Mineo Niwa

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

Accelerated evolution of crotalinae snake venom gland serine proteases

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein‐coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

Involvement of an intercellular adhesion molecule 1-dependent pathway in the pathogenesis of secondary changes after spinal cord injury in rats

Abstract: The intercellular adhesion molecule 1 (ICAM‐1) plays an important role in immune responses by promoting infiltration of neutrophils into tissues; however, its implication in the secondary destructive pathomechanism after the initial mechanical injury to the spinal cord has not been clarified yet. This study was conducted to examine the role of ICAM‐1 in this process after spinal cord injury (SCI) in rats. The expression of ICAM‐1 mRNA was investigated by the reverse transcription‐PCR method and the effect of monoclonal antibody (mAb) to ICAM‐1 on SCI was evaluated by measuring various parameters. ICAM‐1 mRNA expression correlated with the severity of injury and reached its maximum level 6 h after SCI. Intravenous injection of ICAM‐1 mAb (1 mg/kg) 30 min after SCI reduced motor disturbance and enhanced recovery. Moreover, it significantly suppressed myeloperoxidase activity by 43.0% and spinal cord edema by 1.1% in the injured spinal cord tissue. The posttraumatic drop in spinal cord blood flow was also improved. These results suggest that ICAM‐1 is deeply involved in the secondary self‐destructive process after mechanical injury of the spinal cord and should be an effective target for developing a pharmacological treatment for SCI.

Roles of nitric oxide in compression injury of rat spinal cord

Nitric oxide (NO) was measured directly after spinal cord injury (SCI) in rats by an ESR spin-trapping technique using Fe2+ and diethyldithiocarbamate (DETC). The levels of NO and lipid peroxides expressed as thiobarbituric acid reactive substances (TBARS) were increased by SCI in the injured region and the adjacent central region. Pretreatment with 30 mg/kg of NG-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase, accelerated increases of the TBARS level and myeloperoxidase (MPO) activity in the injured tissue and caused deterioration of hind limb motor function after SCI, suggesting that NO formation by constitutive NO synthase (c-NOS) has a protective effect against cellular damage resulting from ischemia-reperfusion after SCI. Though c-NOS mRNA expression was not altered after SCI, inducible NO synthase (i-NOS) mRNA expression increased to a maximum of 24 h after SCI with progress of motor dysfunction. Intravenous injection of L-NAME (0.1 mg/kg) 6, 24, 48, and 72 h after SCI reduced the motor disturbance. These results indicate that NO induced by i-NOS may be neurotoxic in the subacute phase after SCI.

Induction and its spread of apoptosis in rat spinal cord after mechanical trauma

This study was undertaken to examine the spinal cord of rats after trauma for the induction of apoptosis. DNA ladder formation was demonstrated by gel electrophoresis of the DNA obtained from the spinal cord at the site of injury after trauma from 12 h to 8 days and was most significant on Days 3 and 4. As for the longitudinal spread of the injury in the spinal cord, the ladder formation was observed in the two segments adjacent to the site of trauma 3 days after trauma. Histological examination using in situ end labeling also demonstrated the appearance of apoptosis. These results provide the evidence for the induction of apoptosis in the spinal cord after trauma in rats.

Preparation of Peptide Thioesters using Fmoc-Solid-Phase Peptide Synthesis and its Application to the Construction of a Template-Assembled Synthetic Protein (TASP)

Preparation of peptide thioesters was conducted through peptide chain construction with Fmoc-solid-phase peptide synthesis on a 2-chlorotrityl resin followed by coupling with HS-(CH2)2-COOEt and deprotection with 95% aqueous CF3COOH. The peptide thioester corresponding to a transmembrane segment of the calcium channel (S4 in repeat IV) thus obtained was introduced onto a peptide template to give an artificial four-helix-bundle protein.

Effects of exogenous transforming growth factor-β1 on spinal cord injury in rats ☆

This study was undertaken to examine the effect of transforming growth factor-beta 1 (TGF-beta 1) administered into the subarachnoid space after spinal cord injury (SCI) on the increased production of inducible-nitric oxide synthase (i-NOS) in the injured spinal cord in rats. The expression of i-NOS mRNA after SCI was remarkably down-regulated by TGF-beta 1 in vivo. Rats treated with TGF-beta 1 showed a better outcome regarding hindlimb motor dysfunction in the first 5 days after injury compared to the saline-treated rats. However, the final outcome was not better and fibrous scar formation in the injured spinal cord was more evident, which was demonstrated as increased immunoreactivity of fibronectin in the later stage after SCI. These results provide evidence of both positive and negative contributions of TGF-beta 1 to the pathology associated with SCI.

A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli

Abstract We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1–27), (ii) a small subunit of the acylase (positions 28–190), designated as α, (iii) a spacer peptide (positions 191–198), (iv) a large subunit (positions 199–718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1–32) of native acylase is replaced by MFPTT.

Inducible nitric oxide synthase in uterine smooth muscle.

The expression of inducible nitric oxide synthase (iNOS) mRNA in rat uterus upon in vivo stimulation with lipopolysaccharide (LPS) was studied by reverse transcription and polymerase chain reaction. The injection of LPS induced an increase in mRNA levels of a macrophage-type iNOS. In unstimulated rats, low levels of iNOS mRNA was detected in the uterus and lungs, but absent or negligible in the kidneys and liver. NO was produced in the LPS-treated uterus by addition of 1 to 1000 microM L-arginine. The production of NO in uterine tissue that faces the outside of the body may provide a bacteriocidal protective function against microorganisms in physiological condition. However, NO produced in a large amounts by cytokine and LPS may play some pathological reaction during septic shock or infection.

Nucleotide sequence and expression in Escherichia coli of the Cephalosporin acylase gene of a Pseudomonas strain

The gene encoding cephalosporin acylase, which hydrolyzes 7-beta-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA) and glutaric acid, was cloned from a Pseudomonas sp. strain V22 and expressed in Escherichia coli, in a two-cistron system, and the enzyme was purified and characterized. The purified enzyme was composed of two non-identical subunits, their molecular weights were estimated by SDS-PAGE to be 40,000 and 22,000, and had a pI of 4.6. The amino acid sequence of the enzyme, deduced from the nucleotide sequence, showed high similarity (97%) with that of a previously reported acyI-encoded cephalosporin acylase. Cephalosporin acylase also resembles the bacterial gamma-glutamyl transpeptidases (GGTs) with respect to their molecular organization and amino acid sequence, but differs from them with respect to catalytic and immunological properties. Purified enzyme exhibited not only cephalosporin acylase activity, but also GGT activity. The Km values of the enzyme for GL-7ACA and L-gamma-glutamyl-p-nitroanilide were 6.1 and 3.8 mM, respectively. Cephalosporin acylase was not recognized by antibodies prepared against bacterial GGTs.