Does ATP cross the cell plasma membrane.

Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes.     

Inhibition of neutrophil sulfhydryl groups by choloromethyl ketones. A mechanism for their inhibition of superoxide production

The inhibition of O2- production by serine protease inhibitors such as chloromethyl ketone derivatives, has been used as evidence to indicate that protease activity is essential for the production of O2- by neutrophils. However, chloromethyl ketones are potent inhibitors of sulfhydryl groups. This study demonstrates that chloromethyl ketones inhibited non-protein sulfhydryl groups as well as O2- production by human neutrophils stimulated with phorbol myristate acetate (PMA). Their inhibition of O2- production could be prevented by reduced glutathione. The results suggest that inhibition of O2- production by chloromethyl ketones is largely due to their inhibition of sulfhydryl groups.     

High avidity binding to DNA protects ubiquitylated substrates from proteasomal degradation

Protein domains that act as degradation and stabilization signals regulate the rate of turnover of proteasomal substrates. Here we report that the bipartite Gly-Arg repeat of the Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 acts as a stabilization signal that inhibits proteasomal degradation in the nucleus by promoting binding to cellular DNA. Protection can be transferred by grafting the domain to unrelated proteasomal substrates and does not involve changes of ubiquitylation. Protection is also afforded by other protein domains that, similar to the Gly-Arg repeat, mediate high avidity binding to DNA, as exemplified by resistance to detergent extraction. Our findings identify high avidity binding to DNA as a portable inhibitory signal that counteracts proteasomal degradation.     

B cell abnormalities in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.     

Beta-alanine but not taurine can function as an organic osmolyte in preimplantation mouse embryos cultured from fertilized eggs

Early preimplantation mouse embryos are susceptible to the detrimental effects of increased osmolarity and, paradoxically, their in vitro development is significantly compromised by osmolarities near that of oviductal fluid. In vitro development can be restored, however, by several compounds that are accumulated by 1-cell embryos to act as organic osmolytes, providing intracellular osmotic support and cell volume regulation. Taurine, a substrate of the beta-amino acid transporter that functions as an organic osmolyte transporter in other cells, had been proposed to function as an organic osmolyte in mouse embryos. Here, however, we found that taurine is neither able to provide protection for in vitro embryo development against increased osmolarity nor is it accumulated to higher intracellular levels as osmolarity is increased, indicating that it cannot function as an organic osmolyte in early preimplantation embryos. In contrast, beta-alanine, the other major substrate of the beta-amino acid transporter, both protects against increased osmolarity and is accumulated to somewhat higher levels as osmolarity is increased, indicating that it is able to function as an organic osmolyte in embryos. However, we also found that beta-alanine is displaced from embryos by glycine-the most effective organic osmolyte in embryos previously identified-and beta-alanine does not increase protection above that afforded by glycine at concentrations near those in vivo. Thus, the beta-amino acid transporter is likely present in early preimplantation embryos to supply beta-amino acids such as taurine for purposes other than to serve as organic osmolytes.     

Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behavior — it has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid–lipid and protein–lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein–lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.     

Initiation of high-voltage discharge in air by a plasma filament produced by an intense femtosecond laser pulse

High-voltage discharge initiated in air by a plasma filament produced by an intense femtosecond laser pulse was studied experimentally. It is found that the threshold of a laser-induced discharge decrease three-fold as compared to that of a discharge in undisturbed air. It is shown that the formation time of a laser-induced discharge decreases by almost three orders of magnitude as the applied voltage increases by a factor of 2. A numerical model of the discharge process is developed that adequately describes the experimental results. In particular, simulations reproduce the experimentally observed steep dependence of the formation time of a laser-induced discharge on the applied voltage, as well as typical values of the electric field required for such a breakdown.     

Resistin release by human adipose tissue explants in primary culture

Resistin, also known as Fizz3 or ADSF, is a protein found in murine adipose tissue and inflammatory lung exudates. The present studies found that resistin was released by explants of human adipose tissue but the release was quite variable ranging from 3 to 158 ng/g over 48 h. The release of resistin was 250% greater by explants of omental than by explants of human subcutaneous abdominal adipose tissue. Resistin release by adipocytes was negligible as compared to that by the non-fat cells of adipose tissue. Leptin formation by adipocytes was 8-fold greater than its formation by the non-fat cells, while the formation of PAI-1 by adipocytes was 38% of that by the non-fat cells. The conversion of glucose to lactate as well as the formation of PGE(2) and IL-8 was approximately 15% of that by the non-fat cells. In contrast the release of IL-6 and IL-1beta by adipocytes was 4-7% of that by the non-fat cells while the formation of resistin and IL-10 by adipocytes was 2% of that by non-fat cells. The release of adiponectin by explants ranged from 1000 to 5000 ng/g over 48 h but did not correlate with that of resistin. The present data suggest that resistin release by explants of human adipose tissue in primary culture is largely derived from the non-fat cells present in the explants.     

Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.     

Membrane Raft Disruption Promotes Axonogenesis in N2a Neuroblastoma Cells

Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1. Special issue article in honor of Dr. George DeVries.     

n-Propanol as a substrate for assaying the ligninperoxidase activity of Phanerochaete chrysoporium.

The steady state kinetics of ligninperoxidase catalysed reaction using n-propanol as the organic substrate and monitoring the formation of propanaldehyde at lambda = 300 nm spectrophotometerically as functions of different reaction parameters has been studied. It has been concluded that n-propanol can be used as a substrate for analysing the activity of ligninperoxidase. The turnover number of ligninperoxidase of Phanerochaete chrysosporium using n-propanol as substrate has been found to be higher approximately by a factor of 10(3) as compared to that using veratryl alcohol as the substrate. The method works in assaying the activity of ligninperoxidase produced by Aspergillus fumigatus indicating that it can be used for assaying the ligninperoxidase activities produced by other microorganisms also and is not limited to assaying the ligninase activity produced by Phanerochaete chrysosporium alone. Under identical experimental conditions, horseradish peroxidase does not show peroxidase activity using n-propanol as substrate indicating that the method does not interfere with the activities of other peroxidases.     

Effector proteins encoded by Salmonella pathogenicity island 2 interfere with the microtubule cytoskeleton after translocation into host cells

The facultative intracellular pathogen Salmonella enterica has evolved strategies to modify its fate inside host cells. One key virulence factor for the intracellular pathogenesis is the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2). We have previously described SPI2-encoded SseF and SseG as effector proteins that are translocated by intracellular Salmonella . Detailed analysis of the subcellular localization of SseF and SseG within the host cell indicated that these effector proteins are associated with endosomal membranes as well as with microtubules. Specific association with microtubules was observed after translocation by intracellular Salmonella as well as after expression by transfection vectors. In epithelial cells infected with Salmonella , both SseF and SseG are required for the aggregation of endosomal compartments along microtubules and to induce the formation of massive bundles of microtubules. These observations demonstrate that SPI2 effectors interfere with the microtubule cytoskeleton and suggest that microtubule-dependent host cell functions such as vesicle transport or organelle positioning are altered by intracellular Salmonella .     

Influence of the carbon source on glycerol kinase activity in Neurospora crassa.

The level of glycerol kinase activity in Neurospora crassa was shown to change in response to resuspension of sucrose-grown mycelia in fresh medium containing a new carbon source: the magnitude of the change depended on the new carbon source provided. Certain carbon sources, such as glucose and fructose, inhibited the small increase that occurred in the absence of any carbon source. Others, and in particular deoxyribose, galactose, glycerol and ribose, greatly enhanced this increase. The activity induced by deoxyribose and galactose had the same stability, both in vivo and in vitro, as that induced by glycerol, and as that induced by incubation of Neurospora cultures at low temperatures. The inhibitory carbon sources, such as glucose and fructose, also restricted the increases induced by deoxyribose, galactose and glycerol: they had more effect on the increases induced by glycerol and deoxyribose than on that induced by galactose. The increase in activity that occurs at low temperature was also inhibited by glucose and sucrose.     

p53 blocks RuvAB promoted branch migration and modulates resolution of Holliday junctions by RuvC

The Holliday junction is the central intermediate in homologous recombination. Branch migration of this four-stranded DNA structure is a key step in genetic recombination that affects the extent of genetic information exchanged between two parental DNA molecules. Here, we have constructed synthetic Holliday junctions to test the effects of p53 on both spontaneous and RuvAB promoted branch migration as well as the effect on resolution of the junction by RuvC. We demonstrate that p53 blocks branch migration, and that cleavage of the Holliday junction by RuvC is modulated by p53. These findings suggest that p53 can block branch migration promoted by proteins such as RuvAB and modulate the cleavage by Holliday junction resolution proteins such as RuvC. These results suggest that p53 could have similar effects on eukaryotic homologues of RuvABC and thus have a direct role in recombinational DNA repair.     

Sustainable Herd Management and the Tragedy of No Man's Land: An Analysis of West Himalayan Pastures Using Remote Sensing Techniques

Using ground truth and remote sensing data this paper tries to demonstrate once again that commons as well as privately owned pastures are managed by their appropriators in such a manner that sustainability as a goal can be approached, while areas not recognized as controlled by one or more legitimate persons and hence open to all may well be overexploited. The analysis of pastures which are recognized as commons in the western Liddar valley in Kashmir (India) shows that these lands are not overexploited by the nomadic and transhumant pastoralists using them. On the whole, individually owned pastures were also not found to be overstocked. Those plots, however, through which all herders transit on their bi-annual migration and which are not recognized as being owned by any authority are highly degraded.     

Cohesin's concatenation of sister DNAs maintains their intertwining

The contribution of DNA catenation to sister chromatid cohesion is unclear partly because it has never been observed directly within mitotic chromosomes. Differential sedimentation-velocity and gel electrophoresis reveal that sisters of 26 kb circular minichromosomes are held together by catenation as well as by cohesin. The finding that chemical crosslinking of cohesin's three subunit interfaces entraps sister DNAs of circular but not linear minichromosomes implies that cohesin functions using a topological principle. Importantly, cohesin holds both catenated and uncatenated DNAs together in this manner. In the vicinity of centromeres, catenanes are resolved by spindle forces, but linkages mediated directly by cohesin resist these forces even after complete decatenation. Crucially, persistence of catenation after S phase depends on cohesin. We conclude that by retarding Topo II-driven decatenation, cohesin mediates sister chromatid cohesion by an indirect mechanism as well as one involving entrapment of sister DNAs inside its tripartite ring.     

Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.     

Where is Darwin 200 years later?

The theory of evolution is perceived by many people, particularly but not only in the United States, as a controversial theory not yet fully demonstrated. Yet, that living organisms, including humans, have evolved from ancestors who were very different from them is beyond reasonable doubt, confirmed by at least as much evidence as any other widely accepted scientific theory. I argue that Darwin’s contribution to science goes much beyond the theory of evolution in itself. The theory of natural selection explains the adaptations of organisms, their ‘design’. The ‘Copernican Revolution’ brought the phenomena of the physical universe into the realm of science: explanations by natural causes that can be tested by observation and experiment. However, the scientific revolution that occurred in the 16th and 17th centuries had left the living world out of scientific explanations, because organisms seemingly show that they are ‘designed,’ and thus call for an intentional designer. It was Darwin’s greatest contribution to science, to demonstrate that the adaptations of organisms, their apparent ‘design’, can be explained by natural processes governed by natural laws. At that point, science came into maturity, because all natural phenomena in the universe, living as well as nonliving, could be investigated scientifically, and explained as matter in motion governed by natural laws.     

VEGF selectively induces Down syndrome critical region 1 gene expression in endothelial cells: a mechanism for feedback regulation of angiogenesis?

The Down syndrome critical region 1 (DSCR1) gene (also known as MCIP1, Adapt78) encodes a regulatory protein that binds to calcineurin catalytic A subunit and acts as a regulator of the calcineurin-mediated signaling pathway. We show in this study that DSCR1 is greatly induced in endothelial cells in response to VEGF, TNF-alpha, and A23187 treatment, and that this up-regulation is inhibited by inhibitors of the calcineurin-NFAT (nuclear factor of activated T cells) signaling pathway as well as by PKC inhibition and a Ca(2+) chelator. We hypothesized that the up-regulation of DSCR1 gene expression in endothelial cells could act as an endogenous feedback inhibitor for angiogenesis by regulating the calcineurin-NFAT signaling pathway. Our transient transfection analyses confirm that the overexpression of DSCR1 abrogates the up-regulation of reporter gene expression driven by both the cyclooxygenase 2 and DSCR1 promoters in response to stimulators. Our results indicate that DSCR1 up-regulation may represent a potential molecular mechanism underlying the regulation of angiogenic genes activated by the calcineurin-NFAT signaling pathway in endothelial cells.     

Leptin and metabolic control of reproduction

Leptin treatment prevents the effects of fasting on reproductive processes in a variety of species. The mechanisms that underlie these effects have not been elucidated. Progress in this area of research might be facilitated by viewing reproductive processes in relation to mechanisms that maintain fuel homeostasis. Reproduction, food intake, and fuel partitioning can be viewed as homeostatic responses controlled by a sensory system that monitors metabolic signals. These signals are generated by changes in intracellular metabolic fuel availability and oxidation rather than by changes in the amount of body fat or by changes in any aspect of body composition. Leptin might be viewed as either a mediator or as a modulator of the intracellular metabolic signal. Consistent with its purported action as a mediator of the metabolic signal, leptin synthesis and secretion are influenced acutely by changes in metabolic fuel availability, and these changes might lead to changes in reproductive function. The effects of leptin treatment on reproduction are blocked by treatments that inhibit intracellular fuel oxidation. Metabolic signals that inhibit reproduction in leptin-treated animals might act via neural pathways that are independent of leptin's action. Alternatively, both leptin and metabolic inhibitors might interact at the level of intracellular fuel oxidation. In keeping with the possibility that leptin modulates the metabolic signal, leptin treatment increases fuel availability, uptake, and oxidation in particular tissues. Leptin might affect reproduction indirectly by altering fuel oxidation or other peripheral processes such as gastric emptying. Reproductive processes are among the most energetically expensive in the female repertoire. Because leptin increases energy expenditure while simultaneously inhibiting energy intake, it may have limited use as a long-term treatment for infertility.