Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.     

Multipoint mapping studies of six loci on chromosome 11

The six loci, beta-globin (HBBC), parathyroid hormone (PTH), oncogene c-Ha-ras-1 (HRAS1), insulin (INS), calcitonin (CAL) and catalase (CAT) loci, have been mapped to 11p in the order: CAT-CAL-PTH-HBBC-(HRAS1-INS). The purpose of the current study was to examine the linkage relationships, especially the multipoint relationships of these loci in detail. In the 18 families studied, a significant sex difference in recombination was found for the HBBC: HRAS1 linkage with more recombination in the male parent than the female parent (22 vs. 2%). The results of the multipoint analyses provided further evidence for the order CAT-CAL-PTH-HBBC-(HRAS1-INS); however, the order of the last two tightly linked loci is still not clear.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

Flash fluorometer made from off-the-shelf photographic equipment to measure tissue levels of fluorescein

A device to measure fluorescein in tissue has been constructed from standard photographic equipment--an electronic strobe and a flashmeter both covered with interference filters. The instrument works well in the light and need not touch the area being measured, an advantage over existing fluorometers. The instrument has been used to measure the amount of dye in flaps in rats, pigs, and three humans. The results revealed that the amount of dye in a freshly made flap was rarely as much as in normal skin, and skin with less than 20 percent of the dye of control areas usually sloughed, although there were exceptions. In the future the instrument will be improved, and its readings will be compared to those obtained from radioactive microspheres, the present "gold standard" of techniques to measure vascularity. The instrument can be used to estimate the blood supply to any tissue and seems to be as reliable as the dermofluorometers already on the market.     

Chromosome number and DNA content of tobacco cells adapted to NaCl

Cultured tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells were found to have altered DNA contents and chromosome numbers after adaptation to NaCl. Cells adapted to 428 mM NaCl were predominately hexaploid compared to the normal tetraploid 2N(2C)=4X=48 chromosome number of unadapted cells. Enrichment of the cell population for hexaploid cells occurred only after exposure to higher NaCl (428 mM), not lower levels of NaCl (171 mM). The majority of adapted cells remain hexaploid for at least 25 cell generations after removal from NaCl exposure. Adapted cell populations were found to have fewer cells with highly polyploid (2N96) nuclei. Salt tolerance of hexaploid cells was not found to be significantly greater than that of tetraploid cells. Cells with higher ploidy levels were less salt tolerant. It is suggested that high levels of NaCl induce polyploidization and that exposure to NaCl selects against cells with very high ploidy levels.     

Inhibition of glycoprotein oligosaccharide processing in vitro and in influenza-virus-infected cells by alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene.

The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (MMNT) on mannosidases involved in asparagine-linked oligosaccharide processing were investigated. MMNT was found to inhibit the activity of rat liver Golgi alpha-mannosidase I in a concentration-dependent manner (50% inhibition with 0.18 mM-MMNT), whereas rat liver endoplasmic-reticulum alpha-mannosidase appeared to be resistant (less than 5% inhibition at 1 mM-MMNT). Jack-bean alpha-mannosidase was also sensitive to inhibition by MMNT (50% inhibition with 0.32 mM-MMNT). Treatment of influenza-virus-infected chick-embryo cells with 1 mM-MMNT led to a decrease in the formation of complex-type asparagine-linked oligosaccharides and an accumulation of high-mannose-type oligosaccharides with the composition Man8(GlcNAc)2 and Man7(GlcNAc)2 on the viral glycoproteins. The biological activities of influenza-virus haemagglutinin and neuraminidase synthesized in the presence of 1 mM-MMNT remained unchanged, but the virus was less infectious than the control.