Interaction between nitric oxide signaling and gap junctions: effects on vascular function.

Nitric oxide signaling, through eNOS (or possibly nNOS), and gap junction communication are essential for normal vascular function. While each component controls specific aspects of vascular function, there is substantial evidence for cross-talk between nitric oxide signaling and the gap junction proteins (connexins), and more recently, protein-protein association between eNOS and connexins. This review will examine the evidence for interaction between these pathways in normal and diseased arteries, highlight the questions that remain about the mechanisms of their interaction, and explore the possible interaction between nitric oxide signaling and the newly discovered pannexin channels. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

The primary structure of NG2, a novel membrane-spanning proteoglycan.

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.

Gene families from the Arabidopsis thaliana pollen coat proteome.

The pollen extracellular matrix contains proteins mediating species specificity and components needed for efficient pollination. We identified all proteins >10 kilodaltons in the Arabidopsis pollen coating and showed that most of the corresponding genes reside in two genomic clusters. One cluster encodes six lipases, whereas the other contains six lipid-binding oleosin genes, including GRP17, a gene that promotes efficient pollination. Individual oleosins exhibit extensive divergence between ecotypes, but the entire cluster remains intact. Analysis of the syntenic region in Brassica oleracea revealed even greater divergence, but a similar clustering of the genes. Such allelic flexibility may promote speciation in plants.

Expression of vascular endothelial growth factor (VEGF) in normal oral mucosa, oral dysplasia and oral squamous cell carcinoma.

Vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine implicated in tumour vasculogenesis. A significant increase in vascularity occurs during the transition from normal oral mucosa (NOM), through dysplasia, to squamous cell carcinoma (SCC). This study investigated the presence of VEGF in NOM, oral dysplasia and SCC. The correlation between VEGF expression and the grade of dysplasia or differentiation of SCC was also examined. Specimens consisting of NOM, oral dysplastic lesions and oral SCC were stained using standard immunohistochemistry methods to determine VEGF expression. Statistical analysis indicated an up-regulation of VEGF during the transition from NOM, through dysplasia to SCC. There was also a significant difference in expression according to differentiation of SCC, but not grade of dysplasia. As VEGF is a potent mediator of vascular development, these results suggest that VEGF may play an important role in the maintenance of a blood supply for developing pre-cancerous and invasive oral lesions.

AEGIS autonomous targeting for ChemCam on Mars Science Laboratory: Deployment and results of initial science team use.

Limitations on interplanetary communications create operations latencies and slow progress in planetary surface missions, with particular challenges to narrow-field-of-view science instruments requiring precise targeting. The AEGIS (Autonomous Exploration for Gathering Increased Science) autonomous targeting system has been in routine use on NASA's Curiosity Mars rover since May 2016, selecting targets for the ChemCam remote geochemical spectrometer instrument. AEGIS operates in two modes; in autonomous target selection, it identifies geological targets in images from the rover's navigation cameras, choosing for itself targets that match the parameters specified by mission scientists the most, and immediately measures them with ChemCam, without Earth in the loop. In autonomous pointing refinement, the system corrects small pointing errors on the order of a few milliradians in observations targeted by operators on Earth, allowing very small features to be observed reliably on the first attempt. AEGIS consistently recognizes and selects the geological materials requested of it, parsing and interpreting geological scenes in tens to hundreds of seconds with very limited computing resources. Performance in autonomously selecting the most desired target material over the last 2.5 kilometers of driving into previously unexplored terrain exceeds 93% (where ~24% is expected without intelligent targeting), and all observations resulted in a successful geochemical observation. The system has substantially reduced lost time on the mission and markedly increased the pace of data collection with ChemCam. AEGIS autonomy has rapidly been adopted as an exploration tool by the mission scientists and has influenced their strategy for exploring the rover's environment.

TNFalpha and IL10 SNPs act together to predict disease behaviour in Crohn's disease.

BACKGROUND: The cytokines tumour necrosis factor (TNF)alpha and interleukin (IL)10 have been implicated in the pathogenesis of Crohn's disease (CD), with increased concentrations reported in patients with active disease. However, limited data exist on their effects on disease phenotype in the same population. Certain single nucleotide polymorphisms (SNPs) within the promoter region of the IL10 (-1082G/A, -592C/A) and TNFalpha (-308G/A, -857C/T) genes have been associated with altered levels of circulating IL10 and TNFalpha. METHODS: We conducted an Australian based case-control study (304 CD patients; 231 healthy controls) of these four SNPs. Further investigation of two SNPs was conducted using a logistic regression analysis. RESULTS: We identified a possible association of both IL10 SNPs and TNFalpha-857 with CD. Further investigation of a relationship with disease severity showed a significant association of higher producing IL10-1082G and TNFalpha-857C alleles with stricturing behaviour, which was strongest when these alleles were combined and persisted after multivariate analysis (p = 0.007; odds ratio (OR) 2.37, 95% CI 1.26 to 4.43). In addition, the TNFalpha-857CC genotype was independently associated with familial CD (p = 0.03; OR 3.12; 95% CI 1.15 to 8.46). CONCLUSION: These two SNPs may help to predict disease behaviour in CD patients, which may be clinically useful in shaping treatment of the disease at an earlier stage.

Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages.

Serum proteins, acting as opsonins, are believed to contribute significantly to liposome-macrophage cell association and thus regulate liposome uptake by cells of the mononuclear phagocytic system (MPS). We studied the effect of serum protein on binding and uptake of phosphatidylglycerol-, phosphatidylserine-, cardiolipin-, and N,N-dioleyl-N,N-dimethylammonium chloride- (DODAC) containing as well as poly(ethylene glycol)- (PEG) containing liposomes by mouse bone marrow macrophages in vitro. Consistent with the postulated surface-shielding properties of PEG, protein-free uptake of liposomes containing 5 mol% PEG and either 20 mol% anionic phosphatidylserine or 20 mol% cationic DODAC was equivalent to uptake of neutral liposomes. In contrast to previous reports indicating that protein adsorption to liposomes increases uptake by macrophages, the presence of bound serum protein did not increase the uptake of these liposomes by cultured macrophages. Rather, we found that pre-incubating liposomes with serum reduced the uptake of liposomes containing phosphatidylserine. Surprisingly, serum treatment of PEG-containing liposomes also significantly reduced liposome uptake by macrophages. It is postulated that, in the case of phosphatidylserine liposomes, the bound serum protein can provide a non-specific surface-shielding property that reduces the charge-mediated interactions between liposomes and bone marrow macrophage cells. In addition, incubation of PEG-bearing liposomes with serum can result in a change in the properties of the PEG, resulting in a surface that is better protected against interactions with cells.

Membrane glycoprotein and surface free energy changes in hypoxic fibroblast cells.

Hypoxia affects the biochemistry of mammalian cells and thus alters their sensitivity to subsequent chemo- and radiotherapy. When V79 Chinese hamster lung fibroblasts were grown under conditions of extreme hypoxia (less than 10 ppm O2) there was a significant shift in the membrane glycoprotein composition. Scanning electron microscopy revealed altered cell surface morphology including loss of pseudopodial projections. Experiments to determine changes in interfacial free energy of these cells using equilibrium two phase systems of poly(ethylene glycol) (PEG) and dextran were carried out. Test fluid droplets of the denser dextran-rich phase were formed on layers of cells in the PEG-rich phase as the bathing medium, and the contact angles the droplets made with the cell layers were measured from photomicrographs. The contact angles on cells in the plateau phase increased significantly with time of exposure to hypoxia, from 25 degrees (zero time) to 35 degrees (6 h) to 60 degrees (9 h). Contact angles on cells in the exponential phase increased from 80 degrees (zero time) to 150 degrees after 20 h of hypoxia. It appears that the altered contact angles reflect changes in cell surface hydrophobicity that may, in part, reflect alterations in the membrane glycoprotein composition.

Kupffer cells do not play a role in governing the efficacy of liposomal mitoxantrone used to treat a tumor model designed to assess drug delivery to liver.

A tumor model designed to assess liposome-mediated drug delivery to liver has been used in an attempt to better understand the mechanism of activity of liposomal mitoxantrone, a liposomal anticancer drug formulation that appears to be uniquely effective in treating this tumor model. Reductions in liposomal mitoxantrone accumulation in the liver were achieved either by use of poly(ethylene)glycol (PEG)-modified lipids or by methods designed to deplete liver phagocytes, a method referred to as hepatic mononuclear phagocytic system (MPS) blockade. A 2-fold reduction in mitoxantrone delivery to the liver was obtained using a mitoxantrone formulation with PEG-modified lipids, and a 3-fold reduction was obtained when liposomal mitoxantrone was given to animals pretreated to induce hepatic MPS blockade. Results demonstrate that the liposomal mitoxantrone formulation prepared with PEG-modified lipids was significantly less active than the formulations that did not contain PEG lipids, with median survival times of 17 days and 100% 60-day survival, respectively. In contrast, hepatic MPS blockade had no effect on the therapeutic activity of 1,2-dimyristoyl phosphatidylcholine/cholesterol (DMPC/Chol) mitoxantrone (100% 60-day survival). These data suggest that the hepatic MPS does not play a role in mediating the therapeutic activity of DMPC/Chol mitoxantrone in the treatment of liver localized disease. Results with formulations prepared with a PEG-stabilized surface, however, suggest that nonspecific methods to decrease liposome cell interactions inhibit the therapeutic activity of DMPC/Chol mitoxantrone.

General and oral health implications of cannabis use.

Cannabis, commonly known as marijuana, is the most frequently used illicit drug in Australia. Therefore, oral health care providers are likely to encounter patients who are regular users. An upward trend in cannabis use is occurring in Australia, with 40 per cent of the population aged 14 and above having used the drug. There are three main forms of cannabis: marijuana, hash and hash oil, all of which contain the main psychoactive constituent delta-9-tetrahydrocannabinol (THC). Cannabis is most commonly smoked, however it can be added to foods. THC from cannabis enters the bloodstream and exerts its effects on the body via interaction with endogenous receptors. Cannabis affects almost every system of the body, particularly the cardiovascular, respiratory and immune systems. It also has acute and chronic effects on the mental health of some users. Therefore, chronic abuse is a concern because of its negative effects on general physical and mental health. Cannabis abusers generally have poorer oral health than non-users, with an increased risk of dental caries and periodontal diseases. Cannabis smoke acts as a carcinogen and is associated with dysplastic changes and pre-malignant lesions within the oral mucosa. Users are also prone to oral infections, possibly due to the immunosuppressive effects. Dental treatment on patients intoxicated on cannabis can result in the patient experiencing acute anxiety, dysphoria and psychotic-like paranoiac thoughts. The use of local anaesthetic containing epinephrine may seriously prolong tachycardia already induced by an acute dose of cannabis. Oral health care providers should be aware of the diverse adverse effects of cannabis on general and oral health and incorporate questions about patients' patterns of use in the medical history.

The heavy chain of tetanus toxin can mediate the entry of cytotoxic gelonin into intact cells.

An artificial conjugate of the heavy chain of tetanus toxin linked by a disulphide bond to the impermeant ribosome-inactivating protein gelonin is cytotoxic to intact HT29 cells by inhibiting intracellular protein synthesis. Neither toxin nor gelonin alone has any significant effect. This shows that the heavy chain has the ability to mediate internalization of a protein to which it is bound by a disulphide bond. Thus the normal role of the tetanus toxin heavy chain may be to allow entry of the light chain into a cell.

Inactivation of platelet-derived growth factor-BB following modification by ADP-ribosyltransferase.

1. Arginine-specific ADP-ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP-ribose from NAD(+) to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline-inducible expression of human ART. 2. From a panel of growth factors, platelet-derived growth factor-BB (PDGF-BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF-AA was not a substrate. Under conditions of maximum labelling 5 mol ADP-ribose was incorporated per mol of PDGF-BB. 3. Purified (ADP-ribosyl)-PDGF-BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF-BB. 4. PDGF-dependent [(3)H-methyl]-thymidine incorporation was measured in the ART1-transfected fibroblast cell line at physiological concentrations of PDGF-BB, and without addition of extracellular NAD(+). Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF-BB, but not to PDGF-AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). 5. In conclusion, we propose that PDGF-BB-dependent signalling may be regulated by post-translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1-transfected fibroblasts.

Delay, discriminatory, and modality specific neurons in striatum and pallidum during short-term memory tasks.

The function of the striatum and its integrative capacities is addressed. The activity of single neurons in behaving Macaca mulatta is studied. Two new classes of neurons were found. In the striatum, but not in the pallidum, these neurons were visual modality specific. These neurons may represent a conjunction of sensory, mnemonic and motor information, and may be concerned with decisions about the emission or withholding of movements. The frontal dysfunction hypothesis of cognitive deficits in Parkinson's disease is considered.

Enrichment of differentiated, stellate astrocytes in cerebellar interneuron cultures as studied by GFAP immunofluorescence and autoradiographic uptake patterns with [3H]D-aspartate and [3H]GABA.

A study was undertaken to correlate the morphology expressed by astroglial cells in post-natal cerebellar, interneuron-enriched primary cultures, and the ability of these cells to accumulate putative neurotransmitter amino acids. Astroglial cell morphology, as studied by GFAP immunofluorescence staining showed considerable changes during the culture period considered (up to 12 days in vitro). While the total number of GFAP-positive cells decreased with time (cell multiplication was prevented by cytosine arabinoside), a progressive enrichment of stellate astrocytes (cells bearing multiple radially arranged processes) and a striking increase in size of these cells was noted. In 12 DIV cultures stellate astrocytes accounted for 70-80% of the astrocytes present, and could reach a diameter of over 300 micron. The L-glutamate analogue, [3H]D-aspartate, was avidly taken up by all the astrocytes, independently of their shape and stage of differentiation. Astroglial cell morphology as delineated by [3H]D-aspartate autoradiography was identical to that evidenced by GFAP staining. On the other hand, [3H]GABA was accumulated in substantial amounts only by the stellate astrocytes, that is by the cells showing greater morphological differentiation. Astrocytes of other shapes were only lightly labelled by [3H]GABA in 2 DIV and 5 DIV cultures, and even less at later stages. Even within the stellate astrocyte population, the extent of [3H]GABA labelling was very variable, from one cell to another. Autoradiographic examinations and the determination of the IC50s for GABA uptake inhibitors consistently indicated that the GABA transport system present in stellate astrocytes did not have the features generally attributed to a glial transport system. In fact, beta-alanine was a very weak inhibitor, while nipecotic acid and ACHC were strongly inhibitory; DABA inhibitory potency fell somewhere in between. [3H]GABA uptake into the inhibitory interneurons present in the cultures showed similar sensitivity to GABA transport inhibitors.

Cerebellar astroglial cells in primary culture: expression of different morphological appearances and different ability to take up [3H]D-aspartate and [3H]GABA.

In non-neuronal cultures of cells dissociated from postnatal rat cerebellum astrocytes, identified by the presence of the marker protein glial fibrillary acidic protein (GFAP), displayed two distinct morphological forms. One class was stellate in shape with radially distributed fine processes, while the other class was more varied in shape being polygonal or elongate. [3H]thymidine incorporation experiments revealed that cells of both morphologies were able to incorporate this nucleoside, suggesting the capacity for both cell types to undergo cell division. An autoradiographic study of the uptake of [3H]D-aspartate and [3H]GABA revealed that whilst the two classes of astrocytes took up the aspartate to apparently the same extent, only the stellate cells were found to be heavily labeled following incubation with [3H]GABA. A study of the cultures over a 12-day period showed that there was a disappearance of the stellate astrocytes. The time of disappearance was found to be dependent upon the initial plating density; the stellate morphology was apparent longer in lower density cultures. Time lapse studies suggested that one of the reasons for the disappearance of the stellate cells might be that in fact they underwent a change in shape following certain cell-cell interactions, but cell death also has to be considered as a further possibility. The relationships between the two classes of astroglial cells in these cultures is not yet clear. The possibilities are that they represent two different types of astrocytes, or just one type at different stages of differentiation, or maybe a combination of the two possibilities.

Subpopulations of rat cerebellar astrocytes in primary culture: morphology, cell surface antigens and [3H]GABA transport.

Glial fibrillary acidic protein (GFAP)-positive astrocytes in preconfluent cultures derived from postnatal rat cerebellum have been previously shown to display two distinct morphologies, one stellate and the other irregularly epithelioid. The immunofluorescence studies described here showed that these cells also possess unique surface characteristics. In cultures derived from 8-day-old animals stellate cells bound the monoclonal antibody A2B5 whereas the epithelioid cells bound another monoclonal antibody against rat neural antigen-2 (RAN2). Some stellate cells derived from 2-day-old animals also bound tetanus toxin. The A2B5 labelling of the stellate cells made it possible to follow their fate in vitro. In confirmation of previous time-lapse studies, they underwent a shape transformation as confluence was approached, ultimately attaining a form resembling that of the epithelioid cells. Autoradiographic transport studies using two tritiated gamma-aminobutyric acid (GABA) analogues cis-1,3-aminocyclohexane carboxylic acid (ACHC) and beta-alanine revealed further differences between the two types of astrocytes. Whereas [3H]ACHC was taken up solely by the stellate cells [3H]beta-alanine was transported by both cell types. In other experiments in which various inhibitors of [3H]GABA transport were used ACHC virtually eliminated uptake into the stellate astrocyte, but had little effect on the epithelioid ones. The 'neuron-like' [3H]GABA transport process in the stellate astrocytes was confirmed in experiments comparing the effect of another compound which has been proposed as an astrocyte-selective GABA transport inhibitor, 4,5,6,7-tetrahydroisoxazolo-(4,5-C)pyridin-3-ol (THPO). No discrimination was found in its effect on the uptake of [3H]GABA into either neurons or stellate astrocytes. Further autoradiographic studies following the uptake of [3H]GABA by postnatal cerebellar slices showed that astrocytes in all layers of the cerebellar cortex and white matter transported [3H]GABA in contrast to the situation in culture where the amino acid is taken up predominantly by the stellate astrocytes. The possibility is discussed that the stellate astrocytes represent a population of cerebellar fibrous astrocytes whereas the identity of the epithelioid astrocytes is less certain.

Nasal bilevel positive airway pressure therapy in children with a sleep-related breathing disorder and attention-deficit hyperactivity disorder: effects on electrophysiological measures of brain function.

OBJECTIVE: To examine the effect of nasal bilevel positive airway pressure (BiPAP) treatment for concurrent sleep-related breathing disorders (SRBDs) and attention-deficit hyperactivity disorder (ADHD) on electrophysiological measures of spontaneous brain activity and auditory stimulus processing. METHODS: Nineteen children diagnosed with both SRBD and ADHD participated. Electroencephalogram (EEG) activity was recorded during a resting period and an auditory oddball task before beginning BiPAP treatment, after 6 months on treatment, and after a subsequent 1 week non-treatment period. Treatment effects on EEG and event-related potentials (ERPs) to target stimuli were examined via topographic analysis. RESULTS: Thirteen of the initial 19 children completed 6 months of BiPAP therapy, with six lost mainly due to compliance problems. Children on BiPAP therapy showed a significant decrease in slow-wave (delta and theta) and an increase in fast wave (beta) EEG activity. The P3 component of the ERP showed treatment effects in amplitude and latency. CONCLUSIONS: The electrophysiological data suggest that SRBDs may contribute to ADHD symptomatology. Treatment of SRBD with BiPAP therapy in children with concurrent ADHD can lead to significant changes, in the direction of normalization, of the typical electrophysiological features of ADHD.

Symptomatic arachnoid cyst at the clivus drained stereotactically through the vertex.

A 40-year-old man had an acute ischemic event affecting the pons. He had no cardiac illness or vasculities and was not diabetic or hypertensive. The contrast-enhanced computed tomographic scan disclosed nothing abnormal, and a vertebral angiogram showed an avascular mass markedly displacing the basilar artery and pons posteriorly and toward the right. A magnetic resonance image clearly delineated a homogeneous arachnoid cyst containing cerebrospinal fluid. To avoid manipulating posterior fossa arteries, after the recent pontine stroke, this arachnoid cyst with unusually favorable anatomical landmarks was approached stereotactically through the vertex. Fenestration and drainage of the cyst was accomplished under local anesthesia and benzodiazepine sedation with low morbidity. The procedure was well tolerated, and the patient returned to gainful employment shortly afterward. He remains asymptomatic at 3 years' follow-up. The successful outcome of this case suggests that in carefully selected symptomatic arachnoid cysts, stereotactic interventions could become a useful surgical alternative. Stereotaxis may also be helpful in other cumbersome surgical cases in which a second port for contrast injection or simple manipulation would be advantageous.

The different mechanisms of the direct and indirect tilt illusions.

Both the tilt illusion and aftereffect exhibit indirect effects under certain conditions: these are negative (assimilation) effects which occur with large (70-90 deg) angular separations between test and inducing gratings. They are opposite in direction to the positive, and much larger, contrast effects which occur at smaller (10-15 deg) separations. Evidence from six experiments shows that stimulus manipulations which reduce direct effects have little or no effect on indirect effects and vice versa, suggesting that the two effects have different determinants. It is proposed that direct effects arise from lateral inhibitory interactions between populations of neurones in striate cortex and that indirect effects occur at a higher level, possibly in areas concerned with stimulus-specific interactions beyond the classic receptive field. The implications of the data for theories of the tilt illusion are considered.

Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol).

The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the reduction or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biological role. The physical and chemical properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a "dysopsonization" phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.