Changes in activity and structure of jaw muscles in Parkinson's disease model rats.

Parkinson's disease (PD), a major neurological disease, is characterised by a marked loss of dopaminergic neurons in the substantia nigra. Patients with PD frequently show chewing and swallowing dysfunctions, but little is known about the characteristics of their stomatognathic functions. The purpose of this study was to evaluate the influence of PD on jaw muscle fibre and functions. PD model rats were made by means of the injection of 6-hydroxydopamine (6-OHDA) into the striatum of 8-week-old Sprague-Dawley male rats. Five weeks after the injection, a radio-telemetric device was implanted to record muscle activity continuously from the superficial masseter and anterior belly of digastric muscles. Muscle activity was recorded for 3 days and was evaluated by the total duration of muscle activity per day (duty time). After recording the muscle activities, jaw muscles were isolated for immunohistochemical and PCR analyses. In PD model rats, the following findings of the digastrics muscles verify that compared to the control group: (i) the higher duty time exceeding 5% of the peak activity level, (ii) the higher expression of the mRNA of myosin heavy chain type I, and (iii) the tendency for fast to slow fibre-type transition. With respect to the masseter muscle, there were no significant differences in all analyses. In conclusion, PD leads to the changes in the jaw behaviours, resulting in a PD-specific chewing and swallowing dysfunctions.

Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4.

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions.

Down-regulation of norepinephrine transporter expression on membrane surface induced by chronic administration of desipramine and the antagonism by co-administration of local anesthetics in mice.

We have previously shown that chronic administration of the antidepressant desipramine, a norepinephrine transporter (NET) inhibitor to mice markedly enhanced convulsions induced by local anesthetics and that behavioral sensitization may be relevant to decreased [(3)H]norepinephrine uptake by the isolated hippocampus. The co-administration of local anesthetics with desipramine reversed the behavioral sensitization and down-regulation of NET function induced by desipramine. The present study aimed to elucidate whether chronic treatment with desipramine regulates the expression of NET protein examined in membrane fractions in various brain regions and whether co-administration of local anesthetics affects the desipramine-induced alteration of NET expression. Desipramine with or without local anesthetics was injected intraperitoneally once a day for 5 days. The animals were decapitated 48 h after the last administration of drugs and the whole cell fraction, membrane fraction and cell-surface protein fraction were prepared. [(3)H]nisoxetine binding was significantly reduced in the P2 fraction of the hippocampus by chronic administration of desipramine, and the reduction was overcome by co-administration of lidocaine with desipramine. Immunoreactive NET was detected by SDS-PAGE and immunoblotting in the murine hippocampus. NET protein expression in the whole cell fraction and membrane fraction was not affected by treatment with any drugs. However, administration of desipramine significantly reduced the amount of immunoreactive NET in the cell-surface protein fraction. This reduction was blocked by simultaneous injection of lidocaine, bupivacaine or tricaine. These results indicate that the NET down-regulation indicated by the reduction of [(3)H]nisoxetine binding was induced by administration of desipramine via decrease of NET localization on the cell surface. The antagonistic actions of local anesthetics against NET down-regulation by desipramine were related to alterations of the cell-surface localization of NET.

Consolidation of transient ionotropic glutamate signals through nuclear transcription factors in the brain.

Long-lasting alterations of neuronal functions could involve mechanisms associated with consolidation of transient extracellular signals through modulation of de novo synthesis of particular functional proteins in the brain. In eukaryotes, protein de novo synthesis is mainly under the control at the level of gene transcription by transcription factors in the cell nucleus. Transcription factors are nuclear proteins with an ability to recognize particular core nucleotides at the upstream and/or downstream of target genes, and thereby to modulate the activity of RNA polymerase II that is responsible for the formation of mRNA from double stranded DNA. Gel retardation electrophoresis is widely employed for conventional detection of DNA binding activities of a variety of transcription factors with different protein motifs. Extracellular ionotropic glutamate (Glu) signals lead to rapid and selective potentiation of DNA binding of the nuclear transcription factor activator protein-1 (AP1) that is a homo- and heterodimeric complex between Jun and Fos family members, in addition to inducing expression of the corresponding proteins, in a manner unique to each Glu signal in murine hippocampus. Therefore, extracellular Glu signals may be differentially transduced into the nucleus to express AP1 with different assemblies between Jun and Fos family members, and thereby to modulate de novo synthesis of the individual target proteins at the level of gene transcription in the hippocampus. Such mechanisms may be operative on synaptic plasticity as well as delayed neuronal death through consolidation of alterations of a variety of cellular functions induced by transient extracellular signals in the brain.

Analysis of poly(ethyl methacrylate)s by on-line hyphenation of liquid chromatography at the critical adsorption point and nuclear magnetic resonance spectroscopy

Liquid chromatography at the critical adsorption point (LC CAP) with on-line NMR detection (on-line LC CAP NMR) was utilized for analysis of tacticity distribution of stereo-regular poly(ethyl methacrylate)s (PEMAs). The separation of a model PEMA sample composed of four constituents with similar molar masses (Mw = (14-16) x 10(3) g mol-1) differing in their tacticity (rr triad content = 0, 33, 68, and 89%) was achieved by LC CAP with a mixed eluent composed of acetone, acetone-d6, and cyclohexane. The tacticity composition within each peak eluted from the LC CAP column was directly determined by a 750-MHz 1H NMR spectrometer that was used as a real-time detector in the continuous-flow mode. Tacticity distribution in a particular PEMA sample with mm/mr/rr = 2/45/53 and narrow molar mass distribution of Mw/Mn = 1.05 has been revealed by the LC CAP NMR technique.

Differential expression and phosphorylation of particular fos family members by kainate in nuclear and cytosolic fractions of murine hippocampus.

An i.p. injection of kainate resulted in severe losses of neuronal cells stained by Cresyl Violet in the CA1 and CA3 pyramidal layers of the murine hippocampus within two weeks, without affecting those in the dentate granule layer up to six weeks. Immunohistochemical analysis revealed marked and predominant expression of Fos family members, including c-Fos, Fos-B and Fra-2 proteins, in the dentate granule layer of the hippocampus, but not in the pyramidal layers, 2-18h after administration. Immunoblotting experiments showed that kainate induced more potent expression of c-Fos protein in nuclear fractions obtained 2h after injection than those obtained 18h later, with similar expression between cytosolic fractions obtained 2 and 18h after administration. Both Fos-B and Fra-2 proteins were more potently expressed in nuclear and cytosolic fractions 18h after administration than 2h when determined on immunoblotting analysis. Two-dimensional electrophoresis revealed expression of several proteins immunoreactive to the anti-c-Fos antibody with different isoelectric points in response to kainate in nuclear and cytosolic fractions of the hippocampus for 2-18h after a single injection. Immunoprecipitation analysis using the anti-c-Fos antibody showed phosphorylation of c-Fos protein on serine residues in hippocampal nuclear fractions 2h after administration, with additional phosphorylation on tyrosine residues 18h later. Prior treatment of the protein synthesis inhibitor cycloheximide prevented the expression of immunoreactivities to the anti-c-Fos antibody detected on two-dimensional electrophoresis in hippocampal nuclear fractions obtained 2h after administration.These results suggest that in vivo kainate signals may lead to persistent expression of the transcription factor activator protein-1 that consists of different Fos family members, as well as of c-Fos protein phosphorylated on serine and/or tyrosine residues, at an early stage after administration. Such signal consolidation processes could play a role in mechanisms associated with neuronal survival after kainate in the dentate granular layer of murine hippocampus.

Predominant expression of nuclear activator protein-1 complex with DNA binding activity following systemic administration of N-methyl-D-aspartate in dentate granule cells of murine hippocampus.

The systemic administration of N-methyl-D-aspartate (100 mg/kg, i.p.) resulted in preferential but transient expression of the transcription factor activator protein-1 in the granule cell layers of the dentate gyrus in the murine hippocampus by maximally 700% 1 h later, without markedly affecting that in the pyramidal cell layers of the CA1 and CA3 subfields for 4 h. The potentiation was completely prevented by prior administration of the N-methyl-D-aspartate channel blocker dizocilpine at 10 mglkg. By contrast, kainate (40 mg/kg, i.p.) potentiated activator protein-1 DNA binding in adjacent areas around the pyramidal and granule cell layers, in addition to potentiating that in neuronal cell layers of the CA1 and CA3 subfields and the dentate gyrus. Light microscopic analysis revealed that kainate, but not N-methyl-D-aspartate, induced marked losses of the pyramidal cells in the CAI and CA3 subfields, without affecting the dentate granule cells, for 14 days after administration. Limited proteolysis by V8 protease and supershift, as well as immunoblotting assays using antibodies against c-Fos and c-Jun, invariably gave support for differential expression by N-methyl-D-aspartate and kainate of the activator protein-1 complex consisting of different partner proteins. Moreover, two-dimensional electrophoresis followed by immunoblotting analysis revealed the expression of several nuclear proteins immunoreactive with the anti-c-Fos antibody at molecular weights and isoelectric points clearly different from those of c-Fos itself in response to kainate, but not N-methyl-D-aspartate, in the hippocampus. These results suggest that in vivo N-methyl-D-aspartate signals are predominantly transduced into cell nuclei to express activator protein-1 complex through molecular mechanisms different from those for kainate signals in the granule cells of the dentate gyrus in the murine hippocampus.

Sustained potentiation of AP1 DNA binding is not always associated with neuronal death following systemic administration of kainic acid in murine hippocampus.

Mice were intraperitoneally injected with kainic acid (KA), followed by dissection of frozen coronal sections and subsequent punching out of the pyramidal and granular cell layers in the hippocampus under a binocular microscope. Systemic administration of KA resulted in marked and sustained potentiation of binding of a radiolabeled double stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 (AP1) in the pyramidal cell layers of the CA1 and CA3 subfields and the granule cell layers of the dentate gyrus 2-18 h later. Morphological evaluation using cresyl violet revealed marked losses of neuronal layers in the pyramidal CA1 and CA3 subfields, but not in the granular dentate gyrus, within 6 weeks after administration. Supershift analysis using antibodies against different Jun and Fos family members differentiated between AP1 DNA binding in hippocampal nuclear extracts obtained 2 and 18 h after the administration of KA. These results suggest that neuronal death may not always follow modulation of de novo synthesis of particular proteins through sustained potentiation of AP1 DNA binding which involves expression of different Jun and Fos family members in response to systemic administration of KA in murine hippocampus.

Differential inhibition by ferrous ions of [3H]MK-801 binding to native N-methyl-D-aspartate channel in neonatal and adult rat brains.

In vitro addition or pretreatment with >/=1 microM ferrous chloride markedly inhibited in a concentration-dependent manner [3H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [3H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [3H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.

Sensitization by prolonged glutathione depletion of kainic acid to potentiate DNA binding of the nuclear transcription factor activator protein-1 in murine hippocampus.

In four of four mice intracerebroventricularly injected with the inhibitor of glutathione synthesis L-buthionine-[S,R]-sulfoximine (BSO) 2 days before, an intraperitoneal injection of kainic acid (KA) invariably led to marked potentiation of DNA binding activity of the nuclear transcription factor activator protein-I (AP1) in the hippocampus at a dose which was ineffective in animals previously injected with vehicle alone. However, KA failed to potentiate binding in animals injected with BSO 1 day before. The intracerebroventricular injection of BSO induced marked and prolonged depletion of a total glutathione content in murine hippocampus for 1-2 days after administration. These results suggest that prolonged depletion of endogenous glutathione for a period longer than 1 day may lead to sensitization of KA signals to potentiate AP1 DNA binding in cell nuclei and thereby modulate de novo synthesis of particular proteins at the level of gene transcription in murine hippocampus.

High-resolution analysis of polyprenols by supercritical fluid chromatography.

A high-resolution analysis of polyprenol mixtures was achieved by supercritical fluid chromatography (SFC). The separation of polyprenols was examined on an octadecylsilane-packed column with liquid carbon dioxide as the mobile phase and ethanol as modifier. Using this chromatography system, the resolution of separation (Rs) between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) was two times higher than that using conventional reversed-phase high-performance liquid chromatography. Our SFC technique allows the advantage of baseline separation of polyprenol samples containing hydrophobic components such as terpenes or fatty acids that are unfavorable for good separation. This method is very useful for the analysis of structurally close polyprenol analogues of rubber plant metabolites.

Analysis of long-chain polyprenols using supercritical fluid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The separation of long-chain polyprenols was successfully achieved using supercritical fluid chromatography (SFC). Each 100-mer greater component was separated using tetrahydrofuran as a mobile phase modifier. The molecular mass distributions derived from SFC analyses agreed with the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses. The number-average molecular mass calculated by MALDI-TOF-MS data were also in accord with the results of quantitative 1H-NMR analysis of terminal groups. A combination of SFC and MALDI-TOF-MS analyses is a powerful tool for the elucidation of the complicated structures of natural polyprenols.

Isolation of hemicellulose from a sorghum, Andropogon sorghum Brot, Kumadake no. 263, and determination of its constituent sugars.

Hemicellulose was isolated from the stem of sorghum, Andropogon sorghum Brot, Kumadake no. 263, and the constituent sugars of this isolate were determined. The xylose concentration in the hemicellulose of SV263 was extremely high.

The occurrence of geometric polyprenol isomers in the rubber-producing plant, Eucommia ulmoides Oliver.

The chain length and geometric isomerism of polyprenols from Eucommia ulmoides Oliver were analyzed using supercritical fluid chromatography. After intensive effort to establish separation conditions for geometric isomers, a phenyl-bonded silica gel-packed column was found that cleanly separated poly-trans and -cis prenols. The presence of long-chain poly-trans prenols (>9 mers) was confirmed for the first time in plants. Trans isomers were found in the leaf, seed coat, and root, but not in the bark and seed. Poly-trans prenols in this plant may act as intermediates for trans-polyisoprene biosynthesis.

[Measurement of serum insulin-like growth factor-I by enzyme-linked immunosorbent assay in uremic patients and uninephrectomized patients].

Using the enzyme-linked immunosorbent assay (ELISA), we determined the serum insulin-like growth factor-I (IGF-I) levels in the patients on hemodialysis and patients who had undergone unilateral nephrectomy. This ELISA system was able to detect IGF-I over the range of 1.6 to 50 ng/ml. The serum IGF-I levels in 45 male and 34 female dialytic patients (12.7 +/- 7.4 ng/ml and 29.0 +/- 15.9 ng/ml) were significantly low (P < 0.01) compared with 50 normal adult men and 53 normal adult women (34.9 +/- 11.4 ng/ml and 44.5 +/- 20.5 ng/ml). The serum IGF-I level tended to decrease with aging. Furthermore, we determined the IGF-I levels in 5 donors for living-related kidney transplantation and 7 patients with upper urinary tract tumor before and after unilateral nephrectomy in order to examine the endocrine effect of IGF-I. The IGF-I level in the donors decreased significantly (P < 0.05) on the fifth postoperative day and returned to the preoperative level on the 14th postoperative day. The IGF-I level in the tumor patients decreased significantly on the fifth postoperative day (P < 0.01) and remained at a low level up to the 14th postoperative day. However, the changes in the postoperative IGF-I level in the patients who had undergone surgical operations other than unilateral nephrectomy showed patterns similar to those in the kidney transplantation donors. These findings suggest that the postoperative fall in the serum IGF-I is affected by either fasting after surgery or consumption due to healing of the operative wound.

[A case of renal subcapsular hematoma caused by paravertebral muscular injection of local anesthetics].

We report a case of left renal subcapsular hematoma caused by paravertebral muscular injection of acetylcholine chloride for the treatment of lumbago in a 42-year-old man. Since the patient suffered from left lumbago after paravertebral muscular injection, he consulted us. Excretory pyelography showed a left poor visualizing kidney and computed tomography demonstrated left renal subcapsular hematoma. Conservative treatment was done. After two weeks, drip intravenous pyelogram was normal and after four months computerized tomographic scan demonstrated no renal subcapsular hematoma. The main causes of renal subcapsular hematoma are renal injury, nephritis, renal tumor and renal biopsy. Renal subcapsular hematoma caused by paravertebral muscular injection is rare and three cases of hematoma including this case have been reported in the Japanese literature.

[A case of fibroma of the spermatic cord].

A case of fibroma of the spermatic cord is presented. A 26-year-old man was admitted with the complaint of a painless swelling in the left scrotum. On physical examination, the tumor seemed to be in the left spermatic cord. It was removed surgically on May 2, 1983. Microscopic examination of the specimen revealed that the tumor was composed of collagen fibers and fibroblasts. This histological finding was compatible with that of fibroma. Seven cases of fibroma of the spermatic cord so far reported in Japan, including this case, were reviewed.

[A case of spontaneous rupture of renal cell carcinoma].

A case of spontaneous rupture of renal cell carcinoma is reported. A 53-year old man was admitted with the chief complaint of sudden gross hematuria and right flank pain on December 28, 1979. On the following day, the clinical impression was right ruptured kidney, and therefore right nephrectomy was done. Pathological diagnosis was renal cell carcinoma. He received the post-operative irradiation of a total of 5,000 rads. He was seen five years later, at which time there was no evidence of local recurrence or distant metastasis of cancer. Thirty three cases of spontaneous rupture of renal cell carcinoma were collected from Japanese and English literature. Most common chief complaint is abdominal or flank pain. Excretory urography, ultrasonography, CT scan and angiography are useful, but it is difficult to diagnose preoperatively when the tumor is small. Therefore, it is important to suspect occult cancer when a reasonable cause of rupture is undetermined. In these indeterminate cases primary nephrectomy should be considered strongly.

[A case of ischias as a complication of internal iliac artery embolization].

A case of severe right radicular ischias following embolization is reported. Gelfoam powder and pledgets were used for right internal iliac artery embolization in the case of intractable bladder cancer hemorrhage. It is supposed that previous radiotherapy and fine embolic material may be the contributing factors to this complication.

[A case of primary localized amyloidosis of the urinary bladder].

A case of primary localized amyloidosis is reported. The patient was a 73-year-old female who suffered from miction pain and consulted our department. There was a 1.5 x 1.5 cm slightly red, nonpapillary tumor around the right ureteral orifice in cystoscopy. The diagnosis was amyloidosis with cystitis hemorrhagica histopathologically. After the treatment with antibiotics for about one month there were no symptoms and no tumors in the urinary bladder cystoscopically. The clinical course was relatively good. The treatment varies from transurethral resection to total cystectomy with urinary diversion. This case was cured by non-operative treatment, but close follow-up of the patient is necessary because of the frequency of multiple recurrence.