longitudinal-measurement-extracellular-matrix-rigidity-3d-tumor

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

University Of Massachusetts Boston

There is considerable evidence that mouse fertilization requires the binding of sperm to two of the three glycoproteins that form the zona pellucida (ZP), ZP3 and ZP2. Despite the biologic importance of this binding, no one has demonstrated that sperm express separate, saturable, and specific binding sites for ZP3 and for ZP2. Such a demonstration is a prerequisite for defining the distribution, numbers, affinities, and regulation of function of ZP3 and ZP2 binding sites on sperm. The experiments reported herein used fluorochrome-labeled ZP3 and ZP2 and quantitative image analysis to characterize the saturable binding of ZP3 and ZP2 to distinct sites on living, capacitated, acrosome-intact mouse sperm. Approximately 20% of the ZP3 binding sites were found over the acrosomal cap, and the remaining sites were located over the postacrosomal region of the head. In contrast, ZP2 binding sites were detected only over the postacrosomal region. Saturation analysis estimated numbers and affinities of the binding sites for ZP3 (B(max) approximately 185 000 sites per sperm; K(d) approximately 67 nM) and ZP2 (B(max) approximately 500 000 sites per sperm; K(d) approximately 200 nM). Use of unlabeled ZP3, ZP2, and ZP1 as competitive inhibitors of the binding of fluorochrome-labeled ZP3 and ZP2 demonstrated that ZP3 and ZP2 bound specifically to their respective sites on sperm. Finally, we demonstrate that extracellular calcium as well as capacitation and maturation of sperm are required for these sites to bind their respective ligands.

Characterization of zona pellucida glycoprotein 3 (ZP3) and ZP2 binding sites on acrosome-intact mouse sperm.

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.

Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm.

The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le(x); Gal beta 4[Fuc alpha 3]GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888-1895). This glycan is recognized by approximately 70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le(a); Gal beta 3[Fuc alpha 4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770-777). Herein, we test the hypothesis that Le(x)- and Le(a)-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le(x)-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le(a)-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le(x)-containing neoglycoproteins. In contrast, neoglycoproteins containing beta4-lactosamine (Gal beta 4GlcNAc), Lewis B (Fuc alpha 2Gal beta 3[Fuc alpha 4]GlcNAc), and sialyl-Le(x) glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le(x)- and Le(a)-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le(x)- and Le(a)-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G(i) protein. Thus, Le(x)-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le(a)-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.

Lewis X-containing neoglycoproteins mimic the intrinsic ability of zona pellucida glycoprotein ZP3 to induce the acrosome reaction in capacitated mouse sperm.

Sepsis remains an important challenge in pediatric critical care medicine. This review provides an appraisal of adjunctive therapies for sepsis and highlights opportunities for meeting selected challenges in the field. Future clinical studies should address long-term and functional outcomes as well as acute outcomes. Potential adjunctive therapies such as corticosteroids, hemofiltration, hemoadsorption, and plasmapheresis may have important roles, but still require formal and more rigorous testing by way of clinical trials. Finally, the design of future clinical trials should consider novel approaches for stratifying outcome risks as a means of improving the risk-to-benefit ratio of experimental therapies.

Article	Year
Characterization of zona pellucida glycoprotein 3 (ZP3) and ZP2 binding sites on acrosome-intact mouse sperm. Biology of reproduction\| PubMed ID: 12021035	2002
Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm. Biology of reproduction\| PubMed ID: 15128590	2004
Lewis X-containing neoglycoproteins mimic the intrinsic ability of zona pellucida glycoprotein ZP3 to induce the acrosome reaction in capacitated mouse sperm. Biology of reproduction\| PubMed ID: 15128591	2004
Pediatric sepsis: challenges and adjunctive therapies. Critical care clinics\| PubMed ID: 23537672	2013

William Hanna

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