Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells

Podocalyxin is a major membrane protein of the glomerular epithelium and is thought to be involved in maintenance of the architecture of the foot processes and filtration slits characteristic of this unique epithelium by virtue of its high negative charge. However, until now there has been no direct evidence for podocalyxin's function. Podocalyxin is a type 1 transmembrane sialoprotein with an N-terminal mucin-like domain. To assess its function, we cloned rat podocalyxin and examined the effects of its expression on the cell adhesion properties of stably transfected Chinese hamster ovary (CHO)-K1 and Madin-Darby canine kidney (MDCK) cells and inducible ecdysone receptor-expressing (EcR)-CHO cells. In a cell aggregation assay, CHO-K1 cells expressing high levels of podocalyxin showed complete inhibition of cell aggregation, and MDCK transfectants showed greatly reduced aggregation ( approximately 60-80%) compared with parental cells. In EcR-CHO cells, the expression level of podocalyxin induced by increasing levels of ecdysone analogue correlated closely with the antiadhesion effect. The inhibitory effect of podocalyxin was reversed by treatment of the cells with Arthrobacter ureafaciens sialidase, indicating that sialic acid is required for inhibition of cell adhesion. Overexpression of podocalyxin also affected transepithelial resistance and the distribution of junctional proteins in MDCK cells by an unknown mechanism that may involve interaction with the actin cytoskeleton. These results provide direct evidence that podocalyxin functions as an antiadhesin that maintains an open filtration pathway between neighboring foot processes in the glomerular epithelium by charge repulsion.     

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

Longitudinal assessment of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon responses during the first year of life in HIV-1-infected infants

Human immunodeficiency virus type 1 (HIV-1) infection results in different patterns of viral replication in pediatric compared to adult populations. The role of early HIV-1-specific responses in viral control has not been well defined, because most studies of HIV-1-infected infants have been retrospective or cross-sectional. We evaluated the association between HIV-1-specific gamma interferon (IFN-gamma) release from the cells of infants of 1 to 3 months of age and peak viral loads and mortality in the first year of life among 61 Kenyan HIV-1-infected infants. At 1 month, responses were detected in 7/12 (58%) and 6/21 (29%) of infants infected in utero and peripartum, respectively (P = 0.09), and in approximately 50% of infants thereafter. Peaks of HIV-specific spot-forming units (SFU) increased significantly with age in all infants, from 251/10(6) peripheral blood mononuclear cells (PBMC) at 1 month of age to 501/10(6) PBMC at 12 months of age (P = 0.03), although when limited to infants who survived to 1 year, the increase in peak HIV-specific SFU was no longer significant (P = 0.18). Over the first year of life, infants with IFN-gamma responses at 1 month had peak plasma viral loads, rates of decline of viral load, and mortality risk similar to those of infants who lacked responses at 1 month. The strength and breadth of IFN-gamma responses at 1 month were not significantly associated with viral containment or mortality. These results suggest that, in contrast to HIV-1-infected adults, in whom strong cytotoxic T lymphocyte responses in primary infection are associated with reductions in viremia, HIV-1-infected neonates generate HIV-1-specific CD8+-T-cell responses early in life that are not clearly associated with improved clinical outcomes.     

Variation in the degree of coupling between delta13C of phloem sap and ecosystem respiration in two mature Nothofagus forests

Day-to-day variability in the carbon isotope composition of phloem sap (delta13Chd) and ecosystem respiratory CO2 (delta13CR) were measured to assess the tightness of coupling between canopy photosynthesis (delta13Chd) and ecosystem respiration (delta13CR) in two mature Nothofagus solandri (Hook. f.) forests in New Zealand. Abundant phloem-tapping scale insects allowed repeated, nondestructive access to stem phloem sap 1-2 m above ground. delta13Chd was compared with delta13C predicted by an environmentally driven, process-based canopy photosynthesis model. Keeling plots of within-canopy CO2 were used to estimate delta13CR. By including a lag of 3 d, there was good agreement in the timing and direction of variation in delta13Chd and predictions by the canopy photosynthesis model, suggesting that delta13Chd represents a photosynthesis-weighted, integrative record of canopy photosynthesis and conductance. Significant day-to-day variability in delta13CR was recorded at one of the two forests. At this site, delta13CR reflected variability in delta13Chd only on days with <2 mm rain. We conclude that the degree of coupling between canopy photosynthesis and ecosystem respiration varies between sites, and with environmental conditions at a single site.     

Relative humidity- and ABA-induced variation in carbon and oxygen isotope ratios of cotton leaves

Cotton (Gossypium hirsutum L. cv. CS50) plants were grown at two levels of relative humidity (RH) and sprayed daily with abscisic acid (ABA) at four concentrations. Plants grown at lower humidity had higher transpiration rates, lower leaf temperatures and lower stomatal conductance. Plant biomass was also reduced at low humidity. Within each humidity environment, increasing ABA concentration generally reduced stomatal conductance, evaporation rates, superficial leaf density and plant biomass, and increased leaf temperature and specific leaf area. As expected, decreased stomatal conductance resulted in decreased carbon isotope discrimination in leaf material ( Δ ¹³C_l). Plants grown at low humidity were more enriched in ¹⁸O than those grown at high RH, as theory predicts. Within each humidity environment, increasing ABA concentration increased oxygen isotope enrichment of leaf cellulose ( Δ ¹⁸O_c) and whole‐leaf tissue ( Δ ¹⁸O_l). Values of Δ ¹³C_l and Δ ¹⁸O_l predicted by theoretical models were close to those observed, accounting for 79% of the measured variation in Δ ¹³C_l and 95% of the measured variation in Δ ¹⁸O_l. Supporting theory, Δ ¹³C_l and Δ ¹⁸O_l in whole‐leaf tissue were negatively related.     

A microstructural model for the anisotropic drained stiffness of articular cartilage

A constitutive model for articular cartilage is developed to study directional load sharing within the soft biological tissue. Cartilage is idealized as a composite structure whose static mechanical response is dominated by distortion of a sparse fibrous network and by changes in fixed charge density. These histological features of living cartilage are represented in a microstructural analog of the tissue, linking the directionality of mechanical stiffness to the orientation of microstructure. The discretized 'model tissue' is used to define a stiffness tensor relating drained stress and strain over a regime of large deformation. The primary goal of this work was to develop a methodology permitting more complete treatment of anisotropy in the stiffness of cartilage. The results demonstrate that simple oriented microscopic behaviors can combine to produce complicated larger scale response. For the illustrative example of a homogeneous specimen subjected to confined compression, the model predicts a nonlinear anisotropic drained response, with inherent uncertainty at cellular size scales.