Immunological studies in multiple low dose streptozotocin induced diabetes in mice

1. Multiple low doses of streptozotocin (MSZ) treatment successfully induced diabetes in male TO, MFI and HO lean mice. In contrast however, BALB/c mice failed to develop persistent hyperglycaemia. Single streptozotocin (SSZ) treatment also produced diabetes in TO mice. SSZ treatment however, produced severe weight loss and atrophy of the lymphoid organs. MSZ treatment on the other hand, was not cytotoxic towards lymphoid organs and, whilst there was no loss of body weight, growth rates were reduced in MSZ treated mice. 2. Following sheep red blood cell (SRBC) immunisation of MSZ-treated mice, haemagglutination titres, and numbers of antigen reactive cells and plaque forming cells were all significantly lower than control values. 3. In vitro proliferation of spleen cells in response to phytohaemagglutinin (PHA) and conconavalin A (ConA) was found to be significantly depressed in MSZ treated mice. However, T-lymphocyte responses were intact when the mice were not overtly hyperglycaemic. In contrast, however, T cell independent responses to lipopolysaccharide (LPS) were generally intact throughout the study period. 4. Cell mediated immunity, as assessed by measurements of delayed (Type IV) hypersensitivity, was also depressed in MSZ treated mice. This suppression could be reversed by insulin therapy. 5. Both natural killer cell activity and antibody dependent cell mediated cytotoxicity were found to be significantly increased in MSZ treated mice. 6. Histological examination of the pancreas showed the presence of insulitis, in MSZ treated mice, and cytotoxic effector cells against obese mice islet cells (as assessed by 51Cr release) and HIT-T15 cells (as assessed by insulin secretion) were found to be significantly increased. Furthermore, these effector cells were also found to show increased proliferation in the presence of homogenates prepared from HIT-T15 cells. Examination of the Sera from MSZ treated mice showed that islet cell surface antibodies were present.

A study of the calcium-induced morphological transistions of human erythrocytes

An examination was made of the morphological transitions induced in human erythrocytes by the elevation of cytosolic calcium, and of the biochemical mechanisms responsible. The loss of the discocyte morphology and the sequential progression of cells through the echinocyte stages 1, 2, 3 and sphereo-echinocyte was found to occur in both a calcium concentration- and a time-dependent manner. SDS-PAGE analysis of cytoskeletal proteins prepared from intact cells loaded with 150uM or 1mM calcium revealed the partial proteolytic loss of proteins 2.1, 2.2 and 4.1. The rate of proteolysis was not paralleled by that of echinocytosis, making a causative relationship unlikely. Cytoskeletal integrity did appear to influence shape reversal from the echinocyte to the discocyte morphology after removal of the calcium and ionophore A23187. The loss of 80% protein 4.1, 40% 2.1 and 30% 2.2 was associated with, although not necessarily the sole cause, of irreversible sphereo-echinocytosis. Pre-treatment of cells with wheat germ agglutinin preserved the discocyte morphology despite continued cytoskeletal proteolysis during calcium-loading. All observations were made on cells incubated either in the presence or absence of glycolytic substrates, effectively altering cell metabolic status. This influenced the rate of progression of cells through the echinocyte stages, the rate of proteolysis of cytoskeletal proteins, and the extent and kinetics of shape reversal from cells transformed to the sphereo-echinocyte morphology. The stage 1 to discocyte transition was the rate limiting step of this shape recovery. In contrast the rate of loss of the discocyte morphology was independent of cell metabolic status during exposure to calcium, as was the extent of restoration of the discocyte morphology from cells transformed to stage 1 echinocytes. An hypothesis is presented that echinocytosis is a discontinuous process with discrete steps initiated by different biochemical mechanisms varying in their dependence on metabolic energy.

Polyunsaturated fatty acids in tumour-induced cachexia

A transplantable murine colon adenocarcinoma (MAC16) was utilised as a model of human cancer cachexia. This tumour has been found to produce extensive weight loss, characterised by depletion of host body protein and lipid stores at a small tumour burden. This weight loss has been found to be associated with production by the tumour of a lipolytic factor, activity of which was inhibited in vitro by the polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA). EPA has also been shown to possess anti-tumour and anti-cachectic activity in vivo, leading to the hypothesis that fatty acids mobilised by the lipolytic factor supply a growth requirement of the MAC16 tumour. In this study mobilisation and sequestration of fatty acids by the tumour was found to be non-specific, although a relationship between weight loss and arachidonic acid (AA) concentration was found in both tumour-bearing mice, and human cancer patients. The anti-tumour effect of EPA, which was found to be associated with an increase in cell loss, but not its anti-cachectic activity, was reversed by the administration of the PUFAs oleic acid (OA) and linoleic acid (LA). LA was also found to be capable of stimulating tumour growth. Inhibition of either the cyclooxygenase or lipoxygenase pathways was found to result in reduction of tumour growth, leading to the implication of one of the metabolites of LA or AA in tumour growth and cachexia. The ethyl ester of EPA was found to be inactive against the growth and cachexia of the MAC16 tumour, due to its retarded uptake compared with the free acid. The anti-proliferative agent 5-fluorouracil was found to cause tumour growth inhibition, and when given in combination with EPA, reduced the phase of tumour regrowth observed after 4 to 5 days of treatment with EPA.

Inhibitors of lipolysis in tumor-induced cachexia

The MAC16 tumour produces a factor which exhibits lipid-mobilizing activity in vitro in addition to causing extensive depletion of host lipid stores. The mechanism of the anti-lipolytic effect of two anti-cachectic agents, eicosapentaenoic acid, an ω-3 polyunsaturated fatty acid (PUFA), and N-(3-phenoxycinnamyl)acetohydroxamic acid (BW A4C), a 5-lipoxygenase inhibitor, has been investigated. These two agents reduce tumour growth and reverse the weight loss which accompanies transplantation of the MAC16 murine colon adenocarcinoma into NMRI mice. Mice transplanted with the MAC16 tumour exhibited weight loss which was directly proportional to the serum lipolytic activity measured in vitro up to a weight loss corresponding to 16% of the original body weight. After this time, an inverse relationship between weight loss and lipolytic activity was observed. Body composition analysis revealed a large decrease in body fat relative to other body compartments. The anti-tumour/anti-cachectic effect of EPA did not appear to be due to its ability to inhibit the production of prostaglandin E2. The MAC16 lipolytic factor increased adenylate cyclase activity in adipocyte plasma membranes in a concentration-dependent manner. EPA inhibited the production of cAMP attributed to this lipid-mobilizing factor. EPA produced alterations in Gi , the guanine nucleotide binding protein which mediates hormonal inhibition of adenylate cyclase, in addition to altering cAMP production in adipocyte plasma membranes in response to hormonal stimulation. The alterations in adenylate cyclase activity were complex and not specific to EPA. EPA stimulated adenylate cyclase activity when in a relatively high fatty acid : membrane ratio and inhibited activity when this ratio was lowered. The inhibitory effect of EPA on adenylate cyclase activity may be the underlying mechanism which explains its anti-lipolytic and anti-cachectic effect. The inability of the related ω-3 PUFA, docosahexaenoic acid (DHA), to inhibit cachexia may be due to a difference in the metabolic fates of these two fatty acids. BW A4C inhibited lipolysis in isolated adipocytes which suggests that this compound may possess the potential for an anti-cachectic effect which is independent of its inhibitory effect on tumour growth.

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

BACKGROUND: Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. METHODS: In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. RESULTS: WF decreased the viability of C2C12 myotubes, especially at concentrations of 20-25 mug.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. CONCLUSION: These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model. © 2008 Yano et al; licensee BioMed Central Ltd.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Low-loss waveguides fabricated by femtosecond chirped-pulse oscillator

Recent results on direct femtosecond inscription of straight low-loss waveguides in borosilicate glass are presented. We also demonstrate lowest ever losses in curvilinear waveguides, which we use as main building blocks for integrated photonics circuits. Low-loss waveguides are of great importance to a variety of applications of integrated optics. We report on recent results of direct femtosecond fabrication of smooth low-loss waveguides in standard optical glass by means of femtosecond chirped-pulse oscillator only (Scientific XL, Femtolasers), operating at the repetition rate of 11 MHz, at the wavelength of 800 nm, with FWHM pulse duration of about 50 fs, and a spectral widths of 30 nm. The pulse energy on target was up to 70 nJ. In transverse inscription geometry, we inscribed waveguides at the depth from 10 to 300 micrometers beneath the surface in the samples of 50 x 50 x 1 mm dimensions made of pure BK7 borosilicate glass. The translation of the samples accomplished by 2D air-bearing stage (Aerotech) with sub-micrometer precision at a speed of up to 100 mm per second (hardware limit). Third direction of translation (Z-, along the inscribing beam or perpendicular to sample plane) allows truly 3D structures to be fabricated. The waveguides were characterized in terms of induced refractive index contrast, their dimensions and cross-sections, mode-field profiles, total insertion losses at both 633 nm and 1550 nm. There was almost no dependence on polarization for the laser inscription. The experimental conditions – depth, laser polarization, pulse energy, translation speed and others, were optimized for minimum insertion losses when coupled to a standard optical fibre SMF-28. We found coincidence of our optimal inscription conditions with recently published by other groups [1, 3] despite significant difference in practically all experimental parameters. Using optimum regime for straight waveguides fabrication, we inscribed a set of curvilinear tracks, which were arranged in a way to ensure the same propagation length (and thus losses) and coupling conditions, while radii of curvature varied from 3 to 10 mm. This allowed us to measure bend-losses – they less than or about 1 dB/cm at R=10 mm radius of curvature. We also demonstrate a possibility to fabricate periodical perturbations of the refractive index in such waveguides with the periods using the same set-up. We demonstrated periods of about 520 nm, which allowed us to fabricate wavelength-selective devices using the same set-up. This diversity as well as very short time for inscription (the optimum translation speed was found to be 40 mm/sec) makes our approach attractive for industrial applications, for example, in next generation high-speed telecom networks.

Role of Ca2+ in proteolysis-inducing factor (PIF)-induced atrophy of skeletal muscle

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C2C12 murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca2 +i, which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-a (TNF-a) and lipopolysaccharide (LPS) also induced a rise in Ca2 +i, but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca2 +i induced by PIF and AngII was completely attenuated by the Zn2 + chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn2 +. The Ca2 +i rise induced by PIF was independent of the presence of extracellular Ca2 +, and attenuated by the Ca2 + pump inhibitor thapsigargin, suggesting that the Ca2 +i rise was due to release from intracellular stores. This rise in Ca2 +i induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca2 +i, which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.

Role of the dsRNA-dependent protein kinase (PKR) in the attenuation of protein loss from muscle by insulin and insulin-like growth factor-I (IGF-I)

Proteolysis-inducing factor (PIF), a tumour-produced cachectic factor, induced a dose-dependent decrease in protein synthesis in murine myotubes, together with an increase in phosphorylation of eucaryotic initiation factor 2 (eIF2) on the alpha-subunit. Both insulin (1 nM) and insulin-like growth factor I (IGF-I) (13.2 nM) attenuated the depression of protein synthesis by PIF and the increased phosphorylation of eIF2alpha, by inhibiting the activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) by induction of protein phosphatase 1. A low-molecular weight inhibitor of PKR also reversed the depression of protein synthesis by PIF to the same extent, as did insulin and IGF-I. Both insulin and IGF-I-stimulated protein synthesis in the presence of PIF, and this was attenuated by Salubrinal, an inhibitor of phospho eIF2alpha phosphatase, suggesting that at least part of this action was due to their ability to inhibit phosphorylation of eIF2alpha. Both insulin and IGF-I also attenuated the induction of protein degradation in myotubes induced by PIF, this effect was also attenuated by Salubrinal. These results suggest an alternative mechanism involving PKR to explain the effect of insulin and IGF-I on protein synthesis and degradation in skeletal muscle in the presence of catabolic factors.

Mechanism of induction of muscle protein loss by hyperglycaemia

Treatment of murine myotubes with high glucose concentrations (10 and 25 mM) stimulated protein degradation through the ubiquitin–proteasome pathway, and also caused activation (autophosphorylation) of PKR (double-stranded-RNA-dependent protein kinase) and eIF2a (eukaryotic initiation factor 2a). Phosphorylation of PKR and eIF2a was also seen in the gastrocnemius muscle of diabetic ob/ob mice. High glucose levels also inhibited protein synthesis. The effect of glucose on protein synthesis and degradation was not seen in myotubes transfected with a catalytically inactive variant (PKR?6). High glucose also induced an increased activity of both caspase-3 and -8, which led to activation of PKR, since this was completely attenuated by the specific caspase inhibitors. Activation of PKR also led to activation of p38MAPK (mitogen activated protein kinase), leading to ROS (reactive oxygen species) formation, since this was attenuated by the specific p38MAPK inhibitor SB203580. ROS formation was important in protein degradation, since it was completely attenuated by the antioxidant butylated hydroxytoluene. These results suggest that high glucose induces muscle atrophy through the caspase-3/-8 induced activation of PKR, leading to phosphorylation of eIF2a and depression of protein synthesis, together with PKR-mediated ROS production, through p38MAPK and increased protein degradation.

Fluctuation-induced traffic congestion in heterogeneous networks

In studies of complex heterogeneous networks, particularly of the Internet, significant attention was paid to analyzing network failures caused by hardware faults or overload, where the network reaction was modeled as rerouting of traffic away from failed or congested elements. Here we model another type of the network reaction to congestion - a sharp reduction of the input traffic rate through congested routes which occurs on much shorter time scales. We consider the onset of congestion in the Internet where local mismatch between demand and capacity results in traffic losses and show that it can be described as a phase transition characterized by strong non-Gaussian loss fluctuations at a mesoscopic time scale. The fluctuations, caused by noise in input traffic, are exacerbated by the heterogeneous nature of the network manifested in a scale-free load distribution. They result in the network strongly overreacting to the first signs of congestion by significantly reducing input traffic along the communication paths where congestion is utterly negligible. © Copyright EPLA, 2012.

Loss of Akt activity increases circulating soluble endoglin release in preeclampsia:identification of inter-dependency between Akt-1 and heme oxygenase-1

Aims - Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-ß (TGF-ß) signalling, which is known to be elevated in preeclampsia. Methods and results - Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion - The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly

The molecular chaperone, Hsc70, together with its cofactor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map,we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Low-loss waveguides fabricated by femtosecond chirped-pulse oscillator

Recent results on direct femtosecond inscription of straight low-loss waveguides in borosilicate glass are presented. We also demonstrate lowest ever losses in curvilinear waveguides, which we use as main building blocks for integrated photonics circuits. Low-loss waveguides are of great importance to a variety of applications of integrated optics. We report on recent results of direct femtosecond fabrication of smooth low-loss waveguides in standard optical glass by means of femtosecond chirped-pulse oscillator only (Scientific XL, Femtolasers), operating at the repetition rate of 11 MHz, at the wavelength of 800 nm, with FWHM pulse duration of about 50 fs, and a spectral widths of 30 nm. The pulse energy on target was up to 70 nJ. In transverse inscription geometry, we inscribed waveguides at the depth from 10 to 300 micrometers beneath the surface in the samples of 50 x 50 x 1 mm dimensions made of pure BK7 borosilicate glass. The translation of the samples accomplished by 2D air-bearing stage (Aerotech) with sub-micrometer precision at a speed of up to 100 mm per second (hardware limit). Third direction of translation (Z-, along the inscribing beam or perpendicular to sample plane) allows truly 3D structures to be fabricated. The waveguides were characterized in terms of induced refractive index contrast, their dimensions and cross-sections, mode-field profiles, total insertion losses at both 633 nm and 1550 nm. There was almost no dependence on polarization for the laser inscription. The experimental conditions – depth, laser polarization, pulse energy, translation speed and others, were optimized for minimum insertion losses when coupled to a standard optical fibre SMF-28. We found coincidence of our optimal inscription conditions with recently published by other groups [1, 3] despite significant difference in practically all experimental parameters. Using optimum regime for straight waveguides fabrication, we inscribed a set of curvilinear tracks, which were arranged in a way to ensure the same propagation length (and thus losses) and coupling conditions, while radii of curvature varied from 3 to 10 mm. This allowed us to measure bend-losses – they less than or about 1 dB/cm at R=10 mm radius of curvature. We also demonstrate a possibility to fabricate periodical perturbations of the refractive index in such waveguides with the periods using the same set-up. We demonstrated periods of about 520 nm, which allowed us to fabricate wavelength-selective devices using the same set-up. This diversity as well as very short time for inscription (the optimum translation speed was found to be 40 mm/sec) makes our approach attractive for industrial applications, for example, in next generation high-speed telecom networks.

Aβ(1-42) induced metabolic effects in a stem cell derived neuron and astrocyte network.

Alzheimer’s Disease (AD) is the most common form of dementia currently affecting more than 35 million people worldwide. Hypometabolism is a major feature of AD and appears decades before cognitive decline and pathological lesions. This has a detrimental impact on the brain which has a high energy demand. Current models of AD fail to mimic all the features of the disease, which has an impact on the development of new therapies. Human stem cell derived models of the brain have attracted a lot of attention in recent years as a tool to study neurodegenerative diseases. In this thesis, neurons and astrocytes derived from the human embryonal carcinoma cell line (NT2/D1) were utilised to determine the metabolic coupling between neurons and astrocytes with regards to responses to hypoglycaemia, neuromodulators and increase in neuronal activity. This model was then used to investigate the effects of Aß(1-42) on the metabolism of these NT2-derived co-cultures as well as pure astrocytes. Additionally primary cortical mixed neuronal and glial cultures were utilised to compare this model to a widely accepted in vitro model used in Alzheimer’s disease research. Co-cultures were found to respond to Aß(1-42) in similar way to human and in vivo models. Hypometabolism was characterised by changes in glucose metabolism, as well as lactate, pyruvate and glycogen. This led to a significant decrease in ATP and the ratio of NAD+/NADH. These results together with an increase in calcium oscillations and a decrease in GSH/GSSG ratio, suggests Aß-induces metabolic and oxidative stress. This situation could have detrimental effects in the brain which has a high energy demand, especially in terms of memory formation and antioxidant capacity.