The -6.1-kilobase chicken lysozyme enhancer is a multifactorial complex containing several cell-type-specific elements.

In the chromatin domain of the chicken lysozyme gene of myeloid and oviduct cells, which both have the potential to activate the gene, a developmentally stable DNase I-hypersensitive site is formed around 6.1 kb upstream of the gene. This implies that this DNA region, which has previously been demonstrated to function as a transcriptional enhancer element in myeloid cells, is intimately involved in the cell-type-specific activation of the lysozyme gene locus. Deletion analysis identifies a 157-bp minimal fragment that confers the same promacrophage-specific enhancer activity as the originally described 562-bp -6.1-kb enhancer fragment. By introducing specific point mutations, we demonstrate in transient gene transfer experiments that the minimal fragment consists of at least six adjacent elements, each substantially contributing to enhancer function. The compact multifactorial enhancer complex includes a nuclear factor I (NF-I)/TGGCA binding site, homologies to AP1, and octanucleotide or enhancer core consensus motifs. Point mutation of the NF-I binding site results in the loss of NF-I binding in vitro and enhancer activity in vivo after gene transfer. Surprisingly, four overlapping oligonucleotides, each consisting of at least two elements of the -6.1-kb enhancer, confer myeloid-cell-specific enhancer activity. We found several myeloid-cell-specific DNA-binding proteins interacting with the -6.1-kb enhancer, a result consistent with that described above. Therefore, we suggest that more than a single trans-acting factor mediates the cell type specificity of the -6.1-kb enhancer.

Comparing non-invasive scapular tracking methods across elevation angles, planes of elevation and humeral axial rotations.

Altered scapular motions premeditate shoulder impingement and other musculoskeletal disorders. Divergent experimental conditions in previous research precludes rigorous comparisons of non-invasive scapular tracking techniques. This study evaluated scapular orientation measurement methods across an expanded range of humeral postures. Scapular medial/lateral rotation, anterior/posterior tilt and protraction/retraction was measured using an acromion marker cluster (AMC), a scapular locator, and a reference stylus. Motion was captured using reflective markers on the upper body, as well as on the AMC, locator and stylus. A combination of 5 arm elevation angles, 3 arm elevation planes and 3 arm axial rotations was examined. Measurement method interacted with elevation angle and plane of elevation for all three scapular orientation directions (p < 0.01). Method of measurement interacted with axial rotation in anterior/posterior tilt and protraction/retraction (p < 0.01). The AMC had strong agreement with the reference stylus than the locator for the majority of humeral elevations, planes and axial rotations. The AMC underestimated lateral rotation, with the largest difference of ∼2° at 0° elevation. Both the locator and AMC overestimated posterior tilt at high arm elevation by up to 7.4°. Misestimations from using the locator could be enough to potentially obscure meaningful differences in scapular rotations.

Identification of a gamma-interferon-responsive element in the promoter of the human macrophage scavenger receptor A gene.

In the present study, we demonstrate gamma-interferon (gamma-IFN)-inducible scavenger receptor A (SR-A) mRNA expression during the early stages of THP-1 and blood monocyte differentiation. Predominant induction of SR-A type II mRNA parallels the increased accumulation of cholesteryl esters under these conditions. A potential signal transducer and activator of transcription (STAT1) binding site (gamma-interferon activation site) in the SR-A promoter demonstrates gamma-IFN-inducible DNA binding activity and is most likely responsible for the gamma-IFN-dependent expression of an SR-A promoter-luciferase fusion construct. In contrast, gamma-IFN inhibits SR-A expression in mature macrophages as well as after prolonged gamma-IFN incubation of THP-1 monocytes. Taken together, these results demonstrate opposite effects of gamma-IFN on SR-A expression and activity during the early versus late stages of monocyte maturation. gamma-IFN-induced STAT1 activation, leading to increased SR-A expression, could therefore play an important role in the initial steps of foam cell formation and xanthomatosis.

The p38-MK2-HuR pathway potentiates EGFRvIII-IL-1ß-driven IL-6 secretion in glioblastoma cells.

The microenvironment of glioblastoma (GBM) contains high levels of inflammatory cytokine interleukin 6 (IL-6), which contributes to promote tumour progression and invasion. The common epidermal growth factor receptor variant III (EGFRvIII) mutation in GBM is associated with significantly higher levels of IL-6. Furthermore, elevated IL-1ß levels in GBM tumours are also believed to activate GBM cells and enhance IL-6 production. However, the crosstalk between these intrinsic and extrinsic factors within the oncogene-microenvironment of GBM causing overproduction of IL-6 is poorly understood. Here, we show that EGFRvIII potentiates IL-1ß-induced IL-6 secretion from GBM cells. Importantly, exacerbation of IL-6 production is most effectively attenuated in EGFRvIII-expressing GBM cells with inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and MAPK-activated protein kinase 2 (MK2). Enhanced IL-6 production and increased sensitivity toward pharmacological p38 MAPK and MK2 inhibitors in EGFRvIII-expressing GBM cells is associated with increased MK2-dependent nuclear-cytoplasmic shuttling and accumulation of human antigen R (HuR), an IL-6 mRNA-stabilising protein, in the cytosol. IL-1ß-stimulated activation of the p38 MAPK-MK2-HuR pathway significantly enhances IL-6 mRNA stability in GBM cells carrying EGFRvIII. Further supporting a role for the p38 MAPK-MK2-HuR pathway in the development of inflammatory environment in GBM, activated MK2 is found in more than 50% of investigated GBM tissues and correlates with lower grade and secondary GBMs. Taken together, p38 MAPK-MK2-HuR signalling may enhance the potential of intrinsic (EGFRvIII) and extrinsic (IL-1ß) factors to develop an inflammatory GBM environment. Hence, further improvement of brain-permeable and anti-inflammatory inhibitors targeting p38 MAPK, MK2 and HuR may combat progression of lower grade gliomas into aggressive GBMs.

Activation of Raf-1 is defective in annexin 6 overexpressing Chinese hamster ovary cells.

Annexin 6 is a Ca2+-dependent phospholipid-binding protein involved in membrane trafficking. In this study we demonstrate the association of Raf-1 with recombinant rat annexin 6. Raf-annexin 6 interaction was shown to be independent of cell activation by epidermal growth factor (EGF) or phorbol esters (12-O-tetradecanoyl-phorbol-13-acetate (TPA)). A stable Chinese hamster ovary (CHO)-anx6 cell line overexpressing annexin 6 was established to examine the function of annexin 6. In these cells, no increase of Ras-GTP levels, induced by EGF or TPA, was detected. In addition, the activity of Raf was completely inhibited, whereas the mitogen-activated protein kinase-P was unaffected.

Evidence for targeted vesicular glutamate exocytosis in osteoblasts.

Regulated intercellular signaling is essential for the maintenance of bone mass. In recent work we described how osteoblasts and osteoclasts express functional receptors for the excitatory amino acid, glutamate, indicating that a signaling pathway analogous to synaptic neurotransmission exists in bone. Here, we show that osteoblasts also express the essential molecular framework for regulated glutamate exocytosis to occur as is present in presynaptic neurons. A combination of reverse transcription-polymerase chain reaction (RT-PCR) and northern and western blotting is used to show expression of the target membrane-SNARE (soluble NSF attachment protein receptor), proteins SNAP-25 and syntaxin 4 and the vesicular-SNARE protein VAMP (synaptobrevin), the minimum molecular requirements for core exocytotic complex formation. Immunofluorescent localizations reveal peripheral SNAP-25 expression on osteoblastic cells, particularly at intercellular contact sites, colocalizing with immunoreactive glutamate and the synaptic vesicle-specific protein, synapsin I. We also identify multiple accessory proteins associated with vesicle trafficking, including munc18, rSec8, DOC2, syntaxin 6, and synaptophysin, which have varied roles in regulated glutamate exocytosis. mRNA for the putative Ca(2+)-dependent regulators of vesicle recycling activity, synaptotagmin I (specialized for fast Ca(2+)-dependent exocytosis as seen in synaptic neurotransmission), and the GTP-binding protein Rab3A are also identified by northern blot analysis. Finally, we demonstrate that osteoblastic cells actively release glutamate in a differentiation-dependent manner. These data provide compelling evidence that osteoblasts are able to direct glutamate release by regulated vesicular exocytosis, mimicking presynaptic glutamatergic neurons, showing that a process with striking similarity to synaptic neurotransmission occurs in bone.

Desialylated LDL uptake in human and mouse macrophages can be mediated by a lectin receptor.

We have compared the uptake of desialylated low density lipoprotein (LDL) with other modified forms of LDL in mouse peritoneal macrophages and PMA-activated human U937 monocytes. Neuraminidase-treated LDL (NT-LDL) caused significant cholesterol ester accumulation in both cell types, although the efficiency relative to loading with acetylated LDL (AcLDL) was markedly different, suggesting a very different complement of receptors in the cells. We therefore determined the effect of PMA-activation on lipoprotein receptor expression in U937 cells and found that while scavenger receptor concentration was elevated after PMA-activation, there was no significant change in the expression of the LDL receptor. Receptor specificity of NT-LDL uptake was examined by competition experiments using the degradation assay. This showed that 125I-labelled NT-LDL uptake in U937 cells could largely be accounted for by the persistent expression of the LDL receptor in these cells. In contrast, in mouse peritoneal macrophages where LDL receptor expression is very low, 125I-labelled NT-LDL degradation was also effectively competed by asialofetuin. Surprisingly, 125I-labelled NT-LDL degradation was also effectively competed by AcLDL. Measurement of sialic acid content of AcLDL showed that approximately 14% of the LDL sialic acid, equivalent to 2 to 3 residues per particle, was lost during acetylation of LDL with acetic anhydride. Thus competition between 125I-labelled NT-LDL and AcLDL could be due to lectin receptor binding rather than competition for scavenger receptor binding.

Role of the macrophage galactose lectin in the uptake of desialylated LDL.

Desialylated low density lipoprotein (LDL) is rapidly taken up and accumulated by both peripheral blood monocytes and cells isolated from human arterial intima consisting predominantly of smooth muscle cells. It is shown that thioglycollate (TG)-elicited mouse macrophages and mouse peritoneal macrophages stimulated with lipopolysaccharide (LPS) show increased expression of a membrane-bound, galactose-specific lectin that could be responsible for this uptake. In LPS-stimulated macrophages accumulation of desialylated LDL is increased ca. 2.6-fold. Accumulation of acetylated LDL in the same cells is reduced, suggesting that the galactose-specific lectin might be responsible for the uptake of desialylated LDL. Transfection of cells with the mouse macrophage Gal/GalNAc-specific lectin (MMGL) increased their capacity to take up asialofetuin (ASF) and, to a smaller extent, desialylated LDL. The uptake of desialylated LDL was small, most likely due to the high k(d) of MMGL for biantennary oligosaccharides as found on LDL, and low concentration of LDL achieved in tissue culture experiments. The data suggest that the expression of galactose-specific lectins can be elevated under inflammatory conditions, and that these receptors could contribute to foam cell formation under conditions of high desialylated LDL concentration, as might be found in arterial intima.

Expression of gamma-IFN responsive genes in scavenger receptor over-expressing monocytes is associated with xanthomatosis.

We have recently described an inherited over-expression of the macrophage scavenger receptor (SR) in blood monocytes from members of a kindred, only two of whom displayed extensive xanthomatosis. Using mRNA differential display we demonstrated abnormally high expression of the signal transducer and activator of transcription (STAT1alpha) in monocytes from the proband II-2. Expression of gamma-interferon inducible protein 10 (IP-10), a STAT1alpha-responsive gene and mediator of inflammatory response, was also abnormally expressed in the monocytes from II-2. Over-expression of both genes was restricted to monocytes from II-2 and was not observed in monocytes from the clinically unaffected family members, unlike that of SR. Gel retardation assays with THP-1 cell extracts identified gamma-IFN inducible DNA binding activity to three potential STATI DNA binding elements in the human IP-10 promoter region from nucleotides - 245 to - 188. Taken together these results suggest that gamma-interferon mediated cell activation is responsible for STAT1alpha-induced transcription of the IP-10 gene in THP-1 macrophages as well as in monocytes from II-2. Analysis of monocytes from familial hypercholesterolemic (FH) subjects, who frequently develop xanthomatosis, revealed a significant number of subjects with elevated STAT1alpha and IP-10 expression. Our data suggest that the inflammatory effects of gamma-IFN signaling could play a role in foam cell formation and xanthomatosis.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli.

A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59-241 has been cloned and expressed in Escherichia coli. A 1.0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a beta-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The beta-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-amino-benzyl-1-thio-beta-D-galactopyranoside agarose.

Inheritance of resistance to covered smut in barley and development of a tightly linked SCAR marker.

Inheritance of resistance to covered smut in the barley line Q21861 was studied using a doubled-haploid population produced by crossing Q21861 with the line SM89010. Based on 3 years of screening in the field and two seasons in the greenhouse, segregation for resistance/susceptibility fits a one-gene ratio, indicating a single major gene for resistance in Q21861. Of 440 random 10-mer primers tested using bulked segregant analysis, one primer (OPJ10) resulted in a reproducible polymorphic band. RAPD marker OPJ10(450) co-segregated in repulsion with the covered smut resistance. This marker was converted to a sequence-characterized amplified region (SCAR) marker linked in coupling (5.5 cM) with the covered smut resistant gene in Q21861. The SCAR marker was amplified in the line TR640 which is also resistant to covered smut, but not in the other resistant lines. The SCAR marker will be useful for marker-assisted selection for covered smut in barley breeding programs.

Effect of administration of a novel recombinant bovine interferon on length of oestrous cycle in cattle.

In ruminants, extensive reproductive loss occurs during the process of maternal recognition of pregnancy and it has been suggested that trophoblast interferons may be potential therapeutic agents. This paper reports results from a trial using eight first lactation Holstein-Friesian heifers to test the efficacy of a novel recombinant bovine interferon produced in bacteria in extending the life of the corpus luteum. Oestrus was synchronised in these animals and 0.1 mg of this non-glycosylated interferon was infused into the uterus twice daily for 13 days starting approximately 12 days after oestrus. This treatment resulted in an extension of the lifespan of the corpus luteum by 5.5 days (P=0.028) compared with untreated controls. In these animals the interovulatory period was extended by 6.4 days (P=0.009). Administration of this protein did not have any adverse effects either on body temperature or on daily milk yields. The results indicate that this novel interferon may have potential therapeutic application for reducing embryo mortality.

Effect of a novel recombinant bovine interferon and trophoblast secretory products on protein metabolism by endometrial explants from cattle and sheep.

The effect of a novel recombinant bovine interferon on the rate of protein synthesis, as measured by the rate of leucine incorporation, in endometrial explants from cows and ewes was investigated and compared with the effect of trophoblast secretory proteins and of a recombinant bovine interferon-alpha1. Exposure of cultured bovine endometrial tissue to the novel recombinant bovine interferon mimicked the effect of pregnancy in reducing the rate of protein synthesis in both caruncular and intercaruncular endometrium. This effect was similar to that seen with naturally produced trophoblast secretory proteins and it was more pronounced than that seen when endometrial tissue was treated with a related recombinant bovine interferon-alpha1. Ovine endometrial cultures were unaffected by exposure to this novel recombinant bovine interferon. This novel recombinant interferon exhibited potent antiviral properties (an activity of 1.6 x 10(10) international reference units/mg protein) and may prove to be a valuable reagent in studying the maternal recognition of pregnancy in the cow and reducing embryo mortality.

Interleukins in glioblastoma pathophysiology: implications for therapy.

Despite considerable amount of research, the poor prognosis of patients diagnosed with glioblastoma multiforme (GBM) critically needs new drug development to improve clinical outcomes. The development of an inflammatory microenvironment has long been considered important in the initiation and progression of glioblastoma; however, the success of developing therapeutic approaches to target inflammation for GBM therapy has yet been limited. Here, we summarize the accumulating evidence supporting a role for inflammation in the pathogenesis of glioblastoma, discuss anti-inflammatory targets that could be relevant for GBM treatment and provide a perspective on the challenges faced in the development of drugs that target GBM inflammation. In particular, we will review the function of IL-1ß, IL-6 and IL-8 as well as the potential of kinase inhibitors targeting key players in inflammatory cell signalling cascades such as JAK, JNK and p38 MAPK.

Annexin A6 is a scaffold for PKCα to promote EGFR inactivation.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.

Annexin A6 inhibits Ras signalling in breast cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca(2+)- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca(2+)- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.