Many interactions that occur among leukocytes, platelets, and endothelial cells in the vasculature have been shown to be mediated by the selectin family of cell adhesion molecules.

The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin MATERIALS AND METHODSMaterials. The sources of most of the materials used are noted in the individual methods sections or in the figure legends. All other chemicals used were of reagent grade and/or the highest quality available.SELEX for Isolation of L-Selectin Ligands. L-selectin receptor globulin (LS-Rg) was prepared as described (31),

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe x ) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe x in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics. ( J. Clin. Invest. 1996. 98:2688-2692.)

The selectins are adhesion receptors that play key roles in leukocyte trafficking. Each has an N-terminal C-type lectin domain that binds to specific carbohydrates in a calcium-dependent manner. L-selectin recognizes sulfated, sialylated ligands on lymph node high endothelial venules. This recognition is abolished by strong periodate oxidation under conditions that destroy oligosaccharides. In contrast, mild periodate oxidation, which selectively oxidizes the side chain of sialic acid residues without affecting the underlying oligosaccharide, markedly enhances this interaction. The enhancement is calcium dependent, indicating that lectin recognition is maintained. Reduction of the sialic acid aldehydes generated by mild periodate to alcohol groups abolishes this effect. Covalent cross-linking of the oxidized ligand to L-selectin can be demonstrated, suggesting Schiff base formation between lysine residues of the selectin and the newly formed aldehydes. Such selectively oxidized sialylated ligands could be used to probe the lectin domains of the selectins and to identify lysine residues near the binding site. Also, this approach could be used to design drugs for disrupting leukocyte-endothelial interactions leading to pathological inflammation.The selectins are a family of adhesion receptors involved in leukocyte migration into lymphoid tissues and areas of inflammation (1-7). Each selectin has an N-terminal C-type lectin domain that binds specific carbohydrate ligands in a calcium-dependent manner. These domains also contain 10-14 lysine residues (8), which may be involved in ligand binding. Naturally occurring selectin ligands contain sialylated fucosylated lactosamine-type oligosaccharides (9)(10)(11). Sulfation of these and/or other oligosaccharides may also be involved in binding (7,9,(12)(13)(14)(15). Two specific ligands have been more precisely identified. The L-selectin binds certain sulfated and sialylated glycoproteins from lymph node high endothelial venules (HEVs) (13, 16), and P-selectin binds a minor 120-kDa neutrophil sialoglycoprotein (17). In both instances, sialic acids are absolutely required for binding.Periodate oxidation has been used to destroy carbohydrates and to demonstrate biological functions, including lymphocyte-endothelium recognition (18). Such studies used strong periodate oxidation (e.g., 50 mM; pH 5.0), which causes cleavage between cis-diols throughout oligosaccharides and destroys them. In contrast, mild periodate oxidation (e.g., 1-2 mM; pH 7.0) selectively oxidizes only the side chain of terminal sialic acids (19)(20)(21)(22), leaving the rest of the oligosaccharide intact. Conditions can be adjusted to selectively eliminate only the C9 carbon or both the C8 and C9 carbons (Fig. 1, structures B and C). The resulting aldehyde can be reduced, leaving a truncated side chain with a primary alcohol group (Fig. 1, structures D and G). These reactions have been used to explore the importance of the sialic acid side chains in interactions involving viral hemagglutinins, lec...

ObjectivesHepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization.MethodsWe developed a pilot PT together with the Dutch EQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM.ResultsThe pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07 ± 0.24 nmol/L.ConclusionsWe established a framework for future PT to enable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.

These authors contributed equally to this work. ‡ These authors share senior authorship. SUMMARYAuxin polar transport mediated by a group of Pin-formed (PIN) transporters plays important roles in plant root development. However, the mechanism underlying the PIN expression and targeting in response to different developmental and environmental stimuli is still not fully understood. Here, we report a previously uncharacterized gene SSR1, which encodes a mitochondrial protein with tetratricopeptide repeat (TPR) domains, and show its function in root development in Arabidopsis thaliana. In ssr1-2, a SSR1 knock-out mutant, the primary root growth was dramatically inhibited due to severely impaired cell proliferation and cell elongation. Significantly lowered level of auxin was found in ssr1-2 roots by auxin measurement and was further supported by reduced expression of DR5-driven reporter gene. As a result, the maintenance of the root stem cell niche is compromised in ssr1-2. It is further revealed that the expression level of several PIN proteins, namely, PIN1, PIN2, PIN3, PIN4 and PIN7, were markedly reduced in ssr1-2 roots. In particular, we showed that the reduced protein level of PIN2 on cell membrane in ssr1-2 is due to impaired retrograde trafficking, possibly resulting from a defect in retromer sorting system, which destines PIN2 for degradation in vacuoles. In conclusion, our results indicated that SSR1 is functioning in root development in Arabidopsis, possibly by affecting PIN protein expression and subcellular targeting.

Summary In Arabidopsis , the initiation and proliferation of stomatal lineage cells is controlled by SPEECHLESS ( SPCH ). Phosphorylation of SPCH at the post‐translational level has been reported to regulate stomatal development. Here we report that IDD 16 acts as a negative regulator for stomatal initiation by directly regulating SPCH transcription. In Arabidopsis , IDD 16 overexpression decreased abaxial stomatal density in a dose‐dependent manner. Time course analysis revealed that the initiation of stomatal precursor cells in the IDD 16 ‐ OE plants was severely inhibited. Consistent with these findings, the transcription of SPCH was greatly repressed in the IDD 16 ‐ OE plants. In contrast, IDD 16‐ RNA i transgenic line resulted in enhanced stomatal density, suggesting that IDD 16 is an intrinsic regulator of stomatal development. Ch IP analysis indicated that IDD 16 could directly bind to the SPCH promoter. Furthermore, Arabidopsis plants overexpressing IDD 16 exhibited significantly increased drought tolerance and higher integrated water use efficiency ( WUE ) due to reduction in leaf transpiration. Collectively, our results established that IDD 16 negatively regulates stomatal initiation via trans‐repression of SPCH , and thus provide a practical tool for increasing plant WUE through the manipulation of IDD 16 expression.