Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.     

Changes in regional cerebral blood flow and oxygen metabolism following ventrolateral thalamotomy in Parkinson syndrome as revealed by positron emission tomography

A case with unilateral symptoms of Parkinson syndrome is presented in which interesting changes in the topographic patterns of cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) were observed by positron emission tomography. This case was associated with severe tremor at rest exclusively in the left extremities. The parietal CBF and CMRO2 for the affected hemisphere were apparently lower than those for the nonaffected hemisphere preoperatively. After thalamotomy involving the right nucleus ventralis lateralis, including the ventralis intermedius, concomitant with complete disappearance of the tremor, the parietal CBF and CMRO2 for the affected side increased and even exceeded those for the nonaffected side.     

Cell death in late embryogenesis of the leech Helobdella

Cell death was characterized during stages 8 and 9 in the leech Helobdella with a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Using confocal analysis, the positions of dying cells were compared to rows of cells expressing the leech engrailed protein ht-en and to fluorescently marked cell lineages. Dying cells were present in diverse tissues. Some dying cells were in no obvious pattern, and others were in segmentally iterated patterns. Particular attention was paid to the ectoderm and mesoderm, where most of the cells examined died over a period equivalent to 1–4 h at 25°C. Segmentally iterated rows of dying cells were observed in the mesoderm just beneath the nf-derived ht-en expressing cell rows at a time when ht-en expressing cells were beginning to disappear. The position of these dying cell rows was consistent with a role in the partial deterioration of the septum. Received: 12 October 1998 / Accepted: 8 February 1999     

Responses of raphe-spinal neurons to stimulation of the pontine parabrachial region producing behavioral nociceptive suppression in the cat

Cholinergic stimulation of the pontine parabrachial region (PBR) produces behavioral nociceptive suppression in the awake cat. This report shows that electrical stimulation of both PBR sites (verified to be associated with behavioral nociceptive suppression on cholinergic stimulation) and the periaqueductal gray (PAG) excites raphe-spinal neurons which have been implicated in descending nociceptive suppression. Although several lines of evidence have strongly indicated that pathways from the PBR and PAG for nociceptive suppression are anatomically as well as neurochemically distinct, the results of the present study appear to suggest that certain components of the pathways from the PBR may be synergic in function with those from the PAG with regard to the activity of raphe-spinal neurons.     

Emotional slow negative potential shift (CNV) in the thalamus.

In order to know the functional relationship between CNV recorded at the vertex and activity of the thalamic nucleus, the CNV at the vertex and the intrathalamic slow potentials responding to an S1-S2-R paradigm were recorded during thalamotomy under local anesthesia. It might be concluded that the activity of the medial thalamus and medial parts of the subthalamic area not only generate slow potential shifts corresponding to S1-S2-R, but also play an important role in controlling the CNV at the vertex.     

Effects of thalamic sensory relay nucleus stimulation on the jaw-opening reflex in response to tooth-pulp stimulation in the cat

The effects of stimulation of the thalamic sensory relay nucleus (TSRN, nucleus ventralis posteromedialis) on the jaw-opening reflex (JOR) in response to tooth pulp stimulation were compared with the effects of stimulation of the periaqueductal gray (PAG) and nucleus raphe magnus (NRM) in the cat. After stimulation of the TSRN, PAG and NRM, the JOR was inhibited. However, while the inhibitory effects of PAG and NRM stimulation lasted for more than 500 ms and were antagonized by the opiate antagonist, naloxone, the inhibitory effects of TSRN stimulation lasted for approximately 100 ms and were resistant to naloxone. These findings suggest that although TSRN stimulation exerts descending inhibitory effects on segmental nociceptive activity, similarly to PAG or NRM stimulation, the descending inhibitory pathways mediating the effect of TSRN stimulation may be largely distinct physiologically as well as pharmacologically from those mediating the effect of PAG and NRM stimulation.     

Spore germination and phytochrome biosynthesis in the fern Lygodium japonicum as affected by gabaculine and cycloheximide

We investigated whether the gradual increase in phytochrome content in the fern Lygodium japonicum (Thunb.) Sw. during dark imbibition results from hydration or from biosynthesis of phytochrome. Addition of gabaculine or cycloheximide to the culture medium caused inhibitions of both red light-induced spore germination and of the appearance of phytochrome in the spores. Fifty percent inhibition of both red light-induced germination and of the appearance of phytochrome in the spores occurred at ca 10⁻7 M cycloheximide. Red light-induced germination and phytochrome appearance were markedly inhibited by 10⁻4 M and completely by 10⁻3 M gabaculine, but germination induced by gibberellic acid was unaffected. Phytochrome was not detected in spores after forced hydration. These results suggest that the increase in phytochrome during imbibition was mainly due to de novo synthesis of the phytochrome apoprotein and to synthesis of the chromophore and/or proteins required for phytochrome formation, rather than to hydration of preexisting phytochrome molecules.