Effects of high temperature and CO2 on intracellular DMSP in the cold-water coral Lophelia pertusa

Significant warming and acidification of the oceans is projected to occur by the end of the century. CO2 vents, areas of upwelling and downwelling, and potential leaks from carbon capture and storage facilities may also cause localised environmental changes, enhancing or depressing the effect of global climate change. Cold-water coral ecosystems are threatened by future changes in carbonate chemistry, yet our knowledge of the response of these corals to high temperature and high CO2 conditions is limited. Dimethylsulphoniopropionate (DMSP), and its breakdown product dimethylsulphide (DMS), are putative antioxidants that may be accumulated by invertebrates via their food or symbionts, although recent research suggests that some invertebrates may also be able to synthesise DMSP. This study provides the first information on the impact of high temperature (12 °C) and high CO2 (817 ppm) on intracellular DMSP in the cold-water coral Lophelia pertusa from the Mingulay Reef Complex, Scotland (56°49' N, 07°23' W), where in situ environmental conditions are meditated by tidally induced downwellings. An increase in intracellular DMSP under high CO2 conditions was observed, whilst water column particulate DMS + DMSP was reduced. In both high temperature treatments, intracellular DMSP was similar to the control treatment, whilst dissolved DMSP + DMS was not significantly different between any of the treatments. These results suggest that L. pertusa accumulates DMSP from the surrounding water column; uptake may be up-regulated under high CO2 conditions, but mediated by high temperature. These results provide new insight into the biotic control of deep-sea biogeochemistry and may impact our understanding of the global sulphur cycle, and the survival of cold-water corals under projected global change.

Detection of a variable intracellular acid-labile carbon pool in Thalassiosira weissflogii (Heterokontophyta) and Emiliania huxleyi (Haptophyta) in response to changes in the seawater carbon system

Accumulation of an intracellular pool of carbon (C(i) pool) is one strategy by which marine algae overcome the low abundance of dissolved CO2 (CO2 (aq) ) in modern seawater. To identify the environmental conditions under which algae accumulate an acid-labile C(i) pool, we applied a (14) C pulse-chase method, used originally in dinoflagellates, to two new classes of algae, coccolithophorids and diatoms. This method measures the carbon accumulation inside the cells without altering the medium carbon chemistry or culture cell density. We found that the diatom Thalassiosira weissflogii [(Grunow) G. Fryxell & Hasle] and a calcifying strain of the coccolithophorid Emiliania huxleyi [(Lohmann) W. W. Hay & H. P. Mohler] develop significant acid-labile C(i) pools. C(i) pools are measureable in cells cultured in media with 2-30 µmol/l CO2 (aq), corresponding to a medium pH of 8.6-7.9. The absolute C(i) pool was greater for the larger celled diatoms. For both algal classes, the C(i) pool became a negligible contributor to photosynthesis once CO2 (aq) exceeded 30 µmol/l. Combining the (14) C pulse-chase method and (14) C disequilibrium method enabled us to assess whether E. huxleyi and T. weissflogii exhibited thresholds for foregoing accumulation of DIC or reduced the reliance on bicarbonate uptake with increasing CO2 (aq) . We showed that the C(i) pool decreases with higher CO2 :HCO3 (-) uptake rates.

Targeted mutation reveals a central role for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol

Scavenger receptor BI (SR-BI) is a cell surface receptor that binds high density lipoproteins (HDL) and mediates selective uptake of HDL cholesteryl esters (CE) in transfected cells. To address the physiological role of SR-BI in HDL cholesterol homeostasis, mice were generated bearing an SR-BI promoter mutation that resulted in decreased expression of the receptor in homozygous mutant (designated SR-BI att) mice. Hepatic expression of the receptor was reduced by 53% with a corresponding increase in total plasma cholesterol levels of 50–70% in SR-BI att mice, attributable almost exclusively to elevated plasma HDL. In addition to increased HDL-CE, HDL phospholipids and apo A-1 levels were elevated, and there was an increase in HDL particle size in mutant mice. Metabolic studies using HDL bearing nondegradable radiolabels in both the protein and lipid components demonstrated that reducing hepatic SR-BI expression by half was associated with a decrease of 47% in selective uptake of CE by the liver, and a corresponding reduction of 53% in selective removal of HDL-CE from plasma. Taken together, these findings strongly support a pivotal role for hepatic SR-BI expression in regulating plasma HDL levels and indicate that SR-BI is the major molecule mediating selective CE uptake by the liver. The inverse correlation between plasma HDL levels and atherosclerosis further suggests that SR-BI may influence the development of coronary artery disease.

Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma

Apolipoprotein E- (apoE) deficient (E−/−) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (E−/−hTgE+/0), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (E+/+), E−/−, and E−/−hTgE+/0 mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify 3H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic α or γ mobilities. Similar particles were also present in E−/−hTgE+/0 plasma. Depending on incubation time, E−/− plasma released 48–74% less 3H-labeled cholesterol from fibroblasts than E+/+ plasma, whereas cholesterol efflux into E−/−hTgE+/0 plasma was only 11–25% lower than into E+/+ plasma. E−/−hTgE+/0 plasma also released 10% more 3H-labeled cholesterol from radiolabeled J774 macrophages than E−/− plasma. E+/+ and E−/−hTgE+/0 plasma each esterified significantly more cell-derived 3H-labeled cholesterol than E−/− plasma. Moreover, E−/− plasma accumulated much smaller proportions of fibroblast-derived 3H-labeled cholesterol in fractions with electrophoretic γ and α mobility than E+/+ and E−/−hTgE+/0 plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells.

Aberrant intracellular localization of Varicella-Zoster virus regulatory proteins during latency

Varicella-Zoster virus (VZV) is a herpesvirus that becomes latent in sensory neurons after primary infection (chickenpox) and subsequently may reactivate to cause zoster. The mechanism by which this virus maintains latency, and the factors involved, are poorly understood. Here we demonstrate, by immunohistochemical analysis of ganglia obtained at autopsy from seropositive patients without clinical symptoms of VZV infection that viral regulatory proteins are present in latently infected neurons. These proteins, which localize to the nucleus of cells during lytic infection, predominantly are detected in the cytoplasm of latently infected neurons. The restriction of regulatory proteins from the nucleus of latently infected neurons might interrupt the cascade of virus gene expression that leads to a productive infection. Our findings raise the possibility that VZV has developed a novel mechanism for maintenance of latency that contrasts with the transcriptional repression that is associated with latency of herpes simplex virus, the prototypic alpha herpesvirus.

Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12°C). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.

Delayed loss of cholesterol from a localized lipoprotein depot in apolipoprotein A-I-deficient mice

The anti-atherogenic role of high density lipoprotein is well known even though the mechanism has not been established. In this study, we have used a novel model system to test whether removal of lipoprotein cholesterol from a localized depot will be affected by apolipoprotein A-I (apo A-I) deficiency. We compared the egress of cholesterol injected in the form of cationized low density lipoprotein into the rectus femoris muscle of apo A-I K-O and control mice. When the injected lipoprotein had been labeled with [3H]cholesterol, the t½ of labeled cholesterol loss from the muscle was about 4 days in controls and more than 7 days in apo A-I K-O mice. The loss of cholesterol mass had an initial slow (about 4 days) and a later more rapid component; after day 4, the disappearance curves for apo A-I K-O and controls began to diverge, and by day 7, the loss of injected cholesterol was significantly slower in apo A-I K-O than in controls. The injected lipoprotein cholesterol is about 70% in esterified form and undergoes hydrolysis, which by day 4 was similar in control and apo A-I K-O mice. The efflux potential of serum from control and apo A-I K-O mice was studied using media containing 2% native or delipidated serum. A significantly lower efflux of [3H]cholesterol from macrophages was found with native and delipidated serum from apo A-I K-O mice. In conclusion, these findings show that lack of apo A-I results in a delay in cholesterol loss from a localized depot in vivo and from macrophages in culture. These results provide support for the thesis that anti-atherogenicity of high density lipoprotein is related in part to its role in cholesterol removal.

The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈Azz〉 element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Increase of intracellular Ca2+ and relocation of E-cadherin during experimental decompaction of mouse embryos

To determine the role of intracellular Ca2+ in compaction, the first morphogenetic event in embryogenesis, we analyzed preimplantation mouse embryos under several decompacting conditions, including depletion of extracellular Ca2+, blocking of Ca2+ channels, and inhibition of microfilaments, calmodulin, and intracellular Ca2+ release. Those treatments induced decompaction of mouse morulae and simultaneously induced changes in cytosolic free Ca2+ concentration and deregionalization of E-cadherin and fodrin. When morulae were allowed to recompact, the location of both proteins recovered. In contrast, actin did not change its cortical location with compaction nor with decompaction-recompaction. Calmodulin localized in areas opposite to cell–cell contacts in eight-cell stage embryos before and after compaction. Inhibition of calmodulin with trifluoperazine induced its delocalization while morulae decompacted. A nonspecific rise of intracellular free Ca2+ provoked by ionomycin did not affect the compacted shape. Moreover, the same decompacting treatments when applied to uncompacted embryos did not produce any change in intracellular Ca2+. Our results demonstrate that in preimplantation mouse embryos experimentally induced stage-specific changes of cell shape are accompanied by changes of intracellular free Ca2+ and redistribution of the cytoskeleton-related proteins E-cadherin, fodrin, and calmodulin. We conclude that intracellular Ca2+ specifically is involved in compaction and probably regulates the function and localization of cytoskeleton elements.

Intracellular gene transfer in action: Dual transcription and multiple silencings of nuclear and mitochondrial cox2 genes in legumes

The respiratory gene cox2, normally present in the mitochondrion, was previously shown to have been functionally transferred to the nucleus during flowering plant evolution, possibly during the diversification of legumes. To search for novel intermediate stages in the process of intracellular gene transfer and to assess the evolutionary timing and frequency of cox2 transfer, activation, and inactivation, we examined nuclear and mitochondrial (mt) cox2 presence and expression in over 25 legume genera and mt cox2 presence in 392 genera. Transfer and activation of cox2 appear to have occurred during recent legume evolution, more recently than previously inferred. Many intermediate stages of the gene transfer process are represented by cox2 genes in the studied legumes. Nine legumes contain intact copies of both nuclear and mt cox2, although transcripts could not be detected for some of these genes. Both cox2 genes are transcribed in seven legumes that are phylogenetically interspersed with species displaying only nuclear or mt cox2 expression. Inactivation of cox2 in each genome has taken place multiple times and in a variety of ways, including loss of detectable transcripts or transcript editing and partial to complete gene loss. Phylogenetic evidence shows about the same number (3–5) of separate inactivations of nuclear and mt cox2, suggesting that there is no selective advantage for a mt vs. nuclear location of cox2 in plants. The current distribution of cox2 presence and expression between the nucleus and mitochondrion in the studied legumes is probably the result of chance mutations silencing either cox2 gene.

Trafficking of spontaneously endocytosed MHC proteins

Class I MHC protein primarily presents endogenous antigen but also may present exogenous antigen. Here, we investigated the intracellular pathway of spontaneously internalized class I MHC protein by confocal microscopy. β2-microglobulin (β2m), labeled with a single fluorophore, was exchanged at the surface of B cell transfectants to specifically mark cell surface and endocytosed class I MHC protein. Intracellular β2m colocalized with fluorophore-conjugated transferrin, implying that class I MHC protein endocytosed into early endosomes. These endosomes containing fluorescent β2m were found close to or within the Golgi apparatus, marked by fluorescent ceramide. Even after 24 hr of incubation, very little fluorescent β2m was found in intracellular organelles stained by DiOC6, marking the endoplasmic reticulum, or fluorophore-conjugated low density lipoprotein, marking late endosomes and lysosomes. Fluorophore-conjugated superantigens (staphylococcal enterotoxin A and B), presumed to enter cells bound to class II MHC protein, also were found to endocytose into β2m-containing early endosomes. Staining with mAb and use of transfectants expressing MHC protein attached to green fluorescent protein confirmed the presence of intracellular compartments rich in both class I and II MHC protein and demonstrated that class I and II MHC protein also colocalize in discrete microdomains at the cell surface. These cell surface microdomains also contained transferrin receptor and often were juxtaposed to cholesterol-rich lipid rafts. Thus, class I and II MHC protein meet in microdomains of the plasma membrane and endocytose into early endosomes, where both may acquire and present exogenous antigen.