Experimental techniques for developing new drugs acting on dementia (2)--Primary culture of mature and functional hypothalamic neurons

It has been difficult to put mature neurons of mammalian central nervous system (CNS) in vitro. Only a few reports have dealt with culture methods so far. They employed non-neuronal cell layers to make postnatal neurons attach better to a culture dish. We established a novel method for culturing mature functional hypothalamic neurons derived from 21 postnatal day rat brain without any help of non-neuronal cell layers. Nerve cells with a few processes could be isolated from sliced hypothalamic tissues by means of enzymatic and mechanical treatments. A new cell-collecting method was developed for these vulnerable cells by allowing the dissociated cell suspension to stand in a vertically held, wide-tipped syringe so that the cells were concentrated near the bottom liquid surface, from which they could be easily dropped into the medium, leaving most of the small debris in the syringe. Astrocyte conditioned medium prolonged neuronal survival, concentration dependently, for up to 28 days in vitro. This culture system may make it possible to study functions of single hypothalamic neurons.     

Atherosclerosis regression in the elderly--correlation between centrally depressed lesions and risk factors

Atherosclerotic plaque with central depression (depressed lesion) may indicate regression of atherosclerosis in the aorta. Aortic depressed lesions have a solitary elevated area of plaque with a sharply-bordered roung depression in its center and no area ulceration. This may be interpretable as a sign of regression of atherosclerosis. To clarify the pathogenesis of depressed lesion, we studied clinical risk factors such as hypercholesterolemia in patients with depressed lesions that were confirmed at autopsy. The patients were divided into 3 groups according to their total cholesterol level at autopsy: a high-risk group (> or = 220 mg/dl), a moderate-risk group (180-220 mg/dl), and a low-risk group (< or = 180 mg/dl). Depressed lesions were found in 16.4% of those in the high-risk group, in 14.6% of those in the moderate-risk group and in 69.0% of those in the low-risk group. Severe aortic atherosclerosis was found in 69.8% of the patients; 50.9% of those with severe disease were in the-low risk group. Depressed lesions were also found in those with low levels of low-density lipoprotein cholesterol (< or = 140 mg/dl), 58.8% of whom were found to have severe atherosclerosis. There was no relationship between total cholesterol level and the presence of depressed lesions. However, a clinical prevention trial may result in sufficient control of ahterosclerosis among those in the high-risk group and may also lead to regression of aortic lesions.     

Immobilization stress increases hepatic IL-6 expression in mice

Male Djungarian hamsters (Phodopus sungorus; 45 animals per group) were either sham-exposed or exposed to a sinusoidal magnetic field for 56 days (Experiment 1: 50 Hz, 450 microTesla peak; max. dB/dt = 140 mTesla s(-1); 24 hrs day(-1)). Except for day 7, no effects were observed with respect to body weights during exposure. However, testicular cell numbers were significantly increased by exposure (tetraploid (4C): p=0.022; diploid (2C): p=0.039). Rectangular magnetic fields (Experiment 2: 360 microTesla; max. dB/dt = 2.5 Tesla s(-1)) caused a significant (p<0.001) but transient suppressing effect on body weights. Significant increases were also observed in testicular cell numbers (4C: p=0.034; haploid (1C): p=0.014) and in serum melatonin (p=0.001). It is concluded that weak magnetic fields may affect reproductive and physiological functions in the mammalian species tested and that the degree of these effects depends upon the fields' gradients.     

3-D device modeling for SRAM soft-error immunity and tolerance analysis

Soft-error tolerance of static random-access memory (SRAM) devices has been predicted by using three-dimensional (3-D) and time-dependent device simulation in conjunction with circuit simulation. An inverter model developed for 3-D device simulation is described, along with the analysis of the inverters device response as a function of time. The output thus obtained was applied as an input voltage source in circuit simulation of unit SRAM cell and the stability of this bistable circuit is studied on that basis. The effects on soft-error immunity of changes in alpha-particle injection conditions and in load resistance and capacitance are described. The validity of the presented model is examined through comparison of the bit-error-rate dependence on incident angle of alpha particles to that of measured rates. To simulate the angular dependence, we introduce statistical distribution models for alpha-particle energy, position of incidence on the device surface, and angle of incident. Results of device/circuit simulation carried out with many sets of energy, position, and angle are presented. Reasonable agreement between results of simulation and experimental data without the use of adjustment parameters is demonstrated. A map of soft-error tolerance on the CR plane with critical charge Q/sub c/ as a parameter is presented and its derivation explained. An analytic expression for the tolerance is clarified by proposing an equivalent circuit model for the simulation of alpha-particle injection at the output node in an inverter circuit. Inverter modeling is shown to be essential to obtaining SRAM soft-error tolerance to high degrees of accuracy.     

Malignant histiocytosis-like B-cell lymphoma, a distinct pathologic variant of intravascular lymphomatosis: a report of five cases and review of the literature

Malignant histiocytosis (MH)-like B-cell lymphoma (BCL) is a neoplastic proliferation of large B cells clinically characterized by fever, hepatosplenomegaly, haemophagocytosis and abnormal laboratory data, without lymphadenopathy or skin lesions. Interestingly, most cases have been reported in Asian patients, and it is unclear whether MH-like BCL is biologically distinct from conventional large B-cell lymphomas. We report five Japanese patients with MH-like BCL. Biopsied specimens of bone marrow, liver and/or spleen showed infiltration of neoplastic B cells accompanied by haemophagocytosing histiocytes. Lymphoma cells were positive for CD19, CD20 and HLA-DR surface antigens, and negative for CD5 and CD10. In four cases elevated serum levels of interleukin (IL)-6 and the soluble IL-2 receptor isoform were noted, but not IL-1beta, IL-2 or tumour necrosis factor-alpha. Autopsies of two cases were pathologically diagnosed as intravascular lymphomatosis (IVL). Based on these observations, the current and nine previous cases reported as MH-like BCL in Japan were re-evaluated. They appear to form a peculiar variant of IVL, characterized by bone marrow involvement at presentation, haemophagocytic syndrome, and a rapidly aggressive clinical course, but rarely neurological complications or skin lesions. This variant may merit separate consideration because of the problems posed in the initial diagnosis and therapeutic approaches.     

Synthesis of nitrogen-rich B–C–N materials from melamine and boron trichloride

Nitrogen-rich B–C–N materials have been prepared by the reaction between melamine and boron trichloride at different temperatures. The composition of the materials was dependent on the synthesis and annealing temperatures: C6N10.8-11H9.4B1.5-1.7 (for products synthesized and annealed at 673 K), C6N9.3-9.4H3.8-3.9B2.2-2.5 (for those synthesized at 673 K and annealed at 873 K) and C6N9.2H3.6B1.2-1.3 (for those synthesized and annealed at 873 K). Fourier transform infrared spectroscopy and 13C nuclear magnetic resonance showed that the s-triazine rings from the melamine molecules were preserved in materials synthesized and annealed at 673 and 873 K. The sample obtained at 873 K had a graphite-like structure as suggested by X-ray and electron diffraction studies. The s-triazine rings were decomposed in the materials synthesized or annealed at 1223 K and the main product obtained was turbostratic boron nitride. © 1998 Chapman & Hall     

In vivo efficacy of SM-20302, a GP IIb/IIIa receptor antagonist, correlates with ex vivo platelet inhibition in heparinized blood but not in citrated blood

This study analyzed temporal changes of striatal dopamine-D2 receptor binding during the course of different extrapyramidal movement disorders using 123I-iodobenzamide (IBZM) SPECT. METHODS: Eighteen patients (9 with Parkinson's disease, 9 with parkinsonian plus syndrome) were followed for 11-53 mo. Dopamine-D2 receptor binding was assessed using 123I-IBZM SPECT at the beginning and at the end of the follow-up period. SPECT data were acquired 120 min postinjection of 3-5 mCi 123I-IBZM. A semiautomated algorithm was applied to the raw data for semiquantitative evaluation of regional cerebral receptor binding. RESULTS: Intraobserver (r = 0.992) and interobserver (r = 0.930) variance was low for the semiautomated interpretation of the SPECT examination of the dopaminergic D2 receptor binding, reflecting a highly reproducible SPECT algorithm. Mean specific dopamine-D2 receptor binding was lower in patients with parkinsonian plus syndrome compared to patients with Parkinson's disease on the initial (p < 0.001) as well as the follow-up study (p < 0.001). In patients with Parkinson's disease, we observed an unaffected receptor binding compared to a reduced binding of radiotracer in patients with parkinsonian plus syndrome during the course of the disease (p < 0.001). CONCLUSION: During the follow-up, patients with Parkinson's disease showed a constant dopamine-D2 receptor binding. In contrast, patients with parkinsonian plus syndrome revealed a decline of the binding of dopamine-D2 receptor. These findings are in agreement with histopathological data that demonstrated a preserved dopamine-D2 receptor status in patients with Parkinson's disease and a decline of the dopamine-D2 receptors in patients with parkinsonian plus syndrome. SPECT examinations using 123I-IBZM are useful for assessing dynamic changes of dopamine-D2 receptors in extrapyramidal movement disorders. Semiquantitative SPECT evaluations may provide valuable information for clinical management and prognosis of the patient with extrapyramidal movement disorders.     

AS-186 compounds, new inhibitors of acyl-CoA: cholesterol acyltransferase from Penicillium asperosporum KY1635

AS-186a, b, c, d, and g were isolated from the cultured broth of Penicillium asperosporum KY1635 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). IC50 values for the effect of AS-186a, b, c, d, and g against ACAT activity of the microsomes from cholesterol-fed rabbit liver were calculated to be 22.9, 8.2, 11.5, 12.4, and 13.9 microM, respectively. Although AS-186a, and b were identical to penicillide and purpactin A, respectively, AS-186c, d, and g were found to be new compounds.     

Filter cake forming and hot isostatic pressing for TZP-dispersed hydroxyapatite composite

The combination of a filter cake forming process and hot isostatic pressing was applied to prepare hydroxyapatite composites containing dispersed tetragonal zirconia polycrystal (TZP) with high strength and toughness. Fine TZP powder was dispersed into as-synthesized hydroxyapatite slurry, formed with the filter cake process and hot isostatically pressed at 800–1150 °C at 100 MPa for 2 h. The temperature needed for densification increased with increasing TZP content; 1100 °C was needed to fully densify the composite with 26.8 wt% TZP. No phase change was found in TZP nor in the hydroxyapatite phase up to the maximum temperature examined in hot isostatic pressing. Significant phase change was found in specimens annealed in air at 1200 °C. The strength and toughness achieved were respectively 190 MPa and 2.3 MPa m^1/2. These values were approximately 20% and 100% higher than the corresponding values for hydroxyapatite ceramics without TZP particle dispersion.     

Detection of human papillomavirus DNA sequences in oral squamous cell carcinomas and their relation to p53 and proliferating cell nuclear antigen expression

BACKGROUND: The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. METHODS: Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. RESULTS: Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Human papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-16 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. CONCLUSIONS: These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes.