Involvement of the Thalamus, Hippocampus, and Brainstem in Hypsarrhythmia of West Syndrome: Simultaneous Recordings of Electroencephalography and fMRI Study.

BACKGROUND AND PURPOSE: West syndrome is a developmental and epileptic encephalopathy characterized by epileptic spasms, neurodevelopmental regression, and a specific EEG pattern called hypsarrhythmia. Our aim was to investigate the brain activities related to hypsarrhythmia at onset and focal epileptiform discharges in the remote period in children with West syndrome using simultaneous electroencephalography and fMRI recordings. MATERIALS AND METHODS: Fourteen children with West syndrome underwent simultaneous electroencephalography and fMRI at the onset of West syndrome. Statistically significant blood oxygen level-dependent responses related to hypsarrhythmia were analyzed using an event-related design of 4 hemodynamic response functions with peaks at 3, 5, 7, and 9 seconds after the onset of each event. Six of 14 children had focal epileptiform discharges after treatment and underwent simultaneous electroencephalography and fMRI from 12 to 25 months of age. RESULTS: At onset, positive blood oxygen level-dependent responses were seen in the brainstem (14/14 patients), thalami (13/14), basal ganglia (13/14), and hippocampi (13/14), in addition to multiple cerebral cortices. Group analysis using hemodynamic response functions with peaks at 3, 5, and 7 seconds showed positive blood oxygen level-dependent responses in the brainstem, thalamus, and hippocampus, while positive blood oxygen level-dependent responses in multiple cerebral cortices were seen using hemodynamic response functions with peaks at 5 and 7 seconds. In the remote period, 3 of 6 children had focal epileptiform discharge-related positive blood oxygen level-dependent responses in the thalamus, hippocampus, and brainstem. CONCLUSIONS: Positive blood oxygen level-dependent responses with hypsarrhythmia appeared in the brainstem, thalamus, and hippocampus on earlier hemodynamic response functions than the cerebral cortices, suggesting the propagation of epileptogenic activities from the deep brain structures to the neocortices. Activation of the hippocampus, thalamus, and brainstem was still seen in half of the patients with focal epileptiform discharges after adrenocorticotropic hormone therapy.

The pathogenesis of experimental membranous glomerulonephritis induced with homologous nephritogenic tubular antigen.

The renal tubular epithelial antigen (Tub-Ag) of rats was solublized by Pronase and purified by gel filtration and acrylamide gel electrophoresis. Purified Tub-Ag was a glycoprotein with S20,W value of 8.4. Utilizing radiolabeled Tug-Ag, a sensitive radioimmunoassay for Tub-Ag and homologous antibody (anti-Tub-Ag) was developed. Tub-Ag activity associated with a protein of the same molecular size was demonstrated in the serum, as well as in Pronase extracts of all the organs tested, including kidney, liver, lung, spleen, intestine, stomach, and heart. The physiochemical properties of the Tub-Ag of rats and its distribution were essentially the same as the Tub-Ag of humans, which had been found in immune deposits in the kidney of some patients with idiopathic membranous glomerulonephritis. Rats were immunized with the purified Tub-Ag emulsified in Freund's complete adjuvant and followed for Tub-Ag and anti-Tub-Ag in the serum, as well as for proteinuria and immunohistological changes in the kidney. Serum Tub-Ag dropped sharply after 20 days, when anti-Tub-Ag appeared in the circulation. Persistent, massive proteinuria appeared still later, more than 30 days after injection, when anti-Tub-Ag disappeared and Tub-Ag reappeared in the serum of some of those rats. In others, anti-Tub-Ag in the serum persisted throughout the observation period of 90 days. The pathology of the kidney of the rats with proteinuria was that of a typical membranous glomerulonephritis; thickening of glomerular capillary walls with granular deposits of gamma-globulin and Tub-Ag was observed. On the basis of these results, Tub-Ag in the serum, probably released from cellular membranes of various organs as a physiological metabolite, is considered to maintain the pathological process in the kidney by providing the antigen continuously to form immune complexes.

Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae.

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene.

We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on beta-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--AGCTGCTTC--CGCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants delta gal14 and delta gal180 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstream activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.

Two alternative pathways of transcription initiation in the yeast negative regulatory gene GAL80.

The yeast GAL80 gene, encoding a negative regulatory protein of galactose-inducible genes, shows both constitutive and galactose-inducible expression. The inducible transcription is under the control of Gal4p, a common activator for the galactose-inducible genes, which binds to an upstream activation sequence, called UASG, spanning between -105 and -89 in the 5'-flanking region of GAL80. Here we demonstrate that the constitutive transcription started at +1, whereas the inducible transcription occurs from a set of downstream sites at +37, +47, +56, and +67. Both transcriptions were enhanced 10-fold by another UAS, whose 5' boundary is located between -195 and -185. Gal4p stimulated transcription, which depends on the TATA box located at -20, from all the downstream sites. By contrast, the constitutive transcription depended on a small region of less than 16 bp long encompassing the +1 site, which directed transcription even in the absence of both the TATA box and the UASs. When a fragment covering that region was inserted immediately upstream of the open reading frame of HIS3, the resulting gene fusion, if introduced into a his3 yeast strain, supported growth on histidine-lacking medium. We detected by gel retardation assay a protein specifically interacting with this fragment. All the transcriptions observed in the in vivo experiments were faithfully reproduced in a cell-free transcription system. From these results, we suggest that initiation of GAL80 transcription involves two alternative pathways; one is initiator dependent, and the other is Gal4p regulated and TATA dependent.

GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae.

Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p.

The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.

White Matter Abnormality Correlates with Developmental and Seizure Outcomes in West Syndrome of Unknown Etiology.

BACKGROUND AND PURPOSE: West syndrome is an epileptic encephalopathy characterized by epileptic spasms, a specific pattern on electroencephalography of hypsarrhythmia, and developmental regression. Our aim was to assess white matter abnormalities in West syndrome of unknown etiology. We hypothesized that diffusion tensor imaging reveals white matter abnormalities, especially in patients with poor seizure and developmental outcomes. MATERIALS AND METHODS: We enrolled 23 patients with new-onset West syndrome of unknown etiology. DTI was performed at 12 and 24 months of age. Fractional anisotropy images were compared with those of controls by using tract-based spatial statistics. We compared axial, radial, and mean diffusivity between patients and controls in the fractional anisotropy skeleton. We determined correlations of these parameters with developmental quotient, electroencephalography, and seizure outcomes. We also compared DTI with hypometabolism on fluorodeoxyglucose positron-emission tomography. RESULTS: At 12 months of age, patients showed widespread fractional anisotropy reductions and higher radial diffusivity in the fractional anisotropy skeleton with a significant difference on tract-based spatial statistics. The developmental quotient at 12 months of age correlated positively with fractional anisotropy and negatively with radial and mean diffusivity. Patients with seizure and abnormal findings on electroencephalography after initial treatments had lower fractional anisotropy and higher radial diffusivity. At 24 months, although tract-based spatial statistics did not show significant differences between patients and controls, tract-based spatial statistics in the 10 patients with a developmental quotient of <70 had significant fractional anisotropy reduction. In patients with unilateral temporal lobe hypometabolism on PET, tract-based spatial statistics showed greater fractional anisotropy reduction in the temporal lobe ipsilateral to the side of PET hypometabolism. CONCLUSIONS: Diffuse abnormal findings on DTI at 12 months of age suggest delayed myelination as a key factor underlying abnormal findings on DTI. Conversely, asymmetric abnormal findings on DTI at 24 months may reflect underlying focal pathologies.

Synergism of protein kinase A, protein kinase C, and myosin light-chain kinase in the secretory cascade of the pancreatic beta-cell.

Protein phosphorylation by myosin light-chain kinase (MLCK), protein kinase A, and protein kinase C (PKC) plays a positive role in insulin secretion from the pancreatic beta-cell. To investigate the underlying mechanisms, we examined intracellular distribution of the insulin granules and MLCK by immunofluorescence and immunoelectron microscopies and also investigated intracellular traffic of the granules in cultured beta-cells (MIN6) by video microscopy. Considerable parts of MLCK immunoreactivity were colocalized with the insulin granules. Subcellular fractionation of MIN6 cell extracts revealed that myosin light chain (MLC) may be distributed with the insulin-rich fractions, and immunofluorescence staining using specific antibodies against mono- and diphosphorylated MLCs depicted presence of phosphorylated MLCs in the cytoplasm, in part, with colocalization with the insulin granules. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) caused a shift of both insulin granules and MLCK to the cell periphery, which was not reproduced by the adenylate cyclase activator, forskolin. In contrast, forskolin, but not TPA, increased the granule movement. Costimulation of the beta-cell by TPA and forskolin induced drastic translocation of insulin granules and MLCK to the cell periphery, resulting in enormous potentiation of insulin release. These findings suggest that these protein kinases increase insulin granules in the ready-releasable pool by acting on different steps in the secretory cascade.

Isolation and characterization of SGE1: a yeast gene that partially suppresses the gal11 mutation in multiple copies.

Recessive mutations of GAL11 in Saccharomyces cerevisiae cause pleiotropic defects that include weak fermentation of galactose, alpha-specific sterility and slow growth on nonfermentable carbon sources. Recent experiments suggest that Gal11P functions as a "co-activator" that links transcriptional activators, such as Gal4p and Grf1p/Rap1p/Tuf1p, with the basic transcription machinery. In the present experiments we isolated a gene, SGE1, that suppresses gal11 for growth on ethidium bromide/galactose agar when the gene was present in two or more copies. The other gal11 phenotypes were not suppressed by SGE1 in the multiple-copy state. Multiple copies of SGE1 increased expression of galactose-inducible genes in gal11 yeast, presumably at the level of transcription. When SGE1 was disrupted in wild-type yeast, the expression of galactose-inducible genes decreased to 50-60% of the wild-type level, presumably due to effect on transcription. Complete DNA sequence analysis revealed that SGE1 encodes a predicted protein of 543 amino acids. SGE1-specific mRNA of 1.8 kilonucleotides was detected by Northern analysis along the direction of the open reading frame. The gene mapped near RAD56, at the right end of chromosome 16.

A genome-wide analysis of transcriptional effect of Gal11 in Saccharomyces cerevisiae: an application of "mini-array hybridization technique".

The Gal11 protein is a subunit of the Mediator complex. Biochemical as well as genetic studies have strongly suggested that Gal11 is a positive global regulator of transcription. Some reports argue that Gal11 is a negative regulator, however. Here we have adopted the "Mini-array membrane hybridization" to analyze the effect of Gal11 in a genome-wide fashion. This technique has been demonstrated to be reliable to identify genes whose expression is controlled by a specific set of genetic and/or physiological signals. Our experiments indicate that this technique is applicable to profile the gene expression in yeast grown in rich medium. Thus mRNAs of 40% of significantly expressed genes are reduced more than two fold in gal11null yeast, in which only 3% of mRNAs are increased more than two fold. These results strongly suggest that Gal11 functions globally as a positive regulator in vivo.

Controlled transcription of the yeast regulatory gene GAL80.

Transcription of the tightly linked genes GAL7-GAL10-GAL1 encoding three galactose-metabolizing enzymes in Saccharomyces cerevisiae is regulated by an interplay of the positive regulatory gene GAL4 and the negative regulatory gene GAL80. The concentration of GAL80 mRNA (determined by a quantitative blot hybridization) was nearly comparable to that of URA3 mRNA, in the wild-type yeast grown in medium with glucose or glycerol. The intracellular concentration of GAL80 mRNA increased by changing the carbon source to galactose by a factor of more than 5, in contrast to a GAL4 mRNA concentration which is essentially unaffected by galactose [Laughon and Gesteland, Proc. Natl. Acad. Sci. USA 79 (1982) 6827-6831]. The inducible expression of the GAL80 gene was suggested to involve its own product and also the product of GAL4 by using various mutations in those genes.

Transcription of the human adenovirus E1a gene in Saccharomyces cerevisiae.

The early region 1a (E1a) and its flanking sequences of human adenovirus type 5 (Ad5) have been cloned in the yeast-Escherichia coli shuttle vector YEp13 and transferred into the yeast Saccharomyces cerevisiae. The E1a-specific RNAs were produced in the transformed yeast cells. The 5' ends of these transcripts were capped but were lacking 10 to 45 nucleotides from the 5' end of the proper E1a mRNA. These transcripts terminated approx. 1000 nucleotides downstream from the proper 3' end. No splicing of the E1a-specific RNA could be detected in the yeast cells.

Expression of the human adenovirus E1a product in yeast.

We synthesized the 13S mRNA-encoded protein of the early region 1a (E1a) of human adenovirus in Saccharomyces cerevisiae under the control of the yeast GAL7 gene promoter. Similar to the case in HeLa cells, the E1a protein in yeast was phosphorylated and formed multiple bands on sodium dodecylsulfate-polyacrylamide gel electrophoresis. These bands migrated more slowly than expected from the Mr calculated on the basis of the nucleotide sequence of the gene. Synthesis of the E1a protein caused induction of a specific family of heat-shock proteins (Hsp70), which, however, did not confer heat resistance to the yeast. In addition, the E1a production resulted in an elongation of the generation time of yeast from 2.4 h to 3.9 h, which was attributed specifically to elongation of the G1 interval in the cell cycle. In the light of these findings, we suggest that the E1a protein synthesized in yeast exerts a specific function.

Core promoter elements are essential as selective determinants for function of the yeast transcription factor GAL11.

The GAL11 gene product, which copurifies with RNA polymerase II holoenzyme, is necessary for full expression of many, but not all, genes in yeast. Here we shows that the GAL11 dependence of a gene for expression is determined by the core promoter structure. In the GAL80 gene, a gal11 null mutation caused reduction of TATA-dependent transcription, but exerted no effect on initiator-mediated transcription. GAL11 stimulated TATA-dependent transcription, but did not affect the TATA-independent transcription in HIS4. GAL11 was also required for transcription mediated by a canonical TATA sequence but not by a nonconsensus TATA sequence of HIS3. These results suggest that GAL11 is specifically involved in the transcription machinery formed on the TATA element.

Yeast GAL11 protein stimulates basal transcription in a gene-specific manner by a mechanism distinct from that by DNA-bound activators.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many, if not all, genes of the yeast Saccharomyces cerevisiae. We have recently shown that GAL11-encoded protein (Gal11p) enhances basal transcription from the CYC1 promoter in a cell-free transcription system [(1993) Proc. Natl. Acad. Sci. USA 90, 8382-8386]. Here we indicate that Gal11p stimulates basal transcription in a gene-specific manner in vitro. We further suggest that the mechanism underlying the transcriptional stimulation by Gal11p is distinct from that by DNA-bound activators, since Gal11p stimulated transcription in a reaction system where activators were unable to enhance transcription due to the lack of intermediary factors.

Annexin XI may be involved in Ca2+ - or GTP-gammaS-induced insulin secretion in the pancreatic beta-cell.

The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic beta-cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin-O-permeabilized MIN6 cells was inhibited by anti-annexin XI antibody, when the release was stimulated by either Ca2+ or GTP-gammaS, but not by a protein kinase C-activating phorbol ester. Inhibition of insulin release by anti-annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic beta-cells.