Visceral Adipose Tissue Immune Homeostasis Is Regulated by the Crosstalk between Adipocytes and Dendritic Cell Subsets

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ATP- and Gap Junction–dependent Intercellular Calcium Signaling in Osteoblastic Cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP₃)- sensitive intracellular calcium stores. In one model, IP₃ traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P₂ purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y₂ (P_2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P_2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P_2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P_2U purinergic receptors, but not gap junctional communication. ROS/P_2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction–independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P_2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP₃. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP₃ well enough to elicit release of intracellular calcium stores in neighboring cells.     

Extracellular Water and Blood Pressure in Adults with Growth Hormone (GH) Deficiency: A Genotype-Phenotype Association Study

Objectives

Growth hormone deficiency (GHD) in adults is associated with decreased extracellular water volume (ECW). In response to GH replacement therapy (GHRT), ECW increases and blood pressure (BP) reduces or remains unchanged. Our primary aim was to study the association between polymorphisms in genes related to renal tubular function with ECW and BP before and 1 year after GHRT. The ECW measures using bioimpedance analysis (BIA) and bioimpedance spectroscopy (BIS) were validated against a reference method, the sodium bromide dilution method (Br⁻).

Design and Methods

Using a candidate gene approach, fifteen single-nucleotide polymorphisms (SNPs) in nine genes with known impact on renal tubular function (AGT, SCNN1A, SCNN1G, SLC12A1, SLC12A3, KCNJ1, STK39, WNK1 and CASR) were genotyped and analyzed for associations with ECW and BP at baseline and with their changes after 1 year of GHRT in 311 adult GHD patients. ECW was measured with the Br⁻, BIA, and BIS.

Results

Both BIA and BIS measurements demonstrated similar ECW results as the reference method. At baseline, after adjustment for sex and BMI, SNP rs2291340 in the SLC12A1 gene was associated with ECW volume in GHD patients (p = 0.039). None of the SNPs influenced the ECW response to GHRT. One SNP in the SLC12A3 gene (rs11643718; p = 0.024) and three SNPs in the SCNN1G gene [rs5723 (p = 0.02), rs5729 (p = 0.016) and rs13331086 (p = 0.035)] were associated with the inter-individual differences in BP levels at baseline. A polymorphism in the calcium-sensing receptor (CASR) gene (rs1965357) was associated with changes in systolic BP after GHRT (p = 0.036). None of these associations remained statistically significant when corrected for multiple testing.

Conclusion

The BIA and BIS are as accurate as Br⁻ to measure ECW in GHD adults before and during GHRT. Our study provides the first evidence that individual polymorphisms may have clinically relevant effects on ECW and BP in GHD adults.     

Important foliar traits depend on species-grouping: analysis of a remnant temperate forest at the Keerqin Sandy Lands, China

Foliar traits are often interpreted to reflect strategies for coping with water and nutrient supply limitations. In this study, we measured several important leaf traits for 147 species sampled from a remnant, temperate deciduous broad-leaved forest in Keerqin Sandy Lands, Northeast China to test whether these traits are ‘invariant’ or dependent on water supply limitations. Our data show that average specific leaf area (SLA), nitrogen (N) and phosphorus (P) concentrations, leaf C/N, C/P and N/P were 273 cm²?g^?1, 18.1 mg?g^?1, 1.60 mg?g^?1, 28.2, 343 and 12.4, respectively. However, most of these traits were significantly different (P?<?0.05) for different species groupings based on growth forms, phylogenetic history, photosynthetic pathways, or habitats. SLA was positively correlated with leaf P concentration across the broad spectrum of 118 species and most species functional groupings. However, SLA was not correlated with N concentration across all species or within each species functional group. SLA and N and P concentrations in dry habitats were lower than those in wet habitats, whereas leaf C/N, C/P, and N/P had the opposite trend both across all species and within major species functional groupings (herb, monocots and C3 species). Our data indicate that SLA vs. leaf N and SLA vs. P relationships may be regulated differentially for different species functional groupings and that water limitation may have a greater influence than nutrient limitation for plant growth.     

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.     

Inhibitory Activity of the Isoflavone Biochanin A on Intracellular Bacteria of Genus Chlamydia and Initial Development of a Buccal Formulation

Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump inhibitors, but their therapeutic use is limited by poor water-solubility and intense first-pass metabolism. Here, we report on effects of isoflavones against C. pneumoniae and C. trachomatis and describe buccal permeability and initial formulation development for biochanin A. Biochanin A was the most potent Chlamydia growth inhibitor among the studied isoflavones, with an IC₅₀ = 12 µM on C. pneumoniae inclusion counts and 6.5 µM on infectious progeny production, both determined by immunofluorescent staining of infected epithelial cell cultures. Encouraged by the permeation of biochanin A across porcine buccal mucosa without detectable metabolism, oromucosal film formulations were designed and prepared by a solvent casting method. The film formulations showed improved dissolution rate of biochanin A compared to powder or a physical mixture, presumably due to the solubilizing effect of hydrophilic additives and presence of biochanin A in amorphous state. In summary, biochanin A is a potent inhibitor of Chlamydia spp., and the in vitro dissolution results support the use of a buccal formulation to potentially improve its bioavailability in antichlamydial or other pharmaceutical applications.     

Irradiation to the immature brain attenuates neurogenesis and exacerbates subsequent hypoxic‐ischemic brain injury in the adult

Cranial radiotherapy is common in pediatric oncology. Our purpose was to investigate if irradiation (IR) to the immature brain would increase the susceptibility to hypoxic‐ischemic injury in adulthood. The left hemisphere of postnatal day 10 (P10) mice was irradiated with 8 Gy and subjected to hypoxia‐ischemia (HI) on P60. Brain injury, neurogenesis and inflammation were evaluated 30 days after HI. IR alone caused significant hemispheric tissue loss, or lack of growth (2.8 ± 0.42 mm³, p < 0.001). Tissue loss after HI (18.2 ± 5.8 mm³, p < 0.05) was synergistically increased if preceded by IR (32.0 ± 3.5 mm³, p < 0.05). Infarct volume (5.1 ± 1.6 mm³) nearly doubled if HI was preceded by IR (9.8 ± 1.2 mm³, p < 0.05). Pathological scoring revealed that IR aggravated hippocampal, cortical and striatal, but not thalamic, injury. Hippocampal neurogenesis decreased > 50% after IR but was unchanged by HI alone. The number of newly formed microglia was three times higher after IR + HI than after HI alone. In summary, IR to the immature brain produced long‐lasting changes, including decreased hippocampal neurogenesis, subsequently rendering the adult brain more susceptible to HI, resulting in larger infarcts, increased hemispheric tissue loss and more inflammation than in non‐irradiated brains.     

Wind-induced stresses in cherry trees: evidence against the hypothesis of constant stress levels

We calculated the wind-induced bending moments and stresses generated in the stems of five Prunus serotina conspecifics differing in height and canopy shape and size (based on detailed measurements of stem projected area and location with respect to ground level) to test the hypothesis that wind-loads generate uniform and constant stress levels along the lengths of tree twigs, branches, and trunks. These calculations were performed using five different wind speed profiles to evaluate the relative importance of the shape of wind speed profiles versus the ’geometry’ of tree shape on stem stress distributions and magnitudes. Additionally, we evaluated the effect of absolute tree size and stem taper on wind- induced stresses by scaling the size of smaller conspecifics to the absolute height of the largest of the five trees yet retaining the original stem proportions (i.e., diameter relative to stem length) for each plant. Finally, we also determined how the factor of safety for wind-loading (i.e., the quotient of stem yield stress and wind-load stress) changed as a function of tree size (and, presumably, age). Our results indicate that wind-load stress levels (1) vary along stem length even for the same wind speed profile and the same maximum wind speed; (2) would increase to dangerous levels with increasing tree height if it were not for ontogenetic changes in stem taper and canopy shape that reduce stress intensities to manageable levels; (3) tend to be more dependent on stem taper and canopy shape and size than on the shape of the wind speed profile; and (4) the factor of safety against wind-induced mechanical failure decreases as trees get larger, but varies along the length of large trees such that preferential stem failure is likely and functionally adaptive. We thus (1) reject the hypothesis of constant wind-induced stress levels; (2) support the view that size-dependent changes in stem taper are required to maintain wind-load mechanical reliability; and (3) suggest that certain portions of mature trees are ’designed’ to fail under high winds speeds, thereby reducing drag and the bending moments and stresses experienced by trunks. Received: 24 May 1999 / Accepted: 8 October 1999