An Attempt to Estimate the Charge Distribution in Sigma-Bond Systems

A classical inductive-effect model has been utilized in an attempt to estimate the change distribution in relatively complicated σ-bond systems. It is assumed that the formal charge on an atom is given as a vectorial sum of the polarities of the bonds attached to that atom and, further, that each bond induces a constant fraction of its own polarity in all of its neighboring bonds. Significance of the model has been tested for several model compounds which permit the closed-form solutions for the distributions of the internally self-consistent bond polarities. The model has been applied to the calculations of the charge distributions in alkanes, ｫ-alkyl halides and hydrated metallic ions ; the results are found to be compatible with observations

Germline mutagenesis mediated by Sleeping Beauty transposon system in mice

Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice

Activation of cAMP-dependent Protein Kinase in Epidermis by the Compounds which Increase Epidermal cAMP

Pig epidermal slices were incubated with various compounds which increased epidermal cAMP (adenosine 3',5'-monophosphate), and the change in cAMP-dependent protein kinase activity ratio was studied by the method of Cherrington et al (J Biol Chem 251:5209–5218, 1976) with modification.Epinephrine (5 × 10−5 m), histamine (10−4 m) and adenosine (10−3 m), potent agonists of epidermal adenyl cyclase, fully activated the protein kinase (PK) during an incubation of 30 to 45 seconds, that was much shorter than that required for maximal cAMP accumulation under the same conditions (5 min). With such a brief stimulus, the epidermal cAMP-PK system did not become refractory and responded to repeated stimuli. Prostaglandin E2 (PGE2) and isobuthylmethylxanthine (IBMX) and ethanol only partially activated the enzyme. Prostaglandin F2α. (PGF2α) and theophylline which were much less effective in increasing epidermal cAMP, activated the enzyme to the same extent as PGE2 and IBMX respectively.These results suggest that protein kinase activation takes place in response to a cAMP increase in small locus of the cell. Such an increase in cAMP can be very small or even not measurable when measured as total cAMP in the tissue homogenate. Also, increases above this level may not be physiologic.It is concluded that measurement of cAMP-dependent protein kinase activity ratio is a more direct and more sensitive way to study the effect of compounds which act through cAMP mediated mechanism

Phosphorylation of Pig Epidermal Soluble Protein by Endogenous cAMP-Dependent Protein Kinase

The distribution of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase and its substrate proteins was analyzed using soluble and particulate fractions of pig epidermal homogenates. When histone was used as a substrate for this enzyme reaction, protein kinase activity was distributed almost equally between the soluble and particulate fractions. However, the effect of exogenously added cAMP was confined almost exclusively to the soluble enzyme. Endogenous protein phosphorylation in the absence of exogenous histone was higher in the particulate fraction than in the soluble fraction, but the stimulating effect of cAMP was observed only in the soluble fraction. These results indicate that cAMP-dependent protein kinase is predominantly localized in the soluble fraction and phosphorylates soluble epidermal proteins. The particulate fraction contains protein kinase which is cAMP-independent and phosphorylates particulate-bound proteins as well as histone. Based on these observations, the soluble fraction was incubated with [γ-32P]-ATP in the presence or absence of cAMP, and phosphorylated protein was analyzed by SDS disc- or slab-gel electrophoresis followed by autoradiography. Among many proteins whose phosphorylation was slightly increased by cAMP, a protein with Mr ∼45,000 was found which was markedly phosphorylated in the presence of cAMP. Although this protein corresponds to one of the richest proteins in the epidermal soluble fraction, an important physiologic role for this phosphorylation has not been clarified

Cyclic AMP-Dependent Protein Kinase Isozymes of Pig Skin and Human Skin from Normal and Psoriatic Subjects

Cyclic AMP-dependent protein kinase isozymes of pig and human skin (epidermis) were separated by DEAE- cellulose column chromatography after micromodification for small biopsy samples. Clear-cut separations of type I and type II isozymes, winch were of about equal amounts, could be obtained only when the ischemia effect was avoided by in vivo freezing of skin and homogenization for less than 10 s. Intradermal injections of epinephrine caused dose-dependent activation of type I isozyme, but not of type 11. Injections of other skin adenylate cyclase stimulators such as histamine, adenosine, and prostaglandin E2 elevated the local cyclic AMP levels to not more than 5 pmol/mg protein and also stimulated only the type I isozyme. Incubation of keratome-sliced pig skin under various conditions caused both activation by dissociation and inactivation by dissociation of the subunits, which appeared to be dependent on the cyclic AMP content. Epinephrine added to the incubation medium led to complete activation of both type I and type II isozymes (the intraepidermal cyclic AMP contents ranged from 20–50 pmol/mg protein). The isozymes of normal skin and involved skin of psoriatics showed identical peaks of type I and type II Isozymes of equal amounts. The data indicate that protein kinase in the involved skin is not in an activated (by cyclic AMP) state

Identification of a Cytokine-induced Antiapoptotic Molecule Anamorsin Essential for Definitive Hematopoiesis

Many growth factors and cytokines prevent apoptosis. Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. The expression of Anamorsin was completely dependent on stimulation with growth factors such as interleukin 3, stem cell factor, and thrombopoietin in factor-dependent hematopoietic cell lines, and forced expression of Anamorsin conferred resistance to apoptosis caused by growth factor deprivation in vitro. Furthermore, Anamorsin was found to act as an antiapoptotic molecule in vivo because Anamorsin−/− mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (FL). Although the number of hematopoietic stem/progenitor cells in the FL did not decrease in these mice, myeloid, and particularly erythroid colony formation in response to cytokines, was severely disrupted. Also, Anamorsin−/− erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the FL of Anamorsin−/− mice. Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines

Raf-1 regulates Rho signaling and cell migration

Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1–deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-α to the plasma membrane. Raf-1 physically associates with Rok-α in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration

後筋筋電図法

The posterior cricoarytenoid muscle (PCA) is the major laryngeal vocal cord abductor, and electromyography (EMG) of this muscle plays an important role in investigating the mechanism of speech and respiration. However, the EMG study of this muscle has been limited, because it's location makes it difficult to record a signal from the muscle. Different PCA recording techniques have been developed. The approach to the muscle developed along three main lines: per oral, percutaneous and per nasal approach. Three kinds of electrodes; a bipolar needle electrode, a surface electrode and a hooked wire electrode have been used for the recording. Techniques of electrode placement in the PCA are reviewed