Partially folded state of the disulfide-reduced form of human serum albumin as an intermediate for reversible denaturation.

The conformation of the fully disulfide-reduced state of human serum albumin was investigated by tryptophan fluorescence spectrum, CD analyses, and size-exclusion chromatography. Both the reduction of the native disulfide-bonded form under nondenaturing conditions and the refolding of the urea-denatured disulfide-reduced form under reduced conditions yielded almost exactly the same disulfide-reduced state with partially folded unique conformation that was clearly distinguished from either the native or fully denatured state. In addition, the interconversion between the urea-denatured reduced form and the partially folded reduced form was reversible with each other; by reoxidation, the partially folded reduced form was converted to the disulfide-bonded form. The conformation of disulfide-reduced serum albumin was highly variable depending on pH and ionic strength conditions. Thus, we concluded that the disulfide-reduced state with partially folded variable conformation is involved in the reversible interconversion between the denatured reduced form and the native disulfide-bonded form of human serum albumin.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

Local formation and degradation of endothelin-1 in guinea pig airway tissues

Endothelin(ET)-1 and big ET-1 caused potent and sustained constriction of isolated guinea pig bronchus. The response to ET-1 was enhanced by phosphoramidon in a simple dose-related manner (0.01-1000 microM), while the response to big ET-1 was enhanced at lower doses (0.01-0.1 microM) but was suppressed at higher doses (100-1000 microM) of phosphoramidon. Big ET-1, when given intravenously (i.v.) to anesthetized guinea pigs, increased both bronchopulmonary inflation pressure and mean arterial blood pressure (2.5, 5, 10 nmol/kg i.v.). The pressor response to big ET-1 was attenuated by phosphoramidon dose-relatedly, while the pulmonary response was modified in a complex fashion composed of delayed onset and prolonged duration of action. These results suggest that ET converting as well as degrading enzymes coexist in the airway tissue and both enzymes are sensitive to phosphoramidon, so that phosphoramidon acts bifunctionally to reduce and stimulate the airway responses to big ET-1.     

Flow cytometric analysis of lipid droplet formation in cells of the human monocytic cell line, U937.

Active uptake of a free fatty acid, oleate, and its incorporation into triacylglycerol and phospholipid in the human monocytic cell line, U937, was identified using 14C-labelled oleate. Excess triacylglycerol accumulates in the form of lipid droplets in the cytoplasm. The extent of lipid droplet formation in each cell can be assessed in the absence of any staining by 90 degrees light scatter intensity, one of the basic parameters of flow cytometry.     

Canopy structure,nitrogen distribution and whole canopy photosynthetic carbon gain in growing and flowering stands of tall herbs

Using a combination of mathematical modeling and field studies we showed that in dense stands of growing herbaceous plants the vertical pattern of leaf nitrogen distribution resembles the pattern of mean light attenuation in the stand and hence tends to maximize total daily photosynthetic carbon gain of the whole stand. Flowering represents a strong sink of nitrogen away from the photosynthetic apparatus and in herbs like Solidago altissima it induces leaf shedding. We studied both the effect of nitrogen reallocation and leaf shedding on the whole canopy photosynthesis and changes in leaf nitrogen distributions in stands moving from the growing to the flowering stage. Despite a decrease in leaf area index and total nitrogen available for photosynthesis in the flowering stand, the leaf nitrogen distribution here also leads to an almost maximum canopy photosynthesis. In both the growing and the flowering stands the leaf area index was higher than calculated optimum values. It is pointed out that this should not necessarily be interpreted as non-adaptive.     

Crystallization of and preliminary crystallographic data for the N-terminal half-molecule of ovotransferrin

The N-terminal half molecule of ovotransferrin has been crystallized from a polyethylene glycol 6000 solution by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with cell dimension of a = 47.0, b = 90.2, and c = 76.2 A. The crystals diffract X-rays to a resolution limit of at least 2.0 A and are resistant to X-ray radiation damage. They appear to be suitable for X-ray structure analysis.     

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.