The pharmacokinetics and safety profiles of belimumab after single subcutaneous and intravenous doses in healthy Japanese volunteers.

WHAT IS KNOWN AND OBJECTIVES: Belimumab is a recombinant human monoclonal antibody that binds and antagonizes the biological activity of soluble B-lymphocyte stimulator (BLyS) protein. BLyS appears to play a role in the pathogenesis of systemic lupus erythematosus, and the biological profile of belimumab suggests that it may have a therapeutic benefit in the treatment for the disease. In this healthy Japanese subjects study, we investigated the pharmacokinetics and safety of a single subcutaneous and intravenous injection of belimumab administered as a 200 mg/mL liquid formulation. METHODS: This was an open-label, randomized, parallel-group, single-dose study in healthy Japanese subjects. Each subject received a single intravenous infusion or a subcutaneous injection of 200 mg belimumab. The pharmacokinetic parameters and safety parameters including local tolerance (injection site), biomarkers, immunogenicity and adverse events were evaluated up to 70 days post-dosing. RESULTS: After a single intravenous or a subcutaneous administration of 200 mg belimumab, all 16 subjects completed the study. There were no serious adverse events or adverse events related to injection site reactions. All seven adverse events were considered mild or moderate in intensity and deemed unrelated to belimumab except for cellulitis following intravenous administration. The bioavailability of the single subcutaneous dose of 200 mg belimumab in the subjects was estimated to be 77·5%. Time to the maximum serum concentration after subcutaneous injection was 6·5 days (median). The geometric mean terminal half-life was comparable between the two administration routes (17·7 days intravenous and 15·9 days subcutaneous). Serum immunoglobulin G level decreased slightly after each treatment. No subjects were found to produce antibelimumab antibodies. WHAT IS NEW AND CONCLUSIONS: A favourable absolute bioavailability in healthy Japanese subjects was seen following a subcutaneous injection of 200 mg belimumab. Considering the intersubject variability, exposures were consistent with those previously observed in healthy non-Japanese subjects. Safety and biomarker data were also consistent with previous non-Japanese clinical studies.

Cyclin-dependent kinase 4 and cyclin D1 are required for excitotoxin-induced neuronal cell death in vivo.

Systemic administration of the glutamic acid analog kainic acid (KA) causes neuronal cell death in brain-vulnerable regions, such as the piriform cortex, hippocampus, and amygdala in rats. We investigated the relationship between the KA-induced neuronal apoptosis and expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1, key regulators of cell cycle progression. Expression of CDK4 and cyclin D1 was upregulated in neurons of the rat piriform cortex and amygdala 1-3 d after KA administration in vivo. CDK4 and cyclin D1 proteins were induced in the cytoplasm and nuclei of neurons, with a concomitant increase of CDK4- and cyclin D1-positive microglia in the affected areas. Continuous infusion of 100 microm CDK4 or cyclin D1 antisense oligonucleotides into the lateral ventricle using mini-osmotic pumps suppressed the excitotoxin-induced neuronal cell death in the piriform cortex and basolateral amygdaloid nucleus, whereas sense oligonucleotides exhibited no such effect. Although KA administration causes prolonged c-Fos expression in the vulnerable regions that preceded the induction of neuronal apoptosis, the CDK4 or cyclin D1 antisense oligonucleotides exhibited no suppressive effect on c-Fos levels. Our results suggest that CDK4 and cyclin D1 are essential for KA-induced neuronal apoptosis in vivo.

Promoter region of the rat phosphoribosylpyrophosphate synthetase-associated protein 39.

The 5' region of the 39-kDa rat phosphoribosylpyrophosphate synthetase-associated protein (PAP39) gene was isolated and sequenced. The promoter region of the rat PAP39 is GC-rich and contains potential binding sites for regulatory factors. Its promoter activity was demonstrated by transfection of the promoter region in fusion with the chloramphenicol acetyltransferase gene into rat pheochromocytoma PC12 cell.

Direct DNA sequencing from colony: analysis of multiple deletions of mitochondrial genome.

We have developed a rapid and efficient nucleotide sequencing technique, named the colony direct sequencing method, which combines both the conventional cloning method for picking up a single gene and the polymerase chain reaction (PCR) method for amplifying the gene directly from a colony. In the present study, the colony direct PCR product was used both for identification of the DNA insert and for nucleotide sequencing by an automated DNA analysis system. A nucleotide sequence of 300 to 400 bp could be determined within 13 h after picking the bacterial colonies on LB medium plates. We applied this method to sequencing of junctional regions of multiple deleted mtDNAs in two siblings with inherited recurrent myoglobinuria. Mitochondrial DNA fragments with deletions were amplified by PCR and then cloned into plasmids. Among 48 white colonies propagated on LB medium plates, nine different clones were identified by PCR directly from colonies. Determination of six different junctional sequences disclosed involvement of directly repeated sequences of 2 to 12 bp in length on each side of the deletions. We believe that the colony direct sequencing method will be a powerful tool in molecular genetics for identification of a single gene among polymorphic DNAs.

Clinical features of hypertrophic cardiomyopathy caused by a Lys183 deletion mutation in the cardiac troponin I gene.

BACKGROUND: Mutations that cause hypertrophic cardiomyopathy (HCM) have been identified in 9 genes that code proteins in the sarcomere. Previous reports have demonstrated that cardiac troponin I (cTnI) gene mutations may account for familial HCM; however, the clinical characteristics and prognosis of patients with HCM caused by cTnI gene mutations are not known. METHODS AND RESULTS: We analyzed cTnI gene mutations in 130 unrelated probands with HCM and their families to clarify the genotype-phenotype correlations. We identified 25 individuals in 7 families with a Lys183 deletion (Lys183 del) mutation in exon 7 of the cTnI gene. The disease penetrance in subjects aged >20 years was 88% by echocardiography and 96% by ECG. Sudden death occurred in 7 individuals of 4 families at any age. Overall, 7 (43.8%) of 16 individuals aged >40 years had left ventricular systolic dysfunction, and 3 (18.8%) displayed dilated cardiomyopathy-like features. Of affected individuals, 4 of 5 individuals aged >40 years followed by echocardiography showed septal thinning and decreased fractional shortening during >5 years of follow-up. CONCLUSIONS: The Lys183 del mutation in the cTnI gene in patients with HCM is associated with variable clinical features and outcomes. HCM caused by the Lys183 del mutation has a significant disease penetrance. This mutation is associated with sudden death at any age and dilated cardiomyopathy-like features in those aged >40 years. However, it remains unclear whether screening of families with HCM for this mutation will be useful in patient management and counseling.

Left ventricular functional reserve in patients with syndrome X: evaluation by continuous ventricular function monitoring.

OBJECTIVES: The objective of this study was to evaluate cardiac functional reserve in patients with syndrome X. BACKGROUND: Syndrome X is characterized by stress-induced anginal pain and ST segment depression, normal findings on coronary angiography and normal left ventricular function at rest. Reduced coronary vasodilative reserve and abnormal myocardial lactate metabolism have been described in such patients. METHODS: To assess left ventricular functional reserve in patients with syndrome X, continuous radionuclide monitoring of left ventricular end-diastolic volume, end-systolic volume and ejection fraction was performed in 12 patients and 13 normal control subjects during supine bicycle ergometer exercise. RESULTS: In control subjects, end-diastolic volume increased at peak exercise from 100% to 106.5% (p < 0.01), end-systolic volume decreased from 39.1% to 22.6% (p < 0.01) and ejection fraction increased from 60.9% to 78.6% (p < 0.01). In patients with syndrome X, end-diastolic volume increased at peak exercise from 100% to 106% (p < 0.01), and end-systolic volume decreased at ST segment depression < or = 0.5 mm (the ST point) from 37% to 28.8% (p < 0.01) but increased at peak exercise to 44.7% (p < 0.01 vs. the ST point). Thus, ejection fraction increased at the ST point from 63% to 72.7% (p < 0.01) but decreased at peak exercise to 57.7% (p < 0.01 vs. the ST point and control subjects) in proportion to the degree of ST segment depression. In nine patients (75%), ejection fraction at peak exercise was lower than baseline values. All patients and control subjects showed a rapid ejection fraction increase just after exercise during the recovery period. The degree of ejection fraction "overshoot" in patients was similar to that in control subjects, but the interval from the end of exercise to the overshoot in patients was significantly longer than that in control subjects (118 vs. 65 s, p < 0.01). CONCLUSIONS: In patients with syndrome X subjected to exercise stress, left ventricular function remained normal before the onset of ST segment depression. Once ST segment depression appeared, left ventricular function deteriorated in proportion to the degree of depression, and reduced left ventricular function persisted into the recovery period. Continuous ventricular function monitoring is thus a useful predictor of reduced left ventricular functional reserve in patients with syndrome X.

Effects of short-term aminophylline administration on cardiac functional reserve in patients with syndrome X.

OBJECTIVES: This study sought to evaluate the effect of adenosine receptor blockade by aminophylline on cardiac functional reserve in patients with syndrome X. BACKGROUND: Aminophylline may have a potentially antiischemic effect through the inhibition of adenosine and, thus, the coronary steal phenomenon in patients with syndrome X. METHODS: A single-blind, placebo-controlled study of an intravenous infusion of aminophylline (6 mg/kg body weight over 15 min) or placebo (20 ml of saline solution over 15 min) was performed during continuous radionuclide monitoring of left ventricular ejection fraction in 12 patients performing supine bicycle ergometric exercise. RESULTS: Aminophylline increased exercise time (aminophylline 400 s vs. placebo 355 s, p < 0.01), decreased degree of ST segment depression (aminophylline 1.6 mm vs. placebo 2.4 mm, p < 0.01) and either abolished (seven patients) or diminished (five patients) chest pain during exercise. Aminophylline also increased left ventricular ejection fraction at rest (aminophylline 66.5% vs. placebo 62.3%, p < 0.05) but did not improve its deterioration at peak exercise (aminophylline 60.1% vs. placebo 56.6%, p = NS) or shorten the abnormally prolonged interval between the end of exercise and the overshoot (aminophylline 115 s vs. placebo 130 s, p = NS). CONCLUSIONS: Aminophylline infusion increases ischemic threshold and prolongs exercise duration in patients with syndrome X. It is hypothesized that aminophylline acts by inhibiting the coronary steal phenomenon through adenosine receptor blockade. It does not improve the deterioration in left ventricular function at peak exercise or the delayed response in ejection fraction in the recovery period, presumably because the beneficial effects of aminophylline that result from the redistribution of coronary blood flow are limited.

Left ventricular systolic dysfunction during exercise and dobutamine stress in patients with hypertrophic cardiomyopathy.

OBJECTIVES: We sought to characterize stress-induced left ventricular systolic dysfunction in patients with hypertrophic cardiomyopathy (HCM). BACKGROUND: Myocardial ischemia and diastolic dysfunction occur in patients with HCM. We hypothesized that, in the setting of transient myocardial ischemia, left ventricular systolic dysfunction occurs during exercise and dobutamine stress. METHODS: We studied 39 patients with HCM but without obstructive symptoms at rest or coronary artery disease. A continuous ventricular function monitor equipped with cadmium telluride detectors (VEST) was used to evaluate left ventricular function during supine bicycle ergometer exercise. Dobutamine stress echocardiography (DSE) was also performed. The left ventricular ejection fraction (LVEF) and regional wall motion were determined from echocardiographic images. RESULTS: Changes in the LVEF correlated between exercise and dobutamine stress (r = 0.643, p < 0.0001). The LVEF decreased more than 5% at peak exercise in 17 of patients (group II), while the other patients had normal responses (group I). New regional wall motion abnormalities during dobutamine infusion were detected in 18 of 110 (16.4%) segments in group I and 42 of 85 (49.4%) segments in group II. Decreased or unchanged regional wall motion occurred more frequently in hypertrophied segments than in nonhypertrophied segments (p < 0.0001). There were significant inverse correlations between the LVEF responses during both stresses and the number of abnormal segments noted during dobutamine stress in all patients (VEST: p < 0.005; DSE: p < 0.0005). Signs of left ventricular obstruction were observed in 11 of 39 patients during DSE. However, there was no significant correlation between the LVEF response and the dobutamine-induced left ventricular pressure gradient. CONCLUSIONS: Exercise-induced systolic dysfunction occurred in 50% of patients with HCM. In these patients, regional wall motion abnormalities were present in hypertrophied segments.

PRKAR1A gene mutation in patients with cardiac myxoma.

BACKGROUND: PRKAR1A gene encodes the type 1A regulatory subunit of protein kinase A. The mutation of this gene causes Carney complex which is an autosomal dominant multiple neoplasia syndrome characterized by spotty pigmentations, endocrine overactivity and cardiac myxoma. We hypothesized that cardiac myxoma may be associated with PRKAR1A gene mutation and determined whether mutation in the PRKAR1A gene is the cause of familial and sporadic cardiac myxoma. METHODS: We studied seven patients (three males and four females) with cardiac myxoma. Two of them had familial cardiac myxoma complicated with Carney complex. The other five patients were characterized as sporadic cardiac myxomas. We analyzed the PRKAR1A gene of all patients by the polymerase chain reaction (PCR)-single-strand conformation method, followed with direct sequence analysis. RESULTS: We identified a novel mutation (494delTG) in exon 4A of the PRKAR1A gene in the patients with Carney complex. A 16-year-old proband had a left atrial myxoma, pituitary adenoma and skin pigmentation. His father also had left atrial myxoma and skin pigmentation. In contrast, no mutations in the PRKAR1A gene were identified in the other five patients with sporadic cardiac myxomas. CONCLUSIONS: These results suggest that mutation of the PRKAR1A gene may be associated with familial cardiac myxoma in Carney complex but may not be associated with sporadic cardiac myxoma.

Promoter region of the mouse cyclin-dependent kinase 5-encoding gene.

While cyclin-dependent kinases, such as CDC2 and CDK2, are key regulators of cell-cycle progression, cyclin-dependent kinase 5 (CDK5) is highly expressed in mature neurons with no evident cell-cycle regulation. The 5'-region of the mouse CDK5 gene was isolated and sequenced. The isolated clone included exons 1 through 7. The 5'-flanking region has a high G+C content. There is no TATA box around the transcriptional start points (tsp), as determined by primer extension analysis. One CCAAT box, one AP-1-binding site, two AP-2-binding sites, and one cAMP-responsive element are located upstream from the tsp. Promoter/cat fusion assays showed that the 5.8-kb fragment of this 5'-flanking sequence possessed the promoter activities expressing cat in rat PC12 pheochromocytoma cells. The effect of deletions of the promoter suggested the presence of two negative control elements located from -2.9 kb to -546 bp, and from -212 to -155 upstream from the 5' end of the tsp. Two positive elements from bp -300 to -212 and from -155 to -41 were also detected. In the element from bp -300 to -212, there was a putative NF-IL6-binding sequence. Thus, the CDK5 promoter region contains multiple positive and negative cis-acting regulatory elements, an arrangement which suggests that the regulation of transcription of CDK5 is under complex control.

Undifferentiated carcinoma of the vulva mimicking epithelioid sarcoma.

We report an undifferentiated sweat gland carcinoma of the vulva in an 80-year-old woman. The tumor, which was located in the right labium majus, resembled an epithelioid sarcoma histologically; it had a granulomatous appearance with multiple tumor nodules containing epithelioid tumor cells. The tumor also contained rhabdoid cells; a large cluster of them showed histological features indistinguishable from those of a malignant rhabdoid tumor. Immunohistochemically, the tumor cells reacted not only for epithelial markers such as cytokeratins, EMA, and CEA, which are known to be expressed by epithelioid sarcoma, but also for CA125 and with monoclonal antibodies recognizing sweat gland structures--namely, EKH5 and EKH6. For comparison, two epithelioid sarcomas and two extrarenal malignant rhabdoid tumors were also studied. Of these tumors, only one extrarenal rhabdoid tumor reacted with EKH5, and none reacted for CA125. Electron-microscopic examination of the present tumor showed the presence of discontinuous basal laminae and tonofibril-like structures as well as primitive cell junctions and interdigitating filopodia. From these findings, we conclude that the tumor was an undifferentiated sweat gland carcinoma mimicking an epithelioid sarcoma. Findings in this case support the idea of the diverse histogenesis of extrarenal malignant rhabdoid tumors and indicate that electron microscopy is important for differentiating epithelioid sarcoma from skin adnexal carcinoma.

Differential screening-selected gene aberrative in neuroblastoma protein modulates inflammatory pain in the spinal dorsal horn.

Differential screening-selected gene aberrative in neuroblastoma (DAN) belongs to a novel gene family that includes the Xenopus head-inducing factor, Cerberus and the dorsalizing factor, Gremlin. It has been suggested that members of this family control diverse processes in growth, development and the cell cycle.Here, we demonstrate that the DAN protein is produced in the small neurons of the dorsal root ganglion and is transported to the nerve terminals in the spinal dorsal horn in adult rats. Furthermore, intrathecal injection of an antibody to the DAN protein suppressed inflammatory pain caused by the introduction of complete Freund's adjuvant or carrageenan into the rat hindpaw. The amount of mRNA for DAN in dorsal root ganglion neurons and of its expressed protein in the spinal dorsal horn were both increased in inflammatory models.Together, these data suggest that the DAN protein may be a novel neuromodulator in primary nociceptive nerve fibers.

Light-induced apoptosis in the neonatal mouse retina and superior colliculus.

UNLABELLED: PURPOSE. Apoptosis occurs naturally in the rodent retina and superior colliculus (SC) during the neonatal period. The authors used mice to demonstrate the dependency of this apoptosis on the light stimulation and the developmental period. METHODS: A number of apoptotic cells were counted in the retina and SC from a group of newborn mice reared in constant darkness (DD group), a group reared in normal light and dark conditions (LD group), and a group reared in constant darkness up to P7 and then transferred to normal condition (DD-to-LD group). Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling (TUNEL) was used for visualization of the apoptotic cells. RESULTS: In the LD group, apoptotic cells significantly increased in the retinal nuclear layers, including both the outer and inner nuclear layers, the retinal ganglion cell layer, and SC at postnatal day 1 (P1) and postnatal day 2 (P2). The number of apoptotic cells in the ganglion cell layer and SC reached the maximum level at P1. In contrast, in the DD group, an increase in the number of apoptotic cells was not observed. At P9, no significant increase in the number of apoptotic cells was observed in the outer nuclear layer, ganglion cell layer, and SC either in the LD, DD, or DD-to-LD groups, but the LD and DD-to-LD groups showed a significant increase in the inner nuclear layer compared to the DD group. CONCLUSIONS: Apoptosis during the neonatal period in the mouse visual system is induced by a light stimulus. This apoptosis was not induced after P7 in the retinal ganglion cell layer and SC, even if excessive cells survived.

Cardiac characteristics and postoperative courses in Cushing's syndrome.

To assess the cardiac characteristics and postoperative courses in patients with Cushing's syndrome, electrocardiography and echocardiography were performed to study 12 consecutive, unselected patients, and results were compared with those of essential hypertension and primary aldosteronism. Eleven patients had hypertension and 7 had diabetes mellitus. Before adrenalectomy, common electrocardiographic abnormalities consisted of high-voltage QRS complexes (10 patients) and negative T waves (7 patients). Echocardiograms showed left ventricular hypertrophy in 9 patients, and all the patients had evidence of asymmetric septal hypertrophy. In patients with left ventricular hypertrophy, the thickness of the interventricular septum ranged from 16 to 32 mm, whereas the ratio of the thickness of interventricular septum to that of the posterior wall ranged from 1.33 to 2.67. The interventricular septum in Cushing's syndrome was extremely thicker and asymmetric septal hypertrophy occurred more often than essential hypertension and primary aldosteronism. Nine patients could be followed up after operation. In these patients abnormal electrocardiographic findings had normalized, the thickness of interventricular septum had decreased and asymmetric septal hypertrophy had disappeared except in 1 patient. The reason why left ventricular hypertrophy in Cushing's syndrome is severe is still unknown. Because left ventricular hypertrophy is more severe and the frequency of asymmetric septal hypertrophy much greater in Cushing's syndrome than in essential and other secondary hypertension, it is thought that not only increased aortic pressure but excessive plasma cortisol may be etiologic factors in the progression of left ventricular hypertrophy in Cushing's syndrome.

Acute increases in plasma oxidized low-density lipoprotein immediately after percutaneous transluminal coronary angioplasty.

To investigate the acute changes in plasma oxidized low-density lipoprotein before and immediately after coronary angioplasty, we studied 132 consecutive patients who successfully underwent this procedure. Plasma oxidized low-density lipoprotein levels were significantly increased immediately after coronary angioplasty in patients with stable angina pectoris as well as in those with acute coronary syndromes.

Expression of proliferating cell nuclear antigen (PCNA) in the adult and developing mouse nervous system.

Proliferating cell nuclear antigen (PCNA) is essential for the function of DNA polymerases delta and epsilon. Because proliferating cell nuclear antigen is required for DNA replication and repair, PCNA is abundantly expressed in proliferating cells. Interestingly, PCNA mRNA has also been detected in the adult mouse brain by Northern blot analysis. In this study, two monoclonal antibodies against PCNA, PC10 and 19F4, were used for Western blot analysis. Monoclonal antibody PC10, but not 19F4, detected a band in the adult mouse brain extract. This PC10-reactive protein in the brain displayed a more acidic isoelectric point than PCNA by two-dimensional gel electrophoresis. In situ hybridization showed that PCNA mRNA was abundantly expressed in the adult mouse subventricular zone. Additionally, relatively low levels of PCNA mRNA expression were also found in neurons throughout the central nervous system, however, no hybridization was observed in the white matter. Immunohistochemistry was also performed using 19F4 and PC10, and staining of progenitor cell nuclei in the subventricular zone was observed with both antibodies. Whereas 19F4 immunostaining was restricted to progenitor cells, PC10 immunostaining was also found in postmitotic nonproliferating cell nuclei. In the cortical neuroepithelium of developing mice, the distribution of PC10 immunoreactivity was wider than that of 19F4 immunoreactivity and PCNA mRNA expression. These results suggest that proliferating cell nuclear antigen mRNA is expressed not only in proliferating cells but also in nonproliferating cells such as neurons. The protein recognized only with PC10 may be a modified, most probably a phosphorylated PCNA.

Collagen remodelling in myocardia of patients with diabetes.

AIMS: To investigate collagen remodelling in the interstitium of the heart in patients with diabetes. METHODS: Immunohistochemical study of the biopsied myocardium using type specific anticollagen antibodies (I, III, IV, V, VI) was performed in 12 patients with non-insulin dependent diabetes mellitus and six non-diabetic patients. There was no history of hypertension or coronary artery stenosis in any of the patients. RESULTS: Noticeable accumulations of collagen types I, III, and VI in the myocardial interstitium were recognised in both groups, but little accumulation of types IV or V was found. Types I and III mainly stained in the perimysium and perivascular region, while type VI predominantly stained in the endomysium. There was no disease specific accumulation of collagen in diabetes mellitus. The percentage of total interstitial fibrosis in the myocardium was significantly higher in the diabetic group than in the control group (p < 0.05). Although the percentages of collagen types I and VI did not differ between the two groups, the percentage type of III was significantly higher in the diabetic group than in the controls (p < 0.01). CONCLUSIONS: Collagen remodelling mainly as a result of an increase in collagen type III in the perimysium and perivascular region, occurs in the hearts of patients with diabetes.

Neonatal capsaicin treatment decreased substance P receptor immunoreactivity in lamina III neurons of the dorsal horn.

Using immunohistochemistry and in situ hybridization technique, the distribution of substance P (SP) and SP receptors was studied in the dorsal horn of the rat spinal cord after neonatal capsaicin treatment. Sprague-Dawley rats administered 100 mg/kg of capsaicin subcutaneously within 24 h after birth were examined at 8 weeks of age. In the capsaicin administered rats, slight reduction of SP immunoreactivities in lamina I, and severe decrease in lamina II were observed. In the control group, SP receptor-mRNA was observed in all laminae, and SP receptor-immunoreactivities were seen to be intense in laminae I and III. In contrast, in the capsaicin administered rats, the SP receptor-mRNA expression was low in laminae II-V, and SP receptor immunoreactivities decreased in laminae III-V. Furthermore, the density of the SP receptor immunoreactivities was considerably decreased in the nerve cells of lamina III. We concluded that elevation of the threshold to painful stimulation in rats was as a result of the decrease in SP immunoreactive afferent fibers in laminae I and II, decrease of the SP receptor-mRNA in the laminae II-V, or the decrease in SP receptor immunoreactive neurons in laminae III-V.

Characterization of binding sites for spider toxin, [3H]NSTX-3, in the rat brain.

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.

Intracellular localization of cyclin-dependent kinase 5 (CDK5) in mouse neuron: CDK5 is located in both nucleus and cytoplasm.

Cyclin-dependent kinase 5 (CDK5) is one of the cyclin-dependent kinases, and is expressed in mature neurons. CDK5 has been postulated to be a neurofilament or tau protein kinase, because it phosphorylates neurofilaments and tau protein (microtubule-associated protein tau) in vitro. It has been reported that CDK5 was immunohistochemically detected only in axons of neurons. We here report the immunohistochemical study of CDK5 using two distinct antibodies, one recognizing the N-terminal of CDK5 and the other the C-terminal. Immunoreactivity of CDK5 was found not only in axons, but also intensively in nuclei, though not in nucleoli, of neurons in the mouse central and peripheral nervous systems. The nuclear CDK5 possibly has a physiological function distinct from the neurofilament or tau protein kinase.