Cell-associated ovalbumin is cross-presented much more efficiently than soluble ovalbumin in vivo

To better understand the antigenic requirements for cross-presentation, we compared the in vivo efficiency of presentation of cell-associated vs soluble OVA with the OT-I (CD8) and OT-II (CD4) TCR transgenic lines. Cross-presentation of cell-associated OVA was very efficient, requiring as little as 21 ng of OVA to activate OT-II cells and 100-fold less to activate OT-I cells. In contrast, soluble OVA was presented inefficiently, requiring at least 10,000 ng OVA for activation of either T cell subset. Thus, cell-associated OVA was presented 500-fold more efficiently than soluble OVA to CD4 T cells and 50,000-fold more efficiently to CD8 T cells. These data, which represent the first quantitative in vivo analysis of cross-presentation, show that cell-associated OVA is very efficiently presented via the class I pathway.     

Interleukin-6 biology is coordinated by membrane bound and soluble receptors

Cytokine receptors exist in membrane bound and soluble form. Both forms bind their ligands with comparable affinity. While most soluble receptors are antagonists in that they compete for the ligands with their membrane counterparts, some soluble receptors are agonists. In this case, the complex of ligand and soluble receptor binds on target cells to a second receptor subunit and initiates signal transduction. Soluble receptors of the IL-6 family of cytokines are agonists. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. This process has been named transsignaling. We have shown that in several chronic inflammatory diseases like chronic inflammatory bowl disease, peritonitis and rheumatoid arthritis, transsignaling via the soluble IL-6R complexed to IL-6 is a crucial point in the transition from the acute to the chronic state of the disease. The mechanism by which the IL-6/ soluble IL-6R complex regulates the inflammatory state is discussed.     

Two offshore coral species show greater acclimatization capacity to environmental variation than nearshore counterparts in southern Belize

Coral Reefs - Coral reefs are enduring decline due to the intensifying impacts of anthropogenic global change. This widespread decline has resulted in increased efforts to identify resilient coral...     

Nonencapsulated or nontypeable Haemophilus influenzae are more likely than their encapsulated or serotypeable counterparts to have mutations in their fucose operon

Population biology of Haemophilus influenzae can be studied by multilocus sequence typing (MLST), and isolates are assigned sequence types (STs) based on nucleotide sequence variations in seven housekeeping genes, including fucK. However, the ST cannot be assigned if one of the housekeeping genes is absent or cannot be detected by the current protocol. Occasionally, strains of H. influenzae have been reported to lack the fucK gene. In this study, we examined the prevalence of this mutation among our collection of H. influenzae isolates. Of the 704 isolates studied, including 282 encapsulated and 422 nonencapsulated isolates, nine were not typeable by MLST owing to failure to detect the fucK gene. All nine fucK-negative isolates were nonencapsulated and belonged to various biotypes. DNA sequencing of the fucose operon region confirmed complete deletion of genes in the operon in seven of the nine isolates, while in the remaining two isolates, some of the genes were found intact or in parts. The significance of these findings is discussed.     

Structure of bacteriocin AS-48: from soluble state to membrane bound state

The bacteriocin AS-48 is a membrane-interacting peptide, which displays a broad anti-microbial spectrum against Gram-positive and Gram-negative bacteria. The NMR structure of AS-48 at pH 3 has been solved. The analysis of this structure suggests that the mechanism of AS-48 anti-bacterial activity involves the accumulation of positively charged molecules at the membrane surface leading to a disruption of the membrane potential. Here, we report the high-resolution crystal structure of AS-48 and sedimentation equilibrium experiments showing that this bacteriocin is able to adopt different oligomeric structures according to the physicochemical environment. The analysis of these structures suggests a mechanism for molecular function of AS-48 involving a transition from a water-soluble form to a membrane-bound state upon membrane binding.     

Multiple forms of glycosyltransferases in Golgi complex membrane fractions

The distribution of multiple forms of galactosyltransferase (EC 2.4.1.22) and sialyltransferase (EC 2.4.99.1) from the microsomes and Golgi complex membrane fractions of rat liver was investigated. Three fractions of Golgi membranes, namely GF1, GF2, and GF3, differing in their morphology and marker enzyme activity, were obtained. A simultaneous increase of glycosyltransferases under study was observed in fractions GF3 less than GF2 less than GF1. Using isoelectrofocusing, the presence of at least 6-8 forms of galactosyl- and sialyltransferases in the microsomes and Golgi fraction was revealed. The distribution patterns of multiple forms along the pH gradient for each membrane fraction were found to be identical. However, the ratios of highly active and low active forms were specific for each fraction. The similarity of multiple form spectra for galactosyl-and sialyltransferase suggest their tight functional interaction and a possible "en block" packing of membrane glycosyltransferases.     

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.     

Encephalomyocarditis viruses with short poly(C) tracts are more virulent than their mengovirus counterparts.

We have constructed three cDNA clones of encephalomyocarditis virus strain R (EMCV-R) with poly(C) tracts of C4, C9, and C20. RNA transcribed from these cDNAs was infectious to HeLa cells, and the resultant viruses grew well in this system, albeit with plaque sizes that were proportional to the poly(C) length. When injected into mice, the progeny viruses were only slightly less pathogenic than EMCV-R, and the observed degree of attenuation was not nearly as dramatic as for equivalent mengoviruses with similar short poly(C)s. Short-tract poly(C)-mediated attenuation is therefore highly dependent on viral genomic context.     

The inhibitory effect of tetramethylethylene diamine on water soluble and membrane bound acetylcholinesterase activity

• 1.1. The inhibitory effect of N,N,N′,N′-tetramethylethylene diamine (TEMED) on water soluble (WSAChE) and membrane bound (MBAChE) acetylcholinesterase was investigated.
• 2.2. TEMED (0.5–4.0 mM) reversibly inhibited WSAChE activity (18–62%) and MBAChE (20–61%) in a concentration dependent manner.
• 3.3. The IC₅₀ being about 2.8 mM for WSAChE and 2.6 mM for MBAChE.
• 4.4. Lineweaver-Burk plots indicated that the nature of inhibition is noncompetitive for both water soluble and membrane bound acetylcholinesterase, with K_m values 68 μM and 123 μM respectively.
• 5.5. An Arrhenius plot showed that the transition temperature (TT) is unaffected in the presence of TEMED.
• 6.6. The activation energy was increased below and above TT in the case of WSAChE only.
• 7.7. On the basis of this behaviour of TEMED with AChE. it can be proposed that it can be used as an eluting agent for the bounded AChE to affinity ligand and may have beneficial action on the reactivatability of irreversibly-inhibited AChE due to its structure.
• 8.8. Moreover there is a possibility that it can be used as a therapeutic agent for the treatment of Alzheimer's disease, myasthenia gravia and glaucoma like some other inhibitors of AChE.

     

Parthenogenetic populations of the freshwater snail Campeloma limum occupy habitats with fewer environmental stressors than their sexual counterparts

1. Sexual organisms should have half the reproductive rate of their parthenogenetic counterparts (i.e. twofold cost of sex), so the plethora of sexual species relative to parthenogenetic species remains an evolutionary paradox. The rarity of parthenogenesis may in part be due to the accumulation of deleterious mutations. Indeed, parthenogenetic populations of the freshwater snail Campeloma limum have a greater mutation load relative to sexual populations of C. limum, although this does not directly affect their reproductive fitness. We hypothesise that although parthenogenesis has no direct effect on fitness in C. limum, mutation accumulation and environmental stress act synergistically to limit the distribution of parthenogenetic populations. 2. We evaluated this hypothesis, predicting that parthenogenetic populations of C. limum would inhabit sites with fewer environmental stressors than their sexual counterparts. We collected water quality, population density and individual size data at multiple time points from eight parthenogenetic and five sexual populations in the south‐eastern United States (Georgia and South Carolina). 3. Consistent with our hypothesis, sexual populations of C. limum inhabited poorer‐quality areas (sites with significantly lower dissolved oxygen and significantly more faecal coliform bacteria) than parthenogenetic populations. Despite these stressors, sexual populations still exhibited significantly higher population density than parthenogenetic populations. 4. Our findings support the hypothesis that mutation‐laden parthenogenetic C. limum populations occupy habitats with fewer environmental stressors relative to their sexual counterparts. Moreover, sexual C. limum populations inhabit lower‐quality habitats where they can presumably evade the twofold cost of sex in the absence of competition from their parthenogenetic counterparts.     

Indigenous microbes and their soluble factors differentially modulate intestinal glycosylation steps in vivo

It has been shown that Bacteroides thetaiotaomicron, a representative member of the gut microflora, signals intestinal epithelial cells both in vivo and in vitro and modulate specific glycosylation processes that may mediate intestinal functions. However it is not known whether these modulations depend on the presence of live bacteria or may be elicited by soluble factors produced in vitro by this bacterium. We used lectins and an histochemical approach to survey tissue sections prepared from various cellular compartments of the small and large intestine of NRMI/KI mice grown under gnotobiotic conditions. We compared the results obtained with bacterial culture supernatant and live B. thetaiotaomicron to those obtained from germ-free mice or mice having a conventional microflora. This approach allowed us to conclude that (1) a small but specific number of glycan patterns were restored after treatment with bacterial culture supernatant and (2) the B. thetaiotaomicron associated mice restored a larger number of patterns, however, the complete conventional mice pattern must be a function of the whole microflora in the gut. The possibility to modulate this complex glycosylation pattern by introducing exogenous bacteria and bacterial products should be considered as a promising approach towards understanding the molecular basis of microbial-host interactions.     

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.     

Monocyte suppressor factor is plasminogen activator inhibitor inhibition of membrane bound but not soluble IL-1

Human monocytes cultured in monolayer for 6 days were found to secrete a factor that suppressed the T cell proliferative response to soluble Ag and to alloantigens. The elaboration of this monocyte suppressor factor (MSF) was not inhibited by indomethacin. It has an apparent Mr of 50 to 60 kDa. It does not inhibit soluble IL-1 in the murine thymocyte costimulator assay but does inhibit the activity of membrane bound IL-1, which we observed to be almost exclusively IL-1 alpha. MSF contains elevated amounts of plasminogen activator inhibitor (PAI) when measured either as bioactivity or in an ELISA. Its immunosuppressive properties are inhibited by anti-PAI antibody. Furthermore, the eluate but not the effluent of an anti-PAI immunoabsorbent column contains all of the immunosuppressive activity. Based on these data we suggest that MSF is, in fact, PAI and postulate that the mechanism of action is inhibition of the plasmin cascade, thereby preventing the release of membrane bound IL-1. This suggests that monocytes possess an autoregulatory circuit that may have implications for the kinetics of the inflammatory response.     

A comparison of the effects of soluble stimuli on free cytoplasmic and membrane bound calcium in human neutrophils

Calcium dynamics in human neutrophils have been studied using Quin 2 fluorescence as a measure of free cytoplasmic calcium and chlortetracycline fluorescence as an indicator of membrane-bound calcium. The results show that 1) FMLP-induced increased cytoplasmic calcium likely comes from at least two different pools. Calcium is released from one only after a high affinity receptor interaction and from the second also after a lower affinity interaction. The initial increment in cytosolic calcium does not appear to originate in the pool(s) reflected by CTC fluorescence. 2) Cytochalasin B strikingly alters the FMLP effect on membrane associated calcium, inducing a marked “recovery” phase which could be a reflection of fusion of granule membranes with the plasma membrane. 3) PMA, at concentrations inducing extensive specific granule release (≤ 10 ng/ml) has no measurable direct effect on membrane-bound or cytosolic calcium. However, PMA inhibits a subsequent CTC fluorescence response to FMLP and following the ionophore, A23187, it induces a clear decrease in cytosolic calcium. These indirect effects may be explained in terms of PMA's activation of protein kinase C.