Visual methods to assess cold fingers and experimental verification.

Cold fingers is complaint of many people. To independently assess actual finger temperature, this paper uses prototype sensors to capture blood vessel width and blood flow rates. We verify their feasibility for future home healthcare use along with far infrared camera outputs. We elucidate the impact of three remedies, massage, hot cocoa, and shoulder exercises, on 7 subjects.

Molecular cloning and characterization of AtTERT, a telomerase reverse transcriptase homolog in Arabidopsis thaliana.

On the basis of its predicted homology to human telomerase reverse transcriptase (hTERT), a cDNA for Arabidopsis thaliana TERT (AtTERT) has now been isolated from cultured cells. The cDNA contains an open reading frame of 3372 bp, encoding a protein with a predicted size of 131 kDa and isoelectric point of 9.9. The AtTERT protein contains the conserved reverse transcriptase motifs 1, 2 and A-E as well as the TERT-specific T motif. Reverse transcription-polymerase chain reaction analysis and an assay of telomerase activity revealed that both AtTERT mRNA and telomerase activity are abundant in shoot apical meristems but are not detectable in rosette leaves.

Histochemical study of rectal aminergic nerves in type I familial amyloid polyneuropathy.

We found degeneration of aminergic nerves in nine patients with type I familial amyloid polyneuropathy by histochemical study of rectal mucosa obtained by biopsy. There was prominent degeneration of aminergic nerves in four patients with uncontrollable alternating constipation and diarrhea, but aminergic nerves were relatively preserved in two patients with intermittent constipation or diarrhea. Sympathetic denervation of the gastrointestinal tract was probably important in causing bowel symptoms.

Gastrointestinal amyloid deposition in familial amyloid polyneuropathy.

We found degeneration of enteric nerve plexuses in two patients with type I familial amyloid polyneuropathy. Amyloid deposition was more severe in the wall of the stomach than in the rectum. Hypomotility of the upper gastrointestinal tract, resulting from both amyloid deposition in the stomach and upper bowel and degeneration of the intrinsic autonomic nerves, may be responsible for anorexia, nausea, and vomiting. Diarrhea and constipation may be caused by degeneration of the enteric nerve plexuses. Gastric biopsy is valuable and safe in the diagnosis to type I familial amyloid polyneuropathy.

Adult fucosidosis: histochemical and ultrastructural studies of rectal mucosa biopsy.

Three sisters with adult fucosidosis showed prominent psychomotor retardation, gargoyle features, and angiokeratoma corporis diffusum, meeting the criteria for type II fucosidosis. By histochemical and ultrastructural studies, biopsy of rectal mucosa revealed many abnormal macrophages that were filled with fucose-rich granules, and electronmicroscopic examination showed several types of characteristic inclusions in the endothelial cells, fibroblasts, and Schwann's cells.

Role of calcium ions in dopamine release induced by sodium cyanide perfusion in rat striatum.

We have previously reported a transient and remarkable increase in dopamine (DA) release in the rat striatum during application of 2 mM sodium cyanide (NaCN) through a brain microdialysis membrane. In the present study we examined the involvement of extracellular Ca2+ in this response. Rats were divided into 4 groups. In the NaCN group a microdialysis probe inserted into the striatum was perfused with Ringer's solution containing 2 mM NaCN for 60 min. The Ca2+ free + NaCN group was subjected to perfusion with NaCN dissolved in Ca2+ free Ringer's solution, and the CdCl2 + NaCN group with the same plus 0.3 mM CdCl2 (a non-specific Ca2+ channel blocker). In the NaCN and Ca2+ free + NaCN groups DA levels in the dialysates increased to 36- and 44-fold of the control level, respectively, while this was suppressed to only a 16-fold increase in the CdCl2 + NaCN group. In response to a 100 mM KCl perfusion given 3 hr later DA levels were increased (22-fold) in the control group. On the other hand this response was inhibited in the NaCN group (3-fold), but not in the other two groups. An in vitro study with striatal slices showed a gradual increase in intracellular Ca2+ during incubation with 2 mM NaCN. These results suggest that excessive influx of extracellular Ca2+ during NaCN perfusion may contribute partly to the increase in the extracellular DA level in the striatum, and also to the suppression of a DA increase in response to high K+ stimulation observed 3 hr later.

Clinical course of cardiovascular involvement in the mucocutaneous lymph node syndrome. Relation between clinical signs of carditis and development of coronary arterial aneurysm.

Seventy-nine patients with mucocutaneous lymph node syndrome were evaluated prospectively by clinical examination, electrocardiography, chest radiography, M mode and two dimensional echocardiography and thallium-201 myocardial scanning. Serial changes were categorized according to the duration of illness: stage I (1 to 10 days), stage II (11 to 20 days), stage III (21 to 30 days), stage IV (31 to 60 days) and stage V (61 days to 40 months). The presence of myocarditis in stages I and II was suggested in 40 of 79 patients (50.6 percent) by electrocardiographic, echocardiographic, radiographic and clinical abnormalities. Myocarditis was accompanied by pericarditis in six patients and by both endocarditis and pericarditis in one patient. These signs of inflammation were resolved by stage III in all but three patients with electrocardiographic abnormalities. In the active stage, large coronary arterial lesions were suspected only because of an abnormal spherical echo-free space in the region of the coronary arteries on two dimensional echocardiograph as well as electrocardiographic evidence of deep Q waves in leads II, III and aVF. One or more coronary aneurysms developed in 11 patients, primarily in stage II; regression of the aneurysm was noted in 5 of these patients during stages III, IV and V. Aneurysm regression demonstrated by angiography did not correlate with echocardiographic changes in aneurysm size in one patient. Moreover, the occurrence of coronary aneurysm did not correlate with the presence of signs of carditis, because the frequency of carditis was the same in patients with and without aneurysm.

Primary adenocarcinoma of the seminal vesicle.

A rare case of primary adenocarcinoma of the seminal vesicle is examined in an 87-year-old Japanese man. Neoplastic cells, especially poorly differentiated cells, showed a positive reaction with periodic acid-Schiff reagent and anti-carcinoembryonic antigen. Neoplastic cells invaded the bladder wall but not the prostate. No other primary tumor was demonstrated.

Behavioral stimulation without alteration of beta and 5-HT receptors and adenylate cyclase activity in rat brain after chronic sertraline administration.

Effects of chronic treatment with selective 5-HT reuptake inhibitors (SSRIs) on the monoaminergic functions have not been much investigated in compared with tricyclic antidepressants. Therefore, we compared the effects of 3-week treatment with sertraline, a potent SSRI, to those of imipramine (10 mg/kg, IP, twice a day), on monoamine receptors and adenylate cyclase (AC) activity in rat brain. Two-week treatment with both sertraline and imipramine reduced immobility in the water wheel test to the comparable extent. Sertraline treatment did not affect Kd and Bmax of [3H]CGP12177 and [3H]ketanserin bindings or cAMP, accumulation by norepinephrine, isoproternol, 5'-guanylylimidodiphosphate [Gpp(NH)p] and forskolin in the cortical membrane compared with vehicle-treated rats. On the other hand, imipramine treatment decreased Bmax of both bindings and norepinephrine- or isoproternol-stimulated cAMP accumulation. Treatment with either antidepressant induced no apparent changes in [3H]8-OH-DPAT [2-(N, N-dipropylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene] binding in the hippocampal membrane. These results suggested that chronic treatment of sertraline induced little effect on monoamine receptors and AC activity in the brain and that the alteration of these functions may not be primarily involved in antidepressive effects of antidepressants, at least of SSRIs.

Methyl-4-phenylpyridinium (MPP(+))-evoked dopamine release from rat striatal slices: possible roles of voltage-dependent calcium channels and reverse dopamine transport.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.

Regulation of dopamine uptake by basic fibroblast growth factor and epidermal growth factor in cultured rat astrocytes.

We examined the characteristics of dopamine (DA) uptake and its regulation by neurotrophic factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in cultured rat astrocytes. In the presence of inhibitors of monoamine oxidase (MAO) and catechol-O-methyl-transferase (COMT), astrocytes took up DA by Na(+)-dependent and Na(+)-independent mechanisms that were sensitive to a reduction in temperature. The Na(+)-dependent and Na(+)-independent components increased linearly with increasing [3H]DA concentration (1-1000 microM), and showed no saturation. Na(+)-dependent DA uptake was significantly inhibited by ouabain, a Na(+)-K+ ATPase inhibitor. In bFGF-treated astrocytes, [3H]DA uptake increased in a time-dependent manner until 48 h, and declined after 72 h in both the presence and absence of Na+. In EGF-treated astrocytes, [3H]DA uptake increased in a time-dependent manner until 72 h in both the presence and absence of Na +. This enhancement of DA uptake induced by EGF or bFGF was significantly inhibited when the cells were cultured with actinomycin D, cycloheximide, or brefeldin A. Actinomycin D and brefeldin A also significantly inhibited the basal uptake of [3H]DA into astrocytes. These results suggest the existence of Na(+)-dependent and Na(+)-independent DA uptake in cultured rat astrocytes, and that EGF or bFGF might stimulate the expression and translocation of the extraneuronal DA transporter.

Protective effect of citalopram against the attenuation of the alpha 1-potentiation of cAMP formation in Fischer 344 strain rats.

We investigated the effects of citalopram, a selective serotonin reuptake inhibitor (SSRI), using an animal model for a depressive state. In Fischer 344 rats, known as emotional animals, repeated stress by twice-daily intraperitoneal (i.p.) saline injections for 14 days elicited a depressive state characterized by a decreased open-field activity and a prolonged immobility during the tail-suspension test. Concomitantly, suppression of norepinephrine (NE)-induced cAMP formation was found in the cerebral cortical slices of the stress-exposed rats without changes in adrenergic alpha 1- or beta-receptors. The difference in cAMP formation between the intact and the stress groups was totally abolished under the blockade of the alpha 1-receptor system or by the stimulation with isoproterenol or forskolin, whereas the suppressed response in the stress group was also observed in combination with isoproterenol and phenylephrine. From these results, we confirmed that the potentiation of the beta-receptor-stimulated cAMP formation by the alpha 1-receptor is attenuated following repeated stress. Chronic i.p. administration of citalopram dissolved in saline improved both the suppressed open-field activity and the prolonged immobility in the tail-suspension test. The animals treated with citalopram exhibited a comparable alpha 1-potentiation effect as observed in the intact rats. However, another SSRI, paroxetine, was less effective on the attenuation of the alpha 1-potentiation in spite of its behavioral improvement in the depressive state. These findings suggest that citalopram has a protective effect against the repeated stress-induced depressive state by mechanisms besides the serotonin reuptake inhibition.

Ca(2+)-dependent enhancement of [3H]noradrenaline uptake in PC12 cells through calmodulin-dependent kinases.

Ca(2+)-dependent regulation of [3H]noradrenaline ([3H]NA) uptake through the NA transporter was studied using PC12 cells. Preincubation for 10 min in the presence of 0.3-10 mM ca2+ in Krebs-Ringer (KR) buffer induced marked enhancement of the uptake (at 1 mM Ca2+, 6.6 times greater than that observed in the absence of Ca2+), which reflected both an increase in Vmax and a decrease in K(m) of the uptake process. Preincubation with 1 mM Ca2+ also induced a significant increase in the Bmax and Kd of [3H]desipramine binding. The uptake was still enhanced after washing cells with Ca(2+)-free buffer following preincubation with 1 mM Ca2+. 1-[N, O-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-c hlo rocinnamyl) -N-methylbenzylamine (KN-93) (inhibitors of Ca2+/calmodulin-dependent kinase II), N-(6-aminohexyl)-5-chloro-1-naphthalenesulonamide (W-7) (a calmodulin antagonist), wortmannin (a myosin light chain kinase inhibitor) significantly reduced Ca(2+)-dependent enhancement of the uptake. Mycalolide B (an inhibitor of actin-myosin interaction) also inhibited the enhancement. Although calphostin C (a protein kinase C (PKC) inhibitor) did not affect the enhancement, 12-o-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake. A synthetic peptide with a sequence (KKVIYKFFS579 IRGSLW) contained in the intracellular COOH-terminal domain of a rat NA transporter was phosphorylated by purified brain Ca2+/calmodulin-dependent protein kinase II. These results suggest that Ca(2+)-dependent enhancement of the [3H]NA uptake in PC12 cells are mediated by activation of calmodulin-dependent protein kinases, probably through stimulation of translocation of the NA transporter to the plasma membrane and/or direct phosphorylation of the transporter itself.

Changes in glutamate receptors, c-fos mRNA expression and activator protein-1 (AP-1) DNA binding activity in the brain of phenobarbital-dependent and -withdrawn rats.

We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [3H(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin e (MK-801), an antagonist of N-methyl-D-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [3H]MK-801 binding in the hippocampus and [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.

Possible involvement of calmodulin-dependent kinases in Ca(2+)-dependent enhancement of [3H]5-hydroxytryptamine uptake in rat cortex.

Effects of Ca2+ on [3H]5-hydroxytryptamine (5-HT) uptake into rat cortical synaptosomes were studied. The uptake was enhanced in the presence of Ca2+ in Krebs-Ringer medium and the uptake at 0.3-5 mM Ca2+ was 2.4-2.7 times greater than that observed in the absence of Ca2+. The maximal increase at the concentration of 1 mM Ca2+ was achieved after 2 min preincubation. Ca(2+)-dependent enhancement of the [3H]5-HT uptake reflected an increase in Vmax of the uptake process. However, Kd and Bmax values for [3H]paroxetine were not significantly changed in the presence of 1 mM Ca2+ compared with Ca(2+)-free condition. On the other hand, uptake was still enhanced after synaptosomes were washed with Ca(2+)-free after preincubation with 1 mM Ca2+. Staurosporine (a protein kinase C inhibitor) and wortmannin (a myosin light chain kinase inhibitor) did not affect Ca(2+)-dependent enhancement of the uptake, whereas 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazin e (KN-62, an inhibitor of Ca2+ /calmodulin-dependent kinase II) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a calmodulin antagonist) significantly reduced it. Moreover, L-type, but not P- or N-type, voltage-dependent Ca(2+)-channel blockers suppressed enhancement of the uptake. These results indicate that Ca(2+)-dependent enhancement of [3H]5-HT uptake is mediated by activation of calmodulin-dependent protein kinases, suggesting a possibility of calmodulin-dependent regulation of in vivo 5-HT uptake.

Blood coagulability and fibrinolysis in streptozotocin-induced diabetic rats.

Changes in coagulation and fibrinolysis in the plasma (in vivo) and hepatocytes (ex vivo) were studied using hyperglycemic rats. Hyperglycemia was induced by intravenous injection of 50 mg/kg streptozotocin (STZ). Eight weeks after the injection, we observed increases in thrombin-antithrombin III complex and tissue type plasminogen activator activity, decreases in plasma levels of antithrombin III, plasminogen and alpha2-plasmin inhibitor, and significant shortening of activated partial thromboplastin time. In freshly isolated or cultured hepatocytes from STZ-induced hyperglycemic rats, concentrations of proteins related to coagulation were increased. An increase in alanine-aminotransferase leakage and decreases in the levels of amylase, triglycerides and phospholipids were observed in the culture medium of hepatocytes from STZ treated rats. In vivo study revealed that STZ-induced subchronic diabetes induced imbalance between coagulation and fibrinolysis, and ex vivo study in hepatocytes from STZ-treated rats showed membrane degeneration and reduction in amylase synthesis, while protein synthesis related to coagulation was not inhibited. These results suggest that, despite vulnerability of liver cells from STZ treated rats, coagulation activity in the liver is retained and rather enhanced in STZ-induced hyperglycemic rats, which may contribute to the promotion of atherosclerosis.

Effects of imipramine and sertraline on protein kinase activity in rat frontal cortex.

Three-week administration of sertraline or imipramine to rats (10 mg/kg, intraperitoneally, twice a day) increased ex vivo cyclic AMP-dependent protein kinase activity in the soluble but not in the particulate fraction of the frontal cortex. However, cyclic AMP-dependent protein kinase activity was not affected in either fraction of the parietotemporal cortex and hippocampus. Neither antidepressant altered protein kinase C activity in the soluble and particulate fractions or Ca2+/calmodulin-dependent protein kinase II activity in the frontal cortex. Therefore, sertraline and imipramine both selectively enhance cyclic AMP-dependent protein kinase activity in the frontal cortex. This enhancement might be involved in their biochemical mechanisms.

Effects of various dopamine uptake inhibitors on striatal extracellular dopamine levels and behaviours in rats.

In vivo central effects of some dopamine uptake inhibitors were evaluated in both brain microdialysis and behavioural studies in rats, and compared with their in vitro affinities to dopamine uptake sites. IC50 values of GBR12909 (1-[2- bis(4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl)piperazine), diclofensine, mazindol, amfonelic acid and nomifensine for inhibiting 1 nM [3H]GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) binding to rat striatal membrane were 7.0, 36, 81, 187 and 290 nM, respectively. In the brain microdialysis study, dopamine levels in the striatal dialysates were increased to 16.3- (GBR12909), 14.1- (nomifensine), 4.8- (diclofensine) and 1.9-fold (amfonelic acid) the respective basal levels 40-60 min after i.p. administration (0.1 mmol/kg) and thereafter decreased slowly but remained at the elevated levels for a further 3 h, while mazindol gradually increased dopamine levels though less pronouncedly than others (1.7-fold 200 min after administration). Remarkable and comparable stereotyped behaviours (licking and forepaw treading) were continuously observed at least for 3 h after administration of GBR12909, nomifensine and amfonelic acid, while stereotypies induced by diclofensine and mazindol were moderate and marginal, respectively. In vivo potencies of dopamine uptake inhibitors to increase the extracellular dopamine levels in the striatum tended to correlate with their in vitro affinities to dopamine uptake sites except in the case of nomifensine, and correlated significantly with their potencies to induce stereotyped behaviours except in the case of amfonelic acid. Based on these findings, pharmacological characteristics of these dopamine uptake inhibitors are discussed.

Regulation of serotonin transporter gene expression in human glial cells by growth factors.

The aims of this study were to identify monoamine transporters expressed in human glial cells, and to examine the regulation of their expression by stress-related growth factors. The expression of serotonin transporter mRNA was detected by reverse transcriptase-polymerase chain reaction in normal human astrocytes, whereas the dopamine transporter (DAT) and the norepinephrine transporter (NET) were not detected. The cDNA sequence of the "glial" serotonin transporter in astrocytes was consistent with that reported for the "neuronal" serotonin transporter (SERT). Moreover, we also demonstrated SERT expression in glial fibrillary acidic protein-positive cells by immunocytochemical staining in normal human astrocytes. Serotonin transporter gene expression was also detected in glioma-derived cell lines (A172, KG-1-C and KGK). Addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) for 2 days increased serotonin transporter gene expression in astrocytes and JAR (human choriocarcinoma cell line). Basic fibroblast growth factor, but not epidermal growth factor, increased specific [3H]serotonin uptake in astrocytes in a time (1-4 days)- and concentration (20-100 ng/ml)-dependent manner. The expression of genes for basic fibroblast growth factor and epidermal growth factor receptors was detected in astrocytes. These findings suggest that the expression of the serotonin transporter in human glial cells is positively regulated by basic fibroblast growth factor.

Glutamate-stimulated proliferation of rat retinal pigment epithelial cells.

We investigated the effects of glutamate on cell proliferation and the expression of basic fibroblast growth factor (bFGF) and its receptor (FGF-R1) mRNA in cultured rat retinal pigment epithelial (RPE) cells. The number of primary RPE cells was significantly higher after treatment with 0.2 to 1.0 mM glutamate (maximum at 1.0 mM) for 7 days than in controls. Glutamate-stimulated cell proliferation was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not by 6,7-dinitroquinoxaline-2,3-dione or L(+)-2-amino-3-phosphonopropionic acid. Proliferation was increased to a similar extent by N-methyl-D-aspartate (NMDA), but not by kainate, alpha-amino-3-hydroxy-3-methyl-4-isoxazolepropionic acid or trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. NMDA-receptor-like immunoreactivity was detected in most cells cultured. Treatment of cells with glutamate increased the level of bFGF mRNA and, to a lesser extent, that of FGF-R1 mRNA, which peaked 2 and 4 days, respectively, after glutamate was added. The increase in bFGF mRNA induced by glutamate was inhibited by MK-801. These findings suggest that glutamate might stimulate proliferation of RPE cells through activation of NMDA receptors and expression of bFGF and further suggest that glutamate may be involved in the proliferative changes of RPE cells in retinal wound healing.