Shinichiro Hori

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

Abstract: : The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two‐site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3–9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3–6 h after operation, and even unilateral re moval of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi‐than iN the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did re moval of the glands. These results suggest that the endoge nous NGF that accumulates in the SCG is mostly synthe sized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.

Detection of dystrophin on two-dimensional gel electrophoresis.

A protein with MW approximately 350 k daltons and pI approximately 5.5, which was deleted in the dystrophic mouse (C57BL/10ScSn-mdx), was detected on two-dimensional gel electrophoresis with silver staining. Deletion of this protein was uniformly observed in the dystrophic mouse extensor digitus longus, soleus and cardiac muscle. This protein specifically reacted with the monoclonal antibody against the chemically synthesized N-terminal fragment of human dystrophin. The protein reacting with this monoclonal antibody was also detected in rabbit back-muscle, rat extensor digitus longus and human skeletal muscle at the same position as the mouse muscle protein, on the two-dimensional gel electrophoresis. Our results show that dystrophin is solubilized in 8M guanidine HCl and that the modified two-dimensional gel electrophoresis can be applied to separate dystrophin.

Ca2+ enrichment in culture medium potentiates effect of oligonucleotides

Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca2+ enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ∼100 nm in size are found in Ca2+-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory.

A case of Duchenne muscular dystrophy with truncated dystrophin : significance of a cysteine-rich domain for functional expression of dystrophin protein

A Duchenne muscular dystrophy case showed truncated dystrophin (320 kDa) with an isoelectric point slightly shifted towards a more alkaline pH. From the polymerase chain reaction and immunochemical analysis data, the expressed dystrophin protein was predicted to lack the portion comprising the tail of the rod-like domain, the cysteine-rich domain, and the head of the C-terminal domain. These results indicated the functional importance of the cysteine-rich domain in the dystrophin protein.

Multiplicity of abnormal dystrophin in Becker muscular dystrophy: A Becker muscular dystrophy gene frequently produced two smaller sizes of dystrophin

Dystrophin is a muscle cytoskeletal protein with a molecular mass (MM) of approximately 420 kDa and an isoelectric point (pI) of approximately 5.5, which is abnormal in size and/or abundance in Becker muscular dystrophy (BMD). We investigated the abnormality of dystrophin molecule in muscles biopsied from 23 BMD patients using the two-dimensional gel electrophoresis (TDGE). We found 7 protein spots which reacted specifically with the monoclonal anti-dystrophin antibody (mAb) A1C raised against N-terminal domain of the normal dystrophin. These spots were focused on the two-dimensional gel at the same position as the normal dystrophin (#1), at the position with MM approximately 480 kDa/pI approximately 5.35 (#2), the position with MM approximately 400-330 kDa/pI approximately 5.51-5.47 (#3), the position with MM approximately 300 kDa/pI approximately 5.4 (#4), the position with MM approximately 235-250 kDa/pI approximately 5.53-5.5 (#5), the position with MM approximately 165 kDa/pI approximately 6.0 (#6), and the position with MM approximately 160 kDa/pI approximately 5.75 (#7). These spots were classified into five patterns in individuals, that is, #1 alone in 3 patients, #3 alone in 1, the combination of #3 and 5 in 17, the combination of #1, 3 and 5 in 1 and the combination of #1, 2, 4, 6 and 7 in 1. The combination of #3 and 5 was observed in 17 of 23 patients (75%).(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of high-molecular mass RNAs by high-performance liquid chromatography on hydroxyapatite.

High-molecular-mass RNAs [transfer-(t-), 5S-, 18S- and 28S-RNA] in 25 mM sodium acetate buffer (pH 6.0) were separated by high-performance liquid chromatography (HPLC) on hydroxyapatite using a linear gradient (120 min-duration) from 0.03 to 0.147 M of phosphate buffer (pH 7.0) containing 0.3 M potassium chloride and 1 mM sodium azide with a slope of 2 mM/ml at a flow-rate of 0.5 ml/min. When the RNAs were dissolved in 4 M guanidine isothiocyanate-25 mM sodium acetate buffer (pH 6.0)-0.1 M beta-mercaptoethanol (4 M GIT), t-, 5S- and 18S- or 28S-RNAs but not 18S- and 28S-RNAs were separated. RNAs extracted from rat superior cervical ganglia with 4 M GIT could be separated. Thus, HPLC on hydroxyapatite is a rapid and accurate means of quantifying and/or preparing high-molecular-mass RNAs such as t- and ribosomal RNAs.

Equilibrium of octadecylsilica gel with sodium dodecyl sulphate

Abstract The retention times of tyrosine (Tyr), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylethylamine (DA), norepinephrine (NE), epinephrine (E), homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxymandelic acid (VMA), tryptophan (Trp), 5-hydroxytryptophan (5-HTP), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) on octadecylsilica (ODS) gel gradually changed when the ODS gel column was washed with buffer containing sodium dodecylsulphate (0.003%). To reach the steady state, 3200 column volumes of buffer were required. The retention properties of this column correlated with the hydrophobicity of the retained materials, including Tyr, DOPA, DA, NE, E, HVA, DOPAC, VMA, Trp, 5-HTP, 5-HT and 5-HIAA. These observations are of practical importance in preparing reproducible ODS gel columns for the determination of trace amounts of materials in biological fluids.

Kinetic Properties of Bovine Pineal Tryptophan‐5‐Monooxygenase Activated by an Endogenous Activating Substance

Abstract: We previously reported that an endogenous activating substance different from bovine serum albumin, phospholipids and heparin, exists in the extract from bovine pineal glands and that this substance interacts with tryptophan‐5‐monooxygenase under reducing conditions with sulfhydryl reagents, to stimulate monooxygenase activity. The present paper reports that the activating substance is of peptide nature; that it is sensitive to trypsin‐digestion; and that it does not change the apparent Kms for substrates, L‐tryptophan and oxygen, and coenzyme, reduced biopterin or DMPH4: but that it increases the Vmax 1.5‐ to 2.3‐fold. These results suggest that an activating protein, present in some particles of the cell structure, activates tryptophan‐5‐monooxygenase under the regulation of a sulfhydryl compound. The apparent Kms for reduced biopterin and DMPH4 were 77.2μM and 294 μM, respectively. The apparent Kms for L‐tryptophan and oxygen with reduced biopterin were 15.0 μM and 4.7%, respectively: with DMPH4, they were 11.0 μM and 8.5%, respectively. Significant inhibition of both L‐tryptophan and oxygen was observed with reduced biopterin, but not with DMPH4 (at the tested concentrations of up to 0.5 MM and 20%, respectively).