Electrophysiological correlates of associative visual agnosia lesioned in the ventral pathway

Visual agnosia has been well studied by anatomical, neuropsychological and neuroimaging studies. However, functional changes in the brain have been rarely assessed by electrophysiological methods. We carried out electrophysiological examinations on a 23-year-old man with associative visual agnosia, prosopagnosia and cerebral achromatopsia to evaluate the higher brain dysfunctions of visual recognition. Electrophysiological methods consisted of achromatic, chromatic and category-specific visual evoked potentials (CS-VEPs), and event-related potentials (ERPs) with color and motion discrimination tasks. Brain magnetic resonance imaging revealed large white matter lesions in the bilateral temporo-occipital lobes involving the lingual and fusiform gyri (V4) and inferior longitudinal fasciculi due to multiple sclerosis. Examinations including CS-VEPs demonstrated dysfunctions of face and object perception while sparing semantic word perception after primary visual cortex (V1) in the ventral pathway. ERPs showed abnormal color perception in the ventral pathway with normal motion perception in the dorsal pathway. These electrophysiological findings were consistent with lesions in the ventral pathway that were detected by clinical and neuroimaging findings. Therefore, CS-VEPs and ERPs with color and motion discrimination tasks are useful methods for assessing the functional changes of visual recognition such as visual agnosia.     

Molecular mechanisms for activation of voltage-independent Ca2+ channels by endothelin-1/endothelin-A receptors

Endothelin-1 (ET-1) activates two types of Ca2+- permeable non-selective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in Chinese hamster ovary cells expressing endothelin-A receptors (CHOETAR), which couple with Gq, Gs and G12. The purpose of this study was to identify the G proteins involved in the activation of these Ca channels, using mutated ETARs with coupling to either Gq or Gs/G12 (designated ETAR(Delta)385 and SerETAR, respectively) and a dominant negative mutant of G12 (G12G228A). ETAR(Delta)385 is truncated downstream of Cys385 in the C-terminal as palmitoylation sites, whereas SerET(A)R is unpalmitoylated because of substitution of all the cysteine residues to serine (CysCys --> SerSer). ET-1 activated SOCC in CHO-ET(A)R(Delta)385. In CHO-SerET(A)R or CHO-ET(A)R pretreated with U73122, an inhibitor of phospholipase C, ET-1 activated NSCC-1. ET-1 activated SOCC in CHO-ETAR microinjected with G12G228A. Moreover, ET-1 activated NSCC-1 in CHO-ETAR treated with LY 294002, the phosphoinositide 3-kinase inhibitor. These results indicate that NSCC-1 is activated via a G12-dependent pathway, NSCC-2 via Gq/phospholipase C-dependent and G12-dependent pathways, and SOCC via a Gq-phospholipase C-dependent pathway. In addition, NSCC-2 and SOCC are stimulated by ET-1 via a phosphoinositide 3-kinase-dependent cascade, whereas NSCC-1 is stimulated via a phosphoinositide 3-kinase-independent cascade.     

Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes

The effects of polysorbate 80, a non-ionic surfactant widely used in pharmaceutical products, on rat thymocytes were examined to reveal its toxic property at the cellular level. Polysorbate 80 at concentrations of 1-100 microg/ml did not significantly affect the cell viability. This surfactant at 30 microg/ml or more augmented the intensity of fluo-3 fluorescence, indicating the increase in intracellular Ca(2+) concentration. Such an augmentation of fluo-3 fluorescence by polysorbate 80 was not seen under the Ca(2+)-free condition, suggesting that polysorbate 80 increased membrane Ca(2+) permeability. The concentration-dependent polysorbate 80 at 10 microg/ml or more attenuated the intensity of 5-chloromethylfluorescein, indicating a decrease in cellular content of glutathione by polysorbate 80. Furthermore, the agent at 1 microg/ml or more attenuated the intensity of bis-(1,3-dibutylbarbituric acid) trimethine oxonol fluorescence, being independent from the changes in membrane potential. This phenomenon indicates that polysorbate 80 at 1 microg/ml or more may attenuate the incorporation of anionic compounds into the membranes. It can be suggested that polysorbate 80 modifies some of membranes and intracellular physiological parameters without affecting the cell viability.     

Effects of fluvastatin on endothelium-derived hyperpolarizing factor- and nitric oxide-mediated relaxations in arteries of hypertensive rats

Endothelial cells release endothelium-derived hyperpolarizing factor (EDHF), as well as nitric oxide (NO). It has recently been suggested that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) improve NO-mediated endothelial function, partially independently of their cholesterol-lowering effects. It is, however, unclear whether statins improve EDHF-mediated responses. Eight-month-old stroke-prone spontaneously hypertensive rats (SHRSP) were treated with fluvastatin (10 mg/kg per day) for 1 month. Age-matched, normotensive Wistar Kyoto (WKY) rats served as controls. Both EDHF- and NO-mediated relaxations were impaired in SHRSP compared with WKY rats. Fluvastatin treatment did not affect blood pressure and serum total cholesterol. The acetylcholine (ACh)-induced, EDHF-mediated hyperpolarization in mesenteric arteries did not significantly differ between fluvastatin-treated SHRSP and untreated SHRSP and the responses in both groups were significantly smaller compared with those of WKY rats. Endothelium-derived hyperpolarizing factor-mediated relaxations, as assessed by the relaxation to ACh in mesenteric arteries contracted with noradrenaline in the presence of N(G)-nitro-l-arginine and indomethacin, were virtually absent and similar in both SHRSP groups. In contrast, NO-mediated relaxation, as assessed by the relaxation in response to ACh in rings contracted with 77 mmol/L KCl, was improved in fluvastatin-treated SHRSP compared with untreated SHRSP (maximum relaxation in control and fluvastatin groups 42.0 +/- 5.2 and 61.2 +/- 3.8%, respectively; P < 0.05). Hyperpolarization and relaxation in response to levcromakalim, an ATP-sensitive K(+) channel opener, were similar between the two SHRSP groups. These findings suggest that fluvastatin improves NO-mediated relaxation, but not EDHF-mediated hyperpolarization and relaxation, in SHRSP. Thus, the beneficial effects of the statin on endothelial function may be mainly ascribed to an improvement in the NO pathway, but not EDHF.     

Establishment of a new animal model of metabolic syndrome: SHRSP fatty (fa/fa) rats

1. We established a new animal model of metabolic syndrome, SHRSP fatty (fa/fa) rats, by crossing stroke-prone spontaneously hypertensive rats of the Izumo strain (SHRSP/Izm) to Zucker fatty (ZF) (fa/fa) rats. 2. The SHRSP fatty (fa/fa) rats have a missense mutation of the leptin receptor gene and plasma leptin concentrations are augmented. The SHRSP fatty (fa/fa) rats develop obesity and hypertension simultaneously. 3. Plasma metabolic parameters, including glucose, insulin and total cholesterol and triglyceride levels, were markedly elevated in SHRSP fatty (fa/fa) rats compared with SHRSP/Izm rats. Plasma triglyceride concentrations in SHRSP fatty (fa/fa) rats were significantly elevated compared with those in ZF (fa/fa) rats. The weight of adipose tissues in SHRSP fatty (fa/fa) rats was greater than that of SHRSP/Izm rats. The phenotype of SHRSP fatty (fa/fa) rats is similar to that of human metabolic syndrome.     

Identification of polycavernoside A as the causative agent of the fatal food poisoning resulting from ingestion of the red Alga Gracilaria edulis in the Philippines

Outbreaks of seaweed poisonings are widely spread over the pacific area. Fatal glycosidic macrolides, polycavernosides, and potent tumor promoters, aplysiatoxins, have been previously isolated from edible seaweed. During 2002-2003, three fatal poisoning incidents occurred resulting from ingestion of two edible red alga, Acanthophora specifera and Gracilaria edulis, in Philippines causing eight deaths among 36 patients. Analytical methods for polycavernosides and aplysiatoxins were first developed, and the causative toxin from G. edulis, collected during the second poisoning event on December 2, 2002, was then investigated. The semipurified toxic fraction obtained from this alga based on mouse bioassay was applied to LC-diode array detection (LC-DAD) and LC/electrospray-MS (LC/ESI-MS) analyses. Both LC-DAD and LC/MS chromatograms of this fraction suggested the presence of polycavernoside A (PA) by comparison with the authentic PA. The amount of PA in the alga was estimated as 84 and 72 nmol/kg, using the standard calibration curves for LC-DAD and for LC/ESI-MS in single ion monitoring (SIM) mode, respectively. Other polycavernoside congeners, A2, A3, and B2, and aplysiatoxin and debromoaplysiatoxin were less than the detection limit (2 nmol/kg alga, signal-to-noise ratio: 3) by LC/ESI-MS SIM analysis. In ESI-MS/MS, authentic polycavernosides showed the daughter ions corresponding to a sequential loss of fucosylxylose residues. These fragmentations were applied to LC/ESI-MS/MS for polycavernosides in selective reaction monitoring (SRM) mode. On SRM mass chromatograms, the toxic fraction from the alga showed the peaks corresponding to PA, supporting the identification of PA as the cause of poisoning of G. edulis in Philippines.     

Patterns of histopathological change determined by the point counting method and its application for the hazard assessment of respirable dust

We evaluate the morphometric point counting method (PCM) for qualitatively analyzing pulmonary inflammation and collagen deposits (i.e., fibrosis) in the assessment of the biological hazards of inhaled respirable particles at a realistic dose comparable to that of exposure in the work environment. Rats were exposed by intratracheal instillation to a 2-mg dose, which is close to the estimated overdose at which macrophage clearance is impared, of each of 3 kinds of particulate matter: crystalline silica, crocidolite asbestos, and titanium dioxide. The lung tissue was evaluated at 3 days, 1 wk, and 1, 3, and 6 mo after exposure. Digital images taken of the lung tissue after processing and staining of the lung sections were examined by the PCM under light microscopy. Evidence of inflammation along with progressive inflammatory changes occurred with crystalline silica and crocidolite, which are well-known hazardous particle types. In contrast, lung tissue from rats exposed to titanium dioxide particles demonstrated a decreasing pattern of histopathological change with increasing retention time. Differences in repair patterns of TiO(2) versus crocidolite and silica following the 2-mg dose exposure suggest that the PCM scoring system may be a useful and sensitive tool for qualitatively evaluating the biological hazards of new particle types, for which no toxicological information exists for low-dose exposure, by using the results from assessment of fibrogenic particle types (such as crocidolite and crystalline silica) as well as particle types with low toxicity (such as TiO(2)) as reference points.     

Interindividual variability in 2-hydroxylation, 3-sulfation, and 3-glucuronidation of ethynylestradiol in human liver

In the current study, we investigated interindividual variability of the 2-hydroxylation, 3-glucuronidation, and 3-sulfation of ethynylestradiol (EE2) using human liver microsomes and cytosol. Km values for the 2-hydroxylation and 3-glucuronidation in pooled liver microsomes and for the 3-sulfation in pooled liver cytosol were 3.34, 23.3, and 2.85 microM, respectively. Vmax/Km (ml/min/g liver) was highest for the 3-sulfation, followed by 2-hydroxylation, suggesting that 3-sulfation is the major metabolic pathway of EE2 in human liver. All further studies were performed at a substrate concentration of 0.1 microM. Microsomal 2-hydroxylation and 3-glucuronidation activities ranged from 0.21 to 5.02 (2.04+/-1.34, mean+/-S.D., n=35) and 0.20 to 4.84 (1.20+/-1.00, n=35) pmol/min/mg protein, respectively. Cytosolic 3-sulfation activity ranged from 4.2 to 24.3 (11.8+/-4.4, n=21) pmol/min/mg protein. All the measured enzyme activities were neither gender-related nor age-dependent, except that 2-hydroxylation was significantly higher in females than in males (p<0.05). The relative contribution of CYP3A to the 2-hydroxylation in liver microsomes was estimated from the degree of inhibition by 1 microM ketoconazole. The degrees of inhibition were between 17.8 and 78.0% (51.6+/-16.0%, n=27). These results indicate that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of EE2 and the relative contribution of CYP3A to the 2-hydroxylation of EE2 in human liver.