Gamma Knife Irradiation of Injured Sciatic Nerve Induces Histological and Behavioral Improvement in the Rat Neuropathic Pain Model

We examined the effects of gamma knife (GK) irradiation on injured nerves using a rat partial sciatic nerve ligation (PSL) model. GK irradiation was performed at one week after ligation and nerve preparations were made three weeks after ligation. GK irradiation is known to induce immune responses such as glial cell activation in the central nervous system. Thus, we determined the effects of GK irradiation on macrophages using immunoblot and histochemical analyses. Expression of Iba-1 protein, a macrophage marker, was further increased in GK-treated injured nerves as compared with non-irradiated injured nerves. Immunohistochemical study of Iba-1 in GK-irradiated injured sciatic nerves demonstrated Iba-1 positive macrophage accumulation to be enhanced in areas distal to the ligation point. In the same area, myelin debris was also more efficiently removed by GK-irradiation. Myelin debris clearance by macrophages is thought to contribute to a permissive environment for axon growth. In the immunoblot study, GK irradiation significantly increased expressions of βIII-tubulin protein and myelin protein zero, which are markers of axon regeneration and re-myelination, respectively. Toluidine blue staining revealed the re-myelinated fiber diameter to be larger at proximal sites and that the re-myelinated fiber number was increased at distal sites in GK-irradiated injured nerves as compared with non-irradiated injured nerves. These results suggest that GK irradiation of injured nerves facilitates regeneration and re-myelination. In a behavior study, early alleviation of allodynia was observed with GK irradiation in PSL rats. When GK-induced alleviation of allodynia was initially detected, the expression of glial cell line-derived neurotrophic factor (GDNF), a potent analgesic factor, was significantly increased by GK irradiation. These results suggested that GK irradiation alleviates allodynia via increased GDNF. This study provides novel evidence that GK irradiation of injured peripheral nerves may have beneficial effects.     

Partial Purification and Some Properties of a Benzhydrylamide-bond Hydrolyzing Enzyme in Pseudomonas diminuta

An enzyme in Pseudomonas diminuta showed hydrolyzing activity of a benzhydrylamide ( = diphenylmethylamide) bond in S-benzylcysteinylglycine benzhydrylamide. The enzyme was purified 225-fold by precipitation with ammonium sulfate, and column chromatography with ECTEOLA-cellulose, DEAE-cellulose and hydroxyapatite. It showed an optimum pH of 6 to 8 and it was markedly inhibited by Hg^{2 +} or p-chloromercuribenzoate. The preparation was more specific against S-benzylcysteinylglycine benzhydrylamide than other substrates tested.     

A Computational Model of Afterimage Rotation in the Peripheral Drift Illusion Based on Retinal ON/OFF Responses

Human observers perceive illusory rotations after the disappearance of circularly repeating patches containing dark-to-light luminance. This afterimage rotation is a very powerful phenomenon, but little is known about the mechanisms underlying it. Here, we use a computational model to show that the afterimage rotation can be explained by a combination of fast light adaptation and the physiological architecture of the early visual system, consisting of ON- and OFF-type visual pathways. In this retinal ON/OFF model, the afterimage rotation appeared as a rotation of focus lines of retinal ON/OFF responses. Focus lines rotated clockwise on a light background, but counterclockwise on a dark background. These findings were consistent with the results of psychophysical experiments, which were also performed by us. Additionally, the velocity of the afterimage rotation was comparable with that observed in our psychophysical experiments. These results suggest that the early visual system (including the retina) is responsible for the generation of the afterimage rotation, and that this illusory rotation may be systematically misinterpreted by our high-level visual system.     

Adult onset globoid cell leukodystrophy (Krabbe disease): analysis of galactosylceramidase cDNA from four Japanese patients

We examined galactosylceramidase (GALC) cDNA in four Japanese patients with adult onset globoid cell leukodystrophy (Krabbe disease; AO-GLD) by polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis, subsequent sequence determination, and restriction enzyme digestion of PCR products. Initial symptoms were the onset of slowly progressive spastic paraplegia from the middle of the second decade, and all patients had diminished GALC activity in their leukocytes. We identified three missense mutations (I66M, G270D, L618S) and one exon-6 skipping (535– 573del). Two of the patients had only the I66M mutant mRNA, and one only the G270D mutant mRNA. The fourth patient carried a compound heterozygous mutation of 535–573del and L618S. To determine the enzymatic activities produced by these mutations, we constructed mutated GALC cDNAs and expressed them in COS-1 cells. Three mutations, viz., G270D, L618S, and exon-6 skipping (535–573del), produced diminished GALC activity as expected. The I66M mutation in the wild-type GALC cDNA(I289) had normal activity, but when this mutation and the V289 polymorphism were introduced into the same allele, it had decreased activity. Thus, the combination of a unique mutation and polymorphism causes conformational change in the GALC enzyme, resulting in low enzymatic activity. AO-GLD mutations, including those found here, are located in the N-terminus (I66M, G270D, 535–573del) or C-terminus (L618S) of the GALC enzyme, whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the clinical features of GLD. Received: 4 February 1997 / Accepted: 28 April 1997     

In vitro synthesis of superoxide dismutases of rat liver

The syntheses of copper, zinc-superoxide dismutase (Cu,Zn-SOD) and manganese-superoxide dismutase (Mn-SOD) in vitro were studied. Both Cu,Zn-SOD and Mn-SOD were preferentially synthesized by free polysomes. Mn-SOD was synthesized as a large precursor (26,000 daltons), which was processed to the mature size (22,500 daltons) by in vitro incubation with a rat liver mitochondrial fraction. On the other hand, Cu,Zn-SOD was synthesized as the mature size product. It was shown that Cu,Zn-SOD and Mn-SOD synthesized in vitro represented 0.018% and 0.016% of the total translation products of free polysomes, respectively.     

Enzymatic Properties of Purified Alkaline Proteinase from Aspergillus sojae†

Some enzymatic properties of purified alkaline proteinase from Aspergillus sojae were investigated. The optimum pH for casein digestion was 11.0. The enzyme activity was almost completely lost at 60°C within ten minutes. At low temperature, the enzyme was highly stable at the range of pH 4.5 to 10.0. At 50°C, the most stable pH was around 6.0. None of metallic ions tested promoted the activity, but Hg²⁺ showed a remarkable inhibition. The Hg²⁺-treatment seemed to cause a large unfolding of the enzyme molecule. The enzyme was inhibited by potato inhibitor and a number of animal sera. Metal chelating reagents and sulfhydryl reagents tested had no effect on the activity, but DFP caused a marked inhibition. The sensitivity to DFP of the enzyme was about 1/300 of that of α-chymotrypsin. The enzyme was inhibited neither by TPCK nor by TLCK. As the result it was assumed that the structure of the active site of the enzyme is fairly different from that of trypsin, or of chymotrypsin.     

Hepatotrophic growth factor in blood of mice treated with carbon tetrachloride

The presence of a human hepatocyte growth factor (hHGF)-like DNA-synthesis promoter in platelet-poor serum of mice with liver injury was examined. Activity of the serum for stimulating DNA synthesis in cultured rat hepatocytes was low in untreated or vehicle-treated mice, but markedly increased 24 h after carbon tetrachloride administration and then dropped to normal levels prior to the peak of liver DNA synthesis. The effect of the serum was additive with the maximal effects of mouse and human epidermal growth factors, but not with that of hHGF. The growth-stimulating factor in the mouse serum, like hHGF, had affinity for heparin and was heat-labile. These results indicate that the level of a serum hHGF-like hepatocyte growth factor increased in mice treated with carbon tetrachloride prior to liver regeneration.     

Anomalous volume change of gramicidin A in ethanol solutions

Gramicidin A (GA) in absolute ethanol (AE) at concentrations (c), below 0.01 g/100 ml, failed to sediment but sedimented normally in ethanol-water mixtures (EWM). The apparent partial specific volume, φ, increased on decreasing c in AE but no increase was observed in EWM, where it remained around φ=0.83. These results indicate that GA solutions consist of an equilibrium system containing species differing in volume. This behavior may help to explain the biological mechanism of action of GA.