Isolation of a Peptidyl Factor, α Substance-IA,Inducing Sexual Agglutinability in Saccharomyces cerevisiae

After a eleven-steps purification, a peptidyl factor named α substance-Ia was isolated in pure form from a culture filtrate of α type cells of the heterothallic Saccharomyces cerevisiae. The substance induced the sexual agglutinability in a haploid cells belonging to the opposite mating type at concentrations from 0.4 to 0.8 ng/ml.     

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.     

Genetic Analysis of Connective Tissue Growth Factor as an Effector of Transforming Growth Factor β Signaling and Cardiac Remodeling

The matricellular secreted protein connective tissue growth factor (CTGF) is upregulated in response to cardiac injury or with transforming growth factor β (TGF-β) stimulation, where it has been suggested to function as a fibrotic effector. Here we generated transgenic mice with inducible heart-specific CTGF overexpression, mice with heart-specific expression of an activated TGF-β mutant protein, mice with heart-specific deletion of Ctgf, and mice in which Ctgf was also deleted from fibroblasts in the heart. Remarkably, neither gain nor loss of CTGF in the heart affected cardiac pathology and propensity toward early lethality due to TGF-β overactivation in the heart. Also, neither heart-specific Ctgf deletion nor CTGF overexpression altered cardiac remodeling and function with aging or after multiple acute stress stimuli. Cardiac fibrosis was also unchanged by modulation of CTGF levels in the heart with aging, pressure overload, agonist infusion, or TGF-β overexpression. However, CTGF mildly altered the overall cardiac response to TGF-β when pressure overload stimulation was applied. CTGF has been proposed to function as a critical TGF-β effector in underlying tissue remodeling and fibrosis throughout the body, although our results suggest that CTGF is of minimal importance and is an unlikely therapeutic vantage point for the heart.     

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.     

Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF·FVIIa complex and by FXa in the ternary TF·FVIIa·FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF·FVIIa-Xa complex normally but were severely impaired in binary TF·FVIIa·PAR2 signaling. The residues identified were located in the model-predicted S2′ pocket of FVIIa, and complementary PAR2 P2′ Leu-38 replacements demonstrated that the P2′ side chain was indeed crucial for PAR2 cleavage by TF·FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF·FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.     

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.     

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.     

The Rate of Human Germline Mutations—Variable Factor of Evolution and Diseases

Russian Journal of Genetics - The emergence of genetic diseases and evolutionary processes are associated with the flow of genetic information from one generation to another, in which genetic...     

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.     

β-aminoisobutyric Acid,l-BAIBA,Is a Muscle-Derived Osteocyte Survival Factor

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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.