell adhesion to GAG-depleted cartilage explants. Bovine cartilage explants were treated with hyaluronidase to deplete GAG content and then seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was assessed after 4 hr and 7 days. The effect of hyaluronidase treatment on GAG content, chondrocyte viability, and biochemical composition of the extracellular matrix was also determined. Hyaluronidase treatment reduced the GAG content of cartilage explants by 78 ± 3% compared with that of controls (p <0.0001). GAG depletion was associated with significantly more HMEC-1 adherence on both the surface (superficial zone) and the underside (deep zone) of the explants (both p <0.0001). The latter provided a more favorable environment for extended culture of HMEC-1 compared with the articulating surface. Hyaluronidase treatment altered the immunostaining for chondroitin sulfate epitopes, but not for lubricin. Our results support the hypothesis that articular cartilage GAGs are antiadhesive to endothelial cells and suggest that chondroitin sulfate and/or hyaluronan are responsible. The loss of these GAGs in osteoarthritis may allow osteochondral angiogenesis resulting in disease progression.

Ability of dyslexic and control teenagers to sustain attention and inhibit responses

Dyslexia and attentional difficulty have often been linked, but little is known of the nature of the supposed attentional disorder. The Sustained Attention to Response Task (SART: Robertson, Manly, Andrade, Baddeley and Yiend, 1997) was designed as a measure of sustained attention and requires the withholding of responses to rare (one in nine) targets. To investigate the nature of the attentional disorder in dyslexia, this paper reports two studies which examined the performance of teenagers with dyslexia and their age-matched controls on the SART, the squiggle SART (a modification of the SART using novel and unlabellable stimuli rather than digits) and the go-gap-stop test of response inhibition (GGST). Teenagers with dyslexia made significantly more errors than controls on the original SART, but not the squiggle SART. There were no group differences on the GGST. After controlling for speed of reaction time in a sequential multiple regression predicting SART false alarms, false alarms on the GGST accounted for up to 22% extra variance in the control groups (although less on the squiggle SART) but negligible amounts of variance in the dyslexic groups. We interpret the results as reflecting a stimulus recognition automaticity deficit in dyslexia, rather than a sustained attention deficit. Furthermore, results suggest that response inhibition is an important component of performance on the standard SART when stimuli are recognised automatically.

Acanthamoeba polyphaga trophozoite binding of representative fungal single cell forms

Acanthamoeba polyphaga trophozoites bind yeast cells of Candida albicans isolates within a few hours, leaving few cells in suspension or still attached to trophozoite surfaces. The nature of yeast cell recognition, mediated by an acanthamoebal trophozoite mannose binding protein is confirmed by experiments utilizing concentration dependent mannose hapten blocking. Similarly, acapsulate cells of Cryptococcus neoformans are also bound within a relatively short timescale. However, even after protracted incubation many capsulate cells of Cryptococcus remain in suspension, suggesting that the capsulate cell form of this species is not predated by acanthamoebal trophozoites. Further aspects of the association of Acanthamoeba and fungi are apparent when studying their interaction with conidia of the biocontrol agent Coniothyrium minitans. Conidia which readily bind with increasing maturity of up to 42 days, were little endocytosed and even released. Cell and conidial surface mannose as determined by FITC-lectin binding, flow cytometry with associated ligand binding analysis and hapten blocking studies demonstrates the following phenomena. Candida isolates and acapsulate Cryptococcus expose most mannose, while capsulate Cryptococcus cells exhibit least exposure commensurate with yeast cellular binding or lack of trophozoites. Conidia of Coniothyrium, albeit in a localized fashion, also manifest surface mannose exposure but as shown by Bmax values, in decreasing amounts with increasing maturity. Contrastingly such conidia experience greater trophozoite binding with maturation, thereby questioning the primacy of a trophozoite mannose-binding-protein recognition model.

The biology of the lichen Rhizocarpon geographicum: symbionts, growth, ecology, and lichenometry

Rhizocarpon geographicum (L.) DC. is one of the most widely distributed species of crustose lichens. This unusual organism comprises yellow-green ‘areolae’ that contain the algal symbiont which develop and grow on the surface of a non-lichenized, fungal ‘hypothallus’ that extends beyond the margin of the areolae to form a marginal ring. This species grows exceptionally slowly with annual radial growth rates (RGR) as low as 0.07 mm yr-1 and its considerable longevity has been exploited by geologists in the development of methods of dating the age of exposure of rock surfaces and glacial moraines (‘lichenometry’). Recent research has established some aspects of the basic biology of this important and interesting organism. This chapter describes the general structure of R. geographicum, how the areolae and hypothallus develop, why the lichen grows so slowly, the growth rate-size curve, and some aspects of the ecology of R. geographicum including whether the lichen can inhibit the growth of its neighbours by chemical means (‘allelopathy’). Finally, the importance of R. geographicum in direct and indirect lichenometry is reviewed.

Discriminating antigen and non-antigen using proteome dissimilarity II:viral and fungal antigens

Immunogenicity arises via many synergistic mechanisms, yet the overall dissimilarity of pathogenic proteins versus the host proteome has been proposed as a key arbiter. We have previously explored this concept in relation to Bacterial antigens; here we extend our analysis to antigens of viral and fungal origin. Sets of known viral and fungal antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we could not determine a threshold able meaningfully to separate non-antigen from antigen. We conclude that viral and fungal antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to mammalian genomes.

Fungal infection of the eye (ocular mycosis)

Although not as important as bacteria or viruses as a cause of eye infection, a large number of fungal species have now been recorded in association with the eye. In addition, several species have been implicated as a cause of eye infection (‘ocular mycosis’) and some may even cause life-threatening conditions. Ocular mycoses are being reported more frequently as a consequence of new medical practice and the increased numbers of immuno-compromised patients in the population, e.g., patients receiving radiation treatment or chemotherapy. This article describes the most common conditions caused by fungi which can affect the different structures of the eye, the importance of fungal contamination of materials as a source of eye infection, and the methods available for treatment.

Celiac disease-specific TG2-targeted autoantibodies inhibit angiogenesis ex vivo and in vivo in mice by interfering with endothelial cell dynamics

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility. © 2013 Kalliokoski et al.

Carbon monoxide expedites metabolic exhaustion to inhibit tumor growth

One classical feature of cancer cells is their metabolic acquisition of a highly glycolytic phenotype. Carbon monoxide (CO), one of the products of the cytoprotective molecule heme oxygenase-1 (HO-1) in cancer cells, has been implicated in carcinogenesis and therapeutic resistance. However, the functional contributions of CO and HO-1 to these processes are poorly defined. In human prostate cancers, we found that HO-1 was nuclear localized in malignant cells, with low enzymatic activity in moderately differentiated tumors correlating with relatively worse clinical outcomes. Exposure to CO sensitized prostate cancer cells but not normal cells to chemotherapy, with growth arrest and apoptosis induced in vivo in part throughmitotic catastrophe. CO targeted mitochondria activity in cancer cells as evidenced by higher oxygen consumption, free radical generation, and mitochondrial collapse. Collectively, our findings indicated that CO transiently induces an anti-Warburg effect by rapidly fueling cancer cell bioenergetics, ultimately resulting in metabolic exhaustion. ©2013 AACR.

Studies in fungal biology

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A bio-adhesive formulation for the delivery of anti-fungal agents to the oesophagus

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Heterodimerisation between VEGFR-1 and VEGFR-2 and not the homodimers of VEGFR-1 inhibit VEGFR-2 activity

Vascular endothelial growth factor (VEGF) signaling is tightly regulated by specific VEGF receptors (VEGF-R). Recently, we identified heterodimerisation between VEGFR-1 and VEGFR-2 (VEGFR1–2) to regulate VEGFR-2 function. However, both the mechanism of action and the relationship with VEGFR-1 homodimers remain unknown. The current study shows that activation of VEGFR1–2, but not VEGFR-1 homodimers, inhibits VEGFR-2 receptor phosphorylation under VEGF stimulation in human endothelial cells. Furthermore, inhibition of phosphatidylinositol 3-kinase (PI3K) increases VEGFR-2 phosphorylation under VEGF stimulation. More importantly, inhibition of PI3K pathway abolishes the VEGFR1–2 mediated inhibition of VEGFR-2 phosphorylation. We further demonstrate that inhibition of PI3K pathway promotes capillary tube formation. Finally, the inhibition of PI3K abrogates the inhibition of in vitro angiogenesis mediated by VEGFR1–2 heterodimers. These findings demonstrate that VEGFR1–2 heterodimers and not VEGFR-1 homodimers inhibit VEGF-VEGFR-2 signaling by suppressing VEGFR-2 phosphorylation via PI3K pathway.