Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. © 2004 European Molecular Biology Organization.

Delivery and stability of antisense oligonucleotide conjugates in vitro

The efficacy of antisense oligonucleotide (ODN) therapy is dependent on four major parameters: delivery to cells, intracellular stability and localisation and efficient action at the target site.The aim of this project was to study the delivery of ODNs to macrophages and to assess the stability of two ODN conjugates, in vitro. The first conjugate aimed to improve uptake of ODNs via mannose receptor mediated delivery, the second investigated the improved delivery of ODN conjugates via non-specific lipophilic interaction with the cell membrane. A mono-mannose phosphoramidite derivative was designed and synthesised and a mono-mannose ODN conjugate synthesised by standard phosphoramidite chemistry. Delivery of this conjugate was enhanced to RAW264.7 and J774 macrophage cell lines via a mechanism of receptor mediated endocytosis. The delivery of three lipophilic ODN conjugates, cholesterol (cholhex), 16-carbon alkyl chain (C16) and hexa-ethylene glycol (HEG) moieties and an unconjugated ODN were assessed in RAW264.7 macrophages. All three conjugates increased the lipophilicity of the ODN as assessed from partition coefficient data. Both the cholhex and unconjugated ODNs were found to have higher degrees of cellular association than the C16 and HEG conjugates. Cellular uptake studies implicated internalisation of these ODNs by an adsorptive endocytosis mechanism. Following endocytosis, ODNs must remain stable during their residence in endosomal/lysosomal compartments prior to exiting and exerting their biological action in either the cytosol or nucleus. Assessment of in vitro stability in a lysosomal extract revealed the cholhex conjugate and unconjugated ODNs to have a longer half-life than the C16 and HEG conjugated ODNs, highlighting the influence of conjugate moieties on lysosomal stability. The effects of base composition and length on stability in a lysosomal extract revealed the longest half-life for homo-cytidine ODNs and ODNs over 20 nucleotides in length. These studies suggest that the above conjugates can enhance cellular association and delivery of antisense ODNs to cultured macrophages. This may lead to their use in treating disorders such as HIV infection, which affects this cell type.

Drug targeting with phagocytic polymorphonuclear leucocytes

1. Phagocytic polymorphonuclear leucocytes (PMNLs) or neutrophils have a marked avidity for the uptake of particulate material and are the first cell type to respond to inflammatory stimuli in vivo. 2. By harnessing these pathophysiological characteristics the inherent targeting capacity of the PMNL could be exploited to carry drug loaded particles to these sites. 3. In vitro chemotaxis of PMNLs was studied in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in the Blindwell chamber assay. 4. After phagocytosis of 1.1m polystyrene latex (PSL) beads at a range of incubation concentrations (5,10,20, and 30 beads/cell) the migration of the PMNL population was not significantly different from control, without beads. 5. The distribution of the beads within the filter showed that a disproportionately large number of PSL (50%) were associated with the cells on the surface of the filter that had not penetrated the filter. Eighty per cent of the PMNL population migrated and despite containing less PSL beads/cell, 50% of the dose was carried into the filter. Between 5 and 10% of these PSL were carried beyond 60m in the assay. 6. These results suggested heterogeneity of the PMNL population and to achieve efficient targeting with these cells preferential selection of the migratory sub-population would be needed. 7. The air-pouch model was then developed to study the focal accumulation of PMNLs in vivo. The PMNL isolated did not survive long enough in the circulation due to the trauma of the isolation procedure used; an alternative method will have to be employed.

Production and action of local mediators in the rat gastric mucosa

This study concerns the production and action of the local mediators nitric oxide (NO) and prostaglandin E2 (PGE2) in the rat gastric mucosa. The major objectives were: (i) to determine which mucosal cell type(s) contained NO synthase activity, (ii) to establish the functional role(s) of NO in the gastric mucosa and (iii) to investigate regulation of gastric PGE2 production. Gastric mucosal cells were isolated by pronase digestion coupled with intermittent calcium chelation and were separated by either density-gradient centrifugation or by counterflow elutriation. The distribution of Ca2+ -dependent NO synthase activity, measured via the conversion of [14C]-L-arginine to [14C]-L- citrulline, paralleled the distribution of mucous cells in elutriated fractions. Pre-treatment of rats with lipopolysaccharide caused the induction of Ca2+ -independent NO synthase in the elutriator fractions enriched with mucous cells. Incubation of isolated cells with the NO donor isosorbide dinitrate (ISDN) produced a concentration-dependent increase in the guanosine 3',-5'-cyclic monophosphate (cGMP) content which was accompanied by a concentration-dependent increase in release of immunoreactive mucin. Intragastric administration of ISDN of dibutyryl cGMP in vivo increased the thickness of the mucus layer overlying the gastric mucosa. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent inhibition (IC50 247 μM) of histamine-stimulated aminopyrine accumulation, a measure of secretory activity, in cell suspensions containing > 80% parietal cells. SNAP increased the cGMP content of the suspension but did not decrease cellular viability, glucose oxidation or adenosine 3',5'-cyclic monophosphate content. The inhibitory effect of SNAP was observed in permeabilised cells stimulated with ATP and was stereospecifically blocked by preincubation with Rp-8-bromoguanosine 3'-5'-monophosphorothioate, which inhibits activation of cGMP-dependent protein kinase. Stimulation of PGE2 release by bradykinin in a low density cell fraction, enriched with parietal cells and devoid of vascular endothelial cells and macrophages, involved a bradykinin B1 receptor. In summary, NO synthase activity is probably present in gastric mucous epithelial cells. NO may promote mucus secretion by elevation of cGMP. NO donors inhibit acid secretion at a specific site and their action may involve cGMP. The bradykinin B1 receptor is involved with PGE2 production in the gastric mucosa.

Palmitate induces insulin resistance and a pro-atherogenic phenotype in monocytes

The present study investigated the effect of the two most abundant FFA in plasma – palmitate and oleate – on insulin sensitivity and vascular function (monocyte phenotype and adhesion to endothelium) using in vitro cell culture models and Wistar rats. Palmitate at 300µM for 6h induced insulin resistance in THP-1 monocytes and L6 monocytes. The ceramide synthesis pathway partly accounted for the palmitate-induced insulin resistance in THP-1 monocytes but not for L6 myotubes. Oleate treatment did not induce insulin resistance in either cell type and co-incubation with oleate protected cells from palmitate-induced insulin resistance. Palmitate at 300µN for 24h significantly increased cell surface CD11b and CD36 expression in U937 monocytes. The increase in CD11b and CD36 expression was effectively inhibited by Fumonisin B1, an inhibitor of ceramide synthesis. Oleate treatment did not show any effect on CD11b and CD36 expression and co-incubation with oleate antagonised the effect of palmitate on CD11b and CD36 expression in U937 monocytes. The increase in CD11b expression did not affect U937 monocyte adhesion to ICAM-1. Treating Wistar rats with palmitate for 6h caused a transient delay in glucose disposal and an increase in adhesion of U937 monocytes to the aortic endothelium, particularly at bifurcations. In conclusion, the present study demonstrates that the saturated free fatty acid palmitate induces insulin resistance and a pro-atherogenic phenotype for monocytes, whereas the unsaturated free fatty acid oleate does not. In vivo studies also confirmed that palmitate induces insulin resistance and an increase in monocyte adhesion to aorta.

NT2 derived neuronal and astrocytic network signalling

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

Regulation of signal transduction by calcitonin gene-related peptide receptors

Calcitonin gene-related peptide (CGRP) plays a pivotal role in migraine, activating its cognate receptor to initiate intracellular signalling. This atypical receptor comprises a distinct assembly, made up of a G protein-coupled receptor (GPCR), a single transmembrane protein, and an additional protein that is required for Ga(s) coupling. By altering the expression of individual receptor components, it might be possible to adjust cellular sensitivity to CGRP. In recognition of the increasing clinical significance of CGRP receptors, it is timely to review the signalling pathways that might be controlled by this receptor, how the activity of the receptor itself is regulated, and our current understanding of the molecular mechanisms involved in these processes. Like many GPCRs, the CGRP receptor appears to be promiscuous, potentially coupling to several G proteins and intracellular pathways. Their precise composition is likely to be cell type-dependent, and much work is needed to ascertain their physiological significance.

The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice

STUDY DESIGN: The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. OBJECTIVE: To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. SUMMARY OF BACKGROUND DATA: Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. METHODS: AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of ß-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS.: Four weeks after injection, the retrograde delivery of the LacZ marker gene was identified in cervical spinal neurons and some glial cells, including oligodendrocytes in the white matter of the spinal cord, in both the twy/twy mouse and the heterozygous Institute of Cancer Research mouse (+/twy). In the compressed spinal cord of twy/twy mouse, AdV-BDNF gene transfection resulted in a significant decrease in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells present in the spinal cord and a downregulation in the caspase apoptotic pathway compared with AdV-LacZ (control) gene transfection. There was a marked and significant increase in the areas of the spinal cord of AdV-BDNF-injected mice that were NF- and NG2-immunopositive compared with AdV-LacZ-injected mice, indicating the increased presence of neurons and oligodendrocytes in response to BDNF transfection. CONCLUSION: Our results demonstrate that targeted retrograde BDNF gene delivery suppresses apoptosis in neurons and oligodendrocytes in the chronically compressed spinal cord of twy/twy mouse. Further work is required to establish whether this method of gene delivery may provide neuroprotective effects in other situations of compressive spinal cord injury.

Influence of Egr-1 in cardiac tissue-derived mesenchymal stem cells in response to glucose variations

Mesenchymal stem cells (MSCs) represent a promising cell population for cell therapy and regenerative medicine applications. However, how variations in glucose are perceived by MSC pool is still unclear. Since, glucose metabolism is cell type and tissue dependent, this must be considered when MSCs are derived from alternative sources such as the heart. The zinc finger transcription factor Egr-1 is an important early response gene, likely to play a key role in the glucose-induced response. Our aim was to investigate how short-term changes in in vitro glucose concentrations affect multipotent cardiac tissue-derived MSCs (cMSCs) in a mouse model of Egr-1 KO (Egr-1-/-). Results showed that loss of Egr-1 does not significantly influence cMSC proliferation. In contrast, responses to glucose variations were observed in wt but not in Egr-1 -/- cMSCs by clonogenic assay. Phenotype analysis by RT-PCR showed that cMSCs Egr-1-/- lost the ability to regulate the glucose transporters GLUT-1 and GLUT-4 and, as expected, the Egr-1 target genes VEGF, TGFβ-1, and p300. Acetylated protein levels of H3 histone were impaired in Egr-1-/- compared to wt cMSCs. We propose that Egr-1 acts as immediate glucose biological sensor in cMSCs after a short period of stimuli, likely inducing epigenetic modifications. © 2014 Daniela Bastianelli et al.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

The development of functional astrocyte-neuron networks derived from stem cells

There is currently great scientific and medical interest in the potential of tissue grown from stem cells. These cells present opportunities for generating model systems for drug screening and toxicological testing which would be expected to be more relevant to human outcomes than animal based tissue preparations. Newly realised astrocytic roles in the brain have fundamental implications within the context of stem cell derived neuronal networks. If the aim of stem cell neuroscience is to generate functional neuronal networks that behave as networks do in the brain, then it becomes clear that we must include and understand all the cellular components that comprise that network, and which are important to support synaptic integrity and cell to cell signalling. We have shown that stem cell derived neurons exhibit spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling (1). Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, astrocytes exhibit morphology and functional properties consistent with this glial cell type. Astrocytes also respond to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. Astroctyes also generate propagating calcium waves that are gap junction and purinergic signalling dependent. Our results show that stem cell derived astrocytes exhibit appropriate functionality and that stem cell neuronal networks interact with astrocytic networks in co-culture. Using mixed cultures of stem cell derived neurons and astrocytes, we have also shown both cell types also modulate their glucose uptake, glycogen turnover and lactate production in response to glutamate as well as increased neuronal activity (2). This finding is consistent with their neuron-astrocyte metabolic coupling thus demonstrating a tractable human model, which will facilitate the study of the metabolic coupling between neurons and astrocytes and its relationship with CNS functional issues ranging from plasticity to neurodegeneration. Indeed, cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose (3). Both co-cultures of neurons and astrocytes and purified cultures of astrocytes showed a significant decrease in glucose uptake after treatment with 2 and 0.2 μmol/L Aβ at all time points investigated (p <0.01). In addition, a significant increase in the glycogen content of cells was also measured. Mixed neuron and astrocyte co-cultures as well as pure astrocyte cultures showed an initial decrease in glycogen levels at 6 hours compared with control at 0.2 μmol/L and 2 μmol/L P <0.01. These changes were accompanied by changes in NAD+/NADH (P<0.05), ATP (P<0.05), and glutathione levels (P<0.05), suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. As numerous cell types interact in the brain it is important that any in vitro model developed reflects this arrangement. Our findings indicate that stem cell derived neuron and astrocyte networks can communicate, and so have the potential to interact in a tripartite manner as is seen in vivo. This study therefore lays the foundation for further development of stem cell derived neurons and astrocytes into therapeutic cell replacement and human toxicology/disease models. More recently our data provides evidence for a detrimental effect of Aβ on carbohydrate metabolism in both neurons and astrocytes. As a purely in vitro system, human stem cell models can be readily manipulated and maintained in culture for a period of months without the use of animals. In our laboratory cultures can be maintained in culture for up to 12 months months thus providing the opportunity to study the consequences of these changes over extended periods of time relevant to aspects of the disease progression time frame in vivo. In addition, their human origin provides a more realistic in vitro model as well as informing other human in vitro models such as patient-derived iPSC.

Annotated record of the detailed examination of Mn deposits from COCOTOW Expedition stations

The cores and dredges described in this report were taken on the COCOTOW Expedition in September until December 1974 by the Scripps Institution of Oceanography from the R/V Spencer F. Baird. A total of 75 cores and dredges were recovered and are available at Scripps for sampling and study.

Observation of manganese nodules and manganese coated coral in the West Northern Atlantic ocean

Photography has become an integral part of submarine geological and biological investigations of the ocean bottom. The underwater cameras used to make these photographs were designed by Harold Edgerton. The pictures were taken from 1960 to 1962, from ships of the Woods Hole Oceanographic Institution. They show that life occurs even in the deepest trenches, and that sedimentary and biological processes in deep water do not differ in kind from those in shallow water.

Observation of manganese coating and encrustation in different environments of the North-Western Atlantic ocean

The major topographic features, or provinces, beyond the continental slope off the Atlantic coast of the United States are (1) Sohm Plain, (2) Hatteras Plain, (3) Nares Plain, (4) Blake Basin, (5) Blake Plateau-Bahama Banks, and (6) Bermuda Rise. The whole of the described area is commonly referred to as the North American Basin. This basin is bounded on the north by Newfoundland Ridge and on the south by Puerto Rico Trench. Topographic features of note within the basin are the divide and the area of depressions between Sohm and Hatteras Plains, the sharply crested Blake Ridge, and the Puerto Rico Ridge. Recently accumulated data on deep-sea oores has given good evidence that the silt and sand covering the abyssal plains are displaced continental sediments in a virtually quartz-free oceanic environment. These sediments were deposited on a primary volcanic bottom. The primary or volcanic bottom is characterized by abyssal hills and seamounts, and the sediment bottom is characterized by abyssal plains, which extend seaward from the continental margins. On the Blake Plateau, bottom photographs and dredge hauls in the axis of the stream show that locally sediment has been removed and the bottom is paved with crusts and nodules of manganese. Photographs and dredged samples from the outer part of the New England Seamount, Chain and Caryn Peak also indicate extensive encrustations of manganese oxide which acts as a binding agent in areas of ooze or other organic debris and thus helps to stabilize the bottom.

Annotated record of the detailed examination of Mn deposits from R/V Caryn Cruise 25 stations

The cores described are taken during the R/V Caryn Cruise 25 in August-September 1951 by the Woods Hole Oceanographic Institution. A total of 11 cores were recovered and are available at Woods Hole Oceanographic Institution for sampling and study.