Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins.

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.

A controlled study of additional sr-L-dopa in L-dopa-responsive restless legs syndrome with late-night symptoms.

OBJECTIVE: To investigate whether a combination treatment of regular-release levodopa (rr-L-dopa) and sustained-release levodopa (sr-L-dopa) compared with monotherapy of rr-L-dopa improves sleep quality and reduces periodic limb movements (PLM) in patients with restless legs syndrome (RLS) and problems with maintaining sleep. BACKGROUND: Reappearance of RLS symptoms during the second half of the night while being treated with rr-L-dopa is a common problem in the treatment of sleep disturbances caused by RLS. METHODS: A randomized, controlled, double-blind crossover trial was undertaken. Eligible patients fulfilled the diagnostic criteria of the International RLS Study Group, and met an actigraphically confirmed higher number of PLM per hour time in bed (PLM index) during the second half compared with the first half of the night under treatment with rr-L-dopa. During the crossover periods the patients received 100 to 200 mg rr-L-dopa plus either placebo or 100 to 200 mg sr-L-dopa at bedtime for 4 weeks each period. RESULTS: Thirty patients with RLS (11 men and 19 women) were assessed by actigraphy and subjective sleep quality, and showed a significant improvement in PLM index (p < 0.0001), in "time in bed without movements" (p < 0.0001), and in subjective sleep quality (p < 0.001). Eight of 30 patients reported an altered pattern of RLS symptoms, characterized by a time shift of RLS symptoms into the afternoon or evening, five of these during monotherapy with rr-L-dopa. CONCLUSIONS: A combination therapy of rr-L-dopa and sr-L-dopa is better than monotherapy with rr-L-dopa in reducing the frequency of PLM and problems maintaining sleep, even in patients who are severely affected.

Rapid onset of action of levodopa in restless legs syndrome: a double-blind, randomized, multicenter, crossover trial.

OBJECTIVE: To investigate the efficacy and safety of levodopa plus benserazide in the treatment of restless legs syndrome (RLS), in terms of the frequency of periodic limb movements (PLMs), objective and subjective criteria of sleep, onset of action, and withdrawal effects. DESIGN: A randomized, double-blind, placebo-controlled, multicenter, crossover trial, with two 4-week treatment periods. SETTING: Outpatient units of three specialist centers in Germany. PATIENTS: Eligible patients had to fulfill the diagnostic criteria of the International RLS Study Group and have sleep disturbances and PLMs during sleep shown on polysomnography at screening. Thirty-five patients were recruited, of whom 32 (13 men, 19 women) completed the study. INTERVENTIONS: Patients received a single dose of standard-release levodopa/benserazide 100/25 mg or placebo at bedtime each night for 4 weeks, before crossing over to receive the alternative treatment for a further 4 weeks; the dose could be doubled if required. The average dosages were 159 +/- 31 mg of levodopa and 1.56 +/- 0.29 capsules of placebo. RESULTS: Levodopa/benserazide significantly reduced the number of PLMs per hour (p<0.0001), increased the time in bed without limb movements (p<0.0001), and improved subjective quality of sleep (p=0.0004). The onset of action was rapid after the first dose, and full efficacy was achieved within the first few days of therapy; these improvements disappeared immediately when treatment was discontinued. Levodopa/benserazide treatment was well tolerated and safe. CONCLUSIONS: Levodopa/benserazide is effective and safe in the treatment of RLS. Objective and subjective measures of sleep improved rapidly after the first dose. RLS symptoms recurred immediately after treatment was discontinued.

L-dopa therapy of uremic and idiopathic restless legs syndrome: a double-blind, crossover trial.

We report the effects of a single bedtime dose of L-dopa 100-200 mg on sleep quality, frequency of periodic leg movements (PLM) and daily living in patients with idiopathic and uremic restless legs syndrome (RLS). Seventeen patients with idiopathic and 11 with uremic (on continuous hemodialysis) RLS were evaluated comparatively by polysomnography, actigraphy and subjective ratings in a randomized, controlled and double-blind crossover trial with L-dopa and placebo for 4 weeks each. Neurophysiologic assessments showed significant reduction of the number of periodic leg movements (p = 0.003) and the PLM-index (p = 0.005) most pronounced during the first 4 hours of bedtime after L-dopa (p = 0.001). Subjective evaluation confirmed improvement of sleep quality (p = 0.002) and showed significantly higher quality of life during daytime (p = 0.030) while the patients received L-dopa therapy. We conclude that L-dopa 100-200 mg proved to be effective in idiopathic RLS and for the first time under controlled conditions in uremic RLS without any severe side effects.

New actigraphic assessment method for periodic leg movements (PLM).

A new actigraphic method by which periodic leg movements (PLM) can be measured is presented. Data acquistion and analysis were brought into line to distinguish short-lasting repetive leg movements from random motor restlessness. The definition of PLM follows the generally accepted criteria for PLM scoring. Thirty restless legs patients, all also suffering from PLM, were investigated three times by polysomnography, including tibialis anterior surface electromyography and actigraphy. A high correlation (reliability) was found for the number of PLM per hour spent in bed between the two methods. Furthermore, the actigraph records PLM specifically. An index of random motor restlessness is not sufficient for a reliable PLM according. In addition, periodic movements in sleep (PMS) and PLM show comparable variability in general. The actigraphic assessment of PLM, however, gives a better measure because PMS recordings may result in a substantial underestimation of PLM when sleep efficiency is reduced. This method is an ambulatory assessment tool that can also be used for screening purposes.

[Treatment of idiopathic and uremic restless legs syndrome with L-dopa--a double-blind cross-over study]. / Behandlung des idiopathischen und urämischen Restless-legs-Syndrom mit L-Dopa--Eine doppelblinde Cross-over-Studie.

In a randomized, controlled and double-blind cross-over trial with L-Dopa and placebo we evaluated the effects of a single bedtime dose of L-Dopa 100 to 200 mg on sleep quality and frequency of periodic leg movements (PLM) in 17 patients with idiopathic and in 11 patients with uremic restless legs syndrome (RLS) by polysomnography, actigraphy, and subjective ratings. Treatment with L-Dopa showed significant reduction of the number of periodic leg movements and the PLM-index most pronounced during the first 4 hours of bedtime. Subjective evaluation confirmed improvement of sleep quality. L-Dopa proved to be effective in idiopathic and uremic RLS without any severe side effects.

Structure of the murine homeobox gene cdx-2. Expression in embryonic and adult intestinal epithelium.

To gain insight into the mechanisms which govern cellular identity in the intestinal epithelium we have begun a detailed study of the murine cdx-2 homeobox gene. We isolated and sequenced both cDNA and genomic clones in order to define the open reading frame and mature transcript. A detailed analysis of cdx-2 transcript levels late in embryogenesis showed that they increased 6-7-fold at a time when the gut undergoes a major developmental transition. In the adult, cdx-2 was expressed in colon in a region-specific manner with transcripts some 5-fold more abundant in the cecum as compared with the rectum. In situ hybridization and immunohistochemical experiments showed cdx-2 transcripts and protein were present in all epithelial cells in the proximal colon irrespective of their degree of differentiation. In distal colon, however, transcripts were most abundant in undifferentiated cells at the bottom of crypts, whereas the highest protein levels were present in mature cells in the upper half of crypts. We conclude that cdx-2 is expressed specifically in gut epithelium where it is not restricted to a particular cell lineage. Rather, the rostrocaudal expression gradient suggests that it may play a role in specifying positional identity.

Isolation and characterization of the murine homeobox gene Cdx-1. Regulation of expression in intestinal epithelial cells.

The murine homeobox gene Cdx-1 is postulated to play an important role in the process of cellular commitment in the intestinal epithelium based upon its graded, tissue-specific expression pattern in the adult animal. We have isolated and sequenced both cDNA and genomic clones in order to understand how this expression pattern is regulated and to determine the amino acid sequence of the encoded protein. Our studies show that the Cdx-1 gene contains 3 exons and 2 introns and encodes a 268-amino acid protein which is translated from a 1.7-1.8-kilobase mRNA. Transcription of the Cdx-1 gene initiates some 30 base pairs downstream from a typical TATA box which, together with the 5' end of the gene, is located within a CpG island. Transient transfection experiments have shown three regions which regulate Cdx-1 gene expression. These include a typical silencer element at -589 to -380, an element at -887 to -1040 which blocks the effect of the silencer and a positive element at -47 to +66; the only one which shows epithelial cell specificity. Using gel shift and footprinting assays we have detected proteins in nuclear extracts prepared from colonic epithelial cells which bind to the silencer element. These initial results suggest that Cdx-1 gene expression is regulated by multiple positive and negative transcription factors.

Homeobox gene expression in the intestinal epithelium of adult mice.

Using a polymerase chain reaction-based strategy, we have detected the expression of nine different homeobox genes in adult mouse intestine. Included among these are the recently described intestine-specific Cdx-1 gene and a new, related gene, Cdx-2. Southern blot experiments show that Cdx-2 is present in a single copy in the mouse genome. Of several adult mouse tissues assayed, intestine was the only one that contained detectable levels of Cdx-2 mRNA. Expression of all nine homeobox genes in different regions of the intestine was quantitated by RNase protection analysis, which revealed a unique expression profile for each gene. These observations suggest that homeobox gene expression may play an important role in cellular differentiation in the adult intestine.

T-cell nuclei contain a protein that binds upstream of the murine granulocyte-macrophage colony-stimulating factor gene.

Stimulation of a murine T-cell line (O18A) by Con A has been shown to cause a large increase in the cytoplasmic granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA level. Using run-on transcription experiments in isolated nuclei, we have shown that some of this response is from enhanced transcription of the GM-CSF gene. Changes in the transcription rate of this gene can be seen as early as 30 min after adding the Con A. With a DNA fragment mobility-shift assay on nuclear extracts from these cells we detected a protein that binds upstream of the murine GM-CSF gene. Partial purification and concentration of this protein by DEAE-Sephacel and phosphocellulose chromatography enabled us to examine its interaction with DNA in more detail. Competition and methylation interference experiments have shown that the protein binds to the sequence 3'-TCCATCAAGGGG-5' (positions -90 to -82). This sequence is contained within a region found to be involved in regulating inducible GM-CSF transcription in a human T-cell line [Miyatake, S., Seiki, M., Yoshida, M. & Arai, K. (1988) Mol. Cell. Biol. 8, 5581-5587].