Simultaneous loss of phospholipase Cδ1 and phospholipase Cδ3 causes cardiomyocyte apoptosis and cardiomyopathy.

Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover. Among 13 PLC isozymes, PLCδ1 and PLCδ3 share high sequence homology and similar tissue distribution, and are expected to have functional redundancy in many tissues. We previously reported that the simultaneous loss of PLCδ1 and PLCδ3 caused embryonic lethality because of excessive apoptosis and impaired vascularization of the placenta. Prenatal death of PLCδ1/PLCδ3 double-knockout mice hampered our investigation of the roles of these genes in adult animals. Here, we generated PLCδ1/PLCδ3 double-knockout mice that expressed PLCδ1 in extra-embryonic tissues (cDKO mice) to escape embryonic lethality. The cDKO mice were born at the expected Mendelian ratio, which indicated that the simultaneous loss of PLCδ1 and PLCδ3 in the embryo proper did not impair embryonic development. However, half of the cDKO mice died prematurely. In addition, the surviving cDKO mice spontaneously showed cardiac abnormalities, such as increased heart weight/tibial length ratios, impaired cardiac function, cardiac fibrosis, dilation, and hypertrophy. Predating these abnormalities, excessive apoptosis of their cardiomyocytes was observed. In addition, siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 increased apoptosis in differentiated-H9c2 cardiomyoblasts. Activation of Akt and protein kinase C (PKC) θ was impaired in the hearts of the cDKO mice. siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 also decreased activated Akt and PKCθ in differentiated-H9c2 cardiomyoblasts. These results indicate that PLCδ1 and PLCδ3 are required for cardiomyocyte survival and normal cardiac function.

Br-DIF-1 accelerates dimethyl sulphoxide-induced differentiation of P19CL6 embryonic carcinoma cells into cardiomyocytes.

BACKGROUND AND PURPOSE: Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells. EXPERIMENTAL APPROACH: P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca(2+) channel blocker. KEY RESULTS: Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca(2+) channel blocker; 100 nM) and a small interfering RNA for the T-type Ca(2+) channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. CONCLUSIONS AND IMPLICATIONS: Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca(2+) channel gene.

Cerebrospinal fluid metabolite and nigrostriatal dopaminergic function in Parkinson's disease.

OBJECTIVES: To evaluate the association between cerebrospinal fluid (CSF) homovanillic acid (HVA) concentrations and nigrostriatal dopaminergic function assessed by positron emission tomography (PET) imaging with carbon-11-labeled 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane ((11)C-CFT), which can measure the dopamine transporter (DAT) density, in Parkinson's disease (PD). METHODS: (11)C-CFT PET scans and CSF examinations were performed on 21 patients with PD, and six patients with non-parkinsonian syndromes (NPS) as a control group. RESULTS: In the PD group, CSF HVA concentrations were significantly correlated with the striatal uptake of (11)C-CFT (r = 0.76, P < 0.01). However, in the NPS group, two indices were within the normal range. CONCLUSIONS: In PD, CSF HVA concentrations correlate with nigrostriatal dopaminergic function. Therefore, CSF HVA concentrations may be an additional surrogate marker for estimating the remaining nigrostriatal dopaminergic function in case that DAT imaging is unavailable.

Significance of radio-pathological correlations: differentiating severe central nervous system infection from acute embolic infarction.

We describe two educational autopsy cases of severe central nervous system (CNS) infection and septic emboli, such cases having been difficult to differentiate from acute infarctions via emergency MR imaging studies. We briefly discuss the pathology and MR findings along with radiopathological correlation.

[Quantitative evaluation of the burden of those giving home care for senile dementia of Alzheimer type subjects using the burnout scale of pines].

We quantitatively measured the physical and psychological burden of caregivers of 25 patients with senile dementia of Alzheimer type (SDAT). The Barthel Index (BADL, full score: 20 points) and the caregiver burden in terms of physical symptoms correlated well (r = -0.964, p < 0.001), as did the degree of abnormal behavior and caregiver burden in terms of psychological symptoms (r = 0.946, p < 0.001). The correlation with the burnout scale (BOS) of Pines was best when both factors of psychological and physical symptoms were included. The correlation between BOS and the caregiver burden in terms of both physical and psychological symptoms was r = 0.874, p < 0.001, and the correlation between BOS and "the degree of abnormal behavior" +(20- "BADL") was r = 0.853, p < 0.001. The burden in terms of physical symptoms increased as the BADL score decreased, but the burden in terms of psychological symptoms increased initially and decreased in the last phase of the disease. We conclude that the BOS score of SDAT caregivers was stable in the initial phase, then increased rapidly, thereafter preserved high, and dropped rapidly as the BADL score decreased.

[Antihypertensive therapy in patients with stroke].

Most patients with acute ischemic stroke do not need antihypertensive therapy, because the rapid lowering of blood pressure (BP) may reduce cerebral blood flow due to impaired cerebral autoregulation. In patients with severe hypertension, or associated with other complications (hemorrhagic transformation, myocardial infarction, renal failure or dissection of the aorta), antihypertensive therapy should be done cautiously. In chronic phase, the optimal BP level for the prevention of stroke recurrence remains unclear. The presence of the J-curve phenomenon is still controversial. The several large scale trials are now in progress to determine the optimal BP level for the secondary prevention in stroke patients.

The effects of short-term blood pressure variability and nighttime blood pressure levels on cognitive function.

We investigated the relationship between 24-h blood pressure (BP) and cognitive function. We performed the Hasegawa Dementia Scale Revised (HDSR), the Mini-Mental State Examination (MMSE), and the Raven's Coloured Progressive Matrices Test (RCPM) in 88 subjects (71+/-9 years) with no history of stroke. Ambulatory BP was non-invasively measured using a TM2421 for 24 h in all patients. Whereas 90% of the scores converged into a narrow range between 25 and 30 points in the HDSR and the MMSE tests, the RCPM score was widely distributed, ranging from 9 to 36 points. The subjects were therefore divided into three groups of > or =25, 26-30, and 31-36 according to their RCPM scores. Subjects with lower scores were significantly associated with increased short-term BP variability during the daytime (p<0.05) and had a tendency toward higher nighttime SBP (p=0.05) compared with those with higher scores. Increased short-term variability of daytime BP and high nighttime systolic BP were associated with cognitive impairment as assessed by the RCPM. The RCPM, which can assess the capacity for judgment through visual information processing, may detect earlier stages of cognitive impairment related to high BP. To prevent a deterioration of cognitive function, strict control of nighttime BP and suppression of short-term BP variability are thus necessary.

NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia.

Although diverse peptides are known to affect invertebrate cardiac activity, the peptidergic regulation of the cardiovascular system of Aplysia is still poorly understood. Asn-D-Trp-Phe-NH(2) (NdWFamide) is a recently purified cardioactive peptide in Aplysia. Pharmacological experiments showed that NdWFamide was one of the most potent cardioexcitatory peptides among the known endogenous cardioactive peptides in Aplysia. NdWFamide-immunopositive neuronal processes were abundant in the cardiovascular region of Aplysia, and many of them originated from neurosecretory cells in the abdominal ganglion (R3-R13 cells). The data suggest that NdWFamide is a cardioexcitatory peptide utilized by R3-R13 cells of Aplysia.

Tri-n-butyltin-induced change in cellular level of glutathione in rat thymocytes: a flow cytometric study.

Since some of organotins, accumulated in edible mollusks of aquatic environments, exert a variety of toxic actions on experimental animals, it causes concern for the health of humans. We examined the effects of tri-n-butyltin chloride (TBT) and other organotins (triethyltin chloride, trimethyltin chloride, triphenyltin chloride and tetrabutyltin) on cellular content of glutathione (GSH) in rat thymocytes using a flow cytometer to further characterize the toxicity of TBT. When the cells were incubated with TBT at concentrations of 3 nM or more for 15 min, the cellular content of GSH dose-dependently decreased. However, it completely or partly recovered until 180 min even in the continued presence of TBT. This recovery was temperature-sensitive, suggesting an involvement of metabolic process. The efficacy of TBT to decrease the cellular content of GSH was greater than those of other organotins. Results suggest that TBT and some organotins at environmentally relevant (nanomolar) concentrations significantly reduce the cellular content of GSH, suggesting that they increase the vulnerability to some biological and chemical insults.

Impairment and restoration of the endothelial blood-brain barrier in the rat cerebral infarction model assessed by expression of endothelial barrier antigen immunoreactivity.

Endothelial barrier antigen (EBA) can be used to detect the blood-brain barrier in the central nervous system of rats. This study investigated the temporal profile of antigen expression in cerebral vessels after infarction and assessed the relationship between re-establishment of this antigen in newly formed vessels and astrocytes around these vessels. Rats were subjected to cerebral ischemia for 2 h by the intraluminal thread method, then killed after 1, 3, 7, 14 and 28 days. Perfusion-fixed paraffin-embedded brains were immunostained for detection of EBA and glial fibrillary acidic protein (GFAP) by the streptavidin-biotin-peroxidase complex method. EBA immunostaining in vessels in the infarcted lesion was reduced at day 1 and had almost disappeared by day 3. Newly formed vessels were found from day 3, but were not stained at day 7. However, these new vessels were weakly stained at day 14 and definitely stained at day 28. GFAP immunostaining was completely negative around these proliferating vessels. The temporal profile of disappearance and re-expression of EBA in cerebral infarcted lesion may be associated with aggravation and improvement of brain edema, although barrier permeability was not explored in this study. The expression of this antigen has no relationship to the formation of astrocyte/endothelial contacts.

Biochemical and physiological properties of pedicellarial lectins from the toxopneustid sea urchins.

Pedicellarial lectins (SUL-I, SUL-II, and TGL-I) were purified from the toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla using gel filtration chromatography, affinity chromatography, and reverse-phase HPLC. SUL-I (Nakagawa et al., 1996) and SUL-II from the large globiferous pedicellariae of T. pileolus are D-galactose-binding lectins with molecular masses of 32 kDa and 23 kDa, respectively; while TGL-I from the globiferous pedicellariae of T. gratilla is a Ca(2+)-independent heparin-binding lectin with a molecular mass of 23 kDa. SUL-I induced mitogenic stimulation on murine splenocytes but TGL-I did not. At higher dose ranges SUL-I exhibited inhibitory effects on the cells. The dual response to SUL-I was effectively inhibited by D-galactose. SUL-I enhanced norepinephrine-induced contraction of isolated rat mesenteric artery with endothelium. When endothelium was removed from the artery, acetylcholine did not relax the norepinephrine-induced contraction. In the same artery the enhancing effect of the contraction by SUL-I was abolished, suggesting that SUL-I acts on the endothelium of mesenteric artery, and may release prostanoids. The present results suggest an extracellular function for SUL-I that may have wide-ranging effects in physiological processes. The primary role of pedicellarial lectins from T. pileolus and T. gratilla might be defense against a foreign body.

Exposure of rat thymocytes to hydrogen peroxide increases annexin V binding to membranes: inhibitory actions of deferoxamine and quercetin.

Effects of hydrogen peroxide (H(2)O(2)) on rat thymocytes were examined, using a flow cytometer and three fluorescent probes, annexin V-fluorescein isothiocyanate (annexin V-FITC) for detecting phosphatidylserine expressed on the membrane surface, ethidium bromide for estimating dead cells, and fluo-3-acetoxymethyl ester (fluo-3-AM) for monitoring changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), to characterize H(2)O(2)-induced cytotoxicity. Exposure to H(2)O(2) (30 microM or more) increased the number of annexin V-positive live cells dose- and time-dependently while the number of dead cells increased at concentrations of 1 mM or more. H(2)O(2) (30 microM or more) increased [Ca(2+)](i) in a dose-dependent manner. Threshold concentration of H(2)O(2) to increase [Ca(2+)](i) was similar to that to increase annexin V binding to membranes. The H(2)O(2)-induced change in cell membranes was attenuated under Ca(2+)-free conditions. Therefore, it is likely that Ca(2+) is involved in the H(2)O(2)-induced cytotoxicity. Deferoxamine was effective to protect the cells suffering from H(2)O(2)-induced oxidative stress, suggesting a contribution of hydroxyl radicals generated by the Fenton reaction. Quercetin also exerted a potent protective action on cells suffering from H(2)O(2)-induced oxidative stress. The results indicate that the exposure of rat thymocytes to H(2)O(2) at micromolar concentrations increases annexin V binding to cell membranes in a Ca(2+)-dependent manner, suggesting the possibility that the oxidative stress caused by H(2)O(2) (and/or hydroxyl radicals) induces apoptosis via increasing [Ca(2+)](i).

Cytotoxic actions of FTY720, a novel immunosuppressant, on thymocytes and brain neurons dissociated from the rat.

Effects of FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol HCl), a novel immunosuppressant, were examined on neurons and thymocytes respectively dissociated from rat brains and thymus glands using a flow cytometer to see if FTY720 exerts cytotoxic actions not only on spleen cells as previously reported but also on the other cells. FTY720 at a concentration of 10 microM deteriorated almost all of the thymocytes, while it was not the case for brain neurons. FTY720 increased the intracellular concentration of Ca2+ ([Ca2+]i) of thymocytes in both the presence and absence of external Ca2+, although the [Ca2+]i increased by FTY720 in the presence of external Ca2+ was much greater than that in the absence of external Ca2+. Thus, FTY720 may increase the membrane permeability of Ca2+ and release Ca2+ from intracellular Ca2+ stores in thymocytes. Furthermore, the number of thymocytes stained with ethidium, a dye impermeant to intact membranes, time-dependently increased after drug application. Therefore, FTY720 at concentrations of 3 - 10 microM non-specifically increases the membrane permeability of thymocytes, resulting in necrotic cell death, although FTY720 at micromolar concentrations was reported to induce apoptosis of spleen cells.

Cerebrospinal fluid homovanillic acid levels are not reduced in early corticobasal degeneration.

To investigate the contribution of nigral degeneration to the development of parkinsonism in the early stages of corticobasal degeneration (CBD), we measured the cerebrospinal fluid (CSF) levels of homovanillic acid (HVA) in patients with early CBD (n = 5), and compared the levels with those in patients with early Parkinson's disease (n = 11) and in normal subjects (n = 13). The mean CSF HVA level in the early CBD group (33.1 +/- 6.0 ng/ml) did not differ significantly from that in the control group (37.1 +/- 12.7 ng/ml), whereas that in the early Parkinson's disease group (19.0 +/- 7.6 ng/ml) was reduced significantly (P < 0.001). This result suggests that neuronal cell loss in the substantia nigra and presynaptic nigrostriatal dopaminergic neuron dysfunction are mild in the early stages of CBD.

[Gait disturbance without motor and sensory involvement in cervical myelopathy--clinical course and radiological findings].

Among 100 patients with cervical myelopathy, we found 7 patients with gait disturbance in which motor or sensory involvements were absent (group A). In these patients, the gait was unsteady, but not ataxic nor spastic. Some of them showed mild hyperreflexia, but no one had Romberg's sign. We compared the effect of neck traction and radiological findings in group A with these in 25 patients who had gait disorders as well as other symptoms associated with cervical myelopathy (group B). Mean age in group A (mean 83.9 +/- 7.9 ys) was older than that in group B (mean 76.6 +/- 5.7 ys). Gait disorders improved in 6 cases of group A (86%), and 5 cases in group B (20%) by conservative therapy of Glisson's traction. On plain X-ray examination, the physiologic lordosis of cervical spine was preserved, the cervical canal diameters from C2 to C7 were more wide, and the number of intervertebral excessive mobility had tendency to be less in group A than in group B. Cervical MRI indicated that the number of intervertebral spinal compression was less in group A than in group B. In the cervical myelopathy, there was a type showing only gait disturbance which was characterized by the good response to Glisson's neck traction, and by the preserved physiological lordosis, relatively wide spinal canal, and slight intervertebral compression.

[Familial parkinsonism: different clinical phenotype between a mother and her daughter].

We reported a familial case of parkinsonism in which clinical features are different between a mother and her daughter. The mother developed rigidity and bradykinesia at age 76. Her symptoms were slowly progressive, and were responsive to levodopa. Thus, she was diagnosed as having Parkinson's disease. In contrast, her daughter developed postural instability as well as rigidity and bradykinesia at age 53 in rapid progression. In addition, she also developed pyramidal signs and autonomic failures, including orthostatic hypotension and urinary incontinence. The disease phenotype of the daughter resembled that of striatonigral degeneration (SND). However, PET studies showed normal binding of 11C-N-methyl-spiperone (NMSP) in the striatum, suggesting that the daughter was also affected with Parkinson's disease.

Synthesis and secretion of active alpha 1-antichymotrypsin by murine primary astrocytes.

Activated astrocytes have been identified as the main source of the serine protease inhibitor alpha 1-antichymotrypsin (ACT), an acute phase protein that is tightly associated with amyloid plaques in Alzheimer's disease (AD) and in normal aged human and monkey brain. We analyzed the synthesis of ACT by cultured murine astrocytes in vitro. The murine astrocytes expressed an ACT-like antigen that crossreacted with antibodies to human ACT. The murine ACT-like protein is secreted by the astrocytes and is able to form an SDS-resistant complex with the serine protease cathepsin G, indicating that the secreted ACT is biologically active. We conclude that cultured primary astrocytes synthesize and secrete murine ACT in an active form. We, therefore, suggest that the ACT present within AD plaques is locally derived from plaque-associated activated astrocytes as a part of a glia-mediated local inflammatory response that is associated with the neurodegeneration seen in AD.