Yeast sterol esters and their relationship to the growth of yeast.

Variation in the percentage of sterols esterified to long-chain fatty acids during cellular growth has been examined. Under all conditions, a constant percentage of sterol esters was maintained during exponential growth. This maintenance level was found to vary with different growth conditions. A sharp increase in the rate of esterification was observed upon entry of the culture into the stationary growth phase. The minor cellular sterol components were found to accumulate after this period of rapid sterol ester synthesis, with a relative decrease in the size of the ergosterol pool. Evidence is presented that sterol esters of ergosterol precursors are unable to be metabolized to ergosterol. Once esterified, the fatty acids do not appear to be scavenged during starvation conditions.     

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper­ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.     

Metabolism of high density lipoprotein subfractions.

Over the past few years, new experimental approaches have reinforced the awareness among investigators that the heterogeneity of HDL particles indicates significant differences in production and catabolism of HDL particles. Recent kinetic studies have suggested that small HDL, containing two apolipoprotein A-I molecules per particle, are converted in a unidirectional manner to medium HDL or large HDL, containing three or four apolipoprotein A-I molecules per particle, respectively. Conversion appears to occur in close physical proximity with cells and not while HDL particles circulate in plasma. The medium and large HDL are terminal particles in HDL metabolism with large HDL, and perhaps medium HDL, being catabolized primarily by the liver. These novel kinetic studies of HDL subfraction metabolism are compelling in-vivo data that are consistent with the proposed role of HDL in reverse cholesterol transport.     

Phytochrome A regulates red-light induction of phototropic enhancement in Arabidopsis.

Phytochrome A (phyA) and phytochrome B photoreceptors have distinct roles in the regulation of plant growth and development. Studies using specific photomorphogenic mutants and transgenic plants overexpressing phytochrome have supported an evolving picture in which phyA and phytochrome B are responsive to continuous far-red and red light, respectively. Photomorphogenic mutants of Arabidopsis thaliana that had been selected for their inability to respond to continuous irradiance conditions were tested for their ability to carry out red-light-induced enhancement of phototropism, which is an inductive phytochrome response. We conclude that phyA is the primary photoreceptor regulating this response and provide evidence suggesting that a common regulatory domain in the phyA polypeptide functions for both high-irradiance and inductive phytochrome responses.     

A chloroplast DNA Phylogenetic study of the eastern Asia-eastern North America disjunct section Rytidospermum of Magnolia (Magnoliaceae)

Chloroplast DNA restriction site variation of ten endonucleases was examined among all ten species or varieties of the eastern Asia–eastern North America disjunct section Rytidospermum of Magnolia. Representatives from seven of the other ten sections of Magnolia and four related genera (Liriodendron, Manglietia, Michelia, and Talauma) were also included in the survey. A cladistic analysis of 200 variable sites using Wagner parsimony yielded 11 equally most parsimonious trees with a consistency index of 0.793 and a retention index of 0.870. The section Rytidospermum is polyphyletic in these trees. Magnolia tripetala from the southeastern U.S. is the only American species that has a sister relationship to the Asian taxa, M. hypoleuca, M. officinalis var. officinalis, M. officinalis var. biloba, and M. rostrata. Other American taxa in the section fall into two lineages, with M. macrophylla var. macrophylla, M. macrophylla var. ashei, and M. macrophylla var. dealbata in one, and M. fraseri var. fraseri and M. fraseri var. pyramidata in the other. They are not related to the Asian species as previously believed. The relationships revealed here are in agreement with morphological, allozymic, and cross compatibility data. These results demonstrate that a robust phylogenetic hypothesis is an important prerequisite for understanding biogeographic patterns.     

Spotlight on phytochrome nomenclature

These recommendations for genes encoding phytochromes were developed independently by Quail et al., but are broadly consistent with the Commission's guidelines. Their original article, kindly provided in advance of publication, appeared as a Letter to the Editor inPlant Cell (6:468–471, 1994) and is published with permission of the American Society of Plant Physiologists.     

A rapid and sensitive micro-assay for the enzymatic determination of plasma and lipoprotein cholesterol

A rapid and inexpensive micro-assay for determining cholesterol in plasma and isolated lipoprotein fractions has been established which utilizes a commercially available enzymatic reagent with semi-automated instruments and microtiter plates. The assay is sensitive, precise, and easy to perform. The color development is linear from 0.4 to 20 micrograms cholesterol/well, with sample volumes of 2 to 100 microliters. Inter- and intra-assay variability yielded coefficients of variation (CV) of 2.75% (n = 51) and 1.09% (n = 32), respectively. The concentrations of total plasma and lipoprotein cholesterol (d greater than 1.006 g/ml) obtained with this method were compared with those analyzed in a lipid laboratory standardized to the Centers for Disease Control. The correlation coefficients between the two methods were 0.976 and 0.964, respectively. For total high density lipoprotein (HDL) and the HDL3 subfraction, inter-assay variability was 4.12% and 6.33% (n = 27), respectively; the intra-assay variability was 2.79% and 4.19% (n = 12).     

The discovery of tertiary-amine LXR agonists with potent cholesterol efflux activity in macrophages

The liver X receptors (LXR) play a key role in cholesterol homeostasis and lipid metabolism. SAR studies around tertiary-amine lead molecule 2, an LXR full agonist, revealed that steric and conformational changes to the acetic acid and propanolamine groups produce dramatic effects on agonist efficacy and potency. The new analogs possess good functional activity, demonstrating the ability to upregulate LXR target genes, as well as promote cholesterol efflux in macrophages.     

Characterization of a defect in the pathway for converting 5'-deoxy-5'-methylthioadenosine to methionine in a subline of a cultured heterogeneous human colon carcinoma

5'-Deoxy-5'-methylthioadenosine (methylthioadenosine) is cleaved to adenine and 5-methylthioribose-1-phosphate (methylthioribose-1-P). Methylthioribose-1-P is converted to 2-keto-4-methylthiobutyrate ( ketomethylthiobutyrate ) which is transaminated to methionine. We report that one subline of a heterogeneous human colon carcinoma, DLD-1 Clone D, only forms methylthioribose-1-P from methylthioadenosine or 5'-deoxy-5'-methylthioinosine (methylthioinosine), a deaminated derivative of methylthioadenosine, whereas Clone A converts methylthioadenosine and methylthioinosine to methionine, as shown by growth studies in culture of Clone A and Clone D cells and radioactive studies utilizing [methyl-14C]methylthioadenosine or [methyl-14C]methylthioinosine in the presence of extracts of these cells lines. To characterize this defect, we utilized three protein fractions isolated from rat liver which together convert methylthioribose-1-P to ketomethylthiobutyrate . Addition of only Fraction A to Clone D sonicates restores its ability to convert methylthioadenosine to methionine. This fraction is responsible for converting methylthioribose-1-P to 5- methylthioribulose -1-phosphate; radioactive studies confirm this observation. Thus, Clone D is deficient in an enzyme contained in Fraction A; this represents a qualitative biochemical difference between the two clones derived from a single human tumor.