Hyperspectral and Physiological Analyses of Coral-Algal Interactions

Space limitation leads to competition between benthic, sessile organisms on coral reefs. As a primary example, reef-building corals are in direct contact with each other and many different species and functional groups of algae. Here we characterize interactions between three coral genera and three algal functional groups using a combination of hyperspectral imaging and oxygen microprofiling. We also performed in situ interaction transects to quantify the relative occurrence of these interaction on coral reefs. These studies were conducted in the Southern Line Islands, home to some of the most remote and near-pristine reefs in the world. Our goal was to determine if different types of coral-coral and coral-algal interactions were characterized by unique fine-scale physiological signatures. This is the first report using hyperspectral imaging for characterization of marine benthic organisms at the micron scale and proved to be a valuable tool for discriminating among different photosynthetic organisms. Consistent patterns emerged in physiology across different types of competitive interactions. In cases where corals were in direct contact with turf or macroalgae, there was a zone of hypoxia and altered pigmentation on the coral. In contrast, interaction zones between corals and crustose coralline algae (CCA) were not hypoxic and the coral tissue was consistent across the colony. Our results suggest that at least two main characteristic coral interaction phenotypes exist: 1) hypoxia and coral tissue disruption, seen with interactions between corals and fleshy turf and/or some species of macroalgae, and 2) no hypoxia or tissue disruption, seen with interactions between corals and some species of CCA. Hyperspectral imaging in combination with oxygen profiling provided useful information on competitive interactions between benthic reef organisms, and demonstrated that some turf and fleshy macroalgae can be a constant source of stress for corals, while CCA are not.     

Lines of mice with chronically elevated baseline corticosterone levels are more susceptible to a parasitic nematode infection

Chronically elevated circulating plasma glucocorticoid concentrations can have suppressive effects on immune function in mammals. House mice (Mus domesticus) that have been selectively bred for high voluntary wheel running exhibit chronically elevated (two-fold, on average) plasma corticosterone (CORT) levels and hence are an interesting model to study possible glucocorticoid-induced immune suppression. As an initial test of their immunocompetence, we compared the four replicate high runner (HR) lines with their four non-selected control (C) lines by subjecting them to infection by a parasitic nematode, Nippostrongylus brasiliensis. At generation 36 of the selection experiment, 10 adult males from each of the eight lines were inoculated subcutaneously with approximately 600 third-stage larval N. brasiliensis, and then sacrificed 12 days after injection. Neither spleen mass nor number of adult nematodes in the small intestine differed significantly between HR and C lines. However, the eight lines differed significantly in nematode counts, and the line means for nematode infestation were significantly positively related to baseline circulating CORT concentration measured in males from generations 34 and 39. Therefore, although selective breeding for high locomotor activity may not have resulted in a generally compromised immune response, results of this study are consistent with the hypothesis that glucocorticoids can have immunosuppressive effects.     

The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors

Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes ( murA and murZ ) encoding UDP- N -acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro , consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA , but remained almost unchanged following inactivation of murZ . Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA . Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.     

Solubilized phenyl-pyrazole ureas as potent, selective 5-HT2A inverse-agonists and their application as antiplatelet agents

Potent 5-HT_2A inverse-agonists containing phenyl-pyrazole ureas with an amino side chain were identified. Optimization of this series resulted in selective compounds that proved effective in modulating 5HT-induced amplification of ADP-stimulated human platelet aggregation.     

ATR and Chk1 Suppress a Caspase-3–Dependent Apoptotic Response Following DNA Replication Stress

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR- or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and -deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.