Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan-containing coats around normal human mesothelial cells

The composition and permeability of the pericellular coat surrounding normal human mesothelial (NHM) cells have been studied in vitro. NHM cells were grown in the presence of ³H-glucosamine and the amount of label recovered in hyaluronan and chondroitin sulfate was determined after selective enzymatic digestion of the polysaccharides in medium, pericellular, and intracellular pools. For comparison a similar analysis was carried out on mesothelioma cells (Mero-14). Of the labeled polysaccharides in the medium and pericellular pools of NHM cells about 80–90% could be ascribed to hyaluronan and only 3–5% to chondrojtin sulfate. In contrast, Mero-14 synthesized only minute amounts of hyaluronan whereas chondroitin sulfate corresponded to 61% of the total glycosaminoglycans in the culture. The results exclude a structure of the pericellular layer of NHM cells similar to the hyaluronan-proteoglycan aggregates found in cartilage. The permeability of the pericellular layer was tested by the exclusion of polystyrene microspheres and bacteria of diameter 0.1–3.0 μm, as well as erythrocytes of diameter 7 μm. While the erythrocytes were excluded the smaller particles penetrated the coat. By adding 0.5 mg/ml of aggregating cartilage proteoglycan to the medium particles of 0.3 μm or larger were also excluded. Thus exogenous proteoglycans can reinforce the structure of the pericellular layer. © 1995 Wiley-Liss Inc.     

Different Methods Yielded Two-Fold Difference in Compliance with Physical Activity Guidelines on School Days

Introduction

The aim was to compare the average and the days method in exploring the compliance of children with physical activity guidelines and describe their physical activity patterns in different school day segments.

Methods

Physical activity was objectively measured in 472 children aged 6–13 for one school week. Children were compliant when fulfilling PA recommendations 1) on average over all measured days (average method) or 2) on at least four measured days (days method). To explore the difference in moderate to vigorous physical activity (MVPA) minutes between compliant and non-complaint children (using both the average and days method) in various day segments, linear mixed models was used.

Results

Compliance with physical activity guidelines was significantly higher with the average compared to the days method (51.7% and 23.7%, respectively). In segmented-day analysis, compliant children accrued more MVPA minutes in all day segments, especially during after-school. Gender differences appeared only during the in-school segments, where girls spent less time in MVPA (average method: -4.39 min, 95% CI = -5.36,-3.42, days method: -4.45 min, 95%CI = -5.46,-3.44). Older children accrued more MVPA minutes during physical education classes, but less during breaks, compared to younger children.

Conclusions

The used methods yielded remarkably different prevalence estimates for compliance to physical activity recommendations. To ensure comparability between studies, interventions and reports, there is a need for internationally agreed operationalization and assessment methods of physical activity guidelines. As non-compliant children had lower MVPA during all day segments, greater efforts should be made to provide physical activity opportunities both during and after school.     

Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12-37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.     

Chick development in free-living great tits Parus major in relation to calcium availability and egg composition

Evidence from a number of avian studies suggests that limitation of exogenous calcium (Ca) may reduce egg quality and retard nestling growth. However, it is poorly understood whether reduction in chick growth in Ca-poor areas is due to insufficient intake of dietary Ca or caused by maternal Ca limitation mediated through subtle changes in composition of eggs. In this study, we provide new evidence that Ca availability during egg formation may indeed affect egg composition and influence chick development of the great tit Parus major at early developmental stages. Ca-supplemented birds breeding in base-poor pine forests produced eggs with elevated yolk Ca concentration compared with controls, while no such effect of supplementation was detected in case of eggshell thickness. Nestling tarsus length in the first half of the nestling period was positively influenced by both yolk dry mass and yolk Ca concentration. The effect of supplementary Ca did not persist throughout the nestling period; initial effects of egg components disappeared as nestlings aged. We conclude that apparently normal eggs may harbor poor nutrient and mineral conditions for chick growth. Such subtle changes in composition of eggs can depress chick growth, especially in harsh years.     

Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate

The inhibition effect of cellobiose on the initial stage of hydrolysis when cellobiohydrolase Cel 7A and endoglucanases Cel 7B, Cel 5A, and Cel 12A from Trichoderma reesei were acting on bacterial cellulose and amorphous cellulose that were [(3)H]- labeled at the reducing end was quantified. The apparent competitive inhibition constant (K(i)) for Cel 7A on [(3)H]-bacterial cellulose was found to be 1.6 +/- 0.5 mM, 100-fold higher than that for Cel 7A acting on low-molecular-weight model substrates. The hydrolysis of [(3)H]-amorphous cellulose by endoglucanases was even less affected by cellobiose inhibition with apparent K(i) values of 11 +/- 3 mM and 34 +/- 6 mM for Cel 7B and Cel 5A, respectively. Contrary to the case for the other enzymes studied, the release of radioactive label by Cel 12A was stimulated by cellobiose, possibly due to a more pronounced transglycosylating activity. Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme-substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzyme-substrate complex is nonproductive.     

Dual Function of UNC-51-like Kinase 3 (Ulk3) in the Sonic Hedgehog Signaling Pathway

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.     

Hematological health state indices predict local survival in a small passerine bird, the great tit (Parus major)

In birds, it has been shown that reproductive effort may impair parental condition, while the relation of different condition indices to subsequent survival is still poorly understood. In this study, we measured body mass and various hematological condition indices in breeding great tits in relation to local survival. Number and quality of nestlings and the occurrence of second broods, potentially reflecting parents' breeding effort, were also considered in analyses. The great tits, both male and female, that returned the following year had had a higher albumin/globulin ratio, lower plasma globulin concentration, and a lower heterophile/lymphocyte ratio during breeding in the preceding year, compared to those who did not return. Surviving males (but not females) also had had a higher level of circulating lymphocytes, compared to nonsurvivors. There was no correlation between breeding effort and survival. We conclude that better immunological state and lower stress in great tits during breeding were positively related to their survival probability.