Neuronal sodium-channel alpha 1-subunit mutations in generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by the presence of febrile and afebrile seizures. The first gene, GEFS1, was mapped to chromosome 19q and was identified as the sodium-channel beta1-subunit, SCN1B. A second locus on chromosome 2q, GEFS2, was recently identified as the sodium-channel alpha1-subunit, SCN1A. Single-stranded conformation analysis (SSCA) of SCN1A was performed in 53 unrelated index cases to estimate the frequency of mutations in patients with GEFS+. No mutations were found in 17 isolated cases of GEFS+. Three novel SCN1A mutations-D188V, V1353L, and I1656M-were found in 36 familial cases; of the remaining 33 families, 3 had mutations in SCN1B. On the basis of SSCA, the combined frequency of SCN1A and SCN1B mutations in familial cases of GEFS+ was found to be 17%.

Identification and validation of candidate Myb target genes

While a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene-c-myc-that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region. (C) 2001 Academic Press.

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for CA-binding protein (GABP)

Within steroid receptor heterocomplexes the large tetraticopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (HspSO) and act coordinately with HspSO to modulate receptor activity. The reversible nature of the interaction between the immunophilins and HspSO suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a fi-kilobase (kb) 5 ' -flanking region of the human gene and demonstrated that a similar to 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GAFF is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. (C) 2001 John Wiley & Sons, Inc.

Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

Background: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. Methods: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. Results: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. Conclusions: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLR It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.

Relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass and muscle strength in elderly men and women: The health ABC study

Background. A decline in muscle mass and muscle strength characterizes normal aging. As clinical and animal studies show it relationship between higher cytokine levels and low muscle mass, the aim of this study was to investigate whether markers, of inflammation are associated with muscle mass and strength in well-functioning elderly persons. Methods. We Used baseline data (1997-1998) of the Health, Aging, and Body Composition (Health ABC) Study on 3075 black and white men and women aged 70-79 years. Midthigh muscle cross-sectional area (computed tomography), appendicular muscle mass (dual-energy x-ray ab absorptiometry). isokinetic knee extensor strength (KinCom). and isometric inip strength were measured. plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were assessed by enzyme-linked immunosorbent assay (ELISA). Results. Higher cytokine levels were generally associated with lower muscle mass and lower muscle strength. The most consistent relationship across the gender and race groups was observed for IL-6 and grip strength: per SD increase in IL-6, grip strength was 1.1 to 2.4 kg lower (p < .05) after adjustment for age, clinic Site. health status, medications, physical activity. smoking. height. and body fat. Ail overall measure of elevated cytokine level was created by combining the levels of IL-6 and TNF-alpha. With the exception of white men, elderly persons having high levels of IL-6 (> 1.80 pg/ml) as well as high levels of TNF-alpha (> 3.20 pg/ml) had a smaller muscle area, less appendicular mass. a lower knee extensor strength. and a lower grip strength compared to those with low levels of both cytokines. Conclusions. Higher plasma concentrations of IL-6 and TNF-alpha are associated with lower muscle mass and lower muscle strength in well-functioning older men and women. Higher cytokine levels. as often observed in healthy older persons. may contribute to the loss Of muscle mass and strength that accompanies aging.

Phase and microstructural evolution during the heat treatment of Sm-Ca-alpha-sialon ceramics

The phase and microstructural evolution of multi-cation Sm-Ca-alpha-sialon ceramics was investigated. Six samples were prepared, ranging from a pure Sm-sialon to a pure Ca-sialon, with calcium replacing samarium in 20 eq% increments, thus maintaining an equivalent design composition in all samples. After pressureless sintering at 1820 degreesC for 2 It, all samples were subsequently heat treated up to 192 h at 1450 and 1300 degreesC. The amount of grain boundary glass in the samples after sintering was observed to decrease with increasing calcium levels. A M-ss' or M-ss',-gehlenite solid solution was observed to form during the 1450 degreesC heat treatment of all Sm-containing samples, and this phase forms in clusters in the high-Sm samples. The thermal stability of the alpha-sialon phase was improved in the multi-cation systems. Heat treatment at 1300 degreesC produces SmAlO3 in the high-Sm samples, a M-ss',-gehlenite solid solution in the high-Ca samples, and a Sm-Ca-apatite phase in some intermediate samples. (C) 2002 Elsevier Science Ltd. All rights reserved.

Thermal stability of mixed-cation alpha-sialon ceramics

A series of alpha-sialon (alpha') compositions containing mixed stabilising cations were prepared, by introducing additional CaO to a basic Sm alpha-sialon compositions. The thermal stability of these Sm-Ca-containing alpha-sialon phases was investigated using XRD, SEM and EDXS techniques. It was found that the addition of calcium into the Sm alpha-sialon systems greatly improved the stability of the alpha-sialon phases. Calcium was found to be incorporated into the alpha-sialon structure, coexistent with the samarium, and partitioning of the calcium and samarium was observed between the alpha' phase and grain boundary phases. This indicates a technique which may be used to improve the thermal stability of the alpha' phase while maintaining good refractory phases at the gialon grain boundaries. (C) 2003 Elsevier Science B.V. All rights reserved.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in

Sodium channel alpha 1-subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms

Background: Mutations in SCN1A, the gene encoding the alpha1 subunit of the sodium channel, have been found in severe myoclonic epilepsy of infancy (SMEI) and generalized epilepsy with febrile seizures plus (GEFS(+)). Mutations in SMEI include missense, nonsense, and frameshift mutations more commonly arising de novo in affected patients. This finding is difficult to reconcile with the family history of GEFS(+) in a significant proportion of patients with SMEI Infantile spasms (IS), or West syndrome, is a severe epileptic encephalopathy that is usually symptomatic. In some cases, no etiology is found and there is a family history of epilepsy. Method: The authors screened SCN1A in 24 patients with SMEI and 23 with IS. Results: Mutations were found in 8 of 24 (33%) SMEI patients, a frequency much lower than initial reports from Europe and Japan. One mutation near the carboxy terminus was identified in an IS patient. A family history of seizures was found in 17 of 24 patients with SMEI. Conclusions: The rate of SCN1A mutations in this cohort of SMEI patients suggests that other factors may be important in SMEI. Less severe mutations associated with GEFS(+) could interact with other loci to cause SMEI in cases with a family history of GEFS(+). This study extends the phenotypic heterogeneity of mutations in SCN1A to include IS.

alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT(3) chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had 25-fold lower activity. These results indicate that as yet unidentified factors influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.