Selective isolation of hyaluronan by solid phase adsorption to silica.

A solid phase adsorption method for selective isolation of hyaluronan (HA) from biological samples is presented. Following enzymatic degradation of protein, HA can be separated from sulfated glycosaminoglycans, other unsulfated glycosaminoglycans, nucleic acids, and proteolytic fragments by adsorption to amorphous silica at specific salt concentrations. The adsorbed HA can be released from silica using neutral and basic aqueous solutions. HA ranging in size from ∼9 kDa to MDa polymers has been purified by this method from human serum and conditioned medium of cultured cells.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies. During cytokinesis in somatic cells, the midbody and subsequent assembly of the midbody remnant, a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the midbody and midbody remnant in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic midbodies (mMB) and mMB remnants using mouse oocytes and demonstrate that mMBs have a specialized cap structure that is orientated toward polar bodies. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies (PB). During cytokinesis in somatic cells, the midbody (MB) and subsequent assembly of the midbody remnant (MBR), a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the MB and MBR in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic MBs (mMB) and mMB remnants (mMBRs) using mouse oocytes and demonstrate that mMBs have a specialized meiotic mMB cap structure that is orientated toward PBs. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

Distinct Aurora B pools at the inner centromere and kinetochore have different contributions to meiotic and mitotic chromosome segregation.

Proper chromosome segregation depends on establishment of bioriented kinetochore-microtubule attachments, which often requires multiple rounds of release and reattachment. Aurora B and C kinases phosphorylate kinetochore proteins to release tensionless attachments. Multiple pathways recruit Aurora B/C to the centromere and kinetochore. We studied how these pathways contribute to anaphase onset timing and correction of kinetochore-microtubule attachments in budding yeast meiosis and mitosis. We find that the pool localized by the Bub1/Bub3 pathway sets the normal duration of meiosis and mitosis, in differing ways. Our meiosis data suggests that disruption of this pathway leads to PP1 kinetochore localization, which dephosphorylates Cdc20 for premature anaphase onset. For error correction, the Bub1/Bub3 and COMA pathways are individually important in meiosis but compensatory in mitosis. Finally, we find that the haspin and Bub1/3 pathways function together to ensure error correction in mouse oogenesis. Our results suggest that each recruitment pathway localizes spatially distinct kinetochore-localized Aurora B/C pools that function differently between meiosis and mitosis.

Distinct Aurora B pools at the inner centromere and kinetochore have different contributions to meiotic and mitotic chromosome segregation.

Proper chromosome segregation depends on the establishment of bioriented kinetochore-microtubule attachments, which often requires multiple rounds of release and reattachment. Aurora B and C kinases phosphorylate kinetochore proteins to release tensionless attachments. Multiple pathways recruit Aurora B/C to the centromere and kinetochore. We studied how these pathways contribute to anaphase onset timing and correction of kinetochore-microtubule attachments in budding yeast meiosis and mitosis. We find that the pool localized by the Bub1/Bub3 pathway sets the normal duration of meiosis and mitosis, in differing ways. Our meiosis data suggests a model that disruption of this pathway leads to PP1 kinetochore localization, which dephosphorylates Cdc20 for premature anaphase onset. For error correction, the Bub1/Bub3 and COMA pathways are individually important in meiosis but compensatory in mitosis. Finally, we find that the haspin and Bub1/3 pathways function together to ensure error correction in mouse oogenesis. Our results suggest that each recruitment pathway localizes spatially distinct kinetochore-localized Aurora B/C pools that function differently between meiosis and mitosis.

Role of Hyaluronan in Inflammatory Effects on Human Articular Chondrocytes.

Hyaluronan (HA) fragments have been proposed to elicit defensive or pro-inflammatory responses in many cell types. For articular chondrocytes in an inflammatory environment, studies have failed to reach consensus on the endogenous production or effects of added HA fragments. The present study was undertaken to resolve this discrepancy. Cultured primary human articular chondrocytes were exposed to the inflammatory cytokine IL-1ß, and then tested for changes in HA content/size in conditioned medium, and for the expression of genes important in HA binding/signaling or metabolism, and in other catabolic/anabolic responses. Changes in gene expression caused by enzymatic degradation of endogenous HA, or addition of exogenous HA fragments, were examined. IL-1ß increased the mRNA levels for HA synthases HAS2/HAS3 and for the HA-binding proteins CD44 and TSG-6. mRNA levels for TLR4 and RHAMM were very low and were little affected by IL-1ß. mRNA levels for catabolic markers were increased, while type II collagen (α1(II)) and aggrecan were decreased. HA concentration in the conditioned medium was increased, but the HA was not degraded. Treatment with recombinant hyaluronidase or addition of low endotoxin HA fragments did not elicit pro-inflammatory responses. Our findings showed that HA fragments were not produced by IL-1ß-stimulated human articular chondrocytes in the absence of other sources of reactive oxygen or nitrogen species, and that exogenous HA fragments from oligosaccharides up to about 40 kDa in molecular mass were not pro-inflammatory agents for human articular chondrocytes, probably due to low expression of TLR4 and RHAMM in these cells.