Putative calcium-binding domains of the Caenorhabditis elegans BK channel are dispensable for intoxication and ethanol activation.

Alcohol modulates the highly conserved, voltage- and calcium-activated potassium (BK) channel, which contributes to alcohol-mediated behaviors in species from worms to humans. Previous studies have shown that the calcium-sensitive domains, RCK1 and the Ca(2+) bowl, are required for ethanol activation of the mammalian BK channel in vitro. In the nematode Caenorhabditis elegans, ethanol activates the BK channel in vivo, and deletion of the worm BK channel, SLO-1, confers strong resistance to intoxication. To determine if the conserved RCK1 and calcium bowl domains were also critical for intoxication and basal BK channel-dependent behaviors in C. elegans, we generated transgenic worms that express mutated SLO-1 channels predicted to have the RCK1, Ca(2+) bowl or both domains rendered insensitive to calcium. As expected, mutating these domains inhibited basal function of SLO-1 in vivo as neck and body curvature of these mutants mimicked that of the BK null mutant. Unexpectedly, however, mutating these domains singly or together in SLO-1 had no effect on intoxication in C. elegans. Consistent with these behavioral results, we found that ethanol activated the SLO-1 channel in vitro with or without these domains. By contrast, in agreement with previous in vitro findings, C. elegans harboring a human BK channel with mutated calcium-sensing domains displayed resistance to intoxication. Thus, for the worm SLO-1 channel, the putative calcium-sensitive domains are critical for basal in vivo function but unnecessary for in vivo ethanol action.

The burrowing behavior of the nematode Caenorhabditis elegans: a new assay for the study of neuromuscular disorders.

The nematode Caenorhabditis elegans has been a powerful model system for the study of key muscle genes relevant to human neuromuscular function and disorders. The behavioral robustness of C. elegans, however, has hindered its use in the study of certain neuromuscular disorders because many worm models of human disease show only subtle phenotypes while crawling. By contrast, in their natural habitat, C. elegans likely spends much of the time burrowing through the soil matrix. We developed a burrowing assay to challenge motor output by placing worms in agar-filled pipettes of increasing densities. We find that burrowing involves distinct kinematics and turning strategies from crawling that vary with the properties of the substrate. We show that mutants mimicking Duchenne muscular dystrophy by lacking a functional ortholog of the dystrophin protein, DYS-1, crawl normally but are severely impaired in burrowing. Muscular degeneration in the dys-1 mutant is hastened and exacerbated by burrowing, while wild type shows no such damage. To test whether neuromuscular integrity might be compensated genetically in the dys-1 mutant, we performed a genetic screen and isolated several suppressor mutants with proficient burrowing in a dys-1 mutant background. Further study of burrowing in C. elegans will enhance the study of diseases affecting neuromuscular integrity, and will provide insights into the natural behavior of this and other nematodes.

Autoantibodies to NMDA receptor in patients with chronic forms of epilepsia partialis continua.

BACKGROUND: Antibody-mediated and cytotoxic T cell-mediated pathogenicity have been implicated as the autoimmune pathophysiologic mechanisms in Rasmussen's encephalitis. METHODS: The authors investigated autoantibodies against the NMDA glutamate receptor (GluR) epsilon2 subunit and their epitopes in serum and CSF samples from 15 patients with chronic epilepsia partialis continua (EPC), 17 with West syndrome, 10 with Lennox-Gastaut syndrome, and 11 control subjects. RESULTS: In 15 patients with chronic EPC, we detected NMDA-type GluR epsilon2 autoantibodies in histologically proven Rasmussen's encephalitis (3/3 patients), clinical Rasmussen's encephalitis (6/7 patients), acute encephalitis/encephalopathy (2/3 patients), and nonprogressive EPC (2/2 patients). Serum IgM autoantibodies were found in the early phase of EPC and became negative later in four patients. The autoantibodies were not detected in West syndrome, Lennox-Gastaut syndrome, or controls. Among 10 patients with histologically proven or clinical Rasmussen's encephalitis, epitope analyses showed that the autoantibodies were predominantly against C-terminal epitopes and rarely against N-terminal epitope, with inconsistency in profile during the courses of disease. Epitope recognition spectrum of autoantibodies was broader in CSF than in serum, and the serum or CSF profile showed an increase in number of epitopes as disease progressed in some patients. CONCLUSIONS: The presence of autoantibodies against NMDA GluR epsilon2 suggests autoimmune pathologic mechanisms but is not a hallmark of Rasmussen's encephalitis. Patients with Rasmussen's encephalitis may have autoantibodies against several neural molecules, and these autoantibodies may be produced in the CNS after cytotoxic T cell-mediated neuronal damage.

Frequent mutations of SCN1A in severe myoclonic epilepsy in infancy.

Mutations in the neuronal voltage-gated sodium channel alpha-subunit type I gene (SCN1A) were found responsible for severe myoclonic epilepsy in infancy (SMEI). The authors describe novel mutations of SCN1A in Japanese patients with SMEI. They screened 12 unrelated patients and a pair of monozygotic twins and detected 10 mutations that lead to truncation of the protein.

The homeobox gene lim-6 is required for distinct chemosensory representations in C. elegans.

The ability to discriminate between different chemical stimuli is crucial for food detection, spatial orientation and other adaptive behaviours in animals. In the nematode Caenorhabditis elegans, spatial orientation in gradients of soluble chemoattractants (chemotaxis) is controlled mainly by a single pair of chemosensory neurons. These two neurons, ASEL and ASER, are left-right homologues in terms of the disposition of their somata and processes, morphology of specialized sensory endings, synaptic partners and expression profile of many genes. However, recent gene-expression studies have revealed unexpected asymmetries between ASEL and ASER. ASEL expresses the putative receptor guanylyl cyclase genes gcy-6 and gcy-7, whereas ASER expresses gcy-5 (ref. 4). In addition, only ASEL expresses the homeobox gene lim-6, an orthologue of the human LMX1 subfamily of homeobox genes. Here we show, using laser ablation of neurons and whole-cell patch-clamp electrophysiology, that the asymmetries between ASEL and ASER extend to the functional level. ASEL is primarily sensitive to sodium, whereas ASER is primarily sensitive to chloride and potassium. Furthermore, we find that lim-6 is required for this functional asymmetry and for the ability to distinguish sodium from chloride. Thus, a homeobox gene increases the representational capacity of the nervous system by establishing asymmetric functions in a bilaterally symmetrical neuron pair.

[Role of pediatrician in surgical treatment of children with intractable seizures].

Surgery is a useful strategy in the management of intractable and disabling seizures in childhood. Identification of suitable candidates for epilepsy surgery and consideration of the timing of surgery are an important role of pediatrician in surgical treatment of epileptic children. To discuss the timing of surgery, we analyzed clinical courses of 126 patients with TLE and 33 patients with FLE who underwent epilepsy surgery. In many of them seizures continued without remission before surgery. Surgery might have been considered earlier in these cases. Thirty-five (27.8%) of 126 patients with TLE and 10 (30.3%) of 33 patients with FLE had seizure remission before surgery. In TLE, seizure remission was rare in the patients who had convulsive seizures at the onset. In conclusion, the timing of surgery should be determined on individual basis in cases with more than one remission. If deletorious effects of seizures on patient's biological and psychosocial state are evident, surgery should be considered earlier.

Tensile stress induces bone morphogenetic protein 4 in preosteoblastic and fibroblastic cells, which later differentiate into osteoblasts leading to osteogenesis in the mouse calvariae in organ culture.

Mechanical stress is an important factor controlling bone remodeling, which maintains proper bone morphology and functions. However, the mechanism by which mechanical stress is transduced into biological stimuli remains unclear. Therefore, the purpose of this study is to examine how gene expression changes with osteoblast differentiation and which cells differentiate into osteoblasts. Tensile stress was applied to the cranial suture of neonatal mouse calvaria in a culture by means of helical springs. The suture was extended gradually, displaying a marked increase in cell number including osteoblasts. A histochemical study showed that this osteoblast differentiation began in the neighborhood of the existing osteoblasts, which can be seen by 3 h. The site of osteoblast differentiation moved with time toward the center of the suture, which resulted in an extension of osteoid. Scattered areas of the extended osteoid were calcified by 48 h. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that tensile stress increased bone morphogenetic protein 4 (BMP-4) gene expression by 6 h and it remained elevated thereafter. This was caused by the induction of the gene in preosteoblastic cells in the neighborhood of osteoblasts and adjacent spindle-shaped fibroblastic cells. These changes were evident as early as 3 h and continued moving toward the center of the suture. The expression of Cbfa1/Osf-2, an osteoblast-specific transcription factor, followed that of BMP-4 and those cells positive with these genes appeared to differentiate into osteoblasts. These results suggest that BMP-4 may play a pivotal role by acting as an autocrine and a paracrine factor for recruiting osteoblasts in tensile stress-induced osteogenesis.

Tensile stress induced osteoblast differentiation and osteogenesis in mouse calvarial suture in culture: possible involvement of BMP-4 and other genes.

Mechanical stress is one of the most potent inducer of bone formation. The mechanism by which cells receive and transduce the signal into osteogenesis, however, remains unknown. Previous studies have demonstrated that mechanical stress causes changes in expression levels of many genes in osteoblasts and osteocytes both in vivo and in vitro. However, none of these changes are specific to bone cells. Moreover it is not clear which types of cells contributed to the increased osteoblasts induced by mechanical stress. The purpose of this study, therefore, was to identify which cells differentiate into osteoblasts and to examine how the expression of genes that are specific to osteogenic cells changes.

The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.

To investigate the behavioral mechanism of chemotaxis in Caenorhabditis elegans, we recorded the instantaneous position, speed, and turning rate of single worms as a function of time during chemotaxis in gradients of the attractants ammonium chloride or biotin. Analysis of turning rate showed that each worm track could be divided into periods of smooth swimming (runs) and periods of frequent turning (pirouettes). The initiation of pirouettes was correlated with the rate of change of concentration (dC/dt) but not with absolute concentration. Pirouettes were most likely to occur when a worm was heading down the gradient (dC/dt < 0) and least likely to occur when a worm was heading up the gradient (dC/dt > 0). Further analysis revealed that the average direction of movement after a pirouette was up the gradient. These observations suggest that chemotaxis is produced by a series of pirouettes that reorient the animal to the gradient. We tested this idea by imposing the correlation between pirouettes and dC/dt on a stochastic point model of worm motion. The model exhibited chemotaxis behavior in a radial gradient and also in a novel planar gradient. Thus, the pirouette model of C. elegans chemotaxis is sufficient and general.

[Clinical evaluation of sisomicin following intravenous drip infusion in respiratory tract infections].

The efficacy, safety and utility of sisomicin (SISO) followed intravenous infusion were evaluated in 35 cases with various respiratory infections. For many cases, SISO was given at a daily dosage of 100 mg, and a single dose was infused over about 1 hour. Clinical efficacy was evaluable in 28 cases including pneumonia (14 cases), bronchitis (8 cases), bronchiectasis (4 cases), pulmonary suppuration (1 case) and pulmonary abscess plus pyothorax (1 case). Almost cases had diagnosis of serious infection associated with various diseases. Clinical efficacy was evaluated as "excellent" in 2 cases, "good" in 15 cases, "fair" in 5 cases and "poor" in 6 cases, and efficacy rate in total case was 60.7%. Efficacy rate stratified by disease was calculated as 57.1% in pneumonia, 87.5% in bronchitis, 50.0% in bronchiectasis. Responses against pulmonary suppuration or pulmonary abscess with pyothorax were little or not. Bacteriologically, organisms isolated from sputum cleared in 7 out of 15 evaluable cases, thus the responses rate was 46.7%. Adverse reaction probably due to treatment observed in 2 cases with hepatic dysfunction. Blood levels of SISO at the end of infusion were ranged from 2.1 to 6.4 micrograms/ml, and no tendency of accumulation in blood after repeated infusion was showed.

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter.

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.

Gangliosides of various rat tissues: distribution of ganglio-N-tetraose-containing gangliosides and tissue-characteristic composition of gangliosides.

Gangliosides were extracted from various tissues of rat (Wistar strain, male, 3 months old) and their structures were elucidated by enzymatic and chemical procedures including the analysis by negative ion fast atom bombardment mass spectrometry. All tissues analyzed contained gangliosides in various but characteristic concentrations. GD1a was detected in the various extraneural tissues (erythrocytes, buffy coat, bone marrow, testis, spleen, and liver) in amounts corresponding to more than 30% of total lipid-bound sialic acid, and surprisingly, it was the sole ganglioside found in buffy coat. The extraneural tissues were classified into several categories according to the nature of the asialo-oligosaccharides of gangliosides as follows: (1) gangliosides with ganglio-N-tetraose were exclusively present (buffy coat and erythrocytes), (2) the concentration of ganglio-N-tetraose-containing gangliosides was higher than that of lactose-containing gangliosides (testis and bone marrow), (3) ganglio-N-tetraose-containing gangliosides amounted to 25-30% of lactose-containing gangliosides (liver and spleen), (4) ganglio-N-tetraose-containing gangliosides amounted to 7-11% of lactose-containing gangliosides (lung and stomach), (5) more than 90% of gangliosides were lactose-containing gangliosides (heart, intestine, and kidney). In addition, the following gangliosides were characteristically detected in high concentration in the following tissues: GM4 in kidney. GM2 in bone marrow, fucosyl GM1 and GM1 in erythrocytes and GM3 with 2-hydroxy fatty acid, phytosphingosine and N-glycolylneuraminic acid in intestine.

Differential reactivities of fucosyl GM1 and GM1 gangliosides on rat erythrocyte membrane revealed by analysis with anti-fucosyl GM1 and GM1 antisera.

Rat erythrocytes contained ganglio-series gangliosides, GM1, fucosyl GM1, and GD1a, in a high concentration. The concentrations of GM1, fucosyl GM1, and GD1a in rat erythrocyte ghosts were 889.0 nmol, 470.6 nmol, and 462.0 nmol per g dry weight, respectively, and the molar ratio of lipid-bound sialic acid, cholesterol and lipid-bound phosphorus was 3.1:73.9:100.0. The reactions of fucosyl GM1 and GM1 on rat erythrocytes with rabbit anti-fucosyl GM1 and anti-GM1 antisera were measured by means of haemolysis in the presence of complement and a binding assay of antibodies with a FACS after staining erythrocytes by the indirect membrane immunofluorescence technique. When measured by ELISA, anti-fucosyl GM1 antiserum was found to react almost exclusively with fucosyl GM1 with a slight cross-reaction with GM1, but anti-GM1 antiserum cross-reacted to a significant extent with asialo GM1. Rat erythrocytes were haemolyzed specifically with anti-fucosyl GM1 antiserum, but not with antisera to GM1, asialo GM1, asialo GM2, Forssman and globoside, and the haemolysis was proved to be definitely caused by the specific recognition of fucosyl GM1 on rat erythrocytes by anti-fucosyl GM1 antibody according to the haemolysis inhibition reaction using various glycosphingolipid-containing liposomes as inhibitors. In addition, although the binding of anti-fucosyl GM1 antibody on rat erythrocytes was clearly demonstrated with a FACS, anti-GM1 antibody did not bind. The observations that the haemolysis of rat erythrocytes and the binding of antibody to rat erythrocytes were found only with anti-fucosyl GM1 antiserum, and not with anti-GM1 antiserum, but that nevertheless the titer of anti-GM1 antiserum was higher than that of anti-fucosyl GM1 antiserum and GM1 on rat erythrocytes was more abundant in concentration than fucosyl GM1, seem to be a matter of great importance in assessing the specificity of anti-ganglioside antibody and the surface distribution of gangliosides on the cell.