Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.     

Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria

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Two-State Folding of Lysozyme Versus Multiple-State Folding of α-Lactalbumin Illustrated by the Technique of Disulfide Scrambling

The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.     

Transforming Growth Factor β Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin

Transforming growth factor β (TGF-β) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3′ untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-β/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-β induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-β-dependent P-body formation and promoted growth inhibition by TGF-β. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-β/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-β''s antiproliferative properties and identify TGF-β as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.     

A Non-Native α-Helix Is Formed in the β-Sheet Region of the Molten Globule State of Canine Milk Lysozyme

The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.     

Mutants of SACCHAROMYCES CEREVISIAE Resistant to the α Mating-Type Factor

Mutants that are resistant to α-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified α-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as nonmating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to α-factor by cells of mating-type a is essential for mating.     

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF₁) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF₁. Both the 64-kD protein and the 61-kD CF₁ α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF₁ α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m⁻² s⁻¹) and declined in response to low irradiance (80 μmol photons m⁻² s⁻¹) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF₁ α-subunit found only in monocots. Analysis of purified CF₁ complexes showed that the 64-kD protein represented up to 15% of the total CF₁ α-subunit.     

Crystal Structure of the α Subunit of Human Translation Initiation Factor 2B

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the α subunit of human eIF2B at 2.65 Å resolution. The eIF2Bα structure consists of the N-terminal α-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 Å in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 Å in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Bα. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Bα. Interestingly, the yeast eIF2Bα missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Bα missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The β and δ subunits of eIF2B are homologous to eIF2Bα and may have tertiary structures similar to the present eIF2Bα structure. The sulfate-ion-binding residues of eIF2Bα are well conserved in eIF2Bβ/δ. The abovementioned yeast and human missense mutations of eIF2Bβ/δ were also mapped on the eIF2Bα structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.     

Hemolytic Disease of the Newborn Due to Sensitization to the Blood Factor hr′

The case reports come from the files of the State Department of Public Health which, together with the California Medical Association, now sponsors the statewide studies of all maternal mortalities. Selected Cases are to be presented here from time to time as a matter of interest and illumination to all physicians concerned with the practice of obstetrics. It is hoped that a review of such significant cases will help to improve the welfare of future California mothers.     

Factor analysis of the impact of the environment on microbial communities in the tvärminne area, southern coast of Finland

Data already examined by regression analysis were subjected to factor analysis to scrutinize the effects of environmental factors on microbial populations in the brackish waters of the Tv?rminne archipelago on the southern coast of Finland. Water samples were collected from 1.0-m depth at one point in Tv?rminne Storfj?rd, 71 times over about 2 years. Twenty-six parameters were determined on each sample, 10 of environmental and 16 of microbiological type. The correlations between the parameters were factorized using the principal axis solution, and eight factors chosen for further consideration were rotated by the varimax method. The major part of the variance (about 90% of the total communality) of the microbiological parameters was covered by five factors, interpreted as phytoplankton blooms, the periods before and after the blooms, freshwater outflows, and water temperature. Wind variables were components in the factors interpreted as freshwater outflows. Rainfall played a minor part in the total variance of the microbial community, but it washed yeasts and proteolytic bacteria from the land into the study area. The eight factors selected covered about 60 to 98% of the variance of the microbiological parameters. The highest values (above 90%) were obtained for direct counts of bacteria, for plate counts of mesophilic and polymyxin-resistant bacteria, and for the two community respiration parameters; the lowest values (60 to 75%) were obtained for H(2)S-producing and proteolytic bacteria.     

The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the �� Subunit of Eukaryotic Initiation Factor 2

UV light induces phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2α in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2α phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2α phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2α after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2α phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N^G-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2α phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2α phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.UV irradiation inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α).² Two eIF2α kinases, double strand RNA-dependent protein kinase-like ER kinase (PERK) and general control of amino acid biosynthesis kinase (GCN2), are known to phosphorylate the serine 51 of eIF2α in response to UV irradiation (1–4). However, the one or more upstream pathways that activate eIF2α kinase(s) upon UV irradiation are not known. In this report, we provide evidence that UV-induced nitric-oxide synthase (NOS) activation and nitric oxide (NO^•) production regulate both PERK and GCN2 activation upon UVB irradiation.Expression of inducible nitric-oxide synthase in a mouse macrophage cell line leads to the phosphorylation of eIF2α and inhibition of translation (5). In cultured neuronal and pancreatic cell lines, production of NO^• and peroxynitrite (ONOO⁻) induces endoplasmic reticulum (ER) stress, which activates PERK and results in cell dysfunction and apoptosis (6–9). Cytokine-stimulated inducible nitric-oxide synthase activation in astrocytes depletes l-arginine and activates GCN2, which phosphorylates eIF2α (10). UV irradiation also activates NOS and elevates cellular NO^• (11–13). However, the UV-induced NOS activation and NO^• production have never been shown to be related to the activation of eIF2α kinase(s). Now we demonstrate that UV-induced activation of NOS mediates the activation of both PERK and GCN2, which coordinately regulate the phosphorylation of eIF2α.