The Wakayama epileptic rat (WER), a new mutant exhibiting tonic-clonic seizures and absence-like seizures.

A new mutant, the Wakayama epileptic rat (WER), exhibiting both spontaneous absence-like behavior and tonic-clonic convulsions, was identified in a colony of Wistar rats. To determine clear seizure characteristics of this mutant strain, we analyzed the mode of inheritance of the convulsion and observed patterns of electroencephalogram (EEG) during the seizures. F1 progeny were produced between the founder male and normal females of the same colony. Animals were monitored through the inbreeding course to analyze genetic control of epileptic behavior. EEGs were recorded using affected animals in the F3-4 and post F13 generations. After the F2 generation, affected rats spontaneously exhibited both absence-like immobile behavior and tonic-clonic convulsions. The absence-like seizures were characterized by motor arrest and head droop. The tonic-clonic convulsions began with neck and forelimb clonus, wild jumping/running, and opisthotonic posturing, and evolved to tonic, then clonic convulsions. Most convulsion onsets occurred between 25-70 days of age. Mating experiments revealed that 0%(0/18) of the animals in F1, 10%(3/26) in F2, 17%(1/6) in backcross progeny and 86% (100/116) in progeny of crosses between epileptic rats showed tonic-clonic convulsions. Ictal cortical EEGs were characterized by 4-6 (5.1 +/- 0.4, mean +/- SD) Hz spike-and-wave complexes in the absence-like seizures and by low-voltage fast waves in the tonic-clonic convulsions. This new mutant rat spontaneously exhibited both absence-like and tonic-clonic seizures. The tonic-clonic seizure was inherited as an autosomal recessive trait with 86% incidence. Thus, the new mutant rat may become a useful model for studying human inherited epilepsies.     

Molecular phylogenetic analyses of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva

In order to elucidate the species composition of free‐floating Ulva that cause green tide in several bays in Japan, and to clarify the generic status of Ulva and Enteromorpha (Ulvales, Ulvophyceae), the nuclear encoded internal transcribed spacer (ITS) region including the 5.8S gene and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/ oxgenase (rbcL) gene sequences for 15 species were determined. Both ITS and rbcL analyses indicate that free‐floating Ulva samples are divided into four different lineages that correspond to Ulva lactuca Linnaeus, U. pertusa Kjellman, U. armoricana Dion etal. and U. fasciata Delile. These four species are distinguished by cell morphology including the arrangement of cells, the shape and size of cells and the position of chloroplasts. Molecular data also indicated that Ulva and Enteromorpha are not separated as respective monophyletic groups within a large monophyletic clade and congeneric as shown by previous molecular studies using the ITS sequences alone. This strongly suggests that these genera are congeneric and Enteromorpha should be reduced to the synonym of Ulva.     

Local blood circulation among knee extensor synergists in relation to alternate muscle activity during low-level sustained contraction.

The relation between local circulation and alternate muscle activity among knee extensor synergists was determined during low-level sustained knee extension at 2.5% of maximal voluntary contraction for 60 min in seven subjects. Blood volume of rectus femoris (RF) and vastus lateralis (VL) was assessed by using near-infrared spectroscopy. Surface electromyogram (EMG) was recorded from RF, VL, and vastus medialis (VM). Alternate muscle activity was observed between RF and either VL or VM. Cross-correlation analysis was used to investigate the relation between blood volume and integrated EMG (iEMG) sequences throughout the task. One negative peak in the cross-correlation function was seen between the iEMG and blood volume with time lag of 30-60 s, indicating that muscle activity increases (or decreases) with the decrease (or increase) in local circulation with the corresponding time lag. Two cases in the emergence of alternate muscle activities, i.e., an increase in the EMG of RF accompanied by a decline of EMG in VL (case I) and vice versa (case II) were further analyzed. The time lag between iEMG and blood volume was longer in case I than that in case II. These results were statistically significant in the RF but not in the VL. It is concluded that even during low-level sustained contraction, local circulation is modulated by the alternate muscle activity of knee extensor synergists, and a negative correlation between the muscle activity and blood volume sequences was found in only RF but not in VL.     

Diatom (Bacillariophyceae) assemblages in salt marshes of south‐central Chile: Relations with tidal inundation time and salinity

To reconstruct sea‐level history from changes in tidal environments using diatom assemblages, we need to better understand the relations among brackish diatom assemblages and changing environments along elevational gradients from diverse coastal sites. Our statistical analysis reveals relations between environmental variables and brackish benthic diatom assemblages in the little studied region of south‐central Chile. Along four transects across salt marshes at two sites, we identified 224 diatom taxa in 112 samples. Detrended canonical correspondence analysis showed that tidal exposure time index and salinity were appropriately regressed against the abundance of diatom species using unimodal‐based methods. Our tests of classical and inverse regressions of weighted average and weighted averaging partial least squares (WA‐PLS) showed that WA‐PLS resulted in the highest coefficient of determination and the lowest root‐mean square of the error of prediction. Our regression will be useful in reconstructing environmental variables from fossil diatom assemblages in Chile.     

Establishment of Vero E6 cell clones persistently infected with severe acute respiratory syndrome coronavirus

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.     

Calcium signaling and synaptic modulation: regulation of endocannabinoid-mediated synaptic modulation by calcium

Postsynaptic Ca2+ signal influences synaptic transmission through multiple mechanisms. Some of them involve retrograde messengers that are released from postsynaptic neurons in a Ca2+-dependent manner and modulate transmitter release through activation of presynaptic receptors. Recent studies have revealed essential roles of endocannabinoids in retrograde modulation of synaptic transmission. Endocannabinoid release is induced by either postsynaptic Ca2+ elevation alone or activation of postsynaptic Gq/11-coupled receptors with or without Ca2+ elevation. The former pathway is independent of phospholipase Cbeta (PLCbeta) and requires a large Ca2+ elevation to a micromolar range. The latter pathway requires PLCbeta and is facilitated by a moderate Ca2+ elevation to a submicromolar range. This facilitation is caused by Ca2+-dependency of receptor-driven PLCbeta activation. The released endocannabinoids then activate presynaptic cannabinoid receptor type 1 (CB1), and suppress transmitter release from presynaptic terminals. Both CB1 receptors and Gq/11-coupled receptors are widely distributed in the brain. Thus, the endocannabinoid-mediated retrograde modulation may be an important and widespread mechanism in the brain, by which postsynaptic events including Gq/11-coupled receptor activation and Ca2+ elevation can retrogradely influence presynaptic function.     

Up-regulated synthesis of mature-type adrenomedullin in coronary circulation immediately after reperfusion in patients with anterior acute myocardial infarction

OBJECTIVE: Levels of adrenomedullin (AM), a potent vasodilatory peptide, have been shown to increase in the early stage of acute myocardial infarction (AMI). The purpose of this study was to determine whether coronary sinus-aortic step-up of mature forms of AM is accelerated in patients with AMI after reperfusion. METHODS: The subjects were 29 consecutive patients with a first episode of anterior AMI and 10 normal controls. All patients with AMI underwent balloon reperfusion therapy within 24 h after symptom onset. Plasma levels of two molecular forms of AM (an active, mature form [AM-m] and an intermediate, inactive glycine-extended form [AM-Gly]) in the aorta and coronary sinus (CS) were measured by specific immunoradiometric assay after reperfusion. RESULTS: Plasma levels of AM-m and AM-Gly in the aorta and CS were higher in AMI patients than in controls. CS-aortic step-up of AM-m, which is an index of myocardial production of AM-m, was significantly greater in AMI patients than in controls (1.7 +/- 1.4 vs. 0.4 +/- 0.3 pmol/L, P < 0.01). However, there was no significant difference in CS-aortic step-up of AM-Gly (P = 0.30). AMI patients with left ventricular dysfunction (n = 10) had a significantly higher CS-aortic AM-m step-up than AMI patients without left ventricular dysfunction (n = 19). AM-m in the aorta and CS negatively correlated with the left ventricular ejection fraction (r = -0.50, r = -0.48, P < 0.01). CONCLUSIONS: Myocardial synthesis of AM-m is accelerated in patients with reperfused AMI, especially in patients with critical left ventricular dysfunction. Increased myocardial synthesis of active AM may protect against cardiac dysfunction, myocardial remodeling, or both after the onset of AMI.     

Hybrid lethality of cultured cells of an interspecific F1 hybrid of Nicotiana gossei Domin and N. tabacum L.

Cultured cells were established from the hypocotyl of F₁ hybrid seedlings of Nicotiana gossei Domin and N. tabacum L. The cultured cells started to die at 26°C, but not at 37°C, which is similar to what occurred in cells of the original hybrid plants. An increase in the number of cells without cytoplasmic strands and acidification of the cytoplasm followed by decomposition of the mitochondria and chloroplasts indicated that vacuolar collapse plays a central role in the execution of cell death. Oxygen but not light was required for cell death. Cellular levels of the superoxide anion and hydrogen peroxide temporarily increased during the early phase at 26°C, while no such oxidative burst was observed at 37°C. The reactive oxygen intermediates are potentially involved in the death of the hybrid cells.     

Purification, characterization, and antifungal activity of chitinases from pineapple (Ananas comosus) leaf

Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.