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text:
Shit has really hit the fan. No need to rehash what's gone down, because I am sure everyone already knows. Unless you live under a rock. Which in that case, I envy you.
I see so many companies sharing endless content about their home offices. If you've seen one home office post then you've seen them all. Honestly, it's a little lazy to include that in your marketing and branding strategy when everyone else is doing it.
What I'm missing from my feed is content featuring companies and its employees sharing how their shared values are shining a light through the dark tunnel we are all currently in.
As we are all forced to abandon our offices and work from home, now is the time for those values to take center stage and lead us to the other side of this crisis. They're what motivates us and keeps us going when everything is so up in the air.
The other day I saw a comment on a LinkedIn post that stopped me in my tracks. It was from a hospitality worker at one of Norway's largest hotel chains. He shared that he had just been laid off and there were no hard feelings towards his employer. He loves his company and knows that they had to do what they had to do in order to survive.
It was such an impactful comment. Without ever having worked at this hotel chain, I gained an immediate understanding of what kind of work environment they had. It was employer branding at its best.
At CompEdge, our own values were quickly put to the test as we packed up our things and retreated to our makeshift home offices.
I won't lie. At first, when things started to shut down and we were told to stay at home, I had a pit in the bottom of my stomach. Too many worries and thoughts hit me all at once. I didn't know what the future would look like and was concerned about how we would do business if B2B Norge was shuttering its doors.
Things seemed bleak for a hot minute.
Then, Adrian came up with an action plan and reminded us of who we are and why we are at CompEdge. To build Norges Råeste Marketing Agency. Even in the middle of a crisis. We were going to not only keep fighting for ourselves, but for the whole B2B industry, in the only way we know how: loud and real.
If that's not rått og ekte, then I don't know what is.
Gathering 17 people who are used to collaborating in person, onto an online space with only a day's notice was not an easy feat. But we did it and have learned how to interact in new ways, like over our morning video meetings that I've affectionately dubbed: "The CompEdge Morning Show".
Over the past week we have mobilized to think differently and keep delivering to our existing clients and bringing in new ones. It's not in our orange blood to give up when facing adversity. And it's not in our DNA to accept the status quo.
This was made possible all because we're being guided what was already in place: our values and our why.
So many other companies that I've seen in both traditional media and social media, both here in Norway and in the US; were left scrambling as this crisis hit. They are companies that already had a bad reputation as an employer (I won't name names). Some even refused to let their employees work from home until the government forced their hands by implementing extraordinary measures. Do you think those employees are going to want to stick around once this pandemic blows over?
I have no doubt that the Corona Virus inevitably made each and every one of us at CompEdge that more loyal to our cause. We could've given up and bowed out to wait for the storm to pass. But instead, we are dancing in the rain and gaining momentum like never before.
Storgata 50155 OsloTelefon: +47 99108488

subset: commoncrawl


text:
Go Old School With Double Gold Slots
Playing all the new video slots that are highly interactive and have multiple screens, bonuses, animation and soundtracks is a lot of fun, but sometimes you want that old school, real-world casino experience. If you are craving this, then Double Gold Slots should be your first choice in slot machine play. You will actually feel like you are sitting at a slot, and you will see the two neighboring machines, and even get glimpses between of the casino floor. The paytable is on the top of the machine, and the reels and controls are just like sitting on your stool at your favorite casino.


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Selectins are carbohydrate-binding cell adhesion molecules that play a key role in the initiation of inflammatory responses. Several studies have suggested that the sialylated, fucosylated tetrasaccharide sialyl Lewis X (sLex) is an important component of leukocyte ligands for E- and P-selectin. We have identified a stable variant of the HL60 cell line, HL60var, which displays a nearly complete absence of staining with several mAb directed against sLex and/or sLex-related structures. HL60var also exhibits a concomitant increase in reactivity with mAb directed against the unsialylated Lewis X (Lex/CD15) structure. Despite this sLex deficiency, HL60var binds well to both E- and P-selectin. No significant differences in expression of α1,3-fucosyltransferases, C2GnT (Core2 transferase), or P-selectin glycoprotein ligand-1 between HL60var and typical sLexhigh HL60 cells were detected. Although the precise molecular basis for the sLex−/low phenotype of HL60var remains uncertain, flow cytometric analysis with the sialic acid-specific Limax flavus lectin revealed a sharp reduction in HL60var surface sialylation. Thus, the loss in mAb reactivity may result from a loss of sialic acid residues from the mAb carbohydrate epitope. However, binding of HL60var to E- and P-selectin remains sensitive to neuraminidase treatment. Taken together, these data indicate that high levels of surface sLex and/or related epitopes are not essential for interactions with vascular selectins, implying that as yet unidentified sialylated, fucosylated structures serve as physiologically relevant ligands for E- and P-selectin.
E- and P-selectin are members of the selectin family of cell adhesion molecules, which are essential for leukocyte recruitment in response to tissue injury or inflammation (1, 2). Both E- and P-selectin are expressed on activated endothelium, and both bind carbohydrate ligands on the surface of leukocytes. While one glycoprotein (P-selectin glycoprotein ligand-1 (PSGL-1),3 appears to account for all leukocyte interactions with P-selectin (3, 4, 5, 6), the precise molecular identity of the glycoproteins or glycolipids on leukocytes that mediates binding to E-selectin is not known. Similarly, the carbohydrates that function as physiologic ligands for selectins have not been definitively identified (1, 7).
Leukocyte adhesion to both E- and P-selectin is regulated in part by expression of certain glycosyltransferases. In particular, E-selectin binding depends critically on expression of leukocyte α1,3-fucosyltransferases, especially FucT-VII, while P-selectin binding requires both FucT-VII and the Core2 transferase (C2GnT) (6, 8, 9, 10, 11, 12). FucT-VII adds fucose residues to sialylated lactosamine precursors in an α1,3-linkage, and can be biochemically distinguished from the other leukocyte-expressed α1,3-fucosyltransferase, FucT-IV, by this preference for sialylated precursors (13, 14, 15, 16). Expression of FucT-VII has been shown to be a key regulatory step in the synthesis of functional ligands for E- and P-selectin4 (8, 9). C2GnT catalyzes the addition of GlcNAc in a β1,6-linkage to the GalNAc of O-linked carbohydrate chains, resulting in the branching of the oligosaccharide structure, and is essential for binding to P-selectin (11, 12, 17). However, the precise carbohydrate structures that mediate binding of leukocytes to vascular selectins remain to be determined.
A number of studies have suggested that leukocyte binding to both E- and P-selectin may occur through direct interaction with the sialylated, fucosylated tetrasaccharide sialyl Lewis X (sLex) or closely related structures (18, 19, 20, 21, 22). Many of these structures are recognized by several mAbs, including CSLEX1, HECA-452, AM3, and 2F3, and these mAb have been proposed to identify functional selectin ligands (23, 24, 25, 26). However, direct evidence for a requirement of these epitopes in E- or P-selectin binding is lacking, and several studies have suggested that they are not essential for selectin-dependent adhesion. For example, brief neuraminidase treatment abolishes binding of these mAb but does not affect or only partially affects binding to E-selectin (27). T cells lacking detectable CSLEX1 or HECA-452 staining bind well to P-selectin, but not E-selectin (28, 29). In addition, several human B cell lines that lack detectable reactivity with these mAb yet bind well to E-selectin have been identified (10). Finally, HECA-452 mAb only weakly blocks binding of cells to E-selectin (28); blocking studies with CSLEX1 are complicated by the agglutinating nature of this IgM mAb (22). Taken together, these studies indicate that although HECA-452 is an excellent reporter epitope for the glycosyltransferases required to generate selectin ligands, particularly FucT-VII (8, 27), structures identified by HECA-452 and related mAb do not themselves directly interact with vascular selectins. Rather, data from our laboratory and others suggest that high levels of FucT-VII lead to concomitant expression of both functional selectin ligands and sLex epitopes, whereas lower levels, while unable to generate detectable levels of surface sLex, are nonetheless competent to produce functional selectin ligands (8, 10, 27) (see footnote 4).
In the present report, we extend our previous observations on the relationship between leukocyte adhesion to vascular selectins, FucT-VII expression, and sLex expression. We describe the identification and characterization of a stable variant of the myeloid cell line HL60, designated HL60var, which has spontaneously lost reactivity with sLex-directed mAb, yet retains the ability to bind both E- and P-selectin. HL60var exhibits a near total loss of expression of several FucT-VII-dependent mAb epitopes that have been implicated in binding to E- and P-selectin, including HECA-452 and CSLEX1. However, this variant expresses normal levels of FucT-VII, as well as normal levels of FucT-IV, C2GnT, and the P-selectin glycoprotein ligand, PSGL-1. Analysis of HL60var surface carbohydrates using the sialic acid-binding Limax flavus lectin (LFA) (30) suggests that these cells harbor a generalized sialylation defect, which inhibits modification of most surface carbohydrates with sialic acid. Consistent with this hypothesis, HL60var also exhibits increased levels of the unsialylated Lewis X structure (CD15). However, this sialylation defect does not appear to significantly impair selectin-binding ability, as HL60var cells bind well to both E- and P-selectin. In addition, HL60var binding to both selectins remains sensitive to neuraminidase treatment, indicating that the ligands expressed by these cells still require sialylation for function. Taken together, these data provide strong evidence for the existence of as yet unidentified carbohydrate structures that interact with E- and P-selectin, and suggest that sLex, cutaneous lymphocyte antigen, and other related mAb-defined carbohydrates are not essential for binding to vascular selectins.
Materials and Methods
Cell lines and reagents
HL60 cells were maintained at 37°C in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% serum, 1% penicillin/streptomycin, and 2 mM glutamine. COS cells were maintained at 37°C in Dulbecco's MEM containing high glucose (Life Technologies) supplemented with 10% Nu Serum (Collaborative Biomedical Products, Bedford, MA), 1% penicillin-streptomycin, and 2 mM glutamine. Anti-carbohydrate IgM mAb were obtained from various investigators with specificities as described (see below) (27). HECA-452 mAb was a gift of L. Picker (University of Texas, Southwestern Medical Center, Dallas, TX). MMA mAb was obtained from the University of Iowa Developmental Biology Hybridoma Bank. VIM2 mAb was a gift of W. Knapp (University of Vienna, Vienna, Austria). 2F3 mAb was a gift of Dr. Reiji Kannagi (Aichi Cancer Center, Nagoya, Japan). The sialic acid-specific lectin LFA was obtained as a FITC conjugate from EY Labs (San Mateo, CA).
Isolation of variant HL60 cells
HECA-452−/low variants of HL60 were isolated by cloning at limiting dilution a parental HL60 population that spontaneously became bimodal for expression of HECA-452 Ag by flow cytometry. Clones were isolated with both HECA-452−/low and HECA-452high phenotypes. In all experiments "HL60" refers to a HECA-452high clone, while "HL60var" refers to the HECA-452−/low clone. The emergence of the HECA-452−/low population of HL60 cells occurred spontaneously after extended time in culture, and in our hands arises over time in all HECA-452high clones of these HL60 cells thus far examined. Therefore, all comparisons with HL60var were made with cells that expressed homogeneously high levels of HECA-452 Ag.
Flow cytometry
For FACS analysis, 5 × 105 cells were washed twice with RPMI 1640, and pellets were resuspended in 100 μl of primary mAb at the following empirically determined optimal dilutions in FACS buffer (1% FCS/0.1% NaN3/1× PBS): purified HECA-452 (8 μg/ml), CSLEX1 ascites (1:200), AM-3 ascites (1:1000), 2F3 tissue culture supernatant (1:5), MMA ascites (1:1000), VIM2 ascites (1:250), KPL1 (anti-PSGL-1) (34) ascites (1:1000), or isotype-matched control mAb (8 μg/ml 1:500). Cells were incubated for 15 min at 4°C, then washed once with 1 ml FACS buffer. Washed cells were resuspended in 100 μl of FITC-conjugated goat anti-mouse IgM or goat anti-mouse IgG (Biosource, Camarillo, CA), and incubated 15 min at 4°C. Cells were washed with 1 ml FACS buffer and resuspended in 0.5 ml FACS buffer for flow cytometry. For staining with FITC-labeled LFA lectin, 5 × 105 cells were washed twice with RPMI 1640, and resuspended in 20 μg/ml FITC-conjugated LFA or in 1 × PBS alone. Cells were incubated for 15 min at 37°C, and then washed once with 1 ml FACS buffer and finally resuspended in 0.5 ml FACS buffer for flow cytometry. Where indicated, LFA staining was performed after treatment of cells with neuraminidase. Neuraminidase treatment of cells was performed at 37°C for 2 h with 0.2 U/ml Clostridium perfringens neuraminidase (Sigma, St. Louis, MO) in a total volume of 0.5 ml RPMI 1640, pH 5.9. Cells were washed twice with RPMI 1640 before staining with LFA. Flow cytometry was performed on a FACScan (Becton Dickinson, Franklin Lakes, NJ). Data were collected using the CellQuest program to analyze a total of 10,000 light scatter-gated events and are plotted on a log scale as histograms of fluorescence intensity.
Low shear, nonstatic COS cell adhesion assays
Binding to E-selectin and P-selectin was determined in a rocking assay employing transiently transfected COS cells (31, 32). Briefly, COS cells were transfected with cDNA encoding either E-selectin or P-selectin by the DEAE-dextran method in 100-mm tissue culture grade petri dishes. Expression levels of E-selectin and P-selectin on transiently transfected COS cells are approximately equal (33). COS were replated into 35-mm dishes 1 day before the adhesion assay. Adhesion of HL60 or HL60var to transfected COS cells was conducted at 4°C with constant rocking. Each plate of COS cells was washed twice with RPMI 1640, followed by addition of 2 × 106 cells in 0.6 ml RPMI 1640. Cells were incubated at 4°C with rocking for 15 min, and the plates were then washed five times with RPMI 1640 and fixed with ice-cold 0.74% formaldehyde. The mean number of HL60 cells bound per COS cell was determined by counting the number bound to ∼100 COS cells in multiple fields of view. The number of leukocytes bound per COS cell is a function of several variables, especially the available surface area of the COS cell, and the size of the leukocyte. Where indicated, adhesion assays were performed after treatment of cells with neuraminidase, performed as described above, or in the presence of 2.5 mM EGTA.
In vitro rolling assays
The ability of HL60 and HL60var cells to roll on E-selectin or P-selectin was assayed in a parallel plate flow chamber. The parallel plate flow chamber and flow assay have been described elsewhere (8). Rolling was observed on monolayers of stably transfected CHO cells expressing either P-selectin (CHO/P) or E-selectin (CHO/E). Expression levels of selectins are approximately equal for CHO/P and CHO/E (6). Total numbers of rolling cells were determined by digital morphometry (Simple analysis software, Compix, Cranberry Township, NJ). Velocity measurements were made using the semiautomated Celltrak program (Compix). The Celltrak program acquires the digitized images, measures the velocities, compiles statistics, and generates histograms of velocity distribution. The histograms consist of measurements on 1000 to 4000 cell pairs in 100 consecutive fields covering a 2- to 6-min period of observation. "Events" refers to the number of individual measurements made in the 0- to 100-μm/s range. "Mean" refers to the average rolling velocity of the cells. For flow assays, data are represented as mean velocity 22/− SD and mean events +/− SD for two to three independent runs.
Semiquantitative RT-PCR detection of FucT-VII, FucT-IV, and C2GnT mRNA
Total cellular RNA was isolated from 107 cells, and 1 μg total RNA was used as template in a 20-μl reverse transcription reaction, exactly as described (6, 10). PCR amplification of cDNA was conducted with 24 (C2GnT or PGK), 30 (FucT-IV), 35 (ST3 Gal III and ST3 Gal IV), or 36 (FucT-VII) cycles of 1 min at 94°C, 1 min at 66°C, and 1 min at 72°C. Preliminary experiments (not shown) demonstrated that these cycle numbers are well below the plateau phase of PCR product accumulation for each gene. Hence, the amount of the PCR product faithfully reflects the amount of mRNA initially present. Primers were as follows: for detection of FucT-VII mRNA, sense 5′-CCC ACC GTG GCC CAG TAC CGC TTC T-3′ and antisense 5′-CTG ACC TCT GTG CCC AGC CTC CCG T-3′; for FucT-IV detection, sense 5′-CGG GTG TGC CAG GCT GTA CAG AGG-3′ and antisense 5′-TCG GGA ACA GTT GTG TAT GAG ATT-3′; for C2GnT detection, sense 5′-TTT TCW GGC AGT GCC TAC TTC GTG GTC and antisense 5′-ATG CTC ATC CAA ACA CTG GAT GGC AAA; for PGK1 detection, sense 5′-ATG ATT ATT GGT GGT GGA ATG GCT-3′ and antisense 5′-TCA TCC ATG AGA GCT TTG GTT CC-3′; for ST3 Gal III detection, sense 5′-GAT CAA AGG TCA AGA CAA TCT GAT-3′ and antisense 5′-GGT CAC TGC CAC ACT GCC AAG-3′; for ST3 Gal IV detection, sense 5′-TGG GTC AAG ACG CCA TCT GC-3′ and antisense 5′-AGG ATT GAC ATC CCA GAT GAG G-3′. Presence of PCR products was assayed by gel electrophoresis in 1.4% agarose gel, followed by Southern blotting as described below.
Southern blots
PCR products were electrophoresed in 1.4% agarose gels, and then transferred onto nitrocellulose (Schleicher & Schuell, Keene, NH) using upward capillary transfer in 10× SSC. Nitrocellulose filters were UV cross-linked, and prehybridization was conducted at 42°C for 2 to 3 h in a solution of 5× SSPE, 5× Denhardt's, 1% SDS, and 200 μg/ml denatured salmon sperm DNA. Radiolabeled probes were generated with the Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's protocol, using either FucT-VII cDNA, FucT-IV cDNA, C2GnT cDNA, or a PGK1 374-bp fragment (amplified from HL60 cell RNA as described above) as template. Hybridization was conducted at 42°C in a solution of 5× SSPE, 5× Denhardt's, 50% formamide, 0.1% SDS, and 100 μg/ml denatured salmon sperm DNA, to which 106 cpm/ml of the appropriate probe was added. Following hybridization, blots were washed twice in 1× SSPE, 0.1% SDS for 5 min at room temperature, and twice in 0.1× SSPE, 0.1% SDS for 15 min at 42°C. Bound radioactivity was detected by exposure to Hyperfilm (Amersham, Arlington Heights, IL).
SDS-PAGE and Western blotting for PSGL-1
Cell lysates were prepared, run on 6% reducing SDS-PAGE gels, transferred to nitrocellulose, and blotted with the anti-PSGL-1 mAb KPL1 (34) as previously described (34).
Fucosyltransferase enzyme activity assays
Assays for FucT-VII and FucT-IV activity were performed exactly as described (8).
A variant of HL60 cells lacks significant reactivity with HECA-452, but binds well to E- and P-selectin
The myeloid cell line HL60 typically reacts very strongly with HECA-452 mAb, similar to normal neutrophils. However, in a population of HL60 cells cultured for several weeks, we noticed the spontaneous emergence of a small population of HECA-452−/low cells. Subsequent cloning by limiting dilution allowed the isolation of several HL60 cell clones that stably exhibited this sharply reduced expression of the HECA-452 epitope. Figure 1⇓A depicts the FACS profile of one of these clones, HL60var, which expressed a near total (∼33-fold) reduction in reactivity with HECA-452 mAb. Staining of HL60var with HECA-452 was only 1.7 times greater than staining by isotype-matched control mAb (Fig. 1⇓B).
In other studies, HECA-452 reactivity has correlated well with the ability to bind to vascular selectins (8, 23, 28, 35). Thus, we were interested in the ability of HL60var cells to bind to E- and P-selectin. Selectin-binding ability was determined first at relatively low stringency. HL60 or HL60var cells were allowed to adhere with constant gentle rocking to COS cells transiently transfected with E- or P-selectin. Surprisingly, these low shear assays detected no significant differences in the selectin-binding ability of HL60var, as compared with HECA-452high HL60 cells, despite the dramatic reduction in HECA-452 reactivity of HL60var cells (Fig. 1⇑C). Binding of HL60var to E- and P-selectin was abrogated by EGTA (data not shown), or by pretreatment of the HL60 cells with neuraminidase (Fig. 2⇓), as was the residual HECA-452 staining (data not shown). These data demonstrate that adhesion of HL60var to E- and P-selectin remained dependent on both sialic acid and calcium, similar to the parent cell line.
HL60var can bind to and roll on E- and P-selectin at physiologically relevant shear rates
Because the low shear COS cell assay measures adhesion at relatively low stringency, but high sensitivity, we were also interested in determining the ability of HL60var to interact with vascular selectins under more physiologically relevant shear conditions. Thus, HECA-452high HL60 cells and HL60var cells were assayed for their ability to interact with E-selectin or P-selectin expressed by stably-transfected CHO cells in in vitro rolling assays that employ a constant linear shear force of 1.9 dynes/cm2. Even under these more stringent and more physiologic conditions, HL60var interacted well with both selectins (Fig. 3⇓A). Binding to CHO/P monolayers under flow conditions was approximately equivalent between HL60var and wild-type HL60 cells, with the variant rolling only 1.4 times faster and actually exhibiting more interactive events than HECA-452high HL60 cells (Fig. 3⇓). Rolling of HL60var on CHO/E monolayers was slightly less efficient, but still biologically significant, as compared with wild-type HL60 (Fig. 3⇓). HL60var exhibited an approximately twofold greater velocity with slightly fewer interactions than normal HL60 cells. These relative differences between HL60 and HL60var were also found when rolling on E-selectin was analyzed at a higher shear level of 3.8 dynes/cm2 (Fig. 3⇓B), under which conditions the velocities of the two cell lines were almost unchanged. In addition, the combination of HECA-452 plus CSLEX1 at 25 μg/ml each did not affect the binding of either HL60 or HL60var to E-selectin (data not shown). Together, these data indicate that the near total loss of HECA-452 reactivity does not greatly impair the ability of HL60var to interact with either E- or P-selectin, under either low or high shear conditions.
HL60var exhibits selective deficiencies in FucT-VII- and sialic acid-dependent mAb epitopes
To further explore the phenotype of this unique HL60 cell variant, we analyzed HL60var for reactivity with several anti-carbohydrate IgM mAb, some of which have previously been suggested to identify selectin-binding epitopes (18, 19, 23, 24, 25, 26). FACS analysis revealed a generalized deficiency in sialic acid-dependent epitopes. HECA-452 and CSLEX1 reactivity was reduced almost to zero (Fig. 4⇓). Similarly, HL60var exhibited a near total loss of staining by AM3 mAb, and a complete loss of detectable staining by 2F3 mAb (data not shown). In contrast, VIM2 reactivity was reduced only approximately fourfold (Fig. 4⇓). HL60var cells also exhibited a concomitant increase in staining by MMA mAb, directed against the unsialylated Lewis X structure (Lex/CD15). An identical increase in MMA reactivity, with a reciprocal decrease in reactivity with sLex-specific mAb, is observed upon neuraminidase treatment of several cell types, and is consistent with a loss of sialic acid, rather than a modification of sialic acid, which would render it invisible to HECA-452. Thus, these data suggested that the phenotype of this variant cell line may derive from a generalized defect in sialylation of surface carbohydrate structures. However, the mAb epitopes that were most greatly reduced on the variant cell line (i.e., HECA-452, CSLEX1, AM3, and 2F3) were also precisely those that we have previously shown to be associated with expression of the α1,3-fucosyltransferase FucT-VII (27). It was therefore important to determine the relative expression of relevant leukocyte glycosyltransferases in the HL60var cell line.
HL60var expresses normal levels of adhesion-associated glycosyltransferases
We and others have clearly demonstrated the importance of certain glycosyltransferases in the construction of functional ligands for E- and P-selectin (6, 8, 9, 10, 11, 12). For E-selectin binding, it is clear that FucT-VII plays a unique and important role in generating high avidity ligands, while FucT-IV, when present at high enough levels in appropriate cell types, can function in synthesizing lower avidity E-selectin ligands (8, 9, 10) (27). Similarly, studies in transfected cell lines and in normal T lymphocytes have demonstrated a requirement for FucT-VII and C2GnT in binding to P-selectin (6, 11, 12). We therefore examined the levels of these enzymes in the HL60 and HL60var cells.
HL60var expressed high levels of mRNA for FucT-VII, FucT-IV, and C2GnT, as determined by semiquantitative RT-PCR (Fig. 5⇓). Expression of FucT-VII and FucT-IV mRNA by HL60var was indistinguishable from that of HECA-452high clones of HL60, and expression of C2GnT mRNA was only slightly reduced (Fig. 5⇓). Neither wild-type HL60 nor HL60var cells expressed detectable mRNA for FucT-III, FucT-V, or FucT-VI (data not shown). Analysis of FucT enzyme activity in cell lysates revealed that HL60var had higher levels of FucT-IV activity, and equivalent levels of FucT-VII activity (Fig. 6⇓). Therefore, HL60var expresses normal levels of FucT-VII and FucT-IV mRNA and activity, and C2GnT mRNA, suggesting that the defect that gives rise to the sLex deficiency of these cells is not related to a defect in fucosylation or in the ability to generate branched O-linked carbohydrates.
HL60var exhibits a generalized defect in surface sialic acid
The dependence of selectin binding on sialic acid is well established (36, 37, 38, 39). The LFA lectin is specific for sialic acid moieties, and binds these residues regardless of linkage or position on the saccharide chain (30). We therefore used this lectin to determine whether HL60var expressed wild-type levels of surface sialic acid. FACS analysis of HL60 and HL60var cells with FITC-conjugated LFA revealed an ∼60-fold reduction in the LFA reactivity of the variant cells (Fig. 7⇓, A and B). The specificity of the lectin for sialic acid is demonstrated by the ∼100-fold reduction in reactivity observed when cells were treated with neuraminidase (Fig. 7⇓, C and D). Significantly, neuraminidase-treatment of HL60var cells further reduced their LFA reactivity (Fig. 7⇓D), indicating that HL60var does not completely lack surface sialylation. A sialylation defect of HL60var would account for the loss in reactivity with HECA-452 and other mAb recognizing sLex or sLex-like structures, as these epitopes are known to be sialic acid dependent, and is consistent with an increase in expression of the unsialylated Lewis X (CD15) structure.
Although sialyltransferases (ST) specifically responsible for construction of selectin ligands have not been definitively identified, at least two ST that can synthesize sLex in vitro, designated ST3 Gal III and ST3 Gal IV (40), have been molecularly cloned (41, 42). RT-PCR analysis for expression of mRNA encoding these enzymes showed no differences between HL60 cells and HL60var cells (data not shown).
PSGL-1 from HL60var exhibits altered SDS-PAGE mobility
Surface levels of PSGL-1 were identical between HL60 and HL60var cells (data not shown). We analyzed PSGL-1 from HL60 and HL60var by SDS-PAGE (Fig. 8⇓). The generalized loss of sialic acid from HL60var was associated with a decreased SDS-PAGE mobility, compared with HL60 cells (Fig. 8⇓), and this difference was not eliminated by treatment with neuraminidase (data not shown). Although the basis for this difference was not determined, these data indicate that the alterations of glycosylation found in the HL60var cell line are reflected in the biochemical properties of PSGL-1.
The identification and characterization of selectin ligands is an important area of research with potential applications to the treatment of a variety of autoimmune and inflammatory disorders. Previous studies have suggested a strong correlation of selectin-ligand expression with reactivity with several mAb, including HECA-452 and CSLEX1, leading to the conclusion (18, 19, 28, 43) that these mAb identify structures that directly mediate leukocyte binding to vascular selectins. Recently, we demonstrated that transfection of hemopoietic cell lines with cDNA encoding FucT-VII results in induction of staining by the sLex-like mAb HECA-452, CSLEX1, AM3, and 2F3, whereas transfection with FucT-IV cDNA induces expression of CD15 and CDw65 epitopes, as identified by MMA (44) and VIM2 (45) mAb, respectively (8, 10, 27). In parallel, FucT-VII transfection always induced E-selectin binding ability, and conferred P-selectin binding in cell lines expressing PSGL-1 and C2GnT (6, 27) (see footnote 4). However, by several criteria, this FucT-VII-induced ability to bind E-selectin could be dissociated from FucT-VII-induced expression of any of the previously implicated mAb epitopes. The identification of a stable sLex−/low variant of HL60 cells that exhibits high levels of adhesion to vascular selectins reinforces the conclusion that sLex-like mAb do not identify structures that directly mediate interactions with vascular selectins. Since HL60var has nearly completely lost reactivity with CSLEX1 and HECA-452 mAb, but still exhibits high avidity interactions with both E- and P-selectin, it is unlikely that these mAb identify essential selectin-binding carbohydrates. These results, in a human myeloid cell line, argue persuasively against the hypothesis (18, 19, 20, 21) that sLex itself is the principal ligand for E-selectin.
HL60var expressed normal levels of mRNA for several adhesion-associated glycosyltransferases, particularly FucT-VII, FucT-IV, and C2GnT, and normal levels of enzyme activity for at least FucT-VII and FucT-IV. It is likely that the C2GnT mRNA levels are also reflective of enzyme activity, as PSGL-1, which requires C2GnT (11, 12), is perfectly functional in this cell line. These results reinforce an essential role played by these enzymes in the construction of functional E- and P-selectin ligands (6, 8, 9, 10, 11, 12). More importantly, these data show that the sLex−/low phenotype of the HL60var cells is not due to the lack of expression or activity of these enzymes. Rather, the unique phenotype of HL60var appears to result from a sialylation deficiency, which prevents expression of a subset of sialic acid-dependent mAb epitopes. The existence of a generalized sialylation defect in HL60var cells is supported by the sharp reduction in reactivity of HL60var cells with the sialic acid-binding lectin LFA, as well as the increased reactivity of these cells with mAb directed against unsialylated structures (i.e., Lex/CD15). However, this loss of surface sialic acid in HL60var cells is not complete, as LFA reactivity can be further reduced by treatment of HL60var cells with neuraminidase. In addition, this loss is selective, affecting primarily the FucT-VII-dependent mAb epitopes, since the VIM2 epitope, which is also sialic acid dependent, exhibited a significantly milder reduction in expression on HL60var. These results raise the possibility that STs that are responsible for construction of sLex and related epitopes are distinct from those involved in biosynthesis of selectin ligands. Consistent with this hypothesis, no difference was detected in the expression of ST3 Gal III and ST3 Gal IV mRNA between HL60 and HL60var, even though these enzymes appear to be at least partially responsible for biosynthesis of sLex (40, 41, 42).
These findings do not exclude the possibility that carbohydrate residues recognized by HECA-452 and/or CSLEX1 contribute to the actual physiologic ligand. For example, the fucose residue of this epitope could contribute to the formation of a "discontinuous saccharide epitope" (1, 46) recognized by E- or P-selectin. Alternatively, the presence of the fucose could indirectly contribute to the appropriate recognition structure. However, the lack of significant inhibition of CSLEX1 or HECA-452 mAb on rolling of HL60var on the endothelial selectins, and the colinear staining of cells with HECA-452 and soluble recombinant E-selectin (47), argue strongly that the selectins are not seeing the same carbohydrates as HECA-452 mAb. Indeed, it is difficult to envisage how two large proteins (mAb and selectin) could bind to the same two residues (fucose and sialic acid) of the same tetrasaccharide without sterically inhibiting each other. Thus, although sLex epitopes and selectin ligands clearly depend on some of the same enzymatic machinery, particularly FucT-VII (27), our results clearly indicate that the carbohydrate residues seen by the selectin are distinct from the carbohydrate residues seen by sLex-type mAb.
Despite severely reduced levels of sLex-type mAb epitopes and an approximately 60-fold reduction in surface sialic acid, adhesion of HL60var to E- and P-selectin remained equally sensitive to neuraminidase as the parent HL60 cells. While this observation provides further support for the idea that the selectin ligands in this cell line are intact and largely unaltered, the low but detectable levels of staining with LFA lectin or HECA-452 mAb were also sensitive to neuraminidase. These results are therefore consistent with the possibility that the density of selectin-binding carbohydrates required for maximal shear-resistant adhesion to E- and P-selectin may be quite low. The sharp reduction in surface sialic acid on HL60var had no significant effect on rolling interactions with P-selectin and only mild effects on rolling on E-selectin, indicating that the low levels of sialylated structures remaining on the surface of HL60var cells were sufficient to mediate high avidity interactions with both selectins. This hypothesis is consistent with the surprising paucity of sLex-type structures found on the PSGL-1 molecule (48), corresponding to the lack of detectable sLex by Western blotting on PSGL-1 from HL60 cells (data not shown).
The identification and characterization of a spontaneous variant of HL60 cells lacking sLex-type mAb epitopes but retaining high avidity selectin ligands extends our previous work in dissociating epitopes recognized by these mAb from selectin-binding ability. In addition, the HL60 variant described in the present report is clearly distinct from one described by Lowe et al., which also does not express sLex but fails to bind E-selectin (22). Taken together, these data suggest the existence of novel carbohydrate structures, not identified by any existing mAb, which serve as physiologically relevant ligands for E- or P-selectin. Identification of the genes responsible for the phenotypes of these HL60 variants may be quite useful in the identification of physiologic E-selectin ligands and in the further understanding of the process of leukocyte recruitment in normal and pathologic situations.
We thank J. Lowe for providing fucosyltransferase cDNAs; M. Fukuda for providing C2GnT cDNA; L. Picker for providing HECA-452 mAb; and W. Knapp for providing VIM2 mAb. MMA mAb was obtained from the University of Iowa Developmental Biology Hybridoma Bank.
↵1 This work was supported in part by Grant CB-204 from the American Cancer Society, a Falk Award of Excellence from the Arthritis Foundation (to G.S.K.), and National Institutes of Health Grants AI 33189 and HL 31963 (to L.M.S.) G.S.K. is an Established Investigator of the American Heart Association. A.J.W. is a predoctoral fellow of the National Science Foundation.
↵2 Address correspondence and reprint requests to Dr. Geoffrey S. Kansas, Department of Microbiology-Immunology, Northwestern Medical School, 303 E. Chicago Ave., Chicago, IL 60611. E-mail address: gsk{at}nwu.edu
↵3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; sLex, sialyl Lewis X; LFA, Limax flavus; CHO, Chinese hamster ovary; RT-PCR, reverse transcriptase-polymer chain reaction; ST, sialyltransferase; SSPE, standard saline phosphate EDTA.
↵4 R. N. Knibbs, R. A. Craig, P. Maly, P. L. Smith, F. M. Wolber, N. E. Faulkner, J. B. Lowe, and L. M. Stoolman. α1,3–Furosyltransferase-VII-dependent synthesis of P- and E-selectin ligands on cultured T lymphoblasts. Submitted for publication.
5 K. R. Snapp et al. Submitted for publication.
- Received September 2, 1997.
- Accepted January 21, 1998.
- Copyright © 1998 by The American Association of Immunologists


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Yung Padawan
- Dec 3, 2017
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General Behaviour
The NCBA Discord server has a set of rules that help maintain a friendly and high-quality experience. These set of rules are enforced by the staff of NCBA and any infringement or infraction of these rules can subject you with punishment that could result in a kick or ban from the server:
(a1) Remain respectful in the community.
(a1.2) Do not harass members (There is a fine line between joking and harassing someone)
(a1.3) Refrain from any behaviors that can be seen as toxic, malicious or otherwise unwanted
(a1.4) Use common sense
(a1.5) Do not use any users photos, images or being without their written consent. (Not everyone wants their photo being used by others)
(a2) Do NOT spam in any form that includes but is not limited to:
(a2.1) Mass mention (spamming the @person feature)
(a2.2) Chat spam (Spamming text in #chats or private messages)
(a2.3) Ear Rape (Using the microphone in obnoxious mannerisms including soundboards)
(a2.4) Soundboards (Using soundboards to modify your voice or play audio clips in public channel )
Note: You may use soundboards if the channel members agree to use them.
(a3) Keep your nicknames readable and unique (Don't impersonate other members/staff)
(a3.1) Don't use ridiculous and unnecessary nicknames: (Ex. D9!$^$#@)
(a4) No NSFW content of any kind that includes and is not limited to:
(a4.1) Pornographic Content
(a4.2) Mutilation, gore or otherwise distressing images/video
(a4.3) Depictions of Minors in a sexual context (Includes Lolita, Hentai) or otherwise pornographic content that is suggestive
(a5) No Posting Malicious Content:
(a5.1) Links that contain viruses, malware, RATS, etc.
(a5.2) Files and attachments that can harm other members (.bat , .exe , .dll, file forms)
(a5.3) Other users private data (passwords, addresses, and private photos)
(a6) Illegal/malicious actions
(a6.1) Doxxing (Obtaining users data: Addresses, Social Media, City, etc.)
(a6.2) DDossing (Distributed Denial of Service to any NCBA servers)
(a6.3) Any actions that are an infraction of United States Law
Note: Any threats relating to committing the above are taken very seriously and can result in a Report of your profile and banning from the NCBA Discord.
Other Information
(b1) Keep all suggestions within #🗊-suggestions-bugs
(b1.1) Be serious, clear and descriptive (Give as much information as possible)
(b1.2) Do not send the same post twice (It has already been seen)
Ps: Psykosis did JYST3R's Homework


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Dell PowerEdge 6850 Specs. 4×2.6GHz Intel Xeon CPUs Dual Core 8 Cores totals. 16GB of RAM, 16 Slots, 0 Available. 3x146GB 15K SAS RAID. 4PCIe Slots/3 PCI slots. PERC 5i SAS/SATA RAID Controller. Dual Power Supplies Only work on 200-240V. Scratches and small dent on top lid. The item "Dell PowerEdge 6850 Server 4×2.6GHz DC Xeon CPUs 16GB 3x146GB 15K SAS RAID Perc5″ is in sale since Wednesday, January 28, 2015. This item is in the category "Computers/Tablets & Networking\Enterprise Networking, Servers\Servers, Clients & Terminals\Computer Servers". The seller is "move-it-out" and is located in Jackson, New Jersey. This item can be shipped worldwide.
- Form Factor: Rack-Mountable 4U
- Brand: Dell
- Product Line: PowerEdge
- Model: PE6850
- Processor Type: Xeon Dual Core
- Processor Speed: 2.6GHz
- Number of Processors: 4
- Memory (RAM) Capacity: 16GB
- Processor Manufacturer: Intel
- Type: Server


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Cinderella (Spanish/English)
Stock information
General Fields
Special Fields
Striking art gives this classic fairy tale a fresh look that will enchant a new generation of readers. Retold in both Spanish and English, this universally familiar story is now a springboard to inspire readers to recognize words in both languages. The dual-language text makes this book a useful and beautiful addition to both home and classroom libraries.
Author description
Monse I'ransoy (illustrator) was born in Barcelona and studied illustration at the Escuela Profesional de la Dona in Barcelona and at the L'cole des Arts Decoratifs in Strasbourg, France. She has illustrated many children's books in Spain.


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London-based singer-songwriter Lily Moore is starting out her year with a new video for 'Now I Know', a heartfelt song about missed opportunities. Capturing both the pains and wonder of young adulthood, the clip is a perfect window into an act who's spent years working her way up from busking and tiny pubs to a support slot with George Ezra at the Royal Albert Hall, and, soon, a headliner of her own at London's buzzy Village Underground.
"A lot of the video actually happened when I was growing up," she explains. "I wanted it to tell the story of lots of my real memories and what it's like to really fancy someone. There were so many times we would have to sprint for our lives to catch the last bus and you'd catch yourself grabbing hands with the person you quite liked at the time and you'd end up thinking oh that was quite nice actually."
Give it a watch - and find out more from Lily herself - below.
Hey Lily, how's it going? Having a good 2020 so far?
Hello. Yeah, all good. Been actually quite quiet so far but I know it's gonna be a busy year, so I'm not complaining one bit!!!
Have you made any resolutions for this year?
I'm doing veganuary as I thought it was about time I did something healthy and good for the planet, not just me. Otherwise, I haven't made any big plans apart from being happy.
How did you get into making music, then?
I'm not too sure, really. I fell into it as it has always been around me since I was born. I got a cassette recorder for Christmas one year when I was really little maybe about 5 or 6, and I remember I would record all my little songs on it - so maybe I can say then!
Is being a musician living up to the hype so far?
Absolutely. Apart from touring is definitely far, far less glam then it gets made out to be. In reality, it's a lot of sitting in the back of a van and eating food from garages. But I do love it if I'm honest.
Has there been much of a learning curve, going pro?
I think I've just had to work harder, which is a good thing because I'm not very good at being bored.
Tell us about your new single, 'Now I Know'?
'Now I Know' was one of the first songs I ever wrote. It's a song about someone I fancied at the time and never realised until it was too late. I wrote it with Jon Green, who's one of my favourite people to work with and it was such an easy song to write. Sometimes the best songs are the ones that come easily.
What's your songwriting process like?
It depends really on who I'm working with. Some people are really good at production and playing instruments which I'm not so normally I leave that to someone else, and I can focus on the lyrics and the story I'm trying to tell. I like to have something on my mind or an idea though before writing a song - something I need to work out how I feel about or get off my chest.
What do you most enjoy writing about, generally?
I think just being able to vent whatever I'm feeling and turn it into something other people can listen to and hopefully find something in. The best feeling is when I do a gig, and everyone knows all the words.
How did the video for 'Now I Know' come together? Did you enjoy your time filming?
I wanted the video to be as real as possible, so I got my mate to be in it with me, and all the memories in the video are things that actually happened when we were being idiots and growing up.
Music aside, what do you do for fun?
Just normal things a 21-year-old in London does! My friends are all still at uni so sometimes I go visit them in different cities and have a good little night out. Yeah, normal stuff really. By that, I mean the pub. I'm also obsessed with food, so maybe cooking as well.
What would you most like to achieve during your music career?
A couple of months ago I would've said playing the Royal Albert Hall, but I was a lucky lady and got to support George Ezra there back in September, so I'm not sure now. Maybe the Pyramid Stage at Glastonbury if I had to say the best of the best. But overall I just want to be able to do this forever - that's the main goal.
Who do you think is the most exciting band or musician around right now?
Husky Loops.
Lily Moore's single 'Now I Know' is out now. She tours the UK this May, including a night at London's Village Underground.