Direct measurements of the nucleosome-forming preferences of periodic DNA motifs challenge established models

Several periodic motifs have been implicated in facilitating the bending of DNA around the histone core of the nucleosome. For example, di-nucleotides AA/TT/TA and GC at ∼10-bp periods, but offset by 5 bp, are found with higher-than-expected occurrences in aligned nucleosomal DNAs in vitro and in vivo. Additionally, regularly oscillating period-10 trinucleotide motifs non-T, A/T, G and their complements have been implicated in the formation of regular nucleosome arrays. The effects of these periodic motifs on nucleosome formation have not been systematically tested directly by competitive reconstitution assays. We show that, in general, none of these period-10 motifs, except TA, in certain sequence contexts, facilitates nucleosome formation. The influence of periodic TAs on nucleosome formation is appreciable; with some of the 200-bp DNAs out-competing bulk nucleosomal DNA by more than 400-fold. Only the nucleotides immediately flanking TA influence its nucleosome-forming ability. Period-10 TA, when flanked by a pair of permissive nucleotides, facilitates DNA bending through compression of the minor groove. The free energy change for nucleosome formation decreases linearly with the number of consecutive TAs, up to eight. We suggest how these data can be reconciled with previous findings.     

Immobilized antibody-based flow type enzyme immunosensor for determination of human serum albumin

A flow-type enzyme immunosensor was prepared for the electrochemical determination of human serum albumin (HSA). The immunosensor was constructed from the immobilized antibody (anti-HSA IgG) reactor and an oxygen electrode. The immunochemical reaction of catalase-labelled antibody with HSA was completed with 30 min. After the immunochemical reaction, hydrogen peroxide solution was injected into the system and a peak current was obtained within 2 min. A linear relationship was observed between the current increase and the logarithm of HSA concentration in the range 10⁻⁸-10⁻⁶ g ml⁻¹. The minimum measurable concentration was 10⁻⁸ g ml⁻¹. The current increase was reproducible with 10% of the relative errors when a sample solution containing 10⁻⁷ g ml⁻¹ of HSA was used. The minimum measurable concentration increased to 10⁻⁹ g ml⁻¹ when hydrogen peroxide was recycled for 5 min in the reactor system. The immobilized antibody reactor could be reused. HSA in human serum was determined by the system proposed.     

Potentiation of thyrotropin releasing hormone-induced inositol phospholipid metabolism and Ca2+ mobilization by cyclic AMP-increasing agents

Thyrotropin releasing hormone (TRH) caused significant breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cells, but vasoactive intestinal peptide (VIP) did not. However, VIP enhanced the TRH-induced hydrolysis of PIP2, the conversion of phosphatidylinositol 4-phosphate (PIP) to PIP2 and the accumulation of phosphatidic acid (PA). On the other hand, the tumor promoter, tetradecanoyl phorbol acetate (TPA), suppressed the TRH-induced hydrolysis of PIP2. In the membrane fraction, the addition of cAMP inhibited the PI kinase activity in a dose-dependent manner, but stimulated the PIP kinase activity. TPA did not affect the PI and PIP kinase activities at all. VIP enhanced the first spike phase of the TRH-induced increase in the intracellular Ca2+ level, while TPA inhibited such Ca2+ mobilization. These results suggested that cAMP-increasing agents enhanced inositol phospholipid metabolism and Ca2+ mobilization induced by TRH in GH3 cells but that TPA inhibited them.     

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.     

Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo.

After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro. To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was increased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylation of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.     

Two types of phosphatidylinositol 3-kinase from bovine thymus. Monomer and heterodimer form

Two types of phosphatidylinositol (PI) 3-kinase (PI3K) have been purified 6250-fold (PI3KI) and 1250-fold (PI3KII) from the cytosol fraction of bovine thymus. Purified PI3KI and PI3KII were found to have apparent molecular masses of 110 and 190 kDa, respectively, by gel filtration. On the other hand, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the molecular mass of PI3KI was again estimated as 110 kDa, PI3KII showed two bands with apparent molecular masses of 110 and 85 kDa, suggesting a heterodimer form. Peptide mapping analysis demonstrated that the 110-kDa protein in PI3KII was the same protein as PI3KI. The specific activity of PI3KI was calculated as 250 nmol/min/mg of protein, while that of PI3KII was 50 nmol/min/mg of protein. The product of PI phosphorylation by PI3KI and PI3KII were confirmed as phosphatidylinositol 3-phosphate by PartiSphere Sax column chromatography. The results show that there are two types of PI 3-kinase in bovine thymus. One exists as a monomer and the other as a heterodimer form. Furthermore, the biochemical properties of these two PI 3-kinases are markedly different. These two types of PI 3-kinase may be regulated differently under physiological conditions.     

Quantitative changes in polyphosphoinositides 1,2-diacylglycerol and inositol 1,4,5-trisphosphate by platelet-derived growth factor and prostaglandin F2 alpha

We developed a novel method to quantify trace amounts of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) using antibodies against PIP and PIP2. With this method, polyphosphoinositides can be measured in the range from 20 to 500 pmol. We applied the method to quantify changes in PIP and PIP2 levels in Balb/c/3T3 cells stimulated by platelet-derived growth factor (PDGF) and prostaglandin F2 alpha (PGF2 alpha), growth factors that stimulate the hydrolysis of PIP and PIP2. PIP2 content decreased rapidly to about 60% of control within 1 min while PIP content decreased gradually but significantly to 60% (PDGF) or 70% (PGF2 alpha) of control. Simultaneously we measured the mass levels of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (DG). Inositol 1,4,5-trisphosphate levels rapidly increased and reached a maximum at 30 s after PDGF or PGF2 alpha stimulation and then decreased to the control level within 2 min. On the other hand, DG formation showed biphasic changes. In the first phase, DG rapidly accumulated and reached a maximum at 30 s after PDGF or PGF2 alpha stimulation and then quickly decreased. In the second phase, DG accumulated gradually, but very markedly, 2 min after PDGF or PGF2 alpha stimulation. Considering the changes in PIP2, DG in the first phase seems to be derived mainly from PIP2 while most of the DG in the second phase derived from other lipids.     

The autoregulation of nodulation mechanism is related to leaf development

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.     

Toxic isolectins from the mushroom Boletus venenatus

Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS–PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galβ1 → 4GlcNAcβ1 → 4Manβ1 → 4GlcNAcβ1 → 4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats.