Tadahiro Shikimi

Pharmacological significances of peptidase and proteinase in the brain—II: Purification and properties of a bradykinin inactivating enzyme from rat brain

Abstract The enzyme inactivating bradykinin (kininase) in rat brain was partially purified by ammonium sulfate fractionation and chromatography on Sephadex G-100, DEAE-cellulose and CM-cellulose. The final preparation showed a single peak in the ultracentrifuge with a sedimentation constant of 4.55S. This enzyme preparation inactivates bradykin by removing its 8 phenylalanine and C-terminal 9 arginine. Enzyme activity was inhibited by chelating agents and SH-compounds and increased by SH reagents and Ca 2+ . Ovomucoid trypsin inhibitor, tosylphenylalanine chloromethylketone (TPCK) and diisopropylfluorophosphate (DFP) had no effect. Among the analgesic antipyretic drugs tested, acetylsalicylic acid and aminopyrine activated the enzyme slightly.

Human urinary trypsin inhibitor (urinastatin)-like substance in mouse liver

Mouse liver contains a human urinary trypsin inhibitor (urinastatin, UT)-like immunoreactive substance with trypsin inhibitory activity. Northern blot analysis demonstrates the presence of the appropriate 1.3 kb mRNA band in liver tissue but not in kidney or other tissues examined. Administration of hydrocortisone, which is known to increase the urinary excretion of the UT-like substance, increased the levels of UT-like substance in serum and in the liver tissue. In contrast, deoxycorticosterone acetate did not have such an effect. These results suggest that the gene encoding UT-like substance is primarily expressed in the liver of the mouse, and that glucocorticoids play an important role in regulating the hepatic synthesis of UT-like substance. Furthermore, these findings indicate that the mouse is a suitable species for research on the biological function of UT or UT-like substances.

Non-existence of a positive correlation between urinary levels of α1 -microglobulin and ulinastatin in patients with Parkinson's disease

Urinary levels of a,‐microglobulin (αlM) and of ulinastatin (UT) and the αlM/UT ratio did not differ significantly between age‐matched controls and patients with Parkinsons disease, and among subdivided groups based on Yahrs stages in Parkinsons disease. Furthermore, these indexes did not correlate with Yahrs stages. Although αlM and UT levels did not correlate in patients with Parkinsons disease, a positive correlation was observed in the control group. The non‐existence of a positive correlation between αlM and UT levels distinguishes Parkinsons disease from other neuropsychiatric diseases such as dementia (Alzheimer‐type and vascular dementia), schizophrenia and mood disorder.

DETECTION OF BIKUNIN MRNA IN LIMITED PORTIONS OF RAT BRAIN

Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney, testis and adrenal gland, expressed bikunin mRNA. Although signals of bikunin mRNA were faint in the whole brain of rats and mice, distinct signals were found in limited portions of rat brain, such as the hippocampus, cerebral cortex and pituitary, but undetectable in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain and choroid plexus. In three distinct types of cells, such as neurons, astrocytes and meningeal cells, in primary cultures isolated from the cerebral cortex and meninges of 1-day-old newborn rats, only neurons positively expressed bikunin mRNA. These results suggest that, in addition to peripheral tissues, neurons in the hippocampus and cerebral cortex produce bikunin, suggesting a potential role of bikunin/IalphaI family in these brain regions.

Changes in the ratio of urinary α1-microglobulin to ulinastatin levels in patients with Alzheimer-type dementia and vascular dementia

Abstract Relationships between urinary levels of α1‐microglobulin (α1M) and ulinastatin (UT) in patients with dementia were investigated. There were no significant differences in α1M and UT levels and α1M: UT ratios among three groups: age‐matched control subjects, patients with either Alzheimer‐type senile dementia (ATD) or vascular dementia (VD). Although a positive correlation was established between α1M and UT levels in these groups, the regression of the demented patients differed significantly from that of controls (P <0.05). A tendency towards a negative correlation between α1M: UT ratios and the levels of severity or duration of the disease was displayed in the ATD group, whereas a tendency toward a positive correlation between α1M: UT ratios and the levels of severity was observed in the VD group. These results suggest that changes in the relationships between urinary levels of α1M and UT may provide a useful biochemical index for diagnoses of ATD and VD.

Demonstration of a human urinary trypsin inhibitor (urinastatin)-like substance in the murine brain

A human urinary trypsin inhibitor (urinastatin)-like immunoreactive substance with trypsin-inhibitory activities was found in the murine brain. Regional levels of this urinastatin (UT)-like substance in the brain were within 5 ng/mg protein and were expressed in a descending order as follows: cerebral cortex not equal to hippocampus > hypothalamus > mesencephalon not equal to corpus striatum >> medulla oblongata > cerebellum. Mechanical lesioning of the cerebral cortex or hippocampus with a needle induced a rapid and intense appearance of a UT-like immunoreactive substance in the neuronal cells of injured sites. Conditioned fear-stress induction incited a reversible increase in the level of UT-like immunoreactive substance in the hippocampus. These results suggest that the UT-like immunoreactive substance is produced by neurons in response to brain injury and fear-stress stimuli.

Serum Contents of the Free Forms of α1-Microglobulin and Ulinastatin: Relation to Diseased States in Patients with Mood Disorders

We have found previously that the relationship between the urinary contents of α1-microglobulin (α1M) and ulinastatin (UT) in patients with mood disorder differs from that of age-matched healthy subjects. However, it has yet to be determined whether or not the difference in the relation correlates with the contents of the free forms of α1M and UT in serum and whether changes in the existing forms of α1M and UT in serum reflect the actual disease states. The relation between serum contents of the free forms of α1M and UT in 10 patients with mood disorders was different from that of 17 age-matched healthy subjects. The regression plot between scores of the Hamilton Rating Scale for Depression and ratios of the free form content to total content (F/T ratio) of UT was more informative on the depressive state than that of α1M. The F/T ratios of UT may afford a useful objective index in monitoring the diseased state of a patient with mood disorder.

Nullification of a positive correlation between urinary levels of α1-microglobulin and ulinastatin by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice

The relationships between urinary levels of alpha 1-microglobulin (alpha 1M) and ulinastatin (UT) were investigated in C57BL/6J mice, a species which reportedly possesses the gene similar to that of humans for synthesizing the precursor protein of alpha 1M and UT. A positive correlation was established in normal mice. However, repetitive administrations (20 mg/kg, IP, four administrations/12 h) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) nullified the positive correlation. A similar phenomenon was induced by ICV-administered MPTP (18 and 36 micrograms) in the animals. Furthermore, L-dopa administration (50 mg/kg, IV) in MPTP-treated (1 week after the final IP administration of MPTP) mice reversed the tendency of MPTP, although the agent alone did not affect the positive correlation in normal mice. These results suggest that nullification of the positive correlation probably was induced by the central effects of MPTP. We have found previously that the lack of a positive correlation between urinary levels of alpha 1M and UT distinguishes Parkinsons disease from other neuropsychiatric diseases such as dementia (Alzheimer-type and vascular dementia), schizophrenia and mood disorders. Our present results displayed a phenomenon that the lack of correlation between urinary levels of alpha 1M and UT in patients with Parkinsons disease is reproducible in MPTP-treated mice.

Significant Decreases of Ulinastatin-Like Immunoreactive Substance in the Cerebrospinal Fluid of Patients with Dementia

Studies to elucidate changes in the contents of ulinastatin-like immunoreactive substance (UTIRS) and alpha 1-microglobulin-like immunoreactive substance (alpha 1 MIRS) in cerebrospinal fluid (CSF) of patients with dementia were performed. Levels of UTIRS in the dementia group were significantly (p < 0.01) lower than those in dementia-free subjects. In addition, lower levels of UTIRS were registered in subjects afflicted with a higher severity of dementia. However, the levels of alpha 1 MIRS were not affected in the dementia group compared with dementia-free subjects. Neither UTIRS nor alpha 1 MIRS contents were related to the existence and stages of Parkinsons disease. These results suggest that UTIRS levels in the CSF are associated with the severity of dementia.

Impairment of the correlation between urinary contents of alpha-1-microglobulin and ulinastatin is induced by intracerebroventricularly administered interleukin-6 in mice.

We have found previously that the correlation between urinary contents of alpha-1-microglobulin (α1M) and ulinastatin (UT) depends on the type of neuropsychiatric disease. Since interleukin (IL)-1β and IL-6 are closely involved in pathophysiological aspects of various neuropsychiatric diseases, effects of intracerebroventricularly (i.c.v.) administered IL-1β and IL-6 on the correlation between urinary contents of these two glycoproteins were examined in mice, a species in which α1M and UT and also the correlation between the urinary contents thereof are expressed similarly to humans. Indices (volume, contents of creatinine, α1M and UT, and α1M/UT ratio) in urine collected after i.c.v. administrations of 2 and 20 ng of either IL-1β or IL-6 were not statistically different from those of the vehicle-treated (control) groups. Neither IL-1β (2 and 20 ng) nor the lower dose of IL-6 (2 ng) affected the positive correlation between urinary contents of α1M and UT. However, a higher dose of IL-6 (20 ng) nullified the positive correlation for 2 days after administration. Recovery to a positive correlation was thereafter displayed. These findings suggest that central IL-6 plays an important role in correlating urinary contents of α1M and UT without affecting the renal functions.