A serine oligopeptidase from African Trypanosomes.

Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already be a cellular target for trypanocidal drugs. If correct, this may represent an important step towards understanding the biochemical mechanisms of the trypanocidal activity of these drugs, as well as providing valuable clues as to how to improve their efficacy

Stable Small Animal Ventilation for Dynamic Lung Imaging to Support Computational Fluid Dynamics Models

Pulmonary computational fluid dynamics models require that three-dimensional images be acquired over multiple points in the dynamic breathing cycle without breath holds or changes in ventilatory mechanics. With small animals, these requirements can result in long imaging times (∼90 minutes), over which lung mechanics, such as compliance, may gradually change if not carefully monitored and controlled. These changes, caused by derecruitment of parenchymal tissue, are manifested as an upward drift in peak inspiratory pressure (PIP) or by changes in the pressure waveform and/or lung volume over the course of the experiment. We demonstrate highly repeatable mechanical ventilation in anesthetized rats over a long duration for dynamic lung x-ray computed tomography (CT) imaging. We describe significant updates to a basic commercial ventilator that was acquired for these experiments. Key to achieving consistent results was the implementation of periodic deep breaths, or sighs, of extended duration to maintain lung recruitment. In addition, continuous monitoring of breath-to-breath pressure and volume waveforms and long-term trends in PIP and flow provide diagnostics of changes in breathing mechanics

Lack of Sik1 in Mouse Embryonic Stem Cells Impairs Cardiomyogenesis by Down-Regulating the Cyclin-Dependent Kinase Inhibitor p57kip2

Sik1 (salt inducible kinase 1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMP-activated protein kinase family. During murine embryogenesis, sik1 marks the monolayer of future myocardial cells that will populate first the primitive ventricle, and later the primitive atrium suggesting its involvement in cardiac cell differentiation and/or heart development. Despite that observation, the involvement of sik1 in cardiac differentiation is still unknown. We examined the sik1 function during cardiomyocyte differentiation using the ES-derived embryoid bodies. We produced a null embryonic stem cell using a gene-trap cell line carrying an insertion in the sik1 locus. In absence of the sik1 protein, the temporal appearance of cardiomyocytes is delayed. Expression profile analysis revealed sik1 as part of a genetic network that controls the cell cycle, where the cyclin-dependent kinase inhibitor p57Kip2 is directly involved. Collectively, we provided evidence that sik1-mediated effects are specific for cardiomyogenesis regulating cardiomyoblast cell cycle exit toward terminal differentiation

MAPk Activation Modulates Permeability of Isolated Rat Alveolar Epithelial Cell Monolayers Following Cyclic Stretch

We cultured (5 days) rat alveolar epithelial cells to investigate the role of mitogen-activated protein kinase (MAPk) signaling in ventilator induced epithelial barrier dysfunction. Cells were stretched to a magnitude of 12% or 37% change in surface area at a rate of 0.25 Hz with and without pretreatment with either the JNK inhibitor SP600125 or the ERK inhibitor U0126. Following stretch (0, 10, 30, or 60 min), MAPk phosphorylation was examined, monolayer permeability to the uncharged tracer carboxyfluorescein measured (0, 10, 60 min of stretch), and occludin expression determined (0 and 60 min of stretch). Stretch to 12%, previously shown not to increase monolayer permeability, did not alter phosphorylation of any MAPk or occludin expression at any time point. Following stretch to 37%, phosphorylation of JNK, ERK, and p38 was significantly higher by 10 minutes than in unstretched monolayers. Phosphorylation of JNK and p38 subsided as stretch continued, and by 30 minutes returned to unstretched levels. Phosphorylation of ERK remained significantly elevated compared to unstretched levels at all stretch durations. Epithelial permeability increased significantly by 10 minutes of stretch compared to unstretched controls, with further significant increases by 60 minutes. Inhibition with U0126 and SP600125 prevented stretch-induced phosphorylation increases of ERK and JNK, respectively, however neither prevented increases in permeability following 10 minutes. Separately, inhibition of JNK or ERK prevented subsequent additional permeability increases as stretch continued to 60 minute time points. Inhibition of JNK, not ERK, prevented loss of occludin, and minimized loss of cell-cell contact following 60 minutes of stretch. These data suggest that stretch-induced JNK signaling modulates epithelial permeability through regulation tight junction protein expression, and is a potential target for clinical treatments during mechanical ventilation

Gene Expression Meta-Analysis Identifies VDAC1 as a Predictor of Poor Outcome in Early Stage Non-Small Cell Lung Cancer

The bioenergetic status of non-small cell lung cancer correlates with tumour aggressiveness. The voltage dependent anion channel type 1 (VDAC1) is a component of the mitochondrial permeability transition pore, regulates mitochondrial ATP/ADP exchange suggesting that its over-expression could be associated with energy dependent processes including increased proliferation and invasiveness. To test this hypothesis, we conducted an in vivo gene-expression meta-analysis of surgically resected non-small cell lung cancer (NSCLC) using 602 individual expression profiles, to examine the impact of VDAC1 on survival.High VDAC1 expression was associated with shorter overall survival with hazard ratio (HR) = 0.6639 (95% confidence interval (CI) 0.4528 to 0.9721), p = 0.035352 corresponding to 52 versus 101 months. VDAC1 predicted shorter time to recurrence and was shown to be an independent prognostic factor compared with histology, gender, age, nodal stage and tumour stage in a Cox multivariate analysis. Supervised analysis of all the datasets identified a 6-gene signature comprising HNRNPC, HSPA4, HSPA9, UBE2D2, CSNK1A1 and G3BP1 with overlapping functions involving regulation of protein turnover, RAS-RAF-MEK pathway and transcription. VDAC1 predicted survival in breast cancer and myeloma and an unsupervised analysis revealed enrichment of the VDAC1 signature in specific subsets.In summary, gene expression analysis identifies VDAC1 gene expression as a predictor of poor outcome in NSCLC and other cancers and is associated with dysregulation of a conserved set of biological pathways, which may be causally associated with aggressive tumour behaviour

Role of GABA Receptors in Fetal Lung Development in Rats

Fluid accumulation is critical for lung distension and normal development. The multi-subunit γ-amino butyric acid type A receptors (GABAA) mainly act by mediating chloride ion (Cl−) fluxes. Since fetal lung actively secretes Cl−-rich fluid, we investigated the role of GABAA receptors in fetal lung development. The physiological ligand, GABA, and its synthesizing enzyme, glutamic acid decarboxylase, were predominantly localized to saccular epithelium. To examine the effect of activating GABAA receptors in fetal lung development in vivo, timed-pregnant rats of day 18 gestation underwent an in utero surgery for the administration of GABAA receptor modulators into the fetuses. The fetal lungs were isolated on day 21 of gestation and analyzed for changes in fetal lung development. Fetuses injected with GABA had a significantly higher body weight and lung weight when compared to phosphate-buffered saline (control)-injected fetuses. GABA-injected fetal lungs had a higher number of saccules than the control. GABA increased the number of alveolar epithelial type II cells as indicated by surfactant protein C-positive cells. However, GABA decreased the number of α-smooth muscle actin-positive myofibroblasts, but did not affect the number of Clara cells or alveolar type I cells. GABA-mediated effects were blocked by the GABAA receptor antagonist, bicuculline. GABA also increased cell proliferation and Cl− efflux in fetal distal lung epithelial cells. In conclusion, our results indicate that GABAA receptors accelerate fetal lung development, likely through an enhanced cell proliferation and/or fluid secretion

Moderation of Calpain Activity Promotes Neovascular Integration and Lumen Formation during VEGF-Induced Pathological Angiogenesis

Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF

Models of Emergency Departments for Reducing Patient Waiting Times

In this paper, we apply both agent-based models and queuing models to investigate patient access and patient flow through emergency departments. The objective of this work is to gain insights into the comparative contributions and limitations of these complementary techniques, in their ability to contribute empirical input into healthcare policy and practice guidelines. The models were developed independently, with a view to compare their suitability to emergency department simulation. The current models implement relatively simple general scenarios, and rely on a combination of simulated and real data to simulate patient flow in a single emergency department or in multiple interacting emergency departments. In addition, several concepts from telecommunications engineering are translated into this modeling context. The framework of multiple-priority queue systems and the genetic programming paradigm of evolutionary machine learning are applied as a means of forecasting patient wait times and as a means of evolving healthcare policy, respectively. The models' utility lies in their ability to provide qualitative insights into the relative sensitivities and impacts of model input parameters, to illuminate scenarios worthy of more complex investigation, and to iteratively validate the models as they continue to be refined and extended. The paper discusses future efforts to refine, extend, and validate the models with more data and real data relative to physical (spatial–topographical) and social inputs (staffing, patient care models, etc.). Real data obtained through proximity location and tracking system technologies is one example discussed

Does Parenteral Nutrition Influence Electrolyte and Fluid Balance in Preterm Infants in the First Days after Birth?

New national guidelines recommend more restricted fluid intake and early initiation of total parenteral nutrition (TPN) in very preterm infants. The aim was study the effect of these guidelines on serum sodium and potassium levels and fluid balance in the first three days after birth.Two cohorts of infants <28 weeks gestational age, born at the Leiden University Medical Center in the Netherlands, were compared retrospectively before (2002–2004, late-TPN) and after (2006–2007, early-TPN) introduction of the new Dutch guideline. Outcome measures were serum sodium and potassium levels, diuresis, and changes in body weight in the first three postnatal days.In the first three postnatal days no differences between late-TPN (N = 70) and early-TPN cohort (N = 73) in mean (SD) serum sodium (141.1 (3.8) vs 141.0 (3.7) mmol/l) or potassium (4.3 (0.5) vs 4.3 (0.5) mmol/l) were found, but in the early-TPN cohort diuresis (4.5 (1.6) vs 3.2 (1.4) ml/kg/h) and loss of body weight were decreased (−6.0% (7.7) vs −0.8% (8.0)).Initiation of TPN immediately after birth and restricted fluid intake in very preterm infants do not seem to influence serum sodium and potassium levels in first three postnatal days. Further research is needed to see if a decreased diuresis and loss of body weight in the first days is the result of a delayed postnatal adaptation or better energy balance

Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice

Mammals are serially infected with a variety of microorganisms, including bacteria and parasites. Each infection reprograms the immune system's responses to re-exposure and potentially alters responses to first-time infection by different microorganisms. To examine whether infection with a metazoan parasite modulates host responses to subsequent bacterial infection, mice were infected with the hookworm-like intestinal nematode Nippostrongylus brasiliensis, followed in 2–4 weeks by peritoneal injection of the pathogenic bacterium Klebsiella pneumoniae. Survival from Klebsiella peritonitis two weeks after parasite infection was better in Nippostrongylus-infected animals than in unparasitized mice, with Nippostrongylus-infected mice having fewer peritoneal bacteria, more neutrophils, and higher levels of protective interleukin 6. The improved survival of Nippostrongylus-infected mice depends on IL-4 because the survival benefit is lost in mice lacking IL-4. Because mast cells protect mice from Klebsiella peritonitis, we examined responses in mast cell-deficient KitW-sh/KitW-sh mice, in which parasitosis failed to improve survival from Klebsiella peritonitis. However, adoptive transfer of cultured mast cells to KitW-sh/KitW-sh mice restored survival benefits of parasitosis. These results show that recent infection with Nippostrongylus brasiliensis protects mice from Klebsiella peritonitis by modulating mast cell contributions to host defense, and suggest more generally that parasitosis can yield survival advantages to a bacterially infected host